Plant Extracts

Plant Extracts
Biological and
Pharmacological Activity
Edited by
Raffaele Capasso and Lorenzo Di Cesare Mannelli
Printed Edition of the Special Issue Published in Molecules
www.mdpi.com/journal/molecules
Plant Extracts: Biological and
Plant Extracts: Biological and
Editors
Raffaele Capasso
Lorenzo Di Cesare Mannelli
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Raffaele Capasso Lorenzo Di Cesare Mannelli
University of Naples Federico II Universita degli Studi di Firenze
Italy Italy
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Contents
About the Editors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
Raffaele Capasso and Lorenzo Di Cesare Mannelli

Special Issue “Plant Extracts: Biological and Pharmacological Activity”
Reprinted from: Molecules 2020, 25, 5131, doi:10.3390/molecules25215131 . . . . . . . . . . . . . . 1
Fang-Rong Cheng, Hong-Xin Cui, Ji-Li Fang, Ke Yuan and Ying Guo
Ameliorative Effect and Mechanism of the Purified Anthraquinone-Glycoside Preparation from
Rheum Palmatum L. on Type 2 Diabetes Mellitus
Rosa Martha Pérez Gutierrez, Abraham Heriberto Garcı́a Campoy, Silvia Patricia Paredes
Carrera, Alethia Muñiz Ramirez, José Maria Mota Flores and Sergio Odin Flores Valle
3 -O-β-D-glucopyranosyl-α,4,2 ,4 ,6 -pentahydroxy- dihydrochalcone, from Bark of
Eysenhardtia polystachya Prevents Diabetic Nephropathy via Inhibiting Protein Glycation
in STZ-Nicotinamide Induced Diabetic Mice
Reprinted from: Molecules 2019, 24, 1214, doi:10.3390/molecules24071214 . . . . . . . . . . . . . 23
Thilina L. Gunathilaka, Kalpa W. Samarakoon, P. Ranasinghe and L. Dinithi C. Peiris

In-Vitro Antioxidant, Hypoglycemic Activity, and Identification of Bioactive Compounds in
Phenol-Rich Extract from the Marine Red Algae Gracilaria edulis (Gmelin) Silva
Truong Ngoc Minh, Truong Mai Van, Yusuf Andriana, Le The Vinh, Dang Viet Hau,
Dang Hong Duyen and Chona de Guzman-Gelani
Antioxidant, Xanthine Oxidase, α-Amylase and α-Glucosidase Inhibitory Activities of Bioactive
Compounds from Rumex crispus L. Root
Nguyen Van Quan, Tran Dang Xuan, Hoang-Dung Tran, Nguyen Thi Dieu Thuy,
Le Thu Trang, Can Thu Huong, Yusuf Andriana and Phung Thi Tuyen
Antioxidant, α-Amylase and α-Glucosidase Inhibitory Activities and Potential Constituents of
Canarium tramdenum Bark
Thania Alejandra Urrutia-Hernández, Jorge Arturo Santos-López, Juana Benedı́,

Francisco Jose Sánchez-Muniz, Claudia Velázquez-González,
Minarda De la O-Arciniega, Osmar Antonio Jaramillo-Morales and Mirandeli Bautista
Antioxidant and Hepatoprotective Effects of Croton hypoleucus Extract in an Induced-Necrosis
Model in Rats
Lucia Recinella, Annalisa Chiavaroli, Giustino Orlando, Luigi Menghini, Claudio Ferrante,
Lorenzo Di Cesare Mannelli, Carla Ghelardini, Luigi Brunetti and Sheila Leone
Protective Effects Induced by Two Polyphenolic Liquid Complexes from Olive (Olea europaea,
mainly Cultivar Coratina) Pressing Juice in Rat Isolated Tissues Challenged with LPS
v
Jae Sung Lim, Sung Ho Lee, Sang Rok Lee, Hyung-Ju Lim, Yoon-Seok Roh, Eun Jeong Won,
Namki Cho, Changju Chun and Young-Chang Cho
Inhibitory Effects of Aucklandia lappa Decne. Extract on Inflammatory and Oxidative Responses
in LPS-Treated Macrophages
Chukwuemeka R. Nwokocha, Isheba Warren, Javier Palacios, Mario Simirgiotis,

Magdalene Nwokocha, Sharon Harrison, Rory Thompson, Adrian Paredes,
Jorge Bórquez, Astrid Lavado and Fredi Cifuentes
Modulatory Effect of Guinep (Melicoccus bijugatus Jacq) Fruit Pulp Extract on
Isoproterenol-Induced Myocardial Damage in Rats. Identification of Major Metabolites
Using High Resolution UHPLC Q-Orbitrap Mass Spectrometry
Tianshuang Xia, Xin Dong, Yiping Jiang, Liuyue Lin, Zhimin Dong, Yi Shen, Hailiang Xin,
Qiaoyan Zhang and Luping Qin
Metabolomics Profiling Reveals Rehmanniae Radix Preparata Extract Protects against
Glucocorticoid-Induced Osteoporosis Mainly via Intervening Steroid Hormone Biosynthesis
Serena Silvestro, Santa Mammana, Eugenio Cavalli, Placido Bramanti and

Emanuela Mazzon
Use of Cannabidiol in the Treatment of Epilepsy: Efficacy and Security in Clinical Trials
Laura E. Ewing, Charles M. Skinner, Charles M. Quick, Stefanie Kennon-McGill, Mitchell

R. McGill, Larry A. Walker, Mahmoud A. ElSohly, Bill J. Gurley and Igor Koturbash
Hepatotoxicity of a Cannabidiol-Rich Cannabis Extract in the Mouse Model
Mario A. Tan, Mark Wilson D. Lagamayo, Grecebio Jonathan D. Alejandro and

Seong Soo A. An
Anti-Amyloidogenic and Cyclooxygenase Inhibitory Activity of Guettarda speciosa
Uthaiwan Suttisansanee, Somsri Charoenkiatkul, Butsara Jongruaysup, Somying Tabtimsri,

Dalad Siriwan and Piya Temviriyanukul
Mulberry Fruit Cultivar ‘Chiang Mai’ Prevents Beta-Amyloid Toxicity in PC12 Neuronal Cells
and in a Drosophila Model of Alzheimer’s Disease
Thais Biondino Sardella Giorno, Carlos Henrique Corrêa dos Santos,

Mario Geraldo de Carvalho, Virgı́nia Cláudia da Silva, Paulo Teixeira de Sousa Jr.,
Patricia Dias Fernandes and Fabio Boylan
Study on the Antinociceptive Activity and Mechanism of Action of Isolated Saponins from
Siolmatra brasiliensis (Cogn.) Baill
vi
Giustino Orlando, Gokhan Zengin, Claudio Ferrante, Maurizio Ronci, Lucia Recinella,
Ismail Senkardes, Reneta Gevrenova, Dimitrina Zheleva-Dimitrova, Annalisa Chiavaroli,
Sheila Leone, Simonetta Di Simone, Luigi Brunetti, Carene Marie Nancy Picot-Allain,
Mohamad Fawzi Mahomoodally, Kouadio Ibrahime Sinan and Luigi Menghini
Comprehensive Chemical Profiling and Multidirectional Biological Investigation of Two Wild
Anthemis Species (Anthemis tinctoria var. Pallida and A. cretica subsp. tenuiloba): Focus on
Neuroprotective Effects
Phyu Phyu Myint, Thien T. P. Dao and Yeong Shik Kim

Anticancer Activity of Smallanthus sonchifolius Methanol Extract against Human Hepatocellular
Carcinoma Cells
Weihui Deng, Ke Liu, Shan Cao, Jingyu Sun, Balian Zhong and Jiong Chun
Chemical Composition, Antimicrobial, Antioxidant, and Antiproliferative Properties of
Grapefruit Essential Oil Prepared by Molecular Distillation
Seham Abdel-Shafi, Abdul-Raouf Al-Mohammadi, Mahmoud Sitohy, Basma Mosa,

Ahmed Ismaiel, Gamal Enan and Ali Osman
Antimicrobial Activity and Chemical Constitution of the Crude, Phenolic-Rich Extracts of
Hibiscus sabdariffa, Brassica oleracea and Beta vulgaris
Cinzia Pagano, Maura Marinozzi, Claudio Baiocchi, Tommaso Beccari, Paola Calarco,
Maria Rachele Ceccarini, Michela Chielli, Ciriana Orabona, Elena Orecchini,
Roberta Ortenzi, Maurizio Ricci, Stefania Scuota, Maria Cristina Tiralti and Luana Perioli
Bioadhesive Polymeric Films Based on Red Onion Skins Extract for Wound Treatment:
An Innovative and Eco-Friendly Formulation
Anna Kurek-Górecka, Michał Górecki, Anna Rzepecka-Stojko, Radosław Balwierz

and Jerzy Stojko
Bee Products in Dermatology and Skin Care
Justine Bodin, Amandine Adrien, Pierre-Edouard Bodet, Delphine Dufour,

Stanislas Baudouin, Thierry Maugard and Nicolas Bridiau
Ulva intestinalis Protein Extracts Promote In Vitro Collagen and Hyaluronic Acid Production by
Human Dermal Fibroblasts
Yeon-hee Kim, Amy Cho, Sang-Ah Kwon, Minju Kim, Mina Song, Hye won Han,
Eun-Ji Shin, Eunju Park and Seung-Min Lee
Potential Photoprotective Effect of Dietary Corn Silk Extract on Ultraviolet B-Induced
Skin Damage
vii
Md. Adnan, Md. Nazim Uddin Chy, A.T.M. Mostafa Kamal, Md Obyedul Kalam Azad,
Arkajyoti Paul, Shaikh Bokhtear Uddin, James W. Barlow, Mohammad Omar Faruque,
Cheol Ho Park and Dong Ha Cho
Investigation of the Biological Activities and Characterization of Bioactive Constituents of
Ophiorrhiza rugosa var. prostrata (D.Don) & Mondal Leaves through In Vivo, In Vitro, and In
Silico Approaches
Muhammad Taher, Siti Syazwani Shaari, Deny Susanti, Dayar Arbain

and Zainul Amiruddin Zakaria
Genus Ophiorrhiza: A Review of Its Distribution, Traditional Uses, Phytochemistry, Biological
Activities and Propagation
Bahare Salehi, Lorene Armstrong, Antonio Rescigno, Balakyz Yeskaliyeva,

Gulnaz Seitimova, Ahmet Beyatli, Jugreet Sharmeen, Mohamad Fawzi Mahomoodally,
Farukh Sharopov, Alessandra Durazzo, Massimo Lucarini, Antonello Santini,
Ludovico Abenavoli, Raffaele Capasso and Javad Sharifi-Rad
Lamium Plants—A Comprehensive Review on Health Benefits and Biological Activities
Reprinted from: Molecules 10, 24, 1913, doi:10.3390/molecules24101913 . . . . . . . . . . . . . . . 439
Bahare Salehi, Esra Capanoglu, Nabil Adrar, Gizem Catalkaya, Shabnum Shaheen,
Mehwish Jaffer, Lalit Giri, Renu Suyal, Arun K Jugran, Daniela Calina, Anca Oana Docea,
Senem Kamiloglu, Dorota Kregiel, Hubert Antolak, Ewelina Pawlikowska, Surjit Sen,
Krishnendu Acharya, Zeliha Selamoglu, Javad Sharifi-Rad, Miquel Martorell,
Célia F. Rodrigues, Farukh Sharopov, Natália Martins and Raffaele Capasso
Cucurbits Plants: A Key Emphasis to Its Pharmacological Potential
Radu Claudiu Fierascu, Georgeta Temocico, Irina Fierascu, Alina Ortan

and Narcisa Elena Babeanu
Fragaria Genus: Chemical Composition and Biological Activities
Xu Zhang, Xiao Cheng, Yali Wu, Di Feng, Yifan Qian, Liping Chen, Bo Yang
and Mancang Gu
In Vitro and In Situ Characterization of the Intestinal Absorption of Capilliposide B and
Capilliposide C from Lysimachia capillipes Hemsl
Lee Suan Chua, Cher Haan Lau, Chee Yung Chew and Dawood Ali Salim Dawood
Solvent Fractionation and Acetone Precipitation for Crude Saponins from
Eurycoma longifolia Extract
Ya-Han Chen, Dong-Sheng Guo, Mei-Huan Lu, Jian-Ying Yue, Yan Liu, Chun-Ming Shang,
De-Rong An and Ming-Min Zhao
Inhibitory Effect of Osthole from Cnidium monnieri on Tobacco Mosaic Virus (TMV) Infection in
Nicotiana glutinosa
Reprinted from: Molecules 2020, 25, 65, doi:10.3390/molecules25010065 . . . . . . . . . . . . . . . 533
viii
About the Editors
Raffaele Capasso is Associate Professor at the Department of Agricultural Sciences of
the University of Naples Federico II, Portici (Naples), Italy. He holds a degree in
Pharmacy (1996), Master’s in Medicinal Plants (2000), Master’s in Pharmacology (2002), and Ph.D. in
Drug Science (2005). He has taught at the University of Naples Federico II as Professor of
Applied Pharmacognosy (starting from 2006) and Phytotherapy (starting from 2011). His main
research interests are as follows: role of cannabinoid, vanilloid, and kappa-opioid receptors in the
gastrointestinal tract, both in physiological (e.g., intestinal motility) and pathophysiological states
(e.g., inflammation and cancer); ethnopharmacological studies on medicinal plants and their active
ingredients used in traditional medicine; clinical pharmacology of herbal products; and nutritional
pharmacology. He has published 150 articles in peer-reviewed international journals (cited in JCI).
He is the recipient of the Farmindustria Prize (2006), from the Italian Society of Pharmacology, to the
best article published by a researcher under 35 years as a first author.
Lorenzo Di Cesare Mannelli was born in Florence, Italy, on September 29th, 1975. He
obtained his Degree in Pharmacy summa cum laude in 1999 (University of Florence), his Ph.D.
in Pharmacology and Toxicology (2005; University of Florence), and his specialization in Applied
Pharmacology (2006; University of Florence). He received postdoctoral fellowships in the
years 2006–2017. He has been a researcher (RTD-B) in pharmacology at Dept. Neurofarba, University
of Florence, since July 2017 (RTD-A till December 2018, then RTD-B). He became Associate Professor
in Pharmacology in 2016 and Full Professor in Pharmacology in 2018. He received the following
awards: Simpar, Study on multidisciplinary pain research, “Young against Pain” award (2016);
“AlbericoBenedicenti” award (2015); award at the Congress of Pharmacognosy, Italy (2014); travel
fellowship for Worldpharma (2010); award at 34◦ Nat Met Pharmacol (2009); travel fellowship for
VI Summer School of Neuroscience (2008). He organized the meetings “The Pharmacological Basis
of Novel Pain Therapeutics” (2017) and “Glial cells and therapeutic perspectives: from maladaptive
plasticity to neurorestoration” (2018). He was invited to speak at several (30) international scientific
meetings. He is a member of the editorial board or guest editor of the following journals: Frontiers
in Pharmacology, Frontiers in Cell Neuroscience, Molecules, Neural Regeneration Research, Current
Drug Targets, and Journal of Applied Pharmacology and Toxicology. He has received grants from the
“Istituto Toscano Tumori” (2014), the Italian Foundation for Multiple Sclerosis (2017), and Fondazione
Cassa di Risparmio di Firenze (2018); he also received a grant from the European Union for the
project IMI2 “Neuroderisk” (2019). He has collaborated with and received grants from the following
pharmaceutical companies: Apharm, Italy; MDM, Italy; Aboca, Italy, Metys Pharm, Switzerland;
Bioiberica, Spain; Kineta, USA; Daya, USA. His research activity is focused on the physiopathological
mechanisms of pain, cognitive functions, and memory and the relative pharmacological modulation.
In particular, he studies the neurotoxicity of anticancer drugs widely in vitro and in vivo. His research
activity is documented by about 200 full papers published in international peer-reviewed journals.
ix
molecules
Editorial
Special Issue “Plant Extracts: Biological and
Pharmacological Activity”
Raffaele Capasso 1, * and Lorenzo Di Cesare Mannelli 2, *
1 Department of Agricultural Sciences, University of Naples Federico II, 80055 Portici, Naples, Italy
2 Department of Neuroscience, Psychology, Drug Research and Child Health-Neurofarba-Section of
Pharmacology and Toxicology, University of Florence, 50139 Florence, Italy
* Correspondence: rafcapas@unina.it (R.C.); lorenzo.mannelli@unifi.it (L.D.C.M.)
Academic Editor: Derek J. McPhee

Received: 30 October 2020; Accepted: 2 November 2020; Published: 4 November 2020
The use of plant extracts for therapeutic purposes knows a wide diffusion. The vegetal origin of
these products intercepts people’s desire to cure themselves with natural drugs; this aspect, together with
effectiveness and regulatory opportunities, is the base of the modern broad use of medicinal plants.
Traditional uses and novel biological effects allow for availability of an extraordinary high number of
different compounds that may constitute a formidable therapeutic potential. Nevertheless, pitfalls are
hidden behind poor pharmacological and toxicological knowledge of plant extracts, non-standardized
methods of extraction, and undefined and not repeatable qualitative and quantitative composition.
In this context, novel experimental studies on plant products are necessary and appreciated to
reinforce the scientific soundness of phytotherapy. This Special Issue aims to respond to this medical
need, comprehensively highlighting the newest discoveries in vegetal resources with an emphasis on
pharmacological activity. More than 30, highly cited, articles were collected.
Cheng et al. [1] showed the hypoglycemic and anti-dyslipidemic effect of purified anthraquinone-
glycoside from Rheum palmatum L. in a rat model of type 2 diabetes mellitus. The anthraquinone
can reduce oxidative stress and regulate Fas/FasL-mediated apoptosis signaling pathway improving
β-cell function.
The dihydrochalcone 3 -O-β-d-glucopyranosyl α,4,2 ,4 ,6 -pentahydroxy–dihydrochalcone
isolated from Eysenhardtia polystachya was able to protect mice against diabetic nephropathy. It improved
renal dysfunction as well as it reduced glycated hemoglobin and advanced glycation end products in
the presence of significant histological recovery of kidney [2].
Gunathilaka and coworkers [3] reported the hypoglycemic and antioxidant in vitro activities of the
marine red algae Gracilaria edulis. A de-polysaccharide methanol extract of G. edulis was sequentially
partitioned by different solvents. The ethyl acetate fraction exhibited the strongest hypoglycemic and
antiglycation potential. Gas chromatography-mass spectrometry analysis of the ethyl acetate fraction
revealed the presence of several candidate anti-diabetic compounds.
Chrysophanol and physcion, isolated from the root of Rumex crispus L. emerged as relevant in the
medicinal properties of the plant against gout and diabetes. Compounds showed scavenging capacity,
xanthine oxidase and α-glucosidase inhibitory activity [4].
Quan et al. [5] showed the result of a phytochemical and pharmacological study on the Canarium
tramdenum bark. Five different extracts were chemically characterized, revealing that C. tramdenum
fruit possesses phenols and terpenoids, which might contribute to reducing risks from diabetes.
A high quantity of α- and β-amyrins highlighted the potentials of anti-inflammatory, anti-ulcer,
anti-hyperlipidemic, anti-tumor, and hepatoprotective properties of C. tramdenum bark.
An ethanol extract of Croton hypoleucus showed antioxidant and hepatoprotective activity in rats
with liver necrosis. Hepatic damage markers were reduced, whereas SOD and Cat gene expression
were increased, suggesting a control of antioxidant defense levels [6].
Molecules 2020, 25, 5131; doi:10.3390/molecules25215131 1 www.mdpi.com/journal/molecules

Molecules 2020, 25, 5131
Recinella et al. [7] described the antinflammatory effects of polyphenolic liquid complexes from
olive pressing juice with high levels of hydroxytyrosol. In isolated rat colon, liver, heart, and prefrontal
cortex samples, a tissue-dedicated molecular analysis revealed that the products exhibited protective
effects on multiple inflammatory and oxidative stress pathways.
The Korean plant Aucklandia lappa Decne., known as “Mok-hyang” was investigated as ethanol
extract for studying in vitro the anti-inflammatory and antioxidative effects. The extract reduced redox
unbalance and proinflammatory mediators by decreasing the nuclear translocation of p65 and by the
enhanced expression of hemeoxygenase 1 [8].
Nwokocha and collegues [9] reported the cardioprotective effects of the juice of Melicoccus bijugatus
fruit pulp. Using a rat model of myocardial injury, the authors showed that a repeated treatment
reduced blood pressure and heart rate as well as decreased heart to body weight ratio. Vitamin C and
related compounds, phenolic acids, flavonoid, fatty acids and terpene derivatives were individuated
as components.
In vivo and in vitro experiments, as well as a UHPLC-Q/TOF-MS-based metabolomics study,
showed the effect of the dry rhizome of Rehmannia glutinosa Libosch. in preventing osteoporosis and
its underlying mechanisms. Rehmannia glutinosa enhanced bone mineral density and improved the
micro-architecture of trabecular bone via interfering with the steroid hormone biosynthesis [10].
Regarding the nervous system, Silvestro et al. [11] studied the effect of cannabidiol, one of the
cannabinoids with non-psychotropic action extracted from Cannabis sativa, against the severe treatment-
resistant epilepsy in addition to common anti-epileptic drugs. An overview of recent literature and
clinical trials showed that the use of cannabidiol could represent hope for patients who are resistant to
conventional anti-epileptic drugs.
Cannabidiol was also studied for toxicological aspects [12]. In mice, cannabidiol induced signs of
hepatotoxicity, possibly of a cholestatic nature; hepatotoxicity gene expression arrays revealed that it
differentially regulated more than 50 genes, many of which were linked to oxidative stress responses,
lipid metabolism pathways and drug metabolizing enzymes.
Tan and coworkers [13] described the anti-inflammatory and neuroprotective properties of
Guettarda speciosa (chloroform and methanol extracts). G. speciosa was able to inhibit cyclooxygenase
assay (partial selectivity for COX-1), further it reduced amyloid-beta aggregates in the neuronal cell
line, suggesting possible anti-neurodegenerative applications.
Beta-amyloid-induced neurotoxicity was also prevented by an aqueous extract of Morus nigra
‘Chiang Mai’ [14]. High amounts of cyanidin, keracyanin, and kuromanin as anthocyanidin and
anthocyanins were found in the extract. M. nigra promoted neurite outgrowth and improved locomotory
coordination of Drosophila co-expressing human amyloid precursor protein and BACE-1 specifically in
the brain.
Siolmatra brasiliensis (Cogn.) Baill, (“taiuiá”, “cipó-tauá”) and its isolated substances (cayaponoside
A1, cayaponoside B4, cayaponoside D, and siolmatroside I) were studied for relieving pain [15].
Hydroethanol extract, ethyl acetate fraction, and isolated saponins showed analgesic effects and
reduced capsaicin- or glutamate-induced hypersensitivity by a mechanism involving muscarinic and
opioid signaling.
Ethyl acetate, methanol, and aqueous extracts of aerial parts of Anthemis tinctoria var. pallida and
A. cretica subsp. tenuiloba were investigated for their phenol and flavonoid content, antioxidant, and key
enzyme (AChE, BChE. tyrosinase and α-glucosidase) inhibitory potentials [16]. Further, ex vivo
studies highlighted neuroprotective properties after an excitotoxicstimulus promoting LDH level and
5-HT turnover normalization as well as the restoration of proteins involved in neuron morphology
and neurotransmission.
Myint et al. [17] studied the activity of a methanol extract of Smallanthus sonchifolius leaf against a
human hepatocellular carcinoma cell line. The extract reduced cell proliferation and cell migration,
it also induced cell cycle arrest and necrosis in a concentration-dependent manner. Putative active
components were melampolide-type sesquiterpenoids.
2
Molecules 2020, 25, 5131
Antiproliferative properties were also depicted for the grape fruit essential oil [18]. Deng et al.
detected by GC-MS twenty-four components (terpenes and oxygenated terpenes); the light phase oil
displayed inhibitory effects on liver cancer cells proliferation, antimicrobial effects against Bacillus
subtilis, Escherichia coli, Staphylococcusaureus and Salmonella typhimurium as well as antioxidant activity.
Antimicrobial activities were described for crude, phenolic-rich extracts of Hibiscus sabdariffa
Brassica oleracea var. capitata f. rubra and Beta vulgaris [19]. Total anthocyanins, phenols, flavonoids
contents were analyzed. Extracts and isolated compounds showed antimicrobial effects against
pathogenic bacteria and fungi. Electron microscopy analysis revealed bacteria morphological alteration,
indicating death and loss of cell content.
Pagano et al. [20] studied the non-edible outside layers of onion for wound healing. A hydroalcoholic
extract was formulated in auto adhesive, biocompatible and pain-free hydrogel polymeric films,
it showed antioxidant, radical scavenging, antibacterial and anti-inflammatory activities suggesting a
potential dermal application for wound treatment.
Kurek-Gorecka and colleagues [21] reported the beneficial effects of bee products in dermatology
and skin care. Honey, propolis, bee pollen, bee bread, royal jelly, beeswax and bee venom contain
biologically active components, such as flavonoid schrysin, apigenin, kaempferol, quercetin, galangin,
pinocembrin or naringenin. These components justify the use of bee products for medical or cosmetic
skin treatment based on antibacterial, anti-inflammatory, antioxidant, disinfectant, antifungal and
antiviral properties.
A protein fraction from Ulva intestinalis containing 51% of proteins and 22% of polysaccharides was
analyzed and tested for the anti-aging potential, fibroblast proliferation and collagen and hyaluronic
acid production on human fibroblast cell lines. A significant increase in collagen and hyaluronic
acid production per cell, and a reduction in cell proliferation without increasing cell mortality were
demonstrated [22].
UVB-induced skin damage in mice was reduced by dietary corn silk [23]. Oral administration
decreased epidermal thickness, wrinkle formation, and positive staining for PCNA, Ki67, and 8-OHdG,
and increased collagen staining. Pro-inflammatory NF-κB target genes and MMP-9 expressions were
lowered, whereas TGF-β/Smad signaling increased. Low skin lipid peroxidation and blood DNA
oxidation levels and high blood glutathione were detected in parallel with higher levels of catalase,
SOD1 and glutaredoxin.
A wide description of Ophiorrhiza rugosa var. prostrata was performed by Adnan and coworkers [24].
The ethanolic extract of leaves, in three different vivo models, evoked antidiarrheal, anti-inflammatory,
anthelmintic and antibacterial effects. Additionally, ADME and PASS analysis revealed a suitable
profile for future medicinal development.
Almost 50 species of Ophiorrhiza plants were reviewed by Taher and colleagues [25]. The analysis
revealed their wide distribution across Asia and the neighboring countries, whereby they were utilized
as traditional medicine to treat various diseases. Biological activities encompass anti-cancer, antiviral,
antimicrobial, and more. The genus propagation reported could produce a high quality and quantity
of potent anticancer compound, namely camptothecin (CPT).
An updated snapshot of Lamium plants and their biological activities were provided by
Salehi et al. [26]. Botanical, phytochemical and biological characteristics were described, highlighting
antimicrobial, antiviral, anti-inflammatory, cytoprotective, anti-nociceptive properties.
Again, Salehi with an international team provided a deed analysis of the Cucurbita genus [27].
The traditional efficacy against gastrointestinal diseases and intestinal parasites were correlated
with their nutritional and phytochemical composition. Among chemical constituents, carotenoids,
tocopherols, phenols, terpenoids, saponins, sterols, fatty acids, and functional carbohydrates and
polysaccharides were those occurring in higher abundance. More recently, a huge interest in a class of
triterpenoids, cucurbitacins, has been stated.
A deep analysis of the Fragaria genus was presented by Fierascu and colleagues [28]. Strawberries
possess biological properties, including antioxidant, antimicrobial and anti-inflammatory effects,
3
Molecules 2020, 25, 5131
but only a few species represent the subject of the last decade of scientific research. The main
components identified in the Fragaria species were here described.
Zhang et al. [29] determined the processes and mechanisms of intestinal absorption of capilliposide
B and C from Lysimachia capillipes Hemsl. Mechanisms involve processes such as facilitated passive
diffusion, efflux transporters, and enzyme-mediated metabolism. Both capilliposides were suggested
to be substrates of P-glycoprotein and multidrug resistance-associated protein 2. Capilliposide B may
interact with the CYP3A4 system.
A phytochemical analysis on saccharide-containing compounds from Eurycoma longifolia was
perfomed by Chua et al [30]. Non-toxic solvent fractionation increased the total saponin content,
evoking anti-proliferative activity against human breast cancer cells.
Osthole was proposed for the treatment of tobacco mosaic virus [31]. Extracted from Cnidium
monnieri, osthole showed comparable or stronger antiviral activity than eugenol and ningnanmycin.
A direct effect on the viral particles was suggested.
A second edition of this Special Issue is in preparation.
References
1. Cheng, F.-R.; Cui, H.X.; Fang, J.L.; Yuan, K.; Guo, Y. Ameliorative Effect and Mechanism of the Purified
Anthraquinone-Glycoside Preparation from Rheum Palmatum L. on Type 2 Diabetes Mellitus. Molecules 2019,
24, 1454. [CrossRef] [PubMed]
2. Rosa, M.P.G.; Abraham, H.G.C.; Silvia, P.P.C.; José, A.M.R.; Maria, M.F.; SergioOdin, F.V. 3 -O-β-D-
glucopyranosyl-α,4,2 ,4 ,6 -pentahydroxy-dihydrochalcone, from Bark of Eysenhardtiapolystachya Prevents
Diabetic Nephropathy via Inhibiting Protein Glycation in STZ-Nicotinamide Induced Diabetic Mice. Molecules
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Sample Availability: Samples of the compounds . . . . . . are available from the authors.
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article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
6
molecules
Article
Ameliorative Effect and Mechanism of the Purified
Anthraquinone-Glycoside Preparation from Rheum
Palmatum L. on Type 2 Diabetes Mellitus
Fang-Rong Cheng 1 , Hong-Xin Cui 1,2 , Ji-Li Fang 3 , Ke Yuan 3, * and Ying Guo 4
1 College of Pharmacy, Henan University of Chinese Medicine, Zhengzhou 450046, China;
chengfr1963888@126.com (F.-R.C.); cuihongxin1974@163.com (H.-X.C.)
2 Collaborative Innovation Center for Respiratory Disease Diagnosis and Treatment & Chinese Medicine
Development of Henan Province, Zhengzhou 450046, China
3 Jiyang College of Zhejiang Agriculture and Forestry University, Zhu’ji 311800, China; fang_qiao418@163.com
4 Zhejiang Chinese Medical University, Zhejiang, Hangzhou 310053, China; littlegy@163.com
* Correspondence: keyuan@zafu.edu.cn; Tel.: +86-0575-87760143
Academic Editors: Raffaele Capasso and Lorenzo Di Cesare Mannelli

Received: 28 February 2019; Accepted: 4 April 2019; Published: 12 April 2019
Abstract: Rheum palmatum L. is a traditional Chinese medicine with various pharmacological

properties, including anti-inflammatory, antibacterial, and detoxification effects. In this study,
the mechanism of the hypoglycemic effect of purified anthraquinone-Glycoside from Rheum palmatum L.
(PAGR) in streptozotocin (STZ) and high-fat diet induced type 2 diabetes mellitus (T2DM) in rats
was investigated. The rats were randomly divided into normal (NC), T2DM, metformin (Met), low,
middle (Mid), and high (Hig) does of PAGR groups. After six weeks of continuous administration
of PAGR, the serum indices and tissue protein expression were determined, and the pathological
changes in liver, kidney, and pancreas tissues were observed. The results showed that compared
with the type 2 diabetes mellitus group, the fasting blood glucose (FBG), total cholesterol (TC),
and triglyceride (TG) levels in the serum of rats in the PAGR treatment groups were significantly
decreased, while superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) levels were
noticeably increased. The expression of Fas ligand (FasL), cytochrome C (Cyt-c), and caspase-3
in pancreatic tissue was obviously decreased, and the pathological damage to the liver, kidney,
and pancreas was improved. These indicate that PAGR can reduce oxidative stress in rats with
diabetes mellitus by improving blood lipid metabolism and enhancing their antioxidant capacity,
thereby regulating the mitochondrial apoptotic pathway to inhibitβ-cell apoptosis and improve β-cell
function. Furthermore, it can regulate Fas/FasL-mediated apoptosis signaling pathway to inhibit
β-cell apoptosis, thereby lowering blood glucose levels and improving T2DM.
Keywords: Rheum palmatum L.; type 2 diabetes mellitus; oxidative stress; apoptosis
1. Introduction
Diabetes mellitus is an endocrine and metabolic disease characterized by hyperglycemia,
often accompanied by a series of complications, including neuropathy, nephropathy, retinopathy,
and cardiovascular and cerebrovascular disease [1,2]. It is a global disease affecting over 300 million
people and is the fourth cause of death and disability in the world [3]. Notably, 90% of patients are
type 2 diabetes mellitus (T2DM). The metabolic disorder of T2DM is associated with insulin resistance,
which is linked to genetic, environmental interactions, and lifestyle [4]. Insulin resistance and insulin
secretion defect are the two most recognized causes of T2DM [5]. Insulin resistance is caused by a
decrease in the efficiency of insulin-induced glucose uptake and utilization, and insulin secretion

Molecules 2019, 24, 1454
defects are caused by insufficient insulin secretion due to apoptosis or dysfunction of β-cells of the
pancreatic islets, resulting in the insulin which cannot meet normal physiological needs [6]. β-cell can
regulate systemic metabolism by secreting insulin, the body’s only hormone lowering glucose [7].
Studies have shown that the mass of β-cell is lowered in the pancreas when there is an imbalance of
regeneration and apoptosis [8]. There are many mechanisms inducing apoptosis of β-cells, including
hyperglycemia, hyperlipidemia, oxidative stress, and activation of proinflammatory factors [9,10].
Therefore, protecting β-cells in the pancreas, inhibiting β-cell apoptosis, and increasing insulin secretion
are important for T2DM patients [11].
Rheum palmatum L., a traditional Chinese medicine, has antibacterial, heat-clearing, and detoxifying
properties, and has been used for the treatment of constipation and gastrointestinal diseases for more
than 2000 years [12,13]. Although its chemical composition is relatively complex [14], it is known
that anthraquinone, forming 3–5% of its content, is the most important activity component [15].
Modern pharmacological studies have shown that rhubarb-derived anthraquinones have a variety
of physiological functions, such as antioxidant, antiviral, and anti-tumor properties, and protect
cerebral cortex neurons [16–18]. In this paper, we studied the glucose-lowering effect of purified
anthraquinone-Glycoside from Rheum palmatum L. (PAGR) in T2DM rats and explored the potential
mechanism in the context of lipid metabolism, oxidative stress, and apoptosis.
2. Results
2.1. Anthraquinone-Glycosides Content of PAGR

The prepared working solution was analyzed three times under 4.3.1 chromatographic
conditions of high-performance liquid chromatography (HPLC), and the contents of the three
anthraquinone-Glycosides were determined (Figure 1). The concentrations of emodin-8-O-β-d-glucoside,
aloe-emodin-8-O-β-d-glucoside, and chrysophanol-8-O-β-d-glucoside were calculated using the
previously determined standard curve and determined to be 6.36%, 8.13%, and 6.78%, respectively, while
the relative standard deviation (RSD) was 1.87%, 2.02%, and 1.58% respectively. The results showed that
these three anthraquinone-Glycosides make up more than 20% of the total anthraquinone-Glycosides,
and aloe-emodin-8-O-β-d-glucoside was the major anthraquinone-Glycoside among them.
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Molecules 2019, 24, 1454
Figure 1. The structure of the emodin-8-O-β-d-glucoside (I), aloe-emodin-8-O-β-d-glucoside (II),

and chrysophanol-8-O-β-d-glucoside (III) and HPLC chromatogram of purified anthraquinone-
Glycoside from Rheum palmatum L. (PAGR) (a) and mixed Reference (b).
2.2. Acute Toxicity Study

The acute toxicity of PAGR was evaluated at a dose of 2000 mg/kg. There were no deaths
or obvious symptoms of poisoning within the initial 24 h of close observation and no behavioral
abnormalities over the following 14 days. Therefore, the doses of PAGR for the low, middle (Mid),
and high (Hig) groups were selected as 100, 200, and 400 mg/kg, respectively.
2.3. Changes inBody Weight and Fasting Blood Glucose in Rats

Polydipsia, polyuria, polyphagia, weight loss, and hyperglycemia are the most typical symptoms of
T2DM [19]. The changes in rats’ body weight and fasting blood glucose (FBG) during the administration
of streptozotocin (STZ) and PAGR are shown in Figure 2. The body weight of rats treated with STZ and a
high-fat diet was obviously decreased, while FBG levels were markedly increased. After administration
of metformin (Met) and PAGR, the body weight and FBG levels of rats began to recover. Notably,
after six weeks of treatment, the body weight of the rats in the Met and PAGR treatment groups
was significantly higher than that of the T2DM group (p < 0.05), and their FBG concentration was
significantly lower than the T2DM group (p < 0.05). Furthermore, the Hig treatment group had
returned to close to normal levels.
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Molecules 2019, 24, 1454
Figure 2. The changes in body weight (a) and fasting blood glucose (FBG) (b) of rats during the
administration of Met and purified anthraquinone-Glycoside from Rheum palmatum L. (PAGR). NC,
normal group; T2DM, type 2 diabetes mellitus; Met, metformin group; Low, low group (100 mg/kg); Mid,
middle group (200 mg/kg); Hig, high group (400 mg/kg). The data were expressed as mean ± standard
deviation (SD) (n = 10), # p < 0.05 ## p < 0.01 vs.NC group; * p < 0.05 ** p < 0.01 vs. T2DM group.
2.4. Effect of PAGR on Lipid Metabolites and Antioxidant Enzyme Activities in Rats
The effect of PAGR on triglyceride (TG) and total cholesterol (TC) is shown in Figure 3a,b.
Compared with the normal (NC) group, the TC and TG of the T2DM group were significantly increased
(p < 0.01). However, compared with the T2DM group, the TC and TG of the Met and PAGR treatment
groups were significantly decreased in a dose-dependent manner. The specific dates were shown in
Table S.
By measuring the concentration of superoxide dismutase (SOD) and glutathione peroxidase
(GSH-PX) in the serum of rats, it was found that the SOD and GSH-PX levels in the T2DM group
were significantly decreased when compared with the NC group (p < 0.05), while those in the Met
and PAGR treatment groups were markedly increased when compared with the T2DM group, with
significant differences between the Met and PAGR Hig groups (p < 0.05) (Figure 3c,d).
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Molecules 2019, 24, 1454
In the course of the development of T2DM, the renal tissue is damaged, and its functions were
altered due to long-term of dysglycemia [20]. It is shown as Figure 3e,f that the serum creatinine and
blood urea nitrogen (BUN) in the T2DM group were significantly increased compared with the NC
group (p < 0.01), while the serum creatinine and BUN in the Met and PAGR treatment groups were
significantly decreased when compared with T2DM group, indicating that PAGR has a protective effect
on the kidney.
Figure 3. Effects of purified anthraquinone-Glycoside from Rheum palmatum L. (PAGR) on total

cholesterol (TC) (a), triglyceride (TG) (b), superoxide dismutase (SOD) (c), glutathione peroxidase
(GSH-PX) (d), creatinine (e), and BUN (f) in serum of rats. NC, normal group; T2DM, type 2 diabetes
mellitus; Met, metformin group; Low, low group (100 mg/kg); Mid, middle group (200 mg/kg); Hig,
high group (400 mg/kg). The data were expressed as mean ± SD (n = 10), # p < 0.05, ## p < 0.01 vs. NC
group. * p < 0.05, ** p < 0.01 vs. T2DM group.
2.5. The Effect of PAGR on Histopathological Changes
2.5.1. Effects of PAGR on Histopathological Changes in Liver

Hyperglycemia not only affects the transduction of apoptotic signals in islet cells but also damages
tissues and organs [21]. As one of the major metabolic organs, the liver is the main organ damaged
by diabetes. Steatosis, inflammatory cell infiltration, and necrosis are the main characteristics of
hyperglycemia-induced liver injury [22]. As evident in Figure 4, the structure of liver tissue of rats in
the NC group is intact, and the hepatocytes are arranged radially around the central vein. In contrast,
compared with the NC group, the number of hepatocytes in the T2DM group is reduced, the hepatic
cord is disordered, and hepatocytes show obvious degeneration and necrosis. The hepatocytes
surrounding the central vein are filled with lipid droplets, and the infiltration of inflammatory cells is
also evident. Notably, these histopathological changes in the livers of the Met and PAGR treatment
groups were improved to varying degrees when compared with the T2DM group.
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Molecules 2019, 24, 1454
Figure 4. The effects of purified anthraquinone-Glycoside from Rheum palmatum L. (PAGR) on

histopathological changes in the liver of rats. Histological observation, H&E, a–f×200; (a), normal
(NC) group; (b), type 2 diabetes mellitus (T2DM) group; (c), metformin (Met) group; (d), Low group;
(e), middle (Mid) group; (f), high (Hig) group; CV, central veins; , hepatocyte; , inflammatory
cell; , lipid droplet.
2.5.2. Effects of PAGR on Histopathological Changes in Kidney

Reduction of SOD and GSH-PX levels and an increase of lipid peroxide and free radicals in vivo
can all increase the expression of transforming growth factor (TGF)-β1 [23]. TGF-β1 can inhibit cell
proliferation, promote renal cell hypertrophy, and lead to glomerulosclerosis and tubule interstitial
fibrosis [24]. It can be seen in Figure 5 that the renal tissue structure was distinct and clear and the
glomerular structure was normal in the NC group. In contrast, the glomerular volume of the model
group was enlarged, and the basement membrane was thickened when compared with the NC group.
However, compared with the T2DM group, the glomerular volume of the Met and PAGR treatment
groups was smaller, the mesangial matrix had slight hyperplasia, and the histopathological changes
were obviously improved.
Figure 5. Effects of purified anthraquinone-Glycoside from Rheum palmatum L. (PAGR) on

histopathological changes in the kidney of rats. Histological observation, H&E, a-f×400; (a), normal
(e), middle (Mid) group; (f), high (Hig) group; , glomerulus; , base membrane; , mesangial.
12
Molecules 2019, 24, 1454
2.5.3. Effects of PAGR on Histopathological Changes in Pancreas

In addition to persistent hyperglycemia, patients with type 2 diabetes often have accompanying
hyperlipidemia, and long-term exposure to high concentrations of glucose or lipids not only causes
functional disorders of islet cells but also disrupts the islet structure [25]. It can be seen in Figure 6
that NC group rats showed normal histology. The islet shape was regular. Islet cells formed clumps
and were distributed among the pancreas exocrine glands. Compared with the NC group, the islet
structure in the T2DM group showed obvious deformation, fewer islet cells, blurred boundaries,
and the structure was unclear. Compared with the T2DM group, the histopathological changes present
in the islet tissue of the Met and PAGR treatment groups were attenuated to some degree.
Figure 6. Effects of purified anthraquinone-Glycoside from Rheum palmatum L. (PAGR) on

histopathological changes in the pancreas of rats. Histological observation, H&E, a–f×400; (a), normal
(e), middle (Mid) group; (f), high (Hig) group; , islet; , islet cell; , lipid droplet.
2.6. Effect of PAGR on the Expression of Cytochrome C (Cyt-c), Caspase-3, and FasLin Pancreas
As shown in Figure 7, the expression of FasL, Cyt-c, and caspase-3 in pancreatic tissue of rats after
administration of PAGR and metformin was significantly reduced compared with the T2DM group.
Figure 7. The effect of purified anthraquinone-Glycoside from Rheum palmatum L. (PAGR) on the
protein expression of Fas ligand (FasL), cytochrome C (Cyt-c), and Caspase-3 in pancreatic tissue. NC,
normal group; T2DM, type 2 diabetes mellitus; Met, metformin group; Low, low group (100 mg/kg);
Mid, middle group (200 mg/kg); Hig, high group (400 mg/kg).
13
Molecules 2019, 24, 1454
3. Discussions
STZ is the most commonly used hydrophilic compound for inducing T2DM. It can be transported
into the cell membrane through the glutamine transaminase transporter to induce DNA alkylation.
DNA alkylation can induce β-cell death [26], which consequently leads to hyperglycemia. Moreover,
a high-fat diet plays an important role in insulin resistance [27]. The gradual natural progression and
changes in the metabolism of human patients with T2DM are well mimicked by a high-fat diet and
low-dose STZ in rats [28].
There is an inseparable relationship between glucose metabolism and lipid metabolism.
Hyperglycemia can lead to dyslipidemia, while abnormal lipid metabolism is considered a major risk
factor for diabetes and its multiple complications [29]. Lipid metabolites, such as TG and TC, directly
antagonize insulin signaling and are considered the main cause of insulin resistance [30]. When TG
remains high, heparin activates lipoprotein lipase, increasing intravascular lipolysis of TG, thereby
increasing the exposure of tissues to free fatty acids, leading to insulin resistance and impairing β-cell
function [31]. TC is the sum of cholesterol contained in all lipoproteins in the blood and is closely related
to various diabetic complications, including cardio-cerebral vascular disease and neuropathy [32].
Therefore, improving lipid metabolism may ameliorate diabetes and its complications [33].
Hyperglycemia can also induce oxidative stress and lipid peroxidation. Importantly, oxidative
stress can regulate insulin secretion in different ways and accelerate the development of diabetes
mellitus [34]. For example, increased oxidative stress may have a negative effect on the regulation
of blood glucose and cause dysfunction or apoptosis of glucose-regulating cells, such as β-cells,
by stimulating the stress-responsive pathway for regulation [35]. Oxidative stress stimulates
mitogen-activated protein kinase (MAPK) stress signals and causes inhibition of insulin signaling [36].
In addition, oxidative stress can promote the expression of many proinflammatory factors, including
tumor necrosis factor (TNF)-α and interleukin (IL)-6, and significantly decrease insulin sensitivity [37].
Furthermore, studies have shown that there is a direct interaction between oxidative stress and
insulin resistance, and the accumulation of oxidation products may damage critical macromolecules in
insulin-sensitive tissues [38]. Therefore, from the perspective of treatment, reducing oxidative stress in
the body may ameliorate diabetes. SOD and GSH-PX are two important enzymes in the antioxidant
system, which can reflect the body s antioxidant capacity. It was found in our study that PAGR could
improve glucose and lipid metabolism in diabetic rats and reduce oxidative stress. Therefore, it may
act by improving the function of insulin secreting β cells.
Under normal physiological conditions, the number of β-cells in the pancreas is in a dynamic
equilibrium due to the regulation of apoptosis, proliferation of pancreatic islet, and production of new
insulin by secretory tube. However, diabetes can develop when β-cell apoptosis occurs in excess [29].
There are two main pathways of apoptosis, the intrinsic (mitochondrial driven) pathway and the
extrinsic (receptor-mediated) pathway [39]. Both oxidative stress and abnormal lipid metabolism
promote the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS). ROS and
RNS can change the membrane potential of mitochondria, leading to the release of Cyt-c [40]. The release
of Cyt-c activates caspase-3 [41], which is the final effector caspase in the caspase cascade and is the
common downstream effector of multiple apoptotic pathways [42]. Caspase-3 induces apoptosis
and thereby drives the death of β-cells resulting in insufficient insulin secretion. Correspondingly,
the process of apoptosis is accompanied by a change in mitochondrial function and structure, which will
also lead to the leakage of Cyt-c. As a marker of mitochondrial damage, Cyt-c levels can reflect
the degree of mitochondrial structural damage [43]. Mitochondria act as energy transducers for
cells, and their destruction can result in the abnormal function of islet β-cells, as shown in Figure 8.
The expression of Cyt-c and caspase-3 corresponded with the increased levels of SOD and GSH-PX in
the serum. PAGR can improve the antioxidant capacity of rats with diabetes and thus reduce the level
of oxidative stress, regulating the mitochondrial-induced apoptosis pathway and reducing the damage
to the mitochondrial structure, thereby protecting the pancreatic β-cells from apoptosis and restoring
the function of β-cells.
14
Molecules 2019, 24, 1454
Figure 8. The signaling pathway of apoptosis. Hyperglycemia leads to the production of reactive
oxygen species (ROS) and reactive nitrogen species (RNS) which change the membrane potential of
mitochondria, leading to the release of cytochrome C (Cyt-c). It also leads to the expression of Fas
ligand (FasL) and Fas, and they combine to form the Fas-associated death domain protein (FADD).
The Cyt-c and FADD all activate caspase cascade reaction, leading to apoptosis.
High concentration of glucose also promotes the expression of Fas and FasL in pancreatic
tissue [44]. FasL is an important ligand for inducing cell death and is a signaling factor of the extrinsic
apoptosis pathway [45]. When it binds to Fas, which is the death receptor, it induces the assembly of a
series of proteins that induce the death signaling complex in seconds. These proteins then activate
procaspase-8, leading to activation of the caspase cascade, which finally induces the activation of
caspase-3 [46]. When the caspase cascade is activated, it induces apoptosis swiftly [47], resulting in
decreased islet β-cells, which is the basis for the decline of insulin secretion. The results of western
blotting suggested that PAGR could inhibit apoptosis of β-cells and improve insulin secretion by
regulating the Fas/FasL-mediated apoptotic signal pathway.
4. Material and Methods
4.1. Drugs and Chemicals

Rheum palmatum L. medicinal materials were purchased from Zhejiang University of Traditional
Chinese Medicine Chinese Herbal Pieces Co., Ltd. (Zhejiang, China) and identified by the Professor
Jiawei Huang who is from Zhejiang Chinese Medical University. STZ was purchased from Aladdin
Bio-reagent (Shanghai, China). Glucose, Superoxide dismutase, Glutathione peroxidase, Triglyceride,
Total cholesterol, Serum creatinine, Blood urea nitrogen Kits were purchased from Nanjing Jiancheng
Biotechnology Co., LTD (Nanjing, China). The BCA Kit was purchased from Aidlab Biotechnologies Co.,
LTD (Beijing, China). The antibodies specific to FasL, Cyt-c, caspase-3 were purchased from Xinbosheng
Technology Co., LTD (Shanghai, China). Emodin-8-O-β-d-glucoside, aloe-emodin-8-O-β-d-glucoside,
chrysophanol-8-O-β-d-glucoside substances were prepared in the laboratory, the structure was
identified by 1 H NMR, 13 C-NMR, and MS, and the purity was over 98% calibrated by HPLC peak area
normalization method. All other reagents used were also chromatographically or analytically pure.
4.2. The Preparation and Determination of PAGR

Ten kg roots of Rheum palmatum. L. medicinal material was weighed and crushed and passed
through a 60-mesh sieve. Then, it was extracted by reflux extraction three times for 1.5 h each, with a 1:7
ratio of plant material to 80% ethanol, followed by extraction filtration. The filtrate was combined and
15
Molecules 2019, 24, 1454
concentrated under reduced pressure at 60 ◦ C bya rotary evaporator (RV3V, Staufen, Germany) until
no alcohol could be smelled, and the appropriate amount of water was added for ultrasonic dissolution.
A concentrate of 6000 mL was obtained. The concentrate was extracted three times with petroleum
ether, ethyl acetate, and n-butanol at a volume ratio of 2 times, and the extraction liquid was combined.
Then, it was concentrated under vacuum pressure to obtain the dry powder from each extracted
fraction, in which the petroleum ether fraction was 158.4g, the ethyl acetate fraction was 212.7 g,
the n-butanol fraction was 343.6 g, and the water was 425.9 g. The dry powder from the n-butanol
fraction was dispersed in water by ultrasound and enriched and purified using a macroporous resin
column (Diaion HP-20, Tokyo, Japan). First, it was eluted with distilled water, followed by 10%, 20%,
40%, and 60% methanol. Each eluted fraction was collected and concentrated under reduced pressure
to dryness to obtain dry powder from each elution fraction. The total anthraquinone-Glycoside content
in each elution fraction was determined by ultraviolet absorption spectrometry (UV-1801, Beijing,
China) with emodin-8-O-β-d-glucoside as the standard substance, and the content was 4.63%, 8.84%,
21.91%, and 6.32%, respectively, determined by ultraviolet and visible spectrophotometer (UV-1801,
Beijing, China). The anthraquinone-Glycoside was mainly concentrated in the 40% methanol elution
fraction. Therefore, it was selected as the active fraction for the following experiments and is henceforth
referred to as PAGR.
4.3. Determination of Three Anthraquinone-Glycosides in PAGR by HPLC
4.3.1. HPLC Chromatographic Conditions

Agilent 1200 (Santa Clara, CA, USA) High-Performance Liquid Chromatography consists of
a quaternary pump, autosampler, column oven, and VWD detector. Column: Agilent Extend-C18
(4.6 mm × 250 mm, 5 μm). The column temperature was 30 ◦ C, flow rate 1.0 mL/min, detection
wavelength 280 nm. Mobile phase acetonitrile was 0.1% formic acid (A) and acetonitrile (B), 0–10 min,
A: 90%, B: 10%, 10–25 min, A: 88%, B: 12%, 25–30 min, A: 78%, B: 22%, 30–40 min, A: 67%, B: 33%,
and the injection volume was 5 μL.
4.3.2. Preparation of Working Solution and Standard Curve

A certain amount of PAGR was accurately weighed, dissolved by ultrasound, and a working
solution was prepared in methanol at constant volume. Certain amounts of emodin-8-O-β-d-glucoside,
aloe-emodin-8-O-β-d-glucoside, and chrysophanol-8-O-β-d-glucoside were dissolved in methanol to
form a mixed reference solution, which was then diluted with methanol to prepare mixed standard
solutions with concentrations of 0.5200, 0.2600, 0.1300, 0.0625, 0.0163, 0.0081, 0.0040, and 0.0020 mg/mL
respectively. The peak area of the different concentrations of the mixed standard solution was
determined by HPLC, and the standard curve was prepared with the peak area as the ordinate and the
mixed standard solution concentration as the abscissa. It was determined that the regression equation
of emodin-8-O-β-d-glucoside was y = 14932x + 1.496, R2 = 0.999, aloe-emodin-8-O-β-d-glucoside was
y = 96120x + 1.555, R2 = 0.999, and chrysophanol-8-O-β-d-glucoside was y = 26804x − 1.231, R2 = 0.999,
indicating that the three anthraquinone-Glycosides had a good linear relationship in the concentration
range of 0.0020–0.5200 mg/mL.
4.3.3. Stability and Precision

A certain concentration of the mixed reference solution was injected six times within a day
according to the chromatographic conditions to determine the peak area. The RSD of the peak areas
of emodin-8-O-β-d-glucoside, aloe-emodin-8-O-β-d-glucoside, and chrysophanol-8-O-β-d-glucoside
was1.18%, 1.52%, and 2.06%, respectively, which indicated that the precision of the instrument was good.
The reference solution was placed at room temperature and analyzed at 2, 4, 6, 8, and 10 h according
to the chromatographic conditions, and the RSD of the peak area of emodin-8-O-β-d-glucoside,
16
Molecules 2019, 24, 1454
aloe-emodin-8-O-β-d-glucoside, and chrysophanol-8-O-β-d-glucoside was 2.30%, 1.26%, and 1.20%,

respectively, indicating that the sample was stable within 10 h.
4.4. Acute Toxicity Test

A single dose of 2000 mg/kg of PAGR was intragastrically administered in rats. The death and
poisoning symptoms of the rats were observed closely within the first 24 h after administration,
and their behavior was monitored routinely over the next 14 days to evaluate the acute toxicity
of PAGR.
4.5. AnimalsTreatment and Sample Collection

Male Sprague-Dawley (SD) rats (150 ± 10 g) were purchased from the Experimental Animal
Center of Zhejiang Academy of Medical Sciences (SCXK, 2015-0033, Zhejiang, China). All experimental
animals were housed in a standard animal room at a temperature of 23 ◦ C ± 1 ◦ C and humidity of
50–60%. Rats were adapted to the environment for one week before the experiment and were then
randomly divided into six groups (n = 10): NC group, T2DM group, Met treated group, and Low,
Mid, and Hig dose groups of PAGR treated. Except for the NC group, the rats in the other groups
were fed with a high-fat diet (corn starch 60%, casein 20%, soybean oil 20%) [48] for 6 weeks and then
injected intraperitoneally with STZ (30 mg/kg) two times every other day. FBG was measured after
72 h of the last injection and was >11.1 mol/L, indicating that the diabetes model was successful. Next,
the NC and T2DM groups were intragastrically administered with normal saline, Met group was
administered with 100 mg/kg metformin [49], and the Low, Mid, and Hig groups were treated with
100, 200, and 400 mg/kg of PAGR for 6 weeks. They all were injected once a day. Blood samples were
drawn from the orbit to measure FBG every week. After fasting for 12 h after the last administration
of PAGR, rats were anesthetized with 10% chloral hydrate, the blood was taken from the abdominal
aorta, and the liver, kidneys, and pancreas were removed quickly and stored at -80◦ Cfor further study.
All experimental procedures were in accordance with the Guide for the Care and Use of Laboratory
Animals of Zhejiang and were approved by the Committee on the Ethics of Animal Experiments at
Animal Center of Zhejiang Agriculture and Forestry University [Ethics Certificate No. 2014001812184].
4.6. Biochemical Measurements

The SOD, GSH-PX, TG, and TC of serum were measured by Superoxide dismutase, Glutathione
peroxidase, Triglyceride, and Total cholesterol Kits of Nanjing Jiancheng, respectively, according to the
commercial instruction.
4.7. Histopathological Examinations

The liver, pancreas, and kidneys were fixed in 10% formalin solution for 24 h and then
paraffin-embedded. Paraffin sections of 5–7 μm thickness were carved up and stained with hematoxylin
and eosin (H&E), and histopathological changes were observed under a BX20 optical microscope
(Tokyo, Japan).
4.8. Western Blot Analysis

The pancreatic sample was lysed using RIPA lysis buffer on ice and centrifuged at 10,001× g
(4 ◦ C, 10min). The protein concentration of the supernatants was determined by the BCA Kit.
For western blot analysis, the protein was separated by sodium dodecyl sulfate polypropylene
gel electrophoresis (SDS-PAGE) and then electrotransferred to polyvinylidene difluoride (PVDF)
membranes. All membranes were incubated with 50 g/L non-fat milk powder in triethanolamine buffer
for 1 h at room temperature and then with primary antibodies against FasL, Cyt-c, caspase-3 (diluted
1:1000), and β-actin (diluted 1:1500) at 4 ◦ C overnight. The following day membranes were washed
with tris-buffered saline containing Tween-20 (TBST), then incubated with peroxidase-conjugated
17
Molecules 2019, 24, 1454
anti-rabbit secondary antibody for 1 h at room temperature. After washing with TBST three times,
ECL reagent was added, and images were captured using a chemiluminescence detection system
(Amersham Pharmacia, Piscataway, NJ, USA).
4.9. Statistical Analysis

All data were expressed as mean ± SD and analyzed by the SPSS statistical software (SPSS19.0 Inc.,
Chicago, IL, USA). One-way ANOVA with Duncan’s test was used for inter-group comparison.
p-values < 0.05 were considered statistically significant, and p-values < 0.01 were considered
extremely significant.
5. Conclusion
In conclusion, PAGR has glucose-lowering properties and attenuates type 2 diabetes. The potential
mechanism is that PAGR reduces the level of oxidative stress by improving lipid metabolism and
enhancing antioxidant capacity, which reduces damage to mitochondrial structures and downregulates
activation of mitochondrial-induced cell death pathways; thereby inhibiting β-cell apoptosis and
improving β-cell function. It also downregulates Fas/FasL-mediated apoptosis in pancreatic tissue,
further inhibiting apoptosis of β-cells.
Supplementary Materials: The following are available online, Table S: Data of Serum indicators (FBG, TC, TG,
SOD, GSH-PX, creatinine and BUN) and body weight of rats.
Author Contributions: F.-R.C. and K.Y. conceived and designed the experiments, H.-X.C., J.-L.F. and Y.G.
performed the experiments, F.-R.C. and H.-X.C. analyzed the data, J.-L.F. and K.Y. wrote the paper.
Funding: This project was supported by the Zhejiang Provincial Science Foundation of China (Grant
No: LY16H280007).
Conflicts of Interest: The authors have no conflicts of interest to declare. All of the authors have approved the
final article.
Data availability: The data used to support the findings of this study are included in the article.
Abbreviations
PAGR purified anthraquinone-Glycoside from Rheum palmatum L.
STZ streptozotocin
T2DM type 2 diabetes mellitus
NC normal
Met metformin
Mid medium
Hig high
FBG fasting blood glucose
TC total cholesterol
TG triglyceride
SOD superoxide dismutase
GSH-PX glutathione peroxidase
Cyt-c cytochrome C
HPLC High-Performance Liquid Chromatography
BUN blood urea nitrogen
H&E hematoxylin and eosin
SD standard deviation
ROS reactive oxygen species
RNS reactive nitrogen species
MAPK mitogen-activated protein kinase
SDS-PAGE sodium dodecyl sulfate polypropylene gel electrophoresis
PVDF polyvinylidene difluoride
18
Molecules 2019, 24, 1454
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21
molecules
Article
3 -O-β-D-glucopyranosyl-α,4,2 ,4 ,6 -pentahydroxy-
dihydrochalcone, from Bark of Eysenhardtia
polystachya Prevents Diabetic Nephropathy via
Inhibiting Protein Glycation in STZ-Nicotinamide
Induced Diabetic Mice
Rosa Martha Pérez Gutierrez 1, *, Abraham Heriberto García Campoy 1 ,
Silvia Patricia Paredes Carrera 2 , Alethia Muñiz Ramirez 3 , José Maria Mota Flores 1 and
Sergio Odin Flores Valle 4
1 Natural Products Research Laboratory, Higher School of Chemical Engineering and Extractive Industries,
National Polytechnic Institute, Av. Instituto Politécnico Nacional S/N,
Unidad Profesional Adolfo Lopez Mateos, Ciudad de México CP 07708, Mexico;
abrahamhgc27@hotmail.com (A.H.G.C.); josemariamota@yahoo.com.mx (J.M.M.F.)
2 Sustainable Nanomaterials Laboratory, Higher School of Chemical Engineering and Extractive Industries,
National Polytechnic Institute (IPN) Professional Unit Adolfo Lopez Mateos,
S/N Av. Instituto Politécnico Nacional, Ciudad de México CP 07708, Mexico; silviappcar@gmail.com
3 CONACYT/IPICYT-CIIDZA, Camino a la Presa de San José 2055, Col. Lomas 4 Sección,
San Luis Potosí CP 78216, Mexico; alethiamura@gmail.com
4 Green Chemistry Research Laboratory, School of Chemical Engineering and Extractive Industries,
National Polytechnic Institute, Av. Instituto Politécnico Nacional S/N,
Unidad Profesional Adolfo Lopez Mateos, Ciudad de México CP 07708, Mexico; sergioodin@gmail.com
* Correspondence: rmpg@prodigy.net.mx; Tel.: +52-55-5729-6000
Academic Editor: Raffaele Capasso

Received: 5 February 2019; Accepted: 22 March 2019; Published: 28 March 2019
Abstract: Previous studies have shown that accumulation of advanced glycation end products
(AGEs) can be the cause of diabetic nephropathy (DN) in diabetic patients. Dihydrochalcone
3 -O-β-D-glucopyranosyl α,4,2 ,4 ,6 -pentahydroxy–dihydrochalcone (1) is a powerful antiglycation
compound previously isolated from Eysenhardtia polystachya. The aim was to investigate whether
(1) was able to protect against diabetic nephropathy in streptozotocin (STZ)-induced diabetic mice,
which displayed renal dysfunction markers such as body weight, creatinine, uric acid, serum urea,
total urinary protein, and urea nitrogen in the blood (BUN). In addition, pathological changes were
evaluated including glycated hemoglobin (HbA1c), advanced glycation end products (AGEs) in the
kidney, as well as in circulation level and pro-inflammatory markers ICAM-1 levels in diabetic mice.
After 5 weeks, these elevated markers of dihydrochalcone treatment (25, 50 and 100 mg/kg) were
significantly (p < 0.05) attenuated. In addition, they ameliorate the indices of renal inflammation
as indicated by ICAM-1 markers. The kidney and circulatory AGEs levels in diabetic mice were
significantly (p < 0.05) attenuated by (1) treatment. Histological analysis of kidney tissues showed
an important recovery in its structure compared with the diabetic group. It was found that the
compound (1) attenuated the renal damage in diabetic mice by inhibiting AGEs formation.
Keywords: dihydrochalcone; advanced glycation end-product; Eysenhardtia polystachya; diabetic

mice; renoprotective

Molecules 2019, 24, 1214
1. Introduction
Type 2 diabetes mellitus is a cause of mortality due to complications such as diabetic nephropathy
(DN), which is the main cause of end-stage renal failure [1]. DN is characterized by kidney
structural changes, declining glomerular filtration rate, and mesangial sclerosis. Hyperglycemia,
oxidative stress, and dyslipidemia are the main causes of increased advanced glycation end products
(AGEs) and contribute to the development of DN in diabetes [2], which has been demonstrated
in several studies with the treatment of anti-glycation compounds such as alagebrium chloride [3],
benfotiamine [4], pyridoxamine [5], and aminoguanidine [6] attenuated development of diabetic
nephropathy. The generation of AGEs promotes kidney damage by protein cross linking, leading to
changes in structure and function of the proteins [7]; they also generate an increase of the expression
of monocyte chemoattractant protein 1 (MCP-1). Previous studies indicated that endothelial cell
exhibition to a uremic environment augment IL-8, vascular adhesion molecule-1 (VCAM-1) and MCP-1
expression indicating a relationship between systemic inflammation and vascular damage with a
uremic toxicity [8]. Other studies indicated that reduction of AGEs production and accumulation in
the tissues could be an effective strategy to improvement of diabetic complications [9].
In previous studies, we isolated several dihydrochalcones from the Bark of Eysenhardtia
polystachya [10], which showed an efficient inhibition fluorescent and non-fluorescent AGE formation,
reduced level of fructosamine, significantly suppressed oxidation of thiols and protein carbonyl content
in a BSA/glucose system; in addition, inhibited generation of MGO, and the formation of amyloid
cross-β structure. Dihydrochalcone demonstrated inhibition at multiple stages of glycation. The aim
was to isolate a dihydrochalcone and study if it can be renoprotective in diabetic mice by inhibiting
AGEs formation.
2. Results and Discussion
2.1. Identification of 3 -O-β-D-glucopyranosyl α,4,2 ,4 ,6 -pentahydroxy–dihydrochalcone (1)

Dihydrochalcone was first found in the methanol extract of Eysenhardtia polystachya, and its
structure was elucidated by spectroscopic methods (IR, 1 H-NMR, 13 C-NMR, COSY, and HMBC).
Compound 1 shows a molecular formula C21 H24 O12 , and this is suggested by using the positive
HRMS that implied ten degrees of unsaturation. The IR spectrum indicates the presence of hydroxyl at
3448 cm−1 , carbonyl (1645 cm−1 ) and benzene ring (3010, 1586, 1441 cm−1 ) functionalities.
1 H, 13 C-NMR, and DEPT spectra show the presence of five aromatic signals at δH 7.49, δH
6.65, and δH 6.39, and also showed signals for 21 C-atoms, including two methylene (CH2 ), eleven
methylenes (CH) groups, and eight quaternary carbons. In the 1 H-NMR spectrum there are five
aromatic signals at δH 7.49 (2H), δH 6.65 (2H) and δH 6.09 (1H). In the HMBC spectrum correlation
of δH 7.49 with δC 131.6 (C-2, 6), 6.65 with δC 118.2 (C-3, C-5) suggested the presence of one
monosubstituted aromatic ring with a hydroxyl group at C-4 (δC 160.7). The glucopyranosyl moiety
was located at C-3 which was further supported by the HMBC correlation of the anomeric proton
resonating at δH 4.80 (1H, d, J = 7.3 Hz, H-1 ) to C-3 (102.6) together with the coupling constant of the
anomeric proton (d, J = 7.3 Hz) indicated that was β-glucoside.
2 ,4 ,6 -trihydroxy substitution of the dihydrochalcone ring A was determined by evaluating the
1 H coupling pattern and the 13 C-NMR chemical shifts which showed the characteristic pattern of
nethofagin dehydrochalcone skeleton [11] except that (1) differs from nethofagin by the presence of a
hydroxyl group at C-8 (C-α). Aliphatic proton was assigned to ABX system at δH 2.75 (1H, dd, J = 16.5,
13.4 Hz, H-α), 2.36 (1H, dd, J = 16.5, 3.1 Hz, H-α), 5.25 (1H, dd, J = 13.4, 3.1 Hz, H-β), suggested a
linked -CH (α)-CH2( β)- moiety leading to the presence of a -CO-CH (OH)-CH2 - moiety. Therefore,
compound 1 was assigned as 3 -O-β-D-glucopyranosyl-α,4,2 ,4 ,6 -pentahydroxy-dihydrochalcone
(Figure 1).
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Figure 1. (A) Dihydrochalcone 1 isolated from bark of Eysenhardtia polystachya; (B) Heteronuclear
Multiple Bond Connectivity (HMBC) of 1.
2.2. Effect of Dihydrochalcone on Glucose, Water Intake, Body Weight, Kidney Weight, Food Consumption,
Urine Volumen and Urine Protein
As shown in the Figure 2 urine volume (A), food consumptions (B), water intake (C), and body
weight (D) were significantly higher when compared to that of the normal control mice. Treatment
with compound (1) at a dose of 100 mg/kg during a period of 5 weeks significantly decrease the
urine volume by 52.68% (Figure 2A; p < 0.05) and urine protein by 52.22% compared to that diabetic
control group (Table 1). While treatment with (1) food consumptions and water intake decreased by
32.75% (Figure 2B; p < 0.05) and 53.84% (Figure 1C; p < 0.05), respectively, compared to that diabetic
control group. However, using compound (1) during the 5 weeks of treatment did not significantly
modify levels of blood glucose in STZ-induced diabetes mice, which developed a stable increase in
the hyperglycemia. In addition, treatment with compound (1) or metformin did not show significant
changes in body weight (Figure 2D). During the period of experimental treatment with compound
(1), there was an improvement in urine volume, urine protein, food consumption, and water intake in
different degrees when compared to metformin, which was used as standard (Figure 2A–D). Figure 3A
shows kidney size during the experimental period; it was observed that a gain in kidney size in the
diabetic control mice was in contrast to a reduction of kidney size when the diabetic mice were treated
with compound (1) or metformin for 5 weeks.
Kidney weight was significantly increased (p < 0.05) in the diabetic group compared to the control
group, while oral administration with compound 1 (100 mg/kg) and metformin (200 mg/kg) exhibited
a significant reduction (p < 0.05) in kidney weight by 32% and 28.6%, respectively, as compared to
diabetic-STZ mice (Figure 3B).
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Figure 2. (A) examination of urine volume at the fifth weeks; (B) examination of food consumptions at
the fifth weeks; (C) examination of water intake at the fifth weeks; (D) examination of body weigh at
the fifth weeks; Results are expressed as mean ± SD; a p < 0.05 vs. diabetic control.
Figure 3. (A) Images of kidney representative of five weeks of the different treatments; (B) examination
of kidney weight at the fifth weeks; Results are expressed as mean ± SD; a p < 0.05 vs. diabetic control.
It was observed that in the STZ-induced diabetic model that a selective destruction of pancreatic
cells producing insulin led to hyperglycemia. Consequently, we have an experimental diabetic
nephropathy model to study pathological changes in the kidney [12]. Hyperglycemia produces an
increase in urine volume, urine protein, food consumption, water intake, blood glucose level, and
reduction of body weight [13]. Treatment with compound (1) significantly enhances these pathological
characteristics in the DN mice model. The finding indicated that the renoprotective effect of compound
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1 on the DN model is related to the improvement of renal function, and avoids the proteinuria.
However, its effect does not depend on changes in blood glucose levels.
Table 1. Effect of dihydrochalcone (1) on urine protein of diabetic-nephropathy mice.
Urine Protein (mg/dL)

Groups
0 Weeks 5 Weeks
NormaL ControL 0.45 ± 0.03 a 0.44 ± 0.08 a
Diabetic ControL 2.82 ± 0.7 3.61 ± 0.4
D + 1 (25 mg/dL) 2.81 ± 0.9 2.69 ± 0.5
D + 1 (50 mg/dL) 2.88 ± 0.6 2.16 ± 0.7
D + 1 (100 mg/dL) 2.89 ± 1.0 1.72 ± 0.9 a
D + Met (200 mg/dL) 2.80 ± 1.3 1.70 ± 0.6 a
Results are expressed as mean ± SD; a p < 0.05 vs. diabetic control (D).
2.3. Effect of Dihydrochalcone on Kidney Index, Creatinine, Uric Acid, Serum Urea, and Urea Nitrogen in the
Blood (BUN)
The development of diabetic nephropathy can be detected by the elevated level of kidney index
indicators such as uric acid, serum urea, BUN, and creatinine. These renal indexes were significantly
higher in the STZ-induced diabetic mice group (DN) in comparison with normal control. Groups
treated with 25, 50, and 100 mg/kg of the dihydrochalcone showed a significantly decreased (p < 0.05)
in kidney index in comparison to DN control group such as BUN, creatinine, uric acid, urea and urine
protein in a dependent dose manner (Tables 1 and 2). In addition, metformin used as a standard also
significantly reduced (p < 0.05) the elevated level of these biomarkers. STZ-induced diabetic mice
showed a significant increase in volume and kidney mass. However, treatment with compound 1 (25,
50 and 100 mg/kg) or metformin (200 mg/kg) significantly re-established (p < 0.05) both volume and
renal mass look closer to the normal group (Figure 3A,B).
Atrophic changes in the renal tubules and glomeruli in the kidneys were observed in the DN
mice model, producing elevated levels of blood urea nitrogen, urea, uric acid, and creatinine in blood.
An important index of glomerular function is the creatinine generated as a metabolite in the muscle,
excreted through glomerular filtration [14]. In the metabolic process, protein breakdown leads to
the production of urea, which is excreted mainly through the kidneys [15]. Metabolism in humans
serum urea nitrogen level is the main end-product of proteins causing a spike in serum, BUN level
is commonly found in DN or glomerulonephritis patients with inhibition glomerular filtration rate.
The accumulation of uric acid generates the production of monosodium urate crystals that can cause
inflammatory and pain response, leading to renal and hepatic injuries. High levels of serum urea,
BUN, uric acid, and creatinine suggest injuries in the kidney [16]. Thus, it could be used as markers
for diagnosis in DN [17].
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Table 2. Effect of dihydrochalcone (1) on biochemical parameters in serum of diabetic-nephropathy mice.
Groups BUN (mg/dL) Creatinine (mg/dL) Uric Acid (mg/dL) Urea (mg/dL)
0 Weeks 5 Weeks 0 Weeks 5 Weeks 0 Weeks 5 Weeks 0 Weeks 5 Weeks
NormaL ControL 15.89 ± 3.4 a 16.34 ± 2.8 a 0.73 ± 0.03 a 0.073 ± 0.03 a 5.12 ± 1.4 a 5.11 ± 1.0 a 35.06 ± 4.3 a 36.49 ± 1.0 a
Diabetic ControL 36.12 ± 4.1 42.51 ± 2.9 3.01 ± 0.9 3.90 ± 0.7 13.11 ± 3.7 16.20 ± 3.1 80.10 ± 5.9 90.38 ± 6.4
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D + 1 (25 mg/dL) 38.24 ± 2.9 30.47 ± 3.5 a 3.25 ± 1.0 2.47 ± 0.5 13.30 ± 2.8 8.13 ± 2.5 a 82.12 ± 3.7 69.28 ± 5.3 a
D + 1 (50 mg/dL) 37.23 ± 5.3 25.48 ± 4.1 a 3.94 ± 0.8 2.10 ± 0.4 a 12.94 ± 1.7 6.24 ± 2.9 a 83.43 ± 4.2 58.19 ± 6.1 a
D + 1 (100 mg/dL) 38.48 ± 4.6 22.11 ± 3.9 a 3.76 ± 0.6 1.70 ± 0.08 a 13.84 ± 1.5 5.41 ± 1.6 a 81.71 ± 5.3 50.13 ± 3.7 a
D + Met (200 mg/dL) 38.40 ± 5.0 21.43 ± 4.2 a 3.83 ± 0.9 1.55 ± 0.6 a 12.67 ± 3.6 3.43 ± 2.3 a 82.84 ± 3.8 56.22 ± 4.6 a
Results are expressed as mean ± SD; a p < 0.05 vs. diabetic control (D).
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2.4. Effect of Dihydrochalcone on MCP-1

Diabetic nephropathy is considered an inflammatory disease where progressive glomerular
damage is related by infiltration to CD11b-positive macrophages [18] wherein MCP-1 is secreted
from resident glomerular cells. In our study, the diabetic model is related to the increase in MCP-1
and simultaneously with accumulation of macrophages in the cortex. Treatment with compound
(1) and metformin were able to significantly reduce (p < 0.05) the expression of the chemokine
MCP-1 associated with diabetes (Figure 4). The finding suggested that the anti-inflammatory effect of
compound (1) participates in DN attenuation. AGEs-induced chronic inflammation, and subsequently
cells and tissues, were disabled and triggered an increasing inflammatory cytokines or oxidative stress
via interaction between RAGE and AGEs [19].
Figure 4. Examination of inflammation markers ICAM-1 in glomeruli of kidneys from diabetic mice at
the fifth weeks; Results are expressed as mean ± SD; a p < 0.05 vs. diabetic control.
2.5. Effect of Dihydrochalcone on Glycosylated Haemoglobin Concentration (HbA1c)

The level of glycosylated hemoglobin in STZ-induced diabetes mice was significantly increased
(p < 0.05) in comparison with normal control mice. However, HbA1C significantly decreased (p < 0.05)
in groups treated with compound 1 in comparison with diabetic group (Table 3) at a dose of 25, 50, and
100 mg/kg by 19.47%, 33.65% and 54.49% respectively. Metformin also significantly reduced (p < 0.05)
the elevated concentration of glycosylated hemoglobin by 48.14% (Table 3).
Table 3. Effect of dihydrochalcone (1) on glycosylated haemoglobin levels and AGEs in Kidney of
diabetic mice.
Groups Glycosylated Haemoglobin (%) AGEs (RFU/mg Protein)

NormaL controL 3.52 ± 0.7 a 1.71 ± 0.9 a
Diabetic controL 9.45 ± 1.1 a 3.8 ± 0.8
D + 1 (25 mg/dL) 7.61 ± 1.8 3.0 ± 0.7
D + 1 (50 mg/dL) 6.27 ± 0.9 a 2.6 ± 0.9
D + 1 (100 mg/dL) 4.3 ± 0.8 a 2.0 ± 0.6
D + Met (200 mg/dL) 4.9 ± 0.6 a 1.9 ± 0.7
Results are expressed as mean ± SD of five mice in each group to five weeks of experimentation. Values a p < 0.05
vs. diabetic control (D).
In diabetes, the excess of glucose in blood reacts with hemoglobin to form HbA1C, which is an
early glycosylation adduct, and with time undergoes complex and slow rearrangements to generate
AGEs. Thus, glycosylated hemoglobin is used in diabetic patients mostly for prognosticate the
developing of diabetic complications, mainly at long term [20].
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2.6. Effect of Dihydrochalcone on AGEs Kidney

In diabetes hyperglycemic condition leads to excessive accumulation of AGEs participating
in the pathogenesis of diabetic nephropathy, leading to structural abnormality [4]. In this study,
treatment with STZ markedly increased AGEs formation in kidneys of diabetic mice in comparison to
normal control. Groups treated with dihydrochalcone (100 mg/kg) or metformin (200 mg/kg) showed
attenuation in AGEs formation in kidney in comparison to the diabetic control group by 47.73% and
50% respectively (Table 3). Results showed that compound (1) is able to block the accumulation and
formation of AGEs in kidney in nephropathy model streptozotocin induced diabetic mice. Reduction
of the formation of AGEs is considered a potential therapy in diabetic nephropathy.
2.7. AGEs Levels in Plasma

AGEs levels in plasma showed a significant increase in 5 weeks of diabetes induction. However,
treatment of compound 1 to diabetic mice showed progressive decrease circulating AGEs in respect
to diabetic control mice (Figure 5A,B). AF show that are better related to the time of illness than AGI.
Plasma AGE levels circulation stopped incrementing, possibly because it is eliminated from blood by
liver and renal filters. This study showed higher serum AGE level in diabetic mice and an increase
in renal dysfunctions. Oral administration of compound (1) act as AGE-inhibitors near the normal
levels as well as AGE-breakers, reducing the serum AGE level and delay the progression of DN.
In addition, metformin treatment showed AGEs level near to normal levels compared to the diabetic
group (Figure 5A,B); this effect is due to its interaction with dicarbonyl compounds formed throughout
the glycation process [21].
Figure 5. (A) Circulating AGE levels measured as absolute fluorescence at 5 weeks of experimental
period; (B) Circulating AGE levels expressed as advanced glycation index. Results are expressed as
mean ± SD; a p < 0.05 vs. diabetic control.
2.8. Histopathology on in Renal Tissues

The renal tissue of control mice revealed normal parenchyma, renal cortex, renal tubules, and
glomeruli (Figure 6A). Sections of STZ-induced diabetic mice kidney showed perivascular lymphocytic
aggregates, endotheliosis, inflammatory cell infiltration, degenerative changes, fibrotic, interstitial
hemorrhage, and glomerular necrosis (Figure 6B). Administration of 100 mg/kg (Figure 6E) of
dihydrochalcone, or 200 mg/kg of metformin (Figure 6F) to the experimental animals showed
an important recovery in the structure of the kidney compared to the diabetic group. However,
treatment a 25 and 50 mg/kg showed a moderate improvement on normal glomerulus compared to
the diabetic group.
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Figure 6. Kidney sections from mice by light microscopy (H and E stained). (A) Control group,
(B) Diabetic control, (C) D + 1 (25 mg/kg), (D) D + 1 (50 mg/kg), (E) D + 1 (100 mg/kg), (F) D + MET
(200 mg/kg).
3. Conclusions
Our findings suggest that the treatment with dihydrochalcone protects renal function and prevents
kidney injury in STZ-nicotinamide induced diabetic nephropathy, ameliorated markers of DN, as well
as inflammation, HbA1C, AGE-inhibition in kidneys and circulation.
The renoprotective effect of dihydrochalcone isolated from Eysenhardtia polystachya might be
associated in part to its ability to react with reactive carbonyl species and cleavage of pre-formed AGEs
within the kidney by a cross- link breaker inhibiting AGEs-formation.
4. Materials and Methods
4.1. Plant
The specimen was identified and authenticated by Biol. Aurora Chamal, Department of Botany,
Universidad Autonoma Metropolitana-Xochimilco, where a voucher specimen (No. 53290) has been
deposited for further reference.
4.2. Extraction, Isolation and Characterization 3 -O-β-D-glucopyranosyl α,4,2 ,4 ,6 -pentahydroxy–dihydrochalcone (1)
The extraction, isolation, and characterization of dihydrochalcone from the bark of Eysenhardtia
polystachya was carried out as follows. Briefly, the bark (40 kg) was pulverized into powder and
extracted with distilled water and methanol (1:1) two times at room temperature. Both extracts were
combined and concentrated under reduced pressure. The extract was subjected to a silica gel column
eluted with ethyl acetate/methylene chloride (2:9) to yield seven fractions (PA-1 to PA-7). Subfraction
PA-5 was then separated by silica gel chromatography with methanol/acetone/ethyl acetate (1:3:6)
and preparative chromatography eluted with methanol/acetone/ethyl acetate (0.5/3/1.5) to give 4
subfractions (PA5-1 to PA5-4). PA5-3 was purified in a sephadex LH-20 column with a gradient of
water/methanol 1:1 increasing the ratio of water to 100% to obtain compound 1 (630 mg).
3 -O-β-D-glucopyranosyl-α,4,2 ,4 ,6 -pentahydroxy–dihydrochalcone: Is a pale yellow powder, m.p.
143–144 ◦ C; HRMS [M+ ] at m/z 468.4130, C21 H24 O12 requires 468.4110; UV λ max (MeOH) nm 260,
303, 375; IR νmax (KBr) cm−1 : 3448, 2932, 1645, 1586, 1519, 1441, 1182, 1071; The 1 H-NMR espectra
(300 MHz, DMSO-d6) showed the following data: δH 2.75 (1H, dd, J = 16.5, 13.4 Hz, H-α), 2.36 (1H, dd,
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Molecules 2019, 24, 1214
J = 16.5, 3.1 Hz, H-α), 5.25 (1H, dd, J = 13.4, 3.1 Hz, H-β), 7.49 (2H, d, J = 8.2 Hz, H-2, 6), 6.65 (2H, d,
J = 8.2 Hz, H-3, 5), 9.10 (s, OH-4), 6.09 (1H, s, H-5 ), 10.60 (s, OH-4 ), 12.24 (s, OH-2 ), 13.50 (s, OH-6 );
Glu: δH 4.80 (1H, d, J = 7.3 Hz, H-1 ’), 3.87 (1H, d, J = 4.4 Hz, OH-2 ’), 3.54 (1H, d, J = 5.3 Hz, OH-3 ’),
3.28 (1H, dd, J = 12.2, 4.5 Hz, OH-4 ’), 3.20 (1H, d, J = 9.3 Hz, OH-5 ’), 3.76 (1H, dd, J = 11.6, 5.2 Hz,
H-6 α), 3.45 (1H, dd, J = 11.6, 6.0 Hz, H-6 β); 13 C NMR (125 MHz, CDCl3) δC : 128.7 (C-1), 131.6 (C-2,
C-6), 118.2 (C-3, C-5), 160.7 (C-4), 44.30 (C-α), 78.97 (C-β), 192.94 (C-9, C=O); 109.4 (C-1 ), 164.2 (C-2 ),
102.6 (C-3 ), 166.2 (C-4 ), 111.5 (C-5 ), 165.7 (C-6 ); Glu: δC 108.6 (C-1 ), 72.3 (C-2”), 78.0 (C-3 ), 69.7
(C-4 ), 76.8 (C-5 ), 60.4 (C-6 ).
4.3. Animals
The study was conducted on healthy adult male C57BL/6J mice, weighing about 25–30 g. Before
and during the experiment, animals were fed a standard laboratory diet (Mouse Chow 5015, Purina)
with free access to water. Mice were procured from the bioterium of ENCB and were housed in
microloan boxes cages in a controlled environment (temperature 25 ± 2 ◦ C). Animals were allowed to
acclimate for a period of three days in their new environment prior to the study. Before commencing
the experiment, litter in cages was renewed three times a week to ensure hygiene and maximum
comfort for the animals. The experiments reported in this study followed the guidelines stated in
“Principles of Laboratory Animal Care” (NIH publication 85-23, revised 1985 and the Mexican Official
Normativity (NOM-062-Z00-1999). All animal procedures were performed in accordance with the
recommendations for the care and use of laboratory animals (756/lab/ENCB).
4.4. Induction of Mild Diabetes (Type 2)

After 15 min of administrating an intraperitoneal injection with 120 mg/kg nicotinamide (Sigma
Chemical Company, St. Louis, MO, USA), mice were made diabetic by administering a single intraperitoneal
injection of freshly prepared streptozotocin (STZ) (60 mg/kg b.w. i.p.) in 0.1 M citrate buffer at
pH 4.5. The animals were allowed to drink 5% glucose solution overnight to overcome the drug-induced
hypoglycaemia. After 10 days of diabetes development, moderately diabetic mice having persistent
glycosuria and hyperglycaemia (blood glucose > 200 mg/dL) were used for further experimentation [22].
After the diabetic mice were randomly divided into five groups (eight mice per group) matched by body
weight. Normal mice were administered with distilled water.
Forty-eight mice were divided into eight groups of six animals and had free access to water and food,
and then animals were treated for five weeks as follows: Group 1: normal mice, Group 2: diabetic control
mice, Group 3: Dihydrochalcone (25 mg/kg/day p.o) treated diabetic mice, Group 4: Dihydrochalcone
(50 mg/kg/day p.o) treated diabetic mice, Group 5: Dihydrochalcone (100 mg/kg/day p.o) treated
diabetic mice, Group 6: Mentformin (AG) (200 mg/kg/day p.o) treated diabetic mice as a standard drug.
4.5. Preparation of Urine, Serum, and Kidney Homogenate

After 5 weeks of treatment, diabetes mice were placed inside individual metabolic cages for 24 h
and urine was collected. Body weight, kidney weight, food, and water intake were recorded; after
12 h fasting, the blood samples were collected from the tail vein. The animals were then sacrificed by
asphyxiation using carbon dioxide. The serum was centrifuged for 20 min at 877× g and stored at
−80 ◦ C for further assays. After the kidneys were removed, weighed and washed with phosphate
buffer saline (PBS pH 7.4). The right kidneys were homogenized in ice-cold PBS, and stored in liquid
nitrogen for further biochemical and molecular assays. The left kidneys were removed and used for
histopathological examination.
4.6. Measured of Biochemical Parameters in Diabetic Mice

The plasma glucose concentration was determined using an enzymatic colorimetric method using
a commercial kit (Sigma Aldrich, San Luis, MO, USA). Total urinary protein levels were measured using
a Rat Urinary Protein Assay Kit (Chondrex, Redmond, WA, USA). In serum, creatinine was evaluated
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using a QuantiChrom™ Protein Creatinine Ratio Assay Kit (BioAssay Systems, San Francisco, CA,
USA), urea, uric acid and blood urea nitrogen test (BUN) were measured using Assay Kits (Abcam,
Cambridge MA, USA) according to the manufacturer’s instructions. Kidneys were dissected and
weighed (wet weight).
4.7. Glycosylated Hemoglobin

Blood samples were obtained by ocular venipuncture into tubes with EDTA and centrifuged at
800× g at 4 ◦ C for 10 min to remove packed cells and plasma. Using two volumes of water packed cells
were lysed. Then was added one volume of carbon tetrachloride to the hemolysate, and refrigerated at
4 ◦ C overnight. Lysate was centrifuged at 27,000× g at 4 ◦ C, for 30 min supernatant removed and used
to analysis. Colorimetric estimation was measured according to the procedure of Parker et al. [23].
One mLof diluted hemolysate (10 mg of hemoglobin or fructose standard) was mixed with 1 mL of
oxalic acid reagent. The mixture was incubated for 60 min at 124 ◦ C in sealed tubes. After incubation,
the mixture was allowed to cool to room temperature and added 1 mL of the trichloroacetic acid
to each tube, mixed, and filtered through a 15 cm × 0.5 (1.d) glass column with a glass wool plug
in the bottom. To 1.5 mL of the filtering add thiobarbituric acid reagent (0.05 M) and measured the
absorbance at 443 nm of glycated hemoglobin.
4.8. AGEs Levels in Kidney

The kidneys were homogenized in 2 mL of 0.25 M sucrose, followed by centrifugation at 5 ◦ C
with 900 × g and the pellet obtained was resuspended in 2 mL sucrose and centrifuged again then
both supernatant obtained were mixed. The proteins were precipitated by adding trichloroacetic acid
(TCA) in equal volume and centrifugated at 5 ◦ C at 900 × g, then protein pellet was added 1 mL
methanol to eliminate the lipid fraction. Then, washing the insoluble protein using 10% cooled TCA
and centrifuged: after the residue was solubilized in 1 mL of 1 N NaOH and the AGEs concentration
was evaluated fluorometrically with an emission at 440 nm and excitation at 370 nm, and the results
were indicated as relative fluorescence units (RFU)/mg protein [24].
4.9. AGEs Levels in Serum

Mouse plasma was centrifuged to collect supernatants. AGEs levels in plasma were used to
determine Absolute Fluorescence (AF; 80 μL) and Advanced Glycation Index (AGI; 20, 40, 80 μL
diluted in 1 mL of PBS) [25] using a Micro-plate Reader (Thermo Fisher Scientific, Voltam, MA, USA).
AGE fluorescence was indicated at excitation wavelength of 350 nm and emission wavelengths of
450 nm in a spectrofluorometric detector (BIO-TEK, Synergy, Salt Lake City, UT, USA). AF was in
arbitrary units (AU) with respect to protein concentrations. AGI was produced by the three points of
dilution and expressed as the slope of the line.
4.10. Measure of the Expression of Intracellular Adhesion Molecule (ICAM)-1

After 5 weeks of treatment, frozen cortex was chopped on ice, then flushed through a disposable
filter (100-mm; BioScientific Corp, Austin, TX, USA) with cold saline solution and collected.
The solution was then flushed through filter (70-mm). The glomerular solution was obtained by
inversion of the filter (70-mm), and afterward was flushed with a cold saline solution, followed
by spinning at 6000 rpm at 4 ◦ C for 15 min. The supernatant was eliminated and the pellets were
suspended in 1 mL of Trizol. The presence of glomeruli was supported in a microscopy. Protein
obtained from kidney cortex were used to evaluate the level of the expression of (ICAM)-1 (BioScientific
Corp, Austin, TX, USA) according to ELISA kit instructions.
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4.11. Histopathology
Kidney fragments were fixed in 10% neutral buffered formalin solution, dehydrated in ethanol,
embedded in paraffin, and sectioned at 5 μm thickness using a rotary microtome. After dehydration,
sections were stained with hematoxylin and eosin (HE). To evaluate the histopathological damage,
each image of sections was examined for microscopic observations (400; Nikon, Tokyo, Japan).
4.12. Statistical Analyses

The data are presented as mean ± standard deviation. The significance of the differences was
analyzed by one-way ANOVA with the Dunnett’s test using GraphPad Prism 7.0 for Windows
(GraphPad Software Inc., San Diego, CA, USA). The value of statistical significance was established at
p < 0.05.
Author Contributions: Conceptualization of the study and design of the experiments, R.M.P.G.; performed the
experiments A.H.G.C., S.O.F.V., and J.M.M.F.; analyzed the data, A.M.R. and S.P.P.C.; Writing of the manuscript
R.M.P.G., A.M.R, and S.P.P.C.
Funding: This study received no external funding.
Conflicts of Interest: The authors claim no conflict of interest in this study.
References
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stress in cultured NRK-49F cells. Chin. Med. J. 2007, 120, 787–793. [CrossRef] [PubMed]
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419, 63–79. [CrossRef]
21. Ahangarpour, A.; Heidari, H.; Oroojan, A.A.; Mirzavandi, F.; Nasr, K.E.; Dehghan, Z.M. Antidiabetic,
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nicotinamide-streptozotocin induced type 2 model of diabetes in male mice. Avicenna J. Phytomed. 2017, 7,
169–179. [PubMed]
22. Parker, K.M.; England, J.K.; Da-Costa, J.; Hess, R.L.; Goldstein, D.E. Improved colorimetric assay for
glycosylated hemoglobin. Clin. Chem. 1981, 27, 669–672. [PubMed]
23. Kalousova, M.; Skrha, J.; Zima, T. Advanced glycation end-products and advanced oxidation protein
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Sample Availability: Samples of the compounds are not available from the authors.
35
molecules
Article
In-Vitro Antioxidant, Hypoglycemic Activity,
and Identification of Bioactive Compounds in
Phenol-Rich Extract from the Marine Red Algae
Gracilaria edulis (Gmelin) Silva
Thilina L. Gunathilaka 1 , Kalpa W. Samarakoon 2 , P. Ranasinghe 3 and L. Dinithi C. Peiris 1, *
1 Department of Zoology, Faculty of Applied Sciences (Center for Instrumentation Facility & Center for
Biotechnology), University of Sri Jayewardenepura, Nugegoda 10250, Sri Lanka;
gunathilakathilina2@gmail.com
2 National Science and Technology Commission, Dudley Senanayake Mawatha, Colombo 8 00800, Sri Lanka;
kalpa.samarakoon@gmail.com
3 Industrial Technology Institute, Halbarawa Gardens, Malabe 10115, Sri Lanka; pathmasiri@iti.lk
* Correspondence: dinithi@sci.sjp.ac.lk; Tel.:+94-714-018-537
Academic Editor: Lorenzo Di Cesare Mannelli

Received: 4 July 2019; Accepted: 3 October 2019; Published: 15 October 2019
Abstract: Obesity and diabetes are major metabolic disorders which are prevalent worldwide.
Algae has played an important role in managing these disorders. In this study, Gracilaria edulis,
a marine red algae, was investigated for antioxidant and hypoglycemic potential using in vitro models.
De-polysaccharide methanol extract of G. edulis was sequentially partitioned with hexane, chloroform,
ethyl acetate, and antioxidants, and hypoglycemic potentials were evaluated using multiple methods.
High antioxidant potential was observed in the ethyl acetate fraction in terms of ferric reducing
antioxidant power, iron chelating, and DPPH and ABTS radical scavenging activities, while the crude
methanol extract exhibited potent oxygen radical-absorbance capacity. Potent α-amylase inhibitory
activity was observed in the ethyl acetate fraction, while the ethyl acetate fraction was effective against
α-glucosidase inhibition. Glucose diffusion was inhibited by the ethyl acetate fraction at 180 min,
and the highest antiglycation activity was observed in both chloroform and ethyl acetate fractions.
Additionally, gas chromatography-mass spectrometry analysis of the ethyl acetate fraction revealed
the presence of several potent anti-diabetic compounds. In conclusion, G. edulis exhibited promising
antidiabetic potential via multiple mechanisms. The ethyl acetate fraction exhibited the strongest
hypoglycemic and antiglycation potential among the four fractions, and hence the isolation of active
compounds is required to develop leads for new drugs to treat diabetes.
Keywords: Gracillaria edulis; methanol extract; fractionation; α-amylase; α-glucosidase; antiglycation;

glucose diffusion
1. Introduction
The incidences of type 2 diabetes and obesity have increased globally due to rapid urbanization
and unhealthy diets. More than 90% of patients with diabetes mellitus are either overweight or
obese [1]. Diabetes mellitus is a chronic disorder that is linked with persistent hyperglycemia due to
the deficiency of insulin secretion. The World Health Organization (WHO) has estimated that by 2035,
the incidence of diabetes mellitus and impaired glucose tolerance will increase by up to 592 million
and 471 million people, respectively [2]. Type 1 diabetes is widespread among Northern European
countries, while type 2 diabetes is most common in African and South Asian countries. For instance,
type 2 diabetes is prevalent among the Sri Lankan population [3]. According to recent statistics, one

Molecules 2019, 24, 3708
in every five Sri Lankan adults either suffers from diabetes or is in the prediabetes stage [4]. The
consumption of more refined fast-release staple carbohydrate food is considered as the major cause
for the progression of obesity. Carbohydrate-rich diets release glucose quickly into the bloodstream,
thus increasing the levels of blood sugar and insulin [5]. High blood glucose level is linked with
increased risk of hypertension, retinopathy, nephropathy, neuropathy, and macrovascular diseases.
These health complications result in an increased risk of morbidity and mortality, and hence reduce the
life expectancy of diabetic patients [4].
The inhibition of carbohydrate digestive enzymes—α-amylase and α-glucosidase—is one of
the significant alternatives to the management of chronic hyperglycemia in diabetic patients [1].
Polyphenols purified from plants are good inhibitors of vital enzymes responsible for carbohydrate
digestion. The enzymes alpha amylase and α-glucosidase are involved in carbohydrate metabolism,
and act synergistically to digest starch [6]. Alpha amylase hydrolyzes the alpha bonds present in
insoluble starch molecules, while α-glucosidase catalyzes the final step of carbohydrate digestion to
convert disaccharides into glucose. The inhibition of such enzymes leads to a reduction of starch
breakdown and an increase in postprandial blood glucose level; thus, enzyme inhibitors can be used as
therapeutic agents for the development of novel drugs to treat diabetes [6].
Recent investigations have reported an association between obesity and chronic inflammation in
adipose tissues. As a result of obesity, the amount of adipose tissue tends to increase and the tissue
undergoes molecular and cellular alterations. Adipocytokines secreted by the adipocytes in adipose
tissues can induce production of reactive oxygen species, thus leading to oxidative stress [7]. Oxidative
stress is tightly linked with the pathophysiological process of chronic inflammatory conditions such as
diabetes mellitus [8]. Therefore, researchers have primarily focused on natural products to discover
novel preventive and regenerative therapies to combat oxidative stress and postprandial hyperglycemia
with minimum side effects.
Marine seaweeds rich in bioactive metabolites play a significant role in the development of novel
drugs and nutraceuticals. Due to the bioactive compounds they contain—namely polyphenols,
sterols, alkaloids, flavonoids, tannins, proteins, essential fatty acids, enzymes, vitamins, and
carotenoids—marine seaweeds are able to withstand harsh environments [9]. Gracilaria edulis (Gmelin)
Silva is a red algae belonging to the family Gracilariaceae, and it has attracted widespread attention due
to its biological and pharmacological properties and various therapeutic benefits such as antidiabetic,
antioxidant, antimicrobial, anticoagulant, anti-inflammatory, and antiproliferative activities [10].
Shanura et al. [11] established the anti-inflammatory activity of methanol extract and fractions of
G. edulis against lipopolysaccharide-induced inflammatory responses, while Koneri and Jha [12]
documented the antidiabetic potential of methanol extracts of G. edulis against fructose-induced type 2
diabetes mellitus in male rats. Patra and Muthuraman [13] revealed the anticancer activity of ethanol
extracts of G. edulis against ascites tumors in mice. Although past studies focused on several biological
properties of G. edulis, this is the first study carried out in Sri Lanka to investigate the antioxidant
and hypoglycemic activities of G. edulis using multiple in vitro mechanisms. The present study
aimed to appraise the antidiabetic potential of G. edulis through inhibitory activities of carbohydrate
digestive enzymes, glucose diffusion, and protein glycation. We also attempted to identify the bioactive
compounds present in G. edulis that are responsible for the above pharmacological activities.
2. Results
2.1. Quantification of Total Phenolic, Flavonoid, and Alkaloid Contents of G. edulis

Results obtained for the crude methanol extract and four fractions of G. edulis with increasing
polarity were used to evaluate the total phenolic, flavonoid, and alkaloid contents of G. edulis. These
results are shown in Table 1. The alkaloid content in the crude methanol extract and in the hexane and
chloroform fractions was higher than the phenol and flavonoid contents, whereas the phenol content
in the ethyl acetate and aqueous fractions was higher than the flavonoid and alkaloid contents.
38
Molecules 2019, 24, 3708
Table 1. Phenol, flavonoid, and alkaloid contents of crude methanol extract and fractions of
Gracillaria edulis.
TPC TFC Total Alkaloids

Extract/Fraction
(μg GAE/g) (μg QE/g) (μg PE/g)
Crude methanol extract 1007.81 ± 54.21 a 541.02 ± 51.84 a 7177.72 ± 63.04 a
Hexane fraction 760.85 ± 37.75 b 688.60 ± 9.55 a 1656.97 ± 45.80 b
Chloroform fraction 560.85 ± 55.08 c 289.39 ± 9.55 b 2875.54 ± 22.29 c
Ethyl acetate fraction 2414.51 ± 50.34 d 1461.49 ± 75.22 c 1073.75 ± 45.88 b
Aqueous fraction 1704.69 ± 43.16 e 786.95 ± 62.04 d 522.34 ± 67.13 d
TPC: total phenol content; TFC: total flavonoid content; GAE: gallic acid equivalent; QE: quercetin equivalent; PE:
Piperine equivalent. Data presented as mean ± standard deviation (n = 4). Mean values in a column superscripted
by different letters (a–e ) are significantly different at p < 0.05.
The total phenolic content in the ethyl acetate fraction (2414.51 ± 50.34 μg GAE/g) was higher
than that in the crude methanol extract and the hexane, chloroform, and aqueous fractions. The lowest
total phenolic content was observed in the chloroform fraction. Similarly, a significant difference
was observed in the phenolic content of the crude methanol extract and the four fractions (p < 0.05).
The total flavonoid content of the crude methanol extract and all fractions increased in the order
chloroform fraction < crude methanol extract < hexane fraction < aqueous fraction < ethyl acetate
fraction, with respective contents of 289.39 ± 9.55, 541.02 ± 51.84, 688.60 ± 9.55, 786.95 ± 62.04, and
1461.49 ± 75.22 μg QE/g. Among the crude methanol extract and four fractions, total alkaloid content
decreased in the order crude methanol extract > chloroform fraction > hexane fraction > ethyl acetate
fraction > aqueous fraction, with respective values of 7177.72 ± 63.04, 2875.54 ± 22.29, 1656.97 ± 45.80,
1073.75 ± 45.88, and 522.34 ± 67.13 μg PE/g.
2.2. In Vitro Antioxidant Activity

The antioxidant capacity of crude methanol extract and fractions of G. edulis were determined
using 1,1-diphenyl-2-picrylhydrazine (DPPH) and 2,2 -azino-bis (3-ethylbenzothiazoline-6-sulphonic
acid) (ABTS) radical scavenging activities, ferrous ion chelating assay, ferric reducing antioxidant
power, and oxygen radical-absorbance capacity. The results obtained for the antioxidant capacity are
presented in Table 2.
The highest DPPH radical scavenging activity was observed in the ethyl acetate fraction (IC50 :
3.17 ± 0.04 mg/mL) and the crude methanol extract (IC50 : 3.19 ± 0.02 mg/mL). By comparison, the
standard Trolox had free radical scavenging activity of IC50 : 0.011 mg/mL. In this study, the reduction
of DPPH occurred in a concentration-dependent manner, as observed from the high reduction of
DPPH (higher radical activity) at 3.75 mg/mL concentrations (Figure A1). The most potent ABTS
radical scavenging activity was observed in the ethyl acetate fraction (IC50 : 0.41 ± 0.02 mg/mL),
while the lowest activity was observed in the crude methanol extract (IC50 : 0.56 ± 0.01 mg/mL).
Moreover, a significant positive correlation was observed between the radical scavenging activity of
DPPH (r = 0.91) and that of ABTS (r = 0.85) with the total phenol content of the ethyl acetate fraction.
The highest ferrous iron chelating activity (FICA) was observed in the ethyl acetate fraction (IC50 :
2.22 ± 0.01 mg/mL), while the lowest ferrous ion chelating activity was observed in the crude methanol
extract (IC50 : 9.23 ± 0.19 mg/mL); the standard EDTA (ethylenediaminetetraacetic acid) exhibited
a chelating activity of IC50 : 0.019 mg/mL. Ferric reducing antioxidant powder (FRAP) and oxygen
radical absorbance capacity (ORAC) are the most frequent measures used to determine antioxidant
activity, which is expressed as Trolox equivalent antioxidant capacity. The ethyl acetate fraction of
G. edulis showed the highest reducing ability (8.51 ± 0.09 mg TE/g), while crude methanol extract
(1.61 ± 0.19 mg TE/g) showed potent oxygen radical absorbance capacity.
39
Molecules 2019, 24, 3708
Table 2. IC50 values of methanol extract of G. edulis and fractions against antioxidant activity and
activities equivalent to standards.
Activity Equivalent to
Extract/ IC50 (mg/mL)
Standard (mg TE/g)
Fraction
DPPH ABTS FICA FRAP ORAC
Crude methanol extract 3.19 ± 0.02 a 0.56 ± 0.01 a 9.23 ± 0.19 a 0.26 ± 0.03 a 1.61 ± 0.19 a
Hexane fraction 6.22 ± 0.01 b 0.54 ± 0.01 b 2.58 ± 0.03 bc 1.93 ± 0.35 b 0.57 ± 0.07 bc
Chloroform fraction 3.29 ± 0.02 c 0.44 ± 0.01 c 2.43 ± 0.01 c
2.19 ± 0.23 b 0.77 ± 0.05 b
Ethyl acetate fraction 3.17 ± 0.04 a 0.41 ± 0.02 d 2.22 ± 0.01 bc 8.51 ± 0.09 c 1.44 ± 0.29 a
Aqueous fraction 3.91 ± 0.03 d 0.45 ± 0.03 c 2.71 ± 0.02 b 1.23 ± 0.21 d 0.44 ± 0.09 c
Trolox (standard) 0.011 ± 0.00 e 0.008 ± 0.00 e - - -
EDTA (standard) - - 0.019 ± 00 d - -
Results are expressed as mean ± SD; n = 4. DPPH (1,1-diphenyl-2-picrylhydrazine); ABTS (2,2 -azino-bis
(3-ethylbenzothiazoline-6-sulphonic acid)); FICA (ferrous iron chelating activity); FRAP (ferric reducing antioxidant
powder); ORAC (oxygen radical absorbance capacity); EDTA (ethylenediaminetetraacetic acid); TE (Trolox
equivalent). Mean values in a column superscripted by different letters (a–e ) are significantly different at p < 0.05.
2.3. In Vitro α-Amylase Inhibitory Assay

The α-amylase inhibitory potential of G. edulis methanol extract and four fractions was evaluated
using starch as the substrate and acarbose as the positive control. Acarbose, crude methanol extract,
and the four fractions exhibited a dose-dependent enzyme inhibition (Figure A2).
The ethyl acetate fraction (IC50 : 279.48 ± 5.62 μg/mL) of G. edulis exhibited more potent
α-amylase inhibitory activity, whereas the lowest activity was observed in the hexane fraction
(IC50 : 393.04 ± 4.73 μg/mL) (Table 3) compared to the standard acarbose (IC50 : 87.43 μg/mL). The
inhibition of the α-amylase enzyme exhibited by the ethyl acetate fraction varied from 10% at 12.5 μg/mL
to 64% at 400 μg/mL assay concentration.
Table 3. IC50 values exhibited by G. edulis methanol extract and methanol fractions against the inhibitory
activity of the enzymes α-amylase and α-glucosidase and antiglycation activities.
Alpha-Amylase Alpha-Glucosidase Anti-Glycation

Extract/Fraction
(μg/mL) (μg/mL) (μg/mL)
Crude methanol extract 349.59 ± 2.44 a 102.24 ± 0.89 a 702.33 ± 12.72 a
Hexane fraction 393.04 ± 4.73 b 163.90 ± 5.23 b 637.53 ± 6.21 b
Chloroform fraction 322.71 ± 4.80 c 122.65 ± 2.37 c 258.23 ± 3.24 c
Ethyl acetate fraction 279.48 ± 5.62 d 87.92 ± 1.62 d 586.54 ± 4.37 b
Aqueous fraction 376.49 ± 12.14 e 148.57 ± 1.87 e 723.78 ± 12.81 d
Acarbose (standard) 87.43 ± 0.59 f 0.38 ± 0.06 f -
Rutin (standard) - - 11.55 ± 0.82 e
Results are expressed as mean ± SD (n = 4). *p < 0.05 compared with the respective standard. Mean values in a
column superscripted by different letters (a–f ) are significantly different at p < 0.05.
2.4. In Vitro α-Glucosidase Inhibitory Assay

All the fractions and the crude methanol extract of G. edulis exhibited a dose-dependent inhibition
of the α-glucosidase enzyme with different degrees of potential (Figure A3). The inhibitory activity
on α-glucosidase enzymes of the crude methanol extract and the fractions of G. edulis are presented
in Table 3. The ethyl acetate fraction, which exhibited the lowest IC50 of 87.92 ± 1.62 μg/mL, was
considered to be a more potent α-glucosidase inhibitor than the crude methanol extract and the
other three fractions. The inhibition of α-glucosidase exhibited by the ethyl acetate fraction varied
from 6% (4.16 μg/mL) to 68% (133.3 μg/mL). The hexane fraction, which had the highest IC50 of
163.90 ± 5.23 μg/mL, exhibited the weakest α-glucosidase inhibitory activity.
40
Molecules 2019, 24, 3708
2.5. Glucose Diffusion Inhibitory Activity

The inhibitory activity of glucose diffusion was determined using a dialysis tube containing the
sample and glucose that had been soaked in NaCl solution. The diffusion of glucose into the external
solution was measured by glucose oxidase kit every 30 min for 3 h. The effects of the methanol extract and
the four fractions of G. edulis (1000 μg/mL) on glucose diffusion are represented in Figure 1. The inhibitory
activity of acarbose or the reference drug was considered as 100%, while the glucose diffusion of the
control at 180 min was considered as 100% with a glucose concentration of 57.65 ± 1.67 μg/mL in the
external solution, compared to the acarbose standard (22.79 ± 0.47 μg/mL). Among the tested fractions,
the ethyl acetate fractions exhibited the maximum inhibition of glucose diffusion at 180 min, and the
glucose concentration of the external solution was found to be 38.15 ± 1.11 μg/mL. The inhibition of
glucose diffusion by the hexane fraction (52.01 ± 0.96 μg/mL) and aqueous fraction (52.69 ± 1.31 μg/mL)
were similar compared to the glucose concentration in the external solution (Table A1).
Figure 1. Effect of methanol extract and fractions of G. edulis (1000 μg/mL) on glucose diffusion through
dialysis membrane compared to the standard acarbose and control. Data presented as means ± standard
deviation (n = 4).
2.6. Antiglycation Activity

The anti-glycation activity of the crude methanol extract and fractions of G. edulis was determined
using bovine serum albumin as a protein source. The crude methanol extract and all four fractions
showed dose-dependent antiglycation activity (Figure 2). As shown in Table 3, among the tested
fractions, the most potent antiglycation activity was observed with the chloroform fraction (IC50 :
258.23 ± 3.24 μg/mL), followed by the ethyl acetate fraction (IC50 : 586.54 ± 4.37 μg/mL), compared to
the standard rutin (IC50 : 11.55 ± 0.82 μg/mL). The aqueous fraction, which had the highest IC50 value
of 723.78 ± 12.81 μg/mL, exhibited the weakest antiglycation activity.
41
Molecules 2019, 24, 3708
Figure 2. Dose–response relationship of methanol extract and its fractions of G. edulis for antiglycation
activity determined by glucose-induced protein glycation and formation of protein-bound fluorescent
advanced glycation end products. Data presented as mean ± standard deviation (n = 4).
2.7. GC-MS or Gas Chromatography-Mass Spectrometry Analysis of Extract and Solvent Fractions
The ethyl acetate fraction of G. edulis, which showed promising biological activities, was subjected
to gas chromatography-mass spectrometry (GC-MS) analysis. The chromatogram obtained for the
ethyl acetate fraction is presented in Figure 3.
Based on the retention time and molecular weights of the GC-MS chromatogram, six compounds
were identified in the ethyl acetate fraction of G. edulis. As listed in Table 4, these compounds
included 2,5-dimethylhexane-2,5-dihydroperoxide, phthalic acid-6-ethyloct-3-yl 2-ethylhexyl ester,
1H-Indole-2-carboxylic acid, 6-(4-ethoxyphenyl)-3-methyl-4-oxo-4,5,6,7-tetrahydro-isopropylester,
2,3,5-Trichlorobenzaldehyde, Benz(b)1,4-oxazepine-4 (5H)-thione, 2,3-dihydro-2,8-dimethyl, and
2-acetoxymethyl-3-(methoxycarbonyl) biphenylene.
Figure 3. Chromatograms obtained from the gas chromatography-mass spectrometry (GC-MS) analysis
of the ethyl acetate fraction of G. edulis.
42
Molecules 2019, 24, 3708
Table 4. Active compounds identified in the ethyl acetate fraction of G. edulis by gas
chromatography-mass spectrometry (GC-MS) analysis.
Retention Time Molecular Reported

Name Compound Class
and % Formula Biological Activity
17.956 2,5-Dimethylhexane- Anti-inflammatory
C8 H18 O4 Organic compound
(54.27%) 2,5-dihydroperoxide Antioxidant [14,15]
55.726 Phthalic acid-6-ethyloct-3-yl Phthalic acid Anticancer
C26 H42 O4
(11.46%) 2-ethylhexyl ester derivative Antimicrobial [16]
1H-Indole-2-carboxylic acid,6-(4-
66.305 ethoxyphenyl)-3-methyl-4-oxo-
C19 H20 FNO3 Indole derivative Antidiabetic [17,18]
(15.39%) 4,5,6,7-tetrahydro-isopropyl
ester
72.446 1,2-dimethoxy-4 (1,3-dimethoxy- Benzene
C13 H18 O4 Antifungal [19]
(10.93%) 1-propenyl) benzene derivatives
Benz(b)1,4-oxazepine-4
73.130 Benzoxazepine Anti-inflammatory
(5H)-thione, C11 H13 NOS
(1.52%) derivatives Antimicrobial [20]
2,3-dihydro-2,8-dimethyl
76.925 2-acetoxymethyl-3- Biphenyl
C17 H14 O4 Antibacterial [21]
(6.49%) (methoxycarbonyl)biphenylene derivatives
Among these active compounds, 1H-Indole-2-carboxylic acid,6-(4-ethoxyphenyl)-3-methyl-4-oxo-

4,5,6,7-tetrahydro-isopropyl ester was identified as possessing antidiabetic activity through insulin
sensitizing and glucose lowering effects [17]. In addition, most of the indole derivatives act as an
activators of glycogen synthase enzyme, which involved in the glycogen synthesis pathway [18], while
2,5-dimethylhexane-2,5-dihydroperoxide has been identified to have antioxidant properties which
help to prevent the development of oxidative stress related to diabetes [14,15].
3. Discussion
Recently, red marine algae has received significant attention due to its immense therapeutic
benefits. However, only a limited number of studies have been performed on Sri Lankan marine algae.
Therefore, to discover the therapeutic potential of tropical marine algae against diabetes and obesity,
we studied G. edulis, a red algae from the northwestern coast of Sri Lanka to investigate its mechanisms
of action.
Polyphenols, flavonoids, and alkaloids are secondary plant metabolites that have shown
therapeutic benefits and are considered as potential sources of antioxidants. The polyphenol, flavonoid,
and alkaloid contents of plants may vary depending on environmental factors, soil type, sun exposure,
rainfall, etc. [22]. As determined by the present study, the ethyl acetate fraction of G. edulis contained
the highest phenol and flavonoid content, whereas the highest alkaloid content was observed in the
crude methanol extract of G. edulis. The present study found comparatively lower phenolic content
compared to the results of Ganesan et al. [23], who reported 3.98 mg/mL phenolic content. This
variation may be due to the different sample collection locations, temperature conditions, and stress
tolerance [24].
Oxidative stress is linked with the development of diabetes, and increases in accumulated fat in
obese individuals through the activation of nicotinamide adenine dinucleotide phosphate (NADPH)
oxidase and the impaired production of adipocytokines [25]. The antioxidant activity determined by
the DPPH assay revealed high free radical scavenging activities in the ethyl acetate fraction and crude
methanol extract of G. edulis, which occurred in a concentration-dependent manner (Figure A1). In the
present study, strong DPPH free-radical scavenging ability was observed at 950 μg/mL of the ethyl
acetate fraction (21.06%) and crude methanol extract (19.52%). This result contradicts a previous report
in India [23]; compared to the present study, [16] reported lower DPPH free radical scavenging activity
of the ethyl acetate fraction (4.73%) and the crude methanol extract (5.20%). The higher antioxidant
43
Molecules 2019, 24, 3708
activity observed in the present study may be due to the use of different extraction procedures and the
differences in the phenolic compounds responsible for antioxidant activity.
The highest ABTS radical scavenging activity was observed in the ethyl acetate fraction of G. edulis,
with a significant positive correlation being observed with total phenol content (r = 85). The reducing
power of the sample depends on the available phenol and flavonoid contents of the sample [26]. In the
present study, the ethyl acetate fraction exhibited the highest FRAP activity, which can be attributed
to the presence of phenolic or flavonoid compounds with functional groups such as hydroxyl and
carbonyl, which leads to the reduction or inhibition of oxidation. In contrast, Francavilla et al. reported
the highest ABTS radical scavenging activity and ferric reducing antioxidant power in ethyl acetate
fraction (0.43 and 0.809 mmol TE/g) of G.edulis collected seasonally in the Lesina Lagoon in Italy
during the period of July [27]. Similarly, the ethyl acetate fraction of the present study exhibited
higher reducing power compared to the previous study, which was collected in February. Therefore,
differences in reducing power might be due to the seasonal variation.
The chelating power of the sample was determined using ferrozine reagent. Ferrozine can chelate
with Fe2+ , forming a red-colored ferrozine-Fe2+ complex [28]. According to the chelation activity, the
highest FICA was observed in the ethyl acetate fraction. The ORAC assay determines the oxidative
degradation of fluorescein in the presence of a free radical generator, such as an azo compound,
for example, 2-azobis (2-amidinopropane) dihydrochloride [29]. The ORAC assay confirmed the
high antioxidant capacity of the crude methanol extract of G. edulis. The high phenol and flavonoid
contents of the ethyl acetate fraction are responsible for the potent antioxidant activity of G. edulis.
Additionally, the presence of bioactive compounds such as 2,5-dimethylhexane-2,5-dihydroperoxide
may also contribute to the antioxidant activity of G. edulis.
The inhibition of key metabolic carbohydrate-digesting enzymes is one of the main strategies to
determine the antidiabetic activity of medicinal plants [30]. Therefore, natural bioactive compounds
that reduce blood glucose levels by inhibiting the key metabolic enzymes (α-amylase, α-glucosidase)
and glucose absorption can be considered to be useful for the management of diabetes [31]. From the
results, it is evident that the ethyl acetate fraction of G. edulis showed potential inhibitory activities
with an effective dose for inhibition of α-amylase (IC50 : 279.48 ± 5.62 μg/mL) and α-glucosidase (IC50 :
87.92 ± 1.62 μg/mL) enzymes that are comparable to the standard drug acarbose (Table 3). The potential
enzyme-inhibiting activity of the ethyl acetate fraction of G. edulis can be attributed to the presence of
phytochemicals such as phenolic and flavonoid compounds. Senthil and Sudha [32] reported potent
effective doses of inhibitory activity of α-amylase (IC50 : 83 μg/mL) and α-glucosidase (IC50 : 46 μg/mL)
in an aqueous extract of G. edulis collected from India. The difference between the results obtained in
the present study may be due to the different solvents used for the extraction method; in the present
study, polyphenol was initially used to extract methanol, while the study conducted by Senthil and
Sudha used water as a solvent.
The glucose diffusion inhibition test was carried out to evaluate the effect of methanol extract
and fractions of G.edulis, with respect to its glucose retardation activity across the dialysis tube.
The glucose entrapment ability of the crude methanol extract and four fractions were found to be
significantly different at different times. Among them, the ethyl acetate fraction of G.edulis exhibited
a significant glucose entrapment ability, which decreased the glucose movement into the external
solution at 180 min compared to the control. The fact that the ethyl acetate fraction exhibited the highest
inhibition of glucose diffusion may be due to the presence of insoluble fiber particles which entrap
glucose molecules [33,34]. The dialysis tube method is a simple technique, which only determines the
potential effect of methanol extract and fractions of G.edulis to retard the glucose diffusion through
the normal dialysis membrane, whereas in the intestinal tract, transportation of glucose is assisted by
glucose transporters incorporated with other molecules, in addition to the intestinal contractions [35].
Therefore, further in vivo studies should be carried out to determine the real effect of methanol extract
and fractions of G. edulis on glucose diffusion.
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Molecules 2019, 24, 3708
Chronic hyperglycemia in diabetic patients leads to the progression of microvascular and

macrovascular complications. The high level of blood glucose leads to the formation of a complex
between glucose and plasma proteins through non-enzymatic reactions and forms AGEs, which are
associated with the pathogenesis of vascular complications in diabetes, renal failure, Alzheimer’s
disease, aging, and other chronic diseases [27]. The present study is the first to report the inhibitory
activity of the methanol extract and four fractions of G. edulis on the formation of AGE products. The
results of the study show that the lowest effective dose of inhibition of AGE formation was exhibited
by chloroform (IC50 :258.23 ± 3.24 μg/mL) and ethyl acetate fractions (IC50 : 586.54 ± 4.37 μg/mL) of
G. edulis, compared to the standard drug rutin. The antiglycation activity of the chloroform and
ethyl acetate fractions may be due to the presence of phenolic and flavonoid compounds, which are
significantly correlated with anti-glycation activity [36].
Furthermore, GC-MS analysis of the ethyl acetate fraction of G. edulis revealed the
presence of active compounds with strong antioxidant and antidiabetic activity, including
1H-Indole-2-carboxylic acid,6-(4-ethoxyphenyl)-3-methyl-4-oxo-4,5,6,7-tetrahydro-isopropyl ester and
2,5-dimethylhexane-2,5-dihydroperoxide. 1H-Indole-2-carboxylicacid,6-(4-ethoxyphenyl)-3-methyl-
4-oxo-4,5,6,7 tetrahydro-isopropyl ester is an indole derivative, which has insulin sensitizing and
glucose lowering effects [17]. In addition, most of the indole derivatives act as an activator of
the glycogen synthase enzyme, which is involved in the glycogen synthesis pathway [18], while
2,5-dimethylhexane-2,5-dihydroperoxide was identified to have antioxidant properties, which help to
prevent the development of oxidative stress related to diabetes [14,15].
4.1. Chemicals and Reagents

Trolox, DPPH, trichloroacetic acid, ABTS, potassium persulpahte, 2,4,6-tripyridyl-s-triazine
(TPTZ), soluble starch, Folin–Ciocalteu reagent, alpha glucosidase from Saccharomyces cerevisiae, gallic
acid, acarbose, quercetin, aluminum chloride, p-nitrophenyl a-d-glucopyranoside, bovine serum
albumin (BSA), and alpha-amylase were purchased from Sigma Aldrich (Allentown, PA, USA). The
chemicals and reagents used for all experiments were of analytical grade.
4.2. Collection of Algae Sample

Permission to collect an algae sample was obtained from the Department of Wildlife Conservation
(permit number WL/3/280/17). Marine red algae (Gracillaria edulis) was manually collected from
Kalpitiya, Sri Lanka (60 4 54.19” N; 800 8 51.78” E). The samples were identified based on their
morphological characteristics by Dr. Kalpa Samarakoon. The collected samples were cleaned and
washed with fresh water to remove salt, sand, and other debris. The samples were freeze-dried using a
LyoBeta freeze drying unit (Telstar), powdered, and stored at −20 ◦ C until further use.
4.3. Preparation of Gracillaria edulis Extract and Its Solvent Fractions

The extraction procedure was conducted according to the method of Lakmal et al., [37] with some
modifications. Homogenized G. edulis powder was extracted three times using 70% methanol, and was
then subjected to sonication (Clifton, 91695) at 25 ◦ C. Polyphenols were separated using 70% ethanol
(v/w% = 1:25) and allowed to stand overnight. The supernatant was separated by centrifugation
(Centurion K241R, Surrey, UK) at 12,000 rpm before being filtered to separate the polyphenol portion.
A portion of the de-polysaccharide methanol extract was subjected to sequential solvent–solvent
partition with hexane, chloroform, and ethyl acetate, respectively. Finally, the four fractions and crude
methanol extract were dried under vacuum (BUCHI, Rotavapor, R-300, New Castle, DE, USA) and
used to conduct the assays.
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Molecules 2019, 24, 3708
4.4. Quantification of Phenol, Flavonoid, and Alkaloid Contents

The total phenol content of the methanol extract and fractions was evaluated using the
Folin–Ciocalteu method (n = 4). Different concentrations (5, 10, and 20 mg/mL) of methanol extract
and the four fractions were prepared by diluting with distilled water. The sample (20 μL), diluted
Folin–Ciocalteu reagent (110 μl), and 10% sodium carbonate solution (70 μL) were mixed and incubated
at room temperature for 30 min. Absorbance was measured at 765 nm. Gallic acid was used as
the standard, and results were expressed as mg gallic acid equivalent per 1 g of dry weight of the
extract/fraction [38].
The total flavonoid content of the methanol extract and fractions (n = 4) of G. edulis were evaluated
by the aluminium chloride method [39]. Different concentrations (5, 10, and 20 mg/mL) of the methanol
extract and fractions were prepared by diluting with methanol. The absorbance before adding the
2% aluminum chloride was taken at 415 nm. A total of 100 μl of 2% aluminium chloride solution
was added and incubated at room temperature for 10 min. A plate reading was recorded at 415 nm.
Quercetin was used as a standard, and results was expressed as mg quercetin equivalent per 1 g of dry
weight of the extract or reaction.
The total alkaloid content of the methanol extract and fractions (n = 4) of G. edulis was evaluated
by the method described by Sreevidya et al. [40] with some modifications. Different concentrations
(5, 10, and 20 mg/mL) of the methanol extract and fractions of G. edulis were prepared by diluting with
95% ethanol and pH was adjusted to 2–2.5. A total of 100 μL of sample was mixed with 200 μL of
Dragendorff reagent and centrifuged at 5000 rpm for 5 min. The precipitate was separated and washed
with 95% ethanol. A total of 200 μL of 1% disodium sulfide solution was added, and the resulting
brownish-black precipitate was centrifuged at 5000 rpm for 5 min. The supernatant was discarded and
the pellet was dissolved in concentrated HNO3 and diluted up to 1 mL with distilled water. A·total of
100 μL of the solution was pipetted and mixed with 500 μL of 3% thiourea. Absorbance was recorded at
460 nm. Piperine was used as the standard, and the results were expressed as mg piperine equivalent
per 1 g of dry weight of extract or fraction.
4.5.1. DPPH Radical Scavenging Activity

The DPPH scavenging activity of the extracts and fractions of G. edulis (n = 4) was determined
using the method described by Blois [41]. A concentration series (3.75, 1.875, 0.938, 0.469, 0.234, 0.1170,
and 0.058 mg/mL) of algal samples was prepared in methanol. A total of 50 μL of sample was mixed
with methanol and absorbance before adding the DPPH solution was taken at 517 nm. Subsequently,
DPPH solution was added and incubated in a dark area for 15 min at 25 ◦ C. After incubation, a plate
reading was taken at 517 nm. Trolox was used as the standard, and the DPPH scavenging activity was
expressed as mg Trolox equivalent per 1 g of dry weight of extract or fraction.
4.5.2. ABTS+ Radical Scavenging Activity

The ABTS+ scavenging activity was measured using the method described by Re et al. [42]. Before
conducting the experiment, ABTS+ radical was produced by incubating ABTS tablet (10 mg) with
2.5 mM potassium persulpahte solution (2.5 mL). Five different concentrations (0.5, 0.25, 0.125, 0.063,
and 0.031mg/mL) of the methanol extract and fractions (n = 4) were diluted in 50 mM PBS (pH 7.4)
and absorbance before adding ABTS reagent was taken at 734 nm. A total of 40 μL of diluted ABTS+
reagent was added and incubated for 10 min at room temperature. After incubation, a plate recording
was taken at 734 nm. Trolox was used as the standard antioxidant and the results were expressed as
mg Trolox equivalent per 1 g of dry weight of extract or fraction.
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Molecules 2019, 24, 3708
4.5.3. Ferric Reducing Antioxidant Power (FRAP)

Ferric reducing antioxidant power was measured according to the method of Benzie and Szeto [43],
with some modifications. Different concentrations (5, 2.5, 1, and 0.5 mg/mL) of the methanol extract and
four fractions (n = 4) were diluted in 300 mM acetate butter (pH 3.6). Before conducting the experiment,
FRAP reagent was prepared using 300 mM acetate buffer (pH 3.6), 10 mM of 2,4,6-tripyridyl-s-triazine
(TPTZ), and 20 mM FeCl3 solution mixed with a ratio of 10:1:1 and incubated at 37 ◦ C for 10 min. The
FRAP reagent (150 μL), acetate buffer (30 μL), and algae samples (20 μL) were mixed together and
incubated for 8 min at room temperature. After incubation, a plate reading was made at 600 nm. Trolox
was used as the standard and the results were expressed as mg Trolox equivalent per 1 g dry weight of
extract or fraction.
4.5.4. Ferrous Iron Chelating Capacity (FICC)

The ferrous iron chelating capacity (FICC) was measured using the method of Carter [44], with
some modifications. Five different concentrations (6.25, 5, 3.13, 2.5, and 1.56 mg/mL) of the methanol
extract and fractions were diluted in distilled water, 1 mM ferrozine (4,4-disulfonic acid sodium salt)
solution, and 1 mM ferrous sulphate solution (n = 4). Algal samples (100 μL), FeSo4 solution (20 μL),
and distilled water (40 μL) were mixed, and absorbance before adding ferrozine solution was taken at
562 nm. Ferrozine solution (40 μL) was mixed and incubated for 10 min at room temperature. A plate
reading was recorded at 562 nm and the results were expressed as mg EDTA equivalent per 1 g of dry
weight of the extract or fraction.
4.5.5. Oxygen Radical Absorbance Capacity (ORAC)

The ORAC was measured using the method of Ou et al. [45], with modifications. Different
concentrations (5, 2.5, and 1.25 mg/mL) of the extract and fractions were diluted in phosphate buffer
saline (75 mM, pH 7.4). Before conducting the experiment, flourcein (16 mg in 100 mL of phosphate
buffer saline) and AAPH [2,2 -azobis (2-amidinopropane) dihydrochloride] solutions (40 mg in 1 mL
of phosphate buffer saline) were prepared. The reaction mixture (200 μL), containing algal samples
(10 μL), PBS (40 μL) and fluorescein solution (100 μL), was preincubated at 37 ◦ C for 5 min. After
incubation, AAPH solution was added and absorbance recordings were made at excitation and emission
wavelengths of 494 nm and 535 nm, respectively, at 1 min intervals for 35 min. Trolox was used as the
standard, and the results were expressed as mg Trolox equivalent per 1 g dry weight of the sample.
4.6. In Vitro α-amylase and α-glucosidase Inhibitory Assay
4.6.1. Alpha-Amylase Inhibitory Activity

The anti-amylase activity was measured using the method of Bernfeld [46], with modifications
(n = 4). Various concentrations of the algal extract (400, 200, 100, 50, 25, and 12.5 μg/mL) and acarbose
(200, 100, 50, 25, 12.5, and 6.25 μg/mL) were preincubated with starch (40 μL) and sodium acetate
buffer (710 μL) at 40 ◦ C in a shaking water bath (Wise Bath, WSB-30, Wertherim, Germany) for 10 min.
The α-amylase enzyme solution (50 μl:1 mg/mL) was added and incubated again for 15 min. After
incubation, 500 μL of 3,5-dinitrosalicylic acid (DNS) solution was added and boiled for 5 min until
color developed and was then kept in an ice bath to cool. The absorbance was recorded at 540 nm
using a SpectraMax Plus 384 instrument (Molecular Devices, San Jose, CA, USA). Another experiment
was carried out in an identical way by replacing enzyme solution with acetate buffer to determine the
absorbance produced by the sample itself. A control experiment was conducted by replacing extracts
with 100 mM sodium acetate buffer. Acarbose was used as the standard and percentage α-amylase
inhibition was calculated.
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Molecules 2019, 24, 3708
4.6.2. Alpha-Glucosidase Inhibitory Activity

The α-glucosidase inhibition assay was performed according to the method of Matsui et al. [47],
with some modifications. Different concentrations of algal samples (133.3, 66.6, 33.3, 16.6, 8.3, and
4.16 μg/mL) or acarbose (2.5, 1.25, 0.625, 0.313, 0.156, 0.078, and 0.039 μg/mL) in acetate buffer,
and p-nitrophenyl-a-d-glucopyranoside (PNPG) solution were incubated at 37 ◦ C for 5 min. After
measuring the absorbance, 25 mU/mL of α-glucosidase was added and incubated at 37 ◦ C for 35 min.
After incubation, 10% NaCO3 solution was added to stop the reaction. The absorbance was then
recorded at 400 nm. The reaction mixture without extract was used as the control and anti-glucosidase
activity (% inhibition) was calculated.
4.6.3. Glucose Diffusion Inhibitory Activity

Glucose diffusion inhibitory activity was determined using a dialysis bag and a glucose oxidase
kit [48]. Initially, a dialysis membrane (D9777) was activated according to the manufacturer’s
instructions. Briefly, 2 mL of the algae sample (1 mg/mL) was placed in a dialysis tube with 2 mL
of 0.22 mM glucose solution. Subsequently, the dialysis tube was dipped in a beaker containing
80 mL of 0.15M NaCl and 20 mL of distilled water and was shaken constantly at 150 rpm at 37 ◦ C.
The glucose concentration (μg/mL) in the external solution was measured at 30 min intervals for 3 h
using a GAGO-20 glucose oxidase kit (Sigma Aldrich). Acarbose and distilled water were used as the
standard and the control, respectively.
4.6.4. Antiglycation Activity

Antiglycation activity was measured using the method of Matsuura et al. [49], with some
modifications. Algal extracts were prepared by dissolving with phosphate buffer saline. Bovine serum
albumin (80 μL), algae extract (80 μL), 400 mM glucose (360 μL), and 50 mM PBS (pH 7.4, 480 μL) was
incubated at 60 ◦ C for 50 h. After incubation, the solution was cooled and 200 μl of 50% trichloroacetic
acid was added and centrifuged at 15,000 rpm at 4 ◦ C for 4 min. The resulting precipitate was dissolved
in PBS (pH 10) and absorbance values were recorded at an excitation wavelength of 370 nm and
emission wavelength of 440 nm (Amino-Bowman Series 2, Thermo Spectronic, Fitchburd, WI, USA).
A sample negative was carried out in the same way, however without adding glucose. A sample
control was conducted by replacing algal extracts with 50 mM PBS. Rutin was used as the standard
and antiglycation activity (% inhibition) was calculated.
4.7. GC-MS Analysis of Extract and Solvent Fractions

The GC-MS analysis was performed on active fraction (ethyl acetate fraction) using a 5975C
gas chromatograph (Agilent Technologies, Palo Alto, CA, USA) and an HP-5MS capillary column
(30 m × 25 μm with a film thickness of 0.25 μm). Briefly, the sample (1 μL) was injected into the
HP-5MS capillary column and was exposed to a temperature of 70 ◦ C for 2 min; then, the temperature
was increased from 70 ◦ C to 200 ◦ C at a rate of 3 ◦ C/min−1 and held at 200 ◦ C for 15 min. Helium was
used as the carrier gas with a flow rate of 1 mL/min. After obtaining the chromatogram, the mass
spectrum of the unknown component was identified using the NIST (NIST 17) library [50].

Statistical analysis of each experiment was carried out using the Minitab 17 software (Cubic
Computing Pvt. Ltd., Bangalore, India) and Microsoft Excel 2016. All the experiments were performed
using four replicates. Mean and standard deviation were calculated using standard equations. One-way
ANOVA was used to determine the significant differences between the methanol extract and the
fractions of G. edulis. p-values less than 0.05 were considered significant. The Pearson’s correlation
coefficient was used for the correlation analysis.
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Molecules 2019, 24, 3708
5. Conclusions
The present study found that G. edulis exhibited promising hypoglycemic activity by inhibiting key
carbohydrate-digesting enzymes, glucose absorption, and the formation of antiglycation end products.
Although antioxidant activity and enzyme inhibitory activity are stronger in commercial drugs than in
G. edulis methanol extracts and its fractions, the hypoglycemic potential of G. edulis was evident in
the present study. GC-MS analysis further confirmed the presence of bioactive compounds rich in
antioxidants and antidiabetic properties. The ethyl acetate fraction exhibited promising antiglycation,
and hypoglycemic potential. Hence, 3T3-L1 mature adipocytes and animal models are required to
confirm anti-obesity and antidiabetic potentials of G. edulis. Isolation of active compounds for the
development of new drugs is also warranted.
Author Contributions: Conceptualization, L.D.C.P., P.R. and K.W.S.; methodology, L.D.C.P., P.R., and K.W.S.;
software, T.L.G.; formal analysis, T.L.G. and P.R.; investigation, T.L.G. and P.R.; data curation, T.L.G.;
writing—original draft preparation, T.L.G.; writing—review and editing, L.D.C.P., and K.W,S.; supervision,
L.D.C.P., P.R. and K.W.S.; project administration, L.D.C.P.; funding acquisition, L.D.C.P. All authors read and
approved the final manuscript.
Funding: This research was funded by the University of Sri Jayewardenepura, Sri Lanka (ASP/01/RE/SCI/2017/50).
Conflicts of Interest: The authors declare no conflict of interest.
Appendix A
Figure A1. Dose dependent DPPH (1, 1-diphenyl-2-picrylhydrazine) radical scavenging activity of
methanol extract and fractions of Gracillaria edulis. Data presented as mean ± standard deviation (n = 4).
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Molecules 2019, 24, 3708
Figure A2. Percentage inhibition of α-amylase by G. edulis methanol extract and its fractions. Results
are expressed as mean ± standard deviation (n = 4).
Figure A3. Dose–response relationship of methanol extract and its fractions of G. edulis for anti
glucosidase activity. Results are expressed as mean ± standard deviation (n = 4).
Table A1. Effect of methanol extract and its fractions of Gracillaria edulis (1000 μg/mL) on glucose
diffusion.
Time Glucose Concentration (μg/mL)

(min)
Ethyl
Standard Methanol Hexane Chloroform Aqueous
Control Acetate
(Acarbose) Extract Fraction Fraction Fraction
Fraction
30 14.32 ± 0.77 a 1.72 ± 0.34 b 3.51 ± 0.47 b 12.26 ± 1.96 a 7.09 ± 0.82 c 2.96 ± 0.47 b 7.43 ± 0.59 c
60 23.41 ± 1.01 a 5.09 ± 0.72 b 11.77 ± 0.47 c 20.66 ± 1.97 a 15.22 ± 1.13 d 10.67 ± 0.75 c 11.64 ± 1.62 c
90 30.58 ± 1.14 a 8.19 ± 0.47 b 22.73 ± 0.57 c 27.89 ± 1.13 d 22.59 ± 1.12 c 15.91 ± 0.61 e 29.13 ± 0.79 a,d
120 42.43 ± 1.54 a
13.15 ± 0.61 b 33.75 ± 0.79 c 40.57 ± 1.44 a 36.98 ± 1.19 e
23.07 ± 2.02 d 36.09 ± 1.54 c,e
150 48.90 ± 1.22 a
18.87 ± 0.57 b 42.08 ± 1.37 c 46.42 ± 0.91 d 41.25 ± 0.61 c 27.55 ± 0.59 e
47.25 ± 1.35 a,d
180 57.65 ± 1.67 a
22.79 ± 0.47 b 48.36 ± 1.04 c 52.01 ± 0.96 d 49.04 ± 0.67 c 38.15 ± 1.11 e
52.69 ± 1.31 d
Results are expressed as mean ± standard deviation (n = 4). Mean values in a column superscripted by different
letters (a–e ) are significantly different at p < 0.05.
50
Molecules 2019, 24, 3708
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molecules
Article
Antioxidant, Xanthine Oxidase, α-Amylase and
α-Glucosidase Inhibitory Activities of Bioactive
Compounds from Rumex crispus L. Root
Truong Ngoc Minh 1, *, Truong Mai Van 2 , Yusuf Andriana 2,3 , Le The Vinh 1 , Dang Viet Hau 1 ,
Dang Hong Duyen 1 and Chona de Guzman-Gelani 4
1 Center for Research and Technology Transfer (CRETECH), Vietnam Academy of Sciences and Technology,
Hanoi 10072, Vietnam; thevinh3839@gmail.com (L.T.V.); hauhoahock20@gmail.com (D.V.H.);
hongduyen1908@gmail.com (D.H.D.)
2 Graduate School for International Development and Cooperation (IDEC), Hiroshima University,
Higashi-Hiroshima 739-8529, Japan; truongmaivan1991@gmail.com (T.M.V.);
yusufandriana@yahoo.com (Y.A.)
3 Research Center for Appropriate Technology, Indonesian Institute of Sciences, Subang, 41213, Indonesia
4 Department of Chemistry, College of Science and Mathematics, Mindanao State University - Iligan Institute
of Technology, Iligan 9200, Philippines; chona.gelani@g.msuiit.edu.ph
* Correspondence: minhtn689@gmail.com; Tel./Fax: +84-912-990-197

Received: 19 September 2019; Accepted: 22 October 2019; Published: 31 October 2019
Abstract: The root of Rumex crispus L. has been shown to possess anti-gout and anti-diabetic properties,
but the compounds responsible for these pharmaceutical effects have not yet been reported. In this
study, we aimed to isolate and purify active components from the root of R. crispus, and to evaluate
their anti-radical, anti-gout and anti-diabetic capacities. From the ethyl acetate (EtOAc) extract,
two compounds, chrysophanol (1) and physcion (2), were isolated by column chromatography with
an elution of hexane and EtOAc at a 9:1 ratio. Their structures were identified by spectrometric
techniques including gas chromatography-mass spectrometry (GC-MS), electrospray ionization-mass
spectrometry (ESI-MS), X-ray diffraction analyses and nuclear magnetic resonance (NMR). The results
of bioassays indicated that (1) showed stronger activities than (2). For antioxidant activity, (1) and
(2) exhibited remarkable DPPH radical scavenging capacity (IC50 = 9.8 and 12.1 μg/mL), which was
about two times stronger than BHT (IC50 = 19.4 μg/mL). The anti-gout property of (1) and (2) were
comparable to the positive control allopurinol, these compounds exerted strong inhibition against
the activity of xanthine oxidase (IC50 = 36.4 and 45.0 μg/mL, respectively). In the anti-diabetic assay,
(1) and (2) displayed considerable inhibitory ability on α-glucosidase, their IC50 values (IC50 = 20.1
and 18.9 μg/mL, respectively) were higher than that of standard acarbose (IC50 = 143.4 μg/mL).
Findings of this study highlight that (1) and (2) may be promising agents to treat gout and diabetes,
which may greatly contribute to the medicinal properties of Rumex crispus root.
Keywords: bioactive compound; anti-radical; anti-gout; anti-diabetic; Rumex crispus;

chrysophanol; physcion
1. Introduction
Rumex crispus L. is known as the curly dock or yellow dock in most of Europe, North Africa,
Turkey, Northern Iran, Central and East Asia, and North America [1]. The roots of this plant have been
used in traditional medicine as a tonic, laxative, and for hemostasis medication. The fruits (seeds) are
used for the treatment of dysentery. The young leaves of the plant are eaten as servings of mixed greens
and soups. The plant has been utilized for treating the relevant disease, dermatology contaminations,

Molecules 2019, 24, 3899
gastrointestinal tract maladies, upper respiratory tract illnesses, and loose bowels [2,3]. The bioactive
substances detected in this plant include flavonoids (isorientin, vitexin, orientin and isovitexin), lipids,
vitamins, carotenoids, natural acids and minerals. The root of R. crispus may be a wealthy source of
anthraquinones glycosides (chrysophanol and emodin) [4].
Antioxidant prevention agents are common or manufactured substances having the capacity to
hinder or delay oxidation at relatively low concentrations [5]. Antioxidant compounds are known
to combat oxidative stress. When oxidative stress is uncontrolled it is associated with several
pathophysiological processes [6]. In a pharmacological examination of the ether, ethanol, and hot
water extracts of R. crispus, the water extract showed the most elevated antioxidant activity. The
most noteworthy amount of total phenolic compounds was found within the ethanol extract of the
seeds. In regards to the reducing power and DPPH scavenging activity, the ethanol extract of the
seeds was the foremost compelling [7]. The power to quench singlet oxygen and the protective
effects of various extracts (hexane, chloroform, ethyl acetate and butanol) of R. crispus seeds against
photodynamic damage were investigated in a biological system. A higher total phenolic content was
observed for the ethyl acetate (EtOAc) and butanol extracts. The level of in vitro antioxidant action of
the methanol concentrate of R. crispus was determined by measuring its ferric-lessening antioxidant
activity, without DPPH radical searching movement and the ability to impact the lipid peroxidation
in liposomes. It was seen that the methanol concentrate had direct antioxidant prevention agent
movement. In view of the in vivo tests, it was inferred that the dose routine did not impact the degrees
of lipid peroxidation. The methanol concentrate of R. crispus roots displayed DPPH radical searching
(IC50 = 42.86 μg/mL) [7,8]. The ethereal piece of R. crispus can be utilized as a compelling and safe
wellspring of antioxidant prevention agents. However, information on the anti-gout and anti-diabetic
properties of the underground plant parts is still limited.
Xanthine oxidase (XOD) is an important enzyme responsible for hyperuricemia, and a predisposing
factor for gout and oxidative stress-related diseases. This enzyme plays an important role in catalyzing
the oxidation of hypoxanthine to xanthine and xanthine to uric acid [9,10]. Nowadays, few drugs
are commonly used to treat gout including allopurinol and febuxostat. However, some serious and
undesirable effects on skin caused by allopurinol and febuxostat may occur when taking the drug,
including Steven-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN), causing death in up to
39% of cases. Lesions can occur on other organs such as the liver and kidney [11,12]. Many studies have
reported that compounds possessing both antioxidant properties will be effective in gout treatment [13].
On the other hand, antioxidants are able to prevent or slow down oxidation by eliminating free radicals,
which further help prevent oxidative disease and increase people’s lifespan [14]. On the other hand,
xanthine oxidase enzyme inhibitors can reduce the enzyme activity, preventing the formation of urate
salts [15,16].
Diabetes has become a worldwide health problem in developed and developing countries. It is
reported that about 425 million people are suffering from diabetes, which accounts for 12% of global
health expenditure [17]. Among diabetic types, the pathogenesis of type 2 diabetes may launch from
many factors such as genetic predisposition, environment, and pancreatic beta-cell dysfunction [18].
Therefore, one of the diabetes treatments is to inhibit the enzyme activity of α-amylase and α-glucosidase
to minimize the formation of blood glucose [19]. On the other hand, oxidative tension in diabetes
coexists with a reduction in antioxidant status [20], which can increase the deleterious effects of free
radicals. It has also been known that alloxan induces diabetogenic activity mainly by inducing oxygen
free base and thereby damaging the pancreas [21]. Supplementation with non-toxic antioxidants may
have a chemoprotective role in diabetes [22]. Antioxidants as well as vitamins C and E, have been
shown to reduce oxidative stress in experimental diabetes [23]. Supplementation of vitamin C has also
been shown to lower berth glycosylated hemoglobin in diabetic patients [23]. Many industrial plant
excerpts and plant products have been shown to have significant antioxidant activity [24–26].
This study aims to isolate compounds from R. crispus root that can scavenge free radicals and
inhibit xanthine oxidase, α-amylase and α-glucosidase. The results of the study will contribute to a
56
Molecules 2019, 24, 3899
better understanding of the value of medicinal plants, as well as contribute to the database for the
production of gout and diabetes medications. Biological properties of the isolated compounds were
also examined for their efficacy and safety in antioxidant activity and inhibition of xanthine oxidase,
α-amylase and α-glucosidase enzyme by in vitro methods. The obtained compounds were identified by
gas chromatography-mass spectrometry (GC-MS), electrospray ionization (ESI), atmospheric pressure
chemical ionization (APCI), nuclear magnetic resonance (NMR), and X-ray diffraction.
2. Results
2.1. Structure Elucidation of Isolated Fractions

Separation of bioactive compounds from Rumex crispus root (RCR) was conducted following the
procedure illustrated in Figure 1.
Figure 1. Isolation and purification of bioactive compounds from Rumex crispus root.
The isolated metabolites were characterized and identified by GC-MS, ESI-MS, APCI-MS, 1 H- and
13 C-NMR and X-ray analyses (Supplementary Figures S1–S21 and Tables S1–S3).
On the basis of GC-MS data (Table 1), isolated fractions were identified. The structure and formula
of compounds were further confirmed by ESI-MS. Two fractions, C2 and C3, were elucidated by 1 H
NMR and 13 C NMR. Chemical structures of the identified compounds are illustrated in Figures 2 and 3.
Table 1. Bioactive compounds identified in EtOAc extract of Rumex crispus root by GC-MS.
Retention Peak Area Chemical Molecular

Fractions Compounds Similarity
Time (%) Formula Weight
20.70 73.39 Chrysophanol C15 H10 O4 254 90.8
C1
22.98 24.82 Physcion C16 H12 O5 284 91.8
C2 20.71 98.10 Chrysophanol C15 H10 O4 254 98.1
C3 22.99 97.79 Physcion C16 H12 O5 284 97.8
57
Molecules 2019, 24, 3899
Chrysophanol (C1, C2) Physcion (C1, C3)
Figure 2. Chemical structures of bioactive constituents identified in EtOAc extract of Rumex crispus L.
root by single-crystal X-ray.
OH O OH
1 8
9a 8a
2 9
7
3 6
10
H3C 4a 10a R1
4 5
O
1 : R1 = H
2 : R1 = OCH3
Figure 3. Structures of compounds 1 and 2 from Rumex crispus L. root.
2.2. NMR Structural Elucidation

Compound 1 was isolated as an orange needle from the EtOAc extract of RCR. Its molecular
formula, C15 H10 O4 , was deduced from a combined analysis of the positive ESI MS at m/z 255.3 [M +
H]+ (C15 H11 O4 ) and 1 H-, 13 C-NMR, HSQC, and HMBC spectra. The 1 H-NMR spectrum of 1 showed
the presence of a methyl group at δH 2.46 (3H, s, H-11) and five protons of aromatic ring at δH 7.82
(1H, dd, J = 7.0, 1.0 Hz, H-5), 7.67 (1H, t, J = 8.0 Hz, H-6), 7.64 (1H, s, H-4), 7.30 (1H, dd, J = 8.5,
1.0 Hz, H-7), 7.09 (1H, d, J = 0.5 Hz, H-2) and two protons of hydroxyl groups at δH 12.1 (1H, s, OH-8),
11.99 (1H, s, OH-1). The 13 C NMR of 1 displayed 15 carbon signals including a methyl, two ketone
groups, five methines, seven quaternary carbons (two of which are bonded to O atoms) (Table 2).
Assignments of all 1 H and 13 C NMR signals were accomplished by interpretation of HSQC and HMBC
spectra. The melting point of compound was 196 ◦ C. The above spectral data were diagnostic of an
anthraquinone having two hydroxyl groups at C-1, C-8 position, and a methyl group. Moreover,
methyl group and two hydroxyl group positions were determined by the HMBC spectral correlation
between the proton of methyl group H-11 (δH 2.46) with C-2 (δC 124.4)/C-4 (δC 121.4); OH-1 (δH 11.99)
with C-2 (δC 124.4)/C-9a (δC 113.8) and OH-8 (δH 12.1) with C-7 (δC 124.6)/C-8a (δC 115.9). By means
of its spectrometric analysis and comparison with previously published data [27], compound 1 was
determined as chrysophanol or 1,8-dihydroxy-3-methylanthraquinone.
Compound 2 was obtained as orange-yellow needles. The 1 H and 13 C NMR spectra of 2 closely
resembled those of 1, except for the appearance of one methoxy group (δH 3.93, (3H, s), δC 56.0) and
the chemical shift of C-6 (Table 2). This suggested that 2 also has an anthraquinone of 1 and 2 was the
result of replacing the aromatic ring proton at C-6 in 1 by a methoxy group. This was confirmed by
comparison of the NMR data of 2 with those of a known anthraquinone in Reference [28]. Thus 2 was
determined to be 1,8-dihydroxy-3-methoxy-6-methylanthraquinone or physcion.
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Molecules 2019, 24, 3899
Table 2. 13 C-NMR data for compounds 1 and 2.
1 2
Position
δC * δC δC ** δC
1 161.1 162.7 162.5 162.5
2 124.2 124.4 124.5 124.5
3 149.0 149.3 148.4 148.4
4 120.4 121.4 121.3 121.3
5 119.2 119.9 108.2 108.2
6 137.2 136.9 166.5 166.5
7 123.9 124.6 106.8 106.8
8 161.4 162.4 165.2 165.2
9 191.4 192.6 190.8 190.7
10 181.3 182.0 181.5 181.9
4a 132.8 133.3 133.2 133.2
8a 115.7 115.9 110.3 110.3
9a 113.6 113.8 113.7 113.7
10a 133.2 133.7 135.3 135.2
CH3 21.6 22.2 22.1 22.1
OCH3 56.0 56.0
δC * Chrysophanol in DMSO; δC ** Physcion in CDCl3.
2.3. Quantitative Analysis of Fraxetin from Rumex Crispus Root

The content of two pure compounds chrysophanol and physcion in RCR is presented in Table 3.
The quantity of these compounds was determined by the instrumentation used in this study.
Table 3. Quantity of pure compounds from R. crispus root.
Concentration
Fractions Retention Time Compounds
(μg/g DW)
C2 20.70 ± 0.02 Chrysophanol 32.50 ± 0.11
C3 22.99 ± 0.05 Physcion 25.04 ± 0.08
Data express means ± SD (standard deviation).
2.4. Antioxidant Activities of the Isolated Fractions

Antioxidant activities of the isolated fractions were determined using three assays namely DPPH,
ABTS free radical scavenging, and reducing power. BHT was used as a standard for all methods. The
results are summarized in Table 4.
Table 4 summarizes the antioxidant activities of the EtOAc R. crispus root extract. For the DPPH
scavenging assay, the antioxidant activity of C2 was the highest as its IC50 value was the lowest
(10.0 μg/mL), followed by C3 (IC50 = 12.0 μg/mL). Statistically, the DPPH radical scavenging properties
of C2 and C3 were stronger than BHT (IC50 = 19.2 μg/mL). The fraction C1 (IC50 = 35.0 μg/mL) exhibited
intermediate antioxidant capacities. For ABTS (2,2 -azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)),
C2 exhibited the maximum scavenging activity with IC50 value of 34.3 μg/mL, followed by C3 and BHT
(IC50 = 44.8 and 46.9 μg/mL). The lowest antioxidant property was fraction C1 (IC50 = 194.7 μg/mL).
For the reducing power ability, C2 displayed the maximum reducing power with IC50 value of
312.6 μg/mL. Generally, the antioxidant properties of the pure compounds C2 and C3 were higher than
standard BHT.
59
Molecules 2019, 24, 3899
Table 4. Antioxidant activities measured by DPPH, ABTS (2,2 -azinobis-(3-ethylbenzothiazoline-6-

sulfonic acid)) and ferric reducing antioxidant power test (FRAP) of EtOAc extract fractions from R.
crispus root in term of IC50 values.
IC50 (μg/mL)
Fractions
DPPH ABTS FRAP
C1 35.0 ± 1.6a 194.7 ± 2.0a 1366.5 ± 8.4a
10.0 ± 0.3c 34.3 ± 0.7c 312.6 ± 6.3c
C2
(39.4 μM) (135.0 μM) (1230.7 μM)
12.0 ± 0.2c 44.8 ± 0.8b 408.6 ± 6.8b
C3
(42.3 μM) (157.7 μM) (1438.7 μM)
19.2 ± 0.3b 46.9 ± 0.9b 422.1 ± 1.1b
BHT*
(87.1 μM) (212.8 μM) (1915.8 μM)
* Positive control. Values are means ± SD (standard deviation); a,b,c indicate significant differences at p < 0.05.
2.5. In Vitro Inhibition of Xanthine Oxidase (XOD), α-Amylase (AAI) and α-Glucosidase (AGI)
The inhibitory effect of the isolated fractions on xanthine oxidase is presented in Table 5. The results
showed that C1–C3 exhibited a considerable activity against xanthine oxidase (IC50 = 36.4–88.8 μg/mL).
Furthermore, C2 manifested the highest inhibitory activity (IC50 = 36.4 μg/mL) followed by C3
(IC50 = 36.4 μg/mL) while C1 exhibited the least (IC50 = 88.8 μg/mL).
Table 5. Xanthine oxidase, α-amylase and α-glucosidase inhibitory activities of isolated fractions from
Rumex crispus root in term of IC50 values.
IC50 (μg/mL)
Fractions
XOD AAI AGI
C1 88.8 ± 0.9a 199.1 ± 1.4a 91.6 ± 1.4b
36.4 ± 0.6c 117.3 ± 1.0b 20.1 ± 0.6c
C2
(143.3 μM) (461.8 μM) (79.1 μM)
45.0 ± 0.7b 113.3 ± 1.3c 18.9 ± 0.4c
C3
(158.5 μM) (398.9 μM) (66.5 μM)
20.5 ± 0.5d - -
Allopurinol*
(150.6 μM)
- 90.9 ± 0.8d 143.8 ± 2.6a
Acarbose*
(140.8 μM) (222.7 μM)
* Positive control. Values are means ± SD (standard deviation); a,b,c,d indicate significant differences at p < 0.05.
A starch-iodine method was applied to examine the inhibitory effect of isolated fractions on
porcine pancreatic α-amylase. Among isolated compounds, C3 displayed the maximum α-amylase
inhibition with IC50 value of 113.3 μg/mL, followed by C2 (IC50 = 117.3 μg/mL). The α-amylase
inhibition is as follows: acarbose > C3 > C2 > C1 corresponding to IC50 values 90.9, 113.3, 117.3, and
199.1 μg/mL, respectively.
The α-glucosidase inhibitory activity of isolated fractions was assayed using a synthetic substrate
p-nitrophenyl-α-D-glucopyranoside (pNPG). All isolated fractions expressed higher α-glucosidase
activity than that of standard acarbose. (Table 5). Of which, the activity of all isolated fractions
was significantly higher than that of standard acarbose. C3 showed the highest inhibitory activity
(IC50 = 18.9 μg/mL) followed by C2 (IC50 = 20.1 μg/mL). Fraction C1 (IC50 = 91.6 μg/mL) exhibited
intermediate α-glucosidase inhibition.
60
Molecules 2019, 24, 3899
3. Discussion
In this study, two compounds were isolated and identified from the EtOAc extract of R. crispus
root, namely chrysophanol (1) (fraction C2) and physcion (2) (fraction C3). They are biologically
active compounds belonging to the flavonoid group. The radical scavenging activity of the isolated
fractions was examined using DPPH, which is a frequently used method in natural product antioxidant
evaluation [27], and ABTS assays. The results of this present study serve as an additional scientific
finding with regards to the radical scavenging activity of the EtOAc extract of R. crispus root. The IC50
values of (1) (10.0 ± 0.3 μg/mL) and (2) (12.0 ± 0.2 μg/mL) are comparable to the value reported by a
previous study on the acetone extract of the R. crispus root with IC50 of 14.0 μg/mL [28]. Moreover, a
study reported that methanol extract of the fruits of R. crispus exhibited antioxidant activity with IC50
value of 3.7 μg/mL [29]. These findings strengthen the antioxidant potential of the roots of R. crispus
over its fruits. Furthermore, this results shows that the antioxidant activities of the pure compounds
(1) and (2) are higher than the standard BHT (Table 4). The results also show a strong correlation
between the concentration of the pure compound (Table 3) and its antioxidant activity. Compound (1)
(32.50 ± 0.11 μg/g DW) exhibits a stronger radical scavenging activity as seen in both DPPH and ABTS
assays (IC50 = 10.0 ± 0.3 and 34.3 ± 0.7 μg/mL, respectively) than (2) (25.04 ± 0.08 μg/g DW, 12.0 ± 0.2
and 44.8 ± 0.8 μg/mL, respectively). This result is in agreement with the findings of Elzaawely et
al. [30] who reported the correlation between the phenolic content and DPPH assay of the EtOAc
extract of the aerial parts of R. japonicus. The same correlation is observed between the concentration
of the pure compound and its reducing power, in that the higher the concentration, the stronger its
reducing power.
Xanthine oxidase (XO) plays a role in gout formation since it catalyzes the oxidation of xanthine
to uric acid. Compounds that inhibit the activity of XO, therefore, can be used to treat gout [31,32].
Dietary flavonoids have been reported to possess inhibitory activity against free radicals and xanthine
oxidase. The antioxidant property is mainly characterized by flavonoid contents that can effortlessly
release hydrogen donors to naturalize free radicals. Therefore, flavonoids could be a promising remedy
for human gout and ischemia by decreasing both uric acid and superoxide concentrations in human
tissues [13]. According to Mohamed Isa et al. [15], Plumeria rubra contains a high amount of flavonoids
and could be used as a new alternative to allopurinol with increased therapeutic activity and fewer side
effects. The XO inhibitory activity in vitro assay of methanol extract of Plumeria rubra flowers possesses
the highest inhibition effects at IC50 = 23.91 μg/mL. Baicalein, baicalin and wogonin, isolated from
Scutellaria rivularis, have been reported to exhibit a strong xanthine oxidase inhibition as evaluated
by modified xanthine oxidase inhibition methods. The results showed that the order was baicalein >
wogonin > baicalin, IC50 = 3.12, 157.38 and 215.19 μM, respectively [33]. From this present study, (1)
(IC50 = 143.3 μM) exhibited a higher inhibitory effect on XO than wogonin and baicalin, whereas (2)
(IC50 = 158.5 μM) inhibited XO stronger than baicalin and showed comparable inhibitory activity as
wogonin. The XO inhibition activity of (1) and (2) is directly related to their concentration as extracted
from the root of R. crispus (Table 3). Allopurinol showed a much lower IC50 value of 20.5 ± 0.5 μg/mL,
which is why allopurinol is the preferred treatment for gout.
Both α-amylase (AA) (responsible for starch digestion) and α-glucosidase (AG) (produces glucose
in the final step of the digestive process of carbohydrates) are related to type 2 diabetes since these
enzymes are responsible for postprandial blood glucose levels [34]. AA and AG inhibitors are therefore
widely used in the treatment of patients with type 2 diabetes, which is related to elevated postprandial
blood glucose levels. Results of this present study showed consistent inhibition activities of (1) and (2)
in both AA and AG in that (2) exhibited a stronger inhibition than (1) (Table 5). However, in terms of
AGI, (1) and (2) turned out to be much better inhibitors compared to acarbose (Table 5). Both terpenoids
and flavonoids might play crucial roles in α-amylase and α-glucosidase inhibition [35,36]. The pure
compounds (1) and (2) registered a stronger anti-diabetic activity than the mixture C3. Moreover, the
synergic effect of the two pure compounds, C3 exerted stronger inhibition against α-glucosidase than
acarbose (IC50 = 143.8 μg/mL). Although this study to isolate and purify active components from
61
Molecules 2019, 24, 3899
the root of R. crispus, as well as test their biological activities, was successful, in vivo tests should be
considered in order to ascertain the anti-gout and anti-diabetic properties of this prospective plant.
4.1. Materials
Rumex crispus root (RCR) was collected (34◦ 23 28.3 N 132◦ 43 06.0” E) in October 2017.
The specimen with voucher number RCR-M2017 was deposited at the Plant Physiology and
Biochemistry Laboratory (IDEC, Hiroshima University, Japan). The samples were cleaned by 1%
NaOCl. After blotting with tissues, samples were dried at 45 ◦ C in the oven for one week then ground
to a fine powder [37].
4.2. Extraction of Rumex Crispus Root

The RCR powder (1.2 kg) was soaked in MeOH (2 L) to produce 23.8 g of MeOH crude extract.
The crude extract was suspended in water (300 mL), then fractionated in hexane and EtOAc to obtain
6.6, 5.8, and 9.7 g water, hexane and EtOAc extracts, respectively.
4.3. Isolation of Pure Compounds

The hexane extract was dried at room temperature, then was purified by MeOH to obtain fraction
C1 (200 mg).
The EtOAc extract was subjected to normal phase column chromatography (CC) using silica gel
(200 g) of 70–230 mesh ASTM and LiChroprep RP-18 (40–63 mm). All fractions were examined by
thin-layer chromatography (TLC) (Merck, Darmstadt, Germany). Fraction 13–15 (C2) (39 mg), and
fraction 16–18 (C3) (30 mg) were crystallized after separation by CC (Figure 1). Compound C2 (1) and
C3 (2) were obtained as pure compounds, the purity levels were confirmed by GC-MS of 98.10% and
97.79%, respectively.
4.4. Antioxidants Activity

Antioxidant activity of the isolated compounds was determined
using DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay, ABTS
(2,2 -azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)) radical cation decolorization assay, ferric
reducing antioxidant power test (FRAP) following previous study [37–39] with slight modifications.
4.5. Identification and Quantification of Isolated Compounds

Identification and quantification of isolated compounds was by GC-MS following previous
study [40,41]. Therefore, the GC-MS system was equipped with a DB-5MS column (30 m × 0.25 mm
internal diameter × 0.25 μm in thickness (Agilent Technologies, J & W Scientific Products, Folsom,
CA, USA). The carrier gas was Helium and the split ratio was 5:1. GC oven temperatures operated
with the initial temperature of 50 ◦ C without hold time, followed by an increase of 10 ◦ C/min up to a
final temperature of 300 ◦ C and holding time of 20 min. The injector and detector temperature were
programmed at 300 ◦ C and 320 ◦ C, respectively. The MS ranged from 29 to 800 amu. JEOL’s GC-MS
Mass Center System Version 2.65a was used to control the GC-MS system and process the data peak.
ESI-MS analysis was conducted on negative/positive ion mode [42]. APCI-MS analysis was
implemented using a mass spectrometer with an electrospray ion source [43].
X-ray diffraction measurement was conducted with a Bruker APEX-II ULTRA diffractometer
(Bruker Corporation, Billerica, MA, USA). The crystals were irradiated using Mo-Kα radiation at −100 ◦ C.
Cell parameters and intensities for the reflection were estimated using the APEX2 crystallographic
software package, in which data reduction and absorption corrections were carried out with SAINT and
SADABS, respectively. The structures were solved and refined with the aid of the program SHELXL.
The refined structures of crystals were reported as a crystallographic information file (CIF) [44].
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Molecules 2019, 24, 3899
4.6. Nuclear Magnetic Resonance (NMR) Data of Chrysophanol and Physcion

Chrysophanol: 1 H-NMR (500 MHz, CDCl3 ), δ (ppm), J (Hz): 12.1 (1H, s, OH-8), 11.99 (1H, s, OH-1),
7.82 (1H, dd, J = 7.0, 1.0 Hz, H-5), 7.67 (1H, d, J = 8.0 Hz, H-6), 7.64 (1H, d, J = 1.5 Hz, H-4), 7.30 (1H,
dd, J = 8.5, 1.0 Hz, H-7), 7.09 (1H, d, J = 0.5 Hz, H-2), 2.46 (3H, s, H-11). 13 C-NMR (125 MHz, CDCl3), δ
(ppm): 192.6 (C-9), 182.0 (C-10), 162.7 (C-1), 162.4 (C-8), 149.3 (C-3), 136.9 (C-6), 133.7 (C-10a), 133.3
(C-4a), 124.6 (C-7), 124.4 (C-2), 121.4 (C-4), 119.9 (C-5), 115.9 (C-8a), 113.8 (C-9a), 22.2 (CH3 ).
Physcion: 1 H-NMR (500 MHz, CDCl3 ), δ (ppm), J (Hz): 12.28 (1H, s, OH-8), 12.08 (1H, s, OH-1),
7.60 (1H, d, J = 1.0 Hz, H-4), 7.34 (1H, d, J = 2.5 Hz, H-5), 7.06 (1H, d, J = 0.5 Hz, H-2), 6.67 (1H, d, J =
2.5 Hz, H-7), 3.93 (3H, s, OCH3), 2.44 (3H, s, H-11). 13 C-NMR (125 MHz, CDCl3), δ (ppm): 190.7 (C-9),
181.9 (C-10), 166.5 (C-6), 165.2 (C-8), 162.5 (C-1), 148.4 (C-3), 135.2 (C-10a), 133.2 (C-4a), 124.5 (C-2),
121.3 (C-4), 113.7 (C-9a), 110.3 (C-8a), 108.2 (C-5), 106.8 (C-7), 56.0 (OCH3 ), 22.1 (C-11).
4.7. Xanthine Oxidase Inhibition (XOD) Activity

The inhibitory effect on xanthine oxidase (XO) of isolated compounds (C1–C3) was measured
spectrophotometrically according to the method reported previously [10] with minor modifications.
4.8. α-Amylase Inhibition (AAI) Assay

The inhibitory effect of C1–C3 on α-amylase was assessed by a starch-iodine method [34] with
modification as follows: in each well of a microplate (U-shape, Greiner Bio-one, NC, USA), 20 μL of
each isolated compound was pre-incubated with 20 μL of 1 U/mL α-amylase solution (from Aspergillus
oryzae, Sigma-Aldrich, St. Louis, MO, USA) at 37 ◦ C for 10 min. The reaction was initiated by pipetting
30 μL of soluble starch (0.5% in deionized water). After 6 min of incubation at 37 ◦ C, an aliquot of 20 μL
of hydrochloric acid (1 M) was added to stop reaction, followed by 120 μL of 0.25 mM iodine solution.
The absorbance at 565 nm was read by a microplate reader (MultiskanTM Microplate Spectrophotometer,
Thermo Fisher Scientific, Osaka, Japan). The inhibitory activity of C1–C3 on α-amylase was performed
as the inhibition percentage calculated using the formula:
% inhibition = (A − C)/(B − C) × 100
where A is the absorbance of the compound, B is the absorbance of reaction without enzyme, C is the
absorbance of the negative control. A commercial diabetes inhibitor acarbose was used as a positive
reference. Dilutions of test samples and dissolutions of enzyme used 20 mM sodium phosphate
buffer (pH 6.9 comprising of 6 mM sodium chloride). α-Amylase solution and soluble starch solution
were prepared and used on the day of the experiment. The IC50 value was calculated to exhibit 50%
inhibitory activity of C1–C3 against α-amylase.
4.9. α-Glucosidase Inhibition (AGI) Assay

The anti-α-glucosidase activity of C1–C3 was evaluated using a method described previously [35]
with some modifications. In brief, an amount of 20 μL methanolic stock solution of isolated compounds
was pre-mixed with an equal volume of 0.1 M potassium phosphate buffer (pH 7) and 40 μL of
α-glucosidase (from Saccharomyces cerevisiae, Sigma-Aldrich, St. Louis, MO, USA) enzyme solution
(0.5 U/mL in 0.1 M potassium phosphate buffer, pH 7). After 6 min incubation at 25 ◦ C, a 20 μL aliquot
of 5 mM p-nitrophenyl-α-D-glucopyranoside (pNPG) substrate (in 0.1 M potassium phosphate buffer,
pH 7) was added to each reaction and the mixture was incubated for another 8 min. The reaction
was terminated by adding 100 μL of 0.2 M Na2 CO3 , and absorbance was recorded at 405 nm. The
inhibition percentage was calculated by the following equation:
% inhibition = (1 − Asample /Acontrol ) × 100
63
Molecules 2019, 24, 3899
where Asample is absorbance of isolated compound, Acontrol is the abosrbance of positive controls
(acarbose or quercetin).

All obtained data were analyzed by Minitab Software (version 16.0, copyright 2015, Minitab Inc.,
State College, PA, USA). One-way analysis of variance (ANOVA) and Tukey’s post hoc test were
used to identify the significant difference among mean values with p < 0.05. All trials were designed
randomly in triplicate.
5. Conclusions
This study documented that the root of Rumex crispus possessed potent antioxidant, xanthine
oxidase, α-amylase and α-glucosidase inhibitory activity in in vitro assays. More specifically, the
compounds isolated from the EtOAc extracts emerged as a promising source of natural antioxidants,
xanthine oxidase, α-amylase, and α-glucosidase inhibitors. In vivo tests should be conducted to affirm
the bioactivity of the isolated compounds from the root of Rumex crispus for the development of
food additives and supplements to reduce the risks type 2 diabetes and gout. The isolation of novel
constituents, as well as investigations on the potent pharmaceutical properties of the root of Rumex
crispus need to be considered.
Supplementary Materials: The following are available online at http://www.mdpi.com/1420-3049/24/21/3899/s1,

Figures S1–S21 and Tables S1–S3 are provided.
Author Contributions: T.N.M., Y.A., and T.M.V. conceived of the idea and implemented the experiments. T.N.M.,
D.V.H., D.H.D., and T.M.V. analyzed data and wrote the manuscripts. D.V.H. interpreted NMR data. C.d.G.-G.,
L.T.V., T.N.M., and T.M.V. gave advice and revised the manuscript. All authors agreed with the final version of
the manuscript.
Funding: No funding was provided in support of this study.
Acknowledgments: Center for Research and Technology Transfer, Vietnam is appreciated for partly supporting
to this research. We thank Tran Dang Xuan and Nobukazu Nakagoshi for helpful advice on this research.
Conflicts of Interest: All authors have no conflicts of interest.
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66
molecules
Article
Antioxidant, α-Amylase and α-Glucosidase
Inhibitory Activities and Potential Constituents of
Canarium tramdenum Bark
Nguyen Van Quan 1 , Tran Dang Xuan 1, *, Hoang-Dung Tran 2, *, Nguyen Thi Dieu Thuy 1 ,
Le Thu Trang 1 , Can Thu Huong 1 , Yusuf Andriana 1 and Phung Thi Tuyen 3
1 Division of Development Technology, Graduate School for International Development and
Cooperation (IDEC), Hiroshima University, Higashi Hiroshima 739-8529, Japan;
nguyenquan26@gmail.com (N.V.Q.); dieuthuykttb@gmail.com (N.T.D.T.); trangle9872@gmail.com (L.T.T.);
cth1412@gmail.com (C.T.H.); yusufandriana@yahoo.com (Y.A.)
2 Department of Biotechnology, NTT Institute of Hi-Technology, Nguyen Tat Thanh University,
298A-300A Nguyen Tat Thanh Street, Ward 13, District 4, Ho Chi Minh 72820, Vietnam
3 Faculty of Forest Resources and Environmental Management, Vietnam National University of Forestry,
Xuan Mai, Hanoi 156200 Vietnam; phungtuyen@gmail.com
* Correspondence: tdxuan@hiroshima-u.ac.jp (T.D.X.); thdung@ntt.edu.vn (H.-D.T.);
Tel./Fax: +81-82-424-6927 (T.D.X.)
Academic Editors: Raffaele Capasso and Lorenzo Di Cesare Mannelli

Received: 13 January 2019; Accepted: 7 February 2019; Published: 9 February 2019
Abstract: The fruits of Canarium tramdenum are commonly used as foods and cooking ingredients
in Vietnam, Laos, and the southeast region of China, whilst the leaves are traditionally used for
treating diarrhea and rheumatism. This study was conducted to investigate the potential use of this
plant bark as antioxidants, and α-amylase and α-glucosidase inhibitors. Five different extracts of C.
tramdenum bark (TDB) consisting of the extract (TDBS) and factional extracts hexane (TDBH), ethyl
acetate (TDBE), butanol (TDBB), and water (TDBW) were evaluated. The TDBS extract contained
the highest amount of total phenolic (112.14 mg gallic acid equivalent per g dry weight), while the
TDBB extract had the most effective antioxidant capacity compared to other extracts. Its IC50 values
were 12.33, 47.87, 33.25, and 103.74 μg/mL in 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2 -azino-bis
(ABTS), reducing power (RP), and nitric oxide (NO) assays, respectively. Meanwhile, the lipid
peroxidation inhibition of the four above extracts was proximate to that of butylated hydroxytoluene
(BHT) as a standard antioxidant. The result of porcine pancreatic α-amylase inhibition showed
that TDB extracts have promising effects which are in line with the commercial diabetic inhibitor
acarbose. Interestingly, the inhibitory ability on α-glucosidase of all the extracts was higher than that
of acarbose. Among the extracts, the TDBB extract expressed the strongest activity on the enzymatic
reaction (IC50 = 18.93 μg/mL) followed by the TDBW extract (IC50 = 25.27 μg/mL), TDBS (IC50 =
28.17 μg/mL), and TDBE extract (IC50 = 141.37 μg/mL). The phytochemical constituents of the
TDB extract were identified by gas chromatography–mass spectrometry (GC-MS). The principal
constituents included nine phenolics, eight terpenoids, two steroids, and five compounds belonging
to other chemical classes, which were the first reported in this plant. Among them, the presence of
α- and β-amyrins were identified by GC-MS and appeared as the most dominant constituents in
TDB extracts (1.52 mg/g). The results of this study revealed that C. tramdenum bark possessed rich
phenolics and terpenoids, which might confer on reducing risks from diabetes. A high quantity of
α- and β-amyrins highlighted the potentials of anti-inflammatory, anti-ulcer, anti-hyperlipidemic,
anti-tumor, and hepatoprotective properties of C. tramdenum bark.
Keywords: Canarium tramdenum; bark; antioxidants; α-glucosidase inhibitors; diabetes; phenolics;

terpenoids; biological activity

Molecules 2019, 24, 605
1. Introduction
Diabetes or diabetes mellitus has become a burden for the global economy in recent decades.
According to the World Health Organization’s report, this disease and its complications cause
substantial economic loss through direct medical costs and loss of work and wages [1]. Among
diabetes cases, type 2 diabetes is much more common and chiefly occurs in adults; however, it is
being increasingly noted in adolescents [2]. The pathogenesis of type 2 diabetes is currently attributed
to endogenous factors such as genetics and metabolic abnormalities and exogenous factors such as
behavior and environment [3]. The type 2 diabetes increases blood sugar level which is considered as a
typical symptom in diabetic patients. Monitoring and control of hyperglycemia are the most prevalent
methods in the treatment of type 2 diabetes nowadays.
As an endogenous toxin, oxidative stress is considered to be an important determinant of type 2
diabetes complications [4]. The causal relation between oxidative stress and type 2 diabetes has been
elucidated through molecular mechanisms [5], whereby the overproduction of reactive oxygen species
related to hyperglycemia likely leads to an imbalance of the quantity of antioxidants inside the body
and eventually, to oxidative stress. On the other hand, the blood sugar level is crucially determined by
the act of digestive enzymes such as α-amylase and α-glucosidase. While α-amylase is responsible for
breaking down long-chain carbohydrates, α-glucosidase directly converts carbohydrate to glucose in
the small intestine. The inhibition of α-glucosidase has been acknowledged as a therapeutic target for
the control of postprandial hyperglycemia, as well as type 2 diabetes [6,7]. Therefore, simultaneously
providing antioxidants and α-amylase and α-glucosidase inhibitors through nutriments is a potential
and feasible method for the management of type 2 diabetes. However, the origin and dose of
ingredients should be scrupulously studied before application and production. Additionally, natural
products are recommended owing to their long history of medicinal and beneficial effects on human
health [8].
Among natural sources, plants have been the most thoroughly scrutinized thanks to their
vast diversity and wide distribution across the Earth. It is easy to derive antioxidant and nutrient
components from every part of plants as fruits, leaves, stems, and roots which exhibit a wide range
of biological effects such as anti-inflammatory, antibacterial, antiviral, anti-aging, and anticancer [9].
Nonetheless, this is also a reason why the potential of plants in treating certain diseases has not yet been
fully exploited. Canarium tramdenanum Dai & Yakovlev, a synonym of Canarium pimela Koenig, a woody
tree belonging to Burseraceae family, is not an exception. This plant is widely distributed in subtropical
and tropical regions of China and Indochina [10]. In Vietnam, ripe fruits of C. tramdenum are commonly
used as foods and cooking ingredients, whilst leaves are traditionally used for treating diarrhea and
rheumatism [10,11]. In China, C. tramdenum or “Chinese black olive” is used in folk medicine as an
anti-bacterial, anti-viral, anti-inflammatory, and detoxifying substance [3]. Recently, vasorelaxant and
antioxidant activities of the fruits and leaves of this plant have been reported [3,12]. The nutritional
compositions of C. tramdenum kernels were also documented [13]; however, their biological activities
were not investigated. To date, no study on the anti-diabetic property of this plant has been reported.
Hence, in this research, we investigated the antioxidant and potential diabetic inhibitory properties of
C. tramdenum bark extracts through in vitro assays of α-amylase and α-glucosidase suppression.
2. Results
2.1. Extraction Yield and Total Phenolic Contents

From 30 g dry TDB, five extracts with different yields were obtained by using an extraction
method based on various polarity solvents. The yield of TDBS was 20% (6 g), followed by TDBW
(6.67%, 2 g), TDBE (5%, 1.5 g), TDBB (5%, 1.5 g), and TDBH (0.42%, 0.13 g).
The total phenolic contents (TPC) of TDB extracts were shown in Table 1. It is noteworthy that
there was a variation in TPC among extracts which ranged from 20.5 to 112.14 mg GAE/g DW. The total
extract TDBS was found to have the highest TPC (112.14 mg GAE/g DW) compared with fractional
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extracts. The lowest TPC was determined in TDBE extract which accounted for 20.5 mg GAE/g DW.
TPC of three fractional extracts TDBE, TDBB, and TDBW (90.62 mg GAE/g DW) were not equal to
that of the total extract TDBS. This could be explained by the loss in fractionation and filtration steps
(around 21.52 mg GAE/g DW). However, TPC in extracts by higher polar solvents (TDBB and TDBW)
were significantly greater than those of extracts by lower polar solvents (TDBE).
Table 1. Total phenolic contents and antioxidant activities of TDB extracts.
TPC DPPH Assay ABTS Assay RP Assay IC50 βC Assay LPI

Samples
(mg GAE/g DW) IC50 (μg/mL) IC50 (μg/mL) (μg/mL) (%)
TDBS 112.14 ± 1.19 a 15.41 ± 0.10 c 62.21 ± 1.78 b 33.25 ± 0.04 b 86.12 ± 0.98 ab
TDBE 20.50 ± 0.60 c 22.23 ± 0.09 e 76.96 ± 1.04 c 41.60 ± 0.03 d 87.52 ± 0.73 a
TDBB 36.57 ± 0.36 b 12.33 ± 0.02 a 47.87 ± 0.12 a 26.24 ± 0.02 a 86.75 ± 0.84 ab
TDBW 33.55 ± 0.48 b 16.45 ± 0.07 d 45.25 ± 0.17 a 33.25 ± 0.06 b 84.09 ± 0.56 b
BHT - 14.99 ± 0.06 b 80.26 ± 1.11 c 38.34 ± 0.01 c 86.67 ± 0.33 ab
Data express means ± SE (standard error); Different superscript letters (a,b,c,d,e ) in a column indicate significant
differences at p < 0.05; -: not measured; TPC, total phenolic contents; GAE, gallic acid equivalent; DW, dry weight;
DPPH, 2,2-diphenyl-1-picrylhydrazyl; ABTS, 2,2 -azino-bis; RP, reducing power; NO, nitric oxide; βC, β-carotene
bleaching; TDBS, C. tramdenum total extract; TDBE, ethyl acetate extract; TDBB, butanol extract; TDBW, water
extract; BHT: butylated hydroxytoluene.
2.2. Antioxidant Activities

The antioxidant capacities of TDB extracts by DPPH, ABTS, reducing power and β-carotene
bleaching assays are displayed in Table 1.
DPPH is a stable reagent and widely used in most antioxidant tests. The principle of this sensitive
assay is based on the reaction of samples with the organic radical DPPH [14]. The antioxidant ability
of samples can be observed visually by the discoloration of DPPH (from purple to yellow or colorless)
or be determined by the reduced absorbance of the final reaction at 517 nm wavelength. In this
study, TDBB extract showed the strongest anti-DPPH radical activity (IC50 =12.33 μg/mL) which was
significantly higher than standard BHT (IC50 =14.99 μg/mL) and other extracts, see Table 1.
In the case of the ABTS assay, antioxidants in samples reacting with an organic cation radical
named ABTS•+ are the basis for this test. In the mixture, with the presence of antioxidants, the nitrogen
atom of ABTS•+ quenches the hydrogen atom of antioxidants yielding the solution decolorization [15].
The decolorization of ABTS•+ solution illustrates the antioxidant capacity of samples. As shown in
Table 2, except for TDBE extract, all TDB extracts presented more powerful activity on ABTS assay
than the standard BHT. Of which, IC50 values of TDBB and TDBW were 1.7-times higher than that of
BHT. Additionally, the TDBS extract was 1.3-times the antioxidant activity of BHT.
Table 2. α-Amylase and α-glucosidase inhibitory activities of C. tramdenum bark (TDB) extracts.
Sample α-Amylase Inhibition IC50 (μg/mL) α-Glucosidase Inhibition IC50 (μg/mL)

TDBS 359.32 ± 6.73 c 28.17 ± 0.12 c
TDBE 491.23 ± 2.49 d 141.37 ± 0.86 b
TDBB 257.20 ± 1.15 b 18.93 ± 0.07 e
TDBW 555.02 ± 9.10 e 25.27 ± 0.12 d
Acarbose 80.26 ± 0.24 a 145.35 ± 0.62 a
Data express means ± SE (standard error); Different superscript letters (a,b,c,d,e ) in a column indicate significant
difference at p < 0.05; TDBS, C. tramdenum total extract; TDBE, ethyl acetate extract; TDBB, butanol extract; TDBW,
water extract.
Reducing power assay or potassium ferricyanide reducing power is based on the competence
of antioxidants in converting potassium ferricyanide (Fe3+ ) to potassium ferrocyanide (Fe2+ ). The
final reaction with ferric trichloride results in a mixture of Fe3+ and Fe2+ , a blue solution which can
be spectrophotometrically determined at 700 nm [14]. An increase in the absorbance (high content of
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Fe2+ in the final reaction) indicates a strong antioxidant activity. By comparing IC50 values, see Table 1,
the order of the antioxidant capacity of TDB extracts was TDBB (26.24 μg/mL) > TDBS and TDBW
(33.25 μg/mL for both) > TDBE (41.60 μg/mL) whilst the IC50 value of BHT was 38.34 μg/mL.
In the β-carotene bleaching assay using linoleic acid, the oxidized product (linoleate-free radical
and other free radicals) of the linoleic acid peroxidation process can gradually decolorize the β-carotene
color by time. The process can be delayed by the presence of antioxidants and the reaction can be
recorded at 492 nm. It is apparent that, at the same concentration of 1000 μg/mL, all total extract and
fractional extracts of TDB exhibit a similar inhibitory level of lipid peroxidation to the standard BHT,
see Table 1. These results demonstrated that antioxidants involved in TDB extracts could negate the
free radicals in the system, thereby they could protect β-carotene color from the bleaching process [16].
Sodium nitroprusside in phosphate buffer saline (pH 7.2) generate nitric oxide (NO) which can
be spontaneously converted into the more stable forms of nitrate and nitrite ions under the aerobic
reaction with oxygen [15,17]. The Griess reagent is used to detect these ions in the mixture by forming
the conspicuous pink solution that can be measured at 546 nm. Antioxidants can prevent the formation
of nitrate and nitrite ions and, therefore, reduce the absorbance of the reaction. Figure 1 shows that all
TDB extracts possess potential NO scavenging activities which are comparable to the standard gallic
acid. Among fractional extracts, TDBB expressed the highest antioxidant activity (IC50 = 103.74 μg/mL),
followed by TDBW (IC50 = 112.54 μg/mL), and TDBE (IC50 = 131.43 μg/mL). The activity of the total
extract TDBS (IC50 = 116.80 μg/mL) was significantly higher than that of TDBE but lower than those
of TDBB and TDBW. In the body, NO associates with many biological systems including neuronal
messenger, vasodilatation, and antimicrobial and antitumor activities [17]. Additionally, the complex
interplay between NO production and the pathogenesis of diabetic nephropathy and angiopathy has
been interpreted [18,19]. Hence, a nitric oxide scavenging assay is indispensable in the research on the
antioxidant and antidiabetic properties of natural products.
Figure 1. Nitric oxide scavenging activity of TDB extracts. Values are means ± SE (standard
error) (n = 3); Different letters indicate significant difference at p < 0.05; TDBS, C. tramdenum total
extract; TDBE, ethyl acetate extract; TDBB, butanol extract; TDBW, water extract; BHT: butylated
hydroxytoluene; GA: gallic acid.
2.3. α-Amylase and α-Glucosidase Inhibitory Activities

In this study, starch-iodine method was applied to examine the inhibitory effect of TDB extracts
on porcine pancreatic α-amylase. The degradation of starch by enzyme activity was visually observed
or spectrophotometrically measured at 565 nm based on changing dark-blue color to yellow color
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of reaction solution. Accordingly, in mixtures with inhibitors, the solution with higher absorption
(darker color) signifies the higher inhibitory activity. As shown in Table 2, the anti-α-amylase activity
was recorded at all TDB extracts and comparable with the standard acarbose. Among extracts, TDBB
extract manifested the highest inhibitory activity (IC50 = 257.20 μg/mL) while TDBW represented the
lowest one (IC50 = 555.02 μg/mL).
The α-glucosidase inhibitory activity of TDB extracts was assayed using a synthetic substrate
pNPG. In physiological buffer (pH 7), α-glucosidase hydrolyzes pNPG to release p-nitrophenol, a
yellow product that can be measured at wavelength 405 nm [20]. The lower absorbance indicates the
stronger suppression on enzymatic activity. All TDB extracts expressed a remarkable inhibition on
α-glucosidase activity (Table 2). Of which, the activity of TDBS, TDBB and TDBW extracts were
extraordinarily higher than that of standard acarbose. The order of α-glucosidase inhibition is
TDBB > TDBW > TDBS > TDBE > acarbose corresponding to IC50 values 18.93, 27.27, 28.17, 141.37,
and 145.35 μg/mL, respectively.
2.4. Correlations between Total Phenolics and Biological Activities

According to Table 3, there was no significant correlation between the total phenolic contents and
biological activities of TDB extracts. These results indicated that TPC might not be the main contributor
to the antioxidant, anti-α-amylase, and anti-α-glucosidase activities of TDB extracts. On the other
hand, except for the β-carotene bleaching assay, all methods showed a strong correlation with each
other, regardless of whether they were based on different methods (p < 0.05). Especially, antioxidant
assays including DPPH, ABTS, reducing power, and nitric oxide were strongly correlated with the
α-glucosidase inhibitory assay at p < 0.05. Meanwhile, ABTS assay was not significantly associated
with the α-amylase inhibitory assay.
Table 3. Pearson’s correlation coefficients between total phenolics and biological activities.
TPC DPPH ABTS RP NO βC AG

DPPH 0.24 - - - - -
ABTS −0.15 0.68* - - - -
RP 0.10 0.99* 0.72 * - - -
NO 0.08 0.96* 0.86 * 0.97 * - -
βC −0.08 −0.10 −0.52 −0.10 −0.26 -
AG 0.23 0.95* 0.85 * 0.94 * 0.98 * −0.33
AA 0.20 0.85* 0.25 0.84 * 0.70 * 0.30 0.65 *
* a significance at p < 0.05; TPC, total phenolic contents; DPPH, 2,2-diphenyl-1-picrylhydrazyl assay; ABTS,
2,2 -azino-bis assay; RP, reducing power assay; NO, nitric oxide scavenging assay; βC, β-carotene bleaching assay;
AG, α-glucosidase inhibitory assay; AA, α-amylase inhibitory assay.
2.5. GC-MS Results

Phytochemical compositions of TDB extracts analyzed by GC-MS were listed in Table 3.
Twenty-four compounds were identified and classified which might be regarded as antioxidants
and α-glucosidase inhibitors of TDB extracts.
To the best of our knowledge, except for α-cubebene and (-)-spathulenol that were previously
identified in the essential oil of TDB [21], the remaining 22 compounds were the first reported as the
principal component of TDB extracts by this study.
Among the identified compounds, phenolics accounted for the highest amount with nine
compounds, followed by terpenoids (eight compounds), steroids (two compounds), carbohydrates
(glycerol), monosaccharides (levoglucosan), phenylpropanoic acids (ibuprofen methyl ester), carboxylic
acids (acetyltributyl citrate) and one unidentified compound. All phenolic compounds were detected
in fractional extracts TDBB and TDBW while all terpenoids were identified in the TDBE extract.
Although the phenolic components were scant in the total extract TDBS, they were accompanied with
greater amounts in the fractional extracts of TDBB and TDBW. This is reasonable when lower polar
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components have been eliminated through hexane and ethyl acetate solvents. Whereas, 2-propylphenol
and acetyltributyl citrate were only detected in fractional extracts TDBB and TDBE, respectively, but
were not recognized in the total extract TDBS. Significantly, β-amyrin and α-amyrin were the most
dominant compositions in the TDB extract, which showed the highest contents in TDBS (41.45% and
19.17%, respectively) and TDBE (29.51% and 20.18%, respectively) extracts.
2.6. Identification of α- and β-Amyrins by GC-MS

The GC-MS analysis on the crystal mixture isolated from F15–F18 of TDBE extract confirmed
the presence of α- and β-amyrins (with a 3:4 ratio) as the dominant components. MS data showed
that the relative intensity corresponding to the main fragments of α-amyrin were 27, 25, 100, and
13 for peaks at m/z 189, 203, 218, and 426, respectively. Meanwhile, β-amyrin data were 15, 52, 100,
and 5, respectively at m/z values similar to the peaks described above, see Supplementary Materials,
Figure S1. The result was coincident with the results of [22,23].
There has been extensive research that validated the diverse pharmacological activity of
α- and β-amyrins, including anti-inflammatory, anti-ulcer, anti-hyperlipidemic, anti-tumor, and
hepatoprotective actions [24,25]. Also, natural sources of α- and β-amyrins are available which
can be easily extracted and isolated in various plants. The most important sources of α- and β-amyrins
are Mexican copal (5 mg/g) and Nelumbo nucifera (3 mg/g), respectively [25]. Apart from the reported
list of plants containing α- and β-amyrins [25], in this study, we supplemented some other sources
together with their contents and biological activities, see Table 4. Accordingly, the content of isolated
α- and β-amyrins in our study (1.52 mg/g) showed substantial potential in comparison with other
plants. Noticeably, C. tramdenum bark involves a greater amount of these terpenoids than C. subulatum
(0.03 mg/g of β-amyrin), a species that belongs to the same Canarium genus (Table 4).
Table 4. Natural sources of α-amyrin and β-amyrin and their principal biological activities.
Plant Species Plant Parts Quantity Biological Activities References

Mixture of α-,β-amyrins Antinociception against
Protium kleinii Resin [26]
(1:1) 2.4 mg/g the visceral pain in mice
Gastroprotective,
Protium Trunk wood anti-inflammatory and
Mixture of α-,β-Amyrins [24,27,28]
heptaphyllum resin hepatoprotective
properties
Antioxidant and
Symplocos
Leaves β-Amyrin 0.17 mg/g free-radical scavenging [29]
cochinchinensis
effects
Anti-inflammatory
Alstonia boonei Stem barks β-Amyrin 0.08 mg/g [30]
activity
Memecylon α-Glucosidase inhibitory
nm nm [31]
umbellatum activities
Melastoma
Leaves α-Amyrin 0.06 mg/g nm [32]
malabathricum
Maesobotrya
Aerial parts β-Amyrin Antimicrobial activity [33]
barteri
α-Amyrin 0.01 mg/g; α-Amylaseinhibitory
Swertia longifolia Aerial parts [34]
β-Amyrin 0.02 mg/g activity
Canarium
Stem bark β-Amyrin 0.03 mg/g Anti-herpetic activity [35]
subulatum
Canarium Mixture of α-,β-amyrins
Barks nm This study
tramdenum (3:4) 1.52 mg/g
nm, not mentioned
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3. Discussion
Canarium is a genus of approximately 100 species in the Burseraceae family [36]. However,
very few studies on antioxidant and anti-diabetic potentials were conducted on the species of this
genus. Most of them focused on exploiting pharmaceutical and medicinal properties of several species
possessing edible fruits. The extracts of C. album fruits had potent antioxidants as tannins [37] and
phenolics [38] and exhibited effective anti-diabetic properties through antiglycation [39]. The leaf
and fruit extracts of C. odontophyllum exerted inhibitory effects on diabetic and obese rats [40,41]. The
information regarding antioxidant and antidiabetic effects of other inedible parts as stem barks of
Canarium plants was fragmented and scant. Only stem bark extract of C. schweinfurthii was proved to
acquire anti-diabetic effects [42]; however, bioactive components of this object were not described. By
this study, for the first time, we comprehensively assessed the antioxidant and potential anti-diabetic
activities and identified the phytochemicals of C. tramdenum bark’s extracts. All extracts presented a
similar antioxidant level to BHT, see Table 1, a well-known antioxidant compound and also commonly
used in food additives [43]. The TDBB extract was even more active than BHT in DPPH, ABTS,
and reducing power assays, see Table 1. Furthermore, TDB extracts showed a potential effect on
the restraint of α-amylase activity and exerted much more powerful α-glucosidase inhibition than
acarbose—a standard inhibitor that is ordinarily used in the clinical practice of diabetes treatment [44].
The antioxidant property is mainly characterized by phenolic contents [45] which can effortlessly
release hydrogen donors to naturalize free radicals. Meanwhile, in α-amylase and α-glucosidase
inhibition, both terpenoids and phenolics might play crucial roles [46]. In this study, we found that
although the total extract of TDBS showed the highest phenolic content, its biological activities were
not stronger than others, see Tables 1 and 2 and Figure 1. We assumed that phenolics might not
be the only contributors toward the antioxidant and antidiabetic properties of TDB extracts. The
examination of correlations between phenolics and biological activities of TDB extracts, see Table 3,
revealed that the total amount of phenolics might not be the determinant of the biological activities
of TDB extracts. The other factors such as functional groups of individual compounds may play
more important roles in this case. In particular, the number of free hydrogen donors determines
the antioxidant activity, the position of hydroxyl groups, methoxy groups, and lactone rings in the
structure of compounds induce inhibitions of α-amylase and α-glucosidase and even the interaction
among compounds in an extract may result in differences in biological activities. In this study, we
hypothesized that the synergistic interaction between phenolic and terpenoid components might result
in the greater biological activities for TDB extracts. However, the composition of phenolics seemed
to be more essential when TDBB and TDBW extracts contained a high content of phenolics but not
terpenoids and presented stronger antioxidant and anti-α-glucosidase activities than other extracts,
see Table 5. Integrating with correlation results, we suggested that the presence of some phenolics,
apart from other components, might be the major factor that determines the biological activity of
TDB extracts. On the other hand, a previous study demonstrated the potential antihyperglycemic
and hypolipidemic effects of α- and β-amyrins [47]. However, the successful isolation of α- and
β-amyrins should be further approached to investigate the role of the two compounds in the TDB
extracts, see Table 3. Moreover, the high yield of the isolation of these bioactive compounds in this
study suggested the potential practical use of TDB extracts, see Table 5. Therefore, although the
GC-MS results showed most of the major components of TDB extract, more sensitive methods such
as the ultra-high performance liquid chromatography integrated with tandem mass spectrometry
(UPLC-MS-MS) should be conducted to affirm and quantify phenolic compounds and other active
components. Additionally, the subsequent in vivo tests and clinical trials should be implemented in
order to certify the antidiabetic property of this prospective plant.
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Table 5. Major phytochemical components of TDB extracts analyzed by GC-MS.
Identified Chemical Peak Area in Extracts (%)

No. RT (min)
Compounds Classification TDBS TDBE TDBB TDBW
1 Glycerol 4.62 Carbohydrates < - - 37.73
2 2-Methoxyphenol 6.39 Phenolics 1.19 - 1.57 3.63
3 Pyrocatechol 7.88 Phenolics 1.28 - 19.31 6.66
4 4-Methylcatechol 9.21 Phenolics < - 1.67 1.38
5 2,6-Dimethoxyphenol 10.08 Phenolics < - - 1.43
6 2-Propylphenol 10.35 Phenolics - - 3.54 -
7 α-Cubebene 10.58 Terpenoids 0.67 < - -
8 3,4-Dimethoxyphenol 11.05 Phenolics < - - 5.22
9 Levoglucosan 11.82 Monosaccharides < - 4.30 1.92
10 3,7(11)-Eudesmadiene 12.37 Terpenoids 1.10 < - -
11 Ibuprofen methyl ester 12.55 Phenylpropanoic acids 1.20 < - -
12 4-Propylresorcinol 13.18 Phenolics < - 13.90 1.75
13 3,4,5-Trimethoxyphenol 13.24 Phenolics 0.71 - 1.99 5.24
14 β-Guaiene 13.69 Terpenoids 0.78 2.22 - -
15 Homovanillic acid 13.79 Phenolics 1.06 - 13.39 3.26
16 (−)-Spathulenol 15.30 Terpenoids < 0.92 - -
17 Methyl hinokiate 16.62 Terpenoids < 2.05 - -
18 Ylangenol 17.07 Terpenoids < 1.00 - -
19 Acetyltributyl citrate 19.78 Carboxylic acids - 1.89 - -
20 Stigmasterol 27.86 Steroids < 2.41 - -
21 γ-Sitosterol 28.56 Steroids 0.89 6.52 - -
22 β-Amyrin 29.12 Terpenoids 41.45 29.51 - -
23 Not identified 29.32 Not identified 7.41 9.49 - -
24 α-Amyrin 29.72 Terpenoids 19.17 20.18 - -
RT, retention time; -, not detected; <, trace of peak area that was lower than 0.5%.
4.1. Materials and Instrumentations

Stem bark of C. tramdenum (TDB) was collected at 21.07◦ N, 106.10◦ E, Bac Ninh province, Vietnam
in 2016. The species was identified at a study field based on Vietnam Plant Data Center (http://www.
botanyvn.com) and Plants Database Missouri Botanical Garden, United States (TROPICOS—http:
//www.tropicos.org). The specimen with voucher number TDB-J2016 was deposited at the Plant
Physiology and Biochemistry Laboratory, IDEC, Hiroshima University, Japan.
All extraction solvents including methanol, hexane, ethyl acetate, and butanol were purchased
from Junsei Chemical Co., Ltd., Tokyo, Japan. The analytical reagents for antioxidant assays were
acquired from Kanto Chemical Co., Inc., Tokyo, Japan while those of enzymatic assays were procured
from Sigma-Aldrich, St. Louis, MO, USA.
A vacuum evaporator (Rotavapor® R-300, Nihon Buchi K.K., Tokyo, Japan), a microplate reader
(MultiskanTM Microplate Spectrophotometer, Thermo Fisher Scientific, Osaka, Japan), and GC-MS
system (JMS-T100 GCV, JEOL Ltd., Tokyo, Japan) were the main instruments used in this study.
4.2. Sample Preparation and Extraction

After air-drying for 2 days, the outer layer of TDB was scraped to remove all fungi and lichen.
Then, the sample was washed with 1% sodium hypochlorite (NaOCl) and water. After blotting with
tissues, bark samples were dried at 45 ◦ C for 1 week by an oven, then ground to make the powder.
The TDB sample (30 g) was extracted using 1 L of methanol in one week. Afterwards, dry methanol
crude extract (TDBS) was blended with distilled water (200 mL), then successively fractionated by an
equal volume of hexane, ethyl acetate, and butanol. Each fractionation step was conducted three times.
After filtrations, they were separately dried under vacuum by an evaporator at 50 ◦ C. Eventually, five
dried extracts including TDBS (total extract), hexane (TDBH), ethyl acetate (TDBE), butanol (TDBB),
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and water (TDBW) extracts were obtained as fractional extracts. They were preserved in sterilized
vials and kept in a refrigerator (−20 ◦ C) for further tests on the physical properties, biological activities,
and identification of phytochemical components.
4.3. Determination of Total Phenolic Content

The total phenolic content (TPC) was calculated according to the Folin-Ciocalteu method described
previously [48]. Specifically, 20 μL of extract was homogenized with 100μL of Folin-Ciocalteu’s reagent
(10%) and 80μL of 7.5% Na2 CO3 (w/v), respectively. The mixture was then shaken and incubated at
room temperature for 30 min. The absorbance was measured at 765 nm using a microplate reader. TPC
was expressed as mg gallic acid equivalent per g dry weight of sample (mg GAE/g DW).
4.4. DPPH Radical Scavenging Assay

DPPH assay was evaluated following Elzaawely et al. [49] with a slight modification. Fifty
microliters of extract were mixed with 50 μL of DPPH solution (0.2 mg/mL) and 100 μL of 0.1 M
acetate buffer (pH 5.5). Thereafter, the combination was incubated for 20 min in darkness at room
temperature. The absorbance was recorded at 517 nm using a microplate reader. BHT was used as a
standard reference while pure methanol was used as a negative control. The IC50 value was calculated
as the concentration required to reach a 50% reduction of DPPH.
4.5. ABTS Radical Cation Decolorization Assay

2,2 -Azino-bis radical cation (ABTS•+ ) decolorization was measured as described by Pellegrini et
al. [50] with minor modifications. ABTS•+ solution was prepared by mixing aqueous ABTS (7 mM)
solution with 2.45 mM potassium persulfate (1:1 v/v) and incubating in darkness at room temperature
for 16 h. The working solution was then obtained by diluting ABTS•+ solution in methanol to an
absorbance of 0.70 ± 0.05 at 734 nm. In each well of a 96 well-plate, 25 μL of TDB sample was added
to 200 μL of the working solution. After a slight shake, the plate was covered by an aluminum foil and
kept at room temperature for 30 min. Subsequently, the absorbance was recorded by a MultiskanTM
Microplate Spectrophotometer (Thermo Fisher Scientific, Osaka, Japan). The ABTS radical decolorizing
activity was calculated by the following formula
ABTS radical decolorizing activity (%) = (1 − Asample /Acontrol ) × 100
where Asample is the absorbance of reaction with momilactones or positive control (BHT) and Acontrol
is the absorbance of reaction without momilactone or positive control. The IC50 value was determined
as the concentration needed to bleach 50% of ABTS•+ .
4.6. Reducing Power Assay

The reducing capacity was carried out referring to the modified method of Minh et al. [51] with
a 10-times reduction in the volume of each reaction component. Initially, 100 μL of sample, 250 μL
of 0.2 M phosphate buffer (pH 6.6), and 250 μL of potassium ferricyanide (1%, w/v) were combined
and incubated at 50 ◦ C for 30 min. Subsequently, the reaction mixture was stopped by 500μL of
trichloroacetic acid (10%, v/v) and centrifuged for 10 min at 4000 rpm. Eventually, 100μL of the
supernatant was double diluted with distilled water (100 μL) followed by adding 20 μL of ferric
chloride solution (0.1%, w/v). After shaking, the absorbance of the resulting mixture was read at
700 nm by a microplate reader. BHT was used as a standard reference. The IC50 value was calculated
as mentioned above.
4.7. β-Carotene Bleaching Assay

The β-Carotene bleaching activity of TDB extracts was assayed using a β-carotene linoleate
bleaching system [52] with trivial adjustments. Briefly, a mixture of 1mL β-carotene (200 μg/mL in
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chloroform), 20 μL linoleic acid, and 200 mg Tween 40 was evaporated at 40 ◦ C. Afterwards, a volume
of 50 mL of oxygenated water was slowly added and the mixture which was then vigorously shaken
to form a stable emulsion. The emulsion was freshly prepared before each experiment. In each well
of a 96 well-plate, 25 μL of sample or control (1000 μg/mL in methanol) and 200 μL of the emulsion
solution were blended. The reaction was incubated at 45 ◦ C and the absorbance was recorded at
492 nm every 15 min up to 180 min. The lipid peroxidation inhibition (LPI) was calculated as:
LPI inhibition (%) = A180 /A0 × 100
where A0 is the absorbance of reaction at the zero-minute time and A180 is the absorbance of reaction
at the 180-min time. Methanol was used as negative control, whilst BHT was the positive control.
4.8. Nitric Oxide Scavenging Assay

Nitric oxide scavenging was measured following the method of Govindarajan et al. [53] with
some modifications. At the beginning, 100 μL of sample and 100 μL of 5 mM sodium nitroprusside in
0.1 M phosphate buffer saline (pH 7.2) were assimilated and incubated for 30 min at 25 ◦ C. In the next
step, 100 μL of the above mixture was blended with 100 μL of Griess reagent (1% sulfanilamide, and
0.1% naphthyl ethylenediamine dihydrochloride in 2% phosphoric acid). The absorbance decrease of
the resulting purple solution was recorded at 546 nm by a microplate reader. Pure methanol was used
as a negative control while gallic acid was used as a positive reference. The IC50 value was obtained in
the same way as described above.
4.9. Porcine Pancreatic α-Amylase Inhibition Assay

The α-amylase inhibitory activity was assayed based on the starch-iodine method [54]. α-Amylase
solution (2 mg/mL) from porcine pancreas (type VI-B, Sigma-Aldrich, St. Louis, MO, USA) and TDB
extracts were dissolved in 0.2 M phosphate buffer saline (pH 6.9). Iodine (0.25 mM) and soluble
starch (0.5%) solutions were prepared in deionized water. Firstly, 20 μL of α-amylase was mixed
and incubated with 20 μL of the test sample at 37 ◦ C in 10 min. After that, 30 μL of starch solution
was added and then incubated for 8 min. The reaction was suspended by adding 20 μL of 1 M
HCl, followed by 100 μL of iodine solution. The resulting mixture was measured at a wavelength of
565 nm by a microplate reader. The inhibition percentage and IC50 value were calculated as described
previously [54]. Acarbose was used as a positive reference.
4.10. α-Glucosidase Inhibition Assay

The anti-α-glucosidase activity of TDB extracts was measured using a modified version of the
method described by Johnson et al. [55]. In brief, 40 μL of TDB extract in 0.1 M potassium phosphate
buffer (pH 7) were pre-mixed with 40 μL of 0.5 U/mL α-glucosidase (from Saccharomyces cerevisiae,
Sigma-Aldrich, St. Louis, MO, USA). After 6 min of incubation at 25 ◦ C, a 20 μL aliquot of 5 mM
p-nitrophenyl-α-D-glucopyranoside (pNPG) substrate in the buffer was added and the mixture was
incubated for another 8 min. Finally, the reaction was terminated by adding 100 μL of 0.1 M Na2 CO3 ,
and absorbance was recorded at 405 nm. Inhibition percentage was calculated by:
% inhibition = (1 − Asample /Acontrol ) × 100
where Asample is absorbance of the reaction with samples or positive controls (acarbose) and Acontrol
is absorbance of reaction with 10% methanol. The enzymatic inhibitory activity of TDB extracts was
expressed as IC50 value as well.
76
Molecules 2019, 24, 605
4.11. Identification of Phytochemical Component by GC-MS

The phytochemical components of TDB extracts were identified by GC-MS analysis. The GC-MS
system (JMS-T100 GCV, JEOL Ltd., Tokyo, Japan) with an autosampler coupled with a 30 m x 0.25 mm
I.D. x 0.25 μm film thickness DB-5MS column (Agilent Technologies, J & W Scientific Products, Folsom,
CA, USA). A concentration of 1000 μg/mL of each TDB extract was used for the initial injection.
Helium was used as a carrier gas at split ratio 5:1. The GC oven conditions were as follows: The initial
temperature was 50 ◦ C without hold time, the boosted temperature was up to 300 ◦ C at 10 ◦ C/min,
and held for 20 min. The injection port and detector temperature were set at 300 ◦ C and 320 ◦ C,
respectively. The mass range scanned from 29 to 800 amu. The control of the GC-MS system and the
confirmation of analytes were conducted using JEOL’s GC-MS Mass Center System Version 2.65a [56].
4.12. Isolation of Bioactive Compounds α-Amyrin and β-Amyrin from TDBE Extract
By preliminarily screening the GC-MS results, we found that two major compounds in
TDB extracts were tentatively the pentacyclic triterpenes α-amyrin and β-amyrin. Therefore, we
implemented an isolation of these two compounds by column chromatography. In brief, The TDBE
extract (1.2 g) was premixed with 5 g silica gel (70-230 mesh, Merck, Darmstadt, Germany). The
mixture was then loaded onto a normal phase silica gel (40 g) column (2 × 50 cm). The mobile phase
was 100% hexane and hexane:ethyl acetate (98:2, v/v) which yielded 8 fractions (F1–F8) and 12 fractions
(F9–F20), respectively. The elution step for every fraction was 100 mL. Fractions F15–F18 afforded a
mixture of colorless crystal (45.6 mg) which was subsequently identified as α-amyrin and β-amyrin
(75% purity) by GC-MS, see Supplementary Material, Figure S1.

Data were elaborated on the Minitab 16.0 software (Minitab Inc., State College, PA, USA).
All assays were thrice implemented, and results were displayed as means ± standard errors (SE).
Significant differences among tests were determined by one-way ANOVA using Tukey’s test at
p < 0.05. Pearson’s correlation coefficients among total phenolics, antioxidant, anti-alpha amylase, and
glucosidase activities of TDB extracts (n = 3 for each extract) were calculated by using the same software.
5. Conclusions
This study documented that Canarium tramdenum bark possessed potent antioxidant and
α-glucosidase inhibitory activity in in vitro trials. It was found that the fractions of ethyl acetate,
butanol, and water extracts enriched with terpenoids and phenolics appeared as a promising source of
natural antioxidants, α-amylase, and α-glucosidase inhibitors. In vivo tests and clinical trials should
be elaborated to affirm the bioavailability of C. tramdenum bark for the development of food additives
and supplements to reduce the risks from type 2 diabetes. The isolation of novel constituents, as
well as investigations on potent pharmaceutical properties of C. tramdenum bark, should also be
conducted. The contribution of α- and β-amyrins to biological activities of C. tramdenum bark should
be further investigated.
Supplementary Materials: The following are available online, Figure S1: GC-MS chromatography of crystal from
fractions F15–F18.
Author Contributions: N.V.Q. designed the experiments and wrote the manuscript. N.T.D.T., L.T.T and C.T.H.
conducted biological assays. N.V.Q. and Y.A. implemented GC-MS analysis and measured total phenolic contents.
P.T.T. assisted in collection and identification of the species and extraction of samples. T.D.X. and H-D.T. revised
and approved the final submission of the manuscript.
Funding: Nguyen Tat Thanh University Vietnam has partly funded to this research.
Acknowledgments: The authors thank Do Tan Khang, Truong Ngoc Minh and Truong Mai Van for their support
to this research. The Japanese government (Monbukagakusho) is appreciated for providing Nguyen Van Quan
a scholarship.
77
Molecules 2019, 24, 605
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80
molecules
Article
Antioxidant and Hepatoprotective Effects of
Croton hypoleucus Extract in an Induced-Necrosis
Model in Rats
Thania Alejandra Urrutia-Hernández 1 , Jorge Arturo Santos-López 2 , Juana Benedí 2 ,
Francisco Jose Sánchez-Muniz 3 , Claudia Velázquez-González 1 , Minarda De la O-Arciniega 1 ,
Osmar Antonio Jaramillo-Morales 1 and Mirandeli Bautista 1
1 Área Académica de Farmacia, Universidad Autónoma del Estado de Hidalgo, Mariano Abasolo 600,
Colonia Centro, Pachuca, Hidalgo CP 42000, Mexico
2 Departamento de Farmacología, Farmacognosia y Botánica, Facultad de Farmacia,
Universidad Complutense de Madrid, Plaza Ramón y Cajal S/N, 28040 Madrid, Espana
3 Departamento de Nutrición y Ciencia de los Alimentos, Facultad de Farmacia, Universidad Complutense
de Madrid, Plaza Ramón y Cajal S/N, 28040 Madrid, Espana
* Correspondence: mibautista@uaeh.edu.mx; Tel.: +52-(771)-72000 (ext. 4327)
Received: 14 June 2019; Accepted: 9 July 2019; Published: 11 July 2019
Abstract: The aim of this study was to evaluate the antioxidant and hepatoprotective activity
of Croton hypoleucus (EC). The present work reports the first pharmacological, toxicological, and
antioxidant studies of EC extract on liver injury. Liver necrosis was induced by thioacetamide
(TAA). Five groups were established: Croton Extract (EC), thioacetamide (TAA), Croton extract with
thioacetamide (EC + TAA), vitamin E with thioacetamide (VE + TAA) and the positive control and
vehicle (CT). For EC and EC + TAA, Wistar rats (n = 8) were intragastrically pre-administered for
4 days with EC (300 mg/kg.day) and on the last day, EC + TAA received a single dose of TAA
(400 mg/kg). At 24 h after damage induction, animals were sacrificed. In vitro activity and gene
expression of superoxide dismutase (SOD), catalase (Cat), and Nrf2 nuclear factor were measured.
The results show that EC has medium antioxidant properties, with an IC50 of 0.63 mg/mL and a
ferric-reducing power of 279.8 μM/mg. Additionally, EC reduced hepatic damage markers at 24 h
after TAA intoxication; also, it increased SOD and Cat gene expression against TAA by controlling
antioxidant defense levels. Our findings demonstrated the hepatoprotective effect of EC by reducing
hepatic damage markers and controlling antioxidant defense levels. Further studies are necessary to
identify the mechanism of this protection.
Keywords: antioxidant activity; hepatoprotective effect; Croton hypoleucus; oxidative stress
1. Introduction
Medicinal plants play a key role in the human health care system [1]. According to the World
Health Organization, between 65% and 80% of the populations of developing countries currently use
medicinal plants [2], as extracts, infusions, or bioactive compounds to treat primary conditions [3,4].
Several pharmacology studies have shown the role of medicinal plants on the treatment and
prevention of liver diseases [5]. The biological and hepatoprotective activity of plant extracts defends
hepatocytes against lipid peroxidation and other oxidative effects [6] as free radicals, toxic, viral, and
bacterial agents [7]. The hepatoprotective activity of plants has been related to several compounds,
like flavonoids (isoflavones, anthocyanins, catechins, quercetins), saponins, coumarins, alkaloids,
and terpenes [5]. In the complementary traditional medicine, some Croton species are highlighted
due to their anti-inflammatory, antiseptic, antinociceptive, antiplasmodic, antiproliferative, antiviral,

Molecules 2019, 24, 2533
and antibacterial properties, and some compounds, like terpenes, steroids, and flavonoids, have been
identified in the Croton species. These compounds have important biological activities with therapeutic
and medicinal value [3], as anethol from C. zehtneri [8]; triterpenes from C. oblongifolius [9]; and
alkaloids, flavonoids, and glycosides in C. sparciflorus [10]. Croton hypoleucus, known as Palo blanco and
Soliman Liso, is a native shrub of Hidalgo, Mexico. An infusion of its aerial parts is used in treatments
of stomachache and pain. In a preliminary phytochemical screening of EC, we found the presence
of saponins, alkaloids, tannins, flavonoids, sterols, terpenoids, and carbohydrates as they have been
reported before in Croton species [3]. To our knowledge, biological studies of C. hypoleucus have not
been previously reported, but some of its metabolites have been identified in hexane extract, such as
three epoxy-clerodane bearing furan rings, named hypoleins A–C and the Crotonpenes A–B [11].
Liver is the main metabolic and detoxifying organ that first contacts and neutralizes xenobiotic [12]
due to a cellular system of detoxification (cytochrome P450 , flavin-containing monooxygenase,
glutathione transferase), which provides biotransformation of some xenobiotics to toxic intermediates,
leading to liver toxic injury [13]. Acute toxic liver injury is characterized by membrane damage,
massive necrosis of hepatocytes, infiltration of parenchyma by neutrophils, and activation of hepatic
stellate cells, followed by a release of proinflammatory cytokines and the formation of reactive oxygen
species (ROS) as the main factors that damage liver cells [14]. ROS are oxygen-containing molecules,
including superoxide, hydrogen peroxide, and hydroxyl radical, that are highly reactive with other
complex molecules in the cells, such as protein, DNA, and lipids. Endogenous radical scavengers,
like antioxidant enzymes, including superoxide dismutase (SOD) and catalase (Cat), can lead to ROS
degradation [15]. Nuclear factor erythroid 2-related factor 2 (Nrf2) functions as a xenobiotic-activated
receptor to regulate the adaptive response to oxidants and electrophiles [16], and the repair and
removal of damaged proteins [17]. Activation of Nrf2 enhances the levels of antioxidant enzymes and
phase-2- detoxifying enzymes by complex mechanisms, and this may be one of the ways to reduce
oxidative/nitrosative stress and chronic inflammation [18].
Thioacetamide (TAA) is known as a hepatotoxicant, and is used to induce acute and chronic
liver injury due to its effects on protein synthesis, RNA, and DNA [19]. TAA hepatotoxicity requires
metabolic activation by CYP2E1 with the formation of the reactive metabolites, S-oxide (TASO) and S,
S-dioxide (TASO2 ) [20,21]. These active intermediates lead to the formation of adducts of proteins,
lipids, and nucleic acids, as well as the formation of ROS, which promote lipid and protein peroxidation
and mitochondrial damage [22]. The selective destruction of perivenous hepatocytes and proliferative
liver cells allows the TAA model to be used in experimental tests to study the hepatic response against
aggressive attack from xenobiotics and to identify the molecular, biochemical, and physiopathological
mechanisms though which the hepatic lesion develops [5]. Due to taxonomy characteristics and the
pharmacological and chemistry nature of the Croton genus, Croton hypoleucus could be a source of
hepatoprotective compounds. In this sense, the aim of the present work was to evaluate the antioxidant
and hepatoprotective effect of EC in thioacetamide-induced liver damage in a rat model.
2. Results
2.1. Purification of Main Compounds in Dichloromethane Fraction

Two clerodane-type diterpenoids were isolated from the dichloromethane fraction of EC. According
to 1 H and 13 C NMR shifts, they were identified as hypolein B (35 mg) and Crotonpene B (21 mg)
(Figure 1). The 1 H and 13 C NMR shifts are presented.
82
Molecules 2019, 24, 2533
(1) (2)
Figure 1. Clerodane-type diterpenoids identified in the dichloromethane fraction of EC: hypolein B (1)
and Crotonpene B (2).
Hypolein B: Yellow oil. 1 H NMR (400 MHz) CDCl3 : δ 9.92 (s, H-20), 7.96 (s, H-16), 7.37 (dd, J = 1.6,
1.6, H-15), 6.24 (dd, J = 1.6, 0.4, H-14), β2.30, α2.17 (H-12), α1.63 (H-8), α1.66, β1.65 (H-7), α1.77, β1.46
(H-6), α0.95 (H-19), β1.20 (H-18), α1.90, β2.15 (m, H-2), α1.37, β1.53b (m, H-1). 13 C NMR (100 MHz)
CDCl3 : δ 146.75 (C-16), 138.58 (C-15), 124.72 (C-13), 110.86 (C-14), 60.15 (C-3), 53.27 (C-9), 37.27 (C-5,
C-6), 35.29 (C-8), 29.70 (C-11), 27.79 (C-2, C-7), 19.78 (C-18), 17.56 (C-12), 17.45 (C-19), 17.82 (C-17),
15.86 (C-1).
Crotonpene B: Colorless oil. 1 H NMR (400 MHz) CDCl3 : δ 7.96 (s, H-16), 7.37 (dd, J = 1.6, 1.6,
H-15), 6.24 (dd, J = 1.6, 0.4, H-14), 3.63 (br. S, H-20), α2.07, β2.17, H-2), β1.84 (m, H-10), β1.81 (m, H-8),
α1.70 (d, J = 1.2, H-6, H,7), β1.48, α1.58 (dd, J = 2, 1.6, H-1), β1.47 (m, H-6), β1.46 (m, H-7), 1.19
(s, H-18), 1.10 (s, H-17), 0.89 (s, H-19). 13 C NMR (100 MHz) CDCl3 : δ 195.21 (C-12), 173.88 (C-20),
146.75 (C-16), 144.03 (C-15). 128.28 (C-13), 108.87 (C-14), 51.43 (C-20), 43.12 (C-11), 38.40 (C-8). 37.27
(C-5, C-6), 27.97 (C-2), 19.78 (C-18), 18.63 (C-1), 17.98 (C-17), 14.11 (C-19).
2.2. Antioxidant Activity of EC

In this study, the tested doses (2 to 6 mg/mL) were antioxidant dose dependent. The inhibition
percentages were 28.13% and 78.36% to 2 and 6 mg/mL, respectively. According to the dose-response
results, we estimated the IC50 , as the extract concentration required to reduce the initial concentration
of DPPH to 50%. In this case, the EC IC50 was 0.6307 mg/mL. To the FRAP assay, EC had an Fe+3 ion
reducing capacity of 279.8 ± 3.3 μM. Eq. Trolox/mg.
2.3. Acute Toxicity of EC

In the LD50 assay, no animals died during the 14 experimental days after administration of 10, 100,
and 1000 mg EC/kg in the first phase, and 1600, 2900, and 5000 mg EC/kg in the second phase (Table 1).
Table 1. LD50 of EC.
EC Intragastric Doses (mg/kg)

Phase I 10 100 1000
Mortality 0/3 0/3 0/3
Phase II 1600 2900 5000
Mortality 0/3 0/3 0/3
LD50 >5000
2.4. Liver Damage Biomarkers

In order to assess the degree of liver injury, biochemical parameters were estimated. The TAA
effect on transaminases enzymes is shown in Figure 2. Remarkably, the TAA group presented the
highest levels of AST and ALT when they were contrasted with the rest of the treatments. EC and CT
did not show significant differences (p ≤ 0.05) with each other, as well as VE and EC (data not shown).
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Molecules 2019, 24, 2533
In the EC + TAA group, ALT and AST presented a decrease of 65.9% and 75.8%, respectively, against
TAA, with significant differences between groups. A similar behavior was observed for the positive
control (VE + TAA).
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Figure 2. Effect of EC pretreatment on levels of (A) ALT and (B) AST analyzed by Wiener Lab equipment
in the serum of rats intoxicated with a sublethal dose of thioacetamide (TAA). All data are expressed in
U/L. Bars indicate the mean value with SE of two determinations (n = 8). The differences compared
with the vehicle are expressed as “a”; while the differences due to TAA are expressed as “b”, p ≤ 0.05.
The EC + TAA group showed a significant reduction (38.75%) of ALP levels, which was comparable
to the EC and CT groups. A similar tendency was observed in EC + TAA for the T-Bil and D-Bil plasma
concentration, which showed a reduction of 58% and 73%, respectively (Figure 3). The TAA groups
also presented elevated levels of GGT and LDH (1.21 ± 0.24 × 10−4 and 7291 ± 907, respectively).
While, EC + TAA exhibited a reduction of 99% and 68.9% to GGT and LDH, respectively (Figure 4).

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Molecules 2019, 24, 2533

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Figure 3. Effect of EC pretreatment on levels of (A) ALP, (B) T-Bil, and (C) D-Bil analyzed by Wiener
Lab equipment in the serum of rats intoxicated with a sublethal dose of thioacetamide (TAA). In ALP,
the results are expressed in U/L, while T-Bil and D-Bil are expressed in mg/dL of serum. Bars indicate
the mean value with the SE of two determinations (n = 8). The differences compared with the vehicle
are expressed as “a”; while the differences due to TAA are expressed as “b”, p ≤ 0.05.
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Figure 4. Effect of EC pretreatment on the levels of (A) GGT and (B) LDH analyzed by Wiener Lab
equipment in the serum of rats intoxicated with a sublethal dose of thioacetamide (TAA). The results
are expressed in U/L of serum. Bars indicate the mean value with the SE of two determinations (n = 8).
The differences compared with the vehicle are expressed as “a”; while the differences due to TAA are
expressed as “b”, p ≤ 0.05.
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Molecules 2019, 24, 2533
2.5. SOD and Cat Evaluation from Antioxidant Enzyme Activity

To assess the enzymatic antioxidant response of the liver, SOD and Cat activities were measured.
The results showed that compared with the vehicle, the levels of both enzymes in EC + TAA
(35.58 ± 1.7 and 9.09 ± 0.59 U/mg) decreased at 24 h after TAA administration, as TAA did (36.03 ± 1.56
and 8.52 ± 0.10 U/mg) for SOD and Cat, respectively (Figure 5). For Cat, no significant differences
(p ≤ 0.05) were reported between CT and EC + TAA.

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Figure 5. Effect of EC pretreatment on levels of (A) SOD and (B) Cat enzyme activities in rats’ liver
intoxicated by a sublethal dose of thioacetamide (TAA). The results are expressed in U/mg protein. Bars
indicate the mean value with the SE of two determinations (n = 8). The differences compared with the
vehicle are expressed as “a”; while the differences due to TAA are expressed as “b”, p ≤ 0.05.
2.6. MnSOD, CuZnSOD, Cat, and Nrf2 mRNA Expression

Results showed that TAA treatment led to a significant reduction of MnSOD, CuZnSOD, Cat, and
Nrf2 gene expression (Figure 6). On the other hand, the EC + TAA group stimulated relative mRNA
expression of MnSOD, CuZnSOD, and Cat in comparison with its TAA counterparts, but without
reaching similar values to the CT animals. In contrast, the Nrf2 expression displayed a significant
reduction in the EC + TAA group in comparison with both the CT and TAA groups.
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Figure 6. Effect of EC on (A) MnSOD, (B) CuZnSOD, (C) Cat, and (D) Nrf2 expression in homogenated
liver of rats intoxicated with a sublethal dose of thioacetamide (TAA). The results are expressed in
relative expression, arbitrary units (a.u.). Bars indicate the mean value with the SE of two determinations
(n = 8). The differences compared with the vehicle are expressed as “a”; while the differences due to
TAA are expressed as “b”, p ≤ 0.05.
3. Discussion
3.1. Main Compounds in Dichloromethane Fraction

Diterpenoids are characteristic components of the Croton species [3]. They represent 85% of
terpenoid compounds identified in these species [23]. Apart from the Clerodane-type terpenoid,
hypolein B and Crotonpene B have also been reported for Croton hypoleucus by Velazquez et al. [11] in
the hexane extract of the aerial parts, as well as by Sun et al. [24] in the methanol extract of aerial parts
of Croton yanhuii. Diterpenoids are well known as compounds with remarkable biological activities,
such as anti-tumor, anti-malarial, anti-inflammatory, antimicrobial [25], hepatoprotective [26–29], and
cytotoxic [30]. Some diterpenes may be toxic for humans, resulting in acute or chronic impacts on
different tissues and organs. Kubo et al. [31] showed that the nor-diterpene transdehydrocrotonin
isolated from C. cajucara is responsible for its hepatotoxicity, while the extracts of the same plant
showed hypolipidemic activity and an absence of hepatotoxicity in animal models [32]. Also, toxicity
occurs when it is used long term or is taken at high doses [33].
3.2. Antioxidant Activity of EC

The in vitro antioxidant activity of the crude extract was evaluated through 2,2-diphenyl-1-
picrylhydrazyl (DPPH) and the ferric reducing ability of plasma (FRAP) assays. The DPPH molecule
can accept an electron from a hydrogen radical to become itself a stable molecule, and then it reacts
with a reducing agent to form a new bind [21], so the DPPH assay determinates the EC capacity to
scavenge free radicals as an antioxidant power measurement [34]. The percent of inhibition reported
for EC is comparable with the ethanolic extract of C. zambesicus, which presented an inhibition of
72% [35]. On the other hand, IC50 is a widely used parameter to measure the antioxidant activity of
extracts [36]. Croton species leave extracts of C. argyrophyllus and C. heliotropiifolius showed IC50 values
of 0.22 and 0.352 mg/mL, respectively [34], while C. leptstachyus and C. bonplandianum have IC50 values
of 11.6 and 416 μg/mL, respectively. The IC50 was 0.6307 mg/mL, which indicates that a higher EC
concentration was needed to scavenge 50% of the DPPH free radical, as an antioxidant potential [37].
The FRAP assay is based on the reduction, at low pH, of a colorless ferric complex
(Fe3+ -tripyridyltriazine) to a blue ferric complex (Fe2+ -tripiridyl-s-triazine) by the action of electron
donating antioxidants [38]. The reduction is monitored by measuring the change in absorbance at
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593 nm [39]. The extract composition is preponderant for the and the extract is rich in flavonoid-type
phenolic compounds. Flavonoid compounds, such as quercetin, kaempferol, quercitrin, and 3-O-methyl
ether [40], have been recognized in the Croton genus [3]. The participation and synergisms of flavonoid
compounds could contribute to the EC antioxidant capacity [41], and their interactions with other
compounds could potentiate or interfere with the EC antioxidant ability [42]. The antioxidant and
inhibition of free radical production are important for the protection of cells from TAA-induced
hepatotoxicity [43]. Different mechanisms, in which antioxidant compounds perform their scavenging
properties, have been documented: They act as a physical barrier to prevent ROS generation, and
they could access target biological sites, as a chemistry trap catching energy and chelating electrons;
as a reactive species scavenging catalytic system and breaking redox chains, and scavenging radicals;
or binding to targeted metal compounds and avoiding redox chain formation [44]. Most of them
depend on the hydrogen atom transference rate from the compounds to the radicals [45]. The results
of this experiment showed that EC may contain potential compounds able to donate hydrogen atoms
to free radicals to become more stable molecules, and are responsible for the reported antioxidant
activity. The EC capacity to the scavenging DPPH radical and reducing Fe3+ can contribute by reducing
oxidative stress effects and liver damage. The discovery of antioxidant compounds is critical for new
drug research and the treatment of diseases related to oxidative stress.
3.3. Acute Toxicity of EC

Acute toxicity testing is the defining and evaluating of a toxic syndrome produced by a single dose
or a few doses of an extract or drug administered over the course of a day [46]. During the observation
period, rats breathed, ate, and increased body weight normally. Conditions, such as difficulty to
breath, loss of appetite, and death, are signs of toxicity [47]. During the post mortem examination, the
macroscopic morphology of the liver, spleen, lungs, kidneys, and stomach showed normal color and
morphology (data not showed) compared with the vehicle. A lot of Euphorbiaceae species are known
in different countries as being toxic or medicinal plants. Given its therapeutic response, its chemical
diversity can be hypothesized. Compounds, like alkaloids and forbol esters, have been reported in
Croton species [3]. Different extracts and essential oils of Croton species, such as C. membranaceus,
C. sparsiflorus, C. bonplandianum, and C. zehntneri, have been evaluated to find their toxicity level at
different doses from 300 to 5000 mg/kg [10,48,49] and are considered safe. Nevertheless, a dose of
447.18 mg/kg of C. polyandrus essential oil produced the death of mice [50]. The extract toxicity depends
on several factors, such as the chemistry composition, doses, and exposition time. In our study, the
rat’s survival in all evaluated doses during the two study weeks, and a higher 5000 mg/kg suggests
that ethanolic C. hypoleucus extract is not toxic [51].
3.4. Liver Damage Biomarkers

The liver damage induced by xenobiotic agents as TAA is characterized by an increase of serum
liver enzymes. TAA is a toxic agent that causes hepatocytes necrosis and it contributes to cirrhosis
development through multiple action mechanisms, such as oxidative stress, decrease of the antioxidant
system response, and lipid peroxidation [52]. Particularly, AST and ALT transaminases are used
as biomarkers of hepatocellular necrosis. The serum transaminases concentration is referred as an
indicator of the liver damage severity [53–56]. ALT is present in the liver at higher concentrations than
other organs. AST is considered to have a lower specificity for liver damage than ALT due to it is
presence in other organs [57]. Along with TAA metabolism, thioacetamide-s-oxide and reactive species
are produced. The reactive species harm the cell by lipid peroxidation and produce a breakdown and
loss of permeability of the cellular membrane [54]; this is a probable explanation of the increase in
AST and ALT levels from the TAA group. These results are in accordance with several studies on
TAA-induced liver necrosis in experimental animals [58–61]. The present results also demonstrated
the protective role of vitamin E against TAA, and they were in line with a preliminary study [62].
EC + TAA showed a significant decrease in the serum levels of ALT and AST in relation with VE + TAA
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Molecules 2019, 24, 2533
and TAA (p ≤ 0.05). The results in EC + TAA are strongly related to the EC capacity to save the cell
against necrotic damage produced by TAA to reduce the rate of transaminase release and cellular
membrane stabilization [44].
ALP is a hydrolase enzyme, which is eliminated by bile. It is present in cells covering biliary
conducts, as well as other organs, like bone, placenta, kidney, and intestine. Hepatotoxicity leads to an
elevation of normal values due to the body´s excretion inability through bile due to the congestion
or obstruction of the biliary tract, which may occur within the liver, such as was observed in the
TAA group. The result showed for the EC + TAA indices that EC has the ability to reduce the effects
of bile obstruction induced by TAA by decreasing ALP toward vehicle levels (Figure 3). A similar
behavior was reported in bilirubin determination. The bilirubin is a product from regular hemoglobin
breakdown, and it is released into the bile [54]. The T-Bil and D-Bil result to EC + TAA indicates that
EC contributed to bilirubin metabolism after induced damage by TAA.
For D-Bil and T-Bil plasma concentrations, TAA lead to elevated levels of bilirubin. The induced
liver damage by TAA caused the liver to lose its ability to conjugate to bilirubin; thereby, its excretions
are affected, and it causes hyperbilirubinemia in serum. This alteration, along with higher transaminases
levels, is a sign of acute or toxic injury [61] as the TAA group showed. GGT enzyme is localized in the
liver, kidney, and pancreas. It catalyzes the conjugation of electrophilic species from TAA metabolism
with GSH [53]. GGT levels tends to increase due to its release from the hepatocytes to the circulatory
system by changing the membrane permeability. Although the mechanisms for GGT induction are
uncertain, they have been associated with C-reactive protein, a general marker for increased oxidative
stress, which leads to overconsumption of GSH with a compensatory increase in GGT synthesis [62,63].
On the other hand, GGT reflects a state of oxidative stress forward to chronic disease; while
LDH increases its levels as a result of liver diseases [64]. The liver biomarker results describe the
protective ability of EC against free radicals and electrophilic compounds from TAA biotransformation,
which promotes cellular stability, serum transaminases and bilirubin depuration, as well as recovery
competence, thus keeping biomarker levels closer to the vehicle, as C. oblongifolius ethanol extract [9],
C. zehnteneri essential oil [8], C. sparciflorus [10], and C. bonplandianus methanol extract [21] have
shown against necrotic effects of CCl4 , acetaminophen, and N-nitrosodietylamine. The liver
biomarkers’ regulation represents the liver’s recovery to a normal state [65]. To date, the compounds
responsible for the hepatoprotective activity of C. hypoleucus have not been revealed, however, several
studies [8–10] report that flavones, terpenoids, alkaloids, tannins, and saponin may be responsible for
this pharmacological effect.
3.5. SOD and Cat System

Defensive responses of organisms to oxidative stress include the utilization of endogenous
antioxidant enzyme systems, lipid soluble and water-soluble antioxidant molecules, and phytochemicals,
which can be detected through measurement of the total antioxidant capacity. Antioxidants, such as
SOD, Cat enzymes, and GSH, are some of the most important elements that act as a defense against
oxidative damage. They keep ROS at low levels and avoid excessive production [66]. For the purpose of
this investigation, the effect of EC was evaluated on the levels of SOD and Cat as enzymes of the system
of antioxidant defense. SOD is present in the cytoplasm and mitochondria of cells. The SOD molecule
in the cytoplasm contains copper and zinc atoms, while mitochondrial SOD contains manganese. SOD
catalyzes superoxide radical dismutation (·O2 ) into hydrogen peroxide (H2 O2 ); even though H2 O2
is not a radical, it is rapidly converted into hydroxyl radical, which is highly reactive, by means of
the Fenton reaction and Cat enzymatic activity [67]. The regulatory activity of this enzyme enables
mutual protection; when the superoxide radical is produced, it is disabled by Cat, while H2 O2 inhibits
SOD [68]. Hepatotoxicity by TAA requires metabolic activation with the formation of the reactive
metabolites, S-oxide (TASO) and S, S-dioxide (TASO2 ) [21], which bind to microsomal lipids, leading
to is peroxidation, as well as ROS production, such as hydroxyl, peroxide, and superoxide radicals.
ROS affect antioxidant defense mechanisms, and they decrease SOD, Cat, and GPx activity, leading to
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liver damage, cirrhosis, and hepatocellular carcinoma [54]. In our study, the acute liver injury by TAA
was characterized by a reduction in the in vitro activity of SOD and Cat (Figure 5) due to the attack of
superoxide and hydrogen peroxide radicals against the cell [68]. The TAA administration to rats may
cause cellular structure changes, interfere with RNA movement from nuclei to the cytoplasm, and
reduce the number of viable hepatocytes, as well as reduce the oxygen intake rate. TAA prolonged
exposure leads to hyperplastic nodule formation, hepatocellular carcinoma, and cirrhosis. The induced
cirrhosis by TAA in rats has been shown to be an experimental model of disease comparable with
human ethology and pathology [69]. Higher Cat levels compared to SOD means that EC could promote
the antioxidant defense system by increasing Cat activity against H2 O2 and protecting cells against
acute toxic liver damage at 24 h after TAA administration, as shown in Figure 5 shows.
3.6. MnSOD, CuZnSOD, Cat, and Nrf2 mRNA Gene Expression

Gene expression is a process by which DNA instructions are converted into functional products
as proteins. In MnSOD, CuZnSOD, and Cat, the lower gene expression was derived from the process
of TAA detoxification, which produces an attack of hydroxyl radicals and DNA damage. The results
highlight a greater Cat gene expression due to EC + TAA toward basal stages, which means that
EC has an inhibitory capacity against ·O2 and H2 O2 , avoiding peroxidation and DNA damage by
activation of the defense antioxidant system [21]. These results are the opposite to the Nrf2 gene
expression, which was suppressed. Nrf2 plays an important role in the activation of antioxidant
enzymes by regulating their transcription. It is primarily regulated by Keap1 (Kelch-like erythroid
cell-derived protein with CNC homology-associated protein 1) dependent ubiquitination-proteasomal
degradation and is activated by oxidants [70], so Keap1 binds Nrf2 in the cytoplasm and maintains
Nrf2 at a low steady state level [17]. Another mechanism for Nrf2 degradation is phosphorylation by
glycogen synthase kinase 3 (GSK3) via β-transducin repeats-containing protein (β-TrCP)-Cul1-based
ubiquitin ligase [71]. As a consequence, Nrf2 knockout means that the effect could also be mediated by
inflammatory cells [68]. Although Nrf2 activation is generally considered to have a beneficial effect
in liver disease [72], we found that the antioxidant mechanism inducted by EC was independent of
activation of Nrf2 expression. The increase of levels of antioxidant enzymes by activating Nrf2 may
not be enough to decrease oxidative stress and chronic inflammation optimally due to antioxidants,
which tend to decrease in an oxidative environment, and must also be elevated. Besides, the levels of
antioxidant can be increased by supplementation or an Nrf2 independent mechanism [18] as happened
with the EC + TAA group.

The main chemicals used in this study include: CDCl3 , 2,2-Diphenyl-1-picrylhydrazyl, thioacetamide,
tween 80, 2,4,6-Tris(2-pyridyl)-s-triazine, TPTZ (2,4,6-tri (2-pyridyl-s-triazine) 6-hydroxy-2,5,7,8-
tetramethil-chromal-2-carboxylic acid- Trolox, Chloride ferric, Tri-Reagent, and agarose were purchased
from Sigma Chemical Co. (St. Louis, MO, USA); pentobarbital was provided by Pisa (Mexico City,
Mexico). qPCR Master Mix (Nzytech, Portugal); SYBR Green (Biotools, Madrid, Spain); Silica gel 60,
and other chemicals, such as ethanol, methanol, and hydrochloric acid, were reactive grade products
from Merck (Darmstadt, Germany). DNase I RNase-free reagents were bought Thermo Fisher Scientific
(Waltham, MA, USA).
4.2. Preparation of EC
Aerial parts of C. hypoleucus were collected on January 2016, from San Vidal, Tulancingo, Hidalgo
State [20.116002, −98.305734]. The plant was identified by Manuel González Ledezma, taxonomist
of Department of Botany, Autonomous University of Hidalgo State. The specimen, voucher number:
DVM01, was deposited at the Herbarium of Biological Sciences Research Center. Aerial parts were
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dried for a period of 15 days in a light protected area and milled with a commercial grinder. Powdered
plant (500 g) was extracted by maceration with 4 L of ethanol for a week in triplicate. The material was
filtered and concentrated by a rotatory evaporator (Büchi, Switzerland) at 40 ◦ C. The crude extract
obtained was used for in vitro and in vivo analysis.
4.3. Purification of Main Compounds in Dichloromethane Fraction

A sample of 5 g of crude extract was fractionated through a flash column chromatography
performed over silica gel 60 (230–400 mesh). The sample was eluted with 500 mL of hexane,
dichloromethane, ethyl acetate, and methanol to yield 0.3, 1.8, 2.1, and 0.8 g, respectively.
The dichloromethane fraction was dried and supported over silica gel 60 column chromatography and
eluted with a hexane: Dichloromethane (6:4) mixture. In total, 64 fractions of 10 mL were collected
and monitored by TLC on precoated silica gel aluminum sheets. The compounds were visualized
through UV detection and by spraying with vanillin/H2 SO4 /EtOH solution, followed by heating.
The fractions, 48–52, was selected and supported over a preparative layer chromatography with
hexane: Dichlorometane (1:1) which lead to the isolation of two compounds (Figure 1), which were
characterized by 1 H and 13 C NMR spectroscopy using a Spectrometer 400 MHz (Bruker Avance III,
Billerica, MA, USA) and CDCl3 as solvent. The NMR shifts were compared with literature data [11,24].
4.4. Antioxidant Capacity of EC

DPPH Free Radical Scavenging Activity. The DPPH radical scavenging was determined according
to the method of Brand-Williams [73] with slight modifications. From a 50 mg/mL methanol solution
of EC, 50 μL was mixed with 200 μL DPPH reactant (200 μM). The mix of 1 mL methanol and 1 mL
DPPH solution was used as the control. The reaction was carried out in triplicate. The mixture was
kept for 30 min in a 96-well plate. After incubation, the absorbance was measured at 517 nm using
an absorbance plate reader (Fluostar Optima, BMG Labtech, Ortenberg, Germany). The percent of
inhibition was calculated by the following equation:
Abs Control − Abs CE

% inhibition = × 100. (1)
Abs Control
The IC50 or effective concentration values, representing the amount of extract required to decrease
the absorbance of DPPH by 50%, were calculated from the percentage of radical scavenging activity.
Ferric-reducing power FRAP. The ferric-reducing power of the crude extract was determined
according to a modified protocol of Benzie and Strain [74]. The working FRAP solution was prepared
daily by mixing 25 mL of acetate buffer (0.3M pH 3.6), 2.5 mL of 10 mM TPTZ in HCl (40 μM), and
2.5 mL of ferric chloride in distilled water. The working solution was kept at 35 ◦ C and in the dark.
In the test, 30 μL of crude extract in methanol (10 mg/mL) was mixed in 90 μL of distilled water plus
900 μL of FRAP solution. The reaction mixture was incubated for 30 min at 37 ◦ C. The absorbance was
measured at 595 nm with a spectrophotometer (UVikon 930 spectrophotometer, Kontron Instruments
S.A., Madrid, Spain). For the ferric-reducing power determination, ferrous sulfate heptahydrate
solution (200–1000 μM) instead of extract was used as the calibration curve. The results (in triplicate)
are expressed as μM Fe/mg crude extract.
4.5. Experimental Animals

Male Wistar rats weighing 180 to 200 g and aged 7 weeks were used in this study. The animals
were obtained from the vivarium of the Autonomous University of Hidalgo State. They were adapted
according to appropriate protocols prior to commencement of the experiment. The rats were maintained
in clean polypropylene cages in a temperature controlled room and 12:12 h light/dark cycles with ad
libitum access to pellet food and water. After 1 week of acclimation, rats were randomly assigned
to experimental groups. Besides, all the experiments were conducted by approbation of the Internal
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and Ethical Committee for the care and use of experimental animals with the official certificate
No. 5-12-2017 and according to the Official Mexican Norm (NOM 062-ZOO-1999) [75].
4.6. Acute Toxicity

The acute toxicity was evaluated using the Dietrich Lorke assay [52]. This study was conducted
in two phases. In the first phase, three groups (n = 3) of rats were administered intragastrically with a
homogeneous solution of EC in 1% tween 80 at the respective oral doses of 10, 100, and 1000 mg/kg
body weight. The animals were observed frequently for 14 days and any adverse effects (mortality,
body weight, water and food intake) were recorded for the 14 days. In the second phase, three new
groups (n = 3) rats were administered respective oral doses of 1600, 2900, and 5000 mg/kg body weight
of crude extract. In both phases, a vehicle was fed 1 mL of Tween 80.1% intragastrically. The possible
number of deaths was recorded and the LD50 value was determined.
4.7. Thioacetamide-Induced Hepatotoxicity

The thioacetamide-induced damage was performed following a pre-established protocol [56].
Male Wistar rats were randomly distributed into 5 groups (n = 8). Groups EC and EC + TAA received
a single dose intragastrically (i.g.) of crude extract (300 mg/kg body weight) every 24 h for four
days of treatment. At the same time, VE + TAA was administered with 100 mg/kg (i.g.) and groups
Thioacetamide (TAA) and vehicle (CT) were administered i.g. with a tween 80 solution (1% v/v). On the
fourth day, group TAA, EC + TAA, and VE + TAA were administered one dose (400 mg/kg body weight)
of TAA dissolved in 1 mL NaCl (0.9%) intraperitoneally (i.p.). Then, 24 h after TAA administration,
all animals were sacrificed with intramuscular pentobarbital doses (50 mg/kg body weight) via the
i.p. route. Then, through an abdominal dissection, samples of blood were obtained by portal vein
puncture. Serum from blood was separated by centrifugation at 4000 rpm, 10 ◦ C, and 15 min and
analyzed for various biochemical parameters related to liver damage using well-established protocols.
On the other hand, livers were rapidly dissected out and washed using 0.9% NaCl sterile solution and
then immediately stored in a −80 ◦ C freezer until tissue homogenate preparation.
4.7.1. Biochemical Parameters

Quantitative determination of the ALT, AST, ALP, GGT, and DHL enzymes and DB was carried
out with WIENER-Lab optimized equipment (Rosario, Argentina). Quantitative determination of ALT
enzyme was measured by diminution in absorbance to 340 nm at 25 ◦ C, produced by the oxidation
of NADH into NAD+ while pyruvate reduction into lactate was done by lactate dehydrogenase [76].
The activity of AST was measured by the diminution in absorbance at 340 nm and 25 ◦ C produced by
NADH oxidation to NAD+ in the paired reaction of the oxaloacetate reduction into malate by malate
dehydrogenase activity [77]. ALP determination was done through pnitrophenylphosphate (pNPP)
hydrolysis yielding phosphate and p-nitrophenol at alkaline pH and 405 nm. The p-nitrophenolate
production is directly proportional to the enzymatic activity of the sample [78]. GGT was analyzed by
the change in absorbance at 410 and 480 nm due to the formation of 5-amino-2-nitrobenzoate, a product
from a glutamyl group transfer from the donor substrate, gamma-glutamyl-3-carboxy-4-nitroanilide,
to the acceptor, glycylglycine, by GGT [79]. LDH catalytic activity was determined by measuring
the NAD+ formation rate from NADH oxidation in an alkaline pH at 340 nm and 37 ◦ C [80].
The evaluation of bilirubin was performed through production of azobilirubin from the bilirubin
reaction with diazotized sulfanilic acid photocolorimetrically measured at 530 nm [81].
4.7.2. SOD and Cat in Vitro Activity

Homogeneous samples of liver tissue were used for the SOD and Cat biochemical assays. Liver
tissue was homogenized in phosphate-EDTA buffer (0.1 M sodium phosphate and 0.005 M EDTA, pH 8)
at 100 mg/mL, with the addition of 10 μL/mL of perchloric acid. It was then centrifuged at 10,000 rpm
for 10 min at 4 ◦ C. Superoxide dismutase (SOD) activity was measured using the Nitroblue Tetrazolium
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Molecules 2019, 24, 2533
reagent (NBT) method according to Neha and Mishra protocol [82] with modifications. This method is
based on the generation of superoxide radical (O2− ) by autoxidation of hydroxylamine hydrochloride
in the presence of NBT, which gets reduced to nitrite. Nitrite in the presence of EDTA gives a color
measured at 560 nm. Cat activity was measured as described by the Aebi [83] method using hydrogen
peroxide as a substrate. The decomposition of H2 O2 was followed directly by a decrease in absorbance
at 260nm. Enzyme activity was standardized to liver homogenate protein concentrations determined
according to Bradford´s method [84]. Final enzyme activity results are expressed as IU/mg protein.
4.7.3. Extraction and Quantification of RNA by RT-PCR

RNA samples were isolated from 100 mg of liver using TRI-Reagent and treated with DNase I
RNase-free reagents to avoid any contamination with genomic DNA. The yield and quality of RNA
was assessed by measuring absorbance at 260, 280, and 310 nm and by electrophoresis on agarose
gels (1%). Total RNA of sample was reverse-transcribed to first-strand complementary DNA (cDNA)
using Nzytech qPCR Master Mixes. Relative Mn-SOD, CuZn-SOD, Cat, and Nrf2 mRNA levels were
quantified with a LightCycler Real-Time PCR Detection System (Roche Diagnostics, Indianapolis, IN,
USA), using SYBR Green as the fluorescent binding dye. Detection was monitored by measuring the
increase in fluorescence throughout the cycles. The standardization was carried out to the β-actine
value. The results are expressed as fold changes of the threshold cycle (Ct) value relative to the vehicle
using the 2−ΔΔCt method [85]. The PCR protocol was: Preincubation at 95 ◦ C for 10 min followed by
45 cycles of denaturation at 95 ◦ C for 10 s with an annealing temperature of 60 ◦ C for each couple
primer, extension at 72 ◦ C for 15 s, and cooling at 40 ◦ C for 30 s. Primer sequences were as indicated in
Table 2.
Table 2. Primer sequences used in RT-PCR.
Mn-SOD sense: 50-ACTGAAGTTCAATGGCGGG-30 and antisense: 50-TCCAGCAACTCTCCTTTGGG-30

CuZn-SOD sense: 50-CTTCGAGCAGAAGGCAAGCG-30and antisense: 50-GACATGGAACCCATGCTCGC-30
Cat sense: 50-ATCAGGGATGCCATGTTGTT-30 and antisense: 50-GGGTCCTTCAGGTGAGTTTG-30
Nrf2 sense: 50-TTGTAGATGACCATGAGTCGC-30 and antisense: 50-GAGCTATCGAGTGACTGAGCC-30

Significant differences between the results were calculated by variance analysis (ANOVA). One-way
ANOVA was determined by Statgraphics centurion XVII.II version (Statistical graphics Corporation,
Inc., Rockville, MD, USA). A post-hoc Tukey test was performed to identify significant differences
(p ≤ 0.05) between treatments.
5. Conclusions
The current study reports the first antioxidant and hepatoprotective evaluation of ethanolic crude
extract of Croton hypoleucus (EC) in a frame of a thioacetamide-induced (TAA) liver damage model
in rats. The main mechanisms by which EC protects the liver from toxic damage are associated with
its antioxidant properties and its ability to modulate Cat involved in the antioxidant defense system.
Additionally, EC has the ability to recover cell mitochondria and regulate biomarkers of the liver after
TAA injury, thus preventing the development of hepatotoxicity. Nrf2-no dependent catalase activation
revealed the role of antioxidant mechanisms while biochemical parameters were the first sign of its
hepatoprotective activity. Croton hypoleucus could offer a novel alternative to the limited therapeutic
options that exist for the treatment of liver diseases.
Author Contributions: Conceptualization, M.B.; Data curation, O.A.J.-M.; Formal analysis, J.A.S.-L.; Investigation,
M.D.l.O.-A.; Methodology, J.B.; Project administration, C.V.-G.; Supervision, F.J.S.-M.; Writing, review & editing,
T.A.U.-H.
Funding: The present research received a scholarship number 236155 from CONACYT, Mexico.
93
Molecules 2019, 24, 2533
Acknowledgments: We wish to thank the Pharmacology, Pharmacognosy and Botany Department of Pharmacy
school from Complutense University of Madrid for their technical assistance during T.A.U.-H. internship.
Conflicts of Interest: The authors declare no conflicts of interest.
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Protective Effects Induced by Two Polyphenolic
Liquid Complexes from Olive (Olea europaea,
mainly Cultivar Coratina) Pressing Juice in
Rat Isolated Tissues Challenged with LPS
Lucia Recinella 1,† , Annalisa Chiavaroli 1,† , Giustino Orlando 1 , Luigi Menghini 1 ,
Claudio Ferrante 1 , Lorenzo Di Cesare Mannelli 2 , Carla Ghelardini 2 ,
Luigi Brunetti 1, * and Sheila Leone 1
1 Department of Pharmacy, “G. d’Annunzio” University, 66013 Chieti, Italy
2 Department of Neuroscience, Psychology, Drug Research and Child Health - NEUROFARBA -
Pharmacology and Toxicology Section, University of Florence, 50139 Florence, Italy
* Correspondence: luigi.brunetti@unich.it; Tel.: +39 0871 3554758
† These authors contributed equally to the work.
Academic Editor: Derek J. McPhee

Received: 12 July 2019; Accepted: 15 August 2019; Published: 19 August 2019
Abstract: MOMAST(®) HY100 and MOMAST(®) HP30 are polyphenolic liquid complexes from olive
pressing juice with a total polyphenolic content of 100 g/kg (at least 50% as hydroxytyrosol) and
36 g/kg (at least 30% as hydroxytyrosol), respectively. We investigated the potential protective role
of MOMAST(®) HY100 and MOMAST(®) HP30 on isolated rat colon, liver, heart, and prefrontal
cortex specimens treated with Escherichia coli lipopolysaccharide (LPS), a validated ex vivo model
of inflammation, by measuring the production of prostaglandin (PG)E2 , 8-iso-PGF2α , lactate
dehydrogenase (LDH), as well as cyclooxygenase (COX)-2, tumor necrosis factor α (TNFα),
and inducible nitric oxide synthase (iNOS) mRNA levels. MOMAST(®) HY100 decreased
LPS-stimulated PGE2 and LDH levels in all tested tissues. Following treatment with MOMAST(®)
HY100, we found a significant reduction in iNOS levels in prefrontal cortex and heart specimens,
COX-2 and TNFα mRNA levels in heart specimens, and 8-iso-PGF2α levels in liver specimens. On the
other hand, MOMAST(®) HP30 was found to blunt COX-2, TNFα, and iNOS mRNA levels, as well as
8-iso-PGF2α in cortex, liver, and colon specimens. MOMAST(®) HP30 was also found to decrease
PGE2 levels in liver specimens, while it decreased iNOS mRNA, LDH, and 8-iso-PGF2α levels in
heart specimens. Both MOMAST(®) HY100 and MOMAST(®) HP30 exhibited protective effects on
multiple inflammatory and oxidative stress pathways.
Keywords: Cultivar Coratina; inflammation; oxidative stress; hydroxytyrosol
1. Introduction
It has been well established that olive tree (Olea europaea) polyphenols have healthy beneficial effects,
including the prevention of several chronic diseases, such as cancer and aging-associated degenerative
diseases [1,2]. These beneficial properties could be mainly related to the antioxidant activity of
olive tree polyphenols, which were found able to both scavenge free radicals and reactive oxygen
species and activate endogenous antioxidant enzymes, including glutathione peroxidase, glutathione
reductase, and glutathione S-transferase [3–5]. Besides the antioxidant activity, anti-atherogenic,
hepato-protective, hypoglycemic, anti-inflammatory, immunomodulatory, anticancer, and antimicrobic
effects were also suggested for these compounds [5–7]. Hydroxytyrosol (HT) and the secoiridoid
oleuropein (OE) are two abundant phenolic compounds in olives, virgin oil, and waste water from

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olive oil production [8–10]. Particularly, HT has antioxidant and scavenging activities comparable to
oleuropein and catechol [11].
MOMAST(®) HY100 and MOMAST(®) HP30 (Bioenutra, Ginosa, TA, Italy) are polyphenolic liquid
complexes from olive (Olea europaea, mainly Cultivar Coratina) pressing juice with a total polyphenolic
content of 100 g/kg (at least 50% as HT) and 36 g/kg (at least 30% as HT), respectively (Tables 1 and 2).
In addition to HT, both MOMAST(®) HY100 and MOMAST(®) HP30 are also characterized by the
presence of tyrosol and oleuropein.
Table 1. Characteristics of the polyphenolic complex MOMAST(®) HY 100.
Name: MOMAST HY 100

Polyphenolic active complex of hydroxytyrosol—Liquid, with total
Description:
polyphenolic content of 100 g/kg
Source Type: Mainly Cultivar Coratina
Physical State: Liquid
Appearance: Light brown to brown liquid
Moisture: N.A.
Ash: Less than 10% (600 ◦ C)
Total heavy (as Pb): Less than 10 ppm
Total Plate Count: Less than 100 cfu/g
Pesticides: Absence
Polyphenolic content measured through high performance liquid chromatography (HPLC)
Hydroxytyrosol (HPLC): 50 g/Kg
Tyrosol (HPLC) 15 g/kg
Oleuropein (HPLC) 0.5 g/Kg
Total Polyphenols (HPLC) 100 g/Kg
Table 2. Characteristics of the Polyphenolic Complex MOMAST(®) HP 30.
Name: MOMAST HP 30
Polyphenolic active complex from olives’ pressing juice—Liquid, with total
Description:
polyphenolic content of 30 g/kg
Source Type: Mainly Cultivar Coratina
Fisic State: Liquid
Appearance: Brown liquid
Moisture: N. A.
Ash: Less than 10% (600 ◦ C)
Total heavy (as Pb): Less than 10 ppm
Total Plate Count: Less than 100 cfu/g
Pesticides: Absence
Polyphenolic Content
Hydroxytyrosol (HPLC): 15 g/Kg
Tyrosol (HPLC) 3 g/kg
Oleuropein (HPLC) 0.2 g/Kg
Total Polyphenols (HPLC) 30 g/Kg
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Considering the antioxidant effects displayed by both HT and oleuropein, the aim of the present
study was to investigate the putative protective effects of MOMAST(®) HY100 and MOMAST(®) HP30,
both including HT and oleuropein, on the burden of oxidative stress/inflammation occurring on various
isolated rat tissue (i.e., colon, liver, heart, and prefrontal cortex) specimens exposed to Escherichia
coli lipopolysaccharide (LPS), a well-established inflammatory stimulus. Specifically, we studied the
effects of MOMAST(®) HY100 and MOMAST(®) HP30 on multiple inflammatory and oxidative stress
pathways, by measuring the production of prostaglandin (PG)E2 , 8-iso-PGF2α , lactate dehydrogenase
(LDH), as well as cyclooxygenase (COX)-2, tumor necrosis factor α (TNFα), and inducible nitric oxide
synthase (iNOS) mRNA levels. The results support a rational use of these polyphenolic complexes in
the prevention of tissue damage occurring during inflammation.

MOMAST(®) HY100 (10, 50, and 100 μg/mL) and MOMAST(®) HP30 (22, 110, and 220 μg/mL)
were tested in vitro to evaluate their effects on cell viability. We observed that both polyphenolic liquid
complexes were well tolerated by Hypo-E22 and C2C12 cell lines (Supplementary Figures S1–S2).
Particularly, C2C12 and Hypo-E22 cell viability resulted in the limit of biocompatibility (>70 and <130%
compared to the untreated control group) after exposition to polyphenolic extracts, in the respective
tested concentration range, corresponding to identical concentrations of HT (5–50 μg/mL), which were
in agreement with previous in vitro studies [11].
Considering these findings, we performed a second set of experiments aimed to evaluate the
modulatory effects of MOMAST(®) HY100 (10, 50, and 100 μg/mL) and MOMAST(®) HP30 (22, 110,
and 220 μg/mL) supplementation on oxidative stress and multiple inflammatory pathways in colon,
liver, heart, and prefrontal cortex specimens challenged with LPS. As previously reported [12–14],
isolated tissues challenged with LPS is a validated ex vivo experimental model to evaluate the
modulatory effects of herbal extracts and drugs on inflammatory pathways and oxidative stress.
The beneficial effects of plant polyphenols in humans have been confirmed by a large body of
evidence [15–18]. A number of studies confirmed the antioxidant, anti-atherogenic, and protective
effects of olive polyphenols, such as OLE and HT, against coronary artery disease [19–23]. In particular,
HT, deacetoxy oleuropein aglycon, and oleuropein aglycon were classified as the strongest antioxidants
in virgin olive oils [24]. Oxidative stress is defined as an imbalance in the pro-oxidant/antioxidant
homeostasis, where increased production of reactive oxygen/nitrogen species (ROS/RNS) and free
radicals can induce peroxidation reactions on biomolecules including proteins, lipids, and nucleic
acids [25,26]. Oxidative damage is thought to play a key role in the pathogenesis of various
chronic diseases, including cancer, atherosclerosis, cardiovascular diseases, chronic inflammation,
and diabetes [27–29]. 8-Iso-PGF2α , an isomer of prostaglandins produced by free radical-catalyzed
peroxidation of membrane arachidonic acid, is a stable marker of lipid peroxidation and oxidative
stress [30]. We found that MOMAST(®) HP30 (110 and 220 μg/mL) was able to decrease 8-iso-PGF2α
levels on rat prefrontal cortex, colon, liver, and heart tissues, challenged with LPS inflammatory
stimulus (Figures 1–4).
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Figure 1. Effects of MOMAST(® ) HY100 (10, 50, and 100 μg/mL) and MOMAST(® ) HP30 (22, 110,
and 220 μg/mL) on (A) PGE2 levels (pg/mg wet tissue), (B) 8-iso-prostaglandin F2α (8-iso-PGF2α ) levels,
and (C) lactate dehydrogenase (LDH) activity (nmol/L) in rat prefrontal cortex specimens. Data were
reported as means ± SEM. ANOVA, p < 0.01; post-hoc test, * p < 0.05, ** p < 0.01, *** p < 0.001 vs.
lipopolysaccharide (LPS)-treated group.
and (C) lactate dehydrogenase (LDH) activity (nmol/L) in colon specimens. Data were reported as
means ± SEM. ANOVA, P < 0.01; post-hoc test, * p < 0.05, ** p < 0.01 vs. LPS-treated group.
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and (C) lactate dehydrogenase (LDH) activity (nmol/L) in rat liver specimens. Data were reported as
means ± SEM. ANOVA, p < 0.01; post-hoc test, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. LPS-treated group.
and (C) lactate dehydrogenase (LDH) activity (nmol/L) in rat heart specimens. Data were reported as
means ± SEM. ANOVA, p < 0.01; post-hoc test, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. LPS-treated group.
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Moreover, MOMAST(®) HY100 (10, 50, and 100 μg/mL) was effective in inhibiting LPS-induced
8-iso-PGF2α in rat liver specimens (Figure 3B). These effects could be related, at least in part, to the free
radical-reducing and -scavenging properties of HT [31,32], which is found in very high amounts in
MOMAST(®) HY100 and MOMAST(®) HP30 (at least 50% and 30% of the phenolic fraction, respectively).
HT was also shown to decrease low density lipoproteins oxidation [33], platelet aggregation [34],
and 5- and 12-lipoxygenase activity [35] in vitro. However, we cannot exclude that our findings could
also be related to other phenolic compounds which are present, even if in low content, in both liquid
complexes. In this context, the antioxidant activity of OE has been widely confirmed both in vitro
and in vivo [36]. We also investigated the activity of MOMAST(®) HY100 and MOMAST(®) HP30
on LDH level in inflamed tissues. LDH is a cytosolic enzyme, which can be considered a marker of
tissue destruction [37,38]. Additionally, decreased LDH activity after treatment with herbal extracts
has been related to protective effects in chronic inflammatory disorders such as inflammatory bowel
disease (IBD) [39]. Following MOMAST(®) HY100 (10, 50, and 100 μg/mL) treatment, we found
a significant inhibition of LPS-induced LDH level in all tested tissues (Figures 1–4). MOMAST(®)
HP30 (22, 110, and 220 μg/mL) was also able to decrease LDH level induced by LPS in heart specimens
(Figure 7C). Actually, the reduction of LDH level could be related to the presence of HT [40] in both
extracts, and further supports the protective effects induced by MOMAST(®) HY100 and MOMAST(®)
HP30. Finally, we evaluated the modulatory effects of MOMAST(®) HY100 and MOMAST(®) HP30
on pro-inflammatory markers, including PGE2 , COX-2, TNFα, and iNOS. LPS was found to induce
macrophage production of inflammatory cytokines such as TNFα, interleukin-1β (IL-1β), and IL-6,
along with inflammatory mediators including nitric oxide (NO) and PGE2 [41,42]. COX-2, an inducible
enzyme stimulated by mitogenic and inflammatory stimuli, including LPS and cytokines, is known
to be mainly involved in the synthesis of pro-inflammatory PGE2 in both neoplastic and inflamed
tissues [43]. Similarly, iNOS, whose expression is induced by exposure to a number of stimuli, including
LPS and TNFα, is involved in the generation of large amounts of NO, which plays a pivotal role in
acute and chronic inflammation [44–46]. Following LPS inflammatory stimulus, we observed that
MOMAST(®) HP30 was able to reduce COX-2, TNFα, and iNOS mRNA levels in prefrontal cortex,
colon, and liver specimens (Figures 5–7).
and 220 μg/mL) on (A) cyclooxygenase (COX)-2, (B) tumor necrosis factor α (TNFα), and (C) inducible
nitric oxide synthase (iNOS) in rat prefrontal cortex specimens. Data were reported as means ± SEM.
ANOVA, p < 0.01; post-hoc test, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. LPS-treated group.
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nitric oxide synthase (iNOS) in rat colon specimens. Data were reported as means ± SEM. ANOVA,
p < 0.01; post-hoc test, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. LPS-treated group.
nitric oxide synthase (iNOS) in rat liver specimens. Data were reported as means ± SEM. ANOVA,
p < 0.01; post-hoc test, * p < 0.05, *** p < 0.001 vs. LPS-treated group.
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On the other hand, COX-2, TNFα, and iNOS mRNA levels were decreased by MOMAST(®)
HY100 in heart tissue specimens (Figure 8). iNOS mRNA levels were also decreased by MOMAST(®)
HY100 in prefrontal cortex specimens (Figure 5C) and by MOMAST(®) HP30 in heart tissue specimens
(Figure 8C).
and 220 μg/mL) on (A) cyclooxygenase (COX)-2, (B) tumor necrosis factor α (TNFα), (C) and inducible
nitric oxide synthase (iNOS) in rat heart specimens. Data were reported as means ± SEM. ANOVA,
p < 0.01; post-hoc test, ** p < 0.01, *** p < 0.001 vs. LPS-treated group.
As regards PGE2 , we found that while MOMAST(®) HY100 was effective in reducing LPS-induced
PGE2 levels in all tested tissues (Figures 1–4), MOMAST(®) HP30 was able to decrease PGE2 levels only
in liver tissue (Figure 5A). The inhibitory effects induced by MOMAST(®) HY100 and MOMAST(®)
HP30 on PGE2 levels, along with COX-2, TNFα, and iNOS mRNAs, support the protective effects of
both polyphenolic liquid complexes in prefrontal cortex, colon, liver, and heart specimens. Accordingly,
HT was shown to exert anti-inflammatory effects in LPS-stimulated RAW264.7 mouse macrophages by
suppressing nuclear factor-kB (NF-κB) signaling and downregulating gene expression of iNOS, COX-2,
TNFα, and IL-1β, and production of NO and PGE2 [47]. We hypothesize that the protective effects
induced by MOMAST(®) HY100 and MOMAST(®) HP30 could also be related to the presence of other
phenolic compounds, including tyrosol and OE. In this context, tyrosol was found to significantly
inhibit COX-2 gene and protein expression, as well as PGE2 secretion in human glioblastoma cells [48].
Moreover, it has been found that OE significantly downregulated NO, COX-2, iNOS, and TNF-α
in RAW264.7 macrophages following LPS treatment [49]. However, our findings indicate that
MOMAST(® ) HY100 and MOMAST(® ) HP30 could display different effects in tissues. On one hand,
Takeda et al. [50] reported that HT was able to suppress iNOS expression and NO production without
any effect on NF-kB, COX-2, and TNFα expression in mouse peritoneal macrophages challenged with
LPS; on the other hand, Maiuri et al. [51] and Zhang et al. [52] showed that HT was able to inhibit
LPS-stimulated NFκB activation as well as COX-2 gene expression, in J774 murine macrophages and
human monocytic THP-1 cells, respectively.
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In conclusion, both MOMAST(®) HY100 and MOMAST(®) HP30 exhibited protective effects as
indicated by the blunting effect on the tested pro-inflammatory mediators. On the basis of these results,
HT seems to be the main extract component involved in the pharmacological effects. Nevertheless,
considering the inherent limitations of the ex vivo experimental model, further investigations including
oxidative stress and inflammation biomarkers in in vivo studies are needed for a more accurate
evaluation of MOMAST(®) HY100 and MOMAST(®) HP30 efficacy.
3.1. In Vitro Studies

Rat Hypo-E22 cells (Cedarlane Cellution Biosystem) and mouse myoblast C2C12 cell lines (ATCC®
CRL-1772™) were cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% (v/v)
heat-inactivated fetal bovine serum and 1.2% (v/v) penicillin G/streptomycin in 75 cm2 tissue culture flask
(n = 5 individual culture flasks for each condition). The cultured cells were maintained in a humidified
incubator with 5% CO2 at 37 ◦ C. For cell differentiation, Hypo-E22 and C2C12 cell suspensions at
a density of 1 × 106 cells/mL were treated with various concentrations (10, 50, and 100 ng/mL) of
phorbol myristate acetate (PMA, Fluka) for 24 h or 48 h (induction phase). Thereafter, the PMA-treated
cells were washed twice with ice-cold pH 7.4 phosphate buffer solution (PBS) to remove PMA and
non-adherent cells, whereas the adherent cells were further maintained for 48 h (recovery phase).
The morphology of the cells was examined under an inverted phase-contrast microscope. To assess the
basal cytotoxicity of MOMAST(®) HY100 and MOMAST(®) HP30, a viability test was performed on
96-microwell plates, using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test.
Cells were incubated with MOMAST(® ) HY100 (10, 50, and 100 μg/mL) and MOMAST(® ) HP30 (22,
110, and 220 μg/mL), corresponding to HT (5, 25, and 50 μg/mL, respectively), for 24 h. Ten microliters
of MTT (5 mg/mL) were added to each well and incubated for 3 h. The formazan dye formed was
extracted with dimethyl sulfoxide and the absorbance was recorded as previously described [12,52].
The effects on cell viability were evaluated in comparison to the untreated control group.
3.2. Ex Vivo Studies

Male adult Sprague-Dawley rats (200–250 g) were housed in Plexiglass cages
(40 cm × 25 cm × 15 cm), two rats per cage, in climatized colony rooms (22 ± 1 ◦ C; 60% humidity),
on a 12 h/12 h light/dark cycle (light phase: 07:00–19:00 h), with free access to tap water and food,
24 h/day throughout the study, with no fasting periods. Rats were fed a standard laboratory diet (3.5%
fat, 63% carbohydrate, 14% protein, 19.5% other components without caloric value; 3.20 kcal/g).
Housing conditions and experimentation procedures were strictly in accordance with the European
Union ethical regulations on the care of animals for scientific research.
According to the recognized ethical principles of “Replacement, Refinement, and Reduction of
Animals in Research”, colon, liver, heart, and prefrontal cortex specimens were obtained as residual
material from vehicle-treated rats randomized in our previous experiments approved by the Local
Ethical Committee of University “G. d’Annunzio” and the Italian Health Ministry (Italian Health
Ministry authorization N. 880, delivered on 24th August 2015). Rats were sacrificed by CO2 inhalation
(100% CO2 at a flow rate of 20% of the chamber volume per min) and colon, liver, heart, and prefrontal
cortex specimens were immediately collected and maintained in a humidified incubator with 5% CO2
at 37 ◦ C for 4 h, in DMEM buffer with added bacterial LPS (10 μg/mL) (incubation period).
During the incubation period, tissues were treated with scalar concentrations of MOMAST(® )
HY100 (10, 50, and 100 μg/mL) and MOMAST(® ) HP30 (22, 110, and 220 μg/mL). Tissue supernatants
were collected, and the PGE2 and 8-iso-PGF2α levels (ng/mg wet tissue) were measured by
radioimmunoassay (RIA), as previously reported [13,53]. Briefly, specific anti-8-iso-PGF2α and
anti-PGE2 were developed in the rabbit; the cross-reactivity against other prostanoids is <0.3%.
One hundred microliters of prostaglandin standard or sample were incubated overnight at 4 ◦ C with
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the 3H-prostaglandin (3000 cpm/tube; NEN) and antibody (final dilution: 1:120,000; kindly provided
by Prof. G. Ciabattoni), in a volume of 1.5 mL of 0.025 M phosphate buffer. Free and antibody-bound
prostaglandins were separated by the addition of 100 μL 5% bovine serum albumin and 100 μL 3%
charcoal suspension, followed by centrifuging for 10 min at 4,000× g at 5 ◦ C and decanting off the
supernatants into scintillation fluid (Ultima Gold™, Perkin Elmer, Waltham, MA, USA) for β emission
counting. The detection limit of the assay method was 0.6 pg/mL. Additionally, tissue supernatants
were assayed for lactate dehydrogenase (LDH) activity [54]. LDH activity was measured by evaluating
the consumption of nicotinamide adenine dinucleotide dehydrogenase (NADH) in 20 mM HEPES-K+
(pH 7.2), 0.05% bovine serum albumin, 20 μM NADH, and 2mM pyruvate using a microplate reader
(excitation 340 nm, emission 460 nm) according to manufacturer s protocol (Sigma-Aldrich, St. Louis,
MO). LDH activity was measured by evaluating the consumption of NADH in 20 mM HEPES-K+
(pH 7.2), 0.05% bovine serum albumin, 20 μM NADH and 2 mM pyruvate using a microplate reader
(excitation 340 nm, emission 460 nm) according to manufacturer s protocol. In addition, individual
prefrontal cortex, colon, liver, and heart specimens were quickly dissected to evaluate cyclooxygenase
(COX)-2, tumor necrosis factor α (TNFα), and inducible nitric oxide synthase (iNOS) gene expression,
as previously reported [55,56]. Tissue specimens were dissected and stored in RNAlater solution
(Life Technologies, Carlsbad, CA, USA) at −20 ◦ C until further processed. Total RNA was extracted
from the tissues using TRI Reagent (Sigma-Aldrich, St. Louis, MO, USA) according to manufacturer’s
protocol. One microgram of total RNA extracted from each sample in a 20-μL reaction volume
was reverse transcribed using a high capacity cDNA reverse transcription kit (Life Technologies,
Carlsbad, CA, USA). Reactions were incubated in a 2720 thermal cycler (Life Technologies, Carlsbad,
CA, USA) initially at 25 ◦ C for 10 min, then at 37 ◦ C for 120 min, and finally at 85 ◦ C for 5 s.
Gene expression was determined by quantitative real-time PCR using TaqMan probe-based chemistry
(Life Technologies, Carlsbad, CA, USA). Reactions were performed in MicroAmp Fast Optic 96-well
Reaction Plates (Life Technologies, Carlsbad, CA, USA) on an ABI PRISM 7900 HT fast real-time PCR
system (Life Technologies, Carlsbad, CA, USA). PCR primers and TaqMan probes were obtained
from Life Technologies (Assays-on-Demand Gene Expression Products, Rn01483828_m1 for COX-2
gene, Rn01525859_g1 for TNFα, Rn00561646_m1 for iNOS. β-actin (Life Technologies, Carlsbad, CA,
USA, Part No. 4352340E) was used as the housekeeping gene. The real-time PCR was carried out
in triplicate. Data were elaborated with the sequence detection system (SDS) software version 2.3
(Applied Biosystems, Foster City, CA, USA). The comparative 2−ΔΔCt method was used to quantify the
relative abundance of mRNA and then determine the relative changes in individual gene expression
(relative quantification) [57].

Statistical analysis was performed using GraphPad Prism version 5.01 for Windows (GraphPad
Software, San Diego, CA, USA). Means ± S.E.M. were determined for each experimental group and
analyzed by one-way analysis of variance (ANOVA), followed by Newman–Keuls comparison multiple
test. As for gene expression analysis, 1.00 (calibrator sample) was considered the theoretical mean for
the comparison [57]. Statistical significance was set at p < 0.05. The number of animals randomized
for each experimental group was calculated on the basis of the “Resource Equation” N = (E + T)/T
(10 ≤ E ≤ 20) [58–60], according to the guidelines suggested by the National Centre for the Replacement,
Refinement, and Reduction of Animals in Research (NC3RS) and reported on the following web site:
https://www.nc3rs.org.uk/experimental-designstatistics.
Supplementary Materials: The following are available on line: Supplementary Figure S1: Effects of MOMAST(®)
HY100 (10, 50, and 100 μg/mL) and MOMAST(® ) HP30 (22, 110, and 220 μg/mL) on HypoE22 cell line viability.
Supplementary Figure S2: Effects of MOMAST(®) HY100 (10, 50, and 100 μg/mL) and MOMAST(® ) HP30 (22, 110,
and 220 μg/mL) on C2C12 cell line viability.
Author Contributions: Conceptualization, L.R. and L.B.; methodology, A.C., G.O., C.F., S.L.; software, G.O., L.M.;
validation, L.M., C.F., and S.L.; formal analysis, L.D.C.M., C.G.; investigation, A.C., G.O., L.D.C.M., C.G.; resources,
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A.C., L.D.C.M., C.G.; data curation, L.R., L.B., S.L.; writing—original draft preparation, L.R., S.L.; writing—review
and editing, L.R., A.C., L.B., S.L.; visualization, C.F.; supervision, L.B.; project administration, L.R., A.C., L.B., S.L.;
funding acquisition, L.B., S.L. and L.R.
Funding: This research was funded by Bioenutra S.r.l. (Ginosa, TA, Italy) and by grants from the Italian Ministry
of University (FFABR 2017 to S. Leone).
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molecules
Article
Inhibitory Effects of Aucklandia lappa Decne.
Extract on Inflammatory and Oxidative Responses
in LPS-Treated Macrophages
Jae Sung Lim 1,2,† , Sung Ho Lee 3,† , Sang Rok Lee 4 , Hyung-Ju Lim 2,5 , Yoon-Seok Roh 6 ,
Eun Jeong Won 7 , Namki Cho 8 , Changju Chun 8, * and Young-Chang Cho 8, *
1 Department of Biochemistry, Chonnam National University Medical School, Hwasun,
Jeonnam-do 58128, Korea; dr.jslim7542@gmail.com
2 Combinatorial Tumor Immunotherapy Medical Research Center, Chonnam National University Medical
School, Hwasun, Jeonnam-do 58128, Korea; akira0128@naver.com
3 Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA;
puzim23@gmail.com
4 ROK-Biotech, Jeollanamdo Biopharmaceutical Research Center, Hwasun, Jeollanam-do 58141, Korea;
rok94@hanmail.net
5 Department of Microbiology, Chonnam National University Medical School, Hwasun,
Jeonnam-do 58128, Korea
6 College of Pharmacy and Medical Research Center, Chungbuk National University, Cheongju 28160, Korea;
ysroh@cbnu.ac.kr
7 Department of Parasitology and Tropical Medicine, Chonnam National University Medical School, Hwasun,
Jeonnam-do 58128, Korea; Parasite.woni@jnu.ac.kr
8 College of Pharmacy, Chonnam National University, Gwangju 61186, Korea; cnamki@jnu.ac.kr
* Correspondence: cchun1130@jnu.ac.kr (C.C.); yccho@jnu.ac.kr (Y.-C.C.); Tel.: +82-62-530-2944 (C.C.);
+82-62-530-2925 (Y.-C.C.)
† These authors contributed equally to this paper.

Abstract: Aucklandia lappa Decne., known as “Mok-hyang” in Korea, has been used for the alleviation
of abdominal pain, vomiting, diarrhea, and stress gastric ulcers in traditional oriental medicine.
We investigated the anti-inflammatory and antioxidative effects of the ethanol extract of Aucklandia
lappa Decne. (ALDE) in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. ALDE significantly
inhibited the LPS-induced nitric oxide (NO) production and reduced inducible nitric oxide synthase
(iNOS) expression in RAW 264.7 cells. The production of other proinflammatory mediators, including
COX-2, interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α, was reduced by ALDE in
LPS-stimulated RAW 264.7 cells. The mechanism underlying the anti-inflammatory effects of ALDE
was elucidated to be the suppression of LPS-induced nuclear translocation of p65, followed by the
degradation of IκB and the inhibition of the phosphorylation of mitogen-activated protein kinases
(MAPK). In addition, ALDE showed enhanced radical scavenging activity. The antioxidant effect
of ALDE was caused by the enhanced expression of heme oxygenase (HO-1) via stabilization of
the expression of the nuclear transcription factor E2-related factor 2 (Nrf2) pathway. Collectively,
these results indicated that ALDE not only exerts anti-inflammatory effects via the suppression of
the NF-κB and MAPK pathways but also has an antioxidative effect through the activation of the
Nrf2/HO-1 pathway.
Keywords: Aucklandia lappa Decne. extract; lipopolysaccharide; nitric oxide; inducible

nitric oxide; cyclooxygenase-2; nuclear factor-κB; mitogen-activated protein kinase; heme
oxygenase-1; macrophage

Molecules 2020, 25, 1336
1. Introduction
Inflammation is a central feature of various pathological conditions in the host defense against
pathogens and in response to tissue injury. Macrophages are activated in response to various stimuli,
such as LPS, and induce inflammation by producing inflammatory mediators, including nitric oxide
(NO), prostaglandins (PGs), and proinflammatory cytokines, such as interleukin (IL)-1β, IL-6, and tumor
necrosis factor (TNF)-α [1]. Although inflammation is important for the host defense against external
stimuli, excess inflammation leads to severe immune disorders, such as septic shock, rheumatoid
arthritis (RA), systemic lupus erythematosus (SLE), and inflammatory bowel disease (IBD) [2,3]. Thus,
an agent that is able to alleviate the excessive inflammatory response may be a suitable candidate for
the treatment of inflammatory disorders. Although a variety of anti-inflammatory drugs have been
developed, including steroidal drugs and nonsteroidal anti-inflammatory drugs (NSAIDs), owing to
the severe adverse effects of these drugs, natural products and their constituent compounds have been
investigated for the development of new anti-inflammatory drugs.
Aucklandia lappa Decne., referred to as “Mok-hyang” in the 11th edition of the Korean
Pharmacopoeia (KP11), is the root of Saussurea (Aucklandia) lappa Clarke (Chrysanthemum,
Compositae). It contains approximately 1–2.5% of refined oils and has an abundance of sesquiterpenoid
compounds (such as costunolide), which have many pharmacological effects, such as antibacterial [4]
and anti-inflammatory [5] activity and an anti-inhibitory effect on vascular production [6]. Traditionally,
“Mok-hyang” has been used for the treatment of vomiting, gastric pain, abdominal pain, anorexia,
distension, and nausea [7]. Previously, it was reported that Aucklandia lappa Decne. has anti-ulcer [8],
antiviral [9], and anticancer [10] effects. In addition, it has been reported that Aucklandia lappa Decne.
extract (ALDE) inhibited inflammatory chemokine production in HaCaT cells [11] and exhibited
anti-inflammatory effects in RAW 264.7 cells [12]. Thus, although the anti-inflammatory activity of
ALDE has been reported, the mechanisms underlying these anti-inflammatory effects are not well
elucidated. Herein, we investigated the anti-inflammatory and antioxidative effects of ALDE in
LPS-stimulated macrophages and evaluated the associated molecular mechanism in vitro.
2.1. Extraction of ALDE

Aucklandia lappa Decne. was purchased from the Jeonnam Herb Medicine and Agriculture
Cooperative (Hwasun, South Korea). Briefly, air-dried powdered (<0.2 mm) Aucklandia lappa Decne.
(100 g) was extracted with 70% ethanol at approximately 70 ◦ C for 9 h. The resultant ethanolic solution
was filtered, evaporated, and freeze-dried to generate ALDE.
2.2. HPLC Chromatographic Analysis

Chromatographic analysis was performed on a reverse-phase Shimadzu HPLC system (Shimadzu
Corp., Kyoto, Japan) with a Shimadzu LC-20AR solvent pump, coupled to a SPD-20A UV/VIS detector.
Separation was performed on a Phenomenex C18 reverse-phase column (4.6 × 150 mm, 5 μm) using
a gradient solvent system comprising acetonitrile (A) and water (B), with a composition by volume
of 10% A at 0 min and 50% A at 40 min. The flow rate was 2 mL/min; the reaction was monitored
spectrophotometrically at 254 nm.
2.3. Cell Culture

RAW 264.7 cells (ATCC, Manassas, VA, USA), a mouse monocytic cell line, were maintained
in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (both from GE
Healthcare Bio-Sciences, Pittsburgh, PA, USA), 50 U/mL penicillin, and 50 μg/mL streptomycin (Gibco;
Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37 ◦ C in humidified air containing 5% CO2 .
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Molecules 2020, 25, 1336
2.4. Cell Viability Assay

RAW 264.7 cells (4 × 104 /well) were plated in 96-well plates. The cells were treated with various
concentrations (1, 3, 5, 7, 9, 11, and 13 μg/mL) of ALDE for 24 h. Following treatment, cell viability
was measured using an EZ-Cytox Cell Viability Assay kit (Daeil Lab Services Co., Ltd., Seoul, Korea).
Briefly, the cells were incubated with the EZ-Cytox solution (containing a water-soluble tetrazolium
salt) for 2 h at 37 ◦ C. The absorbance of the supernatant at 450 nm was measured using a Synergy H1
Microplate Reader (BioTek Instruments, Inc., Winooski, VT, USA).
2.5. Measurement of NO Production

RAW 264.7 cells (4.0 × 104 cells/well) were plated in 96-well plates. The cells were pretreated
with various concentrations of ALDE (1, 2.5, 5, and 10 μg/mL) for 2 h and subsequently stimulated
by LPS (0.5 μg/mL) for 24 h. The cell supernatants (100 μL) were transferred to new 96-well plates,
and 100 μL Griess reagent (1% sulfanilamide, 0.1% N-1-naphthylethylenediamine dihydrochloride,
and 2.5% phosphoric acid) was added. NaNO2 solutions (2.5, 5, 10, 25, 50, and 100 M) were used to
generate a standard curve to calculate the concentration of NO in the supernatant. The absorbance at
540 nm was measured using a Synergy H1 Microplate reader (BioTek Instruments, Winooski, VT, USA).
2.6. RNA Preparation and cDNA Synthesis

RAW 264.7 cells (8.0 × 105 cells/well) were seeded in 12-well plates. The cells were pretreated
with various concentrations of ALDE (1, 2.5, 5, and 10 μg/mL) for 2 h and then stimulated with
LPS (0.5 μg/mL) for 3 h. Total RNA was extracted using Accuzol (Bioneer Corporation, Daejeon,
Korea) and synthesized into cDNA using a TOPscript cDNA synthesis kit in accordance with the
manufacturer’s instructions.
2.7. Semiquantitative Reverse Transcription (RT)-PCR

The mixture for PCR was subjected to the following thermal profile: 17–25 cycles at 94 ◦ C for
30 s, 60 ◦ C for 30 s, and 72 ◦ C for 30 s using a Bioer thermal cycler (Bioer Technology Co., Hangzhou,
China). Following amplification, the PCR products (10 μL) were separated on a 1.5% (w/v) agarose gel
and stained with ethidium bromide. The following primers were used: Mouse iNOS (sense, 5 -GCA
TGGAACAGTATAAGGCAAACA-3 ; antisense, 5 -GTTTCTGGTCGATGTCATGAGCAA-3 ),
COX-2 (sense, 5 -GCATGGAACAGTATAAGGCAAACA-3 ; antisense, 5 -GTTTCTGGT
CGATGTCATGAGCAA-3 ), TNF-α (sense, 5 -GTGCCAGCCGATGGGTTGTACC-3 ;
antisense, 5- AGGCCCACAGTCCAGGTCACTG-3 ), IL-6 (sense, 5 -TCTTGGGACTGATG

CTGGTGAC-3 ; antisense, 5 -CATAACGCACTAGGTTTGCCGA-3 ), IL-1β (sense, 5 -AGC

TGTGGCAGCTACCTGTG-3 ; antisense, 5 -GCTCTGCTTGTGAGGTGCTG-3 ), and GAPDH (sense,
5 -GTCTTCACCACCATGGAGAAGG-3 ; antisense, 5 -CCTGCTTCACCA CCTTCTTGCC-3 ).
2.8. Western Blotting

The whole-cell lysate was prepared by incubating the cells in a RIPA buffer (50 mM Tris-HCl
pH 8.0, 150 mM NaCl, 0.1% SDS, 0.5% deoxycholate, 1% NP-40, and 1 mM EDTA) with protease
inhibitors (XXX) for 30 min at 4 ◦ C followed by centrifugation (13,200 rpm for 15 min). The supernatant
was denatured in 5× SDS sample buffer (200 mM Tris-HCl pH 6.8, 40% glycerol, 8% SDS, 200 mM
dithiothreitol, and 0.08% bromophenol blue) at 95 ◦ C for 5 min, separated by SDS-PAGE, and then
transferred to nitrocellulose membranes. To block nonspecific binding, we incubated the membranes
in 5% nonfat dry milk in Tris-buffered saline and Tween-20 (25 mM Tris-HCl pH 8.0, 125 mM NaCl, and
0.5% Tween-20) for 1 h at RT. The membranes were incubated with primary antibodies at 4 ◦ C overnight
and then incubated with horseradish peroxidase-conjugated (HRP)-conjugated secondary antibodies
for 1 h at RT. Pierce ECL Western blotting substrate for enhanced chemiluminescence (Thermo Fisher
Scientific, Inc.) was used to detect the HRP-conjugated secondary antibodies. Protein expression was
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Molecules 2020, 25, 1336
analyzed and quantified using LabWorks software version 4.6 (UVP, LLC; Analytik Jena AG, Upland,
CA, USA).
2.9. ELISA
RAW 264.7 cells (4.0 × 104 cells/well) were plated in 96-well plates. The cells were pretreated
with various concentrations of ALDE (1, 2.5, 5, and 10 μg/mL) for 2 h and then stimulated with LPS
(0.5 μg/mL) for 24 h. The expression of the indicated cytokines in the cell supernatant was measured
using an ELISA kit in accordance with the manufacturer’s instructions. Briefly, the culture plates were
incubated overnight with a coating solution at 4 ◦ C, washed three times with 1× PBS/0.05% Tween-20
(PBST), and then incubated with 1× assay diluent (from the ELISA kit) for 1 h at RT. The supernatants
and standard solutions were incubated for 2 h at RT and then washed three times. Next, the plate was
incubated with Ab Detection solution (also from the ELISA kit) for 1 h at RT and then washed three
times. Subsequently, the plate was incubated with a horseradish peroxidase-streptavidin solution
for 30 min at RT and then washed five times. Finally, the plate was incubated with a solution of
3,3 ,5,5 -tetramethylbenzidine for 10 min in the dark; then, 1 N H3 PO4 was added to stop the reaction.
The absorbance at 450 nm was measured spectrophotometrically using a Synergy H1 Microplate reader.
2.10. Subcellular Fractionation

Subcellular fractionation was performed as described previously [13]. Briefly, the cells were
washed twice with ice-cold PBS and lysed with 200 μL of cytoplasmic lysis buffer (10 mM HEPES,
60 mM KCl, 1 mM EDTA, 1 mM DTT, and 1 mM PMSF) on ice for 15 min; subsequently, 10 μL of 0.075%
(v/v) IGEPAL CA-630 (Sigma-Aldrich, St. Louis, MO, USA) was added. After brief centrifugation
(10 s), the supernatants were collected for the cytoplasmic fraction. Next, the pellet was resuspended in
25 μL of a nuclear extraction buffer (20 mM Tris Cl, 420 mM NaCl, 1.5 mM MgCl2 , 0.2 mM EDTA, 1 mM
PMSF, and 25% (v/v) glycerol) on ice for 30 min and vortex mixed every 10 min. After centrifugation
for 30 min at 4 ◦ C, the supernatant was collected to obtain the nuclear fraction. Western blotting was
performed using anti-α-tubulin (cytoplasm) and anti-lamin B1 (nucleus) antibodies to confirm the
cytoplasmic and nuclear extracts, respectively.
2.11. DPPH Free Radical Scavenge Activity

ALDE ethanolic solution was mixed with the same volume of 0.4 mM DPPH ethanolic solution.
The mixture was allowed to react at RT in the dark for 10 min. The absorbance at 517 nm was
measured using a Synergy H1 Microplate reader. The free radical scavenging activity was calculated
as a percentage using the following equation [14].
DPPH free radical scavenging activity (%) = [1 − (Asample /Ablank )] × 100.

The data are presented as the mean ± standard error of the mean. Multiple experimental groups
were compared by one-way analysis of variance followed by Dunnett’s post-hoc test calculated using
GraphPad Prism (version 3.0; GraphPad Software, Inc., La Jolla, CA, USA); p-values < 0.05 were
considered statistically significant.
3. Results
3.1. HPLC and Costunolide-Related Results

Before the investigation of the effects of ALDE on inflammation and oxidative stress, an evaluation
of the major components that exhibit anti-inflammatory and antioxidative effects was required.
Costunolide, a component of ALDE, has been known to inhibit the production of inflammatory
mediators and enhance HO-1 expression [15]. HPLC analysis was performed to show that ALDE and
costunolide exhibited the same retention time. As shown in Figure 1a,b, HPLC analysis of costunolide
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showed a single peak at 40.507 min. One of the major peaks of the ALDE HPLC data (40.587 and
41.626 min) has the same retention time as costunolide. As previously reported, costunolide inhibited
LPS-induced NO production in RAW 264.7 cells and exhibited significant radical scavenging activity
compared with BHA, a positive control (Figure 1c,d). These results indicated that costunolide was a
major component of ALDE and led us to study the anti-inflammatory effects and underlying regulatory
mechanism of action of ALDE in murine macrophages.
Figure 1. HPLC analysis of the ethanol extract of Aucklandia lappa Decne. (ALDE) and costunolide. The
phytochemical characteristics of (a) ALDE and its major component, (b) costunolide, were analyzed
using HPLC. (c,d) NO inhibitory effect of costunolide. Data represent the mean ± SEM of three
independent experiments. # p < 0.05 vs. LPS-untreated control group; ∗ p < 0.05 vs. LPS-treated group.
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(d) DPPH radical scavenging activity of costunolide. Data represent the mean ± SEM of three
independent experiments. ∗ p < 0.05 vs. untreated group. ALDE: Ethanol extract of Aucklandia lappa
Decne.; LPS: Lipopolysaccharide; NO: Nitric oxide.
3.2. ALDE Suppressed the Release of NO in LPS-Stimulated RAW 264.7 Cells

To investigate the effect of ALDE on the viability of RAW 264.7 cells, we treated the cells with
the indicated concentrations of ALDE for 24 h and then quantified the metabolic conversion of a
tetrazolium salt to a formazan dye to determine the percentage of viable cells. ALDE exerted no
significant cytotoxicity in RAW 264.7 cells at concentrations below 13 μg/mL (Figure 2a). Therefore,
subsequent experiments were performed at ALDE concentrations of 1, 2.5, 5, and 10 μg/mL, which
were known to not exert cytotoxic effects. To investigate the anti-inflammatory effects of ALDE,
we examined the effect of ALDE on the production of NO, a well-known proinflammatory mediator,
in LPS-stimulated RAW 264.7 cells. The nitrite level in the culture medium of the RAW 264.7 cells was
significantly increased upon LPS treatment. However, in the cells pretreated with ALDE, a considerable
dose-dependent suppression of LPS-induced NO production was observed (Figure 2b). These results
suggested that ALDE markedly reduced the NO production in LPS-stimulated RAW 264.7 cells.
Figure 2. Effects of ALDE on cell viability and NO production in RAW 264.7 cells. (a) RAW 264.7 cells
were treated with various concentrations of ALDE for 24 h. Subsequently, cell viability was measured
using the EZ-Cytox reagent and compared with that in the untreated group. (b) RAW 264.7 cells were
treated with LPS (0.5 μg/mL) in the presence of ALDE (1, 2.5, 5, and 10 μg/mL) for 24 h. Subsequently,
NO production in the culture supernatant was measured using a Griess assay. NO secretion was
calculated using a standard curve of concentrations of nitrite standard solution. The data presented
are the mean ± SEM of three independent experiments. Differences between groups were analyzed
using the Mann–Whitney U test. # p < 0.05 vs. LPS-untreated control groups; ∗ p < 0.05 vs. LPS-treated
groups. ALDE: Ethanol extract of Aucklandia lappa Decne.; LPS: Lipopolysaccharide; NO: Nitric oxide.
3.3. ALDE Inhibited the Expression of Proinflammatory Enzymes, iNOS and COX-2, in LPS-Stimulated RAW
264.7 Cells
The expression of proinflammatory enzymes, including COX-2 and iNOS, plays an important
role in the immune response from activated macrophages through the production of NO and PGE2,
respectively [16,17]. We investigated the effect of ALDE on the expression of iNOS and COX-2 in
LPS-stimulated RAW 264.7 cells. As shown in Figure 3, the expression of iNOS and COX-2 was
increased markedly in response to LPS treatment. When RAW 264.7 cells were treated with various
concentrations of ALDE, the LPS-induced expression of iNOS and COX-2 was significantly decreased
in a dose-dependent manner (Figure 3a,b). These results indicated that ALDE inhibited the production
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of proinflammatory mediators through the inhibition of the expression of their responsible enzymes,
iNOS and COX-2.
Figure 3. Effects of ALDE on the expression of iNOS and COX-2. RAW 264.7 cells were treated
simultaneously with LPS and ALDE (1, 2.5, 5, and 10 μg/mL). (a) Following stimulation for 6 h, total
RNA was extracted and reverse transcribed to cDNA. mRNA expression of iNOS and COX-2 was
analyzed by RT-PCR. (b) After stimulation for 24 h, the total protein was extracted. The protein
expression of iNOS and COX-2 was detected by Western blotting. The protein β-actin was used as a
loading control for both RT-PCR and Western blotting. The relative density of the mRNA or protein
expression was normalized to that of β-actin and is presented in quantitative graphs. The data presented
are the mean ± SEM of three independent experiments. Differences between groups were analyzed
using the Mann–Whitney U test. # p < 0.05, ## p < 0.01 vs. LPS-untreated control groups; ∗ p < 0.05,
∗∗ p < 0.01 vs. LPS-treated groups. COX-2: Cyclooxygenase-2; iNOS: Inducible nitric oxide synthase.
3.4. ALDE Inhibited the Production of Proinflammatory Cytokines in LPS-Stimulated Macrophages

To investigate whether ALDE affected the expression of proinflammatory cytokines, we performed
Western blotting analysis and RT-PCR. The expression of IL-6, IL-1β, and TNF-α was significantly
increased after treatment with LPS but markedly decreased in a dose-dependent manner after
pretreatment with ALDE (Figure 4a,b). To confirm the inhibitory effect of ALDE on the cytokine
production induced by LPS stimulation, we used ELISA. As shown in Figure 4c–e, LPS-stimulated RAW
264.7 cells treated with ALDE exhibited concentration-dependent inhibition of the proinflammatory
cytokines, such as TNF-α, IL-6, and IL-1β. These results suggested that ALDE exerted anti-inflammatory
effects through the inhibition of proinflammatory cytokines.
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Figure 4. Inhibitory effects of ALDE on the production of proinflammatory cytokines. RAW 264.7
cells were treated with LPS in the presence of ALDE (1, 2.5, 5, and 10 μg/mL). (a) After stimulation for
24 h, the total cellular proteins were extracted. The expression of interleukin (IL)-6, IL-1β, and tumor
necrosis factor (TNF)-α was detected by Western blotting. (b) After simulation for 6 h, total RNA was
extracted. The mRNA expression of IL-6, IL-1β, and TNF-α was analyzed by RT-PCR. β-Actin was
used as a loading control for both RT-PCR and Western blotting. The relative density of the mRNA or
protein expression was normalized to that of β-actin and is presented in quantitative graphs. (c–e) After
stimulation for 24 h, the culture supernatants were collected and analyzed for IL-6, IL-1β, and TNF-α
production by ELISA. The data presented are the mean ± SEM of three independent experiments.
Differences between groups were analyzed using the Mann–Whitney U test. # p < 0.05, ## p < 0.01,
### p < 0.001 vs. LPS-untreated control groups; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. LPS-treated
groups. IL: Interleukin; TNF: Tumor necrosis factor.
3.5. ALDE Suppressed Both NF-κB Activation and MAPK Phosphorylation in LPS-Stimulated Macrophages
The NF-κB and MAPK signaling pathways are the major regulators of the expression of
inflammatory mediators [18,19]. To elucidate the mechanisms underlying the anti-inflammatory effects
of ALDE, we examined the changes in NF-κB translocation into nucleus after treatment with ALDE.
As shown in Figure 5a, the LPS-induced degradation of IκB was significantly suppressed by ALDE
treatment in the cytosolic fraction. In contrast, the level of LPS-induced nuclear NF-κB/p65 protein,
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which is translocated into nucleus after IκB degradation, was decreased by ALDE treatment in RAW
264.7 cells (Figure 5a). Next, we investigated whether ALDE regulated the LPS-induced phosphorylation
of MAPKs. As shown in Figure 5b, LPS treatment significantly induced the phosphorylation of p38,
JNK, and ERK, although ALDE significantly suppressed the phosphorylation of these proteins in a
dose-dependent manner. These results suggested that the anti-inflammatory effects of ALDE were
mediated by the inhibition of the activation of both NF-κB and MAPK signaling.
Figure 5. Inhibitory effects of ALDE on the nuclear translocation of NF-κB and the MAPK signaling
pathway. RAW 264.7 cells were pretreated with ALDE (0, 2.5, and 10 μg/mL) for 2 h and then stimulated
with LPS (0.5 μg/mL) for 15 min. Cytosolic extracts and nuclear extracts were prepared. (a) The
expression of NF-κB/p65 and IκB was detected by Western blotting; α-tubulin was used as a cytosolic
loading control and Lamin B was used as a nuclear loading control. (b) The expression of the proteins
associated with the MAPK signaling pathway (p38, p44/42 ERK, and JNK) was detected by Western
blotting, with β-actin used as a loading control. The relative density of the protein expression was
normalized to each loading control and is presented in quantitative graphs. The data presented are the
mean ± SEM of three independent experiments. Differences between groups were analyzed using the
Mann–Whitney U test. # p < 0.05, ## p < 0.01 vs. LPS-untreated control groups; ∗ p < 0.05, ∗∗ p < 0.01
vs. LPS-treated groups. NF-κB: Nuclear-κB; IκB: Inhibitor of κB; MAPK: Mitogen-activated protein
kinase; p-: Phosphorylated; SAPK/JNK: Stress-associated protein kinase/c-Jun N-terminal kinase; ERK:
Extracellular signal-regulated kinase.
3.6. ALDE Increased the Expression of HO-1 and the Nuclear Translocation of Nrf2 in
LPS-Stimulated Macrophages
To investigate whether ALDE exhibited antioxidative effects, we assayed the radical scavenging
activity. As shown in Figure 6a, ALDE showed significant radical scavenging activity compared with
BHA, a positive control. As the antioxidative effects were mediated by antioxidative regulators, such
as HO-1, the profile of ALDE-mediated HO-1 expression was investigated in LPS-stimulated RAW
264.7 macrophages. Both mRNA expression and protein expression of HO-1 in LPS-stimulated RAW
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264.7 cells were significantly increased by ALDE treatment in a dose-dependent manner (Figure 6b,c).
As Nrf2 is a major regulator of the expression of HO-1 [20], we investigated whether ALDE enhanced
the stability and, subsequently, the expression of Nrf2. We found that the expression of Nrf2 was
increased by ALDE treatment (Figure 6d). These data suggested that ALDE exerts antioxidative effects
through the activation of the Nrf2/HO-1 pathway.
Figure 6. Antioxidative effects of ALDE. (a) DPPH free radical scavenging activity is represented as
the mean ± SEM. ∗ p < 0.01 relative to the MeOH-reacted group. BHA was used as a positive control.
(b–d) RAW 264.7 cells were treated with LPS in the presence of ALDE (1, 2.5, 5, and 10 μg/mL). (b) After
stimulation for 6 h, total RNA was extracted. HO-1 mRNA expression was analyzed by RT-PCR. (c,d)
After stimulation for 24 h, total protein was extracted. The protein expression of HO-1 (c) and Nrf2
(d) was detected by Western blotting. The relative density of the mRNA and protein expression was
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normalized to that of β-actin and is presented by quantitative graphs. The data presented are the
mean ± SEM of three independent experiments. Differences between groups were analyzed using the
Mann–Whitney U test. # p < 0.05 vs. LPS-untreated control groups; ∗ p < 0.05, ∗∗ p < 0.01 vs. LPS-treated
groups. Nrf2: Nuclear factor erythroid 2-related factor; HO-1: Heme oxygenase-1.
4. Discussion
Recently, natural products have been considered important sources of drugs targeting a variety of
diseases, such as cancer and inflammatory disorders [21–24]. In particular, many researchers have
reported that the extracts of natural products, such as fruits, vegetables, plants, and their formulations
have significant anti-inflammatory effects [25–30]. Similarly, as costunolide is a naturally occurring
sesquiterpene lactone that has been extensively studied for its anti-inflammatory activity and is one of
major components of ALDE, we investigated the anti-inflammatory effects of ALDE and the underlying
mechanism of action [15,31,32].
The expression of iNOS is stimulated by not only proinflammatory cytokines, such as TNF-α,
IL-1β, and IL-6 [33], but also bacterial products such as LPS [34]. Therefore, the inhibitory effects of
natural products on NO production that occurs through the inhibition of iNOS expression, suggest that
they may be potent drug candidates for the treatment of inflammatory diseases. TNF-α antagonists,
including anti-TNF receptor antibodies and anti-IL-6 receptor antibodies, are currently used to inhibit
the action of each proinflammatory cytokine for the treatment of RA and Crohn’s disease [35,36]. Thus,
agents that inhibit these proinflammatory cytokines have been suggested as therapeutic candidates
for the treatment of immune diseases. In this study, we examined the inhibitory effects of ALDE
on the production of various LPS-stimulated proinflammatory mediators in RAW 264.7 cells and
found that ALDE significantly inhibited the production of these mediators (Figure 4). These results
suggested that ALDE is a potent anti-inflammatory agent and exerts this action through the inhibition
of proinflammatory responses.
HO-1 expression is enhanced by various proinflammatory stimulators, such as NO, LPS, cytokines,
and other oxidants [17,20,37]. Previous studies have shown that the induction of HO-1 can represent
an efficient antioxidant system and a potential pharmacological target in a variety of oxidant- and
inflammatory-mediated diseases [38–40] and that this was involved in the inhibitory effects on
LPS-induced NO production [41]. In this study, we observed that LPS itself caused a slight increase in
HO-1 expression and that ALDE further enhanced HO-1 expression in LPS-stimulated RAW 264.7
macrophages. These results suggested that the increase in HO-1 expression induced by ALDE could
inhibit NO production in LPS-stimulated RAW 264.7 cells.
The multifunctional regulator nuclear factor erythroid 2-related factor (Nrf2) is considered a
cytoprotective factor that regulates the expression of genes coding for antioxidant, anti-inflammatory,
and detoxifying proteins [20]. The major roles of Nrf2 are mediated by Nrf2-dependent genes and their
encoded proteins, including HO-1, which have important roles in the removal of toxic heme, producing
biliverdin, iron ions, and carbon monoxide. HO-1 and its products exert beneficial effects by protecting
cells from oxidative injury, apoptosis, and inflammation [39]. Thus, the activation of the Nrf2 pathway
is a possible explanation for the increase in HO-1 expression. As the nuclear translocation of Nrf2
allows the activation of the transcription of the HO-1 gene [20], we examined whether ALDE induced
the nuclear translocation of Nrf2 in LPS-stimulated RAW 264.7 cells. We found that ALDE stabilized
the Nrf2 protein expression (Figure 6d). These results suggested that the increase in HO-1 expression
induced by ALDE may be mediated via the Nrf2 pathway.
In conclusion, we showed that ALDE significantly suppressed the production of NO and inhibited
the expression of iNOS, COX-2, and proinflammatory cytokines in LPS-stimulated murine macrophages.
The inhibitory effect was mediated by the inhibition of NF-κB translocation and MAPK phosphorylation.
Moreover, we found that ALDE induced the expression of HO-1 and increased the nuclear translocation
of Nrf2 in LPS-stimulated macrophages. Collectively, our results suggested that ALDE may exert
potent therapeutic effects in various inflammatory diseases.
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Author Contributions: Conceptualization, J.S.L., S.H.L., and Y.-C.C.; methodology, J.S.L. and H.-J.L.; validation,
N.C.; resources, S.R.L.; data curation, Y.-S.R. and E.J.W.; writing—original draft preparation, J.S.L. and S.H.L.;
writing—review and editing, C.C. and Y.-C.C.; visualization, Y.-S.R.; supervision, Y.-C.C.; project administration,
C.C. and Y.-C.C.; funding acquisition, E.J.W. and Y.-C.C. All authors have read and agreed to the published version
of the manuscript.
Funding: This study was financially supported by the Chonnam National University Hwasun Hospital
Institute for Biomedical Science (HCRI 19026) and National Research Foundation of Korea, funded by the
Ministry of Science and ICT (NRF-2019R1F1A1060688) and Ministry of Education (NRF- 2016R1A6A3A11931134;
NRF-2018R1D1A1B07051207).
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of metabolites of tryptophan via indoleamine 2,3-dioxygenase pathway, suppresses inducible nitric oxide
synthase expression by enhancing heme oxygenase-1 expression. Biochem. Biophys. Res. Commun. 2004, 320,
126
molecules
Article
Modulatory Effect of Guinep (Melicoccus bijugatus
Jacq) Fruit Pulp Extract on Isoproterenol-Induced
Myocardial Damage in Rats. Identification of Major
Metabolites Using High Resolution UHPLC
Q-Orbitrap Mass Spectrometry
Chukwuemeka R. Nwokocha 1, *, Isheba Warren 1 , Javier Palacios 2, *, Mario Simirgiotis 3 ,
Magdalene Nwokocha 4 , Sharon Harrison 4 , Rory Thompson 4 , Adrian Paredes 5 , Jorge Bórquez 6 ,
Astrid Lavado 7 and Fredi Cifuentes 7
1 Department of Basic Medical Sciences Physiology Section, Faculty of Medical Sciences, The University of the
West Indies, Mona, Kingston 7, KGN, Jamaica; Warren-Isheba.R@hotmail.com
2 Facultad Ciencias de la Salud, Instituto de EtnoFarmacología (IDE), Universidad Arturo Prat,
Iquique 1110939, Chile
3 Instituto de Farmacia, Facultad de Ciencias, Universidad Austral de Chile, Valdivia 5110566, Chile;
mario.simirgiotis@gmail.com
4 Department of Pathology, Faculty of Medical Sciences, University of the West Indies, Mona Campus,
Kingston 7, KGN, Jamaica; magdanwokocha@yahoo.com (M.N.); sharon.harrison@uwimona.edu.jm (S.H.);
rorykthompson@gmail.com (R.T.)
5 Laboratorio de Química Biológica, Instituto Antofagasta, Universidad de Antofagasta, Antofagasta 1270300,
Chile; adrian.paredes@uantof.cl
6 Departamento de Química, Facultad de Ciencias Básicas, Universidad de Antofagasta, Antofagasta 1270300,
Chile; jorge.borquez@uantof.cl
7 Laboratorio de Fisiología Experimental, Instituto Antofagasta, Universidad de Antofagasta,
Antofagasta 1270300, Chile; astrid.lavado@uantof.cl (A.L.); fredi.cifuentes@uantof.cl (F.C.)
* Correspondence: chukwuemeka.nwokocha@uwimona.edu.jm (C.R.N.); clpalaci@unap.cl (J.P.);
Tel.: +1-876-5895445 (C.R.N.); +56-57-2526910 (J.P.)

Received: 25 December 2018; Accepted: 8 January 2019; Published: 10 January 2019
Abstract: Guinep is traditionally used in the management of cardiovascular ailments. This study
aims to evaluate its medicinal constituents and effects in the management of myocardial injury in
an experimental isoproterenol (ISO) rat model. Sprague-Dawley rats were randomly assigned to four
groups: Group 1 was the control group; Group 2 received M. bijugatus extract (100 mg/Kg; MB) for
six weeks; Group 3 was given ISO (85 mg/Kg) i.p. twice during a 24-hour period; and Group 4 was
given ISO (85 mg/Kg) i.p. and MB extract (100 mg/Kg) for six weeks. The MB was administered
orally by gavage, daily. The blood pressure of conscious animals was measured, while ECG was
performed under anesthesia. Blood and serum were collected for biochemical and hematological
analysis. The ISO group treated with MB showed a significant decrease (p < 0.001) in (SBP), diastolic
(DBP), mean arterial (MAP) and heart rate (HR) compared to the ISO only group. Conversely, MB
treated rats that were not induced with ISO displayed a significant decreases (p < 0.001) in SBP, DBP,
MAP, and HR. ISO significantly elevated the ST segment (p < 0.001) and shortened the QTc interval
(p < 0.05), which were recovered after treatment with 100 mg/Kg of MB. In addition, the results
showed a significant decrease (p < 0.001) in the heart to body weight ratio of the ISO group treated
with MB compared to the ISO only group. Furthermore, the extract normalized the hematological
values depressed by the ISO while significantly elevating the platelet count. UHPLC high-resolution
orbitrap mass spectrometry analysis results revealed the presence of several antioxidants like vitamin
C and related compounds, phenolic acids, flavonoid, fatty acids (oxylipins), and terpene derivatives.

Molecules 2019, 24, 235
The results of this study indicated that Melicoccus bijugatus did display some cardio-protective effects
in relation to myocardial injury.
Keywords: Melicoccus bijugatus; isoproterenol; myocardial infarction; high-resolution orbitrap mass

spectrometry; rat
1. Introduction
Cardiovascular disease (CVD) like acute myocardial infarction (AMI) is one of the leading causes
of death; causing prolonged ischemia of the heart muscle resulting in tissue death or infarction in
the myocardium [1]. This results in edema, a reduction in cardiac output, abnormalities in cardiac
rhythm and transmission blocks that can further impair cardiac function. A reduction in cardiac output
and arterial pressure may stimulate baroreceptor reflexes that lead to the activation of compensatory
mechanisms, such as those of the sympathetic nerves and the renin-angiotensin-aldosterone system [2],
and to elevations in cardiac biomarkers [3].
ISO is a potent non-selective beta-adrenergic receptor (β1 and β2) agonist that causes severe
stress to the myocardium, resulting in infarct-like necrosis of the heart muscle [4]. Its proposed
mechanism of action is through the auto-oxidation and production of free cytotoxic radicals [5], as well
as hyper-stimulation of beta adrenoceptors [6]. These actions lead to the peroxidation of the cellular
membrane, a change in membrane permeability and possible derangement of calcium ion pathway
signaling, hypertrophy and myocardial injury [7,8]. The net effect of these includes a fall in DBP and
MAP while SBP may remain unchanged, rise or fall (depending on the dose). Similarly, cardiac output
may increase because of the positive inotropic and chronotropic effects of the drug, due to a decrease
in peripheral vascular resistance.
Melicoccus bijugatus, known colloquially in Jamaica as Guinep, is a minor member of the
Sapindaceae family [9]. The therapeutic effects of these fruits, including the management of diarrhea,
cardiovascular disease, asthma and constipation, and as an astringent [10], were attributed to the
combination of phenolic compounds and sugars. The phenolic content of this fruit was previously
reported [9,11]. In the seed embryo, flavonoids, epicatechin, catechin, epigallocatechin, B-type
procyanidins, naringenin, naringenin derivatives, phloretin, phloridzin, quercetin, myricetin and
resveratrol, were identified in high amounts. The pulp of the fruit contains phenolic acid derivatives
such as coumaric and ferulic acid derivatives, and hydroxycinnamic and sinapic acid [10,12]. This study
aims to scientifically examine the mechanism of action of M. bijugatus in the management of
cardiovascular ailments like AMI via an experimental rat model.
2. Results
2.1. Blood Pressure Changes and Electrocardiogram (ECG)

Table 1 shows that the extract group displayed a significant decrease (p < 0.001) in the MAP, SBP,
and DBP when compared to the control (normotensive rats). There was also a significant decrease in
the MAP, SBP, and DBP of the ISO group treated with MB compared to the ISO only group (p < 0.001).
The MB group displayed a significantly decreased (p < 0.01) HR when compared to the
normotensive animals (control). There was also a significant decrease (p < 0.001) in the HR of the
ISO plus MB treatment group when compared to the ISO only treatment group (Figure 1, Table 1).
In addition, there was an observable (24.5%) increase in the HR of the ISO only group when compared
to the control group (normotensive). However, this increase was not statistically significant.
Although the PP significantly decreased in all groups,the causes in the MB group (p < 0.001), ISO
group (p < 0.01) and ISO + MB group (p < 0.01) were different compared to that of the control. Table 1
shows that ISO significantly (p < 0.05) increased the DBP, did not increase the SBP, and decreased the
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PP, consistent with decreased left ventricular compliance and increased myocardial stiffness [13]. MB
significantly decreased the blood pressure (MAP, SBP, DBP), HR, and PP, which is proportional to the
stroke volume [14].
Table 1. Effects of Melicoccus bijugatus (MB; 100 mg/Kg) on mean arterial blood pressure (MAP),
systolic blood pressure (SBP), diastolic blood pressure (DBP), pulse pressure (PP), and heart rate (HR)
of normotensive rats and those with myocardial damage with ISO.
Normotensive Myocardial Damage

Control MB ISO ISO + MB
MAP, mmHg 101 ± 3 69 ± 2 *** 113 ± 5 83 ± 6 *,###
SBP, mmHg 131 ± 3 85 ± 2 *** 133 ± 4 106 ± 6 ***,###
DBP, mmHg 87 ± 5 60 ± 3 *** 108 ± 8 * 72 ± 8 ###
PP, mmHg 44 ± 5 27 ± 2 *** 28 ± 2 ** 27 ± 3 **
HR, bpm 246 ± 25 146 ± 11 ** 307 ± 20 181 ± 18 ###
Values are mean ±standard error of the mean of five experiments in mmHg. Statistically significant differences:
* p < 0.05, ** p < 0.01, *** p < 0.001 vs. control; ### p < 0.001 vs. ISO.
Figure 1. Electrocardiograms (ECG) showing the bradycardic effects of M. bijugatus extract (MB,
100 mg/Kg) in myocardial injury. Control shows the normal electrocardiograph. ISO (85 mg/Kg)
shows an elevated ST segment; ISO + MB shows a restored ST segment.
In electrocardiograms from rats and mice, the beginning of the T-wave merges with the end of
the QRS complex without an isoelectric ST segment. The changes, seen in the ECG, that affected the
frequency of the waves can be seen to affect the HR also. ISO significantly elevated the ST segment
(p < 0.001; Figures 1 and 2A) and shortened the QTc interval (p < 0.05; Figures 1 and 2B), which
recovered after treatment with 100 mg/Kg of MB. The M. bijugatus of the plant did not, per se, cause
any change.
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Figure 2. Treatment, with M. bijugatus, of a myocardial injury caused by ISO. The data shows the effects
of MB and ISO on ST segment elevation (A) and the duration of the QTc interval of the ECG (B). ISO
(85 mg/Kg) significantly elevated the ST segment (p < 0.001) and shortened the QTc interval (p < 0.05),
which recovered after treatment with 100 mg/Kg of M. bijugatus. The extract did not, per se, cause any
change. * p < 0.05, *** p < 0.001 vs. control; n = 5.
2.2. Hematological Parameters

There were significant differences (p > 0.05) in the hematological parameters of the treatment
groups when compared to the control group (Tables 2 and 3). ISO significantly reduced the white blood
count (WBC; p < 0.05), red blood count (RBC; p < 0.01), hematocrit (HCT; p < 0.01), mean cell volume
(MCV; p < 0.01) and mean cell hemoglobin values (MCH; p < 0.01). M. bijugatus extract normalized
the hematological values (WBC, RBC, HCT, MCV and MCH) depressed by ISO, while significantly
(p < 0.01) elevating the platelet count.
Table 2. Effect of M. bijugatus on white cell parameters in ISO-induced cardiac injury.

WBC (103 /μL) 9.5 ± 1.2 8.6 ± 1.3 4.1 ± 1.0 * 7.1 ± 1.2
LYM (103 /μL) 5.8 ± 0.6 5.9 ± 1.0 3.0 ± 0.8 4.9 ± 0.8
MID (103 /μL) 1.5 ± 0.2 1.1 ± 0.2 0.62 ± 0.2 * 1.1 ± 0.1
GRA (103 /μL) 2.4 ± 0.5 1.5 ± 0.3 0.5 ± 0.1 ** 1.2 ± 0.3
LYM (%) 61.4 ± 2.9 66.8 ± 2.7 73.9 ± 0.9 ** 69.6 ± 1.1
MID (%) 14.5 ± 0.6 13.9± 1.0 14.5 ± 0.8 13.2 ± 1.5
GRA (%) 27.3 ± 0.9 19.3 ± 2.4 11.6 ± 1.0 *** 17.3 ± 1.4 *
WBC—White blood count, LYM—Lymphocyte, MID—Others white cells, GRA—Granulocyte. Values are mean
±standard error of the mean of five experiments in mmHg. Statistically significant differences: * p < 0.05, ** p < 0.01,
*** p < 0.001 vs. control.
Table 3. Effect of M. bijugatus on red cell and trombocytes parameters in the ISO-induced cardiac injury.

RBC (106 /μL) 6.8 ± 0.2 5.9 ± 0.4 5.2 ± 0.2 ** 6.3 ± 0.42
HGB (g/dL) 14.8 ± 0.3 13.6 ± 0.6 12.5 ± 0.6 14.1 ± 1.0
HCT (%) 40.0 ± 0.8 36.2 ± 1.2 32.6 ± 1.5 ** 34.8 ± 1.4
MCV (fL) 2.4 ± 0.5 1.5 ± 0.3 0.5 ± 0.1 ** 1.2 ± 0.3
MCH (pg) 21.7 ± 0.2 22.2 ± 0.5 24 ± 0.6 ** 22.2 ± 0.4
MCHC (g/dL) 37.1 ± 0.4 37.4 ± 0.6 38.6 ± 0.4 37.7 ± 0.2
RDW 15.0 ± 0.3 15.7 ± 0.5 15.3 ± 1.2 16.6 ± 0.9
PLT (103 /μL) 680.3 ± 31.0 840.0 ± 21.1 * 668.8 ± 49.0 918.8 ± 42.7 **,##
MPV 6.6 ± 0.2 7.7 ± 0.4 7.0 ± 0.2 7.4 ± 0.5
RBC—Red blood count, HGB—Hemoglobin, HCT—Hematocrit, MCV—Mean corpuscular volume, MCH—Mean
corpuscular hemoglobin, MCHC—Mean corpuscular hemoglobin concentration, RDW—Red cell distribution width,
PLT—Platelets, MPV—Mean platelet volume. Values are mean ±standard error of the mean of five experiments in
mmHg. Statistically significant differences: * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control; ## p < 0.01 vs ISO.
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2.3. Histo-Morphological Analysis

The microscopic changes in the muscle fibers of the heart were limited to the group that was
exposed to both ISO and MB. The maximum dimension of the myocardial infarction was of 1.1 mm
and appeared to be in the central region of the left ventricular muscle wall, which is the area furthest
away from both endocardium and epicardium and is most susceptible to infarction from vascular
compromise (Figure 3C).
Figure 3. Histological analysis of myocardial injury. Histomicrograph of transverse sections of the

heart [×200; hematoxylin and eosin stain] taken through the ventricles, just below the atrioventricular
valves of control (A), 100 mg/Kg MB alone (B), ISO treatment alone (C), ISO + MB (D). The ISO group
showed myocardial infarction in the central region of the left ventricular muscle wall (C). The Control
(A) and MB only group (B) displayed no features of myocardial injury. The arrows demonstrated the
area of fibrosis.
Longitudinal and/or transverse sections of the large caliber abdominal blood vessels, i.e., aorta
and caudal vena cava revealed no changes in the intima, media or external layer. The adventitia was
composed primarily of brown fat. No inflammation was appreciated (data not shown).
2.4. Ratio of Heart Weight to Body Weight

The data does not suggest that M. bijugatus is responsible for any significant changes in body
weight that could not be accounted for in the natural growth pattern of the Sprague-Dawley rat. ISO
caused an elevated heart weight/body weight ratio and treatment with MB (100 mg/Kg) caused
a significant decrease (Figure 4).
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Figure 4. Effects of ISO and treatment with M. bijugatus extract on weight/body weight ratio. Depicts
the ISO (85 mg/Kg) induced myocardial damage through an elevation of the heart weight to body
weight ratio. This was significantly reduced in the MB (100 mg/Kg) treated groups. ** p <0.01 vs.
control; ### p < 0.001 vs. ISO, n = 5.
2.5. Identification of the Compounds

Hyphenated UHPLC-MS experiments were employed for the identification of unknown
compounds in the fruits of M. bijugatus since it provides high resolution and an accurate mass
product ion spectra (Figure 5, Table 4 and Supplementary Material). Combining Q-orbitrap HRAM
(high resolution accurate mass) full MS scans and MSn experiments, all compounds were tentatively
identified, including simple phenolic acids, terpenes, fatty acids, and one glycosylated flavonoid.
As far as we know, some of the compounds, for this species, were reported for the first time. Below is
a detailed explanation of the identification.
Figure 5. UHPLC chromatogram total ion current [total ion current (a), UV at 280 nm (b)] of aqueous
extract of M. bijugatus. The details of metabolites are in the Supplementary Material S1.
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Table 4. Identification of metabolites by UHPLC-PDA-OT-MS.

Retention Elemental
Peak Time Composition Theoretical Measured Accuracy MSn Ions
(min) UV Max Tentative Identification [M − H]− Mass (m/z) Mass (m/z) (dppm) (dppm)
1 1.72 220 Citric acid C6 H7 O7 − 191.01863 191.01933 3.68
2 1.82 222 Isocitric acid C6 H7 O7 − 191.01863 191.01955 3.04
3 1.65 - Saccharose C12 H21 O11 − 341.10784 341.10783 2.44
4 2.55 - Glucose C6 H11 O6 − 179.05501 179.05550 2.69
5 3.37 230 Furoic acid C5 H3 O3 − 111.00767 111.00787 3.25
6 6.35 245 Salicilic acid glucoside C13 H15 O8 − 299.07614 299.07712 3.25 137.02440
7 9.80 245–325 Aflavarin C24 H21 O9 − 453.11801 453.11703 −2.1
Trihydroxyoctadecatrienoic
8 10.24 - acid C18 H29 O5 − 325.20205 325.18443 −54.2
163.0291,
145.02870,
9 11.98 275–339 Coumaric acid glucoside C15 H17 O8 − 325.09179 325.09277 3.01 117.03368
163.0291,
145.02870,
10 12.24 275–339 Coumaric acid galactoside C15 H17 O8 − 325.09179 325.09271 2.82 117.03368
147.04449,
11 13.35 275–339 Feruloyl glucoside C16 H19 O9 − 355.10346 355.10336 3.34 193.05058
12 15.02 254–354 Isorhamnetin-3-O-gglucoside C22 H21 O11 − 477.11679 477.11670 −27.12 314.04370
147.04449,
13 15.83 275–339 Feruloyl galactoside C16 H19 O9 − 355.10346 355.10355 3.34 193.05056
14 21.57 236–329 3-O-Caffeoylquinic acid C16 H17 O9 − 353.0878 353.0878 0.53 191.05608
15 21.87 - Rishitin C14 H22 O2 − 221.15361 221.15488 5.74
16 23.54 300 Embelin C17 H26 O4 − 293.17474 293.17587 3.87
17 25.38 235 Sedanenolide C12 H15 O2 − 191.10666 191.10741 3.92
18 26.45 225 Valerenic acid C15 H21 O2 − 233.15470 233.15455 3.87 149.13301
19 27.78 214 Blumenol C C13 H22 O2 − 209.15631 209.15430 3.30
Hydroxyheptadecatrienoic
20 28.32 220 acid C17 H27 O7 − 311.18640 311.16876 −56.6
2.5.1. Simple Organic Acids and Sugars

Peaks 1–5 and 8 were identified as simple organic acids such as vitamin C and sucrose. Peak 1, with
an [M − H]− ion at m/z: 191.01933, was identified as citric acid (C6 H7 O7 − ) and Peak 2 as the isomer
isocitric acid [4]. Peak 3 was identified as saccharose (C12 H21 O11 − ) and Peak 4 as glucose (C6 H11 O6 − ).
Sucrose and glucose were already reported as important constituents of this fruit [10,12]. Peak 5, with
an [M − H]− ion at m/z: 111.00787, was identified as furoic acid (C3 H3 O3 − ) and Peak 16, with an
[M − H]− ion at m/z: 293.17587, was assigned to the bioactive, cell permeable, 1,4-benzoquinone
embelin derivative (C17 H26 O4 − ) [15].
2.5.2. Flavonoids
Peak 12, with an [M − H]− ion at m/z: 477.11670, and the MS2 fragment at m/z: 314.04370 were
identified as Isorhamnetin-3-O-Glucoside (C22 H21 O11 − ) [16].
2.5.3. Phenolic Compounds

Peak 6 was identified as salicilic acid glucoside (C13 H15 O8 − ), the parent ion 299.07712 delivered
a diagnostic salicylic acid ion at m/z: 137.02440 [17], and Peak 7 was identified as the dicoumarin
aflavarin (C24 H21 O9 − ). Peaks 9 and 10, with pseudomolecular ions at m/z: 325.09277 and dioagnostic
MS2 fragments at m/z: 163.02910, 145.02870 and 117.03368, were identified as coumaric acid glucoside
and coumaric acid galactoside, respectively (C15 H17 O8 − ). The presence of coumaric acid glucoside
was already reported in this fruit pulp [10]. In the same manner, Peaks 11 and 13 were identified as the
related compounds feruloyl glucoside and feruloyl galactoside (C16 H19 O9 − ) showing diagnostic MS2
ions at 147.04449 and 193.05056. Peak 14 was identified as chlorogenic acid: 3-O-caffeoylquinic acid
(3-CQA, C16 H17 O9 − ), with a diagnostic quinic acid ion at 191.05608 [18].
2.5.4. Fatty Acids

Two peaks were tentatively identified as fatty acids known as oxylipins [19,20]. Peak 8,
with a pseudomolecular ion at m/z: 325.18443, was identified as a trihydroxy-octadecatrienoic
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acid (C18 H29 O5 − ) and Peak 2, with a pseudomolecular ion at m/z: 311.16876, was identified as
a hydroxyheptadecatrienoic acid (C17 H27 O7 − ) [21].
2.5.5. Terpenes and Related Compounds

Peak 17 was identified as the lactone sedanenolide (C12 H12 O2 − ) [22] and Peak 18 was assigned
to the sesquiterpene valerenic acid (C15 H21 O2 − ) [23]. Peak 15, with a [M − H]− ion at m/z:
221.15488, was identified as the antifungic norsesquiterpene alcohol rishitin (C14 H22 O2 − )[24].
Peak 19, with an [M − H]− ion at m/z: 209.15430, was identified as Blumenol C (C13 H22 O2 − ,
4-(3-hydroxybutyl)-3,5,5-trimethylcyclohex-2-en-1-one) [25].
3. Discussion
This study, for the first time, demonstrated the hypotensive effect of the Melicoccus bijugatus Jacq
when it is administrated orally in normotensive animals. This hypotensive effect was, in part, because
of the decrease in HR. In addition, we demonstrated a partial recovery of ISO-induced myocardial
infarction by the action of M. bijugatus.
ISO is an isopropyl analog of epinephrine that stimulates the α-1 adrenergic receptors [8], oxidative
stress in cardiac myocytes, cell membrane destabilization and damage. It also increased intracellular
adenylyl cyclase in the myocardium, increased lipid deposition in the myocardium, and increased the
heart weight to body weight ratio and myocardial infarction [26]. ISO also significantly elevated the ST
segment and shortened the QTc interval in the experimental animals; this was reversed by treatment
with M. bijugatus extracts. The chronotropic and inotropic actions of ISO also caused an increase in the
SBP. The sum of these cardiovascular changes is in an increase in HR and cardiac output and a decrease
in the MAP, as observed in our study. Treatment of the animals exposed to ISO with M. bijugatus
extracts caused decreases in HR, and MAP. This showed the cardioprotective role of the M. bijugatus.
This increase in the ratio of heart weight to body weight was thought to be a hypertrophic response,
possibly due to an increase in the amount of protein synthesis that was occurring in the damaged
tissue as it attempted to repair itself, as well as an increase the number of inflammatory cells to become
mobilized in response to the damage [27]. Other possible mechanisms include increase in the glucose
uptake in cardiac myocytes along with an increase in oxidative stress with ISO administration [28],
accumulation of fluid in intracellular space of the tissue as well as an increase in the water content of
the cells themselves [29]. Histo-morphological presentations of myocardial infarction to the ISO group
were observed. There were no appreciable changes in the intima, media and adventitia layers of the
aorta, caudal or vena cava (data not shown).
Using UHPLC high-resolution orbitrap mass spectrometry, (UHPLC-OT-HR-MS) we have
identified 20 secondary metabolites in the aqueous extract of M. bijugatus, most of which, as far as we
know, were reported here for the first time. Many of these compounds are simple organic acids, such as
vitamin C, a flavonoid, several phenolic compounds, terpenoids, and two fatty acids. Furthermore,
the results obtained in this study clearly show that the infusion can be a natural source of phenolic
compounds with potential applications in the neutraceutical management of myocardial infarction.
A possible mechanism of action may be through the inhibition of the proliferation of cells of the
vascular smooth muscle by caffeic acid, which is found in the fruit’s pulp tissues [30]. The hypotensive
properties displayed may also be due to the action of coumaric acid derivatives. A derivative of the
sugar of coumaric acid was confirmed to be a major peak in the HPLC fingerprint profile at 280nm [12].
One such derivative of coumaric acid is p-coumaric which has been known to possess antioxidant
effects as well as anti-platelet activity [31].
Increases in serum levels of troponin I, CK-MB, myoglobin, and high sensitivity C-reactive protein
are often associated with myocardial damage [32,33], though with some limitations [3,34]. Our study
showed no significant difference in the serum concentration of CK-MB in rats induced with ISO
compared to the control group (data not shown), this could be due to the timeline of the analysis of
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these biomarkers. Our results are supported by Zhang et al. (2008), who reported a decline in these
biomarkers, down to control levels, after 48 h [35].
Myocardial infarction is often associated with an increase in the white blood cell count as part of
an inflammatory response to the damaged cells that are present due to the necrosis of the tissue [36].
Other hematological features associated with myocardial infarction include an increase in whole blood
viscosity and plasma viscosity, an increase in the white blood cell, leukocyte and neutrophil count [1],
an increase in the erythrocyte count and hemoglobin concentration [37], and an increase in red cell
indices, such as mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration
(MCHC) and mean corpuscular volume (MCV) [4]. Sangeetha and Quine, (2008) reported increases in
hemoglobin, RBC, hematocrit, WBC and platelet counts with ISO induced myocardial infarction in
male Wister rats [37], while Lobo-Fiho et al., (2011) suggested no alterations in terms of the hemoglobin
indices [1]. We observed significant alterations in the hematological parameters in the treatment groups
when compared to the control group. This study showed that ISO significantly reduced the white
blood count (WBC), red blood count (RBC), hematocrit (HTC), mean cell volume (MCV) and mean cell
hemoglobin values (MCH). The extract normalized the hematological values (WBC, RBC, HCT and
MCV) depressed by ISO while significantly elevating the platelet count. The increased production of
platelets suggests that the extract did not present a toxic effect, such as that of Colocasia esculenta (L.)
Schott [38], but presented an increase in platelets similar to Carica papaya Linn. [39].
4.1. Plant Material and Extraction

The fruit of the M. bijugatus tree was collected in September. Mr. Patrick Lewis, Department
of Botany, University of the West Indies, Mona Campus (Kingston, Jamaica), made a botanical
identification of the plant (voucher specimen AN 08, 10/11). The fruit pulp was separated from
the seed by hand using a knife. The pulp was then blended and the juice was squeezed out through
a strainer before being filtered to remove any remaining pulp fibers. Finally, the juice was placed inside
a Freeze Dry machine (Freezone 4.5 L, Labconco, Kansas, MO, USA).

The study was conducted according to the Animal Scientific Procedures Act of 1986 following
the receipt of approval from the UWI/FMS Ethics Committee (AN 06,15/16). Twenty male
Sprague- Dawley rats (8–10 weeks old and 170 g to 230 g) were chosen, housed in plastic cages
at the UWI Mona campus Animal House at room temperature (22–25 ◦ C) with a humidity of 45–51%.
Fresh tap water was available to the rats via a bottle and food was administered ad libitum. Four groups
were identified, each of which consisted of five rats. The first group served as a control group and
did not receive any drugs. Rats from the second group were administered, by gavage, 100 mg/Kg of
M. bijugatus liquid extract only daily for six weeks. The third group of rats received only two doses of
85 mg/Kg b.w. ISO intraperitoneally within a 24-hour period. Finally, rats of Group 4 were injected
intraperitoneally with two doses of 85 mg/Kg b.w. of ISO within a 24-hour period. Following this,
they were given 100 mg/Kg b.w. M. bijugatus extract, by gavage, daily for six weeks. The animals
were sacrificed under anesthesia seven days after the period of treatment. The heart and kidney
tissues were harvested and weighed to obtain a ratio of the heart and kidney weights to body weight.
In addition, a blood sample was collected to conduct biochemical and hematological assessments
among the subject groups.
4.3. Blood Pressure Recordings and ECG

The tail cuff method (CODA) was used to measure the SBP and DBP of the rats once before the
administration of ISO and once per week following the administration of ISO. The pulse pressure
(PP) was calculated using the formula PP = (SBP − DBP). The MAP was calculated using the formula:
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Molecules 2019, 24, 235
MAP = DBP + 1/3(SBP − DBP). For ECG recordings, rats were first anesthetized with ketamine
(42 mg/Kg, i.p.) and xylazine (5 mg/Kg, i.p.). The ECG electrodes (BIOPAC System Inc, California,
CA, USA) were placed subcutaneously in a bipolar configuration (DII). Measurements were done using
the Electrocardiogram Amplifier equipment (ECG100C, BIOPAC System Inc, California, CA, USA)
and tracings were recorded using the AcqKnowledge III computer software program (3.9.1., BIOPAC
System Inc, California, CA, USA). The QT interval was taken as the time from the beginning of the
QRS complex to the end of the T-wave. The RR interval was taken as the time elapsed between two
consecutive maxima of the R-waves. The corrected QT interval (QTc) was calculated in accordance
with the formula [40,41]:
QTc = QT/(RR)1/2
4.4. Biochemical Estimation

Whole blood was collected and assessed using standardized ethylenediaminetetraacetic acid
(EDTA) tubes to obtain a complete blood count (CBC). The Cell Dyn Emerald Hematology Analyzer
(Abbott Laboratories, Chicago, IL, USA) was then used to assess hematological parameters.
4.5. Histo-Morphological Appraisal

Histo-morphologic analyses of the cardiac tissues of the Sprague-Dawley rats were done
using a Nikon Eclipse Ci research microscope (Nikon Instruments Inc., New York, NY, USA).
Mirco-measurements were done via integrated mechanical stages with graduated locator margins and
built in slide holders, as well as X-Y translator knobs. The tissues were harvested and immediately
submerged in 10% neutral buffered formalin for preservation. They were subsequently processed,
embedded in wax, and serial sectioned to a thickness of 4 microns then stained with haematoxylin and
eosin (H and E) stain.
Twelve (12) full-thickness transverse sections from the heart, immediately subjacent to the
atrioventricular valves (three from each group), were analyzed and evaluated for edema, mononuclear
cell infiltration, fibrosis, interstitial hemorrhage and myocyte degeneration. The number of foci
exhibiting degenerative features was recorded and the maximum diameter of the degenerated area
was measured using a stage micrometer (Nikon Instruments Inc., New York, NY, USA).
Sixteen (16) longitudinal and/or transverse sections of the large caliber abdominal blood vessels,
i.e., aorta (two from each group) and caudal vena cava (two from each group), were analyzed and
evaluated for evidence of endothelial injury, degeneration of the intima or elastic lamina, atherosclerotic
change, or any features of adventitial cellular injury.
The Ishak systems were employed to ascribe a stage of fibrosis and to grade the degree
of inflammatory changes (1). In addition to these established, semi-objective parameters,
subjective evaluation was performed for sinusoidal vascular congestion and the degree of nuclear
chromatin density.
4.6. UHPLC-DAD-MS Instrument

The use of the Scientific Dionex Ultimate 3000 UHPLC system (Thermo Fisher Scientific, Bremen,
Germany) hyphenated with a Q exactive focus machine (Thermo Fisher Scientific, Waltham, MA, USA)
was already reported [42]. For the analysis, 5 mg of the lyophilized material were dissolved in 2 mL of
methanol, filtered (PTFE filter) and 10 μL were injected in the instrument, with all specifications set as
previously reported [42].
4.7. LC Parameters and MS Parameters

Liquid chromatography was performed using an UHPLC C18 column (Acclaim,
150 mm × 4.6 mm ID, 2.5 μm, Thermo Fisher Scientific, Bremen, Germany) operated at 25 ◦ C.
The detection wavelengths were 254, 280, 330 and 354 nm, and DAD was recorded from 200 to 800 nm
for peak characterization. Mobile phases were 1% formic aqueous solution (A) and acetonitrile (B).
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Molecules 2019, 24, 235
The gradient program time in minutes, % (B) was: (0.00, 5); (5.00, 5); (10.00, 30); (15.00, 30); (20.00, 70);
(25.00, 70); (35.00, 5) and 12 min for column equilibration before each injection. The flow rate was
1.00 mL min−1 , and the injection volume was 10 μL. The standards and resin extract dissolved in
methanol were kept at 10 ◦ C during storage in the autosampler. The HESI II and Orbitrap spectrometer
parameters were optimized as previously reported [42,43].

The data collected were expressed as mean ±SEM (standard error of mean). The mean values
from the different groups were analyzed using GraphPad Prism Version 6.0 Software (GraphPad
Software, San Diego, CA, USA). One-way or two-way ANOVA were used to compare the means,
followed by the Bonferroni test. A value of p < 0.05 was considered statistically significant [44].
5. Conclusions
In conclusion, we confirmed that Melicoccus bijugatus partially reversed myocardial damage and
injury in an experimental ISO rat model. This was indicated by changes seen in the ECGs and in the
reversal of the increased heart to body weight ratio in animals with ISO induced myocardial injury
and blood pressure changes associated with a normalizing or reversal of symptoms associated with
cardiac injury.
Supplementary Materials: The following are available online, Figure S1: Quadrupole Orbitrap full MS spectra
and structures of all detected compounds in the fruits of Melicoccus bijugatus.
Author Contributions: C.R.N. was the author of the project design, conducted and designed the experiments, and
drafted the manuscript; I.W. performed some experiments; J.P. participated in the design, performed some
experiments, and wrote the manuscript; F.C., A.P. and A.L. participated in the design and conducted the
pharmacological assays. J.B. and M.J.S. performed the isolation and structural elucidation of the pure compounds
for UHPLC-MS; M.N., S.H. and R.T. participated in the design and performed the histopatological experiments.
Funding: This research was funded in part by the World Academy of Science/UNESCO (13–108 RG/BIO/LA)
Grant and UWI Grants to C.R. Nwokocha. Mario Simirgiotis and Jorge Bórquez acknowledge funds from Fondecyt
1180059 Universidad Austral de Chile.
Acknowledgments: The authors wish to express their gratitude to the Rectoria y Vicerrectoria de Investigacion,
Innovacion y Postgrado Universidad de Antofagasta for their financial and technical support.
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Sample Availability: Samples of Melicoccus bijugatus is available from the corresponding author.
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molecules
Article
Metabolomics Profiling Reveals Rehmanniae Radix
Preparata Extract Protects against
Glucocorticoid-Induced Osteoporosis Mainly via
Intervening Steroid Hormone Biosynthesis
Tianshuang Xia 1,† , Xin Dong 1,† , Yiping Jiang 1,† , Liuyue Lin 1 , Zhimin Dong 1 , Yi Shen 1 ,
Hailiang Xin 1, *, Qiaoyan Zhang 1,2, * and Luping Qin 1,2, *
1 Department of Pharmacognosy, Second Military Medical University School of Pharmacy,
Shanghai 200433, China; xiatianshuang@smmu.edu.cn (T.X.); dongxinsmmu@126.com (X.D.);
msjyp@163.com (Y.J.); linliuyue2016@163.com (L.L.); dongzhiminedu@163.com (Z.D.);
18065148122@163.com (Y.S.)
2 Zhejiang Chinese Medical University School of Pharmacy, Hangzhou 310053, China
* Correspondence: hailiangxin@163.com (H.X.); zqy1965@163.com (Q.Z.); qinsmmu@126.com (L.Q.);
Tel.: +86-021-81871309 (L.Q.)
† These authors contributed equally to this work.
Abstract: Rehmanniae Radix Preparata (RR), the dry rhizome of Rehmannia glutinosa Libosch.,
is a traditional herbal medicine for improving the liver and kidney function. Ample clinical
and pharmacological experiments show that RR can prevent post-menopausal osteoporosis
and senile osteoporosis. In the present study, in vivo and in vitro experiments, as well as a
UHPLC-Q/TOF-MS-based metabolomics study, were used to explore the preventing effect of
RR on glucocorticoid-induced osteoporosis (GIOP) and its underlying mechanisms. As a result,
RR significantly enhanced bone mineral density (BMD), improved the micro-architecture of trabecular
bone, and intervened in biochemical markers of bone metabolism in dexamethasone (DEX)-treated
rats. For the in vitro experiment, RR increased the cell proliferation and alkaline phosphatase (ALP)
activity, enhanced the extracellular matrix mineralization level, and improved the expression of
runt-related transcription factor 2 (RUNX2) and osteopontin (OPN) in DEX-injured osteoblasts.
For the metabolomics study, a total of 27 differential metabolites were detected in the DEX group
vs. the control group, of which 10 were significantly reversed after RR treatment. These metabolites
were majorly involved in steroid hormone biosynthesis, sex steroids regulation, and amino acid
metabolism. By metabolic pathway and Western blotting analysis, it was further ascertained that
RR protected against DEX-induced bone loss, mainly via interfering steroid hormone biosynthesis,
as evidenced by the up-regulation of cytochrome P450 17A1 (CYP17A1) and aromatase (CYP19A1),
and the down-regulation of 11β-hydroxysteroid dehydrogenase (HSD11B1). Collectively, these
results indicated that RR had a notable preventing effect on GIOP, and the action mechanism might
be related to steroid hormone biosynthesis.
Keywords: Rehmanniae Radix Preparata; glucocorticoid-induced osteoporosis; metabolomics;

osteoblast; steroid hormone biosynthesis
1. Introduction
Glucocorticoids (GC) are widely used in clinics to treat rheumatoid arthritis, pulmonary,
gastrointestinal, and autoimmune diseases for their excellent anti-inflammatory and immune-modulatory
effects [1]. However, the life-time service of GC usually induces a series of complications, among which

Molecules 2019, 24, 253
osteoporosis is the most devastating one. Currently, glucocorticoids-induced osteoporosis (GIOP) has
become the third most-common etiology of pathological bone loss, only next to senile osteoporosis
and postmenopausal osteoporosis. The underlying pathological mechanism of GIOP mainly lies in the
direct inhibition of the osteoblastic cell cycle, differentiation, and function, and the stimulating action
on endogenous gonadal steroids degeneration [2,3]. Clinical treatment for GIOP mainly includes
calcium and active vitamin D supplementation and oral bisphosphonates, which is similar to that
for postmenopausal osteoporosis and senile osteoporosis, and is incompatible with the pathological
mechanism of GIOP. Besides, these therapeutic regimens may cause some potential adverse reactions,
such as gastroesophageal irritation [4] and osteonecrosis of the jaw [5]. Thus, there is a desperate need
to develop suitable therapeutic alternatives for GIOP with few adverse effects.
Rehmanniae Radix Preparata (RR), prepared from the dry rhizome of Rehmannia glutinosa Libosch.,
has traditionally been used for tonifying kidney essence in China [6]. This herb was first officially
recorded in the Chinese Pharmacopoeia of 1963 version, and up to now, more than 140 compounds have
been isolated and identified, including iridoid glycosides, phenylethanoid glycosides, monoterpenoids,
and triterpenes. In traditional Chinese medicine (TCM), the kidney is in charge of bone, and bone loss
is attributed to the kidney and liver deficiency. According to TCM theory, RR can promote the liver and
kidney function. Hence, RR and TCM formulas containing RR are widely used to treat osteoporosis
patients. In modern pharmacological studies, ample experiments have been used to understand the
effect of RR on preventing osteoporosis. The water extract of RR was proved to improve BMD and
increase the cortical bone thickness and trabeculation of the bone marrow spaces [7]. Liuwei Dihuang
Pill, a traditional Chinese medicine formula mainly containing RR, was also proved to have remarkably
preventive and therapeutic effects on primary osteoporosis through promoting bone formation [8,9].
In addition, it is noteworthy that RR can prevent the decrease of the splenic estrogen receptor and
osteoblastic progestin receptor in aging female mice [10]. Inspired by these findings, we wonder if and
how RR can alleviate bone loss induced by GC.
Metabolomics is a sensitive and unbiased analytical method that is used to comprehensively
characterize the metabolite content of biological samples for understanding disease phenotypes.
Metabolomics is characterized by “integrity and systematization”, which is consistent with the
“multi-component and multi-target” theory in TCM [11]. Analysis of metabolite profiling before
and after treatment with TCM can help explore their comprehensive therapeutic efficacies and action
mechanisms. In this study, an untargeted metabolomics strategy based on UHPLC-Q-TOF/MS was
employed to analyze the metabolic profile of GIOP rats, intending to better understand the action
mechanism of RR on preventing DEX-induced bone loss and provide more promising candidates for
the prevention and treatment for GIOP.
2. Results
2.1. UHPLC-MS Analysis of RR Extract

The UHPLC-MS characteristics of RR extract were detected. As shown in Figure 1, RR extract
contains catalpol, acteoside, and echinacoside.
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Molecules 2019, 24, 253
Figure 1. UHPLC-MS characteristics of RR extract. (A) The representative total ion chromatograms
(TICs) in positive and negative ion mode; (B) catalpol in the RR extract sample; (C) acteoside in the RR
extract sample; (D) echinacoside in the RR extract sample.
2.2. RR Improves the Micro-Architecture and BMD of the Femur in DEX-Treated Rats
As shown in Figure 2A–C, the micro-CT analysis showed a severe impairment of bone
micro-architecture in the femur of DEX-treated rats, and the trabecular region exhibited a small,
thin, and sparse morphology. RR could obviously improve the trabecular micro-architecture, with
a slightly disordered trabecular arrangement and mild expanding medullary cavity. As shown in
Figure 2D–G, the morphologic parameters of trabecular thickness (Tb.Th.) and bone volume fraction
(BVF) decreased significantly, and trabecular separation (Tb.Sp.) and bone surface to bone volume
(BS/BV) increased significantly in the femur of DEX-treated rats compared with those in the normal
group (p < 0.01). RR treatment could significantly reverse the alterations of trabecular morphologic
parameters, increase Tb.Th. and BVF, and decrease Tb.Sp. and BS/BV (p < 0.01). In addition, as shown
in Figure 2K–H, DEX significantly reduced the BMD, bone mineral content (BMC), tissue mineral
density (TMD), and tissue mineral content (TMC) compared with those in the normal group (p < 0.01),
while RR administration significantly enhanced these indices (p < 0.01).
Figure 2. The region of interest (ROI) image and bone parameters analysis in the distal femur in
DEX-treated rats. (A–C) Micro-CT images of ROI region in longitudinal section, transverse section,
and 3-D architecture. (D–K) Trabecular bone parameters analysis of (D) BVF; (E) BS/BV; (F) Tb.Th.;
(G) Tb.Sp.; (H) BMD; (I) BMC; (J) TMD; and (K) TMC in the distal femur region in DEX-treated rats.
Values were expressed as the mean ± SD; n = 7. * p < 0.05, ** p < 0.01 compared with DEX group.
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Molecules 2019, 24, 253
2.3. RR Regulates Bone Metabolism-Related Biochemical Markers in DEX-Treated Rats

As shown in Figure 3A–C, DEX significantly increased the urine calcium (U-Ca) level and
decreased the serum phosphorus (S-P) level compared with those in the normal group (p < 0.01).
RR at low- and medium-doses significantly reduced the U-Ca level, and increased the S-P level,
in DEX-treated rats (p < 0.05). There was no significant difference in the serum calcium (S-Ca) level
between the six groups. Alkaline phosphatase (ALP), bone gla-protein (BGP), deoxypyridinoline
(DPD), and c-terminal telopeptides of type I collagen (CTX-I) are biochemical markers of bone turnover.
As shown in Figure 3D–G, DEX significantly decreased the serum ALP level, and increased the urine
DPD and serum CTX-I levels, compared with those in the normal group (p < 0.01 or p < 0.05). RR at
medium- and high-dosages could significantly increase the ALP level and decrease the DPD level
in DEX-treated rats (p < 0.01 or p < 0.05). RR also insignificantly decreased BGP and CTX-I levels in
DEX-treated rats.
Figure 3. Effects of RR on biochemical markers levels of bone metabolism in DEX-treated rats. (A) U-Ca;
(B) S-Ca; (C) S-P; (D) ALP; (E) BGP; (F) DPD; (G) CTX-I. Values were expressed as the mean ± SD;
n = 7. * p < 0.05, ** p < 0.01 compared with DEX group.
2.4. RR Enhances the Proliferation, Differentiation, and Mineralization Levels of Osteoblasts Injured by DEX
To further validate the in vitro effect of RR on preventing bone loss, the activities of DEX-injured
osteoblasts were assayed. The results showed that DEX significantly decreased the proliferation
(Figure 4A), ALP activity (Figure 4B), and mineralization level (Figure 4C,D) of osteoblasts (p < 0.01).
After treatment, RR at doses of 0.2 mg/L and 1 mg/L significantly increased the cells’ proliferation
compared with that of the DEX-treated control (p < 0.05). RR at all dosages prominently increased
the ALP activity (p < 0.01 or p < 0.05), and the intensity and area of staining, as well as promoted the
formation of mineralized nodules in DEX-treated osteoblasts (p < 0.01). In addition, DEX inhibited
the expression of RUNX2 and OPN compared with that in the control group, while RR treatment
improved the RUNX2 and OPN expression in DEX-injured osteoblasts to some extent (Figure 4E).
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Figure 4. Effects of RR on the proliferation, differentiation, and mineralization levels of DEX-injured

osteoblasts. (A) MTT assay; (B) ALP activity; (C) extracellular matrix mineralization; (D) representative
images of osteoblastic bone mineralization nodule stained with alizarin red; (E) the protein expressions
of RUNX2 and OPN. Values were expressed as the mean ± SD; (A) n = 4, (B–F) n = 3. * p < 0.05,
** p < 0.01 compared with DEX group.
2.5. Metabolomics Analysis

The TICs in both positive and negative ion modes of urine samples from three experimental
groups are shown in Figure S1. The QC superposed graph is shown in Figure S2. To determine whether
metabolite fingerprints in urine differed between the control, DEX, and DEX+RR-M groups in the
metabolomics approach, the partial least squares discriminant analysis (PLS-DA) model was applied.
The metabolic profiles showed that urine samples of the DEX group evidently separated from those of
the normal group. Samples of the DEX+MO group were situated close to those of the control group
and far away from those of the DEX group (Figure 5A,B). Validation of the PLS-DA model exhibited a
good fitting degree (Figure 5C,D).
2.6. Identification of Potential Biomarkers in DEX-treated Rats

Variables far from the origin in the S-plot (Figure 5E,F) with a variable importance plot (VIP)
> 1 and p < 0.05 were applied to select potential biomarkers. As a result, 27 differential metabolites
between rats in the control group and DEX group were selected as potential biomarkers, 23 of which
were the only ones for which the endogenous compound matched with the accurate mass via online
database Metlin and Human Metabolome Database (HMDB), except for dipeptide. These differential
metabolites were mainly involved in amino acid metabolism, fatty acid biosynthesis, steroid hormone
biosynthesis, and arachidonic acid metabolism (Table 1).
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Table 1. Screening of potential biomarkers in rat urine.
Fold Change Value P-Value HMDB/

NO. Metabolite Identification Rt (min) m/z Adduct VIP Related Pathway
DEX/Control DEX+RR/DEX DEX/Control DEX+RR/DEX MELIN ID
1 Glycine 3.818 76.0395 [M+H]+ 1.78 0.491 1.280 0.025 * 0.845 Amino acid metabolism HMDB0000123
2 Benzoic acid 2.449 140.0701 [M+NH4 ]+ 1.23 2.219 0.496 0.006 ** 0.013 * Amino acid metabolism HMDB0001870
3 N-Acetylproline 2.450 158.0807 [M+H]+ 2.29 2.155 0.492 0.008 ** 0.012 * Amino acid metabolism HMDB0094701
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Metabolism of
+ HMDB0012138/
4 Naphthol 0.842 162.0926 [M+NH4 ] 2.20 2.192 0.574 0.047 * 0.158 xenobiotics by
HMDB0012322
cytochrome P450
5 Indoleacetic acid 3.823 198.0513 [M+Na]+ 1.30 0.467 1.280 0.013 * 0.848 Amino acid metabolism HMDB0000197
6 4-Pyridoxic acid 1.156 201.0860 [M+H]+ 1.05 0.373 2.167 0.000 ** 0.014 * Vitamin B6 metabolism HMDB0000017
7 N-Hydroxy-L-tyrosine 1.563 215.1006 [M+NH4 ]+ 2.08 0.307 1.869 0.000 ** 0.312 Amino acid metabolism HMDB0038750
8 N-alpha-Acetylcitrulline 1.154 218.1137 [M+H]+ 1.21 0.396 1.964 0.001 ** 0.057 Amino acid metabolism HMDB0000856
9 3-Oxododecanoic acid 6.690 232.1898 [M+NH4 ]+ 1.24 2.714 0.720 0.010 * 0.442 Fatty acid biosynthesis HMDB0010727
HMDB0013128/
10 Valerylcarnitine/Isovalerylcarnitine 5.306 246.1698 [M+H]+ 1.15 2.136 0.725 0.042 * 0.479 Fatty acid biosynthesis
HMDB0000688
11 N-Phenylacetylaspartic acid 4.080 252.0842 [M+H]+ 1.53 2.064 0.551 0.007 ** 0.022 * Amino acid metabolism HMDB0029355
HMDB0000220/
12 Palmitic acid/Isopalmitic acid 9.939 274.2743 [M+NH4 ]+ 5.52 0.741 1.156 0.013 * 0.463 Fatty acid biosynthesis
HMDB0031068
Steroid hormone HMDB0000031/
13 Androsterone/Epiandrosterone 8.665 313.2156 [M+Na]+ 1.61 3.719 0.592 0.001 ** 0.048 *
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biosynthesis HMDB0000365
Steroid hormone
14 Hydroxypregnenolone 10.545 333.2407 [M+H]+ 1.01 0.414 1.438 0.029 * 0.795 —
biosynthesis
+
15 Galactosylhydroxylysine 6.575 342.1904 [M+NH4 ] 1.01 6.878 0.867 0.006 ** 0.942 Energy metabolism HMDB0000600
Steroid hormone
16 11-Dehydrocorticosterone 9.011 345.2061 [M+H]+ 1.90 0.325 3.389 0.003 ** 0.001 ** HMDB0004029
biosynthesis
+ Steroid hormone
17 Corticosterone 9.537 347.2219 [M+H] 2.53 0.289 4.163 0.128 0.035 * HMDB0001547
biosynthesis
Arachidonic acid HMDB0001043/
18 Arachidonic acid/Arachidonate 10.055 349.2378 [M+FA-H]- 1.87 0.306 0.853 0.014 * 0.989
metabolism HMDB0060102
Table 1. Cont.
Fold Change Value P-Value HMDB/

NO. Metabolite Identification Rt (min) m/z Adduct VIP Related Pathway
DEX/Control DEX+RR/DEX DEX/Control DEX+RR/DEX MELIN ID
HMDB0006528/
19 Docosapentaenoic acid 9.795 353.2475 [M+Na]+ 3.45 3.137 0.413 0.032 * 0.077 Fatty acid biosynthesis
HMDB0001976
+
20 MG(0:0/16:0/0:0) 13.949 353.2667 [M+Na] 2.57 0.670 0.964 0.010 * 0.991 Fatty acid biosynthesis —
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Steroid hormone
21 18-Hydroxycorticosterone 9.008 361.2016 [M-H]- 1.59 0.215 4.840 0.001 ** 0.000 ** HMDB0000319
biosynthesis
Arachidonic acid
22 HETE 8.666 365.2332 [M+FA-H]- 3.04 3.978 0.503 0.000 ** 0.110 —
metabolism
+ Steroid hormone
23 Cortolone 8.667 367.2478 [M+H] 1.40 4.327 0.553 0.000 ** 0.042 * HMDB0003128
biosynthesis
Arachidonic acid
24 Carbocyclic Thromboxane A2 9.793 371.2576 [M+Na]+ 4.34 2.766 0.414 0.048 * 0.078 METLIN-45632
metabolism
Retrograde
25 α,α-Dimethyl anandamide 13.286 376.3186 [M+Na]+ 1.34 1.694 1.017 0.026 * 0.999 endocannabinoid METLIN-36748
signaling
Lysophosphatidic
26 LysoPA(i-14:0/0:0) 8.665 405.2038 [M+Na]+ 2.41 7.529 0.452 0.000 ** 0.027 * HMDB0114765
metabolism
Retrograde
27 O-Arachidonoyl Glycidol 9.789 405.2644 [M+FA-H]- 3.23 3.122 0.286 0.035 * 0.120 endocannabinoid METLIN-44872
signaling
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* p < 0.05, ** p < 0.01 compared with DEX group.
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Figure 5. Multivariate analysis based on the UHPLC-Q/TOF-MS profiling data for samples in the
control, DEX, and DEX+RR groups in positive and negative ion mode (n = 8). (A) PLS-DA score plot in
positive ion mode; (B) validation of PLS-DA model in positive ion mode; (C) S-plot of PLS-DA model
in positive ion mode; (D) PLS-DA score plot in negative ion mode; (E) validation of PLS-DA model in
negative ion mode; (F) S-plot of PLS-DA model in negative ion mode.
2.7. RR Reverses Metabolic Dysregulation in DEX-Treated Rats

The metabolic changes after treatment with RR at 4 g/kg in GIOP rats were studied. Seen in Table 1,
the levels of benzoic acid, N-acetylproline, 4-pyridoxic acid, androsterone, N-phenylacetylaspartic
acid, 11-dehydrocorticosterone, 18-hydroxycorticosterone, cortolone, corticosterone, and lysoPA
were significantly reversed after RR treatment, and these metabolites were majorly involved in
steroid hormone biosynthesis, sex steroids regulation, and amino acid metabolism. The heat map
was constructed based on the normalized data set of the reversed metabolites, and the results
showed that the variation tendency of most metabolites after RR treatment was different from that
in DEX group, while the same as that in the control group (Figure 6A). The ingenuity metabolic
pathway analysis showed that the potential biomarkers were majorly involved in steroid hormone
biosynthesis (Figure 6B). The metabolic pathway map associated with differential metabolites was
depicted based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) (Figure 6C). These results
showed that RR reversed metabolic dysregulation in DEX-treated rats, mainly via intervening steroid
hormone biosynthesis.
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Figure 6. Heat map and metabolic pathway analysis based on the UHPLC-Q/TOF-MS profiling data
for samples in the control, DEX, and DEX+RR groups. (A) Hierarchical clustering heat map of the
differential metabolites before and after RR treatment in DEX-treated rats; (B) summary of ingenuity
pathway analysis with MetPA. The size and color of each circle were based on pathway impact value
and p-value, respectively; (C) construction of the metabolic pathway related to differential metabolites.
The metabolites were labeled in blue (up-regulation after RR treatment) or red (down-regulation after
RR treatment).
2.8. RR Regulates the Expressions of Key Proteins in Steroid Hormone Biosynthesis

To further validate the regulatory effect of RR on steroid hormone biosynthesis, the expression of
key proteins related to steroid hormone biosynthesis was analyzed by Western blotting. As shown in
Figure 7, DEX significantly disturbed the expression of CYP17A1, CYP19A1, HSD11B1, and HSD3B2
(p < 0.01 or p < 0.05). After treatment, RR at all doses prominently increased the expression of
CYP19A1 compared with that in the DEX group (p < 0.01 or p < 0.05). RR at a dose of 0.2 mg/L
prominently increased the expression of CYP17A1 (p < 0.01). RR at doses of 1 mg/L and 5 mg/L
also significantly inhibited the HSD11B1 expression (p < 0.05). However, there was no effect of RR on
HSD3B2 expression.
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Figure 7. Effects of RR on the expression of key proteins related to steroid hormone biosynthesis.
Protein expressions and relative levels of (A) CYP19A1; (B) CYP17A1; (C) HSD11B1; (D) HSD3B2 were
analyzed by Western blotting. All values are expressed as the mean ± SD; n = 3. * p < 0.05, ** p < 0.01
compared with DEX group.
3. Discussion
GIOP, recognized as the most common iatrogenic cause of secondary osteoporosis, has imposed
a serious threat to public health. The present investigation found that RR could prevent GIOP in
both DEX-treated rats and DEX-injured osteoblasts. Our metabolomics analysis showed that DEX
caused great metabolic disorders, while RR could rebalance the metabolic disruptions, mainly via
regulating steroid hormone biosynthesis and amino acid metabolism. Through further pathway and
Western blotting analysis, it was ascertained that RR preventing GIOP was related to intervening
steroid hormone biosynthesis.
Both human and animal studies demonstrated deleterious skeletal effects within weeks
of pharmacological GC administration, as evidenced by the alteration of BMD and the bone
micro-architecture. The histomorphometric parameters of the trabecular bone could predict
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GC-induced osteopenia and the deterioration of bone quality. The present study found that RR
could improve the micro-architecture, enhance the BMD, and increase the trabecular parameters in the
femur in DEX-treated rats, suggesting that administration with RR was effective in both preserving
bone mass and rescuing the deterioration of bone micro-architecture. It is generally assumed that
bone loss in the chronic state of GIOP is mostly attributable to the decrease of osteoblastic bone
formation, and excessive GC can inhibit the osteoblastic proliferation, differentiation, and bone matrix
mineralization [12]. In this study, RR was able to improve the osteoblastic proliferation, ALP activity,
and bone matrix mineralization level, which further prove that RR has a potent anti-GIOP effect.
RUNX2 is a regulator in osteoblast differentiation at an early stage, and plays a crucial role in skeletal
morphogenesis, tooth development, chondrogenesis, and vasculogenesis [13]. It has been proved that
RUNX2 can upregulate the expression of the PI3K-Akt pathway, and enhance its DNA binding ability
in immature mesenchymal stem cells (MSCs) and immature osteoblasts [14]. The increased expression
of bone matrix proteins, such as ALP, BGP, and OPN, also stimulates mineralization and leads to
bone formation [15]. It was found in our study that RR could increase the ALP level in the serum of
DEX-treated rats, and enhance the expression of RUNX2 and OPN in DEX-injured osteoblasts, further
suggesting that RR was able to promote osteoblastic differentiation.
To delineate the mechanisms underlying the preventive effect of RR against GIOP, a metabolomics
profiling of rat urine based on UHPLC-Q-TOF/MS analysis was applied. It is well-known that the
deficiency of androgens or estrogens will induce bone loss [16]. Epiandrosterone is a metabolite of
the most abundant adrenal androgenic steroid dehydroepiandrosterone (DHEA) [17], and androgens
and estrogens are both made from DHEA [18]. It was found in our study that DEX decreased the
epiandrosterone or androsterone level in GIOP rats, while RR could reverse its abnormal descent
after eight-week treatment. It indicated that DEX might cause androgens and estrogens deficiency via
suppressing DHEA secretion, whereas RR made a significant callback of these hormone levels, and
further protected against glucocorticoid-induced bone loss. Pregnenolone is a well-known precursor
for the biosynthesis of various sex hormones, such as estrogen, progesterone, and testosterone [19].
It has been proved that pregnenolone and its heterocyclic analogues have the potential to become
novel anti-osteoporotic agents [20]. Hydroxypregnenolone is converted from pregnenolone by
cytochrome P450, and is also involved in the biosynthesis of gonadal steroid hormones and adrenal
corticosteroids [18,21]. In our study, a significant decrease of hydroxypregnenolone was found in GIOP
rats, and compared with that, there was a rising tendency after RR treatment, indicating that RR could
improve the pregnenolone level, and further sustain bone homeostasis.
Dehydrocorticosterone and corticosterone both belong to adrenocortical steroids. It was reported
that corticosterone and 11-dehydrocorticosterone levels were decreased in GIOP model rats [22],
which was consistent with the finding in the present study. Contrarily, elevated corticosterone,
18-hydroxycorticosterone, and 11-dehydrocorticosterone levels were detected after RR treatment.
In addition, it was found in our study that the correlation coefficient of 18-hydroxycorticosterone
and 11-dehydrocorticosterone vs. ALP and DPD levels was more than 0.6, suggesting that these two
metabolites might be related to bone metabolism. The conversion of inactive 11-dehydrocorticosterone
into active corticosterone is catalyzed by 11β-hydroxysteroid dehydrogenase (HSD11B1), which was
confirmed to play a crucial role in metabolically relevant tissues, such as skeletal muscles [23]. Previous
studies discovered a close relationship between HSD11B1 activity and osteoblast differentiation
after continuous injury of human osteoblasts with DEX, and proved that DEX could induce an
overexpression of HSD11B1 and decrease osteoblast differentiation [24]. In the present study,
an elevated HSD11B1 level was found in DEX-injured osteoblasts, while RR treatment could depress
its overexpression, further indicating that RR preventing bone loss might be related to regulating
HSD11B1 activity and its conversion of inactive 11-dehydrocorticosterone into active corticosterone.
Except for HSD11B1, some other key proteins participating in steroid hormone biosynthesis also
influence bone metabolism. Aromatase, known as cytochrome P450 19A1 (CYP19A1), is closely related
to postmenopausal osteoporosis. The aromatase deficiency can result in estrogen deficiency [25], and
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the estrogen deficiency at menopause accelerated the age-dependent involution of the female skeleton
and contributed to the loss of bone mass, architectural integrity, and strength [26]. CYP17A1 encodes
an enzyme with both 17α-hydroxylase and 17,20-lyase activities, and 17α-hydroxylase is responsible
for hydroxylating pregnelone and progesterone, which are then converted to C19 steroid precursors of
testosterone and estrogen by 17,20-lyase activity [27]. The deficiency of CYP17A1 can result in reduced
growth and osteoporosis. It was found in our study that RR could improve the CYP19A1 and CYP17A1
levels to some extent, further suggesting that RR preventing GIOP was related to intervening steroid
hormone biosynthesis.
In conclusion, pharmacological experiments and UHPLC-Q/TOF-MS-based metabolomics
analysis were used to evaluate the effects and underlying mechanisms of RR on protecting against
GIOP. In GIOP model rats, RR was able to improve the cancellous bone structure, enhance BMD,
and ameliorate bone metabolism homeostasis. In DEX-injured osteoblasts, RR could improve the cell
proliferation, differentiation, and mineralization level, and increase the expression of RUNX2 and OPN.
Metabolomics profiling indicated that RR might prevent DEX-induced bone loss through regulating
sex steroids regulation, steroid hormone biosynthesis and amino acid metabolism. Metabolic pathway
and Western blotting analysis further clarified that RR protected against GIOP, mainly via intervening
steroid hormone biosynthesis. The above results demonstrated, for the first time, that RR helped
protect against GIOP, and provided an excellent candidate for GIOP therapeutics.
4. Methods and Materials

Chemicals and reagents used in this study included chromatographic-grade methanol and
acetonitrile (Merck, Darmstadt, Germany); standard chemicals of catalpol, echinacoside, and acteoside
(HPLC ≥98%, Shanghai Yuan-ye Bio-Technology Co., Ltd., Shanghai, China); and DEX (Dalian Meilun
Biotech Co., Ltd., Dalian, China); ALN (MSD Pharmaceutical Co., Ltd., Hangzhou, China); Ca, P, ALP,
and TRAP assay kits (Nanjing Jian Cheng Bioengineering Institute, Nanjing, China); enzyme-linked
immunosorbent assay (ELISA) kits for BGP, DPD, and CTX-I (Xin Yu Biological Engineering Co., Ltd.,
Shanghai, China); antibodies specific for OPN, CYP17A1, CYP19A1, HSD11B1, and HSD3B2 (Abcom,
Cambridge, UK); antibodies specific for RUNX2, anti-rabbit IgG, anti-mouse IgG, and GAPDH (CST,
Danvers, MA, USA).
4.2. Preparation of RR Extract

RR is the processed product of the dry rhizome of Rehmannia glutinosa Libosch. In this study, RR
was purchased from Shanghai De Kang Pharmaceutical Co., Ltd. and authorized by Professor Qin
Luping, Department of Pharmacognosy, School of Pharmacy of the Second Military Medical University.
The voucher specimen (RR 20170328) was deposited in the herbarium of Second Military Medical
University. Dried RR (350 g) was cut into small pieces and reflux-extracted with 2.8 L distilled water at
60 ◦ C three times. The extract of RR was concentrated with a vacuum evaporator, and dissolved in an
appropriate volume of distilled water, and then adjusted to a concentration of 0.1 g/mL, 0.2 g/mL,
and 0.4 g/mL, equivalent to crude drugs for animal experiments, or 0.2 mg/L, 1 mg/L, and 5 mg/L,
equivalent to crude drugs for in vitro experiments. The dose of the RR extract was converted according
to the clinical dosage of humans.
4.3. UHPLC-MS Analysis of RR Extract

UHPLC-MS analysis was performed on an Agilent 1290 Infinity LC system coupled to an
Agilent 6538 accurate-mass quadrupole time-of-flight (Q-TOF) mass spectrometer (Agilent, Palo Alto,
CA, USA). Chromatographic separations were performed on an Acquity UHPLC HSS T3 column
(2.1 × 100 mm, 2.5 μm, Waters, Milford, MA, USA) at 25 ◦ C, and the injection volume was 3 μL at
a flow rate of 0.4 mL/min. The mobile phase consisted of 0.1% formic acid (A) and ACN modified
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with 0.1% formic acid (B). The gradient program was as follows: 5% B over 0–2 min, 5–95% B over
2–13 min, and 95% B over 13–19 min. The capillary voltage was 4 kV for positive ion mode and 3.5 kV
for negative ion mode. The drying gas flow rate was 11 L/min at 350 ◦ C. The nebulizer pressure was
45 psig, the fragmentor voltage was 120 V, and the skimmer voltage was 60 V. Data were collected in a
centroid mode and the mass range was m/z 100–1100 using an extended dynamic range.
4.4. Animal Treatment and Samples Collection

Female Sprague-Dawley (SD) rats aged 12 weeks and weighing 180–220 g were purchased from
Slacom (Shanghai, China), and maintained at the Experimental Animal Center of the Second Military
Medical University. The animals were housed at 24 ± 0.5 ◦ C with a 12-h light and 12-h dark cycle
with free access to food and water. After seven-day acclimatization, animals were randomly divided
into six groups according to their body weights (n = 8 per group): Normal (saline) group, DEX (DEX
2.5 mg/kg + saline) group, DEX + ALN (DEX 2.5 mg/kg + ALN 1 mg/kg) group, DEX + RR-L (DEX
2.5 mg/kg + RR 1 g/kg) group, DEX + RR-M (DEX 2.5 mg/kg + RR 2 g/kg) group, and DEX + RR-H
(DEX 2.5 mg/kg + RR 4 g/kg) group. At the first week, all rats except for those in the normal group
were injected with DEX daily via the caudal vein. Then, the rats were injected with DEX via the caudal
vein twice a week and orally administered with different dosages of RR daily for eight weeks.
The day before the end of the experiment, rats were housed individually in metabolic cages
for 24 h without providing food to collect urine samples. Then, all rats were anesthetized with
an intraperitoneal (i.p.) injection of 300 mg/kg chloral hydrate. The blood was collected from the
abdomen artery, and centrifuged at 3000 rpm for 10 min to collect the sera. Serum samples were stored
at −80◦ C for biochemical determination and metabolomics profiling. The right femur was prepared
for micro-computed tomography scanning. All studies were conducted in accordance with the NIH
publication and Second Military Medical University principles for laboratory animal use and care.
4.5. Micro-CT Analysis and Biochemical Marker Measurement

The measurement of trabecular micro-architecture was performed on an micro-CT system (eXplore
Locus, GE Healthcare, Boston, MA, USA) with the voltage set at 80 kV, current at 80 μA, angular
rotation at 360◦ , and angular step at 0.4◦ . The femur was scanned from the proximal growth plate in
the distal direction (14 μm/slice). The ROI was selected at a distance of 0.16 mm from the distal end of
the growth plate. BMD, BMC, TMD, TMC, Tb.Th., Tb.Sp., BVF, and BS/BV were measured.
The U-Ca, S-Ca, S-P, ALP, and TRAP levels were measured by standard colorimetric methods
using commercially available assay kits. The BGP, DPD, and CTX-I levels were measured using the
ELISA kits and the micro-plate reader (Thermo Fisher Scientific Inc., Pittsburgh, PA, USA).
4.6. UHPLC-Q-TOF/MS Metabolomics Analysis

Rat urine samples (100 μL) in the control, DEX, and DEX+RR-M groups were added into 300 μL
methanol solutions containing 5 μg/mL 2-chloro-L-phenylalanine as the internal standard. The mixed
solution was homogenized by vortex for 5 min and centrifuged at 13,000 rpm, 4 ◦ C for 15 min. A total of
150 μL supernatant was filtered through a 0.22 μm membrane for metabolomics studies. The method of
UHPLC-Q-TOF/MS metabolomics analysis was the same as that mentioned in Section 4.3. The mobile
phase consisted of 0.1% formic acid (A) and ACN modified with 0.1% formic acid (B), and the gradient
program was as follows: 5% B over 0–2 min, 5–15% B over 2–8 min, 15–30% B over 8–10 min, 30–95% B
over 10–13 min, and 95% B over 13–15 min. The reference ions were 121.0509 and 922.0098 in positive
mode, and 112.9856 and 1033.9881 in negative mode. The QC samples were used every eight urine
samples to evaluate the stability during sequence analysis.
4.7. Data Processing and Multivariate Analysis

All UHPLC-MS raw data were transformed into common data format (.mzdata) files using
Agilent MassHunter qualitative software. The program XCMS (http://metlin.scripps.edu/download/)
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was used for peak alignment, peak extraction, and automatic integration. After mean-centering
and pareto-scaling procedures, the retention time (RT)-m/z pairs, observations, and relative ion
intensities of all detected ions were imported into SIMCA-P 11.0 software package (Umetrics, Umea,
Sweden) for principal component analysis (PCA) and PLS-DA. HMDB (http://www.hmdb.ca/)
and Metlin database (https://metlin.scripps.edu/) were selected for metabolites matching based
on a combination of database queries using exact mass measurements. Additionally, the model
of PLS-DA was evaluated according to the cross-validation of R2 , Q2 value, and permutation test.
An independent sample t-test was performed for statistical analysis using SPSS version 20.0 (IBM,
USA) and p < 0.05 was considered statistically significant. A heat map of the different metabolites
was processed by MEV-MultiExperiment Viewer 4.8.1. The pathway analysis of potential biomarkers
was performed with MetaboAnalyst (http://www.metaboanalyst.ca/) and KEGG pathway database
(http://www.genome.jp/kegg/).
4.8. Osteoblasts Culture

Female Wistar rats aged 24 h were purchased from the Experimental Animal Center of the
Second Military Medical University (Shanghai, China). Primary osteoblasts were prepared from the
calvarias according to literature [28], and cultured in α-MEM supplemented with 10% FBS at 37 ◦ C in
a humidified atmosphere containing 5% CO2 . All studies were conducted in accordance with the NIH
publication and Second Military Medical University principles for laboratory animal use and care.
4.9. Osteoblastic MTT and ALP Activity Assay

Osteoblasts were injured with DEX (10 μM), and at the same time treated with RR (0.2, 1 and
5 mg/L) for 48 h in an MTT assay and for seven days in an ALP activity assay. In the MTT assay, 20 μL
of 5 mg/mL MTT was added into plates after drugs treatment. The medium was discarded and the
formazan crystals that formed in the cells were dissolved in 150 μL of DMSO. Optical density (OD)
was measured at 570 nm. For the ALP activity assay, the cells were lysed after drug treatment, and
the total protein concentration was measured using a BCA-protein assay kit. The ALP activity was
measured according to the conversion of colorless p-nitrophenyl phosphate to colored p-nitrophenol.
4.10. Osteoblastic Bone Matrix Mineralization Assay

Osteoblasts were cultured in osteogenic differentiation medium (α-MEM with 10% FBS, 50 μg/mL
ascorbic acid, and 10 mM β-glycerophosphate) for 12 days. Cells were injured with DEX (10 μM), and
at the same time treated with RR (0.2, 1 and 5 mg/L) for another nine days in osteogenic differentiation
medium. After washing, cells were fixed with ice-cold 4% paraformaldehyde for 10 min. Then, 0.1%
Alizarin Red staining solution (pH 8.3) was added and incubated for 30 min at 37 ◦ C. The staining of
Alizarin Red was incubated with 5% (v/w) cetylpyridinium chloride for 30 min at 37 ◦ C and OD was
measured at 570 nm.

Osteoblasts were injured with DEX (10 μM), and at the same time treated with RR (0.2, 1 and
5 mg/L) for 48 h. Then, cells were lysed and centrifuged at 12,000 rpm, 4 ◦ C for 10 min. The protein
concentration of the supernatants was determined using the BCA kit. Each sample (20 μg protein)
was separated by SDS-PAGE (10% gel) and transferred to polyvinylidene fluoride (PVDF) membranes.
The membranes were then blocked in 5% BSA, and incubated with the primary antibodies overnight at
4 ◦ C. After washing with TBS-T, the membranes were incubated with a second antibody for 1 h at room
temperature. Immunoreaction signals were detected using electrochemiluminescence (ECL) reagent
(Tanon, Shanghai, China), and exposed on a Gel imaging system (Tanon-5200 Multi, Shanghai, China).
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Molecules 2019, 24, 253

The data were expressed as the mean ± standard deviation (SD) and group differences were
determined by one way analysis of variance (ANOVA) with the Turkey test. The analyses were
performed using SPSS version 20.0 and p < 0.05 was considered statistically significant.
Supplementary Materials: The following are available online, Figure S1: Representative total ion chromatograms
(TIC) in metabolomics study; Figure S2: Fig. S2 Quality control (QC) superposed graph in metabolomics study.
Author Contributions: H.X. and L.Q. conceived and designed the research; T.X., X.D. and Y.J. performed
and analyzed the data of the metabolomics study; L.L., Z.D. and Y.S. performed and analyzed the data of
pharmacological experiments; T.X. and Q.Z. wrote the manuscript. All authors read and approved the manuscript.
Funding: This research was funded by National High-tech R&D Program of China (863 Program, Grant no.
2013AA093003), and National Natural Science Foundation of China (Grant no. U1505226, U1603283).
Conflicts of Interest: The authors declared no conflict of interest.
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molecules
Review
Use of Cannabidiol in the Treatment of Epilepsy:
Efficacy and Security in Clinical Trials
Serena Silvestro, Santa Mammana, Eugenio Cavalli, Placido Bramanti and Emanuela Mazzon *
IRCCS Centro Neurolesi “Bonino-Pulejo”, Via Provinciale Palermo, Contrada Casazza, 98124 Messina, Italy;
silvestro9110@gmail.com (S.S.); santa.mammana@irccsme.it (S.M.); eugenio.cavalli@irccsme.it (E.C.);
placido.bramanti@irccsme.it (P.B.)
* Correspondence: emanuela.mazzon@irccsme.it; Tel.: +39-090-6012-8172

Received: 20 March 2019; Accepted: 11 April 2019; Published: 12 April 2019
Abstract: Cannabidiol (CBD) is one of the cannabinoids with non-psychotropic action, extracted
from Cannabis sativa. CBD is a terpenophenol and it has received a great scientific interest
thanks to its medical applications. This compound showed efficacy as anti-seizure, antipsychotic,
neuroprotective, antidepressant and anxiolytic. The neuroprotective activity appears linked to its
excellent anti-inflammatory and antioxidant properties. The purpose of this paper is to evaluate the
use of CBD, in addition to common anti-epileptic drugs, in the severe treatment-resistant epilepsy
through an overview of recent literature and clinical trials aimed to study the effects of the CBD
treatment in different forms of epilepsy. The results of scientific studies obtained so far the use of
CBD in clinical applications could represent hope for patients who are resistant to all conventional
anti-epileptic drugs.
Keywords: cannabidiol; treatment-resistant epilepsy; clinical trials
1. Introduction
Cannabis sativa L. is an ancient medicinal plant wherefrom over 100 cannabinoids are extracted [1].
Among them, the most studied are Δ9 –tetrahydrocannabinol (Δ9 –THC), a psychoactive compound,
and the CBD, a non-psychotropic phytocannabinoid [2]. CBD is a cyclohexene which is substituted
in position 1 by a methyl group, by a 2,6-dihydroxy-4-pentylphenyl group at position 3, and with
a prop-1-en-2-yl group at position 4 (Figure 1). Most cannabinoids exert their action by interacting
with cannabinoid receptors, but CBD shows a low affinity for these receptors. Nevertheless, it
affects the activity of other receptors such as serotonin receptors [5-HT], opioid receptors [ORs],
and non-endocannabinoid G protein-coupled receptors (GPCRs) [3] and other targets (ion channels
and enzymes).
Figure 1. Structure of CBD.

Molecules 2019, 24, 1459
In recent years, the scientific community has shown interest in this compound due to its good
safety profile and neuroprotective properties [4] in several neurodegenerative diseases, including
Amyotrophic Lateral Sclerosis [5], Parkinson’s [6,7], Huntington’s [8] and Alzheimer’s diseases [9–11].
This neuroprotective action is due to its anti-inflammatory [12,13] and antioxidant [14,15] properties.
CBD shows anti-inflammatory properties in several experimental studies, modulating some
pro-inflammatory cytokines such as interleukin-1β (IL-1β ) [16], interleukin-6 (IL-6) [17,18] and tumor
necrosis factor α (TNF-α) [16,18], as well as regulation of cell cycle and immune cells’ functions [19].
Furthermore, another mechanism by which CBD performs its anti-inflammatory action is mediated
by interaction with the Transient Potential Vanilloid Receptor Type 1 (TRPV1). TRPV1 receptor is a
nonselective cation channel that, when activated, allows the influx of Ca2+ . The sensitivity but also
the density of TRPV1 is increased during neuro-inflammatory conditions. The binding of CBD to
TRPV1 leads to a desensitization of these receptors, with a consequent reduction in inflammation [20].
The CBD also carry out a potent antioxidant activity, modulating the expression of inducible nitric
oxide synthase and nitrotyrosine as well as reducing production of reactive oxygen species [21]. CBD is
also generating interest due to its therapeutic properties such as antidepressant [22], antipsychotic [23],
analgesic [24], and antitumor [25]. In addition, it has been shown that CBD can significantly reduce
two important forms of anxiety, namely obsessive-compulsive disorder [26] and post-traumatic stress
disorder [27,28].
Moreover, for a long time, the CBD has been investigated for its anticonvulsant effects [29–31].
Several studies confirmed its efficacy in the treatment of epileptic seizures, especially in pediatric
age [32,33]. In 2016, the first results of phase III clinical trials showed beneficial effects of CBD
(Epidiolex® ; GW Pharmaceutical, Cambridge, UK) in treatment-resistant seizure disorders, including
Lennox-Gastaut Syndrome (LGS) and Dravet syndromes (DS).
Epilepsy is a chronic neurological disorder. About 30% of epilepsy patients are affected by
Treatment-Resistant Epilepsy (TRE) due to the failure of common anti-epileptic therapies [34]. This form
of epilepsy is characterized by recurrent seizures that negatively affect the quality of life.
The purpose of this review is to provide an overview of recent clinical trials registered on
ClinicalTrial.gov. These trials study the use of different CBD formulation in patients affected by
severe forms of drug-resistant epilepsy. Moreover, we have described studies approved by local ethics
committees published in PubMed.
2. Epilepsy
According to the World Health Organization, epilepsy affects more than 50 million people
worldwide. Epilepsy is the most common neurological disorders characterized by recurrent
seizures [35]. A “seizure” is a paroxysmal transient phenomenon determined by an abnormal excessive
or synchronous neuronal activity in the brain [36]. Epilepsy can also cause deficit sensorimotor,
cognitive, compromising quality of life and an increased risk of premature death [37]. The International
League Against Epilepsy, according to the point of onset, classifies epileptic seizures into focal,
generalized and unknown seizures [38]. Focal convulsions caused by an anomalous electrical activity
in a circumscribed part of the brain and are classified into simple and complex. Simple focal convulsions
are characterized by motor, sensory and sensory manifestations without loss of consciousness. On the
contrary, complex focal convulsions involve a loss of consciousness [39]. Generalized seizures begin
in one or more areas of the brain and can then spread to the entire brain. Generalized seizures are
divided into crises absences, characterized by a rapid and transient loss of consciousness; tonic crises
that cause muscle stiffening; atonic crisis, characterized by loss of muscular control; clonic seizures
that cause rhythmic muscle movements; myoclonic seizures, characterized by muscle contraction and
localized tremors. Finally, tonic-clonic seizures represent the most serious type of epileptic seizures,
last about 5–10 min and are characterized by intense generalized contractions to the whole body [39,40].
The unknown seizures are called so when the beginning of a seizure is not known. These seizures
can also be defined as "epileptic spasms" characterized by sudden extension or flexion of the limbs. Is
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Molecules 2019, 24, 1459
defined Secondary Epilepsy when the onset is caused by several factors such as head trauma, infectious
diseases (meningitis, AIDS, viral encephalitis), developmental disorders, alcohol or drug abuse, and
other pathological conditions (brain tumors, stroke).
The most well-known epilepsies are DS, Sturge-Weber Syndrome (SWS), Tuberous Sclerosis
Complex (TSC) and West Syndrome (WS) and LGS. DS is a rare encephalopathy, which has its onset in
the first year of life [41]. DS is associated with the mutation in the gene encoding the α1 subunit of the
voltage gated sodium channel (SCN1A) [42]. SWS is caused by a somatic mutation of the GNAQ gene
(9q21) that encodes the Gq protein, involved in the intracellular signal of several G protein-coupled
receptors that control the function of various growth factors and vasoactive peptides [43]. Patients
manifest neurological abnormalities of variousdegrees, focal epileptic seizures [44]. TSC is an autosomal
dominant disease, caused by a mutation of two genes: TSC1 (localized on chromosome 9p34.3) that
encodes for hamartin and TSC2 (localized on chromosome 16p13.3) that encodes for tubulin. Often TSC
patients present generalized epilepsy. WS or Infantile Spasm (IS) is the epileptic encephalopathy. This
syndrome is characterized by genetic heterogeneity and the mutated gene most frequently observed in
patients with this syndrome is CDKL5 (cyclin-dependent kinase-like 5) [45]. WS is characterized by
the association between axial spasm discharges and psychomotor retardation [46]. LGS is a severe
epileptic encephalopathy of childhood. This syndrome is a rare condition likely associated with a
genes mutation. Nevertheless, to date, it is quite unclear how the involved genes may cause this
syndrome mainly characterized by recurrent seizures from early in life. An epileptic form that does
not respond to therapy with at least two or three appropriately selected anti-epileptic drugs (AEDs) is
defined as TRE and this is estimated to affect 30% of patients [47,48].
3. Common Antiepileptic Drugs

AEDs are the mainstay for the treatment of epilepsy and are intended to mitigate seizures.
Epileptogenic discharges occur as a result of neuronal hyperexcitability caused by voltage-dependent
ion channels and neurotransmitter concentrations alteration. AEDs primarily act reducing neuronal
excitability blocking excitatory neurotransmitter action such as glutamic acid and enhancing inhibitory
neurotransmitters such as γ-aminobutyric acid (GABA). Furthermore, the antiepileptic actions of most
AEDs are due to the modulation of voltage-gated ion channels such as sodium (Na+ ) and calcium
(Ca2+ ). The neuronal Na+ and Ca2+ channels are responsible for the rise of the action potential
and for the intrinsic excitability control of the neuronal system [49]. Some AEDs act inactivating a
single voltage-dependent channel while others instead simultaneously inactivate bothchannels. Both
of these mechanisms result in a reduction for neuronal hyperexcitability. Examples of drugs that
perform interacting with a single channel are phenytoin that selectively blocks the Na+ channel [50]
and ethosuximide that blocks the T-type Ca2+ channel [51]. Instead, carbamazepine, lamotrigine,
oxcarbazepine and zonisamide control seizures blocking both these voltage-dependent ion channels [52].
There are also anti-epileptics that act enhancing the GABAergic system. GABA is the main
inhibitory neurotransmitter of the nervous system that acts on GABA receptors, ligand-dependent
ionic receptors that increase chlorine conductance. AEDs are responsible for increasing GABA
transmission reducing neuronal excitability. Drugs that exert their action through these mechanisms
are benzodiazepines, phenobarbital, stiripentol, tiagabine and vigabatrin. Benzodiazepines (such as
clobazam diazepam, lorazepam, clonazepam) phenobarbital and stiripentol enhance the inhibitory
transmission of GABA by allosteric activation of the GABAA receptor thus increasing the frequency of
chloride (Cl− ) channel openings [53,54]. Vigabatrin, instead, is an inhibitor of GABA transaminase,
the enzyme responsible for the catabolism of GABA [55]. In addition to enhancing the inhibitory
transmission of GABA, other drugs exert their antiepileptic action, also exploiting the blockage of
the Ca2+ and Na+ channels. Among the AEDs that perform their action through these effects are
included, felbamate, lamotrigine and topiramate [56]. Other drugs are valproic acid and levetiracetam
that perform their mechanism of action enhancing the transmission of GABA and blocking Ca2+
channels [57,58].
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Molecules 2019, 24, 1459
The anticonvulsant and neuroprotective efficacy of some drugs is also given by the inhibitory action
of neurotransmitters, such as glutamate. Glutamate (or glutamic acid) is the most common excitatory
neurotransmitter and is responsible for excitatory transmission on neurons. Felbamate and topiramate
also perform their mechanism of action inhibiting glutamate thus decreasing l’ hyperexcitability
neuronal [59,60].
The choice of drugs is mainly linked to the identification of the type of seizure and epileptic
syndrome. For patients with epilepsy, effective seizure control is the determining factor for a good
quality of life. AED dosages must be individualized to maximize therapeutic effects and avoid side
effects. The early childhood epilepsy syndrome such as DS, LGS and WS present no easy medical
management due to the fact that subjects often show convulsion resistant to the available treatment.
Therefore, of safe and effective therapies arenecessary to reduce the risk of neurological sequelae.
The drugs preferentially used in particular forms of pediatric epilepsy are phenobarbital, phenytoin,
benzodiazepine, topiramate, levetiracetam and valproic acid [61].
4. Cannabidiol and Molecular Targets in Epilepsy

CBD shows a low affinity for endocannabinoid receptors and it carries out its mechanisms of
action by interacting with other molecular targets. One of the most important ion channel targets
towards which the CBD shows a high affinity is the Transient Receptor Potential Vanilloid (TRPV).
Specifically, TRPV1 is a non-selective channel that shows a high Ca2+ permeability and is involved
in the modulation of seizures and in epilepsy. In fact, when active, it promotes the release of glutamate
and the increase in Ca2+ , with consequent neuronal excitability [62]. The antiepileptic action of
CBD does not seem to be due to direct interaction with these molecular targets. However, it has
been observed that the CBD agonist action towards TPRV1 determines one a desensitization of
these channels with consequent normalization of intracellular Ca2+ [63]. T-Type Ca2+ , are another
class of ion channels with which CBD interacts. These channels control Ca2+ peaks in neurons
and they are involved in the regulation of cell excitability. The activation of these channels due to
a hyperpolarization of the membranes of neurons determines an increase in the concentration of
intracellular Ca2+ , in this way the T-Type Ca2+ channels increase the excitability of neurons. This
mechanism is often observed in pathophysiological conditions such as epilepsy [49]. The interaction of
the CBD with the T-type Ca2+ channels causes a blockage of these channels, this mechanism could
be responsible for the antiepileptic action, even if there are no studies available that confirm this.
Receptors represent other molecular targets that have been evaluated to describe their potential role in
epilepsy through interaction with CBD. Serotonin receptor (5-hydroxytryptamine [5-HT]) belonging to
the superfamily of the G protein-coupled receptors are divided into seven distinct classes (5-HT1 to
5-HT7 ). These receptors may depolarize or hyperpolarize neurons, modifying the conductance and/or
concentration ionic within the cells. This suggests that 5-HT receptors are involved in epilepsy even
though their role is still not entirely clear [64]. CBD shows a high affinity towards two subtypes of
serotonin receptors: 5-HT1A e 5-HT2A . These receptors can have different functions and regulatory
characteristics, in fact, for example, the activation of 5HT1 receptors in the hippocampus causes an
increase of neurotransmission; in contrast, in raphe nuclei, activation of 5-HT1A receptors produces the
inhibition of serotonergic neurons [65]. The dysregulation of brain neurotransmission mediated by
5-HT2 might results responsible for the pathophysiology of depression and epilepsy [66]. However,
although the role of serotonin receptors in epilepsy is unclear, 5-HT1A e 5-HT2A subtypes may represent
a valid therapeutic target through which CBD can perform its anti-epileptic action [61,67]. Opioid
receptors (OR) are G-protein-coupled receptors involved in a variety of brain disorders, including
epilepsy [68,69]. The CBD at high micromolar concentrations determines the blocking of μ and δ OR,
and this block would seem to generate anticonvulsant actions, even if there are still no studies to
support this theory. The CBD also shows a good affinity towards the orphan G-protein-coupled receptor
(GPR55), a class of receptors involved in the modulation of the synaptic transmission. The agonist
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Molecules 2019, 24, 1459
action of CBD towards these receptors would seem to attenuate synaptic transmission with consequent
antiepileptic effects [70].
An important enzyme target of CBD involved in epilepsy is cytochrome P450 (CYP450). CBD
inhibits CYP450 [71], but this mechanism does not seem to be directly involved in the antiepileptic
mechanism. It seems to be responsible for the hepatic metabolism of a variety of AEDs, as shown by
the combined administration of CBD and clobazam (CLB) [72].
5. Cannabidiol: Clinical Trials for Epilepsy

In the last decades, some clinical studies were conducted in order to investigate the potential
effects of the efficacy of CBD in the management of epilepsy. This review provides an overview of
the studies recorded on http://clinicaltrial.gov, distributing them into complete trials (see Table 1)
and ongoing trials. All trials test the use of CBD as an adjunct to common AEDs and most of them
assess the efficacy and safety of CBD especially in infants, children and teenagers. Trials exploring
the combination of CBD/Δ9 -THC have been excluded. In addition, we included further studies that
have been approved by local ethics committees (see Table 2). In the description of these clinical trials,
attention was also paid to the possible interactions of CBD with other anti-epileptic drugs.
5.1. Completed Clinical Trials

All clinical studies (phases 1, 2 and 3) reported below assess the safety and/or efficacy of CBD in
addition to common AEDs. Most of these studies enrolled pediatric patients (0.5 to 17 years) with
diagnoses of genetically based epilepsy, LGS, DS and WS, resistant to common antiepileptic treatments.
The following trials collect data on administration short-term CBD (from 10 days to 3 months).
A phase 1/2 clinical trial NCT02324673 has evaluated efficacy and safety of the CBD oral solution
at three different doses (10, 20 and 40 mg/kg/day) administered for 10 consecutive days in sixty-one
children (1–17 years) with drug-resistant forms of epilepsy. It was to assess the plasma concentration of
the CBD and its metabolite (7-hydroxycannabidiol, 7-OH-CBD) in blood samples collected at baseline,
at day 1 and at day 10 post-treatment at different time points (1, 2, 4, 8, 12, 24, 48, 72 h). On day one, the
plasma concentrations of CBD and its metabolite increase in a dose-dependent manner. These levels,
instead, decrease at the 10th day. Moreover, both at the first and at the tenth day CBD concentrations
are double those of its metabolite. A negative change in clinical global impression of severity score
at the end of treatment has shown an improvement of illness in all dosage. An improvement, in a
dose-dependent manner, has also been observed in daily seizure activity. Serious Adverse Events
(SAEs) such as apnoea, skin eruption and thrombophlebitis, were observed in 5% of patients that
received medium-dose CBD and in 9.5% that received high-dose. Further no-serious Adverse Events
(AEs), such as anaemia, gastrointestinal disorders (diarrhoea, flatulence, constipation, gastroesophageal
reflux disease, nausea), nervous system disorders (somnolence, psychomotor hyperactivity, seizure,
ataxia) were respectively observed in 65% of patients treated with low-dose CBD, in 45% treated with
medium dose and in 80.95% treated with high-dose. The results of this study showed that CBD can be
considered safe and tolerable even at high concentrations.
A phase 2, multi-center clinical trial (NCT02551731) enrolled 9 children (6 months to 36 months)
with a diagnosis of WS (Infantile Spasms) who have not responded to first-line therapies. CBD in oral
solution was administered at the initial dose of 20 mg/kg/day or 40 mg/kg/day. The protocol provided
the division of the study into two parts: Part A and Part B. In Part A was evaluated the efficacy,
defined as complete resolution of spasms and hypsarrythmia (if present at baseline) confirmed by
video-electroencephalogram (EEG) and safety at day 14 of treatment. Instead, in Part B was evaluated
the efficacy and the long-term safety up to 64 weeks of treatment. In Part A, 14.3% of children showed
complete resolution of spasms and hypsarrhythmia (when present at baseline) on day 14. Only in 33%
showed AEs, while no SAEs were recorded. No results of Part B of the clinical trial were recorded
because only one subject has completed the study. Then, these results confirm the efficacy and safety
of the CBD oral solution after 14 days of treatment.
161
Table 1. Completed cannabidiol clinical trials in epilepsy (https://clinicaltrials.gov/). The table shows the efficacy and safety of CBD in different forms of epilepsy. In
all studies, CBD is used as adjunctive therapy to conventional antiepileptic drugs.
Identifier Study Title Subjects Conditions CBD Dose Concomitant AEDs Efficacy Security Ref
A Study to Evaluate the Safety,
Tolerability and Efficacy of Oral
Administration of PTL101 16 children (2 Pediatric Intractable 25–450
NCT02987114 - - - -
(Cannabidiol) as an Adjunctive to 15 years) Epilepsy mg/kg/day
Molecules 2019, 24, 1459
Treatment for Pediatric

Intractable Epilepsy
Cannabidiol Oral Solution in SAEs in 5% of patients with
61 children (1 Resistant Seizure 10, 20, 40
NCT02324673 Pediatric Participants With - Improvement of illness medium-dose and in 9.5% -
to 17 years) Disorders mg/kg/day
Treatment-resistant Seizure Disorders with high-dose
Cannabidiol Oral Solution for Complete resolution of
9 infants (6 to Refractory Infantile 20–40
NCT02551731 Treatment of Refractory - spasm in 14.3% of children No SAEs were recorded -
36 Months) Spasms mg/kg/day
Infantile Spasms after 14 days of treatment
52 children
Cannabidiol Oral Solution as an SAEs in 77.78% of infants,
and young Treatment-resistant 10, 20, 40
NCT02318602 Adjunctive Treatment for - - in 38.46% of children and in -
adults (1 to 18 Seizure Disorder mg/kg/day
Treatment-resistant Seizure Disorder 0% of adults
years)
GWPCARE2 A Study to Investigate
150 children
the Efficacy and Safety of
and young 10, 20
NCT02224703 Cannabidiol (GWP42003-P) in Dravet Syndrome - - - -
162
adults (2 to 18 mg/kg/day
Children and Young Adults With
years)
Dravet Syndrome
4 children with concomitant
CLB, Valproate, - valproate showed elevate [78]
100 children
Safety, and Tolerability of Epidiolex Levetiracetam, damage of liver function
and young 5–50
NCT02695537 In Patients (Ages 1–19 Years) With Intractable Epilepsy Phenobarbital, Clonazepam,
adults (1 to 18 mg/kg/day Reduction of seizures of
Intractable Epilepsy Phenytoin, Carbamazepine,
years) 63.6% after 12 weeks of Improvement of AE Profile [79]
Lamotrigine,
treatment
Oxcarbazepine,
Ethosuximide, Topiramate, 4 children with concomitant
Vigabatrin, Zonisamide, - valproate showed elevate [78]
University of Alabama at 100 children Eslicarbazepine, Ezogabine, damage of liver function
5–50
NCT02700412 Birmingham (UAB) Adult and adults (15 EpilepsySeizures Pregabalin, Perampanel,
mg/kg/day Reduction of seizures of
CBD Program to 99 years) Rufinamide, Lacosamide
63.6% after 12 weeks of Improvement of AE Profile [79]
treatment
Table 1. Cont.
Identifier Study Title Subjects Conditions CBD Dose Concomitant AEDs Efficacy Security Ref
The median percent
CLB reduction in seizures SAEs were reported in
Efficacy and Safety of GWP42003-P
225 children Valproate frequency from baseline 19.40% of patients at the
for Seizures Associated With Epilepsy Lennox 10, 20
NCT02224560 and adults (2 Lamotringine was 37.2% in the 10 dose of 10 mg/kg/day of the [75]
Lennox-Gastaut Syndrome in Gastaut Syndrome mg/kg/day
to 55 years) Levetiracetam mg/kg/day CBD group; CBD and in 15.85% at the 20
Children and Adults (GWPCARE3)
Molecules 2019, 24, 1459
Rufinamide 41.9% in the 20 mg/kg/day mg/kg/day

CBD group
TEAEs in 5 patients; SAE in
10% of patients at the dose
CLB of 5 mg/kg/day, in 25% at
A Dose-ranging Pharmacokinetics
Valproate the 10 mg/kg/day and in
and Safety Study of GWP42003-P in 34 children (4 5, 10, 20
NCT02091206 Dravet Syndrome Stiripentol - 11.11% at the 20 mg/kg/day [73]
Children With Dravet Syndrome to 10 years) mg/kg/day
Levetiracetam dose.
(GWPCARE1)
Topiramate 6 patients with concomitant
valproate had elevated ALT
or AST
CLB
Antiepileptic Efficacy Study of The median frequency of
120 children, Valproate
GWP42003-P in Children and Young seizures decreased from
NCT02091375 young adults Dravet Syndrome 20 mg/kg/day Stiripentol SAEs in 16.39% of patients [74]
Adults With Dravet Syndrome 12.4 to 5.9, compared to the
(2 to 18 years) Levetiracetam
(GWPCARE1) placebo-treated group
Topiramate
163
A Study to Investigate the Efficacy
CLB Serious TEAEs occurred in
and Safety of Cannabidiol The monthly frequency of
171 children Valproate 4 patients;SAEs in 23.26% of
(GWP42003-P; CBD) as Adjunctive Lennox-Gastaut seizures decreased by a
NCT02224690 and adults (2 20 mg/kg/day Lamotrigine patients. 16 of the 36 [76]
Treatment for Seizures Associated Syndrome median of 43,·9% from
to 55 years) Levetiracetam patients on valproate had
With Lennox-Gastaut Syndrome in baseline in the CBD group
Rufinamide transaminase elevations
Children and Adults (GWPCARE4)
GWPCARE5 - An Open Label CLB
264 children, The monthly frequency of
Extension Study of Cannabidiol Dravet Syndrome Valproate
and adults (2 seizures decreased by a
NCT02224573 (GWP42003-P) in Children and Lennox-Gastaut - Stiripentol SAEs in 29.2% of patients [77]
years and median ranged from 38% to
Young Adults With Dravet or Syndrome Levetiracetam
older) 44%
Lennox-Gastaut Syndromes Topiramate
A Randomized Controlled Trial to
All participants reduced the 2 patients withdrew from
Investigate Possible Drug-drug 20 adults (18 to
NCT02565108 Epilepsy 20 mg/kg/day CLB maintenance dose of CBD the study due to SAEs -
Interactions Between Clobazam and 65 years)
from 10% for the day (seizure cluster)
Cannabidiol
Initial 20
An Open-label Extension Study to mg/kg/d
Investigate Possible Drug-drug 18 adults (18 to titrated to
NCT02564952 Epilepsy CLB - SAEs in 11% of patients -
Interactions Between Clobazam and 65 years) maximum
Cannabidiol dose of 30
mg/kg/day
CBD: Cannabidiol; TEAEs: Treatment-emergent adverse events; SAEs: serious adverse events; AST: aspartate transferase; ALT: alanine transferase.
Table 2. Data obtained from trials authorized by local ethics committees (https://www.ncbi.nlm.nih.gov/pubmed/). The table shows the efficacy and safety of CBD in
different forms of epilepsy. In all studies, CBD is used as adjunctive therapy to conventional antiepileptic drugs.
Study Design Subjects Conditions CBD Dose Concomitant AEDs Efficacy Safety Ref
Treatment-related SAEs were
recorded in 20 patients;
A prospective, 214 children and Initial 2–5 mg/kg/day The median reduction in SAEs were reported in 30% of
Drug Resistant CLB
Molecules 2019, 24, 1459
open-label, expanded adults (1 to titrated to maximum monthly motor seizure was patients. Thrombocytopenia and [83]
Epilepsy Valproate
access study 30 years) dose of 50 mg/kg/day of 36.5% elevated liver function test in
patients with
concomitant valproate
Overall quality of life
Children and Initial 2–5 mg/kg/day, significantly improved in
A prospective, Drug Resistant
adults (1 to titrated to maximum - 48 patients, The median - [86]
open-label study Epilepsy
30 years) dose of 50 mg/kg/day monthly seizures frequency
was 13.9
Epilepsy Dravet CLB
Median monthly
Syndrome CDKL5 Valproic acid
A prospective, 55 children and Initial 5 mg/kg/day convulsive seizure A serious treatment-emergent AEs
deficiency disorder Levetiracetam
multicentre, adults (1 to titrated to maximum frequency decreased from such as status epilepticus (9%) and [87]
Aicardi Doose Rufinamide
open-label study 30 years) dose of 50 mg/kg/day baseline by 51.4% at week respiratory infection (5%)
syndromes Dup15q Felbamate
12 and by 59.1% at week 48
syndromes Topiramate
164
Initial 5 mg/kg/day 12 patients reported 4 patients withdrew from the study
A prospective, 40 children (1 to Drug Resistant
titrated to maximum - substantial improvement of because of AEs; [88]
open-label study 17 years) Epilepsy
dose of 25 mg/kg/day the condition SAEs were reported in 15 patients
A median monthly seizure
607 children Initial 2–10 mg/kg/day frequency of 51% was
A prospective, multiple Drug Resistant SAEs were reported in 33%
(average age to maximum dose of recorded after 12 months of [89]
center, open-label study Epilepsy of patients;
13 years) 50 mg/kg/day treatment and maintained
at weeks 96
Levetiracetam
Valproic acid
Felbamate
CLB 50% of seizures reductions
Expanded access 5 infants (1 to Sturge-Weber Rufinamide in all patients; AEs were recorded during
2–25 mg/kg/day [85]
program 45 months) Syndrome Perampanel Improvements in quality of the study
Clorazepate life in all patients
Oxcarbazepine
Lacosamide
Topiramate
Table 2. Cont.
Study Design Subjects Conditions CBD Dose Concomitant AEDs Efficacy Safety Ref
50% in seizures reduction
in 33% of patients in the AEs in 36% of patients in the CLB
210 children
Retrospective study Epilepsy 2.9, 5.8 mg/kg/day CLB CBD group; in 44% of CBD group and in 7% of patients in [80]
(≤ 19 years)
+ CLB and in 38% of CLB CBD + CLB group
group
Molecules 2019, 24, 1459
Expand access 13 children and

50% of reduction in seizures
investigational new young (4 to Refractory Epilepsy 5–25 mg/kg/day CLB No serious AEs in 77% of patients [72]
in 69.23% of patients
drug (IND) trial 19 years)
CLB
Open-label, 78 healthy Moderate AEs in 8 patients; mild
- 750 mg twice daily Stiripentol - [81]
fixed-sequence trial subjects AEs in most of patients
Valproate
CLB
Lacosamide 4 patients recorded a
18 children and
Tuberous Sclerosis Levetiracetam reduction in seizure rate
Expanded access study adults (2 to 5–50 mg/kg/day AEs in 66.7% of patients [82]
Complex Lamotrigine greater than 80%; 1 patient
31 years)
Valproic acid became seizures-free
Rufinamide
Expanded access 26 children (1 to
Refractory epilepsy 5–25 mg/kg/day CLB A 50% reduction in seizures SAEs in 23.1% of patients [84]
program 17 years)
165
CBD: Cannabidiol; TEAEs: Treatment-emergent adverse events; SAEs: serious adverse events.
Molecules 2019, 24, 1459
The patients that concluded the clinical trials NCT02324673 and NCT02551731 have been involved
in the completed phase 3 open-label clinical-trial NCT02318602. The participants were divided for
an age range into three groups: infants (1 to <2 years of age), children (2 to <12 years of age) and
teenagers (12 to <17 years of age). All individuals continued the treatment with CBD at the same dose
of trials NCT02324673 (10, 20 and 40 mg/kg daily) and NCT02551731 (20 mg/kg daily) for 48 weeks.
The first outcome of this clinical trial was to evaluate the safety of CBD as adjunctive therapy for
children with treatment-resistant convulsive disorders. Patients following treatment with established
AEDs were continued uninterrupted, dose adjustments were allowed if necessary based on safety
concerns or changes in seizure control. SAEs such as seizures, status epilepticus and mental status
changes occurred in 77.78% of infants, in 38.46% of children and 0% of teenagers. No serious AEs
(anemia, diarrhoea, constipation, vomiting, infection of the upper respiratory tract, nasopharyngitis,
otitis media and influenza) occurred in 88.89% of infants, in 92.31% of children and 88.24% of teenagers.
In all patients, no significant changes were observed as respect to baseline in laboratory values or in
vital signs. These results show that while the administration of CBD cannot be considered safe in
infants, but it was generally well tolerated in adults.
The multicenter, open-label clinical trial, NCT03196934, is an extension of the NCT02318602 trial.
The aim of this study is to assess the long-term safety of CBD oral solution as an adjunctive treatment
for pediatric subjects with a treatment-resistant seizure. No result is available today.
Six randomized, double-blind, placebo-controlled studies were funded by GW Pharmaceuticals
for evaluated the activity of the new formulation of purified CBD oral solution (GWP42003-P or
Epidiolex), an epileptic medication and now Food and Drug Administration (FDA) approved for the
treatment of seizures associated with DS and LGS in patients two years of age or older.
The first clinical trial GWPCARE1 was divided into two parts: Part A (NCT02091206) and Part B
(NCT02091375). NCT02091206, a double-blind randomization study (phase 2), to evaluate the safety
of multiple doses of the CBD oral solution (GWP42003-P) in 34 children (4 to 10 years) with DS. All
patients before enrolment had to have stabilized all AEDs at least 1 month before and the therapy
stability had to be maintained during the study. Participants were randomized to one of the three doses
(5, 10 and 20 mg/kg/day) of active drug or placebo at a 4:1 ratio. In addition, patients had to take their
usual dose of antiepileptic drugs 2 h before CBD administration. The primary outcome was to assess the
incidence of Treatment-Emergent AEs (TEAEs). A pharmacokinetic evaluation was also performed by
measuring the plasma concentrations of CBD and its metabolites 7-OH-CBD and 7-carboxycannabidiol
(7-COOH-CBD), and of the most common antiepileptic drugs taken by patients. Serious TEAEs, such
as pyrexia and convulsions, occurred in five patients: one at the dose of 5 mg/kg/day, two at the dose
of 10 mg/kg/day, one at the dose of 20 mg/kg/day and one in the placebo group. No serious TEAEs
(such as pyrexia, somnolence, decreased appetite, sedation, vomiting, ataxia and abnormal behaviour)
reported in 80% of patients at 5 mg/kg/day of CBD, in 62.5% at the 10 mg/kg/day dose, in 77.78% at
the 20 mg/kg/day dose and in 85.71% of placebo group. SAEs such as status epilepticus, convulsion,
parvovirus infection, rash maculopapular, occurred in 10% of patients who received 5 mg/kg daily of
CBD, in 25% at the 10 mg/kg daily, in 11.11% at the 20 mg/kg daily and in the 14.29% in placebo group.
CBD was generally well-tolerated at the 5–20 mg/kg/day dose range. Elevated transaminases (ALT or
AST) were only reported with concomitant use of valproate. The study showed that exposure to CBD
and its metabolites increased in a dose-dependent manner, and 7-COOH-CBD was the most abundant
circulating metabolite at all doses and times. In fact, at the end of treatment, 7-COOH-CBD levels
were 13–17 times higher than those of CBD. The results also showed a pharmacokinetic interaction
of CBD with CLB, resulting in an increment of the metabolite N-desmethylclobazam [N-CLB] in
plasma exposure of the patients. An elevation in N-CLB, was absent in patients co-administered with
stiripentol, possibly reflecting prior inhibition of the CYP2C19 isoenzyme [73].
All doses of CBD were well-tolerated and the 20 mg/kg/day dose was chosen by the for Part B
(NCT02091375) study. NCT02091375 enrolled 120 children (2 to 10 years) with DS and drug-resistant
epileptic seizures. Patients received either the CBD oral solution at a dose of 20 mg/kg/day (n = 61)
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or placebo (n = 59), for 14 weeks, in addition to the standard antiepileptic treatment. During
CBD-treatment, SAEs (status epilepticus, convulsion and somnolence) occurred in 16.39% of patients
and in 5.8% of the placebo group. Instead, non-serious AEs (diarrhoea, vomiting, pyrexia, fatigue,
upper respiratory tract infection, nasopharyngitis, decreased appetite, somnolence, lethargy, headache,
convulsion, cough, irritability, gamma-glutamyltransferase increased, transaminases increased, weight
decreased) occurred in 75.41% of patients who had taken CBD at the dose of 20 mg/kg/day and in
47.46% of the placebo group. The results suggested that, following the administration of CBD, the
median frequency of seizures decreased from 12.4 to 5.9, compared to a decrease from 14.9 to 14.1 in the
placebo-treated group. In 43% of patients treated with CBD and in 27% of patients in the placebo group
occurred a reduction in seizure frequency by 50% or more and 3 patients were free of seizures [74].
Although the administration of CBD has caused high rates of AEs, CBD appears to be efficacy in the
treatment of patients with DS.
Subsequently, GW launched a second Phase 3 trial, GWCARE2 (NCT02224703), to evaluate DS
patients’ responses to either a low (10 mg/kg/day) or a high dose (20 mg/kg/day) of GWP42003-P for
14 weeks. The study, still recruiting, plans to enroll 150 participants, both children and adults (2 to
18 years). The results are not available.
The phase 3 clinical trial NCT02224560 (GWPCARE3) included 225 patients with LGS (2 to
55 years) with two or more seizures/week. This study was to evaluate the safety and efficacy
of the CBD oral solution (GWP42003-P) as an adjunctive treatment of other antiepileptic drugs.
The patients were divided into 3 groups and treated with CBD at the dose of 10 mg/kg/day or
20 mg/kg/day or with placebo for 14 weeks. During the treatment, SAEs such as pneumonia, status
epilepticus, elevated aspartate aminotransferase concentration, elevated alanine aminotransferase
concentration and elevated γ-glutamyltransferase concentration, occurred in 19.40% of patients treated
with 10 mg/kg/day of CBD, in 15.85% treated with 20 mg/kg/day and in 10.53% of the placebo group.
Increases in serum aminotransferase concentrations occurred only in patients treated with placebo.
While, non-serious AEs (diarrhea, vomiting, decreased appetite, pyrexia, fatigue, somnolence, upper
respiratory tract infection, nasopharyngitis) have been observed in 53.73% of 10 mg/kg daily group,
in 76.83% of 20 mg/kg/day group, and in 52.63% of the placebo group. A median percent reduction
from baseline in drop-seizure frequency was 37.2% in the 10 mg/kg/day CBD group, 41.9% in the
20 mg/kg/day CBD group, and 17.2% in the placebo group. The results reported by the authors show
that the addition of the CBD to conventional antiepileptic therapy reduces the frequency of seizures in
a dose-dependent manner [75].
The phase 3 clinical trial NCT02224690 (GWPCARE4) included 171 patients (aged between 2 and
55 years) with a diagnosis of LGS. Participants had to have taken one or more antiepileptic drugs
(the most used was lamotrigine, valproate and CLB) at a stable dose for at least 4 weeks prior to
screening as well as interventions for epilepsy. The first endpoint was to aim the efficacy of the CBD
oral solution (GWP42003-P) as adjunctive treatment in reducing the number of drop seizures when
compared to the placebo. The secondary endpoint was to assess the safety of CBD by measuring AEs
using standard severity measures. Individuals were divided into two groups: 85 received placebo and
86 received a CBD at a dose of 20 mg/kg/day for 14 weeks. SAEs (pneumonia, viral infection, alanine
aminotransferase increased, aspartate aminotransferase increased, γ-glutamyltransferase increased)
occurred in the 23.26% of CBD group and in 4.71% of patients in the placebo group. Serious TEAEs
(increased levels of alanine aminotransferase, aspartate aminotransferase and γ-glutamyltransferase)
occurred in four patients in the CBD group. Instead, the most common no serious-AEs (vomiting,
diarrhoea, loss of appetite and drowsiness) occurred in the 61.63% of CBD group and in 50.59% of
patients in the placebo group. After 14 weeks of treatment, the monthly frequency of seizures decreased
by a median of 43·9% from baseline in the CBD group. A reduction in seizures frequency of 50% or
more, was reported in 44% of patients in the CBD group and in 24% of patients in the placebo group.
The study found that in many patients treated with antiepileptic drugs that included CLB, a higher
onset of somnolence was observed. High levels of transaminases were recorded in patients treated with
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valproate. Nevertheless, the high rate of AEs, the results showed that the administration of long-term
CBD oral solution in patients with LGS determines the reduction in seizure frequency compared to
placebo [76].
Subsequently, all patients who completed the treatment period in NCT02091206, NCT02091375,
NCT02224703, NCT02224560 or NCT02224690 were included in the sixth clinical trial GWPCARE5
(NCT02224573). The results of this study will help to understand the safety of CBD administered over
long periods. Patients received an oral solution of CBD (100 mg/mL), titrated from 2.5 to 20 mg/kg/day
over a 2-week period, in addiction with their existing treatment. The median treatment duration was
274 days. SAEs such as status epilepticus and convulsion occurred in 29.2% of patients. Commonly
reported AEs (diarrhoea, pyrexia, decreased appetite and somnolence) occurred in 93.2% of patients.
17.2% of patients from GWPCARE1 that taking valproic acid, had liver transaminase elevations.
In patients from GWPCARE1 Part B, the monthly frequency of seizures from baseline decreased by
a median of ranged from 38% to 44% in 12-week periods up to week 48.85% of patients reported an
improvement in the overall condition after 48 weeks of treatment. This trial showed that long-term
CBD treatment was safe and efficacy to reduce seizure frequency in patients with treatment-resistant
DS [77].
A randomized controlled trial NCT02565108 (phase 2) included twenty patients (aged 18 to
65 years) with diagnosed epilepsy treated with CLB. This study examined the possible drug-drug
interactions between CLB and CBD. Participants before enrolment followed a stable therapy for at least
a month with antiepileptic drugs, including CLB. Patients received CBD oral solution (GWP42003-P) at
a dose of 20 mg/kg/day after taking CLB for 21 consecutive days. 75% of patients in the CBD group and
50% of patients in the placebo group showed non-serious AEs (diarrhea, nausea, vomiting, dizziness,
somnolence, sedation, dermatitis). Results showed that all participants reduced the maintenance dose
of CBD of 10%/day.
Eighteen participants who completed trial NCT02565108 were transferred to the open-label
extension (OLE) trial NCT02564952. The OLE phase was a safety study. Initially, all participants
received CBD at a dose of 20 mg/kg/day, thereafter the dose was decreased or increased to a maximum
of 30 mg/kg/day. All individuals during the study continued to receive CLB. In addition to CLB,
participants could not take more than two other AEDs during the study. Only seven of the 18 participants
completed the study, 11.11% showed SAEs (status epilepticus, seizure, alanine aminotransferase
abnormal, aspartate aminotransferase abnormal γ-glutamyltransferase abnormal). While, 94.44% of
patients presented no-serious AEs such as diarrhoea, vomiting, headache, hyponatraemia, dizziness,
seizure, somnolence, irritability, respiratory tract infection. The high rate of AEs in the concomitant
use of the CBD and CLB for prolonged periods of time may be unsafe.
The clinical trials of phase I NCT02695537 and NCT02700412, will evaluate prospectively and
longitudinally the safety and tolerability of CBD oral solution (Epidiolex) at various doses, between
5 mg/kg/day and 25 mg/kg/day with additional titration in some cases up to 50 mg/kg/day. These
two trials will enroll both 100 patients with drug-resistant epilepsy. Clinical trial NCT02695537 will
enroll patients aged 1 to 18 years, while the NCT02700412 patients aged 17 to 99 years. However,
Gaston, et al. [78] evaluated possible CBD interactions with antiepileptic drugs typically used in
39 adults and 42 children of these trials. An analysis was carried out to check for non-uniform
changes in both the CBD dose and the dose of other AEDs. In the two combined arms (pediatric and
adult) the results recorded linear increases in serum levels of topiramate, rufinamide and [N-CLB]
and linear decreases in CLB levels correlate with increasing CBD does. However, there were no
significant changes in the levels of other AEDs analyzed (valproate, levetiracetam, phenobarbital,
clonazepam, phenytoin, carbamazepine, lamotrigine, oxcarbazepine, ethosuximide, vigabatrin,
ezogabine, pregabalin, perampanel and lacosamide). During the study, six adults and eight children
showed sedation. The intake of concomitant CBD and valproate resulted in high levels of AST and ALT.
Liver function tests showed elevated damage greater than three times the normal limit in four children
who dropped out of the study, while the damages of about twice the upper normal limit in eight adults
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were resolved with valproate withdrawal. A major onset of somnolence following the concomitant
administration of CBD and CLB and high levels of transaminases following co-administration of CBD
and valproate was also recorded in another study [73] and in clinical trial NCT02224690. In conclusion,
the results obtained by the researchers show that the use of CBD with other drugs can be considered
safe. On the contrary concomitant use of CBD with valproate is not recommended as a significant
liver dysfunction has been observed. Probably because CBD enhances the toxic action of valproate.
The interaction between CBD and CLB was also highlighted. Since both of these drugs are metabolized
of the cytochrome P450 pathway, this interaction can often induce high plasma levels of n-CLB.
Therefore, it is important to monitor this drug-drug interaction. However, as the adverse effects
occurring, in this case, are not serious, the concomitant use of CBD with CLB can be considered safe
and above all effective, especially in pediatric patients with refractory epilepsy. Part of the results
of NCT02695537 and NCT02700412 were described by Szaflarski, et al. [79]. The study showed the
efficacy and safety of Epidiolex in 72 children and 60 adults. The results obtained show an average
reduction of all types of seizures of 63.6% with difference significant between baseline and 12 weeks.
The reduction in seizures seems to have remained stable, in fact, there were no significant differences
between 12 and 24 weeks and between 24 and 48 weeks. The severity of the seizures assessed by the
Chalfont Seizure Severity Scale (CSSS) also showed an improvement from a baseline score of 80.7 to
enroll at 39.3 at 12 weeks with CSSS scores stable even between 12 and 24 weeks and between 24 and
48 weeks. The analysis of AE Profile indicates a significant improvement in the presence/severity of
adverse events between the baseline and 12 weeks with stable AEDs thereafter without significant
differences between 12 and 24 weeks and between 24 and 48 weeks. The results of this study show
significant improvements in the profile of adverse events, in the severity of crises and in reducing
the frequency of seizures as early as 12 weeks; improvements that have been maintained during the
48 weeks of treatment.
A clinical trial of phase II NCT02987114, is an open-label, single-center trial, that recruited 16
children (aged 2-15 years), with intractable epilepsy. The aim of this trial was to evaluate the safety,
tolerability and efficacy of oral administration of PTL101 (formulation of seamless gelatin matrix green
beads containing CBD) as adjunctive therapy for pediatric intractable epilepsy. Patients at least 4 weeks
before enrolment had to have stabilized the doses of antiepileptic drugs. This clinical trial has included
4 weeks of observation of clinical parameters and 13 weeks of CBD treatment at an initial dose of
25 mg/kg daily up to the maximum dose of 450 mg/kg. Subsequently the patients were monitored for
2 weeks. The results of this study, not yet available.
The results obtained from all the completed studies show that CBD is a safe compound when
combined with common AEDs. An aspect of particular interest concerns the association of CBD with
valproate and CLB. In particular, some studies have shown that the association between CBD and
valproate leads to a reduction in liver function related to an increase in transaminases. This alteration
has been shown to be reversible and not to cause permanent liver damage. Pharmacokinetic studies
have shown that CBD determines, as associated with CLB, a plasma increase in the metabolites of
this benzodiazepine. All trials reporting efficacy data show that CBD is able to reduce the frequency
of seizures.
5.2. Ongoing Clinical Trials

In the last 4 years, various tests have been started to evaluate the efficacy and safety of CBD as an
antiepileptic. In this subsection, we have collected the trials that to date have not yet available results
as they are still in the patient recruitment phase.
Two phase 2 clinical trials NCT03355300 and NCT03336242 are expecting to recruit about
30 pediatric patients (3 to 17 years), with treatment-resistant childhood absence seizure. Both studies
will include three experimental treatment cohorts (20, 30 and 40 mg/kg/day). NCT03336242 will assess
efficacy, safety, tolerability and pharmacokinetics of CBD oral solution after 4 weeks of treatment. This
study will include a 4-week screening period and a 5 or 10 day titration period (depending on study
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cohort), a 4-week treatment period followed by 5-day tapering for doses >20 mg/kg/day and a 4-week
follow-up period. Instead, NCT03355300 will evaluate the long-term (up to approximately 54 weeks)
safety and tolerability of the CBD oral solution, monitoring the incidence of SAEs and AEs during and
after treatment. For both trials, the final data collection and the results are expected by the end of the
year 2019.
In the clinical trial NCT03676049, CBD will be administered as an adjunct to all current AEDs
in 5–10 patients (aged between 5 and 19) with refractory epilepsy. The CBD oral solution used
for treatment, with prior approval from the National Institute on Drug Abuse was prepared at the
University of Mississippi, and subsequently received FDA approval for compassionate use. A dosing
titration period will start with 100 mg/day, and will be titrated monthly as tolerated based on clinical
response, up to 300 mg/day. During the treatment period the patients will be subjected to control visits
at the baseline, at the fourth, at the eighth and at the twelfth weeks. During these visits the efficacy of
the treatment will be evaluated, observing the laboratory tests, quality of life of the patient, the profile
of the side effects and the crisis count. Patients who after 3 months of treatment show stability could
continue the use of CBD for another 3 months.
The clinical trial NCT02461706 will assess the safety and efficacy of CBD when administered as
adjunctive therapy in 50 children (2 to 16 years) who have resistant to AEDs. Patients treated with
AEDs were to have stabilized doses at least 4 weeks prior to enrolling. The study established the
starting dose of 25 mg/kg/day. Maximum dose titration should be achieved in most patients within
5 weeks. The patients will be clinically evaluated at baseline, once a month for three months and once
every three months thereafter. In addition, to ensure the safety of the study, all patients who reached
the maximum dose (more than 600 mg of daily) of the CBD will be monitored at least once a month
until the steady state of the maintenance dose was reached.
A double-blind, randomized, placebo-controlled phase 3 trial (NCT02783092), is intended to
evaluate the efficacy of the adjuvant use of CBD oral solution (200 mg/ml dissolved in corn oil), in
patients with epilepsy. The estimated 126 patients (2 to 18 years) will be treated with CBD at the initial
dose of 5 mg/kg/day, up to a maximum dose of 25 mg/kg/day. The primary outcome is to evaluate
whether CBD treatment resulted in a 50% reduction in seizure frequency compared to treatment with
antiepileptic drugs after 30 days. The results of this study (estimated final data collection in August
2020) will make it possible to clarify the efficacy of CBD at different doses.
The phase I clinical trial NCT02286986 is a multi-center study that to investigate the
pharmacokinetics and dose-ranging tolerability, efficacy and safety of CBD (GWP42003-P), in 25 children
and young adults (2 to 25 years) with epilepsy. The study was divided into two parts: Part A and Part
B. Part A was used to evaluate the safety and tolerability of more ascending doses of GWP42003-P
compared to placebo. The best-tolerated dose in Part A of the study was used to treat patients in Part B
for 60 consecutive days. The antiepileptic efficacy of GWP42003-P compared to placebo was evaluated
by monitoring the incidence in convulsions, determining the plasma concentration of GWP42003-P
and its main metabolite following the escalation of multiple doses of GWP42003-P. Furthermore, was
investigated the effect of GWP42003-P on the pharmacokinetics of concomitant and cognitive function,
sleep quality and daytime sleepiness were also observed. The trial is still active and the results have
not been published, yet.
Two phases II clinical trials NCT02607904 and NCT02607891 want to verify the possible drug-drug
interactions between GWP42003-P and two antiepileptic drugs, stiripentol or valproate in patients with
epilepsy. Both trials will enroll patients between 16 to 55 years. In the trials NCT02607904, patients
will be treated up to a maximum dose of 30 mg/kg/day for 12 months. Instead, in NCT02607891
trials, the participants will be randomized into a 4: 1 ratio to receive GWP42003-P or corresponding
placebo. The hypothesis is that levels of stiripentol or valproate may be altered as a result of using
GWP42003-P. During treatment, CBD will be administered at a maximum dose of 20 mg/kg/day for
25 days. Participants had to take stiripentol or valproate and no more than two other AEDs during the
blinded period of the study.
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Two phases III clinical trials NCT02953548 and NCT02954887 intended to evaluate the efficacy and
safety of CBD oral solution (GWP42003-P; GW Pharmaceutical) in infants with WS (Infantile Spasms).
These studies weredivided into 3 phases: a pilot safety phase; a randomized central controlled phase
and an open-label extension phase. The NCT02953548 will be described in the pilot phase. Two cohorts
of five participants will be enrolled sequentially. GWP42003-P will be administered up to a maximum
dose of 40 mg/kg/day for the 2-week treatment period. Instead, clinical trial NCT02954887 will be
an extension trial that will recruit 202 infants (from 1 month to 24 months), for 1 year of treatment.
The results expected from this study will allow observing if the administration of CBD will be effective
in infants with WS.
Two phases III clinical trial NCT02544763 and NCT02544750 (GWPCARE6) will evaluate
respectively, in a double-blinded phase and in an open-label extension phase, the efficacy of the
CBD oral solution (GWP42003-P) as adjunctive therapy by monitoring the frequency of seizures in
patients with TSC. NCT02544763 is expecting to recruit about 210 patients (1 to 65 years). All AEDs or
interventions will be stabilized at least 1 month before the screening and the stability of the therapy will
be maintained during the study. Patients will be treated with CBD at the dose of 25 or 50 mg/kg/day
for 16 weeks. The efficacy of the CBD will be tested by evaluating the change in seizure frequency.
Patients that will complete this blinded phase will be included in the NCT02544750 trial. The safety of
CBD administration will be measured based on the incidence of AEs. All participants will be dosed
up to a maximum of 50 mg/kg/day. From these two trials the results, not yet available, will help to
understand if the administration of CBD can lead to a decrease in the crisis in patients with TSC.
A phase 1/2 clinical trial NCT03014440, aim to determine the safety and tolerability of CBD
(Epidiolex) in addition to the anti-epileptic treatments in use, in patients aged 1 to 20 years with
drug-resistant epilepsy. Antiepileptic therapy followed by patients had to be stable for at least 1 month.
To date, there is no information available regarding the treatment, the doses used and the results.
The NCT02660255 is an observational, open-label, flexible dose study. The aim of this trial is to
evaluate the safety and efficacy of Epidiolex, in addition to common AEDs. The study will be recruited
subjects aged 1-60 years with treatment-resistant epilepsy. Patients prior to enrolment will be treated
with 1–4 AEDs on stable settings from least 1 month. Epidiolex will be administered for 1 year and
9 months. To date, no superior information and results are available, yet.
The clinical trial NCT02397863 is an open-label, multi-center study including patients (1 to 18 years
of age) with drug-resistant epilepsy. Patients are treated with CBD (Epidolex), the daily dosage is up
to 25 mg/kg/day with optional up-titration to a maximal daily dosage up to 50 m/kg/day until the end
of treatment. Treatment was provided for a total of 52 weeks. For this study the results have not been
published, yet.
Clinical trial NCT02332655 (phase 1/2) aims to assess the tolerability and optimal dose of CBD to
be used as a treatment in children and young adults with SWS and drug-resistant epilepsy to define
the optimal dose of Epidiolex. The study involving the recruitment of the 10 patients (aged 1 months
to 45 years) already in treatment with antiepileptic drugs. Patients treated with 1–5 basic antiepileptic
drugs had to have reached stable doses for a minimum of 4 weeks prior to enrolment. Treatment
will start with 2 mg/kg/day. The dose will be increased by 3 mg/kg/day after seven days and then by
5 mg/kg/day every seven days up to a maximum dose of 25 mg/kg/day given for 48 weeks. From the
expected results potential efficacy of CBD in refractory crises in patients with SWS will emerge.
An open-label observational study NCT02556008 will evaluate the efficacy of pure CBD for the
treatment of 25 children (1 to 17 years) with severe refractory epilepsy. The pure CBD used during
treatment is not approved by the FDA, therefore, investigators conducted this study through the
FDA’s expanded access mechanism for compassionate use. CBD will be administered as an adjunct
to all current anti-epileptic therapies. Patients had to undergo therapeutic treatment with 1-3 basic
antiepileptic drugs at stable doses for a minimum of 4 weeks prior to enrolment. The expected dosage
of the study was 2 mg/kg/day for a first week, 3 mg/kg/day for the second week, 5 mg/kg/day for the
third week up to a maximum dose of 25 mg/Kg/day. Seizure frequency will be assessed four weeks
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before the initiation of CBD, the next month, and at least every 3 months thereafter. The results of
efficacy of CBD are not yet available. Data from these studies will be available soon, as the final data
collection for many studies is expected by the end of 2019.
5.3. Clinical Trials Approved by Local Ethics Committees

In this subsection, clinical studies published in indexed journals have been described. Many of
these manuscripts report the results obtained on long periods of treatment with CBD and provide
important efficacy data. In addition, the common antiepileptic drugs taken by patients in association
with CBD are detailed.
The study conducted by Geffrey, et al. [72], approved by Massachusetts General Hospital (MGH)
Institutional Review Board (IRB) evaluated the CBD interaction with CLB. The aim of this study was to
evaluate possible interactions between CBD and CLB, assessing its efficacy, safety and pharmacokinetics.
For this study, 13 patients (4 to 19 years) with refractory epilepsy and treated with CLB were recruited.
Patients started taking CBD at a dose of 5 mg/kg/day and treated up by 5 mg/kg/day each week to
a dose of 25 mg/kg/day, for 8 weeks. CLB was administered daily at a stable dose of 0.5 mg/kg that
was decreased during the study when side effects were observed. The plasma levels of CBD, CLB and
[N-CLB] were measured at baseline and at weeks 4 and 8 of treatment. The results of the efficacy study
showed a 50% convulsion reduction in nine out of 13 subjects, corresponding to a 70% response rate.
In two patients, however, there was an increase in the frequency of seizures during the treatment period,
therefore the dose of CLB was reduced. Increases in plasma levels of CBD, CLB and its metabolite
were recorded. Already in the fourth week, the mean of CBL levels had been an increase of 60 ± 80%,
while the mean in [N-CLB] was an increase of 500 ± 300%. The results of the safety study show that in
77% subjects AEs were reported as somnolence (n = 6), ataxia (n = 2), irritability (n = 2), restless sleep
(n = 1), urinary retention (n = 1), tremor (n = 1) and loss of appetite (n = 1). After adjusting the doses of
CLB all AEs were resolved. Therefore, the results reported by the authors show an interaction between
CBD and CLB, and that CBD influences [N-CLB] levels much more than CLB levels.
The efficacy of the interaction between CBD and CLB was also seen in a study conducted by
Porcari, et al. [80]. This study was approved by the Vanderbilt University Institutional Review Board.
In this retrospective study, the aim is to define the efficacy of CBD alone or in association with CLB in
209 children (≤ 19 years) with epilepsy. The duration of treatment in patients receiving CBD was 1.1
years, while patients received CBD + CLB for 1.3 years, and for 2.5 years received CLB. The reduction
of antiepileptic drugs was seen in 21% of the CBD group, in 26% CBD + CLB and in 18% of CLB group.
No-seizures were observed in 14% of patients in the CBD group, in 9% of patients in the CBD + CLB
group and in 11% of patients in the CLB group. The results reported by the authors show a reduction
of the crises > of 50% in 33% of the CBD group, in 44% of the CBD + CLB group and in 38% of the
CLB group. It was also observed that LGS was the most commonly observed syndrome in all cohorts,
and in these patients, the response rate was 58% with CBD, 52% with CBD and CLB and 40% with
CLB alone. The sedation was the most common AEs reported in 36% of patients in the CLB group, in
7% of patients in the CBD + CLB group and in 0% of CBD group. This retrospective study suggests
that CBD is useful in the treatment of refractory epilepsy with benefits that cannot be attributed to the
interaction with CLB and increased levels of its active metabolite.
Morrison, et al. [81] conducted a pharmacokinetic study that evaluated the possible drug-drug
interactions between CLB, stiripentol or valproate and CBD (Epidiolex). This study was approved by
the Independent Ethics Committee of the Foundation Evaluation of Ethics in Biomedical Research,
Assen, The Netherlands. For this open-label phase I study, were enrolled 78 healthy subjects.
The primary outcome was to evaluate the interaction of multiple CBD administration as a perpetrator
drug with antiepileptic drugs (victim) at steady-state plasma concentrations: CLB (and its active
metabolite, N-desmethylclobazam); stiripentol and valproate (and its potentially hepatotoxic metabolite,
4-ene-VPA). On the contrary, was also evaluated the interaction of CBD (victim drug) and its metabolites
7-OH-CBD and 7-COOH-CBD at steady-state plasmatic concentrations, with multiple doses of CLB,
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stiripentol or valproate as perpetrators drugs. CBD was given at 750 mg twice daily, CLB at 10 mg/kg/day,
stiripentol at 750 mg and valproate at 750 mg twice a day. The results showed a significant interaction
between CBD and CLB. When CLB was used as the victim drug, significant increases in its metabolite
[N-CLB] were recorded. These increases are related to an inhibition of the CPY2C19. In addition, the
concentrations of the active metabolite 7-OH-CBD increased when was co-administered with CLB.
Stiripentol, however, increased by 28% when it is at steady-state plasma concentrations alone, and
by 50% following co-administration with CBD. The 50% increase in stiripentol concentration may be
caused by an inhibition of CPY2C19 by the CBD. Instead, co-administration of stiripentol with CBD
not caused an increase in CBD concentrations, but caused a 29% increase of 7-OH-CBD and 13% of
7-COOH-CBD. The interaction of the CBD and valproate did not affect the pharmacokinetics of the two
drugs. Regarding the safety study, six subjects were withdrawn due to adverse events; three when CLB
was added to the steady-state CBD and three when the valproate was added to the steady-state CBD.
Two subjects reported SAEs when CLB was co-administered to CBD. Moderate AEs were reported in
eight subjects; instead mild AEs were reported in most subjects. The results obtained by the authors can
be concluded by saying that the co-administration of drugs was moderately tolerated. Furthermore,
the drug-drug bidirectional interaction noted when CLB was co-administered with CBD, suggests a
dose reduction for CLB when administered with CBD.
Another study to evaluate the efficacy of the CBD oral solution (GWP42003-P) as a therapy for
drug-resistant epilepsy in TSC was conducted by Hess, et al. [82] (approved by Massachusetts General
Hospital Institutional Review Board and U.S. Food and Drug Administration). Of the 56 patients
enrolled in this study, only 18 patients (aged 2 to 31 years) were evaluated because they were affected
by TSC. At the time of enrolment, patients were taking between one and seven anti-epileptic drugs,
such as lacosamide (n = 14), CLB (n = 10), levetiracetam (n = 7), lamotrigine (n = 5), valproic acid
(n = 3) and rufinamide (n = 3). Treatment started at a dose of 5 mg/kg/day. This dose was increased
by 5 mg/kg/day every week up to the initial maximum dose of 50 mg/kg daily, for 12 months. After
the third month of treatment, doses of the CBD and concomitant AEDs could be adjusted monthly
in almost all patients in order to optimize seizure control. 15 patients achieved the initial maximum
dose of 25 mg/kg/day of CBD, while five achieved the highest dose of 50 mg/kg/day of the CBD, and at
this dose, none reported CBD-related AEs. Instead, six patients decreased the dose of CBD during the
study in order to alleviate AEs and interactions with concurrent AEDs. 66.7% of patients reported AEs
and among them, drowsiness, ataxia and diarrhoea. Three months after the treatment, in four patients
a reduction in seizure rate greater than 80% was recorded and one patient became seizure-free and he
remained free until the twelfth month. The results also show that in patients took CBD and CLB the
response rate after 3 months of treatment was 58.3% against 33.3% in patients who did not take CLB.
Given the results reported by the authors in this study, the CBD can be considered valid and safe in the
treatment of refractory epilepsy in the TSC.
Five patients enrolled in this study were included in another multicentre analysis of CBD
expanded-access conducted by Devinsky, et al. [83] (in 11 epilepsy centers in the USA). The aim of this
study was to assess safe, tolerated and effective of CBD (Epidiolex) in children and young adults with
severe, intractable, treatment-resistant epilepsy (the most common epilepsy syndrome treated were DS
and LGS). This study was approved by the institutional review boards at each study site. CBD was
used in addition to anti-epileptic treatment. For this trial, 214 patients were enrolled (1 to 30 years);
of these 162 patients after the first dose of CBD were monitored for 12 weeks and were included in
the safety and tolerability analysis while 137 patients (64%) were included in the efficacy analysis.
Patients started the treatment with CBD at the initial dose of 2–5 mg/kg daily up to a maximum
dose of 50 mg/kg daily for 12 weeks. In the safety study, SAEs were observed in 30% of patients.
Treatment-related serious AEs, such as status epilepticus, diarrhoea, pneumonia and weight loss were
recorded in 20 individuals. Instead, in 79% of patients showed no serious AEs, the most common were
decreased appetite, fatigue, somnolence, diarrhoea, convulsions, status epilepticus, sedation, lethargy.
After 12 weeks of treatment, results showed a median reduction in monthly motor seizures of 36.5%.
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In the patients with DS (n = 32), the treatment led to a median reduction of monthly motor convulsions
of 49%, in 16 patients a reduction of 50%. Instead, for patients with LGS (n = 30), an average reduction
of 36.8% in motor crises was recorded. Findings obtained from this study showed that CBD seems to
reduce the frequency of seizures and also shows an appropriate safety profile, even in patients with DS
and LGS.
Another study conducted by Sands, et al. [84] assessed the long-term safety, tolerability and
efficacy of CBD to children with refractory epilepsy. This study was approved by the Human Research
Ethics Committee of the UCSF Benioff Children’s Hospital. The CBD oral solution (Epidiolex) was
administered in addition to other anti-epileptic treatments in 26 patients (aged 1 to 17 years). The doses
of concomitant antiepileptic drugs had to be stable during the 4-week of baseline period and had
to remain stable during the first three months of treatment. CBD was administered at the starting
dose of 5 mg/kg daily and subsequently, weekly dosage was measured in increments of 5 mg/kg
daily up to a maximum dose of 25 mg/kg daily. The duration of therapy ranged from 4 to 53 months.
The patients underwent blood tests performed during the baseline period, after 1, 2 and 3 months and
thereafter every 3 months from treatment. Furthermore, the minimum concentrations of antiepileptic
drugs were evaluated. The frequency of seizures and AEs was monitored during the treatment period.
The primary outcome of the study was to test the efficacy of CBD in terms of > 50% reduction in
the frequency of motor seizures. Fifteen of 26 patients discontinued treatment, one due to a status
epilepticus, one for severe weight loss, all others for lack of efficacy. Instead, six patients showed SAEs
as status epilepticus (n = 3), catatonia (n = 2) and hypoalbuminemia (n = 1). 21 out of 26 patients
reported no serious AEs among which the most frequent were: reduced appetite (n = 10), diarrhoea
(n = 9), and weight loss (n = 8). In patients showing significant weight loss, the doses of CBD were
reduced. Changes in the concentrations of antiepileptic drugs were observed in four patients. Three of
them reported increased CLB concentrations, one reported an increment in phenobarbital contractions.
In three patients was observed an increment in aspartate aminotransferase and alanine transferase
levels when CBD was co-administered with valproate. The reduction in the frequency of seizures >
50%, was rediscovered in 38.4% of patients after 3 months of treatment, in 56.7% after 6 months, in
42.3% after 9 months, in 38.4% after 12 months, 42.3% after 18 months and 34.6% after 24 months.
In conclusion, after 24 months of treatment, of the 26 patients enrolled, only nine continued CBD as
adjunctive therapy. Of these patients, seven had a 50% reduction in the frequency of motor crises,
three of which remained completely free of seizures. Only seven of the nine patients who continued
treatment showed a reduction in seizure frequency > 50% after 36 months. The results reported by the
authors showed that long-term CBD results in a clinically significant reduction in seizure frequency,
and a low percentage of SAEs. Moreover, because treatment was stopped after a few months in most
patients, the number of patients exposed to CBD for a long time is low and the rate of adverse effects
over time may be underestimated.
Using the same CBD formulation (Epidiolex) and the same administration doses Kaplan, et al. [85]
conducted a study that was approved by the Federal Drug Administration for the use of Epidiolex
in the treatment of pediatric medically refractory epilepsy in SWS. In this study, five patients (aged
1 months to 45 years) were enrolled. The maximum dose of 25 mg/kg daily was tolerated only by two
patients, while in the other three the maximum tolerated dose was 20 mg/kg per day. Three participants
withdrew from the study, two due to lack of efficacy (week 38 and week 9), while one due to the
temporary increase in seizures during dose titration, but later re-enrolled. Three subjects remain in the
extension phase of the study continued to take CBD for more than a year. All subjects reported at least
one CBD-related adverse event during the study such as temporarily increased seizures, behavioral
issues, increased aspartate aminotransferase and fatigue. All transient AEs resolved spontaneously
after dose changes in concomitant anticonvulsants or CBD. Seizure reduction above 50% was seen
in two patients at weeks 14 and in three patients with bilateral brain involvement. Instead, subjects
reported improvements in quality of life during the treatment. As suggested by the results obtained
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from this study, CBD appears to be well tolerated and a valid candidate as adjunctive therapy for
seizures management in individuals with SWS.
Rosenberg, et al. [86] belonging to the same research group of Devinsky, et al. [83] prolonged the
study for evaluating the Quality of Life of Childhood Epilepsy (QOLCE) before and after treatment
with CBD (Epidolex). The study was approved by the NYU Langone Medical Center institutional
ethics board. For this study were enrolled patients (aged 1–30) with intractable treatment-resistant
epilepsy. In addition to the baseline antiepileptic drugs, patients were given CBD at the initial dose
of 2–5 mg/Kg daily up to the maximum dose of 50 mg/Kg daily for 12 weeks. After 12 weeks of
treatment with CBD, the median monthly seizures frequency was 13.9 and the median percent change
from baseline was –39.4%. In addition, the results indicated an improvement of 8.2 ± 9.9 points in
patient QOLCE. In fact, patients showed an improvement in behaviour, in memory, in energy/fatigue,
in control/impotence, in other cognitive functions and in global quality of life.
The same research group of Devinsky, et al. [87], conducted a study for evaluating the safety
and efficacy of long-term CBD administration in patients with severe childhood-onset epilepsy, and
with CDKL5 deficiency disorder and Aicardi, Doose syndromes and Dup15q syndromes. This study
was approved by the IRB at each institution. For this study, 55 patients aged between 1 and 30
were enrolled (with 55 in the safety group and 50 in the efficacy group). Patients were given a
pharmaceutical compound of Highly purified CBD (Epidolex). Treatment included 144 weeks of
Epidiolex administration in addition to anti-epileptic therapies at the starting dose of 5 mg/Kg per
day. During treatment, an increase of 2–10 mg/kg per day was carried out every two weeks up to the
maximum dose of 50 mg/kg per day. The efficacy study showed that the percent change in median
monthly convulsive seizure frequency for all patients after treatment decreased from baseline of
51.4% to week 12 and of 59.1% to week 48 with a no significant change between weeks 12 and 48.
After 12 weeks of follow-up was reported a decrease of 50% or more of seizures in 50% of patients
and in 57% at 48 weeks. The safety results of the drug showed that of the 55 patients,10 patients
withdrew by week 48, including 5 by weeks 12 and 48 due to lack of efficacy (n = 4) and AEs (n = 1). A
total of 15 (27%) participants withdrew by week 144 of extended follow-up. There were no deaths
during the study. SAEs that occurred during treatment were convulsions (9%), status epilepticus (9%)
and respiratory infection (5%). While other adverse events reported more frequently were diarrhea
(29%), drowsiness (22%) and fatigue (22%). These results can demonstrate the safety and tolerability
of long-term treatment with CBD and the reduction in seizure frequency in these four aetiologies
of epilepsy.
Chen, et al. [88] conducted an open-label study, the aim was to assess the tolerability and safety of
CBD (Epidiolex) in the treatment of drug-resistant epilepsy in children. Sydney Children’s Hospital
Network Human Research Ethics Committee approved the protocol for this study. Children (n = 40;
mean age 8.5 years) with drug-resistant epilepsy and uncountable daily seizures in focal/multifocal
epilepsy, epileptic encephalopathy, LGD and DS, were enrolled. CBD was administrated in addition to
anti-epileptic therapy at the initial dose of 5 mg/Kg daily for 12 weeks. The initial dose was increased
every week by 5 mg/Kg daily up to a maximum dose of 25 mg/Kg daily. During the treatment five
children withdrew from the study, two because he had an increase in the frequency of the seizures,
one because has manifested significant somnolence, one for respiratory depression and one because
their transaminase level was elevated. SAEs occurred in 15 out of patients, the frequent recurring
to treatment were increased seizure number (in eight patients), intercurrent illness (in five patients),
liver function disorder (in all patients), hyperlipidemia (in all patients), severe somnolence with
anorexia and respiratory depression (in one patient). Over-therapeutic phenytoin levels are another
SAEs manifested in two participants were considered related to treatment and occurred at doses
of 10 mg/kg/day and 20 mg/kg/day. All participants showed AEs not all attributable to treatment.
While, AEs that occurred frequently (15 individuals) and linked to the treatment was the drowsiness
(AEs spontaneously resolved the 10 participants), and gastrointestinal disorders (nausea, vomiting,
diarrhoea) in nine patients, somnolence (13 individuals) and increased seizures (two individuals).
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Instead, 12 children showed an improvement in health in general. The results reported in this study
show that Epidiolex can be considered useful as adjuvant therapy. The presence of adverse events and
possible interactions with antiepileptic drugs are important aspects to be taken into consideration.
Szaflarski, et al. [89] conducted an open-label, Expanded-Access Program (EAP) in 25 epilepsy
centers in the USA, and it was approved by an institutional review board at each site. The aim of
this study was to evaluate the safety and efficacy of CBD oral solution (Epidiolex), in addition to
common AEDs in patients with different forms of treatment-resistant epilepsies (TREs). For the study,
607 patients with a mean age of 13 were enrolled. All patients were included in the safety study,
while 508 were included in the efficacy study. Treatment involved a 4-week baseline period followed
by a 96-week treatment period. During treatment, patients received Epidiolex at the initial dose of
2–10 mg/Kg up to a maximum dose of 25-50 mg/Kg daily. 146 patients (mostly due to lack efficacy
[15%] or AEs [5%]) from the safety study group and 136 patients (mostly due to lack efficacy [15%]
or AEs [4%]) from the efficacy group were withdrawn from the study. SAEs were found in 33% of
patients such as convulsion (9%), status epilepticus (7%), pneumonia (5%), and vomiting (3%). Instead,
AEs manifested in 88% of patients the most common were diarrhea (29%), somnolence (22%), and
convulsion (17%). Already after 12 weeks of treatment, the median monthly frequency of seizure
convulsions was reduced by 51% and by 48 % the frequency of total seizures. These reductions
remained stable during the 96 weeks of treatment. Between weeks 12 and 96 the average dose of CBD
was 25 mg/kg daily, 55% of patients at follow-up had reduced the dose. Half of the patients taking
concomitant CLB and valproate reduced the dose compared to baseline during the study. While, most
of those who take simultaneously levetiracetam, had remained at their basal doses. The data obtained
from this study show that CBD as an adjunct treatment to common AEDs can be used in the long-term
effective treatment in patients with TRE.
A very interesting data in these studies is the pharmacokinetics and interaction of CBD with
common AEDs. The interaction of these drugs is very complex and is linked to the individual
metabolites produced and to the possible metabolic pathways that are involved. Specifically, the study
conducted by Geffrey, et al. [72] shows a bidirectional drug-drug interaction when CBD is administered
with the CLB for long period of time. Therefore, CLB determines an increase in serum levels of the
CBD metabolite (7-OH-CBD) and conversely, CBD causes an increase in the metabolite of CLB (n-CLB).
CBD is an inhibitor of CYP2C19, an enzyme involved in the degradation of n-CLB, these explain
how the CBD associated with CLB causes elevated plasma levels of this metabolite. In contrast, CBD
does not influence the pharmacokinetics of valproate and stiripentol when co-administered, moreover,
stiripentol causes a slight decrease in CBD metabolites (7-OH-CBD 7-COOH-CBD) while valproate
causes a slight increase of 7-OH-CBD. The mechanisms by which these interactions take place are not
yet known but do not cause clinically relevant effects. However, the CBD shows good safety profiles,
the interaction with these two drugs does not require the interruption of therapy. The modulation of
the dose of these AEDs will be sufficient to resolve the adverse events. In conclusion, the results of
these studies show that the administration of CBD as an addition to the common AEDs for long periods
of time leads to clinically significant reductions in the frequency of convulsive and total seizures in
different etiologies of epilepsy. Furthermore, an improvement in the quality of life of these patients
was also observed.
6. Conclusions
The CBD is a compound extensively studied for its potential efficacy for the treatment of epilepsy.
In this review, we reported the studies conducted in infants, children and teenagers affected by epilepsy
resistant to common AEDs.
To date, available safety data show that the administration of CBD associated with other AEDs
causes non-serious adverse events, which can be resolved reducing the dose of CBD and/or common
AEDs. In this context, particular attention should be paid when CBD is associated with valproate and
CLB. Specifically, abnormal liver function was noted in participants taking concomitant valproate,
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therefore, it is necessary to monitor serum levels of these compounds and their respective metabolites.
Instead, when CBD is associated with CLB it induces an increase in its metabolites. Since the adverse
effects are not serious, this association can be considered safe.
The available results also highlight the efficacy of CBD as adjunctive to common AEDs.
The mechanism by which CBD interacts with other AEDs is not yet fully known, as many metabolic
pathways involved in this interaction are still unknown. In addition, not all the molecular targets used
by the CBD to exercise its antiepileptic action are yet known. However, the results obtained to date
encourage the use of CBD associated with AEDs.
Author Contributions: S.S. wrote the manuscript; S.M. contributed to bibliographic research; E.C. contributed in
graphical support; P.B. and E.M. revised the manuscript.
Funding: This study was supported by current research fund 2019, Ministry of Health, Italy.
Acknowledgments: This manuscript was supported by grants of the Italian Ministry of Health.
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molecules
Article
Hepatotoxicity of a Cannabidiol-Rich Cannabis
Extract in the Mouse Model
Laura E. Ewing 1,2 , Charles M. Skinner 1,3 , Charles M. Quick 4 , Stefanie Kennon-McGill 1 ,
Mitchell R. McGill 1,2,3 , Larry A. Walker 5,6 , Mahmoud A. ElSohly 5,6,7 , Bill J. Gurley 3,8 and
Igor Koturbash 1,3, *
1 Department of Environmental and Occupational Health, University of Arkansas for Medical Sciences,
Little Rock, AR 72205, USA; leewing@uams.edu (L.E.E.); cmskinner@uams.edu (C.M.S.);
skennonmcgill@uams.edu (S.K.-M.); mmcgill@uams.edu (M.R.M.)
2 Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock,
AR 72205, USA
3 Center for Dietary Supplements Research, University of Arkansas for Medical Sciences, Little Rock,
AR 72205, USA; gurleybillyj@uams.edu
4 Department of Pathology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA;
quickcharlesm@uams.edu
5 National Center for Natural Products Research, University of Mississippi, University, MS 38677, USA;
lwalker@olemiss.edu (L.A.W.); melsohly@olemiss.edu (M.A.E.)
6 ElSohly Laboratories, Inc. (ELI), Oxford, MS 38655, USA
7 Department of Pharmaceutics and Drug Delivery, School of Pharmacy, University of Mississippi, University,
MS 38677, USA
8 Department of Pharmaceutical Sciences, University of Arkansas for Medical Sciences, Little Rock, AR 72223,
USA
* Correspondence: ikoturbash@uams.edu; Tel.: +1-501-526-6638; Fax: +1-501-526-6931
Received: 11 April 2019; Accepted: 29 April 2019; Published: 30 April 2019
Abstract: The goal of this study was to investigate Cannabidiol (CBD) hepatotoxicity in 8-week-old
male B6C3F1 mice. Animals were gavaged with either 0, 246, 738, or 2460 mg/kg of CBD (acute toxicity,
24 h) or with daily doses of 0, 61.5, 184.5, or 615 mg/kg for 10 days (sub-acute toxicity). These doses
were the allometrically scaled mouse equivalent doses (MED) of the maximum recommended human
maintenance dose of CBD in EPIDIOLEX® (20 mg/kg). In the acute study, significant increases
in liver-to-body weight (LBW) ratios, plasma ALT, AST, and total bilirubin were observed for the
2460 mg/kg dose. In the sub-acute study, 75% of mice gavaged with 615 mg/kg developed a moribund
condition between days three and four. As in the acute phase, 615 mg/kg CBD increased LBW
ratios, ALT, AST, and total bilirubin. Hepatotoxicity gene expression arrays revealed that CBD
differentially regulated more than 50 genes, many of which were linked to oxidative stress responses,
lipid metabolism pathways and drug metabolizing enzymes. In conclusion, CBD exhibited clear signs
of hepatotoxicity, possibly of a cholestatic nature. The involvement of numerous pathways associated
with lipid and xenobiotic metabolism raises serious concerns about potential drug interactions as
well as the safety of CBD.
Keywords: cannabidiol; hepatotoxicity; liver injury; natural products; phytochemical
1. Introduction
Cannabidiol (CBD) is a non-psychotropic phytochemical present in Cannabis sativa that has gained
significant popularity over the last decade. It is a major component of EPIDIOLEX® , a drug indicated
for the treatment of drug-resistant epileptic seizures associated with Dravet and Lennox-Gastaut

Molecules 2019, 24, 1694
syndromes [1,2]. CBD has also been proposed as treatment for a number of other neuropsychiatric
disorders for which clinical trials are currently ongoing [3].
CBD has also been marketed for a wide range of other indications, including ‘anti-cancer’,
‘anti-inflammatory’, ‘sleep promotion’, ‘relaxation’, ‘normal cartilage and joint function’, ‘antioxidant
effects’, and ‘pain management’ just to name a few. The vast majority of those effects, however, were
documented either in vitro or in clinical trials with equivocal results [4,5]. Apart from its purported
salutary effects, accumulating evidence from pre-clinical in vivo studies and large-scale clinical trials,
implies that CBD may elicit several potentially negative health outcomes. Specifically, numerous
reports have demonstrated neurological, cardiovascular and reproductive toxicities subsequent to
CBD use [6–14]. The authors of a large clinical trial that utilized CBD (dose regimen 2.5–30 mg/kg/day)
to treat 278 patients with Dravet syndrome reported adverse events in 93% of subjects [15]. Another
recent study inferred a strong genotoxic potential for CBD at concentrations commonly detected in
human blood [16]. Furthermore, CBD may have a high drug interaction potential as it modulates
numerous cytochrome P450 enzymes responsible for xenobiotic metabolism [17–21].
Of particular concern is the risk for CBD-induced hepatotoxicity [22]. Animal studies have
reported increased liver weights in rhesus monkeys and elevated liver enzymes in dogs when CBD was
administered at doses as low as 2 mg/kg of body weight [14,23]. In recent clinical trials, elevated liver
enzymes were observed in 5–20% of patients treated with CBD, and a few patients were withdrawn
due to the threat of fulminant liver failure [1,2,24].
The number of ‘CBD-containing’ products, available mostly online, is growing exponentially.
However, the U. S. Food and Drug Administration (FDA) prohibits sales of CBD as a dietary
supplement or food ingredient on the grounds that any ‘article’ that has been approved as a new
drug or authorized for investigation as a new drug cannot be marketed as an ingredient in dietary
supplements or conventional foods per the Food, Drug, & Cosmetic Act (FDCA) [21 U.S.C. §321(ff)(3)(B)
and 21 U.S.C. §331(II), respectively] [25]. Furthermore, a clear regulatory oversight exists which has
led to an uncontrolled CBD market that, in turn, threatens the health of a trusting general public.
For instance, in a series of tests performed by the FDA on a panel of ‘CBD-containing products’, a
large fraction either did not contain the label-claimed quantity of CBD or they were contaminated
with Δ9-tetrahydrocannabidiol (THC) [26]. Furthermore, a recent independent analysis performed by
CosumerLab.com, revealed that CBD doses in commercially-available products ranged from as little as
2.2 mg to as much as 22.3 mg, further amplifying concerns of potential toxicity [27].
As expansion of the CBD market seems inevitable, additional scientific studies are needed in
order to support any required regulatory actions. For instance, if CBD is to be considered as a food
additive, it will have to be filed as a new dietary ingredient (NDI) or a GRAS (generally recognized as
safe) notice will need to be submitted to FDA. The latter will require a number of toxicity studies, the
majority of which, in the case of CBD, remain to be performed. Analysis of genotoxic potential of CBD,
the first toxicity test recommended by the FDA, was recently performed and the results published [16].
Therefore, we proceeded to the next set of recommended tests designed to address the short-term
toxicity of a CBD-rich extract in a rodent model. Since liver injury is the primary concern for CBD,
this study was designed to investigate the hepatotoxicity potential of CBD. The data collected in this
study will provide important information for both industry and regulatory agencies in regards to the
short-term toxicity of CBD. Furthermore, the results of these studies will aid in selecting appropriate
models and doses for long-term studies (i.e., sub-chronic and chronic toxicity as well as carcinogenicity
and reproductive toxicity studies) as well as the determination of a no observable effect level (NOEL)
for selected endpoints.
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2. Results
2.1. Acute Toxicity Study

For acute toxicity studies, a dose of 246 mg/kg was chosen as an initial dose as this is a MED
analogous to those used in recent clinical trials (MED of 20 mg/kg CBD) [1,2]. Subsequently, we used
doses of 738 and 2460 mg/kg CBD as 3× and 10× doses, respectively.
Mice at 738 mg/kg and 2460 mg/kg groups developed a sub-lethargic condition which presented
as decreased appetite and slow response to exogenous stimuli at 4–5 h after CBD administration.
This was still evident in mice receiving the 2460 mg/kg dose at the 24 h time-point. Administration
of 2460 mg/kg CBD led to marginal body weight loss (9.5% from control) whereas the response in
738 mg/kg mice was mixed, with 4 mice exhibiting patterns of substantial weight loss (11%–15%)
while two gained body weight (~10%) (Figure 1A). Further, 738 mg/kg caused significant increases
in liver-to-body weight ratios (18%, p < 0.005), while 2460 mg/kg was characterized by an uneven
distribution of these values (Figure 1B), even when using pre-gavage body weights to calculate organ
to body weight ratios. No significant differences in kidney- and heart-to-body weight ratios were
observed (Supplementary Figure S1A,B). No appreciable histomorphological differences were observed
between control and experimental mice (Supplementary Figure S2). A small dose-dependent increase
in total glutathione (GSH + GSSG) was observed in mice gavaged with CBD; however, increased
levels of oxidized glutathione (GSSG) and GSSG/GSH ratio were also observed in mice gavaged with
2460 mg/kg CBD (Figure 1C–E).
Figure 1. Effects of single gavage with CBD. Mice were gavaged with 246, 738, or 2460 mg/kg of CBD
in sesame oil with tissues harvested at 24 h. (A) Body weight change, (B) liver to body weight ratios,
intrahepatic concentrations of (C) total glutathione (GSH), (D) reduced glutathione (GSSG), and (E)
GSH/GSSG ratio. Data are presented as mean ± SEM (n = 6). * indicates a significant difference as
calculated with a One-Way ANOVA and Tukey’s post-hoc test, and # indicates a significant difference
as calculated with a Kruskal-Wallis test with a Dunn’s post-hoc test (p < 0.05).
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Clinical biochemistry analysis revealed moderate, but statistically significant (p < 0.01–0.001),
dose-dependent increases in both AST and ALT serum levels (Table 1, Supplementary Figure S3A,B).
Administration of 2460 mg/kg CBD led to marked elevations of total bilirubin (>20-fold, p < 0.001)
(Table 1, Supplementary Figure S3C). No significant differences were observed in ALP or GGT
(Supplementary Figure S3D–E).
Table 1. Clinical chemistry parameters 24 h after dosing with CBD oil. Cells in bold italics are
significantly different from vehicle (One-Way ANOVA, indicated by *, or Kruskal-Wallis with appropriate
post-hoc test). Data presented as mean ± SEM (n = 6/group).
Vehicle 246 mg/kg 738 mg/kg 2460 mg/kg

ALT 27.5 ± 2.7 31.7 ± 2.6 38.7 ± 4.3 * 57.0 ± 8.0 *
AST 51.0 ± 2.0 58.7 ± 1.8 80.3 ± 3.7 * 120.8 ± 14.3 *
ALP 110.8 ± 2.9 106.0 ± 4.5 90.3 ± 8.8 98.8 ± 4.2
GGT 4.7 ± 0.2 4.3 ± 0.5 5.5 ± 0.3 4.2 ± 0.8
Total Bilirubin 0.1 ± 0.0 0.1 ± 0.0 0.13 ± 0.0 2.1 ± 0.1
Gene expression analysis of a panel of cytochromes and UDP-glucuronosyltransferases revealed

significant and mostly dose-dependent increases in many isoforms (Figure 2). Of particular interest
was the striking up-regulation of Cyp2b10 (homologous to human CYP2B6) that exceeded 1000-fold at
2460 mg/kg CBD (p < 0.001). Cyp1a2 and Cyp2e1, major cytochromes involved in the metabolism of
ethanol and acetaminophen (APAP), were increased by 4- and 50-fold, respectively. At the same time,
Cyp2d22, an isoform considered non-inducible, remained unaffected, lending further credence to the
findings (Figure 2).
2.2. Sub-Acute Toxicity Study

Given the overt toxicity observed with 2460 mg/kg, we used a starting CBD dose of 61.5 mg/kg for
this phase of experiments. This dose is an MED of 5 mg/kg CBD, which it turn is analogous to initial
target doses administered in clinical trials for treatment-resistant epilepsy [28]. Subsequently, doses of
184.5mg/kg and 615 mg/kg CBD were considered as 3× and 10× doses, respectively.
Shortly after the second gavage with CBD, overt toxicity manifested as profound lethargy, loss of
appetite, and body weight loss was observed in 33% of animals (2 out of 6) in the 615 mg/kg group. Two
more mice developed similar symptoms after the third gavage. Thus, the remaining four animals were
terminated at the end of day 3 (6 h after the third CBD dose). The remaining mice in the 61.5 mg/kg and
184.5 mg/kg cohorts were gavaged as scheduled for 10 days and exhibited no visible signs of toxicity.
Histopathological evaluation revealed pan-hepatic cytoplasmic swelling in mice gavaged with
615 mg/kg CBD (Figure 3). Foci of cytoplasmic swelling were clearly present in mice gavaged with
184.5 mg/kg, but not in the livers of mice gavaged with 61.5 mg/kg CBD.
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Figure 2. Effects of single gavage with CBD on intrahepatic expression of cytochrome P450s and
UDP-glucuronosyltransferases. Livers were collected at 24 h and gene expression was measured using
the quantitative real-time (qRT) PCR. * - indicate data analyzed by One-Way ANOVA with Tukey’s
post-test, and # indicate non-normal data analyzed with a Kruskal-Wallis and Dunn’s post-hoc test.
Data are presented as mean ± SEM fold changed from vehicle (n = 6), with * or # as p < 0.05; ** or ## as
p < 0.01; *** or ### as p < 0.001; and **** or #### as p < 0.0001.
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Figure 3. Effects of 2-week administration of CBD on liver histomorphology. H&E stained liver sections
from (A) vehicle mice or those gavaged with (B) 61.5 mg/kg, (C) 184.5 mg/kg, or (D) 615 mg/kg CBD in
sesame oil for 2 weeks. Note that 615 mg/kg group was terminated after 2–3 doses due to overt toxicity
elicited by CBD.
Gavaging mice with 615 mg/kg CBD resulted in significant reductions in body weight (10%,
p < 0.05) (Figure 4A). Furthermore, we observed a dose-dependent increase in liver-to- body weight
ratios (5–30% range) (Figure 4B). Kidney-to-body weight ratios were significantly decreased in mice
receiving 615 mg/kg CBD (Supplementary Figure S4). There were no significant differences in the total
glutathione levels in any experimental groups and only modest changes were observed in GSSG and
GSSG/GSH ratios (Figure 4C–E).
Analysis of clinical biochemistry parameters revealed that mice receiving 615 mg/kg CBD had
significantly elevated total bilirubin, and moderately high levels of ALT and AST (Table 2, Supplementary
Figure S3). However, no significant changes in any of these parameters were observed at lower CBD doses.
Gene expression analysis of a panel of cytochrome P450s (CYPs) and UDP-glucuronosyltransferases
(UGTs) revealed similar patterns of response as that observed in the acute study phase. Up-regulation
of CYP and UGT genes appeared dose-dependent, especially in the case of Cyp2b10 and Ugt1a9, with
significant changes occurring, in many instances, after the lowest CBD dose (61.5 mg/kg) (Figure 5).
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Figure 4. Effects of 2-week administration of CBD on: (A) Body weight dynamics, (B) Liver to body
weight ratio. Intrahepatic concentrations of (C) total glutathione (GSH), (D) reduced glutathione
(GSSG), and (E) GSH/GSSG ratio. Data are presented as mean ± SEM (n = 6). # indicates a significant
difference as calculated with a Kruskal-Wallis test with a Dunn’s post-hoc test with # representing
p < 0.05; and ### p < 0.001.
Table 2. Clinical chemistry parameters after dosing with CBD oil for two weeks. Cells in bold italics
are significantly different from vehicle (One-Way ANOVA, indicated by *, or Kruskal-Wallis with
appropriate post-hoc test). Data presented as mean ± SEM (n = 6/group).
Vehicle 61.5 mg/kg 184.5 mg/kg 615 mg/kg

ALT 40.0 ± 3.6 41.3 ± 14.8 32.3 ± 4.3 115.4 ± 51.2
AST 66.7 ± 6.7 74.7 ± 6.7 68.2 ± 3.9 157.0 ± 48.9
ALP 112.2 ± 3.5 112.7 ± 4.6 104.0 ± 2.0 113.2 ± 12.2
GGT 5.2 ± 0.3 4.0 ± 0.4 4.7 ± 0.2 5.0 ± 0.3
Total Bilirubin 0.1 ± 0.0 0.1 ± 0.02 0.23 ± 0.02 1.5 ± 0.7 *
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Figure 5. Effects of a two-week administration of CBD on intrahepatic expression of cytochrome P450s

and UDP-glucuronosyltransferases. Livers were collected 6 h after the last gavage and gene expression
was measured using the quantitative real-time (qRT) PCR. * - indicate data analyzed by One-Way
ANOVA with Tukey’s post-test, and # indicate non-normal data analyzed with a Kruskal-Wallis and
Dunn’s post-hoc test. Data are presented as mean ± SEM fold changed from vehicle (n = 6), with * or #
as p < 0.05; ** or ## as p < 0.01; *** or ### as p < 0.001; and **** or #### as p < 0.0001.
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2.3. Hepatotoxicity Gene Expression Analysis

Given that CBD-induced hepatotoxicity was observed in both study phases, we further investigated
the mechanistic underpinnings of this toxicity. For this purpose, we employed a gene expression
array comprised of 84 genes recognized as biomarkers of liver toxicity. These genes have been linked
to pathologies such as cholestasis, steatosis, phospholipidosis, non-genotoxic hepatocarcinogenicity,
necrosis, and generalized hepatotoxicity. This approach was successful in previous studies into
herb-induced liver injury [29–31].
During the acute phase, 22 genes were significantly (p < 0.05 ANOVA) down-regulated and
26 genes were significantly up-regulated in mice livers. The vast majority of affected genes (32) were
dysregulated in a dose-dependent manner (Table 3). Furthermore, several genes, including Fmo1,
Lgr5, and Lss, exhibited a biphasic response, where the up-regulation observed at lower doses was
succeeded by down-regulation at higher dose(s).
Table 3. Gene-markers of hepatotoxicity affected by CBD administration. Genes that are significantly
up- or down-regulated sorted by greatest fold changed at the highest dose (2460 mg/kg or MED of
200 mg/kg). Cells in bold italics indicate those significantly different from vehicle (One-Way ANOVA,
indicated by *, or Kruskal-Wallis test with appropriate post-hoc comparison). Genes that are commonly
and significantly dysregulated in both the acute and sub-acute studies are highlighted in yellow. Data
presented as mean ± SEM (n = 6/group) fold change from vehicle.
Up-Regulated
Single Dose 2 Week Dosing
Gene 246 mg/kg 738 mg/kg 2460 mg/kg 61.5 mg/kg 184.5 mg/kg 615 mg/kg
Krt8 32.6 ± 18.5 32.2 ± 0.4 46.9 ± 25.1 0.7 ± 0.1 0.8 ± 0.2 8.7 ± 4.2 *
Map3k6 4.9 ± 1.9 1.6 ± 0.2 38.0 ± 17.3 1.7 ± 0.9 0.8 ± 0.1 8.6 ± 2.9
Cdkn1a 0.3 ± 0.1 0.5 ± 0.2 22.7 ± 6.9 0.3 ± 0.1 0.4 ± 0.1 9.3 ± 3.7
Hmox1 0.9 ± 0.1 2.8 ± 0.6 19.0 ± 8.8 * 1.5 ± 0.4 1.5 ± 0.3 2.4 ± 1.2
Nqo1 3.6 ± 0.8 4.92 ± 1.0 * 9.7 ± 1.7 * 1.0 ± 0.1 1.1 ± 0.1 6.2 ± 1.2 *
Ugt1a1 4.8 ± 0.5 7.8 ± 0.6 7.5 ± 1.0 2.7 ± 0.1 4.1 ± 0.2 9.1 ± 0.9
Rplp2 1.0 ± 0.1 1.5 ± 0.2 5.7 ± 0.8 * 0.5 ± 0.1 0.5 ± 0.1 0.7 ± 0.2
Abcb1a 1.5 ± 0.4 1.0 ± 0.2 5.6 ± 0.8 2.1 ± 0.5 2.1 ± 0.2 6.4 ± 1.8 *
Slc39a6 2.0 ± 0.3 1.4 ± 0.1 5.5 ± 1.3 1.1 ± 0.2 0.9 ± 0.1 2.9 ± 1.1
Aldoa 2.8 ± 0.9 2.1 ± 0.2 5.4 ± 1.4 1.3 ± 0.1 1.1 ± 0.1 2.8 ± 0.5 *
Pla2g12a 1.6 ± 0.3 2.0 ± 0.3 5.3 ± 0.6 1.7 ± 0.3 1.4 ± 0.1 3.5 ± 0.3
Ubc 1.3 ± 0.1 1.1 ± 0.2 4.8 ± 0.6 * 0.6 ± 0.1 1.1 ± 0.1 1.6 ± 0.3
Abcc3 2.6 ± 0.6 2.4 ± 0.4 4.5 ± 1.2 1.2 ± 0.2 1.1 ± 0.1 2.2 ± 0.3 *
Abcc2 1.4 ± 0.2 1.6 ± 0.3 3.9 ± 0.3 * 1.2 ± 0.1 0.9 ± 0.0 3.4 ± 1.3
Gsr 1.9 ± 0.5 2.7 ± 0.3 * 3.9 ± 0.4 * 0.8 ± 0.1 0.9 ± 0.1 2.9 ± 0.5 *
Icam1 1.9 ± 0.4 1.2 ± 0.2 3.8 ± 0.9 0.8 ± 0.1 0.7 ± 0.1 1.9 ± 0.6
Cyp3a11 2.9 ± 0.3 * 1.6 ± 0.2 3.6 ± 0.7 * 2.3 ± 0.2 * 1.6 ± 0.2 2.9 ± 0.3 *
Txnrd1 1.3 ± 0.2 1.6 ± 0.3 3.5 ± 0.4 1.4 ± 0.3 1.0 ± 0.2 2.6 ± 1.0
Lss 3.2 ± 0.5 2.2 ± 0.1 0.3 ± 0.0 0.5 ± 0.2 0.4 ± 0.2 * 0.9 ± 0.7
Pgk1 1.7 ± 0.4 1.3 ± 0.1 3.1 ± 0.9 0.9 ± 0.0 1.0 ± 0.1 1.6 ± 0.3
Ddx39 1.1 ± 0.1 2.2 ± 0.6 * 2.6 ± 0.3 * 0.9 ± 0.1 0.6 ± 0.1 3.1 ± 0.7
Psme3 1.2 ± 0.2 1.0 ± 0.1 2.6 ± 0.5 1.0 ± 0.1 0.8 ± 0.1 1.5 ± 0.3
Ipo4 1.1 ± 0.1 1.9 ± 0.3 * 2.5 ± 0.2 * 0.8 ± 0.1 0.9 ± 0.1 1.7 ± 0.4
Osmr 1.4 ± 0.2 1.0 ± 0.2 2.4 ± 0.3 * 0.7 ± 0.1 0.4 ± 0.1 1.5 ± 0.6
Krt18 1.5 ± 0.2 2.0 ± 0.2 * 2.2 ± 0.4 * 0.7 ± 0.1 0.7 ± 0.1 3.2 ± 0.9
Timm10b 1.3 ± 0.1 1.7 ± 0.2 * 1.9 ± 0.3 * 1.0 ± 0.1 1.1 ± 0.2 1.7± 0.4
Tfrc 2.0 ± 0.1 3.3 ± 0.6 1.8 ± 0.1 1.3 ± 0.1 0.7 ± 0.1 2.6 ± 0.3 *
Mrps18b 0.8 ± 0.0 0.9 ± 0.1 1.7 ± 0.2 * 1.0 ± 0.1 0.8 ± 0.1 1.6 ± 0.3
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Table 3. Cont.
Down-Regulated
Single Dose 2 Week Dosing
Gene 20 mg/kg 60 mg/kg 200 mg/kg 5 mg/kg 15 mg/kg 50 mg/kg
Igfals 1.1 ± 0.1 0.5 ± 0.1 * 0.02 ± 0.0 * 0.8 ± 0.1 0.9 ± 0.2 0.2 ± 0.1 *
Lgr5 1.2 ± 0.1 0.5 ± 0.1 * 0.04 ± 0.0 * 0.4 ± 0.0 * 0.3 ± 0.1 * 0.2 ± 0.0 *
Car3 0.8 ± 0.1 0.2 ± 0.0 * 0.1 ± 0.1 * 0.5 ± 0.1 * 0.5 ± 0.0 * 0.1 ± 0.0 *
Atp8b1 1.4 ± 0.3 0.5 ± 0.1 0.2 ± 0.0 0.6 ± 0.1 0.5 ± 0.1 0.4 ± 0.1
Ppara 0.7 ± 0.1* 0.6 ± 0.0 * 0.2 ± 0.0 * 0.5 ± 0.1 * 0.5 ± 0.1 * 0.3 ± 0.1 *
Avpr1a 1.1 ± 0.2 0.8 ± 0.1 0.2 ± 0.0 * 0.5 ± 0.1 * 0.3 ± 0.1 * 0.3 ± 0.1 *
Abcb11 1.1 ± 0.2 0.5 ± 0.0 * 0.2 ± 0.0 * 0.6 ± 0.1 0.7 ± 0.1 0.3 ± 0.1 *
Mcm10 1.9 ± 0.5 1.4 ± 0.1 0.2 ± 0.0 0.9 ± 0.2 0.6 ± 0.1 0.2 ± 0.0 *
Fabp1 0.6 ± 0.0 * 0.4 ± 0.0 * 0.2 ± 0.1 * 0.8 ± 0.1 0.7 ± 0.0 * 0.1 ± 0.0 *
Fads1 0.7 ± 0.1 0.6 ± 0.0 0.2 ± 0.0 0.9 ± 0.1 0.8 ± 0.1 0.6 ± 0.2
Cdc14b 0.9 ± 0.1 0.7 ± 0.1 0.3 ± 0.0 0.6 ± 0.1 * 0.7 ± 0.1 0.4 ± 0.1 *
Mbl2 1.3 ± 0.1 0.7 ± 0.0 0.3 ± 0.0 * 0.9 ± 0.1 0.9 ± 0.1 0.6 ± 0.1 *
Asah1 1.1 ± 0.1 0.9 ± 0.1 0.4 ± 0.1 * 0.8 ± 0.1 0.7 ± 0.1 0.6 ± 0.1
Lpl 0.7 ± 0.0 0.4 ± 0.1 0.5 ± 0.1 0.4 ± 0.1 0.5 ± 0.1 0.2 ± 0.1
Emc9 1.3 ± 0.2 0.7 ± 0.0 0.5 ± 0.0 0.9 ± 0.1 0.9 ± 0.1 0.6 ± 0.1
Rhbg 0.9 ± 0.1 0.5 ± 0.0 * 0.5 ± 0.1 * 0.7 ± 0.1 1.0 ± 0.1 0.6 ± 0.2
L2hgdh 0.9 ± 0.1 0.8 ± 0.0 0.5 ± 0.1 * 0.7 ± 0.1 0.7 ± 0.1 0.4 ± 0.1 *
Cxcl12 1.2 ± 0.2 0.8 ± 0.1 0.5 ± 0.1 0.8 ± 0.0 0.7 ± 0.0 * 0.4 ± 0.1 *
Maob 1.0 ± 0.1 0.8 ± 0.1 0.6 ± 0.0 * 0.8 ± 0.1 0.8 ± 0.1 0.6 ± 0.1 *
Rdx 1.1 ± 0.1 0.8 ± 0.1 0.6 ± 0.0 * 0.7 ± 0.1 0.8 ± 0.1 0.7 ± 0.1
B2m 1.0 ± 0.1 0.6 ± 0.0 * 0.7 ± 0.1 0.9 ± 0.1 0.8 ± 0.1 0.6 ± 0.1 *
Cryl1 1.0 ± 0.2 0.4 ± 0.0 0.7 ± 0.1 0.8 ± 0.1 0.8 ± 0.1 0.6 ± 0.1
Ipo8 1.4 ± 0.1 0.8 ± 0.1 * 0.9 ± 0.1 0.9 ± 0.1 0.7 ± 0.1 0.5 ± 0.2 *
Srebf1 1.2 ± 0.1 0.7 ± 0.1 0.9 ± 0.1 0.8 ± 0.1 0.7 ± 0.1 0.7 ± 0.1 *
Scd1 0.6 ± 0.1 0.3 ± 0.0 * 0.9 ± 0.3 0.5 ± 0.1 0.6 ± 0.0 0.1 ± 0.0
Dnajb11 1.3 ± 0.2 1.1 ± 0.1 1.1 ± 0.2 0.7 ± 0.1 0.4 ± 0.1 * 0.9 ± 0.1
Tagln 1.0 ± 0.1 0.8 ± 0.2 1.2 ± 0.7 0.6 ± 0.1 0.5 ± 0.1 * 0.3 ± 0.1 *
Abcb4 1.1 ± 0.3 0.6 ± 0.1 1.4 ± 0.4 0.8 ± 0.1 0.6 ± 0.1 0.4 ± 0.1 *
Fasn 2.2 ± 0.3 1.4 ± 0.1 1.5 ± 0.5 0.5 ± 0.0 0.4 ± 0.1 * 0.5 ± 0.2
During the sub-acute phase, 21 genes were significantly down-regulated and 12 genes were
significantly up-regulated. Unlike the acute phase, only 15 affected genes were dysregulated in a
dose-dependent manner. Another 15 genes were affected only at the high CBD dose (i.e., 615 mg/kg
CBD).
Expression of a substantial number of genes (21) was affected during both study phases. The largest
subset of dysregulated genes (9) was associated with general hepatotoxicity. Of these, Aldoa, Gsr, Krt8,
Krt18, Nqo1, and Pla2g12a were up-regulated, whereas Avrp1a, Car3, and Igfals were down-regulated.
All the gene expression data is summarized in Supplementary Table S3.
2.4. Dose-Response Analysis

Dose-response was also evaluated with linear and log regression models, with the best fit model
meeting at least three of the following criteria: lowest AICc, lowest standard deviation of residuals,
lowest absolute sum of squares, and/or highest R2 value (at least 0.5) (Supplementary Table S4). In the
24 h acute study, AST, Cyp1a1, Cyp1a2, Cyp2b10, Cyp3a4, Gsr, Ipo4, Nqo1, Timm10b, and Ugt1a1 had an
increasing response with CBD dose (R2 > 0.5) and Abcb11, Car3, Cdc14b, Fabp1, Fads1, Igfals, L2hgdh,
Lgr5, Maob, Mbl2, and Ppara had a decreasing response with CBD dose (R2 > 0.5). Similar responses
for some of the parameters were seen in the two-week acute study: increasing responses in Cyp2b10,
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Ugt1a1, and liver to body weight ratios; and decreasing responses with Atp8b1, Avpr1a, Car3, Cdc14b,
Cxcl12, Fabp1, L2hgdh, Lgr5, Ppara, Scd1, and Tagln. The parameters in common between the two time
points were Cyp2b10, Ugt1a1, Car3, Fabp1, L2hgdh, Lgr5, and Ppara, which, aside from Cyp2b10 and
Ugt1a1, were down-regulated.
3. Discussion
The marketing of products containing CBD, a non-psychotropic constituent of the Cannabis
sativa plant, has grown rapidly in the last five years. It has been successfully utilized for therapy of
treatment-resistant epilepsy and may have a number of other beneficial health effects. However, to
our knowledge, there is a lack of comprehensive toxicological studies devoted to CBD safety that are
critical for further marketing of CBD and CBD-containing products.
In this study, we demonstrated that CBD, when delivered orally to mice in the form of a
concentrated CBD-enriched Cannabis extract, has the potential to cause liver injury. In the acute
toxicity study, the highest CBD dose (2460 mg/kg), exhibited clear evidence of hepatotoxicity as
indicated by marked increases in serum ALT, AST, and total bilirubin as well as increased intrahepatic
concentrations of oxidized glutathione. Interestingly, this dose did not result in consistent increases in
liver-to-body weight ratio; however, a similar response was observed in rhesus monkeys injected with
sub-lethal or lethal doses of CBD [14]. Although 2460 mg/kg (MED of 200 mg/kg CBD) is not applicable
to most real-life scenarios, it does provide critical information regarding the potential consequences of
CBD overdose as well as for doses needed for further sub-chronic and chronic toxicity studies. Single
administration of lower doses (246 mg/kg and 738 mg/kg CBD) caused only increases in liver-to-body
weight ratios among the generally liver-focused toxicological responses measured.
The administration of CBD caused dose-dependent and sometimes dramatic induction of major
cytochromes and UDP-glucuronosyltransferases (Supplemental Table S3). Induction of murine Cyp
isoforms by CBD has been noted previously following sub-chronic dosing [19]. Of particular concern is the
induction of Cyp2e1 and Cyp2b10. The former isoform is a central participant in the biotransformation of
ethanol and APAP, while the latter plays a role in the metabolism of a number of prescription medications
including bupropion, clobazam, cyclophosphamide, ketamine, propofol, and several others. Furthermore,
CYP2B6 and CYP3A4, the human homologues of Cyp2b10 and Cyp3a11 (another CYP induced in this
study), are central in the metabolism (N-demethylation) of clobazam, an anti-seizure medication used in
the treatment of epilepsy. Interestingly, recent clinical studies have noted that serum concentrations of
N-desmethylclobazam, the active metabolite of clobazam, are markedly increased when co-administered
with CBD (Epidiolex® ) [32]. Such clinical observations appear to support the inductive effects of CBD on
CYPs noted in this study. To what extent, however, the induction of murine Cyps by CBD is translatable
to humans remains to be determined. Clearly, additional clinical studies investigating CBD-mediated
drug interactions are needed, especially if Epidiolex® is to be prescribed for other medical conditions, but
more importantly as CBD gains popularity across the U.S. following its deschedulization as a result of the
passage of the Agriculture Improvement Act of 2018, otherwise known as the 2018 Farm Bill [33].
The 10 day sub-acute study also revealed that CBD doses above 50 mg/kg MED, although well
tolerated after single administration, were toxic when repetitively delivered. The observed general
toxicity was, in part, mediated by liver injury as numerous signatures of hepatotoxicity were observed,
including pan-hepatic cytoplasmic swelling, increases in liver-to-body weight ratios, and elevated
ALT, AST, and total bilirubin. No measurable toxicological responses associated with liver injury were
observed in mice gavaged with CBD at 184.5 mg/kg (MED of 15 mg/kg CBD) or lower, however, foci of
hepatocyte cytoplasmic swelling were often detected. These findings are in line with observations from
recent clinical trials in which 5–20% of patients exhibited increases in liver enzymes during chronic
CBD administration at doses of 20 mg/kg [1,2,24]. Taken together, this evidence suggests that, despite
some inter-species differences in CBD disposition, the mouse is a reliable model for assessing the safety
of this popular cannabinoid.
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Another important finding of this study was the wide palette of molecular responses elicited
by CBD, particularly the dysregulation of more than 50 genes involved in hepatotoxicity. To our
knowledge, the magnitude of such a response has not been observed in previous studies utilizing similar
gene expression arrays aimed at examining the hepatotoxicity of bromobenzene, carbon tetrachloride,
dimethyl nitrosamine, or OxyELITE-Pro, a botanical dietary supplement linked to severe liver injury in
humans [29,30,34]. The involvement of numerous enzymatic pathways in response to CBD exposure
suggests that liver injury associated with this cannabinoid occurs via various mechanisms. Of particular
concern was the up-regulation of genes associated with oxidative stress, in particular Hmox1, Nqo1,
and Txnrd1. These findings, coupled with increased levels of oxidized glutathione, infer a strong
pro-oxidant trait to CBD, thereby bringing into question its claimed ‘antioxidant’ properties.
Importantly, a number of genes were differentially regulated at low and high doses of CBD, resulting
in a biphasic or hormetic response. For instance, the expression of lanosterol synthase (Lss), a gene
responsible for the biosynthesis of cholesterol, steroid hormones, and vitamin D, was up-regulated after
246 and 738 mg/kg CBD, but substantially down-regulated with a CBD dose of 2460 mg/kg. Interestingly,
previous studies have also described a biphasic response to CBD, where low doses were stimulatory,
while higher doses were inhibitory [35]. More strikingly, cyclin dependent kinase inhibitor 1A (Cdkn1a)
was down-regulated after exposure to non-toxic doses of CBD, but significantly up-regulated at doses
associated with either overt toxicity (2460 mg/kg CBD) or mortality (615 mg/kg CBD) in acute and sub-acute
studies, respectively. Previous studies have demonstrated that down-regulation of Cdkn1a, also known
as p21, stimulates liver regeneration, while overexpression inhibits this process [36–38]. Furthermore, a
recent report of Cdkn1a up-regulation resulting from acute cholestatic injury was proposed as a biomarker
for impaired liver regeneration [39]. Elevated total bilirubin in conjunction with up-regulation of Cdkn1a
and a number of other gene-markers of cholestatic liver injury (i.e., Abcb1a, Abcc2, Abcc3, Atp8b1, and
Rdx) and down-regulation of fatty acid metabolism-related genes (Car3, Fabp1, and Ppara) observed in our
study, suggest that CBD-induced liver injury may be cholestatic, though ALP and GGT were not elevated.
Future studies are needed to confirm this hypothesis.
In conclusion, the results of these studies demonstrate that, despite the beneficial effects of CBD in
the treatment of certain therapy-resistant seizures, it poses a risk for liver injury. Furthermore, the
probability of CBD-drug interactions appears quite high. Therefore, additional studies are needed to
examine the toxicity of chronic low-dose CBD exposure as well as explore CBD’s potential to interact
with other medications. Such studies will provide important information regarding the range of CBD
doses that can be deemed safe for the purpose of regulatory decision-making.
4.1. CBD Extract Characterization, Dosing Solution, and Dose Calculations

CBD extract was prepared following GMP procedures from the leaves and flowering tops by the
extraction of CBD rich cannabis plant material (5.61% of CBD and 0.2% THC) using hexane as the
extraction solvent. The extract was then evaporated to dryness followed by raising the temperature to
80 ◦ C to effect complete decarboxylation of the extract. The final extract was analyzed using GC/MS
for its cannabinoid content, solvent residue, heavy metals, bacterial and fungal counts and aflatoxin
content following USP procedures. The results showed the following: cannabidiol content 57.9%;
other cannabinoids: cannabichromene 2.03%, Δ9 -tetrahydrocannabinol 1.69%, cannabigerol 1.07%,
Δ8 -tetrahydrocannabinol <0.01%; tetrahydrocannabivarin <0.01%. Residual solvent <0.5%; loss on
drying 0.32%; heavy metals: lead, mercury, cadmium, and arsenic were not detected; aflatoxins: AFB1 ,
AFB2 , AGF1 , AFG2 were not detected.
Doses of the CBD extract were calculated based on the CBD content listed above to deliver the
required dose of CBD. For simplicity, the ‘CBD-rich cannabis extract’ will be referred to as ‘CBD’
throughout this manuscript. The extract was diluted in sesame oil to prepare the gavage solution.
Allometric scaling for CBD mouse equivalent doses (MED) was determined per the recommendation
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of Wojcikowski and Gobe which, in turn, is based upon the FDA Industry Guidance for Estimating
the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Volunteers [40].
The scaling factor of 12.3, commonly used for mice weighing between 11–34 g, was used to calculate
the MED for CBD. The MED was based on the maximum recommended human maintenance dose
of CBD (Epidiolex® ), which is 20 mg/kg. For the 1× dose, the quantity of CBD administered was
20 mg/kg × 0.025 kg (average mouse weight in our study) × 12.3 (scaling factor for mice) = 6.15 mg
total CBD delivered in 300 μL of gavage solution or 246 mg/kg. Consequently, 3× dose = 18.45 mg total
CBD in 300 μL gavage solution or 738 mg/kg), and 10× dose = 61.5 mg total CBD in 300 μL gavage
solution or 2460 mg/kg). In the sub-acute study, the dose of 61.5 mg/kg (MED of 5 mg/kg CBD) was
considered as 1× dose. Consequently, the doses of 184.5 mg/kg (MED of 15 mg/kg CBD) and 615 mg/kg
(MED of 50 mg/kg CBD) were considered as 3× and 10×, respectively. Control mice received 300 μL of
sesame oil.
4.2. Animals
Male B6C3F1 /J mice, 8 weeks of age (standard age of mice used in safety assessment studies), were
purchased from Jackson Laboratories (Bar Harbor, ME, USA) and were housed at the UAMS Division
of Laboratory Animal Medicine (DLAM) facility. B6C3F1 /J mice are characterized by an average
sensitivity to hepatotoxicants and are widely used by both the FDA and pharmaceutical industry to
investigate the potential for xenobiotics to produce hepatotoxicity. Animals were given one week to
acclimate before the initiation of studies. Animal experiments were conducted in two stages. In the
first stage (acute toxicity), mice were gavaged with a single dose of either 246, 738, or 2460 mg/kg
of CBD (MED of 20, 60, and 200 mg/kg, respectively) and 24 h later animals were euthanized and
tissues/organs were harvested. During the second stage (sub-acute toxicity), mice were gavaged with
CBD extract for ten days (Mon-Fri) with (MED of 61.5, 184.5, and 615 mg/kg, respectively) for reasons
explained later in the Results. Mice were terminated six hours after the last gavage.
To avoid any potential fasting-exacerbated toxicity, food and water were provided ad libitum.
Each animal was individually identified with an ear tag. Animal body weights were measured and
recorded twice a week. All procedures were approved by the UAMS Institutional Animal Care
and Use Committee (protocol number: AUP # 3701), and all personnel followed the appropriate
safety precautions.
4.3. Blood Sampling and Clinical Biochemistry

To measure the effects of CBD extract on a panel of liver enzymes characteristic for drug-induced
liver injury, blood was collected at the end of each experimental stage described above. Blood was
collected from the retroorbital plexus with a heparinized micro-haematocrit capillary tube (Fisher
Scientific, Pittsburg, PA, USA) and placed into a 1.1 mL Z-gel microtube (Sarstedt, Newton, NC, USA).
Tubes were kept on ice and centrifuged at 10,000 rpm for 20 min; serum samples were then immediately
aliquoted and delivered to the Veterinary Diagnostic Laboratory at the Arkansas Livestock and Poultry
Commission (Little Rock, AR, USA) on dry ice where the samples were processed the same day.
4.4. Histopathological Assessment

Livers were excised and a 1 mm section was obtained from the left lateral lobe and another from
the right medial lobe. Sections were fixed in 4% formalin for 24 h, then briefly rinsed in PBS and stored
in 70% ethanol for 24 h. Livers were then processed at the UAMS Pathology Core Facility, stained with
hematoxylin eosin, and evaluated by a board-certified pathologist in a blinded fashion.
For histologic assessment purposes, each liver was represented by two sections obtained from
different locations within the liver. Each section was initially evaluated at magnifications of ×40 and
×100. Sections were further evaluated at ×200 and ×400 to check for the presence of mitotic figures,
necrotic foci, and apoptotic bodies.
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4.5. Glutathione Analysis

Glutathione was measured using a modified Tietze assay [41]. Briefly, liver tissue was homogenized
in 3% sulfosalicylic acid. One aliquot was diluted in N-ethylmaleimide (NEM) to mask reduced
glutathione (GSH) and facilitate measurement of oxidized glutathione (GSSG), while another was
diluted in 0.1 M HCl for measurement of total (GSH + GSSG) glutathione. After removal of NEM
by solid phase extraction with a C18 column, glutathione was measured in both aliquots using a
colorimetric glutathione reductase cycling detection method [41].
4.6. Analysis of mRNA Levels of Major Cytochromes and Transporter Genes

Total RNA was extracted from flash frozen liver tissue using the RNeasy Mini Kit (QIAGEN,
Germantown, MD, USA). Following purification, 1000 ng were reverse transcribed with the High
Capacity cDNA Reverse Transcription Kit (Thermo Fisher, Waltham, MA, USA). Primers were added
at a final concentration of 5 μM (Supplementary Table S1). Gene expression values were normalized to
the internal control gene Hprt and expressed as fold change according to the ΔΔCt method.
4.7. Hepatotoxicity Gene Expression Array

Total RNA was extracted as described above. The cDNA was diluted to 5 ng/μL and 105 μL was
mixed with an equal volume of 2× TaqMan® Fast Advanced Master Mix. For real-time PCR, 100 μL of
the mix was applied to each of two channels on a TaqMan Low Density Hepatotoxicity Array (TLDA)
(Supplementary Table S2) (Thermo Fisher, Waltham, MA, USA). Four biological samples were loaded onto
each array with six replicates analyzed per group. Analysis was performed using the ExpressionSuite
Software v1.1 (Thermo Fisher, Waltham, MA, USA).

All statistical analyses were performed with Graphpad Prism 6 software (Graphpad Software,
San Diego, CA, USA). Treatment groups were compared with their respective untreated group using
ANOVA followed by Tukey’s multiple comparison test. In cases where the data was not normally
distributed as indicated by a positive Brown-Forsythe test, a Kruskal-Wallis with Dunn’s multiple
comparison test was used instead. Comparisons were considered significant at p < 0.05. To evaluate
trends in dose-dependent responses, all parameters were analyzed with regression analyses within
Prism using log(dose) as the independent variable. We used three different models to determine best
fit: linear regression with y-intercept constrained to 0, linear regression with unconstrained y-intercept,
and log(agonist) vs response (three parameters). Regression models were excluded if the model was
ambiguous, interrupted, or not converged or if the 95% confidence intervals were calculated to be ‘very
wide’. Models for each response parameter were compared using the goodness of fit parameters R2
(higher being better), standard deviation of residuals (lower being better; noted as Sy.x), AICc (lower
being better), and the absolution sum of squares (lower being better), with the model having best
goodness of fit parameters being considered the better model.
Supplementary Materials: The following are available online, Figure S1: Kidney- and Heart-to Body-Weight Ratio
in Mice Gavaged with Single Dose of CBD, Figure S2: Effects of Single Dose of CBD on Liver Histomorphology,
Figure S3: Kidney- and Heart-to-Body-Weight Ratio in Mice Gavaged with CBD for 10 Days, Figure S4: Kidney-
(A) and heart- (B) to-body-weight ratio in mice gavaged with CBD for 10 days, Table S1: Forward and Reverse
Primer Sequences for Cyp P450s and UGTs, Table S2: Custom Taqman Low Density Hepatotoxicity Array Gene
Targets, Table S3: Commonly dysregulated genes sorted by function, including CYPs, Ugts, and hepatotoxicity
markers, Table S4: Dose-response analysis with linear and log regression models.
Author Contributions: Conceptualization, L.A.W, M.A.E., B.J.G. and I.K.; Data curation, L.E.E. and I.K.; Formal
analysis, L.E.E., C.M.S., C.M.Q., S.K.-M., M.R.M., M.A.E., B.J.G. and I.K.; Funding acquisition, M.R.M. and
I.K.; Investigation, L.A.W., C.M.S., M.R.M., M.A.E., B.J.G. and I.K.; Methodology, M.A.E.; Resources, B.J.G.;
Writing—original draft, M.R.M., M.A.E., B.J.G. and I.K.
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Funding: This work was supported by the National Institute of General Medical Sciences [grant # P20 GM109005
to IK], the American Association for the Study of Liver Diseases (AASLD) Foundation [Pinnacle Research Award
to MRM], and the Arkansas Biosciences Institute.
Acknowledgments: The authors are thankful to Robin Mulkey and Bridgette Engi for excellent animal care at the
UAMS Animal Facility.
Conflicts of Interest: Quick serves as a scientific consultant for Allergen. The other authors have no conflicts of
interest to disclose.
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value. Phytother. Res. 2014, 28, 22–27. [CrossRef]
41. McGill, M.R.; Jaeschke, H. A direct comparison of methods used to measure oxidized glutathione in biological
samples: 2-vinylpyridine and N-ethylmaleimide. Toxicol. Mech. Methods 2015, 25, 589–595. [CrossRef]
[PubMed]
Sample Availability: Tissue samples from experimental animals are available from the authors.
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Communication
Anti-Amyloidogenic and Cyclooxygenase Inhibitory
Activity of Guettarda speciosa
Mario A. Tan 1,2,3,4, *, Mark Wilson D. Lagamayo 2 , Grecebio Jonathan D. Alejandro 2,3,4
and Seong Soo A. An 1, *
1 Department of Bionano Technology, Bionano Research Institute, Gachon University, 1342 Sungnam-daero,
Sujung-gu, Seongnam-si, Gyeonggi-do 461-701, Korea
2 Graduate School, University of Santo Tomas, Manila 1015, Philippines;
mwdlagamayo@gmail.com (M.W.D.L.); gdalejandro@ust.edu.ph (G.J.D.A.)
3 Research Center for the Natural and Applied Sciences, University of Santo Tomas, Manila 1015, Philippines
4 College of Science, University of Santo Tomas, Manila 1015, Philippines
* Correspondence: matan@ust.edu.ph (M.A.T.); seong.an@gmail.com (S.S.A.A.); Tel.: +63-2-7314031 (M.A.T.);
+82-31-750-8755 (S.S.A.A)
Academic Editors: Raffaele Capasso, Lorenzo Di Cesare Mannelli and Nicola Volpi
Received: 23 September 2019; Accepted: 11 November 2019; Published: 14 November 2019
Abstract: Guettarda speciosa is known in traditional folk medicine for treating cough, cold, sore
throat, fever, wounds, epilepsy, and headaches. To discover the scientific pharmacological potential
of G. speciosa, we explore its anti-inflammatory, cytotoxicity, and inhibition of amyloid-beta (Aβ)
aggregation effects. Cyclooxygenase assay of the G. speciosa CHCl3 (GSC) extract and G. speciosa
MeOH (GSM) extract are more selective to COX-1 inhibition with a 50% inhibitory concentration
(IC50 ) of 3.56 μg/mL for the GSC extract and 4.98 μg/mL for the GSM extract. Neuroblastoma SH-SY5Y
inhibition and thioflavin T assay amyloid-beta (Aβ) aggregate inhibition of the GSM and GSC extracts
showed their potential therapeutic effects against Alzheimer’s disease. The putative compounds
from the LC-MS analysis could be responsible for the observed activities. The results suggest that
G. speciosa possesses anti-inflammatory and anti-neurodegenerative properties and a promising lead
as a source of pharmacologically active compounds.
Keywords: Alzheimer’s disease; COX-1; cytotoxicity; Guettarda speciosa; thioflavin T
1. Introduction
Guettarda speciosa L. (Rubiaceae) is a perennial shrub or small tree, which grows in coastal habitats
in tropical areas. This species is the only representative of the genus Guettarda L. in the Philippines [1].
The genus is widely distributed from East Africa to South and Southeast Asia and the South Pacific [2].
It is regarded as a medicinal plant used in traditional folk medicine for treating postpartum infection,
cough, cold, sore throat, dysentery, fever, boils, wounds, epilepsy, and headache [3–6]. In African
medicinal plants, the flower decoction was combined with Ocimum americanum L. and O. gratissimum
L. to treat malaria, while the roots are used for diarrhea (decoction), rheumatism (rubdown on
articulations), and pelvic pain (massage) [7]. These traditional folkloric claims were corroborated by
pharmacological studies including the antiepileptic activity of the inner bark extract from India [5] and
the anti-inflammatory activity in murine macrophages of the methanolic extract from Indonesia [8].
Phytochemical analysis has elaborated the presence of iridoids and their glucosides, phenolics, glycerol
derivatives, steroids, triterpenoids [9,10], and fatty acids [11]. There is limited information on the
biological activities and chemical constituents associated with G. speciosa.
To address this gap, and in the interest of searching for medicinal Rubiaceae plants from the
Philippines with potential anti-inflammatory and anti-neurodegenerative activities [12–15], we herein

Molecules 2019, 24, 4112
describe the acute toxicity, cyclooxygenase inhibition, and anti-amyloidogenic activity of the extracts
of G. speciosa.
2. Results
2.1. Acute Oral Toxicity

No death was observed among the animals over the 14-day period. Hence, assessment of the
acute oral toxicity indicated that the G. speciosa MeOH (GSM) extract was safe and nontoxic up to
2000 mg/kg following the Organization for Economic Co-operation and Development (OECD) 425
guidelines. Prominent signs of toxicity and abnormalities were also not observed. Moreover, the post
toxicity and gross necropsy study showed that all vital organs, such as the liver, kidneys, and stomach,
were comparable to the control group as indicated in the histopathological results (Figure 1) analyzed
by a licensed veterinarian.
Figure 1. Histopathological examination of (a) liver, (b) left kidney, (c) right kidney, and (d) stomach
in normal and G. speciosa extract-treated (GSM) groups. No significant changes were observed in the
examined vital organs of the GSM-treated groups when compared to the normal control group.
2.2. COX-1 and COX-2 Assay

Aerial parts of G. speciosa were extracted with MeOH to afford the crude extract (GSM).Various
extracts of G. speciosa with varying polarity were prepared using solvent partitioning. Nonpolar
compounds are contained in the G. speciosa hexane (GSH) extract, semi-polar compounds in the
G. speciosa CHCl3 (GSC) extract, and polar compounds in the G. speciosa aqueous (GSA) extract. All the
extracts were dried free of solvents prior to use in the succeeding experiments.
The cyclooxygenase screening assay results of the G. speciosa extracts (Figure 2) showed a greater
inhibition to the COX-1 enzyme as compared to the COX-2. The extracts were initially tested at a
concentration of 10 μg/mL. More than 50% inhibition for the COX-1 enzyme was observed for GSC
(66.68% ± 2.77) and GSM (62.25% ± 2.39) extracts. None of the extracts gave a 50% inhibition using
COX-2 enzyme with the GSC extract exhibiting the highest inhibition with 30.85% ± 5.11. Interestingly,
the GSM extract exhibited a negative inhibition with COX-2 (−9.98% ± 5.62). The percentage inhibition
of the extracts had a significant difference when compared to the positive control, indomethacin
(4.0 mM) (85.1–86.3%), at p < 0.05.
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100.00
80.00
60.00
% Inhibition
40.00 COX-1 ACTIVITY

COX-2 ACTIVITY
20.00
0.00
INDO GSA GSC GSH GSM
-20.00
Positive control and Guettarda speciosa extracts

-40.00
Figure 2. In vitro cyclooxygenase screening of Guettarda speciosa (G. speciosa) extracts at a concentration
of 10 μg/mL. The G. speciosa extracts exhibited an inhibition to the COX-1 enzyme with the chloroform
GSC and methanol GSM extracts showing a greater than 50% inhibition. Indomethacin (4.0 mM)
(INDO) was used as the positive control. GSA—G. speciosa aqueous extract; GSC—G. speciosa CHCl3
extract; GSH—G. speciosa hexane extract; GSM—G. speciosa MeOH extract.
Based on the screening results, the 50% inhibitive concentration (IC50 ) of the GSC and GSM
extracts for COX-1 were also determined using seven concentrations (0.5, 1, 5, 10, 40, 70, and 100 μg/mL).
The results indicate an IC50 of 3.56 μg/mL for the GSC extract and 4.98 μg/mL for the GSM extract.
Because of the promising results in the cyclooxygenase assay, the GSM and GSC extracts were further
evaluated via cell viability and thioflavin T assays.
2.3. Cell Viability

The cytotoxicity of the GSM and GSC extracts was explored against neuroblastoma SH-SY5Y
utilizing the ATP luminescence assay. The ATP serves as the cell’s most important chemical energy
storage for all biological processes. When cells are exposed to environmental or metabolic stressors
(depleted iron and carbon sources), their ability to produce ATP is impeded. Hence, cellular ATP
measurement is a good indication of a cell’s metabolic health and subsequent viability. As illustrated
in Figure 3, the GSM extract exhibited a 17% cell growth inhibition using the smallest concentration of
0.39 μg/mL, followed by 25% growth inhibition at 0.78 μg/mL. Surprisingly, an almost similar trend
was observed, using 12.5, 6.25, 3.13, and 1.56 μg/mL concentrations with cell growth inhibitions at
35–38%. The highest concentration at 50 μg/mL showed a cell growth inhibition of 57%. Compared
to the negative control, a significant difference was shown on the cell cytotoxicity of the extracts
at all the given concentrations (p < 0.05). Cytotoxicity was also observed on the GSC extracts in a
concentration-dependent manner from 0.39 to 50 μg/mL as observed in Figure 3. A 62% cell growth
inhibition was noted on the 50 μg/mL, followed by the 25 μg/mL with 50% inhibition. The lowest
concentration, 0.39 μg/mL, showed only a 6% cell growth inhibition with a cell viability of 94%.
Statistical analyses also indicated a significant difference on the percentage cell viability of the
GSC extracts when compared to the control (100% cell viability), with the exception of the lowest
concentration (0.39 μg/mL).
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Figure 3. Effect of Guettarda speciosa MeOH (GSM) and CHCl3 (GSC) extracts on cell viability in
neuroblastoma SH-SY5Y cells. Cell viability was determined using the ATP luminescence assay.
The results indicate % cell viability vs. the negative control (mean ± SD of triplicate measurement).
All of the extracts exhibited a significant difference on the % cell viability on the negative control vs.
the plant extracts at p < 0.05, except for the GSC extract at 0.39 μg/mL).
Through the cell viability ATP luminescence assay, the half maximal inhibitory concentration of
the cell population death was determined. The GSM extract inhibited the growth of SH-SY5Y cells
with an IC50 value of 43.44 μg/mL. The GSC extract was found to be more active with an IC50 of 8.049
μg/mL. SH-SY5Y cells have been used in many neurological studies, such as Parkinson’s disease (PD),
Alzheimer’s disease (AD), and traumatic brain injury (TBI) [16]. The cell viability of the neuroblastoma
SH-SY5Y cells against the GSM extracts may suggest promising leads with respect to the identification
of potential bioactive secondary metabolites in neurological diseases.
2.4. Thioflavin T (ThT) Assay

The abnormal aggregation of β-amyloid (Aβ), tau protein accumulation, decreased acetylcholine,
oxidative stress, and neuroinflammation of the nervous system are some of the pathological hallmarks
associated with Alzheimer’s disease. To address whether the GSM and GSC extracts are capable of
inhibiting the aggregation of Aβ, we also utilized thioflavin T (ThT) assay using phenol red as the
positive control (Table 1). The GSC extract at 50 μg/mL exhibited the highest inhibition of the Aβ
fibril formation with 65.78%. This is also statistically comparable (p < 0.05) to the inhibition of the
positive control at 69.85%. The GSM extract gave a 54.71% inhibition but is statistically different from
the positive control (p < 0.05). The observed activity of the GSC and GSM extracts may be attributed to
the presence of secondary metabolites, which could be capable of inhibiting the Aβ fibril formation.
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Table 1. Thioflavin T (ThT) assay results of Guettarda speciosa extracts.
Aβ1-42 Aggregation Inhibition (%) a

Phenol Red (50 μM) b 69.85 ± 0.29
GSM Extract (50 μg/mL) 54.71 ± 2.92
GSM Extract (5 μg/mL) 17.20 ± 0.85
GSC Extract (50 μg/mL) 65.78 ± 3.12 *
GSC Extract (5 μg/mL) 24.63 ± 1.19
a The values are expressed as mean ± SD of three trial experiments. b The positive control. * No significant difference
with the positive control at p < 0.05. GSM—G. speciosa MeOH extract; GSC—G. speciosa CHCl3 extract.
2.5. Metabolite Profiling

A total of nine putatively identified compounds were determined from the GSM extract using
LC-MS analysis. Optimized run method produced the LC chromatogram as shown in Figure 4,
where the Y-axis represents the % signal intensity and the X-axis is the retention time in minutes.
The compounds presented (Table 2 and Figure 5) are those that fall within the “good match” standards
of the Traditional Chinese Medicine (TCM) library [17,18]. These putatively identified nine compounds
were also previously isolated from the different Guettarda species based on the available literature
data. Loganin (3) was isolated from G. platypoda DC. [19] and G. pohliana Müll. Arg. [20]; rotundic
acid (7) from G. angelica Mart. ex Müll. Arg. [21] and G. platypoda [22]; quinovic acid (9) from G.
angelica [23] and G. platypoda [22,24]; strictosidine (1), sickingine (5), 5-caffeoylquinic acid (6), and
4,5-dicaffeoylquinic acid (8) from G. acreana K. Krause [3]; sweroside (2) from G. platypoda [24] and
G. pohliana [20]; and β-sitosterol (4) from G. platypoda [22]. Interestingly, the putatively identified
compounds exhibit diverse biological activities as reported in the literature (Table 2). The putatively
identified metabolites with their reported activities in the literature may explain the biological activities
exhibited by the extracts in this study. Several compounds were identified to have anti-inflammatory
activity (2, 4, and 6), cytotoxicity (2 and 3), and neuroprotective (3) activity.
Figure 4. Chromatogram of G. speciosa MeOH (GSM) extract. Nine putative compounds were identified
utilizing the UNIFI data analysis software and comparing the acquired MS spectra to library matching
using the Traditional Chinese Medicine (TCM) library that is incorporated in the UNIFI analysis
software. All of these compounds were previously isolated from other Guettarda species. The x-axis is
the retention time in minutes, while the y-axis is the peak % signal intensity.
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Table 2. LC-MS putative compounds from G. speciosa (GSM) extract.
Exact Mass Elemental Error

RT Putative Identity Biological Activity
Calculated Observed Composition (ppm)
1.27 530.22644 530.22500 C27 H34 N2 O9 −2.63 Strictosidine (1) Antimicrobial [25]
Cytotoxic [26];
Antigenotoxic [27];
2.26 358.12638 358.12690 C16 H22 O9 1.45 Sweroside (2)
Antiosteoporotic [28];
Anti-inflammatory [29]
Antigenotoxic [27];
2.35 390.15259 390.15900 C17 H26 O10 2.35 Loganin (3) Neuroprotective [30];
Cytotoxic [31]
Anti-inflammatory [32,33];
2.42 414.38617 414.38650 C29 H50 O 0.72 β-Sitosterol (4) Antipyretic [33] Anthelminthic,
Antimutagenic, Analgesic [34]
3.25 528.21100 528.21050 C27 H32 N2 O9 −0.94 Sickingine (5)
5-Caffeoylquinic Antimicrobial [35]
3.37 354.09509 354.09430 C16 H18 O9 −2.25
acid (6) Anti-inflammatory [36]
4.93 488.35019 488.35210 C30 H48 O5 3.88 Rotundic acid (7) Antimicrobial [37,38]
4,5-Dicaffeoylquinic
15.37 516.45500 516.45460 C25 H24 O12 −0.77 Antipigmentation [39]
acid (8)
17.35 486.33453 486.33290 C30 H46 O5 −3.28 Quinovic acid (9)
RT—retention time in minutes.
Figure 5. Structures of putatively identified compounds from G. speciosa.
3. Discussion.
In our continuous study using endemic Rubiaceae species indigenous to the Philippines,
we described the potential therapeutic effects of G. speciosa extracts. The leaf extracts of G. speciosa,
free from any trace of solvents, have been shown to inhibit in vitro the aggregation of Aβ1–42 ,
cyclooxygenase-1 enzyme, and cell growth of neuroblastoma SH-SY5Y cells. These biological activities
may be ascribed to the compounds that were characterized using untargeted LC-MS. Natural products
from plants continue to be the source of pharmacologically active compounds with diverse structures
in the treatment or prevention of various diseases. In this experiment, these compounds were defined
as indole alkaloids (1, 5), iridoids (2, 3), steroid (4), triterpenoids (7, 9), and polyhydroxy cyclic acids
(6, 8). Pharmacological studies on G. platypoda have reported the synergistic action of quinic acid
glycosides with β-sitosterol and triterpenes with anti-inflammatory activity, while the mechanism
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of β-sitosterol is comparable to hydrocortisone [32]. Most of the Rubiaceae species also contained
iridoids and indole alkaloids [40], which are known to possess anti-inflammatory activities [41,42].
This study also described the inclination of the G. speciosa extracts to COX-1 inhibition as compared
to the COX-2 enzyme. Both COX-1 and COX-2 perform a complex function in the mechanism of central
nervous system (CNS) inflammation [43]. Most studies prefer a COX-2 inhibition, because COX-1 is
involved in the cytoprotective function in the gastrointestinal system. Moreover, the suppression of
COX-1 can result in side effects, including ulcers and bleeding [44]. Several studies have reconsidered
the advantages of selective COX-1 inhibition. As stated, COX-1-dependent prostaglandin synthesis is
implicated in pathological progressions, including atherosclerosis, cancer, endothelial dysfunction,
neuroinflammation, preterm labor, and pain [43,45].
Alzheimer’s disease (AD) is the most common cause of neurodegenerative dementia in elderly
people, often associated witha progressive memory loss and other cognitive impairments [12].
Abnormal β-amyloid (Aβ) deposition, tau protein aggregation, a decreased level of acetylcholine,
oxidative stress, and neuroinflammation of the nervous system are numerous causes associated with
enhanced AD progression [46]. Although there is no cure for AD, currently, only five compounds
(donezipil, tacrine, rivastigmine, galantamine, and memantine) are available and approved in the market
to reduce the symptoms associated with AD [47]. The biological activity of the GSM extracts to inhibit
the Aβ aggregation exhibits the potential of this medicinal plant as a new pharmacologicallyactive
material or therapeutic agent to minimize the effect of AD. To the best of our knowledge, this is the
first report on the SH-SY5Y cytotoxicity, Aβ aggregation prevention, and COX-1 inhibition activities
associated with G. speciosa.
4.1. PlantMaterials
Fresh leaves of G. speciosa were collected from Bantayan Island, Cebu, Philippines (11◦ 12’60.00” N,
123◦ 43’59.99” E) in April 2017. The plant was collected and identified by Grecebio Jonathan Alejandro,
a Philippine Rubiaceae specialist. A voucher specimen was kept at the University of Santo Tomas
Herbarium (USTH 014369).
4.2. Extraction and Fractionation of Extracts

Air-dried, ground leaves of G. speciosa (1.7 kg) were placed in a percolator and extracted with
MeOH. The ground leaves were allowed to soak overnight. The extract was drained, collected,
and concentrated under reduced pressure using a rotary evaporator. A total of 15.0 L MeOH was used
with the extraction process repeated thrice. The procedure obtained 207 g of the MeOH extract (GSM),
and a portion (158 g) was suspended in distilled H2 O (300 mL) and partitioned exhaustively with
hexane (2500 mL). The combined hexane layer was dried with anhydrous Na2 SO4 and concentrated
under reduced pressure to obtain the hexane extract (GSH, 23.2 g). The aqueous layer was partitioned
exhaustively with CHCl3 (2800 mL). The collected CHCl3 layer was dried with anhydrous Na2 SO4 and
concentrated under reduced pressure to obtain the CHCl3 extract (GSC, 10.7 g). The aqueous layer was
freeze-dried using a lyophilizer (Thermo Fisher Scientific, Singapore) to obtain the aqueous extract
(GSA, 18.7 g).
4.3. Animal Study

The experiment protocol (UST-IACUC code number RC2017-890915) was approved by the
Institutional Animal Care and Use Committee (IACUC) at the Research Center for Natural and Applied
Sciences (RCNAS), University of Santo Tomas (UST), and was further issued by the Philippine Bureau
of Animal Industry and Animal Research Permit.
Six female Sprague-Dawley rats were used to assess the acute oral toxicity following the OECD
425 guidelines. The animals were housed in the UST-RCNAS Animal House and acclimatized to
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laboratory conditions for seven days before conducting the experiment. They were fed with standard
rodent pellets and given access to clean drinking water. The laboratory conditions were maintained at
a temperature of 25 ± 3 ◦ C, humidity at 60 ± 4%, and a 12/12 h light/dark cycle.
4.4. Acute Oral Toxicity (OECD 425 Guidelines)

All animals were fasted for 24 h to determine their actual weight before receiving the crude
extract. One rat served as a normal control group and was treated only with water as the vehicle
medium. The animals were given 2000 mg/kg GSM via gastric gavage. The animals were observed
periodically for 24 h for signs of toxicity; thereafter, they were observed daily for 14 days. The animals
were then sacrificed by CO2 inhalation. Gross necropsy, observation of gross pathological changes,
and microscopic examination of all livers, kidneys, and stomachs of the test animals were performed
by a licensed veterinarian.
4.5. COX-1 and COX-2 Assay

The cyclooxygenase assay was based on a previous protocol [15] as follows. The following were
added to 150 μL of 100 mM Tris: 10 μL of 10 ppm plant extracts in DMSO, 10 μL of 1000 μM Hemin,
and 10 μL of 250 U/mL COX-2 or COX-1 enzyme (Cayman Chemicals, Singapore). Indomethacin
(Sigma Aldrich, St. Louis, MO, USA) was used as the positive control, and DMSO served as the negative
control. The mixture was incubated at 25 ◦ C for 15 min. After incubation, 10 μL of 200 μM amplex red
was added to the mixture. Then, 10 μL of 2000 μM arachidonic acid (Sigma Aldrich, St. Louis, MO,
USA) was added, and the reaction fluorescence absorbance was monitored for 2 min using Varioskan
Flash (Thermo Scientific, Waltham, MA, USA) with excitation and emission wavelengths at 535 and
590 nm, respectively. The percent inhibition of the samples and the positive control were determined
based on the averaged slope of each replicate using the following formula:
% Inhibition = [(Slope uninhibited − Slope inhibited)/Slope uninhibited] ∗ 100. (1)
“Slope uninhibited” is the slope of the line from the fluorescence intensity vs. time plot of the
negative control group, and “slope inhibited” is the slope of the line from the fluorescence intensity
vs. time plot of the samples/positive control. The method above was also done for the GSM and GSC
extracts at different effective well concentrations (0.5, 1, 5, 10, 40, 70, and 100 μg/mL) to obtain the
IC50 in μg/mL. Three trials consisting of three replicates per trial were done for each concentration of
each sample.
4.6. Cell Viability Assay

The neuroblastoma cells (SH-SY5Y) were purchased from the American Type Culture Collection
(ATCC, Manassas, VA, USA). SH-SY5Y cells were maintained in Dulbecco’s modified eagle media
(DMEM) supplemented with 10% fetal bovine serum (FBS), 1% kanamycin, and 1% penicillin.
Cell cultures were maintained at 37 ◦ C in 5% CO2 and passaged once per week. The SH-SY5Y cells
were subcultured into a 96-well plate at 2 × 105 cells/well and incubated for 24 h. After incubation,
the cells were treated with plant extracts at different concentrations and incubated for another 72 h.
The medium was removed, and the wells were washed with phosphate-buffered saline (PBS). Fresh
medium (100 μL) was added and incubated for another 30 min. After incubation, CellTiter-Glo®
luminescent reagent (100 μL; Promega, Madison, WI, USA) was added, and the luminescence was
measured using a PerkinElmer Victor-3® multi-plate reader (PerkinElmer, Waltham, MA, USA) [48].
The 50% inhibitive concentrations of the plant extracts were calculated using a nonlinear regression
curve fit (GraphPad Prism ver. 6, San Diego, CA, USA). The values representing cell viability were
expressed as means ± standard deviation (SD) of three trial experiments.
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4.7. Thioflavin T (ThT) Fluorescense Assay

Aβ1-42 (Aggresure™)(10 μM, 30 μL) in PBS (pH 7.4) was incubated with (20 μL) or without the
tested extracts at 37 ◦ C for 24 h in a 384-well plate. Then, 20 μL ThT solution (50 μM) in glycine-NaOH
buffer (pH 9) was added. The fluorescence signal was measured (excitation wavelength, 450 nm;
emission wavelength, 510 nm) using a PerkinElmer Victor-3® multi-plate reader. The percentage of
aggregation inhibition was calculated using the following equation: [(1-IFi /IFc ) ∗ 100%], where IFi and
IFc are the fluorescence absorbance with and without the inhibitors, respectively, after subtracting the
background fluorescence of the ThT solution [46].
4.8. Untargeted LC-MS Metabolite Profiling

Untargeted LC–MS/MS analysis of the GSM extract was performed using a Xevo G2-S Qtof (Waters
Corp., Singapore). The separation was achieved using a BEH HSS T3 column (50 × 2.1 mm internal
diameter). The system delivered a constant flow of 0.4 mL/min, and the mobile phase consisted of 5%
CH3 CN and 0.1% HCOOH. The injection volume was 1 μL. For operation in MS/MS mode, a mass
spectrometer with an electrospray interface (ESI) was used, and the parameters were set as follows:
capillary voltage, 3.0 kV for negative mode; source temperature, 120 ◦ C; desolvation temperature,
400 ◦ C; cone gas flow, 100 L/h; and desolvation gas flow, 1000 L/h. Low collision energy at 6 V, high
collision energy at 20–50 V, and lock mass solution at 1 ng/μL were used to calibrate mass accuracy.
All LC–MS/MS data were processed by the MassLynx version 3.5 NT Quattro data acquisition software
(Milford, MA, USA). For putative compound identification, accurate mass screening was carried out
using the UNIFI data analysis software (San Mateo, CA, USA). The acquired MS spectra were subjected
to library matching using the Traditional Chinese Medicine (TCM) library that is integrated within
the UNIFI analysis software. Annotation of the candidate masses was based on the accurate mass
match, isotopic ratio match, and precursor ion intensity counts. The criteria for a component ID to be
considered a good match were as follows: a mass accuracy error ≤ 5 mDa or ≥ −5 mDa and a response
precursor for precursor ion ≥2000.

All values were reported as mean values with standard deviations (mean ± SD). Statistical
significance of the data was analyzed by one-way ANOVA and Levene’s test followed by Tukey’s
honestly significant difference (HSD) test (GraphPad Prism 5 software package, version 5.02, GraphPad
Software Inc., San Diego, CA, USA), and p < 0.05 was considered statistically significant.
5. Conclusions
This study has demonstrated the first therapeutic potential of G. speciosa on neuroblastoma
cytotoxicity, cyclooxygenase-1 inhibition, and the control of Aβ aggregation. The results of
the untargeted LC-MS metabolite profiling also describe several compounds, which might be
pharmacologically relevant. Hence, deeper understanding of the chemistry and pharmacological
aspect of G. speciosa is warranted as this plant is being utilized in traditional folk medicine. It also
presents its significance as a prospective biologically active material for further development of novel
and safer plant-based agents and/or pharmacologically relevant natural products for anti-inflammatory
or anti-neurodegenerative diseases.
Author Contributions: M.A.T. and S.S.A.A. conceptualized the research and wrote the manuscript; M.A.T. and
M.W.D.L. performed the experiments; G.J.D.A. collected and authenticated the plants.
Funding: This research was funded by the National Research Foundation of Korea (NRF) Grants awarded by the
Korean government (MEST, No. 2017R1A2B4012636). The Philippine DOST-NSC-SEI for the thesis grant was
awarded to M.W.D.L.
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Acknowledgments: The Pascual Pharma Corporation is gratefully acknowledged for the LC-MS analysis. We also
thank Felicidad Christina Ramirez for the help on the statistical analyses.
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Sample Availability: Samples of the plant extracts are available from the authors.
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molecules
Article
Mulberry Fruit Cultivar ‘Chiang Mai’ Prevents
Beta-Amyloid Toxicity in PC12 Neuronal Cells and in
a Drosophila Model of Alzheimer’s Disease
Uthaiwan Suttisansanee 1 , Somsri Charoenkiatkul 1 , Butsara Jongruaysup 2 , Somying Tabtimsri 3 ,
Dalad Siriwan 4, *,† and Piya Temviriyanukul 1, *,†
1 Institute of Nutrition, Mahidol University, Salaya, Phuttamonthon, Nakhon Pathom 73170, Thailand;
uthaiwan.sut@mahidol.ac.th (U.S.); somsri.chr@mahidol.ac.th (S.C.)
2 Office of Sericulture Conservation and Standard Conformity Assessment, The Queen Sirikit Department of
Sericulture, Ministry of Agriculture and Cooperatives, Bangkok 10900, Thailand; butsara_2000@hotmail.com
3 The Queen Sirikit Department of Sericulture Center (Kanchanaburi), Nong Ya, Mueang Kanchanaburi
District, Kanchanaburi 71000, Thailand; yodyingtts@gmail.com
4 Institute of Food Research and Product Development, Kasetsart University, Chatuchak, Bangkok 10900,
Thailand
* Correspondence: dalad.s@ku.th (D.S.); piya.tem@mahidol.ac.th (P.T.)
Abstract: Alzheimer’s disease (AD) is the most common form of dementia, characterized by chronic
neuron loss and cognitive problems. Aggregated amyloid beta (Aβ) peptides, a product of cleaved
amyloid precursor protein (APP) by beta-secretase 1 (BACE-1), have been indicated for the progressive
pathogenesis of AD. Currently, screening for anti-AD compounds in foodstuffs is increasing, with
promising results. Hence, the purpose of this study was to investigate the extraction conditions,
phytochemical contents, and anti-AD properties, targeting Aβ peptides of Morus cf. nigra ‘Chiang Mai’
(MNCM) both in vitro and in vivo. Data showed that the aqueous extract of MNCM contained high
amounts of cyanidin, keracyanin, and kuromanin as anthocyanidin and anthocyanins. The extract
also strongly inhibited cholinesterases and BACE-1 in vitro. Moreover, MNCM extract prevented
Aβ-induced neurotoxicity and promoted neurite outgrowth in neuronal cells. Interestingly, MNCM
extract reduced Aβ1–42 peptides and improved locomotory coordination of Drosophila co-expressing
human APP and BACE-1, specifically in the brain. These findings suggest that MNCM may be useful
as an AD preventive agent by targeting Aβ formation.
Keywords: Morus species; Alzheimer’s disease; anthocyanins; anthocyanidins; amyloid peptides;

beta-secretase 1; Drosophila melanogaster
1. Introduction
Alzheimer’s disease (AD) is the most common form of dementia and a public health concern
worldwide. AD is the fifth leading cause of death among people over 65 years old [1] and growth of
AD prevalence is expected. In the USA, the care cost for dementia patients has been estimated at $290
billion, rendering a huge economic problem for society [2]. AD is a chronic neurodegenerative disorder
that is characterized by the loss of cholinergic neurons, low levels of acetylcholine, and aggregation of
neurotoxic amyloid beta plaque. Loss of cholinergic neurons located in the basal forebrain leads to
reduced production of the neurotransmitter, acetylcholine, which is involved in memory and cognitive
functions. Hence, inhibition of acetylcholine-degrading enzymes, cholinesterases (acetylcholinesterase
(AChE) and butyrylcholinesterase (BChE)), may improve attention span and cognitive ability [3].
The accumulation of amyloid beta or beta amyloid (Aβ) peptides results from the cleavage of

Molecules 2020, 25, 1837
transmembrane amyloid precursor protein (APP) by β-site amyloid precursor protein cleaving enzyme
1 or beta-secretase 1 (BACE-1) as one of the AD hallmarks [4]. The Aβ peptides, either as Aβ1–40 or
Aβ1–42 , are secreted and aggregated as a dense senile plaque due to their hydrophobic properties.
However, familial AD patients usually exhibit a higher ratio of Aβ1–42 in the brain, indicating that
Aβ1–42 may be used as a marker for AD pathogenesis [5]. Therefore, besides cholinesterase inhibitors,
BACE-1 inhibitors could be a target of interest for AD prevention.
Several epidemiological reports have documented that the consumption of fruits and vegetables
may prevent or delay the onset of degenerative diseases, including AD and dementia [6,7].
Anthocyanins are flavonoids and occur in fruits and vegetables, mostly in berries. It has been
shown that anthocyanins prevent streptozotocin-induced sporadic dementia of Alzheimer’s type
by decreasing AChE activity in both the cerebral cortex and hippocampus of rats [8]. Furthermore,
anthocyanins, including cyanidin-3-glucoside, delphinidin-3-glucoside, and petunidin-3-glucoside,
suppressed BACE-1 expression in the hippocampal neurons of Aβ1–42 -treated rats [9]. These data
support the postulation that anthocyanin-rich foodstuffs may exert anti-AD properties and promote
AD prevention and treatment.
The mulberry tree belongs to the family Moraceae. Three important mulberries are widely grown
as Morus alba, Morus rubra, and Morus nigra [10]. Interestingly, M. nigra has been reported to have the
highest amounts of anthocyanins compared to other species [10,11]. M. nigra is generally known as black
mulberry. This plant is cultivated in Africa, South America, and Asian countries, including Thailand.
Almost all parts of M. nigra are utilized for both food and pharmacological properties. Its leaves
have been demonstrated to have antinociceptive, anti-inflammatory, and antidiabetic properties [12],
while the fruits have historically been used as food because they are rich in nutrition elements,
flavonols, and anthocyanins [11,13]. The fruits are also used in traditional medicine as they exert
a wide range of health benefits, such as antimicrobial, anti-inflammatory, and antioxidative stress
properties [12,14]. Evidence showed that compounds isolated from M. alba as artoindonesianin O
and inethermulberrofuran C exhibited anti-AD properties [15,16]; however, little is known about the
anthocyanin-rich M. nigra. Therefore, here, the anti-AD properties of M. nigra were investigated.
A well-characterized Morus cf. nigra ‘Chiang Mai’ (MNCM) that is widely planted in Thailand was
used in this study. The mulberry fruits of the mentioned cultivar were determined for their extraction
conditions, phytochemical contents, antioxidative stress, and inhibitory activity against AChE, BChE,
and BACE-1 in vitro. The extract was also determined for its anti-AD properties, targeting Aβ peptides
in the adrenal phaeochromocytoma (PC12) neuronal cells and in a Drosophila model of AD. These
flies were developed for studying potential therapeutic approaches since human APP and BACE-1
are co-expressed specifically in the central nervous system (CNS), representing the production of Aβ
peptides in humans.
2. Results
2.1. Extraction Optimization of Morus cf. nigra ‘Chiang Mai’ (MNCM)

To optimize the extraction conditions regarding anti-AD properties of MNCM, aqueous ethanol
was utilized as a solvent for anthocyanins extraction. First, the sample (30 mg/mL) was extracted
with 0–100% (v/v) aqueous ethanol at 30◦ C for 30 min. The results suggested that acetylcholinesterase
(AChE) inhibition was optimally achieved when extraction was performed under 0% (v/v) ethanol
(or ultrapure water) (Table 1). Inhibition decreased with an increasing percentage of ethanol to 20%
(v/v), and then completely diminished.
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Molecules 2020, 25, 1837
Table 1. Effects of different percentages of aqueous ethanol on MNCM extraction regarding

AChE inhibition.
Independent Variable Dependent Variable

Controlled Variables
(Solvents, % (v/v) Aqueous Ethanol) % AChE Inhibition
0% (v/v) (ultrapure water) 40.29 ± 1.27 a
20% (v/v) 27.07 ± 1.36 b
40% (v/v) ND * • Extraction temperature 30 ◦ C
60% (v/v) ND * • Shaking time 30 min
80% (v/v) ND * • Extraction concentration 30 mg/mL
100% (v/v) (absolute ethanol) ND *
Values expressed are mean ± standard deviation (SD) of triplicate experiments (n = 3). Lower letter case indicates
significant differences in each column at p < 0.05 calculated by one-way analysis of variance (ANOVA) and Duncan’s
multiple comparison test. * ND = not detected.
The effects of shaking time on AChE inhibition were then investigated by utilizing water extraction
of MNCM. The shaking time varied from 0.5 to 6 h and was applied with fixed conditions of a 30 ◦ C
extraction temperature and 30 mg/mL extraction concentration. The results suggested that AChE
inhibition was continuously elevated with an increased shaking time and achieved the significantly
highest inhibition at the 2-h shaking time (Table 2). However, AChE inhibition started to decline after
reaching this optimal shaking time.
Table 2. Effects of different shaking times on MNCM extraction regarding AChE inhibition.

(Shaking Time, h) % AChE Inhibition
0.5 27.63 ± 0.42 c
1 33.63 ± 0.97 b • Extraction temperature 30 ◦ C
2 43.35 ± 3.32 a • Extraction solvent of water
4 30.18 ± 1.15 c • Extraction concentration 30 mg/mL
6 28.41 ± 0.58 c
multiple comparison test.
The last parameter for MNCM extraction was the extraction temperature. The effect of temperature
(30–90 ◦ C) on AChE inhibition using water extraction conditions of a 2-h shaking time and 30 mg/mL
extraction concentration was investigated. The results indicated that AChE inhibition increased with
increasing extraction temperature and reached optimal inhibition at 50 ◦ C (Table 3). However, when
raising the extraction temperature above 50 ◦ C, AChE inhibition declined to the lowest inhibition
at 90 ◦ C.
Table 3. Effect of different temperatures on MNCM extraction regarding AChE inhibition.

(Temperature, ◦ C) % AChE Inhibition
30 27.89 ± 1.36 b
50 32.20 ± 2.67 a • Extraction solvent of water

70 23.66 ± 2.16 c • Shaking time of 2 h
90 11.29 ± 0.18 d • Extraction concentration 30 mg/mL
multiple comparison test.
Thus, the optimized extraction conditions of MNCM to achieve the highest AChE inhibition were
aqueous-based extraction (ultrapure water) using a 50 ◦ C extraction temperature and 2-h shaking time.
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Molecules 2020, 25, 1837
2.2. Antioxidant Activities of MNCM Extract

Antioxidant activities were determined using MNCM extracted under optimized extraction
conditions as mentioned above. Antioxidant activities determined by the 2,2-diphenyl-1-picrylhydrazyl
(DPPH) radical scavenging assay suggested that MNCM extract exhibited scavenging activity of
0.40 ± 0.03 μmol TE/100 g DW, while the chelating ability of ferrous ion was 21.33 ± 0.35 μmol TE/g
DW as investigated by the ferric ion reducing antioxidant power (FRAP) assay. The antioxidant
capacity measured by the oxygen radical absorbance capacity (ORAC) assay was determined at
132.21 ± 8.88 μmol TE/g DW.
2.3. Phytochemical Analysis

It was found that MNCM extracted under optimized extraction conditions exhibited total phenolic
contents (TPCs) of 6.93 ± 0.58 mg GAE/g DW. The only anthocyanin detected in MNCM extracted under
acidic methanol was cyanidin, with a content of 233.77 ± 24.02 μg/g DW, while anthocyanins were
detected as cyanidin-3-O-rutinoside or keracyanin (610.99 ± 9.17 μg/g DW) and cyanidin-3-O-glucoside
or kuromanin (730.97 ± 3.61 μg/g DW) utilizing high performance liquid chromatography (HPLC)
analysis (Figure S1).
2.4. MNCM Extract Inhibits Cholinesterase and BACE-1 Activities in Vitro

The MNCM extracted under optimized extraction conditions inhibited the key enzymes involved
in AD, including AChE, BChE, and BACE-1, at different percentages. The AChE inhibitory activity
of MNCM extract with 55.36 ± 4.02% inhibition was lower than BChE inhibition with 81.43 ± 4.56%
inhibition at the final extract concentration of 5 mg/mL. Under the same extract concentration, BACE-1
inhibition was reported at 66.34 ± 5.32%.
2.5. MNCM Extract Prevents Aβ Peptide-Induced Toxicity and Promotes Neurite Outgrowth
To investigate the neuroprotective effect of MNCM extract on PC12 neuronal cells, the cytotoxicity
of MNCM extract was studied. PC12 cells were exposed with various concentrations of MNCM extract
(50–200 μg/mL) for 24, 48, and 72 h. Results from the resazurin assay (Figure 1A) displayed that all
concentrations of MNCM aqueous extract were not toxic to PC12 cells even after 72 h of treatment.
We then selected these four concentrations for further analysis.
As mentioned above, MNCM is rich in anthocyanins and anthocyanidins, resulting in antioxidant
activities. In addition, free radicals are also involved in the pathogenesis of AD [17]. Therefore,
the protective effects of MNCM extract against H2 O2 , an oxidative stress inducer, were determined.
Pre-treatment of PC12 cells with MNCM extracts (50–200 μg/mL) for 24 h significantly protected cells
from oxidative stress-induced cell death in a dose-dependent manner compared with non-pretreated
cells, as seen in Figure 1B, confirming the antioxidant activities in vitro.
It is well established that Aβ peptide-induced neuronal toxicity occurs via oxidative stress
induction [18]. As illustrated in Figure 1C, PC12 cells were pre-treated with MNCM extract for
24 h before adding Aβ25–35 peptides. The Aβ25–35 peptides are widely used in AD study. Moreover,
they have short fragments but retain active domains of Aβ1–42 . In addition, the Aβ25–35 and Aβ1–42
peptides induce neural toxicity in a similar fashion [19]. Figure 1C shows that non-pretreated cells
gave approximately 40% cell viability after exposure to Aβ25–35 peptides, whereas MNCM extract
prevented Aβ peptide-induced toxicity in a dose-dependent manner, similar to Figure 1B. It seemed
likely that the 200 μg/mL extract could diminish all adverse effects of Aβ peptides compared with the
DI treatment.
Neurite outgrowth is a vital mechanism in neuronal growth and differentiation, and defects
in the process might lead to neurodegenerative disorders like AD [20]. Therefore, we determined
the effects of MNCM extract on neurite outgrowth. The results in Figure 1D and Figure S2 show
that cells without nerve growth factor (NGF) or MNCM extract contained a lower percentage of
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Molecules 2020, 25, 1837
neurite-bearing cells, whereas nerve growth factor (NGF) stimulated neurite outgrowth as previously
reported. A dose-dependent manner of MNCM extract in stimulating neurite outgrowth was observed.
Intriguingly, a high dose of MNCM extract at 200 μg/mL activated neurite outgrowth similar to the
NGF-treated cells.
In conclusion, aqueous extract of MNCM was not toxic to PC12 cells, prevented H2 O2 or Aβ
peptide-induced cell death, and promoted neurite outgrowth.
Figure 1. (A) Determination of safe doses of MNCM extract on the cell viability of PC12 cells after 24, 48,
and 72 h of MNCM extract treatment, the percentage of cell viability is displayed. (B) Preventive effects
of MNCM extract on H2 O2 -induced cell death, cells were pre-treated with the extract (50–200 μg/mL)
for 24 h, then 300 μM of H2 O2 was added for another 24 h. The percentage of cell viability is illustrated.
(C) Preventive effects of MNCM aqueous extract on Aβ peptide-induced cell death, cells were pre-treated
with MNCM extract (50–200 μg/mL) for 24 h, then 20 μM of Aβ peptides were added for another 24 h.
The percentage of cell viability is illustrated. (D) Effects of MNCM extract (50–200 μg/mL) on neurite
outgrowth compared with the nerve growth factor (NGF, 50 ng/mL). The data are represented as the
percentage of neurite-bearing cells. The bar graphs are representative of three experiments and show
mean ± standard deviation (SD). The one-way ANOVA followed by Tukey’s test was used to determine
the differences between groups. ***, p < 0.001.
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Molecules 2020, 25, 1837
2.6. MNCM Extract Reduces Aβ1-42 by Inhibiting BACE-1 Activity in a Drosophila Model of AD
To further investigate the anti-AD properties of MNCM extract in vivo, we employed a Drosophila
model to our advantage by co-expressing human APP and BACE-1 specifically in the CNS of fly brains,
thereby representing the amyloidogenic pathway. These short memory-deficient AD flies proved to be
a useful tool to delineate the preventive effects of food or phytochemicals on the Aβ pathway [21]. First,
we investigated safe doses of MNCM extract in Drosophila larvae. Larvae were exposed to MNCM
extracts (0–1 mg/mL), and then the hatched flies were scored. As seen in Figure 2A, compared to
the DI treatment, MNCM extracts up to 500 μg/mL were not toxic, whereas toxicity was observed at
1 mg/mL of MNCM extract. Thus, MNCM extracts at 150, 250, and 500 μg/mL were selected and used
for further analysis.
It is known that the cleavage of APP by BACE-1 results in Aβ peptides as AD hallmarks. Hence,
the flies were treated with MNCM extract at the indicated concentration from one day after eclosion,
and donepezil, an AD drug, was used as the control. After 28 days, heads were collected, and the
levels of Aβ1–42 peptides were quantified. The data showed that donepezil and 500 μg/mL of MNCM
extract reduced Aβ1-42 peptide formation by approximately 2 fold compared with DI-treated flies
(Figure 2B). A lesser reduction was also observed at 250 μg/mL of MNCM extract, while MNCM extract
at 150 μg/mL was not potent enough to reduce Aβ1–42 formation, consistent with the cell study.
AD leads to a progressive decline in locomotory coordination. This ability can be measured by the
climbing assay in Drosophila. Therefore, we tested whether MNCM extract ameliorated Aβ1–42 -induced
motor dysfunction in the AD flies. Using the same treatment as above, at day 28, flies were recorded
for their climbing index. As shown in Figure 2C, the DI-treated flies representing AD exhibited an
extremely reduced ability to climb compared to the AD-free flies (elav-GAL4), suggesting severe
locomotory coordination possibly from high amounts of Aβ1–42 peptides (Figure 2B). Interestingly, the
climbing index was rescued in a dose-dependent manner when flies were exposed to MNCM extracts
at 250 and 500 μg/mL and donepezil.
To test whether MNCM extract acts as a BACE-1 inhibitor and leads to a reduction in Aβ1–42
peptides, fly brain lysates at day 28 of treatment were prepared and determined for BACE-1 activity.
It was found that MNCM extract at 150 μg/mL and the DI control had the same BACE-1 activity
(Figure 2D). However, flies treated with donepezil, and 250 and 500 μg/mL MNCM extract showed
significantly decreased BACE-1 activity in AD fly brains. Donepezil is claimed to be a cholinesterase
inhibitor, and its BACE-1 inhibitory activity has been documented [22].
In summary, aqueous extract of MNCM reduced Aβ1–42 formation and improved locomotor
dysfunctions by inhibiting BACE-1 activity in the Drosophila model of AD.
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Figure 2. (A) Determination of safe doses of MNCM extract in fly larvae, the third-instar larvae were
fed with MNCM extract (150 μg/mL–1 mg/mL). The surviving flies were counted within 5 days after the
first eclosion and the percentage of the survival rate was calculated. (B) Effects of MNCM extract on the
accumulation of Aβ1–42 peptides in fly brains. Flies were treated with MNCM extract (150–500 μg/mL)
for 28 days, and after that fly heads were lysed and subjected for ELISA. (C) Effects of MNCM extract
(150–500 μg/mL) on the locomotory coordination (climbing) of AD flies after 28 days of treatment.
(D) Effects of MNCM extract on the BACE-1 activity in fly brains. Flies were treated with MNCM
extract (150–500 μg/mL) for 28 days, then fly heads were lysed and subjected for BACE-1 activity
determination. The data are representative of three replicates and show mean ± standard deviation
(SD). The one-way ANOVA followed by Tukey’s test was used to determine the differences between
groups. ***, p < 0.001.
3. Discussion
AD is a complex and progressive neurodegenerative disorder and an effective therapy is lacking.
Therefore, the identification of novel AD therapeutic agents is urgently required. It is well-documented
that oxidative stress and the expression of cholinesterases and BACE-1 play a vital role in AD initiation
and progression [3,4,17]; thus, an ideal AD therapeutic agent should function against different AD
pathogenic mechanisms [23]. Plants and their bioactive constituents are of great interest due to their
safety and efficacy. Indeed, many plant-derived compounds, including phenolic acids and flavonoids,
have been reported for their anti-AD properties toward oxidative stress, AChE, BChE, and BACE-1
activities [23,24].
Mulberry has been recognized to be rich in anthocyanins as members of the flavonoids, and
especially for M. nigra. Therefore, this project aimed to study the anti-AD properties of aqueous extracts
of M. cf. nigra ‘Chiang Mai’ fruit (MNCM), which is widely grown in Thailand. The major findings
were (i) MNCM extract was rich in anthocyanins and anthocyanidins, especially cyanidin, kuromanin,
and keracyanin, which are probably involved in antioxidative stress; (ii) MNCM extract exhibited up
to 50% inhibitory activity against AChE, BChE, and BACE-1; (iii) MNCM extract protected neuronal
cells from H2 O2 or Aβ peptide-induced toxicity and promoted neurite outgrowth; and (iv) MNCM
extract reduced Aβ1-42 peptides by inhibiting BACE-1 activity in a Drosophila model of AD.
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Previous reports suggested that mulberry exhibited different degrees of phenolics and antioxidant
activities depending on both internal factors (such as cultivars and stages of maturity) and external
factors (such as detection methods and extraction conditions) [25,26]. Comparing water-extracted
MNCM with the TPCs of 6.39 mg GAE/g DW, five cultivars of Korean mulberries (M. alba) extracted
under 70% (v/v) aqueous ethanol exhibited lower TPCs, ranging from 2.2 to 2.6 mg GAE/g DW [25].
Besides, water-extracted MNCM also exhibited higher TPCs than methanolic-extracted M. alba collected
in North Serbia, with the TPCs ranging from 1.05 to 2.16 mg GAE/g DW [27]. As for antioxidant
activities, these values seemed to be greatly affected by both internal and external factors. In comparison
to MNCM with ORAC activity of 132.21 ± 8.88 μmol TE/g DW, it was previously suggested that
juices from different maturity stages of thornless blackberry exhibited ORAC activities ranging from
86.8–204.1 μmol TE/g DW, while those from red raspberry ranged of 40.8–114.9 μmol TE/g DW [28].
In the same study, juices from various cultivars of ripe strawberry exhibited ORAC activities of
120.8–172.3 μmol TE/g DW. The FRAP activity of MNCM (21.33 ± 0.35 μmol TE/g DW) was comparable
to raspberry (28.11 μmol TE/g DW) and cranberry (22.41 μmol TE/g DW) extracted under a mixture of
70% (v/v) aqueous methanol (MeOH) and 70% (v/v) aqueous acetone (1:1, v/v) [29]. Interestingly, the
FRAP activity of MNCM was higher than blackcurrant and blueberry (17.81 and 17.27 μmol TE/g DW,
respectively) but lower than those of blackberry and black chokeberry (11.63 and 33.16 μmol TE/g DW,
respectively) [29]. However, the DPPH radical scavenging activity (0.40 ± 0.03 μmol TE/100 g DW
or approximately 0.44 μmol TE/100 g fresh weight with 90% moisture content) of MNCM was lower
than blackberries, black mulberries (M. nigra), bilberries, and blackthorns ranging from 1.6–8.4 μmol
TE/100 g frozen fruit extracted under acidified MeOH (0.1% HCl) [30].
Interestingly, keracyanin (60%) and kuromanin (38%) were the two main anthocyanins detected
in mulberry [31,32]. Keracyanin (610.90 μg/g DW) and kuromanin (730.97 μg/g DW) detected in
MNCM extract were in the range of those detected in Korean mulberries (30.6–486.7 μg keracyanin/g
DW and 93.2–1364.9 μg kuromanin/g DW) [25]. The aqueous extract of MNCM provided good
inhibitory activities against AChE, BChE, and BACE-1. It was previously reported that 18 commercially
available mulberries (M. alba) in Poland, extracted under 80% (v/v) aqueous methanol, exhibited
AChE inhibitory activity in the range of 2.6–37.9% [33]. However, this paper failed to indicate the
extract concentration in the enzyme assay, and this cannot be used for comparison with MNCM
extract. Considering the predominant anthocyanins and anthocyanidin detected in mulberry, cyanidin
was able to inhibit AChE, with IC50 of 14.43 μM, while its BChE inhibitory activity was slightly
higher [34]. Compared to cyanidin, its anthocyanin glycosides, including keracyanin and kuromanin,
with insignificantly different cholinesterase inhibition, exhibited lower inhibitory activity against AChE
and BChE [34]. However, an in vitro report on the BACE-1 inhibitory activity of these anthocyanidins
and anthocyanidins remains unwritten.
After studying the anti-AD properties in vitro regarding AChE, BChE, and BACE-1, we also
examined the anti-AD effects of MNCM extract on PC12 neuronal cells. As shown in Figure 1B,C,
pre-treatment with MNCM extract evidently protected cells from H2 O2 or Aβ peptide-induced
toxicity. It may be possible that MNCM extract exerted its effective antioxidative properties based
on the high amounts of anthocyanins and anthocyanidins as previously mentioned. Furthermore,
it has been found that Aβ25–35 peptides cause PC12 apoptosis by triggering oxidative stress, lipid
peroxidation, and intracellular calcium ([Ca2+ ]i ), similar to that of H2 O2 [35,36], indicating that
Aβ25–35 peptides lead to mitochondrial dysfunction. Mitochondria is an organelle function in ATP
synthesis and Ca2+ homeostasis, thus its impairment will provoke [Ca2+ ]i release and the apoptotic
protease-activating factor 1 (Apaf1)-mediated intrinsic apoptotic pathway. Indeed, Aβ25–35 peptides
induced Apaf1-mediated cell death, while pre-treatment with ethanolic extract of Chinese M. nigra for
24 h followed by Aβ25–35 exposure downregulated Apaf1 [37]. Previous studies showed that cyanidin
and kuromanin attenuate Aβ-induced PC12 neurotoxicity by maintaining mitochondrial stability [38].
Interestingly, MNCM extract was high in cyanidin, kuromanin, and keracyanin, indicating that MNCM
extract may play a role in maintaining mitochondrial stability, which eventually leads to reduced
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Aβ25–35 -mediated cell death. Additionally, kuromanin has been reported to reverse ethanol-induced
inhibition of neurite outgrowth [39]. Neurite outgrowth is in neuron growth, and poor neurite
outgrowth is observed in AD [20]. MNCM extract activated the neurite outgrowth of PC12 cells
(Figure 1D). A high dose of MNCM extract at 200 μg/mL activated neurite outgrowth better than
that of NGF-exposed cells. This could be because MNCM may enhance the expression of NGF as
demonstrated in M. fructus [40].
To elucidate the anti-AD properties of MNCM extract in depth, a Drosophila co-expressing human
APP and BACE-1 was employed. The fruit fly has emerged as a promising alternative model for AD
drug screening since transgenic flies carrying AD-related genes demonstrated AD characteristics as
in humans [41]. Flies also possess a unique approach for AD study because the elav-GAL4 driver
can drive the expression of AD proteins in the brain at an early stage. This was in accordance with
one of the present strategies for anti-AD agents to target at the earlier stages [42]. As illustrated in
Figure 2B,C,D, MNCM extract inhibited BACE-1 activity, resulting in decreased Aβ1–42 peptides and
improved locomotor functions in AD flies, in a dose-dependent fashion. The data raise the hypothesis
that MNCM extract may penetrate the blood–brain barrier (BBB) and restrain BACE-1 function. It is
important to consider that several neurotherapeutic agents worked well in the cell study but not
in animal models since they failed to cross the BBB. BACE-1 is a rate-limiting enzyme responsible
for amyloid peptide production, thereby making it ideal for AD therapy [43]. Anthocyanins and
anthocyanidins are able to cross the BBB, especially kuromanin, and are located in the brain regions
contributing to cognitive functions [44,45]. Thus, the present data support that MNCM could be further
developed as a potential natural product for AD prevention by targeting BACE-1. Furthermore, in this
study, MNCM was extracted by water, making it useful for further application as a functional food
for neuroprotection.
4.1. Mulberry Collection and Preparation

Morus cf. nigra ‘Chiang Mai’ (MNCM) was sourced from the Queen Sirikit Department of
Sericulture. The sample was identified based on the morphology and nuclear ribosomal internal
transcribed spacer (nITS) (GenBank: MK946679.1) and deposited at the Bangkok Herbarium (BK),
Bangkok, Thailand. The Herbarium voucher specimen is B. Jongruaysup et al. 12-1 (BK). Fruits
of uniform color and ripening stage were selected and cleaned before being freeze-dried. Samples
were then ground to a fine powder and extracted using ultrapure water (Smart2Pure 3 UVTM Water
Purification System, Thermo Fisher Scientific, Waltham, MA, USA) at 50 ◦ C for 2 h.

The in vitro antioxidant activity of the mulberry extract was performed from a well-established
protocol for 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity, oxygen radical absorbance
capacity (ORAC), and ferric ion reducing antioxidant power (FRAP) assays [46–49].
4.3. Total Phenolic Contents, Anthocyanin, and Anthocyanidin Determination

Total phenolic contents (TPCs) were determined using Folin-Ciocalteu reagent as described by
Thuphairo et al. 2019 [24]. Gallic acid was used as a reference matter, and the TPCs were reported as mg
gallic acid equivalent (GAE)/g dried matter (DW) [24]. To determine anthocyanins and anthocyanidins,
the mulberry powder was extracted under acidic conditions. The extracts were collected by filtering
through a 0.45-μm polytetrafluoroethylene (PTFE) syringe filter. The HPLC analysis (an UtiMate
HPLC with an HPG-3400SD pump and a photodiode array detector from DIONEX, Sunnyvale, CA,
USA) was performed using Thermo Scientific Chromeleon Chromatography Data System (CDS)
software (DIONEX, Sunnyvale, CA, USA) and a Reprosil-Pur ODS-3 column (250 mm × 4.6 mm, 5 μm
from Dr. Maisch GmbH, Ammerbuch, Germany). For anthocyanidin analysis, a constant flow rate
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of 1 mL/min at ambient temperature was employed with an isocratic solvent of 82% Milli-Q water
containing 0.4% (v/v) trifluoroacetic acid (TFA) (Solvent A) and 18% acetonitrile containing 0.4% (v/v)
TFA (Solvent B) over 60 min. For anthocyanin analysis, a constant flow rate of 1 mL/min at ambient
temperature was employed. The solvent system is shown in Table 4.
Table 4. Solvent system of anthocyanin analysis using HPLC.
Time (min) Solvent A Solvent B

0 88 12
6 88 12
8 85 15
25 85 15
25 88 12
30 88 12
Solvent A = Milli-Q water containing 0.4% (v/v) TFA; solvent B = acetonitrile containing 0.4% (v/v) TFA.
Samples were kept in the autosampler at 4 ◦ C until injection (20 μL). The presence of
anthocyanins and anthocyanidins was visualized at 525 and 530 nm, respectively. Anthocyanins
(cyanidin-3-O-glucoside (kuromanin), cyanidin-3-O-rutinoside (keracyanin), cyanidin-3,5-O-
diglucoside (cyanin), cyanidin-3-O-galactoside (idaein), pelargonidin-3,5-O-diglucoside (pelargonin),
malvidin-3-O-galactoside (primulin), and petunidin-3-O-glucoside) and anthocyanidins (cyanidin,
delphinidin, pelargonidin, peonidin, petunidin, and malvidin) were used as standards to identify
anthocyanins and anthocyanidins in the sample by comparing their retention times (Rt ) and UV-vis
spectral fingerprints. All chemicals were received from Sigma-Aldrich (St. Louis, MO, USA).
4.4. Determination of Cholinesterases and Beta-Secretase 1 (BACE-1) Inhibitory Activities

Inhibitory activity of MNCM extract against AChE was carried out as previously reported [24,50,51].
In brief, a mixture containing 20 ng of Electrophorus electricus AChE (1000 units/mg, 100 μL), 16 mM
5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB, 10 μL), 0.8 mM acetylthiocholine (40 μL), and the extract
(50 μL) were well-mixed. The initial velocity was measured at 412 nm using a microplate reader
(SynergyTM HT 96-well UV-visible spectrophotometer using Gen5 data analysis software from BioTek
Instruments, Inc., Winooski, VT, USA). Percentage of inhibition was then calculated as follows:

B−b
% inhibition = 1 − × 100, (1)
A−a
where A is the initial velocity of the reaction with enzyme, a is the initial velocity of the reaction without
enzyme, B is the initial velocity of the enzyme reaction with extract, and b is the initial velocity of the
reaction with extract but without enzyme.
Inhibitory activity of MNCM extract against BChE was determined similarly to AChE, except that
100 ng of equine serum BChE (≥10 units/mg protein, 100 μL) and 0.1 mM butyrylthiocholine (BTCh)
were used as the reaction enzyme and substrate, respectively [24,50]. All chemicals and reagents for
cholinesterase inhibitory activities were purchased from Sigma-Aldrich (St. Louis, MO, USA).
The BACE-1 activity was determined using a BACE-1 activity detection kit (Sigma-Aldrich,
St. Louis, MO, USA) following the manufacturer’s instructions and expressed as the percentage of
BACE-1 inhibition.
4.5. Cell Culture and Cytotoxicity Analysis by Resazurin Assay

The PC12 neuronal cells were obtained from the American Type Culture Collection (ATCC,
Manassas, VA, USA) and cultured in Roswell Park Memorial Institute 1640 (RPMI1640), 10% (v/v) fetal
bovine serum (FBS) (Thermo Fisher Scientific, Waltham, MA, USA) and 1% (v/v) penicillin-streptomycin
(Sigma-Aldrich, St. Louis, MO, USA) at 37 ◦ C in a humidified atmosphere containing 5% CO2 .
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For cytotoxicity analysis, each well of a 96-well plate of 1 × 104 exponentially growing PC12
cells was seeded and cultured overnight. Then, the cells were exposed to 50, 100, 150 and
200 μg/mL of MNCM extracts for 24, 48, and 72 h. At the indicated time, 20 μL of resazurin
dye (CellTiter-Blue® , Promega, Madison, WI, USA) was added. One hour later, the fluorescence
(emission = 585 nm, excitation = 570 nm) was measured. Cells treated with deionized water (DI) were
used as a negative control.
4.6. Prevention of Hydrogen Peroxide (H2 O2 ) and Aβ Peptide-Induced Toxicity

Each well of a poly-D-lysine (PDL)-coated 96-well plate of PC12 cells was plated and cultured
overnight at a density of 2 × 104 cells/mL. Then, the cells were treated with MNCM extracts for 24 h
(50, 100, 150, and 200 μg/mL). After that, 300 μM of H2 O2 (Merck, Darmstadt, Germany) or 20 μM of
amyloid peptide (Aβ25–35 ) (Bachem, Bubendorf, Switzerland) were added. Forty-eight hours later, cell
survival was measured by the resazurin assay, as mentioned above.
4.7. Determination of Neurite Outgrowth

Each well of a poly-D-lysine (PDL)-coated 6-well plate of PC12 cells was seeded and cultured
overnight at a density of 1 × 104 cells/mL. Cells were then treated with MNCM extracts at 50, 100, 150,
and 200 μg/mL in RPMI1640 with 0.5% FBS for one week, and the medium was changed every two
days. The nerve growth factor (NGF) (Sigma-Aldrich, St. Louis, MO, USA) at 50 ng/mL was used as a
positive control. Before analysis under a phase-contrast microscope, the neurite-bearing cells were
fixed with 4% paraformaldehyde. The percentage of neurite-bearing cells was expressed. At least three
independent experiments were carried out.
4.8. Drosophila Stocks and Culture

Fly stocks (elav-GAL4 (8760) and UAS-APP-BACE-1) (33798)) were obtained from the Bloomington
Stock Center at Indiana University. After eclosion, F1 progeny flies obtained from the mating between
elav-GAL4 and UAS-APP-BACE-1 were cultured on Formula 4-24 blue® medium (Carolina, Burlington,
NC, USA) containing MNCM extract or DI (control) at 28 ◦ C for 28 days. Flies were transferred to fresh
media every 2–3 days. The Drosophila study was approved by Mahidol University-Institute Animal
Care and Use Committee (MU-IACUC) (COA.No.MU-IACUC 2018/022).
4.9. Climbing Assay

The assay was determined following the published method [52]. At day 28 after treatment, 20 to
30 flies were placed in a transparent tube. After tapping, their rate of climb to the top of the tube was
recorded and analyzed. At least three independent experiments were performed.
4.10. Quantification of Aβ Peptide by Enzyme-Linked Immunosorbent Assay (ELISA)

Quantification of Aβ peptide was performed as reported with slight modification [53]. The heads
of F1 progeny flies at day 28 were collected and homogenized in 5 M guanidine-HCl containing 2X
Halt protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA). Then, the protein
concentration was measured using the BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA,
USA). Before sample loading, a serial dilution of supernatants was made with ELISA diluent buffer
containing protease inhibitor cocktail. After following the manufacturer’s instructions (human Aβ42
ELISA kit (Thermo Fisher Scientific, Waltham, MA, USA)) the samples were measured at 450 nm.
The concentration of Aβ1-42 peptides was calculated and compared with standard recombinant human
Aβ1-42 .
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4.11. Determination of BACE-1 Activity in Fly Brain Lysate

The heads of F1 progeny flies at day 28 were collected and homogenized in T-PER tissue protein
extraction reagent (Thermo Fisher Scientific, Waltham, MA, USA). Then, the protein concentration and
BACE-1 activity were measured as mentioned above within the same day.

Figure S1: High-performance liquid chromatography (HPLC) chromatograms of (A.) cyanidin chloride standard (B.)
kuromanin and keracyanin standard (C.) anthocyanidin analysis of MNCM extract and (D.) anthocyanin analysis
of MNCM extract. The retention times (Rt ) of cyanidin chloride, kuromanin and keracyanin in MNCM extract
was also indicated; Figure S2: Representative images from the neurite outgrowth study showing neurite-bearing
cells of (A.) control, (B.) MNCM extract-treated cells at 50 μg/mL, (C.) MNCM extract-treated cells at 100 μg/mL,
(D.) MNCM extract-treated cells at 150 μg/mL, (E.) MNCM extract-treated cells at 200 μg/mL, and (F.) NGF-treated
cells at 50 ng/mL.
Author Contributions: B.J. and S.T. initial technical support and provided samples. U.S. performed experiments,
interpreted the results and generated the figures and tables, wrote the manuscript. D.S. and P.T. supervised,
interpreted the results and designed the research, wrote the manuscript, reviewed the manuscript. S.C. suggested
and reviewed the manuscript. All authors have read and agree to the published version of the manuscript.
Funding: This study was supported by Biodiversity-Based Economy Development Office (BEDO) (grant
No. 22/2561), and partially supported by Grants-in-Aid from JSPS Core to-Core Program B. Asia-Africa
Science Platforms.
Acknowledgments: We thank Kanchana Pruesapan, Plant Varieties Protection Division, Department of
Agriculture, Bangkok for identification of the sample. We also thank Woorawee Inthachat for artwork editing and
personal assistant.
Conflicts of Interest: All authors declare that there are no conflicts of interest.
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molecules
Article
Study on the Antinociceptive Activity and
Mechanism of Action of Isolated Saponins from
Siolmatra brasiliensis (Cogn.) Baill
Thais Biondino Sardella Giorno 1,2 , Carlos Henrique Corrêa dos Santos 3 ,
Mario Geraldo de Carvalho 3 , Virgínia Cláudia da Silva 4 , Paulo Teixeira de Sousa Jr. 4 ,
Patricia Dias Fernandes 1,2, * and Fabio Boylan 5
1 Federal University of Rio de Janeiro, Institute of Biomedical Sciences, Laboratory of Pharmacology of Pain
and Inflammation, Rio de Janeiro 21941-902, Brazil; thais.sardella.farma@hotmail.com
2 Federal University of Rio de Janeiro, Institute of Biomedical Sciences, Graduate Program in Pharmacology
and Medicinal Chemistry, Rio de Janeiro 21941-902, Brazil
3 Federal Rural University of Rio de Janeiro, Department of Chemistry, Seropédica 23890-000, Brazil;
caio.chcs@msn.com (C.H.C.d.S.); mgeraldo@ufrrj.br (M.G.d.C.)
4 Federal University of Mato Grosso, Department of Chemistry, Cuiabá 78935-901, Brazil;
vcsvirginia@yahoo.com.br (V.C.d.S.); pauloteixeiradesousa@gmail.com (P.T.d.S.J.)
5 Trinity College Dublin, Trinity Biomedical Sciences Institute, School of Pharmacy and Pharmaceutical
Sciences, Dublin 2, Ireland; fabio.boylan@tcd.ie
* Correspondence: patricia.dias@icb.ufrj.br; Tel.: +5521-3938-6442

Received: 4 November 2019; Accepted: 9 December 2019; Published: 14 December 2019
Abstract: Infusions of roots of Siolmatra brasiliensis (Cogn.) Baill, (“taiuiá”, “cipó-tauá”) are used for
toothache pain and ulcers. We aimed to study the antinociceptive effects and identify the possible
mechanism of action of this plant and its isolated substances (cayaponoside A1, cayaponoside B4,
cayaponoside D, and siolmatroside I). Hydroethanol extract (HE), ethyl acetate fraction (EtOAc),
and isolated saponins were evaluated in chemical and thermal models of pain in mice. Animals
were orally pretreated and evaluated in the capsaicin- or glutamate-induced licking and in the hot
plate tests. The antinociceptive mechanism of action was evaluated using the hot plate test with the
following pretreatments: Atropine (cholinergic antagonist), naloxone (opioid antagonist), or L-NAME
(nitric oxide synthase inhibitor). All extracts and isolated saponins increased the area under the curve
in the hot plate test. Tested substances induced a higher effect than the morphine-treated group.
Our data suggest that stems of S. brasiliensis and their isolated substances present antinociceptive
effects. Cholinergic and opioidergic pathways seem to be involved in their mechanism of action.
Taken together our data corroborate the traditional use of the plant and expands the information
regarding its use.
Keywords: Siolmatra brasiliensis; antinociceptive activity; cayaponoside A1; cayaponoside B4;

cayaponoside D; siolmatroside I; pain; analgesia
1. Introduction
Siolmatra brasiliensis (Cogn.) Baill is a climbing plant belonging to the Cucurbitaceae family
that occurs in the central region of Brazil, especially in Cerrado and Pantanal where it is popularly
known as “taiuiá” or “cipó-tauá” [1]. Its roots are considered a purifying and antisyphilis agent [2].
Infusions prepared with roots are widely used in traditional medicine as an analgesic for treatment of
toothache [2] and for the treatment of ulcers [3]. Due to the presence of cucurbitacins, compounds
responsible for the bitter tang and high toxicity, Lima et al. [1] showed some toxicological effects of

Molecules 2019, 24, 4584
S. brasiliensis only at very high doses (i.e., 2 g/kg). In a recent study, Dos Santos et al. [3] performed
a regional ethnopharmacological use of the infusion of S. brasiliensis stems in Mato Grosso (Brazil)
and demonstrated that the crude hydroethanol extract reduced the hyperglycemia and glycosuria
in diabetic mice. On the other hand, our continuous search for evidences for the traditional use of
Brazilian species led us to hear about the popular use of S. brasiliensis to treating pain as a result of
toothache. On that basis, the aim of the present work was to investigate the antinociceptive effect
of S. brasiliensis extract and its previously isolated saponins: Cayaponoside A1, cayaponoside B4,
cayaponoside D, and siolmatroside I, and suggest the mechanism of their antinociceptive activity.
In this regard we used atropine (cholinergic antagonist), naloxone (opioid antagonist), or L-NAME
(nitric oxide synthase inhibitor) to evaluate the participation of these pathways in the antinociceptive
effect of S. brasiliensis. It is important to mention that siolmatroside I (a dammarane type saponin) was
described by our research group for the first time in the plant kingdom.
2. Results
2.1. Assessment of Side Effects and Toxicity

A single oral administration of HE, EtOAc, or the isolated saponins (SI, D, B4, and A1) were
evaluated against a possible toxic effect when administered orally. Doses of 150 mg/kg of extracts or
10 mg/kg of each isolated saponins were used. After periods of 1, 3, 6, and every 24 h until five days
post-oral administration animals were evaluated regarding behaviour alterations. We also evaluated
food and water intake every 24 h until the fifth day. Results obtained indicated that none of the extract
or isolated saponins induced compartmental alterations, did not change the amount of food and water
intake, and did not induce any mucosal lesion five days after their administration, presenting visual
conditions similar to those for the vehicle (supplementary material, Tables S1–S4).
2.2. Antinociceptive Effect of HE, EtOAc, SI, D, B4, and A1 in the Hot Plate Test
The hot plate model was used to evaluate the supraspinal antinociceptive effect of the tested
substances. As it can be observed in Figure 1A and B, all three doses of HE and EtOAc significantly
increased the time necessary for mice to respond to the stimulus. Maximal effects were observed at
150- or 120-min post-administration, respectively, and they returned to basal levels after this period.
When data obtained in time course assays were converted to a graph of area under the curve (AUC) it
could be noted that all doses increased these values. Furthermore, higher doses (30 and 100 mg/kg)
of EtOAc were significantly even when compared to the morphine-treated group (Figure 1C and D).
Although a lower dose (30 mg/kg) of HE presented higher area under the curve values than higher
dose (100 mg/kg) (7001 ± 1260 versus 4697 ± 1483) a statistical significance between both groups was
not observed (Figure 1A and B).
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Molecules 2019, 24, 4584
Figure 1. Effects of hydroethanol extract (HE, graphs A and B) or ethyl acetate fraction (EtOAc, graphs
C and D) of S. brasiliensis in the hot plate model. Animals were orally pretreated with different doses
of HE, EtOAc, morphine (2.5 mg/kg) or vehicle. The results are presented as mean ± SD. (n = 6 per
group) of increase in baseline (graphs A and C) or area under the curve (graphs B and D) calculated
by Prism Software 5.0. Statistical significance was calculated by ANOVA followed by Dunnett’s test.
* p < 0.05 when comparing to vehicle-treated group; # p < 0.05 when comparing treated mice with the
morphine-treated group.
The next step was the evaluation of the saponins isolated from ethyl acetate fraction using this
same model. The doses were chosen based on the yield of each saponin after isolation from the ethyl
acetate fraction. Data shown in Figure 2 demonstrated that doses of 1 and 3 mg/kg of all saponins
presented a significant antinociceptive effect increasing the AUC. It is interesting to note that SI (at the
doses of 1 and 3 mg/kg) presented an effect higher than that observed for the positive control group
(morphine-treated mice).
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Molecules 2019, 24, 4584
Figure 2. Effects of Saponins isolated from the ethyl acetate fraction of the stems of S. brasiliensis:
siolmatroside I (SI), cayaponoside D (D), cayaponoside B4 (B4), and cayaponoside A1 (A1) in the hot
plate model. Animals were orally pretreated with different doses of A1, B4, D, I, morphine (2.5 mg/kg),
or vehicle. The results are presented as mean ± SD. (n = 6 per group) of increase in baseline (graphs A,
C, E, and G) or area under the curve (graphs B, D, F, and H) calculated by Prism Software 5.0. Statistical
significance was calculated by ANOVA followed by Dunnett´s test. * p < 0.05 when comparing to
vehicle-treated group; # p < 0.05 when comparing treated mice with the morphine-treated group.
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Molecules 2019, 24, 4584
2.3. Investigation of the Mechanism of Action of EtOAc, SI, D, B4, and A1 in the Hot Plate Model
As the ethanol extract, ethyl acetate fraction and its isolated saponins (SI, D, B4, and A1) showed
that the significant antinociceptive effect was decided to further investigate the role of different
nociceptive pathways involved in the transmission of nociceptive stimulus or the activation of
pathways involved in the control of nociception. None of the receptor antagonists (atropine and
naloxone) or enzyme inhibitor (L-NAME) demonstrated any antinociceptive effect per se in the hot
plate model (Data not shown). As the intention was to observe an inhibitory effect, we decided to use
the higher dose of the extract, fraction (100 mg/kg), or isolated saponins (3 mg/kg). The pretreatment
with atropine (muscarinic receptor antagonist, 1 mg/kg, i.p.) or naloxone (opioid receptor antagonist,
1 mg/kg, i.p.) reversed the antinociceptive effect of HE and EtOAc (Figure 3A), SI, D, B4, and A1
(Figure 3B). The inhibitor of nitric oxide synthase enzyme (L-NAME, 3 mg/kg, i.p.) reversed the
antinociceptive effect EtOAc (Figure 3A), SI, B4, and A1 (Figure 3B).
Figure 3. Effects of different antagonists on the antinociceptive activity of HE, EtOAc (graph A),
siolmatroside I (SI), cayaponoside D (D), cayaponoside B4 (B4), and cayaponoside A1 (A1) (graph B) in
the hot plate model. Animals were pretreated with atropine (1 mg/kg, i.p.), naloxone (1 mg/kg, i.p.) or
L-NAME (3 mg/kg, i.p.), 15 min prior to oral administration of HE, EtOAc (100 mg/kg), SI, D, B4, and A1
(3 mg/kg). The results are present as mean ± SD. (n = 6 per group) of the area under the curve calculated
by Prism Software 5.0. Statistical significance was calculated by ANOVA followed by Dunnett´s test.
* p < 0.05 when comparing HE, EtOAc, SI, D, B4, and A1-treated mice to the vehicle-treated group;
# p < 0.05 when comparing antagonist or inhibitor pretreated mice with the HE, EtOAc, SI, D, B4, or
A1-treated group.
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Molecules 2019, 24, 4584
2.4. Antinociceptive Effect of HE, EtOAc, SI, D, B4, and A1 in the Capsaicin- or Glutamate-Induced
Nociception
The results depicted in Figure 4A show that HE and EtOAc produced a significantly and
dose-dependent reduction in the capsaicin-induced neurogenic pain. Pretreatment of animals with
HE (at 10, 30, or 100 mg/kg doses) significantly reduced the paw licking induced by capsaicin by
22.2%, 50.4%, and 65.3% (52.1 ± 4.1; 33.5 ± 3.1, and 23.4 ± 1.7 s, respectively). Same doses of EtOAc
demonstrated 40.7%; 54.2% and 67.8% reduction (40 ± 1.5; 30.9 ± 1.5; 21.7 ± 1.2 s, respectively) when
compared to vehicle-treated animals (67.5 ± 1.9 s). Oral treatment of mice with A1 or SI significantly
inhibited the capsaicin-induced licking pain at the doses of 0.3, 1, and 3 mg/kg, with the following
results: 43.1 ± 1.4 (36.1%), 34.9 ± 1.1 (48.3%), 26.4 ± 1.2 (60.9%), and 49.0 ± 1.2 (27.4%), 38.8 ± 1.1
(42.5%), 32.8 ± 2.0 s (51.4%), respectively, when comparing with vehicle-treated mice (67.5 ± 1.9 s).
Only the dose of 3 mg/kg of B4 and D demonstrated a significant effect (Figure 4C).
Figure 4. Antinociceptive effect of HE, EtOAc (graphs A and B), siolmatroside I (SI), cayaponoside
D (D), cayaponoside B4 (B4), and cayaponoside A1 (A1) (graphs C and D) on the licking response
induced by capsaicin or glutamate in mice, respectively. Animals were pretreated with different doses
of HE, EtOAc, SI, D, B4, and A1 or vehicle 60 min before the injection of capsaicin (1.6 ng/paw) or
glutamate (3.7 ng/paw). The results are presented as mean ± SD. (n = 6 per group) of the time that
the animal spent licking the capsaicin-injected paw. Statistical significance was calculated by ANOVA
followed by Dunnett´s test. * p < 0.05 when compared to vehicle-treated mice.
We also observed that HE (at 100 mg/kg, p.o.) produced a significant reduction (41.9%) of
glutamate-induced licking response (24.8 ± 3.2 s) when compared to vehicle-treated mice (42.7 ± 1.8 s).
While EtOAc, at all doses, significantly and dose-dependently inhibited the glutamate-induced pain
behavior (31.9 ± 2.5, 28.4 ± 2.9, and 21 ± 2.1 s, to 10, 30, and 100 mg/kg, respectively) when comparing
with the vehicle-treated group, leading to an inhibition of 25.3%, 33.5%, and 50.8%, respectively
(Figure 4B). Among the isolated saponins, only SI (0.3, 1, and 3 mg/kg p.o.) was effective in reducing
(31.9 ± 2.6, 27.6 ± 3.6, and 23.8 ± 5 s) the glutamate-induced nociceptive response corresponding to
an inhibition of 5.3%, 34.6%, and 44.3%, respectively. The cayaponoside A1 reduced the paw licking
at the doses of 1 and 3 mg/kg (34.0 ± 1.5 and 23.3 ± 2.1 s) producing a reduction of 20.4% and 45.4%,
respectively (Figure 4D).
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3. Discussion
This study investigated the antinociceptive activity of S. brasiliensis confirming its popular use
and contributing to the pharmacological knowledge of this plant.
To the best of our knowledge this is the first study showing that hydroethanol extract (HE) of
S. brasiliensis stems, one of its fraction (EtOAc) and two saponins isolated from this fraction (A1 and SI)
have antinociceptive activity when administered orally in different models of thermal and chemical
nociception in mice.
When a chemical, mechanical, or thermal stimulus occurs in mice paw, there is activation of
nociceptors that transmit nociceptive information to the somatosensory cortex, for example, present in
the central nervous system (CNS) producing an organized response, resulting in an elevation of motor
response and/or licking of the paw [4]. The administration of HE, EtOAc, SI, D, B4, and A1, produced
a rapid effect and time course of action similar to morphine.
Several saponins have already been related to the antinociceptive/anti-inflammatory activity.
Examples to cite are saponins from Ipomoea involucrate [5], Pterodone marnatus [6], and
Xeromphisnilotica [7]. Saponins isolated from S. brasiliensis in this study belong to the cucurbitane (A1,
B4, D) and dammarane (SI) types. Cucurbitanes have been shown to possess a good antinociceptive
and anti-inflammatory action as demonstrated recently in a Momordica charantia review paper [8].
Knowing that several endogenous systems are involved in pain control, the mechanism of
antinociceptive action of HE, EtOAc, SI, D, B4, and A1 was also investigated. Atropine, a non-selective
muscarinic receptor antagonist, was used to assess the involvement of the cholinergic system. Some
studies show that activation of the muscarinic receptor (subtype M2) may reduce the response of
peripheral nociceptors in front of noxious stimuli. The stimulation of the M2 and M4 muscarinic
receptors in the dorsal horn of the spinal cord contributes to the analgesic effect through the release of
inhibitory interneurons, reducing nociceptive transmission [9]. Our results showed the reversal of the
antinociceptive effect of HE, EtOAc, SI, D, B4, and A1 when atropine was used suggesting that the
effects of extract, fraction, or isolated saponins may involve, at least in part, activation of muscarinic
receptors and consequently cholinergic pathway.
Our data also indicated that the opioid system could be involved in the antinociception caused
by HE, EtOAc, SI, D, and B4. Opioid receptors (μ, δ, and κ) can be found in peripheral, spinal,
and supraspinal regions. The downward pain modulation also involves the participation of opioid
receptors, which promotes reduction in synaptic release of γ-aminobutyric acid (GABA) to the spinal
cord rostral medial projections and periaqueductal gray; subsequently leading to spinal disinhibited
projections of adrenergic neurons in the locus coeruleus [10]. In this context, the extract and isolated
substances tested in this work may exert their antinociceptive effects through the opioid system at
the peripheral level, spinal, and/or supraspinal. Literature reviews have already demonstrated that
saponins may present an antinociceptive activity. The investigation of the mechanism of action of
a Polygonum verticillatum methanol extract rich in saponins showed the involvement of the opioid
pathway [11], similarly to what was observed in our study for the ethanol extract of S. brasiliensis, its
EtOAc fraction, two curcubitane, and one dammarane type of saponin.
The knowledge of the dual role of nitric oxide (NO) in nociceptive transmission and its contribution
to antinociception by opioid pain killers and anti-inflammatory drugs motivated us to assess whether
the antinociception of HE, EtOAc, SI, D, B4, and A1 involve nitrergic pathway. NO present in the dorsal
horn of the spinal cord can stimulate the release of neurokinin A, substance P, CGRP, and glutamate by
primary afferent fibers facilitating nociceptive transmission. Activation of NMDA type receptors by
NO is involved in central sensitization mechanism [10]. In this context, our results showed that the
antinociceptive effect of EtOAc, SI, B4, and A1 was reversed by pretreatment with L-NAME suggesting
that the antinociceptive effect of EtOAc and the three substances involves the inhibition of NOS activity
and/or indirect reduction in NO levels.
Hydroethanol extract, ethyl acetate fraction, and the isolated substances SI and A1 also presented
an inhibitory effect against the neurogenic nociception induced by intraplantar injection of capsaicin.
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Molecules 2019, 24, 4584
It is an amine extracted from red pepper that stimulates nerve endings causing intense thermal and
nociceptive pain [9,11,12]. Studies have shown that capsaicin acts through vanilloid receptor type-1
(TRPV-1) expressed on nociceptive fibers [13] and in the dorsal root ganglion of the spinal cord,
trigeminal ganglia, and CNS [14]. The stimulation of TRPV-1 receptors is mediated by the release
of several neurotransmitters, including glutamate and substance P, from the peripheral and central
terminals of primary sensory neurons thus contributing to nociceptive processing [15,16]. Our data
suggest that extracts and isolated saponins reduce licking behavior induced by capsaicin. We have
no means to attest that the observed effect is mediated through a direct action on peripherals and/or
central vanilloid receptors, but we can suggest that part of the effect is mediated by capsaicin pathway.
We also suggest that at least part of the inhibitory effect observed can also occur due to an effect
against the glutamate pathway since it was observed that EtOAc reduced glutamate-induced licking
response. It is most probably that the effect of S. brasiliensis extract, fraction, and SI occurs due to a
direct combination of effects in the capsaicin and glutamate systems. It is also suggested that isolated
saponins D and B4 do not present an effect against both neurogenic substances.
Taken together our data suggest S. brasiliensis and the isolated substances siolmatroside I (SI),
cayaponoside D (D), cayaponoside B4 (B4), and cayaponoside A1 (A1) present significant antinociceptive
effects. Cholinergic and opioidergic pathways seem to be involved in their mechanism of action. We
also demonstrated that the siolmatroside I was the most active saponin identified in the plant. Taken
together our data corroborate the traditional use of the plant and expands the information regarding
its use.
4. Methods
4.1. Plant Material

Stems of S. brasiliensis were collected in Jangada/Mato Grosso State, Brazil and identified by Dr.
Vali Joana Pott (Department of Biology, UFMT, Cuiabá, Brazil). A voucher specimen was deposited at
the herbarium of the UFMT under the number CGMS: 31643.
4.2. Extraction and Isolation

Air dried stems of S. brasiliensis (2.07 kg) were powdered and subjected to extraction with a mixture
of EtOHcH2 O (7:3) (3 × 6 L) (named hydroethanol, HE extract). The HE extract was concentrated under
reduced pressure and circulation oven (45 ◦ C). HE (149 g) was suspended in a mixture of MeOH:H2 O
(1:1) (1 L) and subjected to a liquid–liquid partition between chloroform, ethyl acetate, and n-butanol.
Cayaponoside A1 [17] (A1), cayaponoside B4 [18] (B4), cayaponoside D [17] (D), and siolmatroside I
(SI) were isolated by us from the ethyl acetate fraction, as described previously [3] (Figure 5).
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Molecules 2019, 24, 4584
Figure 5. Saponins isolated from the ethyl acetate fraction of the stems of S. brasiliensis: Cayaponoside
A1, cayaponoside B4, cayaponoside D, and siolmatroside I.
4.3. Animals
Animals used in this study (Swiss Webster mice (25–30g)) were donated by Instituto Vital Brazil
(Niterói, Rio de Janeiro, Brazil) and maintained in a room with light-dark cycle of 12 h, 22 ± 2 ◦ C,
60% to 80% humidity and food and water provided ad libitum. Before each test, the animals were
acclimatized to the laboratory for at least 1 h. Twelve hours before each experiment the animals
received only water in order to avoid food interference with substances absorption. Animals were daily
monitored to assess their physical conditions and that with any signs of suffering were euthanized.
None of the animals used became severely ill or died at any time prior to the experimental endpoint.
At the end of each assay, animals were euthanized following AVMA guidelines. The experimental
protocols used in this work have been carried out in accordance with the Guide for the Care and Use of
Laboratory Animals as adopted and promulgated by the US National Institutes of Health, and were
approved by the rules advocated by Law 11,794, of 8 October 2008 by the National Council of Animal
Experimentation Control (CONCEA) and were approved by the Ethics Committee of Animal Use
(CEUA)/UFRJ and received the number 31/19 and 34/19.
4.4. Extracts, Isolated Substances, and Control Drugs’ Administration

Hydroethanol extract, fractions, and the isolated substances were dissolved in dimethylsulfoxide
(DMSO, Sigma-Aldrich, St. Louis, MO, USA) in order to prepare a stock solution at 200 mg/mL. The
hydroethanol extract (HE) and the ethyl acetate fraction (EtOAc) were tested at doses of 10, 30, or 100
mg/kg. Siolmatroside I (SI), cayaponoside D (D), cayaponoside B4 (B4), and cayaponoside A1 (A1)
were tested at doses of 0.3, 1, or 3 mg/kg. All of them were administered by oral gavage in a final
volume of 0.1 mL per animal randomly divided into groups of 6 to 8 mice. The final amount of DMSO
used in higher dose (100 mg/kg) had no effect per se. Morphine (2.5 mg/kg) was used as a reference
drug and was administered by oral gavage at the intervals indicated in each protocol. Control group
was the given vehicle (ultrapure water with the same amount of DMSO used in higher dose).
4.5. Drugs and Reagents

Morphine sulfate and naloxone hydrochloride (both dissolved in phosphate buffer saline, PBS)
were kindly provided by Cristália (São Paulo, Brazil). Atropine sulfate monohydrate (in PBS), capsaicin
(in DMSO), l-glutamic acid hydrochloride (glutamate, in PBS), l-nitroarginine methyl ester (L-NAME,
in PBS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). All drugs were diluted just before
their use.
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Molecules 2019, 24, 4584
4.6. Acute Toxicity

Acute toxicity parameters were determined following the method described by Lorke [19]. Oral
dose of the HE, EtOAc (150 mg/kg), or isolated saponins (SI, D, B4, A1, 10 mg/kg) were administered
to groups of ten mice (five males and five females). Parameters such as convulsion, sedation, reflex,
hyperactivity, increased or decreased respiration, and food and water intake were observed at 1, 3, 6,
12, and every 24 h over a period of five days to analyze the behaviour of the animals. After that, mice’s
stomachs were removed in order to search for ulcers (single or multiple erosion, ulcer or perforation)
and instances of hyperemia were counted.
4.7. Hot Plate Test

Mice were orally treated with substances, vehicle, or morphine and immediately placed on a hot
plate apparatus (Insight Equipment, Brazil) set at 55 ± 1 ◦ C. The time necessary for mice that licked
their fore- and/or hind-paws (named reaction time) was measured at intervals of 30 min post-oral
administration. Baseline was calculated with the use of measurements obtained at 60 and 30 min before
the administration of the compounds, vehicle, or morphine and considered as a normal reaction of the
animal to the temperature. The calculation of area under the curve (AUC) of responses from 30 min
after drug administration until the end of the experiment was used to indicate the antinociception. The
following formula based on the trapezoid rule was used to calculate the AUC: AUC = 30 × IB ((min 30)
+ (min 60) + . . . + (min 180)/2), where IB is the increase from baseline (in %) [20,21].
4.8. Analysis of the Mechanisms of Action of Hydroethanol Extract (HE), Ethyl Acetate Fraction (EtOAc), and
its Isolated Saponins
To investigate the involvement of the cholinergic, opioid, or nitrergic system in the mechanism of
action of substances, some specific receptor antagonists and/or enzyme inhibitors were used. After
15 min of intraperitoneal administration of atropine (muscarinic receptor antagonist, 1 mg/kg), naloxone
(opioid receptor antagonist, 1 mg/kg), or l-nitro arginine methyl ester (L-NAME, inhibitor of nitric
oxide synthase enzyme, 3 mg/kg) mice received oral administration of HE or EtOAc (100 mg/kg each),
SI, D, B4, or A1 (3 mg/kg each). Based on data from literature [22,23] and previous data from our
own laboratory [24], dose response curves with agonists and respective antagonist were previously
constructed and the dose of antagonist that reduced in 50% the agonist effect was chosen for these
assays. The antinociceptive effect was evaluated in the hot plate test as described above.
4.9. Capsaicin- and Glutamate-Induced Nociception

The animals were pretreated 60 min before the intraplantar injection of capsaicin or glutamate
with substances or vehicle by oral gavage. After treatment, mice received an intraplantar injection of
capsaicin (20 μL, 1.6 μg/paw) or glutamate (20 μL, 3.7 ng/paw). Immediately animals were individually
placed in a transparent box and the time that the animal kept licking the capsaicin- or glutamate-injected
paw was recorded during a period of 5 min [25] and 15 min [26], respectively. The time mice spent
licking the injected paw was recorded and this was considered as the nociceptive reaction.

Experimental groups were composed of 6–8 mice and the results were presented as mean ± S.D.
Statistical significance between groups was determined using the application of analyses of variance
(ANOVA) followed by Dunnett´s post-test. Differences were considered significant (*) when p < 0.05.
The area under the curve (AUC) was calculated using Prism Software 5.0 (Graph Pad Software, La Jolla,
CA, USA).
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Molecules 2019, 24, 4584
5. Conclusion
Our work validated for the first time the antinociceptive activity in peripheral and central
models of analgesia of S. brasiliensis ethanol extract, its ethyl acetate fraction, and its isolated saponins
cayaponoside A1, cayaponoside B4, cayaponoside D, and siolmatroside I. The actions are mediated
by opioid, nitrergic, and cholinergic systems and, in part by vanilloid receptors. Our study confirms
that this species may be used for treating pain processes corroborating with its traditional use and
contributing to the pharmacological knowledge of this Brazilian species.
Supplementary Materials: The following are available online. Table S1: Toxic effects of hydroethanol extract (HE),
ethyl acetate fraction (EtOAc), siolmatroside I (SI), cayaponoside D (D), cayaponoside B4 (B4) and cayaponoside A1
(A1) observed at 1, 3, 6 or 12 hours post-oral administration. Table S2: Toxic effects of hydroethanol extract (HE),
ethyl acetate fraction (EtOAc), siolmatroside I (SI), cayaponoside D (D), cayaponoside B4 (B4) and cayaponoside
A1 (A1) observed at 1, 2, 3, 4 or 5 days post-oral administration; Table S3: Effects of hydroethanol extract (HE),
ethyl acetate fraction (EtOAc), siolmatroside I (SI), cayaponoside D (D), cayaponoside B4 (B4) and cayaponoside
A1 (A1) in water and food intake observed at 1, 2, 3, 4 or 5 days post-oral administration; table S4: Effects of
hydroethanol extract (HE), ethyl acetate fraction (EtOAc), siolmatroside I (SI), cayaponoside D (D), cayaponoside
B4 (B4) and cayaponoside A1 (A1) in the presence of ulcers or hyperemia 5 days post-oral administration.
Author Contributions: Conceptualization, T.B.S.G., M.G.d.C., P.T.d.S., F.B., and P.D.F.; Data curation, T.B.S.G.,
C.H.C.d.S., and P.D.F.; Formal analysis, T.B.S.G., C.H.C.d.S., M.G.d.C., V.C.d.S., F.B., and P.D.F.; Funding acquisition,
M.G.d.C. and P.D.F.; Investigation, T.B.S.G., C.H.C.d.S., and P.T.d.S.; Methodology, T.B.S.G., C.H.C.d.S., and
V.C.d.S.; Project administration, P.D.F.; Resources, V.C.d.S., P.T.d.S., and P.D.F.; Supervision, M.G.d.C. and P.D.F.;
Writing—original draft, T.B.S.G.; Writing—review and editing, M.G.d.C., F.B., and P.D.F.
Funding: This research was funded by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq,
Grant support and fellowship to PDF), Fundação Carlos Chagas Filho de Apoio à Pesquisa do Estado do Rio de
Janeiro (FAPERJ, Grant support and fellowship to PDF and TBSG), Conselho de Administração de Pessoal de
Ensino Superior (CAPES, fellowship to TBSG). FB also wishes to acknowledge the High Education Authority’s
Programme for Research in Third-Level Institutions Cycle 5’s funding support for TBSI and to reinforce the
importance of SFI programme ISCA-Brazil (Grant no. SFI/13/ISCA/2843).
Acknowledgments: Alan Minho for technical assistance and Instituto Vital Brazil (Niterói City, Brazil) for
mice donation.
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Sample Availability: Samples of the compounds hydroethanol extract (HE), ethyl acetate fraction (EtOAc),
siolmatroside I (SI), cayaponoside D (D), cayaponoside B4 (B4) and cayaponoside A1 (A1) are available from
the authors.
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molecules
Article
Comprehensive Chemical Profiling and
Multidirectional Biological Investigation of Two Wild
Anthemis Species (Anthemis tinctoria var. Pallida and
A. cretica subsp. tenuiloba): Focus on
Neuroprotective Effects
Giustino Orlando 1,† , Gokhan Zengin 2,† , Claudio Ferrante 1, *, Maurizio Ronci 3 , Lucia Recinella 1 ,
Ismail Senkardes 4 , Reneta Gevrenova 5 , Dimitrina Zheleva-Dimitrova 5 , Annalisa Chiavaroli 1 ,
Sheila Leone 1 , Simonetta Di Simone 1 , Luigi Brunetti 1 , Carene Marie Nancy Picot-Allain 6 ,
Mohamad Fawzi Mahomoodally 6 , Kouadio Ibrahime Sinan 2 and Luigi Menghini 1
1 Department of Pharmacy, University “G. d’Annunzio” of Chieti-Pescara, 66100 Chieti, Italy
2 Department of Biology, Faculty of Science, Selcuk University, Konya 42130, Turkey
3 Department of Medical, Oral and Biotechnological Sciences, University “G. d’Annunzio” of Chieti-Pescara,
66100 Chieti, Italy
4 Department of Pharmaceutical Botany, Faculty of Pharmacy, Marmara University, Istanbul 34668, Turkey
5 Department of Pharmacognosy, Faculty of Pharmacy, Medical University of Sofia, 1431 Sofia, Bulgaria
6 Department of Health Sciences, Faculty of Science, University of Mauritius, Réduit 80837, Mauritius
* Correspondence: claudio.ferrante@unich.it; Tel.: +39-0871-355-4755

Received: 1 July 2019; Accepted: 14 July 2019; Published: 16 July 2019
Abstract: Ethyl acetate (EA), methanol (MeOH), and aqueous extracts of aerial parts of Anthemis
tinctoria var. pallida (ATP) and A. cretica subsp. tenuiloba (ACT) were investigated for their phenol
and flavonoid content, antioxidant, and key enzyme inhibitory potentials. All extracts displayed
antiradical effects, with MeOH and aqueous extracts being a superior source of antioxidants. On the
other hand, EA and MeOH extracts were potent against AChE and BChE. Enzyme inhibitory effects
against tyrosinase and α-glucosidase were observed, as well. We also studied Anthemis extracts in
an ex vivo experimental neurotoxicity paradigm. We assayed extract influence on oxidative stress
and neurotransmission biomarkers, including lactate dehydrogenase (LDH) and serotonin (5-HT),
in isolated rat cortex challenged with K+ 60 mM Krebs-Ringer buffer (excitotoxicity stimulus). An
untargeted proteomic analysis was finally performed in order to explore the putative mechanism
in the brain. The pharmacological study highlighted the capability of ACT water extract to blunt
K+ 60 mM increase in LDH level and 5-HT turnover, and restore physiological activity of specific
proteins involved in neuron morphology and neurotransmission, including NEFMs, VAMP-2, and
PKCγ, thus further supporting the neuroprotective role of ACT water extract.
Keywords: Anthemis; oxidative stress; neurotransmission; proteomic; phytomedicine
1. Introduction
Anthemis L is the second largest genus in Asteraceae family including more than 210 species,
which are distributed in western Eurasia, Mediterranean and a small part of eastern Africa. According
to the Flora of Turkey, the Anthemideae are divided into three subgenera (Anthemis, Maruta and Cota)
and the subgenus Anthemis includes four sections; Hiorthia, Anthemis, Maruta, and Chia [1–3]. In Turkey,

Molecules 2019, 24, 2582
the genus is represented by 81 taxa belonging to 51 species, 29 (54%) of which are endemic. Species
belonging to Anthemis genus are commonly referred to as “Papatya”, in Turkey [1,4,5].
The species belonging to Anthemis genus are known to possess various biological properties and
have found broad use in pharmaceutics, cosmetics, and food chemistry. The flowers of Anthemis species
are well-documented for their use as antiseptic and healing herbs, with flavonoids, and essential
oils being the main active components [2,6]. Extracts, tinctures, salves, and tisanes are extensively
used as antispasmodic, anti-inflammatory, antibacterial and sedative agents, in Europe [5]. Extracts
are also used to clean wounds and ulcers, and as therapy for irradiated skin injuries, cystitis and
dental afflictions [2]. The antimicrobial activity of essential oils of several Anthemis species have been
previously reported [7–10]. Moreover, Anthemis species are widely used to treat intestinal disorders,
kidney stones, and hemorrhoids in traditional medicine. The plant is also used as antispasmodic
medications and to stimulate menstrual flow. It is documented that the seed oil has been used in the
treatment of earaches and deafness [11–13].
Anthemis genus is mainly characterized by the presence of sesquiterpene lactones, flavonoids and
essential oils. Sesquiterpene lactones belonging to germacranolides, eudesmanolides, and guaianolides
have been gained attention because of their chemo-ecological functions, biological activities and
taxonomic significance. They are the major classes of secondary metabolites in Anthemis genus [2,3,14].
The essential oil compositions of several Anthemis species has also been investigated [2,12,15,16].
A. tinctoria var. pallida (ATP) is a rounded perennial plant measuring between 20 to 45 cm. The
flowers are white or cream [17]. ATP, commonly known as yellow chamomile, produces a yellow dye
used in food industry for production of diary and butchery products. Decoction of ATP flower is
traditionally taken to treat shortness of breath, bronchitis, stomachache, anxiety, and to strengthen
hair [18]. Aerial part of Anthemis species has been reported to exhibit antimicrobial property [17].
However, there is no record of the use of A. cretica subsp. tenuiloba (ACT) by folk populations, in Turkey.
To the best of our knowledge, there are no reports in literature investigating chemical profile and
biological activities of ATP and ACT. Thus, we aimed to determine the chemical characterization and
biological effects of these two Anthemis species. Phytochemical profiles of ethyl acetate (EA), methanol
(MeOH) and aqueous extracts were performed by ultra-high-performance liquid chromatography
coupled with electrospray ionization high resolution mass spectrometry (UHPLC-ESI/HRMS). The
samples were assayed for evaluating antioxidant and enzyme inhibitory potential, as well.
Finally, considering both the traditional antianxiety effect of A. tinctoria, the relationships between
anxiety and brain oxidative/inflammatory stress [19], alongside with the well-established multi-target
protective effects exerted by flavonoid fraction, in the brain [20], we studied the putative protective
role of Anthemis extracts in isolated rat cortex challenged with a neurotoxicity stimulus (K+ 60 mM).
The influence of extract supplementation on the levels of specific biomarkers of oxidative stress and
neurotransmission, including lactate dehydrogenase (LDH) and serotonin (5-HT), was investigated
using validated analytical methods. An untargeted proteomic profile was also performed on rat cortex
homogenate, in order to explore the putative mechanism of action of Anthemis extracts. It is expected
that results presented in this study will support the protective effects of the studied Anthemis extracts
as potential pharmacological agents.
2.1. Total Phenolic and Flavonoid Contents

Phenolic compounds are of increasing interest mainly due to their diverse chemical structure and
wide biological activity valuable in the prevention of some chronic or degenerative diseases. To this
end, the evaluation of the phytochemical profile of plant extracts is important. In the present study,
the total phenol and flavonoid contents of EA, MeOH, and aqueous extracts of ATP and ACT were
illustrated in Table 1. The phenolic content of ATP and ACT ranged from 26.46 to 100.09 mg GAE/g and
21.31 to 47.61 mg GAE/g, respectively. Highest phenolic content was observed in the MeOH extract of
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Molecules 2019, 24, 2582
ATP, followed by its aqueous extract. Whilst for ACT, MeOH extract contained the highest amount of
phenols, followed by EA extract. Regarding the total flavonoid content, the results showed that EA
(ATP: 45.82 an ACT: 46.26 mg RE/g) and MeOH (ATP: 48.54 and ACT: 45.08 mg RE/g) extracts of both
species were rich in flavonoids.
Table 1. Total phenol and flavonoid content of Anthemis extracts *.
Total Phenol Content Total Flavonoid

Plant Names Solvents
(mg GAE/g) Content (mg RE/g)
EA 26.46 ± 1.11 d 45.82 ± 0.40 b
A. tinctoria var. pallida MeOH 100.09 ± 2.83 a 48.54 ± 0.57 a
Aqueous 86.74 ± 1.80 b 23.10 ± 0.13 d
EA 21.31 ± 1.58 e 46.26 ± 0.25 b
A. cretica subsp. tenuiloba MeOH 46.73 ± 0.80 c 45.08 ± 0.26 c
Aqueous 47.61 ± 1.89 c 21.17 ± 0.24 e
* Values expressed are means ± S.D. of three parallel measurements. GAE: Gallic acid equivalent; RE: Rutin
equivalent. Different letters indicate significant differences in the extracts (p < 0.05).
2.2. LC-MS Results

In the present study, 70 compounds were tentatively identified by UHPLC-ESI/MS in both ACT
and ATP extracts. The negative ion mode was used for analysis of acylquinic acids and flavonoids,
while positive ion mode was used for sesquiterpenes determination (Table 2).
2.2.1. Acylquinic Acids

Twenty nine acylquinic acids were identified in tested Anthemis extracts (Table 2). The acylquinic
acids elucidation was based on the hierarchical key developed by Clifford and colleagues [21,22]. Peaks
4, 5, 6, and 7 were identified as 3-O-, 1-O-, 5-O- and 4-O-caffeoylquinic acids ([M − H]− at m/z 353.088),
respectively, according to the relative abundance of the characteristic fragment ions at m/z 191.055
[quinic acid − H]− , 179.034 [caffeic acid − H]− , 173.045 [quinic acid − H − H2 O]− , and 135.044 [caffeic
acid – H − CO2 ]− [21,22]. Compounds 4 and 6 were identified by comparison with neochlorogenic and
chlorogenic acid, respectively. In the same manner, peaks 1, 2, and 3 were assigned as 3-O-, 5-O-, and
1-O-p-coumaroylquinic acids ([M − H]− at m/z 337.093), while peaks 8, 9, 10, and 11 ([M − H]− at m/z
367.103) were assigned as 3-O-, 1-O- 5-O-, and 4-O-feruloylquinic acid (Table 4). With respect to the
diacylquinic acids, peaks 12–15 were related to 3,4-O-, 1,5-O, 3,5-O, and 4,5-O-dicaffeoylquinic acids
([M − H]− at m/z 515.120); 12 and 13 were identified by comparison with standards. The presence of 14
was evidenced by the relative abundance of the ions at m/z 191.055, 179.034, and 135.043 [21–23], while
the ion at 173.044 was prominent for 15. Compounds 16–24, [M − H]− at m/z 529.136 were tentatively
identified as caffeoylferuloylquinic acids [21,22]. Among the tricaffeoylquinic acids, peaks 25–29 were
related to ([M − H]− at m/z 677.152). Compounds 26, 28, and 29 yielded indicative fragment ions at
173.045 deduced 4-substituted CQA [22]. According to the presence of weak signal at m/z 203.034,
the relative intensity of the fragment ion at m/z 335.078, and lipophilicity, peaks 26, 28, and 29 were
tentatively assigned as 1,3,4-O-, 1,4,5-O-, and 3,4,5-O-tricaffeoylquinic acid, while 25 and 27 were
related to 3,4,5-O-tricaffeoylquinic acid and its isomer.
2.2.2. Flavonoids
Based on literature and comparison with standards, 15 flavonoid aglycones 30–44 (most of them
methoxylated), twelve glycosides, and one caffeoyl-O-flavonoid were identified in the studied extracts
(Table 4). Regarding 41–43 ([M − H]− at m/z 345.061), the fragment ion at m/z 287.020, due to consecutive
loss of 2CH3 − and CO is more intense in the product-ion spectra of 43 than 41 and 42. Probably
methoxylation of 43 in both A- and C-rings provides very stable fragments due to concurrent methyl
loss [24]. Fragment ion at m/z 121.028 (1,2 B) (for 41 and 42) were attributed to the Retro-Diels Alder
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Molecules 2019, 24, 2582
(RDA) cleavages of the flavonoid skeleton specific for 3 ,4 -dihydroxy flavonols [25]. Thus, according
to literature, 41–43 were tentatively identified as eupatolitin, spinatoside, and spinacetin, respectively.
The fragmentation fingerprints of 52 and 56 were associated with isorhamnetin derivatives,
witnessed by the abundant fragment ion at m/z 315.051 supported by the ions at m/z 300.027 and
133.028 [24]. Fragmentation patterns and monoisotopic profiles of 52 was in good agreement with
those of caffeoyl-O-isorhamnetin. The fragmentation of [M − H]− at m/z 609.1472 (56) yielded abundant
ion at m/z 315.0517 ([M − H − 294.095]− indicating the loss of hexose and pentose moieties.
2.2.3. Sesquiterpenes
Thirteen sesquiterpene lactones including one eudesmanolide, three germacranolides, and nine
guaianolides, were tentatively identified in both ACT and ATP extracts. Concerning compound 58 ([M
+ H]+ at m/z 229.122), its fragmentation pattern involved losses of 18 Da (H2 O), 28 Da (CO) and 46 Da
(CO2 H) suggesting chamazulene carboxylic acid, a degradation product of proazulenic sesquiterpene
lactones, e.g., matricarin [25]. Similar fragmentation patterns were observed in spectra of 59 and 60. In
addition, a loss of 44 Da (CO2 ) and fragment ions at m/z 185.095 ([M + H – 16 − 44]+ and 95.049 (C6 H7 O)
due to the overall fracture of lactone ring, suggested dehydroleucodin or isodehydroleucodin [26].
Accordingly, 61 was assigned as leucodin ([M + H]+ at m/z 247.132), where C-13 was saturated in a
methyl group. 68 and 69 were tentatively identified as matricarin and its isomer, due to the concomitant
loss of (CO2 + H2 O) at m/z 245.117 from the additional acetyl group [3]. Three isobaric sesquiterpene
lactones 63–65 shared the same [M + H]+ at m/z 263.127 (exact mass). Peaks 63–65 demonstrated
difference of 15.995 Da, in comparison to 61, suggesting the presence of an additional hydroxyl group.
Thus 63–65 were tentatively assigned to hydroxyleucodin and its isomers [27]. In the same manner,
peaks 62, isobaric pair 66/67, and 70 were ascribed as parthenolide, stizolin, and ludalbine, respectively,
previously identified in Anthemis species [3].
2.3. Antioxidant Activity

Oxidative stress-related diseases often arise as a result of the imbalance between the production
of free radicals and reactive oxygen/nitrogen species, and antioxidant defences. These diseases can be
managed/prevented using natural antioxidants that represent promising therapeutic candidates [29].
Different antioxidant assays are needed to obtain certain information regarding antioxidant profile of
herbal extracts. From this point, the antioxidant capacity of different extracts of ATP and ACT were
evaluated using multiple assays based on different mechanisms and the results were presented in
Table 3.
242
Table 2. Peak assessment of compounds in Anthemis extracts.
Accurate Mass Molecular Exact Mass

Peak № MS/MS Data m/z tR min Delta ppm Tentative Structure Ref.
[M − H]− m/z Formula [M − H]− m/z
Acylquinnic acids
Monoacylquinic acids
337.0946 (3.1), 191.0557 (7.7), 173.0453 (2.5), 163.0390 3-p-coumaroyl-quinic
Molecules 2019, 24, 2582
1 337.0946 C16 H17 O8 3.05 337.0929 5.071 [19]

(100), 119.0488 (22.8) acid 1,2,6
337.0932 (7.2), 191.0554 (100), 173.0445 (6.7), 163.0390 5-p-coumaroyl-quinic
2 337.0932 C16 H17 O8 4.07 337.0929 0.829 [19]
(5.8), 119.0489 (5.5), 93.0329 (17.6) acid 1,2,3,5,6
337.0930 (5.9), 191.0554 (100), 173.0446 (1.9), 163.0391
1-p-coumaroyl-quinic
3 337.0930 C16 H17 O8 (2.4), 127.0391 (1.5), 119.0487 (1.4), 111.0439 (1.3), 4.74 337.0929 0.473 [19]
acid 1,2,5,6
93.0331 (5.3), 85.0280 (7.1)
353.0879 (32.4), 191.0553 (100), 179.0341 (60.7), 173.0443 neochlorogenic
4 353.0879 C16 H17 O9 (4.1),161.0233 (3.9), 135.0439 (50.7), 111.0438 (0.9), 2.47 353.0867 0.325 (3-caffeoylquinic) acid *
93.0333 (4.5), 85.0279 (8.7) 1,2,3,4,6
353.0879 (29.9), 191.0554 (47.7), 179.0341 (70.1), 173.0447

1-caffeoylquinic acid
5 353.0879 C16 H17 O9 (100), 135.0439 (57.7), 111.0436 (3.3), 93.331 (22.6), 2.48 353.0867 0.240 1,2,3,4,6 [19]
85.0280 (8.6)
353.0880 (3.8), 191.0554 (100), 179.0338 (1.4), 173.0446 chlorogenic
243
6 353.0880 C16 H17 O9 (0.9), 161.0234 (2.1), 135.0441 (1.1), 111.0435 (1.2), 3.30 353.0867 0.495 (5-caffeoylquinic) acid *
93.0331 (2.7), 85.0279 (0.4) 1,2,3,4,5,6
353.0880 (28.1), 203.6057 (0.1), 191.0554 (100), 179.0341

4-caffeoylquinic acid
7 353.0880 C16 H17 O9 (58.3), 173.0446 (4.6), 161.0233 (3.9), 135.0438 (51.8), 5.92 353.0867 0.665 1,2,3,4,6 [19]
111.0437 (2.2), 93.0330 (4.82), 85.0279 (10.2)
367.1043 (12.5), 193.0500 (100), 173.0453 (3.9), 149.0598
3-feruloylquinic acid
8 367.1043 C17 H19 O9 (2.9), 134.0361 (65.7), 127.0395 (1.0), 111.0439 (1.4), 3.54 367.1034 2.410 1,2,5,6 [19]
93.0331 (2.9)
367.1039 (49.3), 161.0234 (100), 127.0390 (1.7), 85.0281 1-feruloylquinic acid
9 367.1039 C17 H19 O9 3.97 367.1034 1.238 1,2,5,6 [19]
(13.4)
367.1035 (14.1), 193.0499 (6.7), 191.0555 (100), 173.0447
5-feruloylquinic acid
10 367.1035 C17 H19 O9 (18.2), 134.0447 (10.9), 111.0437 (3.9), 93.0331 (26.2), 4.52 367.1034 −0.015 1,2,3,4,5,6
85.0280 (5.3)
367.1030 (11.8), 193.0499 (17.1), 173.0446 (100), 111.0435 4-feruloylquinic acid
11 367.1030 C17 H19 O9 4.78 367.1034 -1.322 1,2,5,6 [19]
(3.1), 93.0331 (24.0)
Table 2. Cont.

Diacylquinic acids
515.1196 (100), 353.0880 (18.5), 335.0788 (6.4), 203.0331
(1.3), 191.0554 (29.5), 179.0341 (58.1), 173.0446 (65.7), 3,4-dicaffeoylquinic
12 515.1196 5.79 515.1184 0.137 *
Molecules 2019, 24, 2582
C25 H23 O12

161.0235 (19.8), 135.0439 (57.5), 127.0389 (3.6), 111.0436 acid 1,2,3,4,5,6
(4.7), 93.0330 (18.7), 85.0277 (4.3)
515.1199 (21.8), 353.0881 (83.1), 335.0782 (2.6), 191.0554
1,5-dicaffeoylquinic
13 515.1199 C25 H23 O12 (100), 179.0341 (48.6), 173.0445 (10.6), 161.0233 (12.4), 5.96 515.1184 *
acid 1,2,3,4,5,6
135.0439 (57.5), 127.0387 (2.8), 93.0332 (6.4), 85.0280 (9.0)
515.1204 (22.6), 353.0880 (87.4), 191.0555 (100), 179.0342
14 515.1204 C25 H23 O12 (52.0), 173.0455 (9.8), 161.0235 (8.9), 135.0438 (51.7), 6.14 515.1184 1.787 [28]
acid 3,4
93.0333 (2.8), 85.0281 (8.6),
515.1199 (75.6), 353.0880 (60.6), 203.0343 (3.1), 191.0555
15 515.1199 C25 H23 O12 (34.7), 179.0342 (72.3), 173.0447 (100), 135.0440 (48.1), 6.34 515.1184 0.720 [28]
acid 1,2,3,4,5,6
127.0384 (1.2), 111.0439 (3.7), 93.0330 (17.2), 85.0280 (4.2)
529.1356 (100), 367.1037 (8.3), 353.0889 (7.4), 349.0935
(5.2), 335.0774 (11.8), 193.0499 (60.5), 191.0555 (9.2),
3-feruloyl-5-caffeoylquinic
16 529.1356 C26 H25 O12 179.0342 (42.1), 173.0446 (48.1), 161.0235 (24.6), 149.0596 6.00 529.1351 0.889 [28]
244
acid 1,2,4,5,6
(1.1), 134.0361 (56.0), 111.0437 (8.9), 93.0331 (14.7),
85.0276 (2.7)
529.1357 (55.4), 367.1038 (25.6), 193.0499 (2.6), 179.0342
17 529.1357 C26 H25 O12 (3.0), 173.0449 (1.9), 161.0234 (100), 135.0441 (12.7), 6.92 529.1351 1.003 [28]
acid 1,2,3,5
134.0367 (4.3), 127.0380 (0.5), 93.0331 (1.1), 85.0279 (3.2)
529.1354 (38.3), 367.1040 (39.6), 353.0883 (44.99),
193.0506 (15.6), 191.0555 (100), 179.0343 (41.61), 173.0454 3-caffeoyl-5-feruloylquinic
18 529.1354 C26 H25 O12 7.00 529.1351 0.436 [28]
(14.8), 161.0239 (13.0), 135.0440 (52.3), 134.0363 (21.5), acid 1,2,3,4
93.0332 (14.2), 85.0281 (7.4)
529.1352 (66.9), 367.1044 (100), 193.0504 (12.1), 179.0333
19 529.1352 C26 H25 O12 (57.2), 173.0447 (76.9), 161.0236 (15.6), 135.0439 (73.7), 7.12 529.1351 0.077 [28]
acid 4,5,6
134.0365 (49.6), 93.0331 (76.7)
529.1361 (14.2), 367.1036 (60.3), 193.0499 (14.5), 173.0447
3-caffeoyl-4-feruloylquinic
20 529.1361 C26 H25 O12 (100), 161.0239 (1.1), 134.0362 (17.5), 127.0392 (1.0), 7.23 529.1351 1.816 [28]
acid 1,2,3,6
111.0436 (3.4), 93.0330 (24.9),
Table 2. Cont.

529.1388 (83.7), 353.0898 (56.5), 191.0550 (75.9), 179.0346
4-caffeoyl-5-feruloylquinic
21 529.1388 C26 H25 O12 (79.9), 173.0448 (100), 161.0237 (11.9), 135.0439 (69.8), 7.28 529.1351 6.880 [28]
acid 4,5,6
93.0330 (38.2)
Molecules 2019, 24, 2582
529.1356 (74.2), 367.1031 (21.5), 349.0925 (2.1), 191.0554

22 529.1356 C26 H25 O12 (1.2), 179.0341 (30.5), 173.0446 (4.94), 161.0234 (100), 7.34 529.1351 0.776 [28]
acid 1,2,3,5
135.0439 (49.6), 93.0331 (1.9)
529.1348 (100), 367.1052 (45.3), 179.0348 (10.3), 173.0449
4-feruloyl-?-caffeoylquinic
23 529.1348 C26 H25 O12 (60.2), 161.0237 (24.0), 135.0441 (16.4), 111.0439 (13.0), 7.44 529.1351 −0.717 **
acid 4,6
93.0331 (77.1)
529.1353 (74.9), 367.1037(19.5), 349.0932 (4.4), 179.0341
24 529.1353 C26 H25 O12 (73.22), 161.0234 (62.3), 135.0439 (100), 134.0364 (12.4), 7.71 529.1351 0.304 **
acid-isomer 1,2,5,6
93.0331 (0.6), 85.0276 (0.5)
Triacylquinic acids
677.1523 (2.9), 515.1105 (44.5), 353.0903 (3.7), 341.0888
(4.4), 191.0552 (9.6), 179.0341 (100), 173.0446 (6.0), 1,3,5-tricaffeoylquinic
25 677.1523 C34 H29 O15 4.85 677.1512 1.634 [28]
161.0230 (4.6), 135.0439 (79.8), 111.0443 (1.0), 93.0331 acid 1,2,4,5,6
(1.6)
245
677.1524 (89.2), 515.1190 (17.3), 353.0879 (25.83),
341.0876 (8.0), 335.0772 (23.0), 323.0791 (5.1), 203.1523
1,3,4-tricaffeoylquinic
26 677.1524 C34 H29 O15 (0.9), 191.0552 (30.6), 179.0341 (71.0), 173.0446 (54.7), 5.26 677.1512 1.812 [28]
acid 1,2,3,5,6
161.0233 (46.0), 135.0439 (100), 127.0388 (4.3), 111.0437
(7.8), 93.0330 (19.3), 85.0280 (1.9)
677.1529 (52.3), 515.1200 (55.7), 353.0878 (26.0), 341.0900
1,3,5-tricaffeoylquinic
27 677.1529 C34 H29 O15 (15.6), 191.0553 (57.3), 179.0338 (75.3), 173.0449 (31.1), 5.37 677.1512 2.447 **
acid 2,5,6 isomer
161.0236 (34.66), 135.0439(100), 93.0328 (7.5)
677.1524 (87.1), 515.1281 (24.3), 353.0878 (26.2), 341.0880
(31.5), 335.0787 (2.3), 323.0779 (10.6), 191.0555 (34.4), 1,4,5-tricaffeoylquinic
28 677.1524 C34 H29 O15 5.66 677.1512 1.812 [28]
179.0342 (94.0), 173.0447 (78.7), 161.0235 (21.9), 135.0439 acid 1,2,5,6
(100), 111.0439 (2.2), 93.0331 (24.8), 85.0279 (4.8)
677.1519 (88.4), 515.1203 (30.3), 353.0881 (55.6), 335.0779
(19.1), 203.0344 (1.3), 191.0554 (57.4), 179.0341 (79.9), 3,4,5-tricaffeoylquinic
29 677.1519 C34 H29 O15 7.85 677.1512 0.985
173.0447 (100), 161.0234 (33.1), 135.0439 (95.5), 111.0437 acid 1,2,3,4,5
(1.1), 93.0331 (28.3), 85.0278 (6.3)
Table 2. Cont.

Flavonoids
269.0457 (100), 151.0027 (6.39), 149.0233 (5.74), 117.0332
30 269.0457 C15 H9 O5 8.73 269.0444 0.644 apigenin 1,2,3,4,5,6 *
(22.24), 107.0124 (5.35)
Molecules 2019, 24, 2582
285.0406 (100), 151.0028 (5.92), 133.0282 (25.08), 107.0126

31 285.0406 C15 H9 O6 7.82 285.0393 0.452 luteolin 1,2,3,4,5,6 *
(3.32)
287.0566 (14.91), 151.0025 (100), 135.0435 (89.99),
32 287.0566 C15 H11 O6 7.53 287.0550 1.842 eriodictyol 1,2,3,4,5,6 [24]
125.0231 (5.03), 107.0124 (13.58)
299.0561 (58.00), 284.0328 (100), 256.0382 (0.86), 227.0350
33 299.0561 C16 H11 O6 8.91 299.0550 0.029 diosmetin 1,2,3,4,5,6 *
(3.45), 211.0393 (2.22)
3,4 ,7-trihydroxy-3 -
299.0562 (95.99), 284.9335 (100), 227.047 (2.78), 151.0033
34 299.0562 C16 H11 O6 9.09 299.0550 0.430 methoxyflavone [23]
(3.67), 107.0123 (2.84) 1,2,3,4,5,6
301.0352 (100), 300.0273 (24.24), 178.9976 (21.40),

35 301.0352 C15 H9 O7 7.83 301.0342 −0.717 quercetin 1,2,3,4,5,6 *
151.0025 (49.72), 121,0282 (16.11), 107.0124 (14.15)
315.0512 (61.55), 300.0279 (100), 271.0252 (28.24), 255.030 Mass
36 315.0512 C16 H11 O7 8.34 315.0499 0.584 nepetin 4,5,6
(11.23), 227.0349 (2.28), 136.9872 (2.51), bank
246
315.0513 (86.15), 301.0315 (11.76), 300.0276 (100), 243.
37 315.0513 C16 H11 O7 7.90 315.0499 0.965 rhamnetin 1,2,3,4,5,6 *
0303 (0.70), 165.9890 (1.63), 136.9868 (9.78)
315.0514 (100), 301.0316 (3.73), 300.0273 (41.59), 243.
38 315.0514 C16 H11 O7 9.26 315.0499 1.156 isorhamnetin 1,2,3,4,5,6 *
0298 (1.08), 151.0025 (7.85), 107.0126 (6.32)
329.0607 (14.45), 314.0436 (100), 299.0198 (25.08), Mass
39 329.0670 C17 H13 O7 9.25 329.0655 0.954 jaceosidin 1,3,4,6
271.0250 (47.23),133.0282 (5.34), 107.2971 (0.52) bank
331.0463 (100), 316.0226 (56.40), 287.0199 (15.97),
40 331.0463 C16 H11 O8 7.80 331.0448 1.086 patuletin 1,3,4,6 [23]
271.0246 (5.47), 270.0176 (4.09), 165.9897 (19.03)
345.0618 (91.18), 330.0384 (100), 315.0150 (50.33),
41 345.0618 C17 H13 O8 8.36 345.0604 0.694 eupatuletin 4,5,6 **
287.0201 (15.30), 121.0280 (1.86)
345.0618 (100), 330.0385 (95.66), 315.0150 (46.41),
42 345.0618 C17 H13 O8 8.40 345.0604 0.694 spinatoside 1,2,3 [23]
287.0198 (14.78), 121.0284 (7.72)
345.0619 (100), 330.0385 (42.35), 315.0145 (4.01), 301.0388
43 345.0619 C17 H13 O8 9.38 345.0604 0.694 spinacetin 1,2,3 [23]
(6.46), 287.0199 (40.72)
359.0775 (100), 344.0539 (49.89), 329.0304 (52.64),
44 359.0775 C18 H15 O8 9.95 359.0761 0.750 jaceidin 1,2,3,4,5,6 [23]
301.0359 (6.67), 287.0139 (4.46)
Table 2. Cont.

apigenin-7-O-glucoside
45 431.0981 C21 H19 O10 431.0981 (100), 269.0440 (27.72), 268.0378 (57.01) 6.17 431.0972 −0.673 1,2,3,4,5,6 *
447.0934 (100), 327.0507 (0.95), 285.0405 (99.96), 151.0030 luteolin-7-O-glucoside

46 447.0934 C21 H19 O11 5.45 447.0921 0.348 *
Molecules 2019, 24, 2582
(7.20), 133.0280 (5.74), 107.0123 (4.57) 1,2,3,4,5,6
461.0725 (42.84), 285.0406 (100), 151.0025 (3.72), 133.0280 luteolin-7-O-glucuronide

47 461.0725 C21 H17 O12 5.46 461.0714 −0.150 1,2,3 *
(10.72), 107.0121 (1.50)
461.1094 (100), 446.0858 (28.76), 299.0554 (12.48),
diosmetin-O-glucoside
48 461.1094 C22 H21 O11 284.0313 (9.73), 283.0250 (21.41), 269.0467 (2.08), 6.37 461.1078 2.220 1,2,3 **
255.0300 (73.76), 227.0345 (0.67), 151,0028 (0.86)
463.0887 (100), 372.1873 (4.84), 301.0360 (93.71), 300.0282
49 463.0887 C21 H19 O12 4.77 463.0871 1.038 isoquercitrin 1,2,3,4,5,6 *
(27.20)
50 463.0903 C21 H19 O12 463.0903 (100), 301.0349 (47.50), 300.0276 (66.34) 5.29 463.0871 4.601 hyperoside 1,2,3,4,5,6 *
477.1041 (100), 315.0496 (14.04), 314.0434 (53.04),
isorhamnetin-7-O-glucoside
51 477.1041 C22 H21 O12 300.0274 (5.68), 285.0411 (6.84), 243.0297 (22.73), 6.10 477.1027 0.442 1,2,3,4,5,6 *
271.0249 (27.98), 151.0032 (3.66)
493.0777 (11.58), 315.0512 (99.41), 314.0435 (100),
247
300.0277 (14.30), 285.0411 (13.30), 243.0293 (34.06), caffeoyl-O-isorhamnetin
52 493.0777 C25 H17 O11 9.12 493.0765 0.194 1,3 **
227.0341 (4.04), 177.0182 (16.10), 151.0030 (4.86),
133.0283 (12.50)
493.0974 (100), 331.0463 (96.71), 316.0224 (22.61), patuletin-O-hexoside
53 493.0974 C22 H21 O13 5.63 493.0976 −0.257 4,5,6 **
287.0196 (26.93), 271.0253 (9.58), 165.9891 (8.24)
593.1504 (97.39), 318.6214 (6.24), 285.0405 (100), 284.0327 luteolin-7-O-rutinoside
54 593.1504 C27 H29 O15 5.71 593.1500 −1.354 1,2,3,4,5,6 *
(60.74), 227.0352 (29.58)
609.1467 (72.52), 343.0477 (1.65), 301.0354 (100), 300.0278
55 609.1467 C27 H29 O16 (38.67), 178.9970 (1.59), 151.0027 (13.46),121.0279 (1.84), 5.17 609.1450 0.923 rutin 1,2,3,4,5,6 *
107.0123 (4.76)
609.1472 (96.84), 411.8945 (5.23), 315.0517 (100), 300.0275 isorhamnetin-O-
56 609.1472 C27 H29 O16 5.43 609.1450 2.149 **
(48.87), 133.0284 (7.86) pentosyl-hexoside 4,5,6
623.1613 (5.57), 315.0514 (100), 301.0306 (1.22), 300.0279 isorhamnetin-3-O-rutinoside
57 623.1613 C28 H31 O16 6.08 623.1606 −0.703 1,2,3,4,5,6 *
(16.19), 151.0025 (7.85), 107.0125 (3.48)
Table 2. Cont.

Sesquiterpenes
229.1220 (100), 211.1116 (12.69), 201.1272 (19.57), chamazulene
58 229.1220 C15 H17 O2 6.10 229.1223 −3.643 [25]
183.1167 (40.99), 91.0548 (6.40), carboxylic acid 1,2,3
Molecules 2019, 24, 2582
245.1170 (100), 227.1067 (44.35), 217.1222 (20.20), dehydroleucodin/

59 245.1170 C15 H17 O3 201.0910 (20.68), 199.1116 (75.08), 185.0959 (28.15), 10.48 245.1172 −0.942 isodehydroleucodin [26]
95.0497 (66.93) 1,2,3
245.1171 (84.77), 227.1065 (44.23), 217.1225 (17.55), dehydroleucodin/

60 245.1171 C15 H17 O3 201.0910 (17.75), 199.1116 (63.86), 185.0960 (18.51), 10.48 245.1172 −0.942 isodehydroleucodin [26]
95.0497 (100) 1,2,3
247.1325 (100), 229.1220 (90.72), 211.1113 (8.83), 201.1271 desacetoxymatricarin

61 247.1325 C15 H19 O3 9.79 247.1328 −1.663 1,2,3,4,5,6 **
(37.99), 187.0752 (37.99), 183.1167 (22.85), 91.0548 (9.16)
249.1481 (100), 231.1378 (30.41), 213.1277 (9.04), 203.1431
62 249.1481 C15 H21 O3 (10.25), 193.0856 (5.87), 189.1272 (11.79), 159.1167 (22.04), 10.42 249.1485 −0.451 parthenolide 1,2,3,4,5,6 **
119.0857 (16.94), 105.0702 (18.62), 95.0860 (10.61)
263.1275 (100), 245.1170 (55.39), 227.1061 (9.88), 219.1021
63 263.1275 C15 H19 O4 (15.59), 217.1223 (47.56), 203.1065 (10.39), 199.1115 10.52 263.1277 −0.316 hydroxyleucodin 1,2,3 **
248
(21.45), 191.0704 (28.72), 95.0497 (96.99)
263.1274 (100), 245.1171 (33.08), 227.1063 (4.95), 219.1012
hydroxyleucodin
64 263.1274 C15 H19 O4 (14.30), 217.1224 (16.65), 203.1065 (10.12), 199.1117 11.22 263.1277 −0.406 **
isomer 1,2,3
(15.19), 191.0702 (79.26), 95.0497 (20.99)
263.1275(100), 245.1170 (51.98), 227.1063 (11.55),
hydroxyleucodin
65 263.1275 C15 H19 O4 219.1014 (34.60), 217.1223 (47.24), 203.1067 (12.57), 11.22 263.1277 −0.406 **
isomer 1,2,3
199.1117 (22.59), 191.0702 (32.46), 95.0497 (81.14)
265.1432 (8.16), 247.1326 (34.45), 229.1221 (100), 211.1115
66 265.1432 C15 H21 O4 (3.94), 201.1273 (20.45), 187.0752 (14.30), 183.1168 (7.67), 6.04 265.1434 −0.813 stizolin 1,3 **
91.0548 (5.66)
265.1429 (24.21), 247.1325 (100), 229.1222 (71.63),
67 265.1429 C15 H21 O4 219.1372 (36.25), 201.1272 (55.14), 187.1111 (17.50), 9.78 265.1434 −1.982 stizolin isomer 1,3 **
183.1174 (17.90), 91.0548 (13.40)
Table 2. Cont.

305.1360 (44.97), 287.1268 (3.27), 269.1206 (1.63),
263.1274 (42.58), 245.1170 (100), 227.1065 (63.42),
68 305.1360 C17 H21 O5 217.1220 (60.59), 109.1116 (23.36), 201.0903 (12.29), 7.05 305.1383 −7.538 matricarin 3 **
185.0958 (19.24), 181.1010 (44.17), 171.1166 (56.51),
Molecules 2019, 24, 2582
105.0703 (50.85), 95.0496 (25.16)

305.1363 (23.50), 263.1279 (20.85), 245.1170 (100),
227.1063 (29.92), 217.1216 (19.51), 201.0911 (2.62),
69 305.1363 C17 H21 O5 5.42 305.1383 −6.654 matricarin isomer 3 **
185.0961 (16.47), 181.1009 (28.17), 171.1167 (31.70),
131.0857 (46.50), 95.0496 (3.40)
307.1544 (2.15), 289.1433 (0.75), 267.2721 (0.18), 247.1325
70 307.1544 C17 H21 O5 9.67 307.1540 1.366 ludalbin 1,2,3 **
(2.63), 229.1219 (100), 211.1115 (0.95), 183.1168 (2.88)
1 -A.
cretica MeOH extract, 2 -A.cretica aqueous extract, 3 -A. cretica EA extract, 4 -A. palida MeOH extract, 5 -A. palida aqueous extract, 6 -A. palida EA extract. *-comparison with standard
substance. **-tentatively identification.
Table 3. Antioxidant properties of Anthemis extracts *.
Metal Chelating
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Phosphomolybdenum CUPRAC
Plant Names Solvents DPPH (mg TE/g) ABTS (mg TE/g) FRAP (mgTE/g) Abilitiy (mg
(mmol TE/g) (mgTE/g)
EDTAE/g)
Ethyl acetate 2.59 ± 0.19 b 40.30 ± 0.78 e 45.52 ± 5.53 f 113.31 ± 2.26 d 47.63 ± 3.77 f 39.01 ± 4.42 a
A. tinctoria var.
Methanol 2.99 ± 0.14 a 407.07 ± 8.88 a 320.11 ± 5.67 a 691.17 ± 12.07 a 362.12 ± 2.63 a 28.28 ± 1.81 c
pallida
Aqueous 2.65 ± 0.02 b 298.40 ± 6.74 b 303.16 ± 8.57 b 584.01 ± 8.71 b 316.34 ± 4.15 b 33.59 ± .16 b
Ethyl acetate 1.69 ± 0.08 cd 45.47 ± 2.16 e 57.13 ± 3.89 e 112.87 ± 4.41 d 55.74 ± 2.27 e 21.90 ± 0.81 d
A. cretica subsp.
Methanol 1.77 ± 0.08 c 97.22 ± 0.22 c 112.41 ± 2.35 d 223.09 ± 6.17 c 143.21 ± 1.77 d 20.93 ± 1.70 d
tenuiloba
Aqueous 1.56 ± 0.05 d 86.74 ± 2.46 d 127.68 ± 0.45 c 214.45 ± 1.39 c 130.86 ± 1.81 c 39.64 ± 1.34 a
* Values expressed are means ± S.D. of three parallel measurements. TE: Trolox equivalent; EDTAE: EDTA equivalent. Different letters indicate significant differences in the extracts
(p < 0.05).
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Based on the experimental results (Table 3), it can be noticed that for both species, MeOH extract
exhibited the highest total antioxidant activity. According to data presented in Table 3, ATP MeOH
extract (DPPH: 407.07 ± 8.88 and ABTS: 320.11 ± 5.67 mg TE/g), followed by the aqueous extract
(DPPH: 298.40 ± 6.74 and ABTS: 303.16 ± 8.57 mg TE/g) showed higher radical scavenging activity
in both assays. Likewise, ACT MeOH (DPPH: 97.22 ± 0.22 and ABTS: 112.41 ± 2.35 mg TE/g) and
aqueous extracts (DPPH: 86.74 ± 2.46 and ABTS: 127.68 ± 0.45 mg TE/g) showed potent radical
scavenging activity.
CUPRAC and FRAP assays were employed to assess the reducing capacity of different extracts.
CUPRAC method evaluates the conversion of Cu (II) into Cu (I) while FRAP assay measures the
reducing potential of an antioxidant reacting with the colourless TPTZ/Fe3+ complex to form a blue
TPTZ/Fe2+ complex at low pH [30]. Remarkable reducing potencies were displayed by MeOH
(CUPRAC: 691.17 ± 12.07 and FRAP: 362.12 ± 2.63) and aqueous (CUPRAC: 584.01 ± 8.71 and FRAP:
316.34 ± 4.15 mg TE/g) extracts of APT. EA extract displayed the lowest reducing capacity. This trend
was also observed as regards ACT extracts (Table 3).
Chelation of pro-oxidant metals is recognized as one of the most important mechanisms of action
of antioxidants. Particularly, iron is the most powerful and abundant pro-oxidant and transition metal
which causes oxidative changes of cellular components, such as, lipids and proteins [31]. Evaluation of
iron chelating activity showed that ATP and ACT extracts possessed notable chelation potential, with
the highest activity displayed by ATP EA extract (39.01 ± 4.42 mg EDTAE/g) and ACT aqueous extract
(39.64 ± 1.34 mg EDTAE/g).
2.4. Enzyme Inhibitory Activity

The enzyme inhibitory effect of ATP and ACT extracts was determined against cholinesterases,
α-amylase, α-glucosidase, and tyrosinase and the results were presented in Table 4. EA and MeOH
extracts of ATP and ACT exhibited potent inhibitory activity against AChE, with the highest activity
observed for ATP MeOH extract (3.28 ± 0.43 mg GALAE/g) and ACT EA extract (4.68 ± 0.21 mg
GALAE/g). On the other hand, ATP EA extract (3.48 ± 0.21 mg GALAE/g), ACT EA extract
(2.51 ± 0.34 mg GALAE/g), and ACT MeOH extract (1.15 ± 0.05 mg GALAE/g) showed inhibitory
effect against BChE. Both AChE and BChE hydrolyze acetylcholine and terminate the synaptic
transmission [31]. While enhanced AChE activity is associated with early stages of AD, BChE activity
is found to increase with the progression of the disease. Therefore, both enzymes are considered as
legitimate therapeutic targets for managing AD [32,33].
Table 4. Enzyme inhibitory activity of Anthemis extracts *.
AChE BChE Amylase Glucosidase

Tyrosinase
Plant Inhibition Inhibition Inhibition Inhibition
Solvents Inhibition
Names (mg (mg (mmol (mmol
(mg KAE/g)
GALAE/g) GALAE/g) ACAE/g) ACAE/g)
Ethyl acetate 1.33 ± 0.03 c 3.48 ± 0.21 a 124.60 ± 0.15 b 0.78 ± 0.05 a 21.94 ± 1.91 b
A. tinctoria
Methanol 3.28 ± 0.43 b na 124.48 ± 1.23 b 0.54 ± 0.02 c 9.15 ± 2.26 c
var. pallida
Aqueous na na 72.10 ± 1.64 d 0.11 ± 0.01 d 4.95 ± 0.25 d
A. cretica Ethyl acetate 4.68 ± 0.21 a 2.51 ± 0.34 b 128.73 ± 0.71 a 0.65 ± 0.01 b 24.16 ± 0.12 a
subsp. Methanol 3.45 ± 0.26 b 1.15 ± 0.05 c 128.85 ± 1.41 a 0.52 ± 0.06 c 4.49 ± 0.93 d
tenuiloba Aqueous na na 88.95 ± 0.49 c 0.09 ± 0.01 d 2.49 ± 0.17 e
* Values expressed are means ± S.D. of three parallel measurements. GALAE: Galatamine equivalent; KAE: Kojic
acid equivalent; ACAE: Acarbose equivalent; na: not active. Different letters indicate significant differences in the
extracts (p < 0.05).
Based on the results (Table 4), it can be observed that the extracts of both species showed remarkable
inhibitory activity against tyrosinase with values ranging from 124.60 ± 0.15 to 72.10 ± 1.64 mg KAE/g
and from 128.85 ± 1.41 to 88.95 ± 0.49 mg KAE/g for ATP and ACT, respectively. The lowest inhibitory
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activity against tyrosinase was detected for the aqueous extracts of both species. Tyrosinase is a key
enzyme in melanin biosynthesis which is responsible for skin pigmentation. However, excessive
melanin production could lead to various skin disorders such as melasma, lentigines, age spots, and
post-inflammatory hyperpigmentation. Thus, tyrosinase inhibitors, used as hypopigmenting agents,
became increasingly important for medicinal and cosmetic products [34].
Type II diabetes is a growing pandemic and poses an enormous public health challenge for almost
every country worldwide. α-Amylase and α-glucosidase are considered as key therapeutic targets
for the management of type II diabetes. α-Amylase and α-glucosidase are carbohydrate hydrolysing
enzymes, responsible for the breakdown of carbohydrates into glucose [35,36]. The present study
showed that all ATP and ACT extracts displayed weak α-amylase inhibitory effects, despite being
active α-glucosidase inhibitors. The highest inhibitory effect against α-amylase (0.78 and 0.65 ACAEs/g
extract, for ATP and ACT, respectively) and α-glucosidase (21.94 and 24.16 ACAEs/g extract, for ATP
and ACT, respectively) was displayed by EA extracts.
2.5. Multivariate Analysis

In an attempt to compare the difference between extract activity, multivariate statistic (sPLS-DA)
was carried out by using principal component analysis (PCA) and partial least squares projection
to latent structures. PCA is very useful in reducing the dimension of data with minimum loss of
information. Figure 1 showed the score plots of projected extracts on principal components PC1
vs. PC2 and PC1 vs. PC3. PCA allowed analysis of biological activities disparity between the
extracts. The first three components defining most of variance were found to be the best to clearly
classify the extracts (Figure 1C). Nevertheless, the result obtained did not facilitate us for a better
discrimination and classification of extracts. Thus, it was considered necessary to apply sPLS-DA, a
supervised multivariate analysis known to offer many advantages over PCA, notably by erecting more
parsimonious and easily interpretable models compared to PCA.
Figure 1. (A): Relationship between total phenol content (TPC), total flavonoid content (TFC) and
biological activities. (B,C): results of preliminary multivariate analysis with PCA (B: Percentage of
explained variance and Eigen value per component, C: PCA sample plot on PC1 vs. PC2 and PC1 vs.
PC3 respectively).
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Firstly, analysed species were used as class membership criteria to assess whether they were
characterized by distinctive biological activities. sPLS-DA samples plot was reported in Figure 2;
as shown, a clear separation between A. cretica subsp. tenuiloba and A. tinctoria var. pallida was
achieved, thus suggesting distinctive biological activities. Afterwards, with the aim to identify the
most discriminant biological activities providing the differences overviewed in the sPLS-DA samples
plot, VIP (variable importance in projection) plot was generated (Figure 2). Five biological activities
including PPBD, DPPH, ABTS, CUPRAC and FRAP possessed a VIP score upper 1, which suggested
them as discriminants for the two species.
Figure 2. Supervised analysis with sPLS-DA. A: sPLS-DA samples plot with confidence ellipse plots
considering the species as class membership criteria. B: Performance of the model (BER) for three
prediction distances using 10 × 5-fold cross-validation. C: VIP score plot displaying the biological
activities having highly contributed to the discrimination of both studied species. D: Factorial plan 1-2
of the sPLS-DA with confidence ellipse plots according to the extraction conditions as class membership
criteria. E: The model performance per component for the three prediction distances using 5-fold
cross-validation repeated 10 times. F: VIP score plot showing the biological activities outlining the
difference between the three extraction conditions.
Secondly, sPLS-DA was performed considering three different extraction conditions, in order to
evaluate the effect of extraction solvents on biological activities. As shown in samples plot (Figure 2) the
subspace formed by the first two components showed that methanol, water and ethyl acetate extracts
were well separated. Next, the prediction performance and the number of components necessary for
the final model were evaluated according to BER (Balanced Error Rate). The performance of our model
reached its best for two components, which suggested ncomp = 2 for a final sPLS-DA model (Figure 2).
Subsequently, the biological activities having highly contributed to the separation of used solvents
were identified. As it could be seen in Figure 2, AChE, BChE, tyrosinase, α-amylase and α-glucosidase
were the most contributing biological activities (Figure 2).
2.6. Multidirectional Biological Evaluation

The biological activity of Anthemis extracts was formerly evaluated through allelopathy assay,
a validated pharmacognostic test for discriminating herbal extract phytotoxicity. Particularly, we
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investigated the effects of scalar extract concentrations (100 μg/mL–10 mg/mL) on seedling germination
of three commercial lettuce varieties, namely Canasta (C), Romana verde (RV), and Romana bionda
(RB). After challenging the seeds with Anthemis water and EA extracts, we observed that germination
process was unaffected in the tested concentration range (Figure 3A–E). Conversely, ATP MeOH extract
displayed concentration-dependent inhibition of seedling germination, in the range 1–10 mg/mL
(Figure 3F). The root elongation rate test revealed evident inhibitory effect, in the range 1–10 mg/mL.
On the other hand, extracts resulted biocompatible at the lowest tested concentration (100 μg/mL),
with percentage elongation rate ≥70% compared to vehicle untreated group. The results of elongation
rate test suggest a further toxicological investigation, with independent methods in order to confirm
the biocompatibility limit, as described below.
Figure 3. Seedling germination and growth of Canasta (C), Romana verde (RV) and Romana bionda
(RB) seeds challenged with A. tinctoria and A. Cretica extracts. Results are expressed as root and
hypocotyl (seedling) length ± SD at different concentrations and mean of GP after the fourth day since
the sowing. (A): Effect of A. cretica water extract on seedling germination. (B): Effect of A. cretica
ethyl acetate (EA) extract on seedling germination. (C): Effect of A. cretica water methanol (MeOH) on
seedling germination. (D): Effect of A. tinctoria water extract on seedling germination. (E): Effect of
A. tinctoria ethyl acetate (EA) extract on seedling germination. (F): Effect of A. tinctoria water methanol
(MeOH) on seedling germination.
The potential toxicity of water, MeOH and EA extracts of Anthemis species (0.1–20 mg/mL) was
also investigated through brine shrimp (Artemia salina Leach) lethality assay. Evaluation of lethality
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induced on brine shrimp, Artemia salina Leach, is considered predictive of cytotoxicity [37]. The results
of this test revealed LC50 values < 10 mg/mL, for all tested extracts.
Additionally, we evaluated the activity of Anthemis extracts on HypoE22 cell line viability.
According to brine shrimp and allelopathy assays, we tested the extracts at 100 μg/mL. MTT test
revealed that Anthemis extracts were well tolerated by HypoE22 cells, with a resulting cell viability
≥70% (Figure 4). This concentration was used for subsequent ex vivo investigations aimed to elucidate
extract neuroprotective effects, as following reported.
Figure 4. Effect of A. tinctoria (A. T.) and A. cretica (A. C.) extracts (100 μg/mL) on HypoE22 cell line
viability (MTT test). Data are means ± SD of three experiments performed in triplicate.
Cortical spreading depression (CSD) is a pathophysiological and mass depolarization of neurons

and glial cells which is characterized by a change in ion and water distribution across neuron
membrane associated with cytotoxic effects, including neuron death [38]. In physiological conditions,
neurotransmitter release is elicited by depolarizing-stimuli (K+ 9–15 mM) which, through the increased
passage of Ca2+ ions across nerve terminals via voltage-sensitive calcium channels (VSCCs), stimulates
classical neurotransmitter exocytosis. On the other hand, in CSD, the neurotransmitter release
increases possibly through additional mechanisms, including membrane transporter reversal [39].
Particularly, excitotoxicity depolarizing-stimuli (K+ ≥ 50 mM) were reported to increase significantly
5-HT overflow [40] which could stimulate neurotransmitter turnover, thus explaining the cortical 5-HT
depletion induced by CSD, in vivo [41]. CSD has been recently described as a potential triggering
mechanism in migraine with aura, via the activation of trigeminal nociceptive system, both peripherally
and centrally [42]. While low 5-HT state could play a pivotal role in migraine attack, through multiple
effects, including the reduction of pain perception threshold, the increased tendency of having headache
and the interference with the control of cerebrovascular nociception [41]. A chronic reduction of 5-HT is
also seen in migraineurs and in depressed patients, while amitriptyline and venlafaxine are first-choice
drugs for treating patients suffering from migraine with comorbid depression [43]. Considering the
role played by 5-HT in anxiety and migraine [41,43], and the traditional use of Anthemis species in
anxiety [18], we tested ATP and ACT extracts (100 μg/mL) in isolated cortex specimens challenged with
an excitotoxicity stimulus constituted by K+ (60 mM) Krebs-Ringer buffer. The results indicated that
ATP and ACT EA extracts and ACT water extracts were able to completely blunt K+ (60 mM)-induced
5HIIA/5-HT ratio (Figure 5), which has long been considered as a valuable index of 5-HT degradation, in
the brain [44,45]. On the other hand, MeOH extracts were ineffective in modifying K+ (60 mM)-induced
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5-HT degradation (Figure 5). Conversely, MeOH extracts revealed a more selective enzyme inhibition
on AChE (Table 4). Recently, pilocarpine, a muscarinic receptor agonist, was able to antagonize CSD
effects, after sub-convulsing dose administration [46], thus suggesting a role played by acetylcholine
signaling stimulation, in CSD. Actually, extract capacity to improve 5-HT and acetylcholine pathways
could be related to their antiradical activity (Table 3) [47]. Previously, antioxidant herbal extracts
were shown to blunt oxidative stress-induced reduction of neurotransmitter level, in the brain [48,49].
Specifically, water Harpagophytum procumbens extract was able to prevent cortex 5-HT depletion induced
by amyloid β-peptide [48], possibly through concomitant antioxidant mechanisms, that have been,
at least partially, displayed by Anthemis extracts, as well. Whereas multiple studies also pointed out
the efficacy of isolated secondary metabolites, including polyphenols and tocopherols, in blunting
oxidative stress-induced monoamine depletion, thus further suggesting a putative role in managing
clinical symptoms related to neurodegenerative diseases [50,51].
Figure 5. Effect of A. tinctoria (A. T.) and A. cretica (A. C.) extracts (100 μg/mL) on serotonin (5-HT)
turnover, expressed as 5HIIA/5-HT ratio. Turnover was evaluated on isolated rat cortex challenged
with basal (K+ 3mM) and depolarizing stimuli (K+ 15 mM; K+ 60 mM). Data are means ± SD of three
experiments performed in triplicate. ANOVA, p < 0.0001; post-hoc, * p < 0.05, *** p < 0.001 vs. K+
60 mM control group.
On the other hand, after evaluating the effects of Anthemis extracts (100 μg/mL) on LDH, a
well-recognized marker of tissue damage [52], we observed that ACT EA and MeOH extracts, alongside
with ATP water and EA extracts, revealed effective in blunting K+ (60 mM)-induced LDH level
(Figure 6). Considering the results of qualitative fingerprint analysis, we could hypothesize that the
observed effects might be related to the presence of flavonoids and terpenes such as apigenin, patuletin,
jaceosidin, quercetin, luteolin, and parthenolide.
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Figure 6. Effect of A. tinctoria (A. T.) and A. cretica (A. C.) extracts (100 μg/mL) on lactate dehydrogenase
(LDH) level, measured on isolated rat cortex challenged with basal (K+ 3mM) and depolarizing stimuli
(K+ 15 mM; K+ 60 mM). Data are means ± SD of three experiments performed in triplicate. ANOVA,
p < 0.0001; post-hoc, * p < 0.05 vs. K+ 60 mM control group.
In agreement with the antiradical activity (Table 2) and blunting effect on 5-HT turnover (Figure 5),
ACT water extract samples has been subjected to a further proteomic study, in order to deepen our
knowledge about the putative mechanism of action related to neuroprotective effects. The deepening
about ACT water extract was performed in comparison with the corresponding ATP extract that,
despite showing a null effect on 5-HT turnover (Figure 5), displayed a significant inhibitory effect on
K+ (60 mM)-induced LDH level (Figure 6).
Particularly, untargeted proteomic analysis showed that K+ 60 mM was able to significantly
downregulate neurofilament (NFEM) proteins (Figure 7A/Supplementary Data 1), expressed along
the axons and involved in axonal diameter regulation. Reduced NFEM levels have long been related
to neurodegeneration [53]. The treatment of isolated rat cortex with ACT water extract was able to
prevent NFEM downregulation, restoring the activity of NEFM proteins during K+ 15 mM physiologic
depolarizing stimulus. While ATP water extract did not exert any relevant effect on NFEM level, in
isolated rat cortex challenged with K+ 60 mM (Figure 7B/Supplementary Data 2). Conversely, K+ 60 mM
stimulus led to significant upregulation of protein C kinase γ (PKCγ) and vesicle-associated membrane
protein-2 (VAMP-2) (Figure 7A/Supplementary Data 2), compared to physiologic depolarizing stimulus
(K+ 15 mM). VAMP-2 is placed on the membranes of neuronal endings’synaptic vesicles, playing
a key role in synaptic vesicle fusion to the presynaptic neuronal ending membrane [54]. Multiple
studies suggested upregulation of VAMP-2 level during hypoxia [55,56], which is strictly related to
high K+ concentration-induced CNS injury [39]. PKCγ plays multiple roles in neuronal cells and eye
tissues, such as regulation of the neuronal receptors GRIA4/GLUR4 and GRIN1/NMDAR1, modulation
of receptors and neuronal functions related to sensitivity to opiates, pain and alcohol, mediation
of synaptic function and cell survival after ischemia, and inhibition of gap junction activity after
oxidative stress. Its level is positively related to migraine pathogenesis [57]. Additionally, PKCγ
gene expression was observed in histidine triad nucleotide-binding protein 1 (Hint1) KO mice, that
also showed increased anxiety-related behavior, compared to wild type control mice [58]. Also in
this case, treatment of isolated rat cortex with ACT water extract was able to restore the activity of
both VAMP-2 and PKCγ during K+ 15 mM-depolarizing stimulus (Figure 7A/Supplementary Data 2),
further supporting the neuroprotective effects of this extract against the burden of oxidative stress and
inflammation occurring in CSD.
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$ %
Figure 7. Panel A: Untargeted proteomic analysis performed on rat cortex challenged with basal
(K+ 3mM) and depolarizing stimuli (K+ 15 mM; K+ 60 mM). The activity of the detected proteins
was calculated in comparison with the calibrator of the experiment (K+ 60 mM). Panel B: Untargeted
proteomic analysis showing the effects of A. tinctoria and A. cretica water extracts (100 μg/mL) on rat
cortex challenged with excitotoxicity depolarizing stimulus (K+ 60 mM). T The activity of the detected
proteins was calculated in comparison with the calibrator of the experiment (K+ 60 mM). In subfigure
A, it is showed that K+ 60 mM depolarizing stimulus downregulated NEFMs and upregulated VAMP-2
and PKCγ levels. On the other hand, as depicted in subfigure B, A. cretica water extract (100 μg/mL) was
able to restore the activity of specific proteins involved in neuron morphology and neurotransmission,
including NEFMs, VAMP-2, and PKCγ. After treating rat cortex with that A. cretica water extract,
the activity of these proteins was similar to that measured after challenging the brain tissue with
physiologic depolarizing stimulus (K+ 15 mM).
3.1. Plant Material and Preparation of Extracts

Sampling of the plant species (Anthemis tinctoria L. var. pallida DC. and A. cretica L. subsp. tenuiloba
(DC.) Grierson) was done in Kastamonu (Hanonu) of Turkey in 2018. The collected plant identity was
confirmed by the botanist Dr. Ismail Senkardes (Marmara University, Faculty of Pharmacy, Istambul,
Turkey). Aerial parts were collected from wild full blooming plants and dried in ventilated oven (in the
dark, temperature 40 ◦ C) until constant weight. Afterwards, dried materials were powdered.
Methanol and ethyl acetate extracts were prepared through maceration method (5 g plant material
in 100 mL solvents for 24 h). After that, extracts were filtered and then concentrated by using one
rotary evaporator under vacuo. Regarding water extracts, infusion method was selected (5 g plant was
kept in 100 mL boiling water in 20 min). The infusions were filtered and then lyophilized. All extracts
were stored at +4 ◦ C, avoiding light exposure.
3.2. Assays for Total Phenolic and Flavonoids

With reference to our earlier report [59], total bioactive components namely total phenols (TPC)
and flavonoids (TFC) were measured by spectrophotometric assays. Gallic acid was the standard for
phenols, while rutin was selected for flavonoids.
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3.3. Antioxidant and Enzyme Inhibition Assays

Antioxidant properties of Anthemis extracts were determined by different in vitro assays namely
FRAP, CUPRAC, DPPH, ABTS, chelating and phospomolybdenum assays. Regarding enzyme
inhibitory properties, some enzymes including tyrosinase, cholinesterase, α-amylase and α-glucosidase
were selected. All experimental procedures were given in our earlier report [59].
3.4. UHPLC-ESI/HRMS Analysis

Neochlorogenic acid (3-CQA) (4), chlorogenic acid (5-CQA) (6), apigenin (30), luteolin (31),
quercetin (35), isoquercitrin (49), hyperoside (50), luteolin-7-O-rutinoside (54), and rutin (55) were
obtained from Extrasynthese (Genay, France). 3,4-O-diCQA (12), 3,4-O-diCQA (13), diosmetin (33),
rhamnetin (37), isorhamnetin (38), luteolin-7-O-glucuronide (47), isorhamnetin-7-O-glucoside (51), and
isorhamnetin-3-O-rutinoside (57) were purchased from PhytoLab (Vestenbergsgreuth, Germany).
The UHPLC-ESI/HRMS analyses were carried out on a Q Exactive Plus heated electrospray
ionization (HESI-II) – high resolution mass spectrometer (HRMS) (ThermoFisher Scientific, Inc.,
Bremen, Germany) equipped with an ultra-high-performance liquid chromatography (UHPLC) system
Dionex Ultimate 3000RSLC (ThermoFisher Scientific, Inc.) [60].
3.5. Statistical Analysis for Antioxidant and Enzyme Inhibitory Assays

To interpret data gathered, R version 3.5.1 software (The R Foundation, St. Louis, MO, USA)
with corrplot and mixOmics packages was used to perform univariate and multivariate statistical
analyses. One way analysis of variance (ANOVA) and Tukey’s post hoc test were employed to
compare bioactive compounds and biological activities between the samples. Also, relationships
between bioactive compounds and biological activities were evaluated by the estimation of Pearson’s
correlation. For multivariate analysis, biological activities of samples were firstly analyses by PCA
to pinpoint similarities or differences between samples. Then sPLS-DA was applied by using the
species and different extraction condition as class memberships respectively. This allowed better
comparison between the two studied species and gauged the effect of the different extraction solvents
on biological activities.
3.6. Pharmacological Assays
3.6.1. Allelopathy Bioassay

Allelopathy bioassay was carried on the seeds of three commercial lettuces [Canasta (C), Romana
verde (RV) and Romana bionda (RB)], because of their fast germination rate and high sensitivity. The
detailed procedure has been extensively reported in our recent paper [61]. Seeds were treated with
scalar Anthemis extract concentrations (0.1–10 mg/mL) and considered germinated for observed root
length ≥ 1 mm, after the third day of treatment.
3.6.2. Artemia salina Lethality Bioassay

Artemia salina lethality bioassay was performed as previously reported [61]. Brielfy, brine shrimp
larvae were bred at 25–28 ◦ C for 24 h in presence of Anthemis extracts (0.1–20 mg/mL) dissolved in
incubation medium (artificial sea water). After incubation period (24 h) with extracts, the number
of surviving shrimps was evaluated and their vitality was compared to untreated control group.
Experiments were carried out in triplicate, and percentage mortality was calculated with the following
equation: ((T − S)/T) × 100, where T and S are the total number of incubated larvae and survival
napulii, respectively.
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3.6.3. In Vitro Studies

Rat hypothalamic Hypo-E22 cells were cultured in DMEM (Euroclone), as previously reported [48].
The effects of Anthemis extracts (100 μg/mL) on Hypo-E22 cell line viability was evaluated through
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test.
3.6.4. Ex Vivo Cortical Spreading Depression Paradigm

Male adult Sprague-Dawley rats (200–250 g) were sacrificed by CO2 inhalation (100% CO2 at a
flow rate of 20% of the chamber volume per min) and cortex specimens were immediately collected
and maintained in thermostatic shaking bath at 37 ◦ C for 1 h (incubation period), in Krebs-Ringer
buffer at different K+ concentrations, as described below:
K+ 3 mM: corresponding to basal condition;

K+ 15 mM: corresponding to physiologic depolarizing-stimulus;
K+ 60 mM: corresponding to excitotoxicity depolarizing-stimulus.
The present experimental paradigm reproduced the neural pathophysiological condition named
cortical spreading depression (CSD), and was designed according to previous ex vivo and in vivo
studies, describing the use of elevated K+ concentrations (up to 50–60 mM) to induce central nervous
system (CNS) injury [38–40]. During incubation, cortex specimens were challenged with water, MeOH
and EA A. tinctoria and A. cretica extracts (100 μg/mL). Afterwards, individual cortex slices were
homogenized in perchloric acid solution (0.05 M) in order to extract and quantify serotonin (5-HT)
and its main metabolite (5-hydroxyindoleacetic acid, 5HIIA) via HPLC coupled to electrochemical
detection, as previously reported [61,62]. The results were expressed as ng/mg wet tissue. Additionally,
we carried out colorimetric evaluation of LDH level [52]. Finally, an untargeted proteomic profile was
performed on rat cortex homogenate, as described below, in order to further elucidate the putative
mechanism of action of Anthemis extracts.
3.7. Protein Extraction and Filter-aided Sample Preparation

After protein quantification, a volume corresponding to 50 ug of proteins was loaded onto a
Nanosep 10-kDa-cutoff filter (Pall Corporation, Michigan city, MI, USA) and digested according to the
protocol we routinely use in our laboratory. Briefly, the sample was washed twice with 200 μL urea
buffer (8 M urea, 100 mM Tris pH 8.5 in milliQ water) to remove the detergents present in the lysis
buffer. The proteins on the filter where subsequently reduced and alkylated by adding 100 μL of DTT
solution (8 mM dithiothreitol in urea buffer) and 100 μL of IAA solution (50 mM iodoacetamide in
Urea buffer). For protein digestion, the buffer was exchanged with 50 mM ammonium bicarbonate,
before adding trypsin to a ratio of 1:50 (enzyme:substrate). The reaction was incubated for 16 h at
37 ◦ C, and the mixture of peptides was collected by centrifugation, acidified with 10% trifluoroacetic
acid and stored at −20 ◦ C until analysis. The detailed description of mass spectrometric analysis is
reported as “Supplementary Proteomic Analysis”.
3.8. Statistical Analysis for Pharmacological Assays

Statistical analysis was performed using GraphPad Prism version 5.01 for Windows (GraphPad
Software, San Diego, CA, USA). Means ± S.E.M. were determined for each experimental group
and analyzed by one-way analysis of variance (ANOVA), followed by Newman-Keuls comparison
multiple test. Statistical significance was set at p < 0.05. As regards the animals randomized for each
experimental group, the number was calculated on the basis of the “Resource Equation” N = (E + T)/T
(10 ≤ E ≤ 20; https://www.nc3rs.org.uk/experimental-designstatistics).
259
Molecules 2019, 24, 2582
4. Conclusions
Results collected in the present study indicated the promising biological effects of ATP and ACT
extracts. As summarized in Figure 8, tested extracts showed significant antioxidant activity and
potent inhibitory effects against key enzymes, involved in Alzheimer’s disease, type II diabetes, and
hyperpigmentation conditions. Particularly, EA and methanol extracts of both species showed higher
enzyme inhibitory activity (at least 1.5 fold: Table 4) compared to water extracts. Conversely, ACT
water extract revealed more significant protective effects, as evidenced by reduced (−74%) cortex 5-HT
turnover and restored activity of key proteins (i.e., NFEMs and PKCγ) involved in neuron morphology
and neurotransmission, in the selected model of neurotoxicity. In this context A. cretica water extract
appears to be a good candidate for future investigations aimed to confirm and characterize the observed
pharmacological effects, possibly through the use of independent experimental methods.
Figure 8. Protective effects induced by A. cretica and A. tinctoria extracts, as evidenced by the present
pharmacological investigation.
Supplementary Materials: The following are available online. Supplementary Data 1; Supplementary Data 2;
Supplementary Proteomic Analysis.
Author Contributions: Conceptualization, G.Z., L.M., G.O. and C.F.; methodology, M.R., D.Z.-D., R.G.; software,
L.M.; validation, C.F., M.R., R.G. and D.Z.-D.; formal analysis, C.F., L.R.; investigation, A.C., S.L., A.M., G.M.,
M.P.D., S.D.S, I.S., C.M.N.P.-A., K.I.S.; resources, G.O., L.M., C.F.; data curation, C.F., G.Z.; writing—original draft
preparation, C.F., G.Z.; writing—review and editing, L.M., M.F.M., G.O.; visualization, L.B.; supervision, L.B.;
project administration, L.R., G.O., C.F.; funding acquisition, G.O., C.F.
Funding: This research was supported by Italian Ministry of University Grant (FAR 2017 granted to Claudio
Ferrante; FAR 2017 granted to Giustino Orlando).
Conflicts of Interest: The authors declare no conflict of interest
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Sample Availability: Samples of the extracts are available from the authors.
263
molecules
Article
Anticancer Activity of Smallanthus sonchifolius
Methanol Extract against Human Hepatocellular
Carcinoma Cells
Phyu Phyu Myint 1,† , Thien T. P. Dao 2,† and Yeong Shik Kim 2, *
1 Department of Chemistry, Loikaw University, Loikaw 09013, Myanmar
2 College of Pharmacy and Natural Products Research Institute, Seoul National University, Seoul 08826, Korea
* Correspondence: kims@snu.ac.kr; Tel.: +82-2-880-2479

Received: 22 July 2019; Accepted: 21 August 2019; Published: 22 August 2019
Abstract: Background: This research aimed to investigate the cytotoxicity of methanol extract of
Smallanthus sonchifolius leaf (YLE) against a human hepatocellular carcinoma cell line (HepG2).
This plant is currently used as a traditional herbal remedy in the treatment of liver diseases in
some rural parts of Myanmar. Methods: The cytotoxic activity of the plant extract against the
cancerous cell line was assessed using an MTT assay. YLE demonstrated a significant effect (IC50
= 58.2 ± 1.9 μg/mL) on anti-cancer activity, which was further investigated using various assays
including an in vitro cell migration assay, a colony formation assay, cell cycle analysis, western blot
analysis, and a ROS assay. The significance of the phytochemical constituents of YLE could be
identified using LC/Q-TOF-MS techniques. Results: We putatively identified the active components
in YLE, which were possibly melampolide-type sesquiterpenoids. YLE showed an inhibitory effect
on HepG2 cell proliferation and cell migration. YLE also induced cell cycle arrest and necrosis in a
dose-dependent manner. Additionally, YLE significantly suppressed ROS formation in HepG2 cells.
Conclusions: These findings suggest that YLE is sufficient for application as a promising anti-liver
drug in herbal medicine.
Keywords: S. sonchifolius leaf; HepG2 cells; MTT assay; cell cycle arrest; anti-liver cancer drug; antioxidant
1. Introduction
In 2018, liver cancer was the sixth most common cancer and the fourth leading cause of cancer
deaths worldwide [1]. The highest incidence of this cancer can be seen in East Asia, Southeast Asia,
and North and Southern Africa [2]. Based on the database of the International Agency for Research on
Cancer (IARC), there were more than 69,000 new cancer cases in Myanmar in 2018 and liver cancers
were in the top 5 in terms of incidence, mortality, and prevalence by cancer site [1]. Currently, the
Ministry of Health and Sports from Myanmar supports the implementation of the National Cancer
Control Plan, focusing on priority activities and maximizing efforts in line with the respective mandates,
priorities, and areas of expertise of the partner and to achieve better results for cancer prevention, care,
and control.
Testing, annual screenings, and early intervention for cancers are currently inadequate on many
accounts, which include the rise in population, an inadequate supply of drugs, the cost of treatments,
the side effects of several synthetic medicines, and increasing resistance to the drugs used. In most
rural areas, herbal medicine has been used for decades by traditional practitioners to treat cancer
problems. Medicinal plants have long been used in the treatment of liver diseases or the maintenance
of a healthy liver. Yacon, or Smallanthus sonchifolius ((Poepp. & Endl.) H. Rob.), is a plant belonging to

Molecules 2019, 24, 3054
the Asteraceae family, native to the Andean regions of South America [3]. The plant contents include
phenolic acids, flavonoids, and sesquiterpene lactones [4,5]. Yacon has been used as a functional
food with multiple beneficial effects on the body, including as an antimicrobial, as an antioxidant,
hypolipidemic effects, and probiotic substances [3,6]. The plant was cultivated in Myanmar in the
2000s. It has become increasingly popular as medicated green tea for diabetes patients and its use
is wide-spread.
In recent years, Yacon has emerged as a potential anti-cancer agent. Previous in vitro studies
indicated that the crude extract of Yacon and the phytochemicals derived from the plants exerted the
cytotoxicity against breast cancer [7], colon cancer [7,8], and cervical cancer [9,10]. The anticancer
property was attributed to sesquiterpene lactones in Yacon [9–11]. In addition, Yacon has been
well-known to have antioxidant effects because of an abundant amount of polyphenols, which are
found at high quantities in leaves or stems of the plant [6]. Recent studies have indicated that
antioxidants might possess anti-tumor and hepatoprotective effects, although the mechanism needs
further investigation [12].
This research aimed to evaluate the effects of Yacon leaf extract (YLE) on liver cancer in vitro
using hepatocellular carcinoma HepG2 cell line, which is the most commonly used in drug metabolism
and hepatotoxicity studies. HepG2 cells are nontumorigenic with high proliferation rates and an
epithelial-like morphology that performs many differentiated hepatic functions [13]. The medicinal
plant is of high pharmacological importance, but it is still not reported for its chemotherapeutic
potential as an alternative medicine for liver cancer disease. Our results may provide scientific evidence
for the therapeutic potential of this plant, as a functional food, on liver cancer.
2. Results
2.1. Cytotoxicity of YLE by MTT Assay

The sample was evaluated for cytotoxic activity on human hepatoma carcinoma cell lines (HepG2),
as presented in Figure 1. The results of the MTT assay showed a dose-dependent reduction in cell
viability of HepG2 cells while YLE did not affect those of non-tumor HEK 239 cells after 24 h treatment.
The calculated IC50 of YLE on HepG2 was 58.2 ± 1.9 μg/mL.
Figure 1. Cell viability of HepG2 and HEK 239 cells after being treated with different concentration of
YLE. Data are presented as means ± standard deviation (S.D) (n = 3); ** p < 0.01 vs. control group.
2.2. YLE Reduces Colony Formation of HepG2 Cells

To determine the effect of YLE on the replicative potential and the longer-term viability of
liver cancer cells under colony-forming culture conditions, we treated HepG2 cells with various
concentrations of the extracts for 24 h or 48 h, then conducted a crystal violet-based clonogenic assay.
Data showed that YLE significantly inhibited colony formation of HepG2 after 14 days. We found
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Molecules 2019, 24, 3054
that cell proliferation rates gradually decreased as the concentration of the extract was increased.
These findings suggest that YLE exerts its strong inhibitory effect on longer-term viability liver cancer
cells in a dose-dependent manner. The full inhibitory effect of YLE on the HepG2 clonogenicity could
be observed within 24 h at 100 μg/mL dose and 48 h at 80 and 100 μg/mL (Figure 2).
Figure 2. HepG2 cells after 24 h and 48 h treatment with YLE were allowed to grow into visible colonies
for an additional 2 weeks. The figure presents one of three independent experiments.
2.3. Effect of YLE on Wound Healing in HepG2 Cells

Migration of cells plays a vital role in cancer cells survival; thus, we conducted a wound-healing
assay to examine the effect of YLE on the healing process in HepG2 cells. In this study, at the start
of in vitro scratch test, there were little or no cells inside the scratch region. After 24 h exposure to
YLE, it was observed that the cell migrated towards the induced gap (Figure 3). The control sample
migration (0 μg/mL) was noted to be the highest in all the cell lines tested. YLE significantly decreased
the migration in HepG2 cells.
Figure 3. Effect of YLE on (A) cell morphology and (B) migration ability of HepG2 cells. The figures
show one of three independent experiments.
2.4. Effect of YLE on ROS Production

To evaluate the influence of YLE on mediating ROS generation in HepG2 cells, we incubated
cells with different concentrations of YLE in indicated times (3 h, 6 h, 9 h). The intracellular ROS was
measured via 2 ,7 -dichlorofluorescein diacetate (DCF-DA) fluorescence. As displayed in Figure 4,
intracellular ROS levels were significantly decreased in a concentration-dependent manner (Figure 4A),
while the cell viability of HepG2 was not affected in short-time treatment (Figure 4B). These data proved
that a reduction of ROS production in HepG2 cells was not due to decreasing numbers of living cells.
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The ROS-inhibitory effect of YLE showed similar patterns after 3 h and 6 h of treatment. The specific
changes could be observed only at high concentrations, such as 80 and 60 μg/mL, respectively. However,
after 9 h of treatment, the ROS production was inhibited notably (>50%) even at the lowest concentration
(40 μg/mL) used in the experiments. These results suggest that the antioxidant effect of YLE possibly
led to induced cytostasis in HepG2 cells after 24 h treatment.
Figure 4. Effect of YLE on ROS formation of HepG2 cells. (A) Antioxidant effect of YLE on HepG2 cells
after 3 h, 6 h, and 9 h. Data are expressed as averages ± S.D (n = 3); * p < 0.05, ** p < 0.01 vs. control
group. (B) Effect of YLE on cell viability. Data indicated no specific change between treatment groups
and the control group after indicated times (3 h, 6 h, 9 h).
2.5. YLE Induces Cell Cycle Arrest in HepG2 Cells

Next, we examined the effect of YLE on the cell cycle of a HepG2 liver cancer cell line after 24 h
treatment using flow cytometry. Figure 5 shows the relative percentages of HepG2 cells in each phase
of the cell cycle following treatment. There was a dose-dependent increase in the percentage of cells in
the G0/G1 phase. The data in this study suggested that YLE could inhibit cell proliferation of HepG2
cells by inducing the cell cycle at the G0/G1 phase.
Figure 5. Effect of YLE on the cell cycle of HepG2 cells. The figure represents triplicate experiments.
2.6. YLE Induces Necrosis in HepG2 Cells

To clarify the mechanism of YLE on inducing cell death in HepG2 cells, we stained the YLE-treated
cells with Annexin V/PI after 24 h exposure and measured the fluorescence by flow cytometry.
The percentage of each subpopulation of cells is described in Figure 6. We found that YLE induced the
loss of cell membrane integrity, which was indicated by the increase in numbers of PI-positive cells.
The proportion of only the PI-positive cell population treated with 100 μg/mL YLE was approximately
20-fold higher than the one in the control group. Additionally, YLE did not trigger caspase activation
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or cleavage of caspase proteins, such as caspase 3 and caspase 8. These data suggest a necrotic mode of
cell death induced by YLE.
Figure 6. Effect of YLE on (A) the cell death mechanism and (B) the expression levels of proteins in
HepG2 cells. The figure shows one of three independent experiments.
2.7. Metabolites Identification of Methanol Extract of YLE

In this study, we confirmed the presence of several metabolites in YLE based on the characteristic
fragments in the MS spectra previously described in the literature [5]. We focused on raw formulas of
several melampolide sesquiterpene lactones, which were previously described as main components in
Yacon, as follows: C23 H28 O8 , C23 H28 O9 , and C23 H28 O10 [5]. Figure 7 displays the total ion current
chromatogram (TIC) of the extract (Figure 7A) and the obtained extracted-ion chromatograms (EIC)
showing the series of peaks with m/z values corresponding to the three selected molecular formulas
(Figure 7B–D). Compound 1 (retention time (rt) 11.94), compound 2 (rt. 12.81 min), and compound 3
(rt.15.13) exhibited a protonated molecular ion [M + H]+ at m/z 465.1657 [C23 H28 O10 + H]+ , m/z 449.1812
[C23 H28 O9 + H]+ , and m/z 433.1788 [C23 H28 O8 + H]+ , respectively. These formulas shared the common
product-ions that were characterized for uvedalin moiety with m/z 213.0901; 241.0815; 273.1809,
putatively identified as an isoform to enhydrin (C23 H28 O10 ), uvedalin (C23 H28 O9 ), and polymatin B
(C23 H28 O8 ) [5] (Figure 7E–G).
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Figure 7. TIC in the positive mode of YLE (A). EIC of m/z 465.1761 (±10.0 ppm) [C23 H28 O10 + H]+ (B);
EIC of m/z 449.1812 (±10.0 ppm) [C23 H28 O9 + H]+ (C); EIC of m/z 433.1862 (±10.0 ppm) [C23 H28 O8
+H]+ (D). Product-ion chromatograms of compound 1 (E), compound 2 (F), and compound 3 (G).
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3. Discussion
Yacon (S. sonchifolius), a common edible plant grown throughout the world, is well known for its
anti-diabetic properties [14]. It is also demonstrated to have several other pharmacological properties,
including anti-inflammatory, anti-oxidant, anti-allergic, and anti-cancer effects [15]. The cytotoxicity
potential of hexane, methanol, and dichloromethane extracts of Yacon leaves was assessed against
MCF-7 and HT-29 cell lines by using the AlamarBlue® assay [7]. Sesquiterpene lactones from Yacon
leaves, such as enhydrin, uvedalin, and their derivatives, also exhibited cytotoxic activity against
MGC80-3 [16], HeLa, HL-60, and B16-F10 cell lines [9]. However, there has been no report yet to
evaluate the anti-liver cancer activity of S. sonchifolius on liver cancer cells. Therefore, this study aimed
to investigate the anti-cancer effect of YLE on HepG2 cells in terms of inhibiting cell proliferation and
migration. In addition, we also examined the YLE effect on cell cycle and ROS generation in this cell line.
In the current study, YLE was found to show a potent inhibitory effect on HepG2 cell survival. Firstly,
we examined the toxicity of YLE on liver cancer HepG2 cells. The calculated IC50 (58.2 ± 1.9 μg/mL)
implied the promising inhibitory effect against these cancer cells. Indeed, the colony formation of
HepG2 cells was significantly suppressed with increasing concentration of YLE up to more than 90% at
100 μg/mL, in comparison with the control, after 24 h or 48 h treatment. Taken together, these results
suggested that YLE demonstrates a long-term suppressive effect on cell proliferation of HepG2 in a
concentration-dependent manner.
It is known that metastasis is one of the leading causes of death from cancer and it is complicated
to examine [17]. During tumor metastasis, malignant cells migrate into neighboring healthy tissues,
contributing to tumor development. In this study, YLE could effectively prevent the migration of
HepG2 in a dose-dependent manner. Therefore, YLE could contribute to hinder metastasis progression
in hepatocellular carcinoma.
To control cancer growth, inhibition of the progression of the cell cycle is one of the essential
strategies [18]. In this study, flow cytometry analysis demonstrated that the extract dose-dependently
increased the percentage of cells of the G0/G1 phase of the HepG2 cell cycle. This observation reveals
that YLE induces cell cycle arrest at the G0/G1 phase of the cell cycle.
In term of inducing cancer cell death, we found that YLS induced necrosis in HepG2 cells.
The number of cells in the necrosis subpopulation significantly increased from 3.88% (control group) to
70.98% (100 μg/mL YLE-treatment group), whereas YLE did not activate caspase 3 or caspase 8, which
regulate apoptosis in cells.
High levels of ROS in cancer cells have been found in almost all cancer cells due to high
metabolomic activity and they have specific functions in cancer cell development [19]. ROS have been
reported to be involved in cell proliferation, cell survival, cell cycle progression, and angiogenesis.
Therefore, suppressing ROS may be a useful strategy in cancer treatment [20]. Since YLE has been
reported to contain a large number of polyphenols [4], we sought to evaluate the antioxidant effect
of YLE on HepG2 cells. YLE could notably reduce ROS in HepG2 in both dose and time-dependent
manners. These observations may support the inhibitory effect of YLE on HepG2 cell proliferation.
The presence of sesquiterpene lactones, such as polymatin B, enhydrin, and uvedalin, was also
confirmed by HPLC coupled with high-resolution mass tandem analysis [5]. These compounds have
been demonstrated to have cytotoxicity as well as to induce apoptosis or necrosis in several cancer cell
lines [11]. In the current study, we partly confirmed the presence of these sesquiterpenoids sharing
the common fragment characteristic of the uvedalin moiety. These compounds may be significant as
chemical defenses for human hepatocellular carcinoma HepG2 cells. Although the effect of individual
constituents of this plant extract on HepG2 cells needs to be further investigated, the findings in
the current study suggest that S. sonchifolius leaf could be recommended as a potential source of a
chemopreventative agent against liver cancer.
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4.1. Preparation of the Samples

The Yacon (Smallanthus sonchifolius ((Poepp. & Endl.) H. Rob.) leaves were collected from Pindaya
Township, Shan State, in the eastern part of Myanmar. The sample was identified at the Department of
Botany, University of Yangon. The leaves were cleaned, carefully dried in shadow, and powdered.
About 2 g of ground powder was soaked in 20 mL methanol and shaken for 8 h at 180 rpm and
37 ◦ C. The soaked substance was filtered throughout 110 mm filter paper (Hyundai, Seoul, South
Korea). The solvent was eliminated with a rotary evaporator (SB-1200, EYELA, Shanghai, China)
at 60 ◦ C. The residue was placed in a freeze drier (Operon, Korea) to dry. The crude extract was
kept in a refrigerator at 4 ◦ C and protected from light. The experiment was performed in triplicate.
Four-hundred mg of the sample extract was dissolved in 1 mL of dimethyl sulfoxide (DMSO) and then
serially diluted to 100, 60, 40, and 20 μg/mL for further biological experiments. All other chemical
reagents were from Sigma-Aldrich Chemical Company (St. Louis, MO, USA) unless otherwise noted.
4.2. Cell Line and Culture Medium

HepG2 and HEK 239 cell lines were obtained from the American Type Culture Collection (ATCC).
Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) media supplemented with 10%
(v/v) fetal bovine serum and 1% (v/v), 100 U/mL penicillin and 100 μg/mL streptomycin. Cells were
cultured in an incubator at 37 ◦ C and 5% CO2 humidified atmosphere. Media were changed every 2 to
3 days. Reagents and media for cell culture were purchased from GenDePOT (Katy, TX, USA).
4.3. Cytotoxicity Assay (MTT Assay)

The cellular toxicity of the YLE on cultured cells was measured using 3-(4,5-dimethylthiazol-2-yl)-2,
5-diphenyl tetrazolium bromide (MTT). Cells were grown in a 96-well plate at a density of 1 × 104 cells
per well. The cells are allowed to grow overnight in a cell culture incubator. Then, we treated cells with
different concentrations of samples (0, 20, 40, 60, and 100 μg/mL) for 24 h. Later, cells were washed
twice with phosphate buffer saline (PBS). MTT solution was added to each well (final concentration of
MTT was 0.5 mg/mL) and the plate was incubated for 3 h. Finally, the medium was replaced by DMSO
to solubilize the formazan crystals. The optical density was measured by absorbance at 595 nm with a
microplate spectrophotometer (SpectraMax 190, Molecular Devices, San Jose, CA, USA). We identified
the IC50 value using the ED50 Plus v1.0 online software (National Institute of Respiratory Diseases
(INER), Mexico) as described elsewhere [21].
4.4. Cell Colony Formation Assay

Cells were seeded at a density of 1 × 103 cells/well in 6-well plates and allowed to attach overnight.
Then, the cells were treated with YLE (0, 40, 60, 80, and 100 μg/mL) for 24 h or 48 h. After treatment, cells
were continuously grown for another 2 weeks. The media were changed every 3 days. After 14 days,
cells were washed with PBS and fixed with 3.7% formaldehyde solution for 30 min before being stained
with 0.5% crystal violet for another 30 min at room temperature (RT). Cells were washed with water to
remove the dye and then photographed.
4.5. Cell Migration Assay (Wound–Healing Assay)

We seeded cells in a 24-well plate (5 × 104 cells/well) and cultured at 37 ◦ C, 5% CO2 for 24 h.
The 80–90% cell confluence was observed before the scratch assay was performed. We stimulated a
wound using a sterile 200 μL pipette tip. After washing with PBS to remove loosened debris, cells
were treated with YLE (0, 40, 60, 80, and 100 μg/mL) for 24 h at 37 ◦ C, 5% CO2 . After treatment,
cells were washed twice with PBS. The images of cells were captured under a CKX41 microscope
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(Olympus, Japan) at 400× magnification. Images were processed with ProgRes Capture Pro software
v.2.8.8 (JENOPTIK Optical Systems, Jena, Germany).
4.6. Reactive Oxygen Species (ROS) Production Assay

The level of intracellular ROS was determined by the change in fluorescence resulting from
the oxidation of the fluorescent probe dichlorofluorescein diacetate (DCF-DA). We seeded cells at a
density of 1 × 106 cells/well in 6 wells plate and then treated them with 0, 40, 60, 80, and 100 ug/mL of
YLE for 3, 6, and 9 h. After indicated periods, cells were washed twice with DPBS and stained with
dichlorofluorescein diacetate (DCF-DA, 10 μM) at 37 ◦ C for 30 min in the dark. After washing twice
with PBS, the fluorescence generation was measured in a microplate reader, (excitation wavelength
(ext.): 485 nm; emission wavelength (emi.): 535 nm). Data were expressed as the percentage of ROS
relative to untreated control groups.
4.7. Cell Cycle Analysis

HepG2 cells at a concentration of 1 × 106 /well cells were cultured in 6-well plates and treated with
different concentrations of YLE for 24h. After treatment, the cells were washed with PBS and harvested
using a centrifuge at 1300 rpm for 3 min. Then, cells were re-suspended and fixed with 70% ethanol at
−20 ◦ C for 1 week. After fixing, we harvested cells by washing with PBS and centrifuging at 2000 rpm
for 3 min. Cells were incubated with RNase (200 μg/mL) at 37 ◦ C for 30 min to remove cellular RNA,
then stained with propidium iodide (PI, 100 μg/mL) for another 10 min at RT in the dark. Finally, the
cells were analyzed by flow cytometry (BD FACSCalibur, BD Biosciences, USA) according to detected
signals in the FL2 channel (ext: 488 nm, emi: 564–606 nm) while data were analyzed with Cell Quest
Pro software (BD Biosciences, San Jose, CA, USA).
4.8. Annexin V/PI Assay

HepG2 cells were seeded in the 6-well plates (1 × 106 cells/well). After overnight incubation,
we treated cells with indicated concentrations of YLE for 24 h. Cells were washed with PBS and
harvested using a centrifuge at 2000 rpm for 3 min for the following analysis. To distinguish apoptotic
and necrotic cell death in HepG2 cells, we used the BD Annexin V: FITC Apoptosis Detection Kit
I (BD Biosciences, San Diego, CA, USA), according to the manufacture’s direction. The cells were
analyzed by flow cytometry (BD FACSCalibur, San Jose, CA). Signals were detected in the FL1 channel
(for Annexin V) and FL3 channel (for PI), while data were analyzed with Cell Quest Pro software.

The HepG2 cells were seeded in a 6-well plate at 1 × 106 cells/well and treated with various
concentrations of YLE for 24 h. The whole cells lysates were prepared by homogenizing cells in a
prepared lysis buffer (20 mM HEPES (pH 7.6), 350 mM NaCl, 20% glycerol, 0.5 mM EDTA, 0.1 mM
EGTA, 1% NP-40, 50 mM NaF, 0.1 mM DTT, 0.1 mM PMSF, and a protease inhibitor cocktail) on
ice for 30 min, followed by collecting the supernatant using a centrifuge at 15,000 rpm for 10 min.
The concentration of protein was identified using a Bradford assay. The equal amounts of proteins
were separated by electrophoresis on 10% SDS gels and transferred to nitrocellulose membranes.
Blots were blocked with 5% BSA for 1 h prior to incubating with the following primary antibodies:
β-actin conjugated with HRP (Santa Cruz, 47778) as1:3000, pro-caspase 3 (rabbit, Santa Cruz H-277:
sc-7148) as 1:1000, cleaved caspase 3 (rabbit, Abcam ab2302) as 1:1000, pro-caspase 8 (rabbit, Santa
Cruz, sc-6134) as 1:1000, and cleaved caspase 8 (rabbit, Cell Signaling Technology, 9496) as 1:1000,
at 4 ◦ C overnight. After washing the membrane and incubating with goat anti-rabbit IgG (H + L)
secondary antibody conjugated with HRP (GeneTex, GTX213110, 1:3000) for 1 h at RT, the bands of
interest were detected using an EZWestern ECL kit (Daeillab Service, Seoul, South Korea) and then
photographed on the LAS-4000 imaging system (GE Healthcare, Chicago, IL, USA).
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4.10. LC-Q-TOF-MS Analysis of YLE

Since the cytotoxicity of YLE was attributed to its active ingredients, which were known to be
sesquiterpenoids, we confirmed the presence of these compounds using liquid chromatography analysis
in an HPLC system (Agilent Technologies, Santa Clara, CA, USA) linked to a G6530A ESI-Q-TOF
MS spectrometer (Agilent Technologies). Chromatographic separations were conducted using a C18
column (2.1 × 150 mm, 3.5 μm, Agilent Technologies). The mobile phases were water with 0.1% formic
acid (A) and acetonitrile with 0.1% formic acid (B). The gradient program consisted of the following:
0–15 min, 5–50% B; 15–20 min, 50% B; 20–30 min, 50–95% B; followed by 95% B for 10 min washing.
The flow rate was 0.3 mL/min and the temperature was kept at 40 ◦ C. The injection volume was 3 μL.
The instrument was operated with an ESI source in positive ion mode. The mass determination was
performed using the following MS conditions: Ion spray voltage of 400 kV; desolvation temperature,
350 ◦ C; desolvation gas flow rate, 10 L/min. The fragmentor was set at 150 V. The full-scan mass spectra
were acquired within an m/z range of 100 to 1200 m/z in the MS mode followed by the target MS/MS
mode. Data acquisition and proceeding were performed using Mass Hunter Qualitative Analysis
software (Agilent Technologies). The compounds were putatively identified based on their mass
tandems in comparison with published data.

In this study, we performed all experiments in triplicate and analyzed results using one-way
ANOVA followed by a Dunnett’s test (SPSS version 25.0; Chicago, IL, USA). Probabilities of p < 0.05
were considered significant.
5. Conclusions
In this study, we partially identified the active ingredients present in the YLE, which are possibly
melampolide sesquiterpene lactones with uvedalin moiety. The results in this study indicate that YLE
appears to be capable of killing malignant liver cancer cells by inhibiting the growth and migration in
addition to inducing necrosis and cell cycle arrest. Furthermore, we also confirmed the antioxidant
effect of YLE on liver cancer cells. To conclude, these findings suggest S. sonchifolius (Yacon) is a
promising potential anti-liver cancer agent in the area of herbal medicine. Further research regarding
the role of each active compound in YLE towards anti-liver cancer activity would be worthwhile.
Author Contributions: Conceptualization, Y.S.K. and P.P.M.; Methodology, T.T.P.D. and P.P.M.; Software, T.T.P.D.;
Validation, T.T.P.D., P.P.M. and Y.S.K.; Formal Analysis, T.T.P.D. and P.P.M.; Investigation, T.T.P.D. and P.P.M.;
Resources, Y.S.K. and P.P.M.; Data Curation, T.T.P.D.; Writing—Original Draft Preparation, P.P.M.; Writing—Review
& Editing, T.T.P.D.; Visualization, Y.S.K. and P.P.M.; Supervision, Y.S.K.; Project Administration, Y.S.K.; Funding
Acquisition, Y.S.K. and P.P.M.
Funding: This research was funded by a grant (NRF-2017R1A2B4009301) from the National Research Foundation
of Korea. P.P.M was supported by the Korea Foundation for Advanced Studies (KFAS). T.T.P.D. was awarded a
fellowship from the BK21 PLUS supported from the Ministry of Education.
Acknowledgments: We appreciated the technical support of Ji Yoon Lee in College of Pharmacy, SNU.
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molecules
Article
Chemical Composition, Antimicrobial, Antioxidant,
and Antiproliferative Properties of Grapefruit
Essential Oil Prepared by Molecular Distillation
Weihui Deng, Ke Liu, Shan Cao, Jingyu Sun, Balian Zhong and Jiong Chun *
National Navel Orange Engineering Research Center, College of Life Sciences, Gannan Normal University,
Ganzhou 341000, China; dwh110by@163.com (W.D.); liuke121602026@126.com (K.L.);
scoral29116@163.com (S.C.); SJYnj_1997@163.com (J.S.); bal.zh@163.com (B.Z.)
* Correspondence: qczx99@163.com; Tel.: +86-797-839-3068
Academic Editor: Raffaele Capasso

Abstract: Grapefruit essential oil has been proven to have wide range of bioactivities. However,
bioactivity of its molecular distillate has not been well studied. In this study, a light phase oil
was obtained by molecular distillation from cold-pressed grapefruit essential oil and GC-MS was
used to identify its chemical composition. The antimicrobial activity of the light phase oil was
tested by filter paper diffusion method, and the anticancer activity was determined by the Cell
Counting Kit-8 (CCK-8) assay. Twenty-four components were detected with a total relative content
of 99.74%, including 97.48% of terpenes and 1.66% of oxygenated terpenes. The light phase oil had
the best antimicrobial effect on Bacillus subtilis, followed by Escherichia coli, Staphylococcus aureus and
Salmonellaty phimurium. DPPH and ABTS assays demonstrated that the light phase oil had good
antioxidant activity. The CCK-8 assay of cell proliferation showed that the light phase oil had a good
inhibitory effect on the proliferation of HepG2 liver cancer cells and HCT116 colon cancer cells.
Keywords: grapefruit essential oil; molecular distillation; GC-MS; antimicrobial; antioxidant;

anticancer activity
1. Introduction
With the frequent occurrence of food safety issues and the toxicity of synthetic chemicals,
the demand for safe and natural alternatives is growing. Plant extracts have been used since ancient
times, and now the focus is on their role in health promotion and their treatment and prevention
properties for various diseases. In the past few decades, plant essential oils (EOs) have attracted a
lot of interest due to their safety and pharmacological properties including bacteriostatic, free radical
scavenging, anti-inflammatory, and inhibitory effects on malignant tumor cell proliferation [1–4].
Citrus Eos are the main aromatic by-products of the juice extraction industry and are widely used
in food, cosmetics and pharmaceutical industry [5–7]. The annual global production of citrus EO is
approximately 16,000 tons, and the cost is about $14,000/ton on the international market. Thus, citrus
EO is of great demand and is one of the more promising market prospects [8]. Grapefruit (Citrus paradisi
Macf.), one of the world s largest production citrus families [9], is famous for its taste and nutritional
value. Grapefruit EO is extracted from grapefruit peel and has been used for a long time as a valuable
ingredient for its characteristic aroma in flavor and fragrance [10,11]. Similar to most citrus EOs,
its major components are terpenes and terpene oxides. Terpene oxides include alcohols, ethers,
aldehydes, ketones, and esters [12–14], which are the main source of the aroma, whereas terpenes
contribute less to the aroma. In spite of extensive studies on the aroma features of grapefruit EO,
in recent years, more and more researchers have become interested in exploring their biological and

Molecules 2020, 25, 217
pharmacological activities. Grapefruit EO has been reported to have a wide range of bioactivities.
It was shown to inhibit the growth of food-borne spoilage bacteria and pathogenic strains [15–17].
Okunowo et al. (2013) found that grapefruit EO obtained from the peel by hydrodistillation exerted
inhibitory effects against bacteria and fungi, andmay be further developed for the treatment of
certain diseases [18]. Grapefruit EO has shown antioxidant activity, which was important for food
preservation and disease prevention [19,20]. Ahmed et al. (2019) reported that grapefruit EO extracted
by hydrodistillation had antioxidant activity by using DPPH and FRAP assays [21]. Grapefruit peel
extracts have been shown to decrease the HL-60 cell viability in a concentration-dependent manner [22].
In fact, grapefruit extracts (a mixture of EO and other nonvolatile phytochemicals) could also inhibit
the growth and proliferation of cancer cells such as neuroblastomas, leukemias, and prostate and lung
cancer lines [23–25]. Cuthrell et al. (2006) reviewed the anticancer activities of phytochemicals found
in grapefruit [26].
Most grapefruit EO samples used for bioactivity studies were made by cold-pressing, steam
distillation, or hydrodistillation methods. Cold-pressing is the predominant method to extract most
citrus peel EOs, including grapefruit EOs. In commercial practice, grapefruitis processed to obtain
juice and other by-products. EO is one of the primary grapefruit by-products. Large-scale grapefruit
EO is mainly prepared by a cold-pressing method based on John Bean FoodTech (JBT) juice extractors
and its technology, which is used by 75% of the world’s citrus juice production [27]. The juice and
EO are extracted separately and simultaneously. The EO was extracted by mechanical rupturing of
the oil sacs in the flavedo, expressing the oil as an aqueous emulsion from which it is separated by
centrifuging. The EO recovery is a physical separation process and no heat is applied throughout
the whole extraction procedure. The operation temperature is much lower than in the distillation
procedure. Thus, the EOs will have characteristics that are closer to those of the essence present
in the grapefruit matrix. Large scale production, low cost and the aroma characteristic remaining
are the big advantages of the cold-pressing method. However, cold-pressed grapefruit oil contains
waxes, pesticide residues, coumarins, carotenoids and other nonvolatile components [28–30], some
of them also have good bioactivities that may cause bias in bioactivity research of EO. César et al.
(2009) found that furanocoumarins isolated from grapefruit peel oil showed potent in vitro inhibitory
activity against intestinal cytochrome P450 3A4, an enzyme involved in “grapefruit/drug” interactions
in humans [28]. Steam distillation or hydrodistillation was carried out at relatively high temperature
which may cause degradation of some thermal sensitive molecules [18,31]. To avoid such problems,
and find a new way to use the commercial available cold-pressed grapefruit EO in biochemistry and
pharmachutical fields, we used a molecular distillation method to prepare grapefruit oil samples for
our bioactivity tests.
Molecular distillation is a special liquid–liquid separation technology under high vacuum, which
is employed as a separation process in the food industry [32,33]. Molecular distillation can divide
the EO mixture into two different phases according to the free path of different molecules at low
temperature. Molecular distillation is prominent with the advantages of low temperature treatment
and high vacuum application, which is very suitable for thermolabile compounds and is used for
concentrating and refining EOs [34,35]. At present, there are seldom reports on the antibacterial
and anticancer activity of Eos obtained by molecular distillation. In this study, the cold-pressed
grapefruit EO was processed by molecular distillation technology and the light phase essential oil
(LPEO) was collected. Its constituents were identified by GC-MS. The activities of LEPO were tested on
microorganisms and malignant proliferating cells (HCT116 colon cancer cells and HepG2 liver cancer
cells) were tested. We expect that this work can stimulate the development of new agents for food
preservation and chemo-preventive anti-cancer treatments.
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2.1. Chemical Composition of the Light Phase Grapefruit Essential Oil

The chemical composition of the grapefruit light phase essential oil (LPEO)was analyzed by
GC-MS. The total ion chromatogram (TIC)of LPEO is shown in Figure 1. The relative content of each
component was calculated by the peak area normalization method. The components were identified
according to retention index and the NIST mass spectral library.
Figure 1. Total ion chromatogram of grapefruit light phase essential oil (LPEO).
As shown in Table 1, twenty-four compounds, accounting for 99.74% of the total oil were identified.
Monoterpenes were the major components, accounting for 96.93% of the total oil. Limonene (93.33%)
was the predominant component of monoterpenes, followed by β-myrcene (2.16%), α-pinene (0.76%),
and sabinene (0.60%). Monoterpene oxide (1.62%) included carvone (0.41%), cis-limonene oxide
(0.43%), and trans-limonene oxide (0.33%). The sesquiterpene (0.55%) included caryophyllene (0.20%),
β-cubebene (0.14%), α-copaene (0.13%), etc. Caryophyllene oxide (0.04%) was the only sesquiterpene
oxide detected. In addition, three linear aldehydes: Octanal (0.36%), decanal (0.19%), and nonanal
(0.05%) were found in LPEO. Pino et al. (1999) reported the chemical composition of grapefruit EO
prepared by steam distillation from solids and effluents produced during commercial oil extraction [31].
The limonene content (70.9%) in steam-distilled oil was much less than LPEO (93.33%); however,
the content of myrcene (13.6%) and α-pinene (3.8%) was much higher than LPEO (myrcene 2.16%
andα-pinene 0.76%). Also, Okunowo et al. (2013) reported the components of grapefruit EO obtained
by hydrodistillation [18]. The content of limonene (75.07%)was closed to that of steam-distilled oil.
Cold-pressed grapefruit oil was shown to have a limonene content of 93.47%, however, the corrected
limonene content became 85.60% when nonvolatiles were excluded [36]. The composition of distilled
samples of grapefruit EO still vary from each other according to genetic differences, soil type, maturity
stages, weather types and culturing conditions etc [37].
Table 1. Chemical composition of grapefruit light phase essential oil (LPEO) by GC-MS.
No. RIa Compounds Composition (%)

1 938 α-Pinene 0.76
2 956 Camphene 0.01
3 977 Sabinene 0.60
4 985 β-Pinene 0.05
5 992 β-Myrcene 2.16
6 1007 Octanal 0.36
7 1049 Limonene 93.33
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Table 1. Cont.
No. RIa Compounds Composition (%)

8 1053 β-Ocimene 0.02
9 1103 Linalool 0.12
10 1108 Nonanal 0.05
11 1127 trans-p-Mentha-2,8-dien-1-ol 0.16
12 1137 cis-Limonene oxide 0.43
13 1141 trans-Limonene oxide 0.33
14 1155 Citronellal 0.04
15 1199 α-Terpineol 0.13
16 1208 Decanal 0.19
17 1251 Carvone 0.41
18 1377 α-Copaene 0.13
19 1388 β-Cubebene 0.14
20 1421 Caryophyllene 0.20
21 1457 Humulene 0.03
22 1482 Germacrene D 0.01
23 1519 δ-cadinene 0.04
24 1566 Caryophyllene oxide 0.04
Total 99.74
Monoterpene hydrocarbons 96.93
Oxygenated monoterpenoids 1.62
Sesquiterpene hydrocarbons 0.55
Oxygenated sesquiterpenes 0.04
others 0.60
RIa , retention indices determined on HP-5 column, using the homologous series of n-alkanes (C8–C20).
2.2. Antimicrobial Activity

Grapefruit EOs prepared by cold-pressing or hydrodistillation using a Clevenger-type apparatus
have shown a wide spectrum of antimicrobial activity in vitro [18,37]. However, antimicrobial activity
of grapefruit EO prepared by molecular distillation has not been well studied. We tested LPEO on five
microorganisms and the results obtained are shown in Table 2. The filter paper diffusion method was
used to test the antibacterial activity of LPEO against different bacteria, and the activity of LPEO was
evaluated according to the diameter of the inhibition zone and the minimum inhibitory concentration
(MIC) values. LPEO exhibited strong antibacterial effects on the four bacteria tested. From the scale of
the inhibition zone, LPEO had the strongest inhibitory effect on B. subtilis with a maximum diameter of
35.59 mm, followed by E. coli, S. aureus, and S. typhimurium. LPEO had no inhibitory activity against
P. aeruginosa with an inhibition zone of 8.57 mm. P. aeruginosa belongs with E. coli and S. typhimurium to
the group of Gram-negative bacteria but exhibits quite a different response to LPEO. This phenomenon
may be partly due to its relatively low outer membrane permeability. LPEO molecules enter the
periplasm by diffusion through the channels of nonspecific porins in the outer membrane, and this
pathway in P. aeruginosa is 10- to 100-fold less efficient than that in E. coli [38]. Regarding MIC values,
LPEO showed the best antimicrobial activity against Bacillus subtilis with a MIC value of 0.78 μL/mL.
Based on the inhibition zone and MIC values, the order of sensitivity of the different bacteria was:
B. subtilis > E. coli > S. aureus > S. typhimurium > P. aeruginosa. Since cold-pressed grapefruit oil
(Citrus paradisi Macf.) has been evaluated as “generally recognized as safe” (GRAS) by the Expert
Panel of the Flavor and Extract Manufacturers Association (FEMA) [39], and LPEO, a distillate from
cold-pressed EO, showed strong sensitivity to most tested microorganisms, it appeared to be suitable
to food applications. These results demonstrate that molecular distillation technology can provide a
grapefruit EO fraction with good antimicrobial activity.
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Table 2. The antimicrobial activity of grapefruit light phase essential oil (LPEO).
Bacterial Strain Diameter of Inhibition Zone (mm) MIC (μL /mL)

Bacillus subtilis (G+) 35.59 ± 1.06 a 0.78
Staphylococcus aureus (G+) 24.34 ± 0.52 c 6.25
Escherichia coli (G-) 26.86 ± 0.17 b 6.25
Salmonella typhimurium (G-) 21.70 ± 0.21 d 12.50
Pseudomonas aeruginosa (G-) 8.57 ± 0.13 e 25.00
Disk diameter is 6.0 mm. Zone of growth inhibition values are presented as mean± standard deviation for at least
three experiments. Different superscript letters represent the significant differences at P < 0.05 according to Tukey’s
multiple range test. The scale of zone of inhibition measurement was the following (disk diameter included):
≥20 mm is strongly inhibitory; <20–16 mm is moderately/mildly inhibitory; <15–10 mm is weak inhibitory; <9–7 mm
is not inhibitory.
Uysal et al. (2011) evaluated the antibacterial activities of grapefruit Eos from solvent-free
microwave extraction (SFME) and hydrodistillation (HD) by the disc-diffusion method [17]. The Eos
obtained from SFME and HD showed the highest activity against S. aureus with inhibition zones of
53 and 41 mm, respectively, higher than LPEO (24.34 mm). The activity against E. coli (30 mm and
28 mm) was close to our result (26.86 mm). Both of their samples and LPEO showed no obvious
activity against P. aeruginosa. LPEO showed better activity against S. typhimurium (21.70 mm) than
their samples (15 mm and 13 mm). Although a lot of plant EOs have shown antimicrobial activity,
the reason of this capacity is not well known. It could be provoked by the major components of the EOs
or due to a synergistic effect among the major components and the minor ones. Different preparation
methods yield EO samples with differences in chemical composition and relative content, and cause
differences in antimicrobial activity.
2.3. Antioxidant Activity

A lot of EOs have been reported to scavenge the free radicals that cause damage to the body and
reduce the risk of many diseases originating from oxidative stress. In order to measure the effect of LPEO
and determine its potential application in food, cosmetic or pharmaceutical industries, we evaluated
its antioxidant activity using two different assays: The 2,2-diphenyl-1-picrylhydrazyl (DPPH)and
2,2 -azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) radical (ABTS) assays. Butylated hydroxytoluene
(BHT) was used as positive control. The IC50 values of BHT in DPPH and ABTS were 0.03 mg/mL and
0.01 mg/mL, which was consistent with the literature [40]. The DPPH and ABTS activities of LPEO
were obtained with IC50 values of 22.06 ± 0.92 mg/mL and 15.72 ± 0.32 mg/mL, respectively. LPEO had
better antioxidant activity than cold-pressed grapefruit EO in the DPPH assay (EC50 > 40 mg/mL) and
hydrodistilled grapefruit EO in the ABTS assay (EC50 = 27.5 mg/mL) [35]. Compared with cold-pressed
orange oil, LPEO had much lower antioxidant activity in the DPPH assay (IC50 = 3.01 ± 0.20 mg/mL)
and better activity in the ABTS assay (IC50 = 23.25 ± 0.84 mg/mL) [41].
2.4. Antiproliferative Activity of LPEO in HepG2 and HCT116 Cancer Cells

The effects of different concentrations of LPEO on the proliferation of HepG2 liver cancer and
HCT116 colon cancer cells were tested by the Cell Counting Kit-8 (CCK-8) method [42,43]. The results
are shown in Figure 2. The viability rate of both cell types decreased with increasing LPEO concentration.
When the concentration of LPEO was less than 0.1 μL/mL, no obvious change of viability of HepG2
cells was observed. However, when the concentration of LPEO was higher than 0.1 μL/mL, the viability
of HepG2 cells significantly decreased; at the LPEO concentration of 0.3 μL/mL, the viability was
7.4%only. LPEO also had a good inhibitory effect on the growth of HCT116 colon cancer cells. At the
concentration of0.05 μL/mL or higher, the viability of HCT116 cells significantly decreased. It was
as low as 7.43% when the concentration of LPEO was 0.5 μL/mL. GraphPad Prism™ (Version 5.00)
software (GraphPad Software, San Diego, CA, USA) was used to calculate IC50 values. IC50 value of
HepG2 and HCT116 was 0.24 and 0.20 μL/mL, respectively. These results indicate that LPEO has a
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significant inhibitory effect on the proliferation of HepG2 hepatoma cells and HCT116 colon cancer
cells in vitro.
Figure 2. Effects on the viability of cancer cellsHepG2 and HCT116 as a function of LPEO concentration.
Significant decreases in cell viability of cancer cells are seen at increasing LPEO concentrations compared
to untreated controls (control group was set to 100%). **—Very significant at p < 0.01, ***—Highly
significant at p < 0.001.
Sun et al. (2002) studied antiproliferative activity of grapefruit fruit extract on the growth
of HepG2 human liver cancer cells in vitro [22]. The extract showed antiproliferative activity in a
dose-dependent manner with the median effective dose (EC50 ) value of 130.09 mg/mL. However,
they did not identify the specific phytochemicals which were responsible for antiproliferative activity.
Manassero et al. (2013) studied the antiproliferative activity of cold-pressed EO from mandarin peel
and its principal component limonene [44]. Mandarin EO and limonene exhibited IC50 of 0.063 μL/mL
and 0.150 μL/mL against HepG2 cells, respectively. The much higher activity of mandarin EO than
LPEO (0.24 μL/mL) may attributed to other high potent phytochemicals in cold-pressed EO. We have
reported antiproliferative activity of the ‘Gannanzao’ orange EO (GOEO) prepared by hydrodistillation,
which exhibited IC50 of 0.29 μL/mL and 0.35 μL/mL against HepG2 cells and HCT116 colon cancer
cells, respectively [43]. LPEO showed a slightly higher activity than GOEO, which may be attributed
to its higher limonene content (LPEO 93.33%, GOEO 88.07%).
The discussion about anticancer activity of some EO components has been made by
Mukhtar et al. [45]. Our study preliminarily tested the inhibitory effect of LPEO on the proliferation
of HepG2 liver cancer cells and HCT116 colon cancer cells. The anticancer activity of LPEO and its
components on cancer cells and their mode of action deserve further study.
3.1. Materials
Cold-pressed Marsh white grapefruit (Citrus paradisi Macf., Lakeland, FL, USA) EO was
purchased from Ungerer Limited. 2,2-diphenyl-1-picrylhydrazyl (DPPH) was purchased from
Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan), 2,2 -azino-bis(3-ethylbenzthiazoline-6-sulfonic
acid) (ABTS), n-alkanes(C8–C20)were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Butylated hydroxytoluene (BHT) was purchased from Macklin, Shanghai, China. The following
microorganisms were purchased from Beijing, China General Microbiological Culture Collection
Center (CGMCC): Escherichia coli (ATCC25922), Staphylococcus aureus (ATCC25923), Bacillus subtilis
(ATCC6633), Salmonella typhimurium (ATCC14028), and Pseudomonas aeruginosa (ATCC9207).
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3.2. Preparation of Grapefruit Light Phase EO Sample

Grapefruit light phase EO(LPEO) was obtained by molecular distillation from cold-pressed
grapefruit EO (Citrus paradisi Macf., Lakeland, FL, USA) using a wiped-film molecular distillation
apparatus (Pope Two Inch Laboratory Scale Wiped-Film Molecular Still & Evaporator, Pope Scientific
Inc., Saukville, WI, USA). The evaporation temperature and operation pressure were 55 ◦ C and 6.0 Torr,
respectively. Cold-pressed grapefruit EO was fed at room temperature and the feeding rate was 3.0
mL/min. The rotational speed of the roller wiper (Pope Scientific Inc., Saukville, WI, USA) was 325 rpm,
and the condenser temperature was 0 ◦ C. The final grapefruit EO sample (LPEO) was obtained from
the light phase outlet with the yield of 86%.
3.3. GC-MS Analyses

The constituents of LPEO were analyzed using an Agilent 7890B gas chromatograph coupled
with an Agilent mass spectrometer detector (Agilent Technologies, Santa Clara, CA, USA). The GC
was equipped with a HP-5 column (30.00 m × 0.25 mm × 0.25 μm). Mass spectra were obtained
by electron ionization (EI) at 70 eV. The injector and detector were operated at 250 ◦ C and 300 ◦ C,
respectively. The temperature program was 80 ◦ C for 4 min, and then increased at 5 ◦ C/min to 250 ◦ C
and held constant for 10 min. The constituents were identified by comparing their mass spectra with
the National Institute of Standards and Technology (NIST, version 2010, U.S. Department of Commerce,
Gaithersburg, MD, USA) data reference. The retention indices (RI) of the constituents were determined
by adding a C8–C20 n-alkanes mixture to the essential oil before injecting in the GC-MS equipment
and analyzing it under the same conditions described above.
3.4. Antimicrobial Activity Assays
3.4.1. Microbial Growth Conditions

The microbial strains were maintained in nutrient agar media at 37 ◦ C. Subsequently, one colony
from each culture was inoculated in liquid medium for 18–24 h with shaking (200 rpm) to obtain
freshly cultured microbial suspensions (>108 CFU mL−1 ) for test.
3.4.2. Determination of Diameter of the Inhibition Zone

LPEO was tested on five microbial strains, using filter paper diffusion method [46]. Briefly,
a suspension of the tested microorganism (106 CFUmL−1 ) was spread on the solid media plates.
The paper discs (Whatman No. 1 filter paper, 6 mm diameter) were impregnated with 20 μL LPEO and
placed on the inoculated agar. The plates inoculated with bacterial strains were incubated for 24 h at
37 ◦ C. After incubation, diameter of the inhibition zone was measured in millimeters. Each test was
performed in triplicates on at least three separate experiments.
3.4.3. Determination of Minimum Inhibitory Concentration (MIC)

MIC values of LPEO against microorganisms were determined by disc-diffusion method [46,47]
Sterile filter paper discs were placed on the surface of Petri dishes and impregnated with 20 μL of EO
at different concentrations (100.00, 50.00, 25.00, 12.50, 6.25, 3.125, 1.56, 0.78, 0.39, and 0.195 mg/mL) in
dimethyl sulfoxide (DMSO). DMSO alone was used as negative control. After staying at 4 ◦ C for 2 h,
all Petri dishes were incubated at 37 ◦ C for 24 h. All determinations were performed in triplicates.
The minimum inhibitory concentration (MIC) values were determined as the lowest concentration of
EOs that inhibited visible growth of the tested microorganism.
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3.5. Free Radical-Scavenging Capacity
3.5.1. DPPH Radical-Scavenging Assay

The free radical-scavenging activity of LPEO was measured using the stable radical
2,2-diphenyl-1-picrylhydrazyl (DPPH) assay [48]. DPPH was dissolved in ethanol at concentration
of 0.1 mmol L−1 . The absorbance of 2.7 mL DPPH solution and 0.3 mL ethanol was measured as the
negative control. A different concentration of the sample solution in ethanol (0.3 mL) was pipetted
into a cuvette with 2.7 mL DPPH solution. The resultant solution was incubated for 30 min at room
temperature in the dark, and then monitored at 517 nm. The DPPH scavenging activity was expressed
according to the following equation:
DPPH scavenging activity (%) = (AC − AS )/AC × 100 (1)
where AC is the absorbance of the negative control, and AS is the absorbance containing 0.3 mL sample
and 2.7 mL DPPH solution. All samples were analyzed in triplicates, and the results are expressed
as the mean ± standard deviation. The scavenging activity was expressed as the 50% inhibitory
concentration (IC50 ), which was defined as the sample concentration necessary to inhibit DPPH radical
activity by 50% after incubation.
3.5.2. ABTS Radical-Scavenging Assay

This method was performed as described by Teles et al. [49], based on the capacity of LPEO to
inhibit the 2,2 -azinobis (3-ethylbenzthiazoline-6-sulfonic acid) radical (ABTS). Twenty-five mL of
ABTS (7 mM) were added to 440 μL of potassium persulfate (K2 S2 O8 , 140 mM), and the solution was
kept in darkness for 12 h at room temperature in order to form the radical. An accurate volume of the
solution was diluted in ethanol until an absorbance of 0.70 at 734 nm. Once the radical was formed,
2 mL of ABTS solution were mixed with 100μL of LPEO and the absorbance measured at734 nm.
ABTS scavenging effect was calculated using the following equation:
ABTS scavenging activity (%) = (AC − AS )/AC × 100 (2)
where AS is the absorbance of the solution when the sample has been added and AC is the absorbance
of the ABTS solution as control. The IC50 was calculated from the graph of scavenging percentage
against LPEO concentration. The results are expressed as the mean ± standard deviation.
3.6. Cancer Cell Culture

HCT116 colon cancer cells and HepG2 liver cancer cells were purchased from Library of Typical
Culture of Chinese Academy of Sciences (Shanghai, China). HCT116 cells were cultured in Dulbecco’s
modified Eagle’s medium (DMEM; Hyclone, UT, USA), supplemented with 10% fetal bovine serum
(FBS) and 1% penicillin/streptomycin (Hyclone, UT, USA). HepG2 cells were cultured in MEM
containing 10% FBS and 1% penicillin/streptomycin (Hyclone, UT, USA). The above-mentioned cells
were maintained in 25 cm2 cell culture flasks in a humidified atmosphere containing 5% CO2 at 37 ◦ C.
Cells were fed until 90% confluence and the confluent cells were washed twice with phosphate buffered
saline (PBS), treated with 0.25% trypsin (Invitrogen, MA, USA) for about 1 min, and incubated at
37 ◦ C. When the cells were contracted and rounded under the microscope, FBS (Hyclone, UT, USA)
containing medium was added, centrifuged at 200× g for 3 min, and subcultured at a split ratio of 1:3.
3.7. Antiproliferative Activity Test of LPEO

The cell proliferation inhibition rate of LPEO was evaluated by CCK-8 assay [42,43]. LPEO (50 μL)
was added to the medium and mixed well. The mixture was diluted in DMSO to prepare solutions
at a concentration of 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, and 0.0 μL/mL, respectively. The cells were placed
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into 96-well plates (3 × 103 cells/well). After 24 h, 100 μL of LPEO at different concentrations was
added and continued to incubate for 48 h at 37 ◦ C in a CO2 incubator, after which the medium in the
96-well plate was disposed. A 100 μL of CCK-8 test solution (DojinDo, Tokyo, Japan) was added and
incubated for 2 h at 37 ◦ C. The optical density (OD) for each well was measured at 450 nm using a
microplate reader (BioTek, Winooski, VT, USA). The cell viability rate at different concentrations of
LPEO treatment was calculated according to the formula:
Viability rate (%) = (ODsample − ODblank )/(ODcontrol − ODblank ) × 100% (3)

The mean and standard deviation of three experiments were determined. Statistical analyses of
the differences between mean values obtained for experimental groups were calculated using IBM SPSS
Statistics 23.0. (IBM Corp. Released 2015. IBM SPSS Statistics for Windows, Version 23.0. Armonk, NY,
USA). p values < 0.05 were regarded as significant, p values < 0.01 as very significant and p values <
0.001 as highly significant.
4. Conclusions
Essential oils are valuable plant extracts used in food, medicine and complementary treatment
strategies [41]. The beneficial role of grapefruit EO has been widely reported. However, the bioactivities
of grapefruit EO prepared by molecular distillation has not been well studied. Molecular distillation is a
very useful technique to separate thermally-sensitive EOs. In our study, molecular distillation was used
to remove undesired components from the cold-pressed grapefruit EO to provide light phase EO (LPEO).
The chemical composition and antimicrobial activity of LPEO were studied. LPEO showed a wide
spectrum of antimicrobial activity against some Gram-positive and Gram-negative microorganisms,
with MIC values ranging from 0.78 to 12.50 μL/mL. LPEO might be used as a novel antimicrobial
agent in the food industry. The antioxidant activity of LPEO by DPPH and ABTS was obtained with
IC50 values of 22.06 ± 0.92 mg/mL and 15.72 ± 0.32 mg/mL, respectively. An in vitro test showed
a dose-dependent antiproliferative activity of LPEO on HepG2 and HCT116 cancer cells. Thus,
LPEO may potentially be used as a new complementary anticancer agent. However, this still needs
further studies.
Author Contributions: J.C. and B.Z. contributed to the conception and design of the study and data analysis;
W.D., K.L., J.S., and J.C. collected the plant material and performed distillation and GC-MS analysis; K.L. and
S.C. carried out bioactivity test; W.D. and J.C. wrote the manuscript. All authors have read and agreed to the
published version of the manuscript.
Funding: This work was supported by Foundation of Jiangxi Province Educational Committee (KJLD14079),
Natural Science Foundation of Jiangxi Province (20141BBG70002), and Innovation Team Plan of Jiangxi Province
(20142BCB24007).
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288
molecules
Article
Antimicrobial Activity and Chemical Constitution of
the Crude, Phenolic-Rich Extracts of
Hibiscus sabdariffa, Brassica oleracea and
Beta vulgaris
Seham Abdel-Shafi 1, *, Abdul-Raouf Al-Mohammadi 2 , Mahmoud Sitohy 3 , Basma Mosa 1 ,
Ahmed Ismaiel 1 , Gamal Enan 1 and Ali Osman 3
1 Botany and Microbiology Department, Faculty of Science, Zagazig University, Zagazig 44519, Egypt;
shefaalab1984@gmail.com (B.M.); ahmedismaiel80@gmail.com (A.I.); gamalenan@ymail.com (G.E.)
2 Department of Science, King Khalid Military Academy, Riyadh 11495, P.O. Box 22140, Saudi Arabia;
almohammadi26@hotmail.com
3 Biochemistry Department, Faculty of Agriculture, Zagazig University, Zagazig 44511, Egypt;
mzsitohy@hotmail.com (M.S.); ali_khalil2006@yahoo.com (A.O.)
* Correspondence: hegazyseham@yahoo.com or sahegazy@zu.edu.eg; Tel.: +20-1289600036

Received: 12 November 2019; Accepted: 22 November 2019; Published: 24 November 2019
Abstract: Crude, phenolic-rich extracts (CPREs) were isolated from different sources, such as Hibiscus
sabdariffa (H. sabdariffa), Brassica oleracea var. capitata f. rubra (B. oleracea) and Beta vulgaris (B. vulgaris)
and characterized. These CPREs showed potential antibacterial and antifungal activities. H. sabdariffa
CPRE (HCPRE) is the most potent, as it inhibited all tested bacteria and fungi. Total anthocyanins
content (TAC), total phenolic content (TPC) and total flavonoid content (TFC) were estimated in all
three CPREs. H. sabdariffa contained 4.2 mg/100 g TAC, 2000 mg/100 g of TPC and 430 mg/100 g of TFC
in a dry weight sample. GC–MS analysis of HCPRE showed 10 different active compounds that have
antimicrobial effects against pathogenic bacteria and fungi, especially alcoholic compounds, triazine
derivatives and esters. Scanning and transmission electron microscopy images of Staphylococcus
aureus DSM 1104 and Klebsiella pneumonia ATCC 43816 treated with HCPRE (50 μg/mL) exhibited signs
of asymmetric, wrinkled exterior surfaces, cell deformations and loss of cell shapes; and adherence
of lysed cell content led to cell clumping, malformations, blisters, cell depressions and diminished
cell numbers. This indicates death of bacterial cells and loss of cell contents. Aspergillus ochraceus
EMCC516 (A. ochraceus, when treated with 100 μg/mL of HCPRE showed irregular cell organelles
and cell vacuolation.
Keywords: Hibiscus sabdariffa; Brassica oleracea; Beta vulgaris; crude phenolic rich extract; anthocyanins;
GC–MS analysis; antimicrobial
1. Introduction
The demand for effective natural antimicrobial compounds free of toxicity and environmental
hazards has enormously increased as a result of the mounting increased drug resistant bacteria,
nullifying drugs’ effectiveness and causing widespread infections [1]. To avoid the increasingly
growing antibiotic resistance, many natural products such as native or modified proteins have been
investigated for their antibacterial actions as possible substitutes for the antibiotics [2–13]. Pathogenic
bacteria and fungi affect agriculture, food industry, consumers and the national economy. The safe,
plant-derived compounds with antimicrobial activity against pathogens are vital. For instance,
carvacrol and cinnamaldehyde reduced Campylobacter jejuni and Salmonella enterica to undetectable

Molecules 2019, 24, 4280
levels at 0.2% concentration. The native cowpea seed proteins 7S and 11S were reported to strongly
inhibit the in vitro growth of Pseudomonas aeruginosa ATCC 26853 and Salmonella typhimurium ATCC
14028 [14]. Additionally, soybean’s glycinin basic subunit was able to inhibit methicillin-vancomycin
intermediate Staphylococcus aureus (MRSA-VISA) while soy glycinin was competent to impede Bacillus
spore germination [15,16].
Anthocyanins are the most important group of water-soluble pigments in nature. The word
“Anthocyanin” is derived from two Greek words ‘anthos’ meaning flower and ‘kyanos’ meaning dark
blue, referring to its important role as a natural colorant [17,18]. Anthocyanins are the polyphenolics
that are responsible for red to purple color in plants. They are members of flavonoid group of
phytochemicals [19,20]. Primary constituents that are present in flavonoid group are anthocyanins,
flavanols, flavones, flavanones, etc. Anthocyanins are the hydroxyl and methoxyl derivatives of
phenyl-2-benzopyrylium salts, regarded as flavonoid compounds [21].
The previous study reported two major anthocyanins, delphinidine-3-sambubioside and
cyanidine-3-sambubioside, and two minor compounds, i.e., delphinidine-3-glu-coside and
cyanidine-3-glucoside, present in the calyces of Hibiscus sabdariffa (roselle) [22]. Approximately 85%
of anthocyanins were delphinidine-3-sambubioside which is the principal source of the antioxidant
capacity of roselle extract [23].
The phenolic structure of anthocyanin stands behind their antioxidant activity; i.e., their capability
to scavenge reactive oxygen species (ROS); i.e., superoxide (O2 − ), singlet oxygen (O2 ), peroxide (ROO),
hydrogen peroxide (H2 O2 ), and hydroxyl radicals (OH) [24]. The herbs’ antioxidant activities may be
attributed to the plant pigments constituting the major components of the herbal extract. Antioxidant
assays in foods and biological systems can be classified in two groups, those based on the evaluation of
lipid peroxidation, and those based on the measurement of free radical scavenging power [25,26].
Roselle is widely used for treating diseases. The aqueous methanolic extract of roselle was
analyzed for its phytochemical constituents, antimicrobial activity and cytotoxicity, revealing the
following components, cardiac glycosides, flavonoids, saponins and alkaloids. It exhibited in vitro
antibacterial activities against Staphylococcus aureus, Bacillus stearothermophilus, Micrococcus luteus,
Serratia mascences, Clostridium sporogenes, Escherichia coli, Klebsiella pneumoniae, Bacillus cereus and
Pseudomonas fluorescence [27].
The in vitro antimicrobial action of roselle extract was ascribed to the flavonoids, which
can establish complexes with the bacterial cell walls, enhancing their permeation to the extract.
The mechanism of action may include some metabolic steps, e.g., inhibition of electron transport
protein translocation, phosphorylation steps, and some other enzyme dependent reactions ending with
raised membrane permeability coupled with the leakage of the bacterial cell constituents [28].
Red cabbage (Brassica oleracea L.) has been extensively studied, due to its distinct color and
potential physiological functions, arising probably from the presence of anthocyanin [29], the major
pigment of this plant [30], which is composed of cyanidin-3-diglucoside-5-glucoside “cores,” that are
non-acetylated, mono-acetylated or di-acetylated with p-coumaric, caffeic, ferulic and sinapic acids.
Anthocyanin was previously extracted from red cabbage using high pressure CO2 [31]. Red cabbage
is one of the most important vegetables belonging to the family Cruciferae. It is an herbaceous plant
characterized by a short stem crowned up with a mass of red leaves (head). It is mainly used as salad,
but can be cooked or pickled. Red cabbage is known for its medicinal properties; e.g., anticancer
activity, due to the presence of indole-3-carbinol. It is an excellent source of vitamin C, vitamin B
complex, potassium and calcium. The purple/red color leaves are due to a pigment belonging to
anthocyanins (flavins). This color varies according to the soil pH, being more reddish in acidic soils,
purple in neutral soils and greenish yellow in alkaline soil. Red cabbage is a rich source of natural
antioxidants such as ascorbic acid, α-tocopherol, β-carotene and lutein [32]; oligosaccharides; and a
some bioactive substances; e.g., flavonols and glucosinolates [33]. Its wide spread use in traditional
medicine were ascribed to its antioxidant, anti-inflammatory and antibacterial properties. It is used
290
Molecules 2019, 24, 4280
for treating symptoms associated with gastrointestinal disorders; e.g., peptic and duodenal ulcers,
gastritis or irritable bowel syndrome [34].
Natural colorants may be promising active biological agents. For example, phycocyanins were
found to have many biological activities [35–37]. Likewise, red beet (Beta vulgaris L.) grows red or purple
tuberous root vegetables, known as beetroot or garden beets, which are a firm, clean, globe-shaped
vegetable with no mucilaginous or watery tissues, and its tubers contain freshly emerged young leaves.
The biological importance of red beet is based on its high red pigment content, (betalain), which
displays excellent values, meeting some applications in food and pharmaceutical products. Among
many plants accumulating betalains, only red beet and prickly pear (Opuntia ficus-indica) are approved
for food and pharmaceutical applications [38]. For example, the use of beet extract as a food colorant
is approved by the US Food and Drug Administration (FDA). As a powerful antioxidant pigment,
betanin may provide protection and reduce risk of cardiovascular disease and cancer [39].
Based on the potentially high content of anthocyanins and other bioactive compounds, three plants
growing in Egypt were selected for this study; Hibiscus sabdariffa (H. sabdariffa), Brassica oleracea
(B. oleracea) and Beta vulgaris (B. vulgaris), as the sources for isolating the crude phenolic rich extract
(CPRE). These extracts (CPRE) were analyzed for total phenolics content and total flavonoids and
evaluated for their antibacterial and antifungal activity by different methods.
2. Results
2.1. Chemical Characterization of Isolated CPRE
2.1.1. Total Anthocyanin, Total Phenolic and Total Flavonoid Contents

Total phenolic contents (TPC) of all samples were assayed by Folin–Ciocalteu’s method, and
found to be varied (Table 1). The highest amount of TPC was observed in H. sabdariffa CPRE (HCPRE)
(2000 mg GAE/100 g dry pigment). B. oleracea showed the lowest amount of TPC (150 mg GAE/100 g dry
pigment). The highest amounts of anthocyanin and flavonoid contents were observed with HCPRE.
Table 1. Chemical characterization of isolated pigments.
Total Anthocyanin Content Total Phenolic Content (mg Total Flavonoid Content (mg
Samples
(mg/100 g Dry Pigment) GAE/100 g Dry Pigment) QE/100 g Dry Pigment)
H. sabdariffa 4.2 2000 430
B. oleracea 2.7 150 50
B. vulgaris 3.8 400 120
GAE: gallic acid equivalent. QE: quercetin equivalent.
2.1.2. Gas Chromatography-Mass Spectrometry (GC–MS) Analysis of HCPRE

The chemical compounds extracted from HCPRE (2000 μg/mL) were obtained by GC–MS analysis
(Table 2; Figure 1). HCPRE contains ten active compounds, most of which have antimicrobial effects
against pathogenic bacteria and fungi, especially alcoholic compounds, triazine derivatives, mercepto
compounds and esters.
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Table 2. The chemical compounds in the Hibiscus sabdariffa pigment (crude, phenolic-rich
extracts—HCPRE) extracted, analyzed by GC–MS.
No Classification M. Formula M. W. Compound Name and Structure

Hydrocarbons
1 (Alkan)-saturated C22 H66 450 CH3 (CH2 )3 OCH3 —Dotriacontan
compounds
Alcoholic
2 C17 H36 O 256
componds
+1 $PLQRFKORUR1HWK\ODPLQH
7ULTD]LQH

Triazine 1 1
3 C5 H8 ClN5 173
derivatives
&O 1+
1

Unsat. alcoholic
4 C19 H38 O 280
compound
14-Methyl-2,15-octadecadien-1-OL
5 Unsaturated ester C17 H22 O2 268
7-Methyl-2-tetradecan-1-OL acetate
Mercepto
6 C16 H34 S 258
compound
1,1-Dimethyl tetradecyl hydrosulfide or tert-headecanethiol
(com.)
7 Alkenes C19 H38 266
1-Nonadecene or Monadeca-1-ene
8 Primary alcohols C37 H76 O 536
1-Heptalriaontanol or Heptatricotanol
9 Unsaturated ester C17 H30 O2 266
7-Methyl hexadeca-3,8-dienoate
Natural product
10 C28 H48 O 400
(Cholesterol)
2-Methylene cholestan-3-oL
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Molecules 2019, 24, 4280
57 60 %

1/

(
7,&06
2OGBNDUNDG
LK

5HODWLYH$EXQGDQFH

7LPH PLQ
Figure 1. The TIC chromatogram of H. sabdariffa using GC–MS. RT—retention Time; SM—signal in
method; NL—noise level.
2.2. Antimicrobial Activity of Crude Phenolic Rich Extract (CPRE) (2000 μg/mL) against Pathogenic Bacteria
The CPREs from H. sabdariffa, Brassica oleracea var. capitata f. rubra and B. vulgaris
(2000 μg/mL) were tested for their antibacterial actions against S. aureus, Streptococcus pyogenes,
Listeria monocytogenes, E. coli, K. pneumonia and Pseudomonas aeruginosa (Table 3). HCPRE
exhibited the highest inhibition zones against the all bacteria, but B. vulgaris pigment had lower
inhibition zones.
Table 3. Antibacterial activity of crude phenolic rich extracts (CPREs) (2000 μg/mL) from three plants
against pathogenic bacteria using agar well diffusion assays.
Inhibition Zone (mm)

Microorganisms
H. sabdariffa B. oleracea B. vulgaris
Gram positive bacteria
S. aureus 48 ± 8.0 38 ± 3.0 a 24 ± 4.0 a
St. pyogenes 40 ± 5.0 b 32 ± 2.0 b 10 ± 2.0 b
L. monocytogenes 32 ± 4.0 c 37 ± 3.0 a 13 ± 3.0 b
Gram negative bacteria
E. coli 46 ± 6.0 a 28 ± 0.50 c 7 ± 0.50 c
K. pneumonia 48 ± 6.5 a 30 ± 2.0 b 18 ± 2.0 a
P. aeruginosa 32 ± 3.0 c 29 ± 0.50 c 7 ± 0.50 c
Means in the same column having different letters are significantly different (p ≤ 0.05).
2.3. Minimum Inhibitory Concentrations (MICs) Values of HCPRE and B. oleracea Pigments against Bacteria
Different concentrations of extracted HCPRE were prepared (0, 25, 50, 100, 200 and 250 μg/mL)
and tested for their antibacterial action (Table 4, Figure S1). The results indicated that the MIC of the
pigment against Gram positive S. aureus, S. pyogenes and L. monocytogenes was 50 μg/mL; the MIC
against Gram negative E. coli and P. aeruginosa was 25 μg/mL; and it was 50 μg/mL for K. pneumoniae.
S. aureus and K. penuomonia are the most sensitive bacteria to HCPRE. Different concentrations of
extracted B. oleracea pigment (BOP) were prepared (0, 25, 50, 100, 200 and 250 μg/mL) and tested for
their antibacterial action against pathogenic bacteria (Table 4). The results showed that the MICs of the
BOP against S. aureus, S. pyogenes and L. monocytogens were 25,100 and 50 μg/mL; and against E. coli,
K. pneumonia and P. aeruginosa were 100, 200 and 100 μg/mL respectively. S. aureus and P. aeruginosa are
the most sensitive bacteria to BOP.
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Molecules 2019, 24, 4280
Table 4. Minimum Inhibitory Concentrations (MIC) values of HCPRE and Brassica oleracea pigments
against pathogenic bacteria using agar well diffusion assays.

Microorganisms
0 25 50 100 200 250
H. sabdariffa pigment
S. aureus −ve −ve 15 ± 3.0 c 20 ± 2.0 b 23 ± 3.0 b 28 ± 4.0 a
St. pyogenes -ve −ve 9 ± 0.5 c 15 ± 1.0 b 20 ± 2.0 a 22 ± 2.0 a
L. monocytogenes −ve −ve 11 ± 1.5 c 22 ± 3.0 b 23 ± 3.0 b 26 ± 3.0 a
E. coli −ve 11 ± 1.5 c 14 ± 2.0 c 20 ± 2.0 b 24 ± 4.0 b 30 ± 5.0 a
K. pneumonia −ve −ve 15 ± 3.0 c 20 ± 2.0 b 25 ± 5.0 a 26 ± 3.0 a
P. aeruginosa −ve 10 ± 1.0 14 ± 2.0 23 ± 3.0 29 ± 6.0 30 ± 4.0
B. oleracea pigment
S. aureus −ve 9 ± 1.0 c 12 ± 2.0 c 16 ± 2.0 b 21±2.0 a 25±3.0 a
St. pyogenes −ve −ve −ve 9 ± 0.5 b 10±1.0 a 11±1.0 a
L. monocytogenes −ve −ve 9 ± 1.0 c 12 ± 1.5 c 23±3.0 b 26±4.0 a
E. coli −ve −ve −ve 10 ± 1.0 b 16 ± 2.0 a 17 ± 2.0 a
K. pneumonia −ve −ve −ve −ve 11 ± 1.0 b 13 ± 1.5 a
P. aeruginosa −ve −ve −ve 11 ± 1.2 c 15 ± 2.0 b 19 ± 2.5 a
Means in the same row having different letters are significantly different (p ≤ 0.05).
2.4. Antifungal Activity of HCPRE against Pathogenic Fungi and MIC Values
HCPRE (2000 μg/mL) strongly inhibited all tested fungi (Table 5). Different concentrations of
HCPRE extract were prepared (0, 100, 200, 300, 400 and 500 μg/mL) and tested for their antifungal
actions against pathogenic fungi. The results showed that the MICs of the pigment against the fungi
(A. ochraceus, F. oxysporum, P. expansum and P. citrinum) were 100 μg/mL, and according to the diameter
of inhibition zones it showed that A. ochraceus was the most sensitive fungus to HCPRE (Table 6;
Figure S2). Moreover, different concentrations of extracted B. oleracea pigment were prepared (0, 100,
200, 300, 400 and 500 μg/mL) and tested for their antifungal actions against pathogenic fungi.
Table 5. Antifungal activities of some plant pigments (2000 μg/mL) against pathogenic fungi using
well diffusion assays.

Microorganismis
H. sabdariffa B. oleracea B. vulgaris
A. ochraceus 45 ± 5.0 a 20 ± 2.0 b −ve
F. oxysporum 40 ± 4.0 b 22 ± 3.0 a −ve
P. expansum 35 ± 3.0 c −ve −ve
P. citrinum 36 ± 3.2 c −ve −ve
Means in the same column having different letters are significantly different (p ≤ 0.05). −ve: No inhibition zone.
The results showed that the MICs of BOP against A. ochraceus and F. oxysporum were 400 and
300 μg/mL, respectively. BOP do not inhibit the growth of P. expansum and P. citrinum (Table 6,
Figure S2).
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Table 6. MICs of H. sabdariffa and B. oleracea pigments against pathogenic fungi using well diffusion assay.

Microorganisms
0 100 200 300 400 500
H. sabdariffa pigment
A. ochraceus −ve 23 ± 2.0 c 30 ± 3.0 bc 34 ± 3.0 b 35 ± 3.0 b 40 ± 5.0 a
F. oxysporum −ve 12 ± 1.0 d 15 ± 1.5 c 20 ± 2.0 b 25 ± 2.0 a 26 ± 2.0 a
P. expansum −ve 9 ± 0.5 c 20 ± 2 b 21 ± 2.0 b 28 ± 2.5 a 32 ± 3.0 a
P. citrinum −ve 13 ± 1.5 d 25 ± 3.0 c 32 ± 3.0 b 33 ± 3.0 b 38 ± 4.0 a
B. oleracea pigment
A. ochraceus −ve −ve −ve −ve 13 ± 2.0 b 15 ± 1.0 a
F. oxysporum −ve −ve −ve 9 ± 1.0 c 11 ± 1.0 b 18 ± 2.0 a
P. expansum −ve −ve −ve −ve −ve −ve
P. citrinum −ve −ve −ve −ve −ve −ve
−ve: No inhibition zone. Mean in the same row having different letters are significantly different (p ≤ 0.05).
2.5. Quantitative Inhibition of Pathogenic Bacteria by Plant Pigments (Bacterial Growth Curve)
Plant pigments (H. sabdariffa and B. oleraceae) were added at their MIC values to test tubes
containing 10 mL NB and inoculated with 10 μl aliquots of bacterial suspensions. Samples and
untreated test tubes (controls) were incubated at 37 ◦ C for 30 h. At appropriate time intervals, 1 mL
aliquots of bacterial suspensions were withdrawn and were analyzed for their turbidity at OD600.
Results are given in (Figure 2).
In the case of treating with H. sabdariffa pigment, almost no growth was shown in bacterial test
tubes treated with pigment. However, bacteria grew rapidly in control tubes (without pigment) and
turbidity went from 0.1 to almost 1.2 at OD600. Distinctive inhibition was observed at OD600, which
increased only ≥0.0 in all of them. Moderate inhibition was observed for growth recorded at OD600
within 30 h in contradicting situations. In case of BOP bacteria grew rapidly in control tubes and
bacterial growth inhibited in treated tubes.
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Figure 2. Quantitative inhibition of Gram-positive and Gram-negative bacteria by MIC of H. sabdariffa

crude phenolic rich extract. (A) S. aureus; (B) E. coli; (C) St. pyogenes; (D) K. pneumonia;
(E) L. monocytogenes; (F) P. aeruginosa.
2.6. SEM and TEM Microscopy Analysis

SEM images showed that the presence of HCPRE (50 μg/mL) on NB media containing S. aureus
affected the bacterial cells and caused cell deformations, wrinkles and loss of cell shapes. The adherence
of lysed cell content led to cell clumping, and this was seen after 18 h of incubation at 37 ◦ C. HCPRE
(50 μg/mL) on nutrient broth (NB) media containing K. pneumonia showed malformations (increases in
length and decrease in width), cell depressions, diminished cell number and observed rectangular
cells as loss of regular cell shapes were detected. That indicates the death of cells and loss of cellular
contents. SEM images of A. ochraceus treated with HCPRE (100 μg/mL) showed destruction of conidia,
failing in conidia formation, thinning and condensation of mycelia; then, malformation and loss of
cell contents.
TEM images showed that S. aureus and K. pneumonia affected by HCPRE (50 μg/mL) on NB media
showed malformed shapes, cell depress, cell vacuolation, blisters and wrinkles.
A. ochraceus, when treated with (100 μg/mL) of HCPRE showed irregular cell organelles and cell
vacuolation (Figure 3A–F).
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(A)
(B)
(C)
(D)
Figure 3. Cont.
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(E)
(F)
Figure 3. (A) SEM of Staphylococcus aureus affected by 50 μg/mL of HCPRE. (B) TEM of S. aureus
affected by 50 μg/mL of HCPRE. (C) SEM of K. pneumonia affected by 50 μg/mL of HCPRE. (D) TEM of
K. pneumonia affected by 50 μg/mL of HCPRE. (E) SEM of A. ochraceus affected by 100 μg/mL of HCPRE.
(F) TEM of A. ochraceus affected by 100 μg/mL of HCPRE.
3. Discussion
Natural colorants obtained from vegetables are more available and healthy than synthetic
colors [40]. The natural pigments are used in medicine and food [41]. Many bacterial organisms have
developed increasing resistance against the frequently used antibiotics [42].
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In this study, the pigments extracted from H. sabdariffa inhibited all tested bacteria and fungi.
The previous studies showed that H. sabdariffa inhibited S. aureus, B. cereus, E. coli, Clostridium sp.,
Klebsiella pneumonia and Pseudomonas fluorescens [42]. Herbal drug formulations composed of medicinal
plants have been inherited from ancient times to treat many diseases, since their antimicrobial properties
suggest them as potentially rich sources of various potent drugs [43]. Natural antimicrobials have
enormous therapeutic potential, since they can probably conduct the required functions without any
posing health hazards often associated with synthetic agents [44]. H. sabdariffa’s aqueous extract has
strong activity against C. albicans [27]. Roselle can be utilized either as a distinct functional food or
as an active ingredient in other functional food potentially applicable in the treatment of various
degenerative diseases [45].
Based on the results, the antibacterial action of anthocyanin was concentration-dependent. HCPRE
contains total anthocyanin content of 4.2 (mg/100 g) in dry pigment. Anthocyanin had relatively higher
antibacterial activity than antifungal activity against the microorganisms investigated. Anthocyanins
were reported to have anticarcinogenic activity against multiple cancer cell lines in vitro and in vivo
tumor types [46]. H. sabdariffa showed antimicrobial activities against some food pathogenic microbial
isolates, e.g., E. coli O157:H7, Salmonella enterica and L. monocytogenes, as well as veterinary, and clinical
isolates. This indicated that HCPRE extract is broadly effective against different microorganisms,
suggesting its application as a potential food-grade antimicrobial [28]. The antibacterial effects of
roselle calyx aqueous and ethanol extracts and protocatechuic acid against food spoilage bacteria
Salmonella typhimurium DT104, E. coli O157:H7, L. monocytogenes, S. aureus and B. cereus were examined
by [47]. The inhibitory activities in a dose-dependent manner against bacteria in ground beef and
apple juice were studied, and it was suggested that they might be potent agents as food additives to
prevent contamination from these bacteria.
The anthocyanins and polyphenols from the H. sabdariffa (roselle) were extracted by an aqueous or
organic solvent. The dried roselle contained total anthocyanins as cyanidine 3-glucoside 622.91 mg/100 g
and 37.42 mg/100 g total phenolic content in dry weight samples [48]. A recent study identified
delphinedine-3-O-sambubioside, delphinidine-3-O-glucoside and cyani-dine-3-O-sambubioside at the
concentrations of 7.03 mg/g, 1.54 mg/g and 4.40 mg/g in the roselle extract. GC–MS analysis showed 10
compounds in HCPRE. All of them have previously been shown to have antimicrobial activity. It is
quite known that many Hibiscus species contain different classes of secondary metabolites, including
flavonoids, anthocyanins, terpenoids, steroids, polysaccharides, alkaloids, sesquiterpene, quinones and
naphthalene groups. Some of these components have antibacterial, anti-inflammatory, antihypertensive,
antifertility, hypoglycemic, antifungal and antioxidative activities [49]. The antioxidant capacity of
anthocyanins is dependent on its basic structural orientation; i.e., the ring orientation will determine
the readiness of a hydrogen atom from a hydroxyl group to be donated to a free radical and the
capability of the anthocyanin to support an unpaired electron [25]. H. sabdariffa is a safe medicinal
plant, having medical compounds with nutritional and medicinal properties [50].
In this study, S. aureus and K. pneumonia were affected by HCPRE (50 μg/mL), showing malformed
shapes, cell depressions, cell vacuolation, blisters and wrinkles. A. ochraceus, when treated with
(100 μg/mL) of HCPRE, showed irregular cell organelles. The anthocyanin-rich blueberry extract was
capable of inhibiting the growth, adhesion and/or biofilm formation of all of the following: P. aeruginosa,
E. coli, P. mirabilis, A. baumannii and S. aureus [51]. Roselle contains proanthocyanidins which combine
or transform the structural entity of P-fimbriae of bacterial cells; thus, inhibiting their adhesion to
the ur-epithelium and formation of biofilms in vitro [45]. The antimicrobial properties of eight food
dyes against 10 bacteria and five fungal organisms were previously investigated, showing that the
red dyes were associated with the best antibacterial activities, while the yellow ones were more
linked to better antifungal activity. Besides the antimicrobial analysis, antioxidant activity, measured
by three different methods, was also investigated. In all the methods, red dye was found to have
greater antioxidant activity. It suggests that the addition of these dyes in food not only enhances
the value addition by making the food more presentable but also shall address the issue of food
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supplementation with substances that are good antibiotics and antioxidants, subsequently proving to
be health benefactors [52].
4.1. Crude Phenolic Rich Extract (CPRE) Preparation

All chemicals used in this work were supplied from Al-Gomhorya Company for chemicals and
public procurement Zagazig city, Egypt. The calyx of H. sabdariffa; the leaves of Brassica oleracea var.
capitata f. rubra (B. oleracea ) and B. vulgaris tuberous root vegetables were obtained from local market
in Egypt). The plants we used were botanically classified by Samer Teleb, Taxonomy and Flora, Botany
and Microbiology Department, Faculty of Science, Zagazig University, Egypt. Milled sample (0.5 g)
was soaked in 50 mL of ethanol for 24 h and the extract obtained was pre-filtered with Whatman
No.4 filter (Whatman®Prepleated Qualitative Filter Paper, Grade 4V, Sigma-Aldrich, USA) before
evaporation using a vacuum rotary evaporator (BüCHI-water bath-B-480, Czech Republic) at 30 ◦ C.
4.2. Crude Phenolic Rich Extract Characterization
4.2.1. Determination of Anthocyanins

Total anthocyanin content was colorimetrically determined according to the procedure described
by [53] where a known volume of the filtered extract was diluted to 100 mL with the extracting solvent
and the resulting color was measured at 520 nm for water and citric acid solution extracts and at
535 nm for acidified ethanol using Spectrophotometer (JENWAY-6405 UV/VIS, Chelmsford, England).
The total anthocyanin content defined as cyanidin-3-glucoside was calculated using the following
Equation (1):

mg Absorbance × dilution factor
Total anthocyanins = × 100. (1)
100 g Sample weight × 55.9
4.2.2. Determination of Total Phenolic Compounds (TPCs)

The TPCs were estimated by Foline–Ciocalteu reagent as described by [54]. One milliliter of
sample (1000 μg in 1 mL) was added to 5 mL of Folin–Ciocalteu reagent (diluted with water 1:10, v/v)
and 4 mL sodium carbonate (75 g/L). The tubes were vortex mixed for 15 s and left stand 30 min at
40 ◦ C, before measuring the absorbance of the developed color at 765 nm. Gallic acid was used to
establish the standard curve (20–200 μg/ mL). The extent of reducing of the Folin–Ciocalteu reagent
by the sample was expressed as mg of gallic acid equivalents (GAE) per g of extract. The calibration
equation for gallic acid was y = 0.001x + 0.0563 (R2 = 0.9792), where y and x are the absorbance and
concentration of gallic acid in μg/mL, respectively.
4.2.3. Total Flavonoids (TFs) Determination

Total flavonoids (TFs) were estimated according to the protocol of [55] by blending 2 mL aliquot
of 20 g/L AlCl3 ethanol reagent with 1 mL of the extract (1000 μg in 1 mL solvent) and measuring the
developed color absorbance at 420 nm after 60 min. Quercetin was used to establish the standard curve
(20–200 μg/mL) and total flavonoid content was expressed as quercetin equivalent (QE), based on the
standard curve. The calibration equation for quercetin was y = 0.0012x + 0.008 (R2 = 0.944), where y is
absorbance and x is concentration of quercetin in μg/mL.
4.2.4. Gas Chromatography–Mass Spectrometry (GC–MS) Analysis

The chemical composition analysis of the samples was carried out using Trace GC1310-ISQ mass
spectrometer (Thermo Scientific, Austin, TX, USA) with a direct capillary column TGram negative5MS
(30 mm× 0.25 mm × 0.25 μm film thickness, Thermo Scientific, Austin, TX, USA). The column oven
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temperature was initially held at 50 ◦ C; then increased by 7 ◦ C/min increments to 200 ◦ C hold for
2 min; and the final temperature at 290 ◦ C was reached by 15 ◦ C/min increments and held for 2 min.
The injector and MS transfer line temperatures were kept at 270 and 250 ◦ C, respectively. Helium,
the carrier gas, was pumped at a constant flow rate of 1 mL/min. The solvent delay was 3 min, and 1 μL
aliquots of the diluted samples were injected automatically using an Autosampler AS1300 coupled
(Thermo Scientific, Austin, TX, USA) with GC. The ion source temperature was set at 200 ◦ C. EI mass
spectra were collected at 70 eV ionization voltages over a range of m/z 45–400 within full scan mode.
The chemical composition of the obtained components was concluded by comparing their retention
times and mass spectra with those of WILEY 09 and NIST 11 mass spectral database.
4.3. Collection of Pathogenic Bacteria and Fungi

S. aureus DSM 1104, St. pyogenes ATCC 19615, L. monocytogenes LMG10470, E. coli LMG 8223,
K. pneumonia ATCC 43816 and P. aeruginosa LMG 8029 were used. Also, pathogenic fungi such as
A. ochraceus EMCC516 were obtained from Egyptian Microbial Culture Collection (Microbiological
Resoures Center MIRCEN, Cairo, Egypt); other fungi were F. oxysporum, p. citrinium and P. expansum.
All bacteria and fungi used in this study were kindly offered by Botany and Microbiology Department
(Laboratory of Bacteriology and Laboratory of fungi), Faculty of Science, Zagazig University, Zagazig,
Egypt. Stock bacterial cultures were routinely kept at −20 ◦ C in glass beads and were sub-cultured
and propagated in brain heart infusion broth (BHIB) (Oxoid). Slope cultures were prepared fresh on
nutrient agar for every experiment [11] and stored at 4 ◦ C throughout the experimental work.
4.4. Antibacterial and Antifungal Activities of the CPREs

The antibacterial and antifungal activities of CPREs (2000 μg/mL) were tested against the
experimental pathogenic bacteria and fungi by agar well-diffusion assays [56].
4.5. MIC Values of H. Sabdariffa CPRE

Pure cultures of bacterial strains were sub-cultured on BHIB at 37 ◦ C. Each strain was spread
uniformly onto an individual plate with a sterile cotton swab. Uniform wells (6 mm diameters) were
made on nutrient agar (NA) plates using a gel puncturing tool. Aliquots (50 μL) of pigment solutions
(0, 25, 50, 100, 200 and 250 μg/mL) were placed into each well. Sterilized distilled water was considered
the negative control. After 24 h incubation at 37 ◦ C, the diameters of the inhibition zones (mm) were
measured using a transparent millimeter ruler. The pure cultures of fungal strains were sub-cultured
on yeast extract agar (YES) at 30 ◦ C. Each strain was spread uniformly onto the individual plates using
sterile cotton swabs. Wells of 6 mm diameter were similarly made on YES plates. Aliquots (50 μL) of
HCPRE and B. oleracea pigment solutions (0, 100, 200, 300, 400 and 500 μg/mL) were placed in each well.
After 4 days’ incubation at 30 ◦ C, the diameters of inhibition zones (mm) were similarly measured.
4.6. Quantitative Inhibition of Pathogenic Bacteria by CPRE (Bacterial Growth Curve)

A series of test tubes each containing 10 mL of nutrient broth (NB) were inoculated with 100 μL
of log phase bacterial suspension and were then treated with 50 μg/mL HCPRE for all bacteria;
and 200 μg/mL B. oleracea pigment for E. coli and 100 μg/mL for other bacteria. Control test tubes
contained NB with bacteria only. Samples and controls were incubated at 37 ◦ C. Growth was determined
at time 0 and after 6, 12, 18, 24, 30, 36, 42 and 48 h of incubation by the turbidity method (OD600) using
a spectrophotometer (JENWAY-6405 UV/VIS, Chelmsford, England).
4.7. Scanning and Transmission Electron Microscopy (SEM-TEM)

S. aureus (Gram positive bacteria) and K. pneumonia (Gram negative bacteria) were selected for
scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Bacteria were
grown on NB media and incubated at 37 ◦ C to reach maximum level of 106 CFU/mL. The MIC values
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of about 50 μg/mL of HCPRE were added to S. aureus and K. pneumonia plates except for controls and
incubated at 37 ◦ C for 18 h. Also A. ochraceus was grown on YSA and incubated at 30 ◦ C for 3 days to
reach the maximum level of growth and MIC value of about 100 μg/mL of H. sabdariffa pigment.
4.7.1. Scanning Electron Microscopy (SEM)

SEM (JEOL-scanning electron microscope JSM-6510 L.V SEM-JAPAN) at electron microscope
(EM) Unit, Mansoura University, Egypt was used to evaluate the morphological changes of tested
microorganisms as described in [2,16].
4.7.2. Transmission Electron Microscopy (TEM)

TEM (JEOL JEM -2100, JAPAN) at EM Unit, Mansoura University, Egypt was used to evaluate
ultrastructural changes of tested microorganisms as described in [14,57].

The collected data were tabulated and analyzed using IBM SPSS software (version 26,
IBM corporation, Chicago, IL, USA). The results were expressed as a means ± standard errors
(SEs) in either tables or figures.
5. Conclusions
According to the obtained results, it can be concluded that H. sabdariffa pigment could be used
as an antibacterial and antifungal agent. It can be efficiently and successfully used as safe, natural
products. It can be prepared with low costs.
Supplementary Materials: The following are available online.

Author Contributions: M.S., G.E., A.-R.A.-M., A.I., and S.A.-S suggested the work protocol, interpreted the
results and revised the manuscript. S.A.-S., A.I., A.O. and B.M. performed the laboratory experiments regarding
microbiological and chemical investigations and prepared the manuscript. A.-R.A.-M. financed the publication fees.
Funding: Zagazig University, Zagazig, Egypt, supported the experimental work. A.-R.A.-M. from the Department
of Science, King Khalid Military Academy, Riyadh 11495, P.O. Box 22140, Saudi Arabia, was responsible for paying
the publication fees.
Acknowledgments: The authors are indebted to Zagazig University, Egypt for facilities throughout the
experimental work, and thank Samer Teleb, Taxonomy and Flora, Botany and Microbiology Department,
Faculty of Science, Zagazig University, Egypt for the botanical classification of the plants. Also, thanks A. H.
Moustafa for his help in GC–MS analysis.
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molecules
Article
Bioadhesive Polymeric Films Based on Red Onion
Skins Extract for Wound Treatment: An Innovative
and Eco-Friendly Formulation
Cinzia Pagano 1,† , Maura Marinozzi 1,† , Claudio Baiocchi 2 , Tommaso Beccari 1 , Paola Calarco 1 ,
Maria Rachele Ceccarini 1 , Michela Chielli 1 , Ciriana Orabona 3 , Elena Orecchini 3 ,
Roberta Ortenzi 4 , Maurizio Ricci 1 , Stefania Scuota 4 , Maria Cristina Tiralti 1 and Luana Perioli 1, *
1 Department of Pharmaceutical Sciences, University of Perugia, via del Liceo 1, 06123 Perugia, Italy;
cinzia.pagano@unipg.it (C.P.); maura.marinozzi@unipg.it (M.M.); tommaso.beccari@unipg.it (T.B.);
paola.calarco@unipg.it (P.C.); chele@hotmail.it (M.R.C.); michela.chielli@virgilio.it (M.C.);
maurizio.ricci@unipg.it (M.R.); maria.tiralti@unipg.it (M.C.T.)
2 Department of Molecular Biotechnology and Health Sciences, Sect. Analytical Chemistry, Via Pietro Giuria 5,
10125 Torino, Italy; claudio.baiocchi@unito.it
3 Department of Experimental Medicine, Sect. Pharmacology, University of Perugia, P.le L. Severi 1, Bld C/4th
floor, 06132 Perugia, Italy; ciriana.orabona@unipg.it (C.O.); elena.orecchini@gmail.com (E.O.)
4 Istituto Zooprofilattico dell’Umbria e delle Marche, via G. Salvemini, 1, 06126 Perugia, Italy;
r.ortenzi@izsum.it (R.O.); s.scuota@izsum.it (S.S.)
* Correspondence: luana.perioli@unipg.it; Tel.: +39-075-585-5133 or +39-075-585-5123

Received: 29 November 2019; Accepted: 10 January 2020; Published: 13 January 2020
Abstract: The onion non-edible outside layers represent a widely available waste material deriving
from its processing and consumption. As onion is a vegetable showing many beneficial properties for
human health, a study aiming to evaluate the use of extract deriving from the non-edible outside layers
was planned. An eco-friendly extraction method was optimized using a hydroalcoholic solution as
solvent. The obtained extract was deeply characterized by in vitro methods and then formulated
in autoadhesive, biocompatible and pain-free hydrogel polymeric films. The extract, very soluble
in water, showed antioxidant, radical scavenging, antibacterial and anti-inflammatory activities,
suggesting a potential dermal application for wounds treatment. In vitro studies showed a sustained
release of the extract from the hydrogel polymeric film suitable to reach concentrations necessary for
both antibacterial and anti-inflammatory activities. Test performed on human keratinocytes showed
that the formulation is safe suggesting that the projected formulation could be a valuable tool for
wound treatment.
Keywords: onion skins extract; hydrogel; polymeric films; anti-inflammatory; antibacterial
1. Introduction
The common onion (Allium cepa L.) is a worldwide cultivated vegetable used both as food and
also in the health field for the presence of bioactive molecules mainly endowed with antioxidant
activity [1–3]. Many scientific studies on the lipid- and glucose-lowering [4–8], anti-inflammatory [9–11],
antioxidant [12–16] and antibacterial [13] properties of onion extracts have been published. Our previous
work in this area reported the pungency determination [17] and the antioxidant and immune modulatory
activities of purple-skin Rojo Duro onion extracts [18].
The non-edible outside layers (hereinafter referred as OL) are the main waste material obtained
from onion processing and consumption. Besides rich in flavonoids [19], they are odorless and can be

Molecules 2020, 25, 318
stored for a long time after the harvesting. The utilization of a biomass endowed with these positive
peculiarities fulfills the principles of circular economy and meets the need of environmentally aware
consumers [20].
The above considerations, along with the availability of onions endowed with short and traceable
production-chain, pushed us to develop an innovative study based on OL use as source of active
ingredients [21–25] for the preparation of dermatological products for wound treatment. The presented
approach is advantageous as it proposes a new formulation by using waste materials reducing the
disposal costs and the consequent environmental impact.
The work was structured as follows: (i) optimization of the extraction method according to green
chemistry principles, (ii) evaluation of the extract activity in terms of antioxidant, radical scavenging,
antibacterial, anti-inflammatory activities and cytotoxicity and (iii) formulation of the extract as an
hydrogel film followed by its activity and safety evaluation.
2. Results
2.1. Optimization of the Extraction Procedure

The first step of the study was the set-up of the best extraction conditions in terms of recovery
material, total phenol content (TPC) and antioxidant activity. On the road of biocompatibility, ultrapure
water and EtOH were chosen as unique extraction solvents and the procedure was tested both at RT
and 60 ◦ C. The obtained results (Table 1) showed that increasing the temperature the recovery yield
increased using both abs and 70% EtOH (Table 1); the presence of water in the extraction medium gave
a higher extract recovery.
Table 1. Recovery extract yield.
Extraction Conditions:
Recovery Yield (%) a
Solvent, Temperature
abs EtOH, RT b 6.4
70% EtOH, RT b 8.7
abs EtOH, 60 ◦ C 7.9
70% EtOH, 60 ◦ C 9.7
a mean of two experiments performed in duplicate. b RT room temperature.
TPC of the extracts obtained by the four different procedures was measured by the Folin–Ciocalteu
method using a calibration curve of gallic acid standard solutions [18]. The calibration curve was fully
validated for intra-day and inter-day precision and accuracy before the use (r = 0.9972). A statistically
significant difference in TPC was observed among the samples resulting from the different extraction
conditions: the use of abs EtOH increased the TPC of the corresponding extracts at both temperatures
(Figure 1A). Although abs EtOH resulted a less efficient extraction solvent in terms of yield, it gave
extracts with higher TPC than those obtained using 70% EtOH. Among the several protocols reported
for assessing the free radical-trapping capability of phytochemicals [26], the ferric ion reducing
antioxidant power (FRAP) and 2,2-diphenyl -1-picrylhydrazyl (DPPH) radical assays were chosen [27].
According to FRAP test results (Figure 1B) the highest total reducing capacity (TRC) was found in
the extract obtained at 60 ◦ C using abs EtOH. The data ranking of the four extracts was perfectly in
accordance with TPC results, hypothesizing that TRC is attributable to the polyphenolic component.
Again, the extract obtained in abs EtOH at 60 ◦ C was endowed with the best performance also in
DPPH assay (Figure 1C) [18]. The three assays therefore confirmed that the antioxidant component
was more soluble in abs EtOH than in hydroalcoholic medium (EtOH 70%) and stable up to 60 ◦ C. Due
to the coherence of the obtained data, the extract obtained by using abs EtOH at 60 ◦ C (hereinafter
referred as OLE, outside layers extract) was selected for the further studies.
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Figure 1. (A) Total phenol content (TPC) of the extracts obtained by the four different extraction
conditions. Data are expressed as mg of GA/mg of freeze-dried extract and represent the mean of six
samples, each measured in triplicate; * p ≤ 0.05 (one-way ANOVA test). (B) Total reducing capacity
(TRC) of the extracts obtained by the four different extraction conditions. Data are expressed as mg of
GA/mg of freeze-dried extract and represent the mean of six samples, each measured in triplicate. * p ≤
0.05. (C) Radical scavenging capacity (RSC) of the extracts obtained by the four different extraction
conditions. Data are expressed as mg of GA/mg of freeze-dried extract and represent the mean of six
samples, each measured in triplicate; * p ≤ 0.05 (one-way ANOVA test).
2.2. Extract Characterization
2.2.1. Fingerprint Analysis of OLE Constituents

The main OLE components were identified. The species more present were the anthocyanidine
cyanidin-3-O-(6 -malonyl-glycoside) (protonated precursor ion m/z = 535.1082) and the flavonol
quercetin (protonated precursor ion m/z = 303.0504). Two other anthocyanidines were present
(responsible together with the previously cited one of the red color of the onion) positional isomers of
cyanidin malonyl glycoside (m/z = 535.1082) and a cyanidin glycoside (m/z = 449.1081). Quercetin is
only present in glycosylated form (m/z = 465.1028) and in much less amount in diglycosilated form
(m/z = 627.1881). In Figure 2A a chromatogram of OLE monitored in full mass is reported showing
the main components identified. The UV spectra (not reported) were highly helpful in substances
identification because their shape were informative about the chemical class the various molecules
belonged to. Figure 2B shows the separation of much more polar substances performed in isocratic
conditions and monitored in ion negative mode. It can be detected that they show very short retention
times. Epigallocatechin has anti-oxidative properties whereas the other three acidic substances possess
anti-bacterial properties. A quantitative analysis for both cyanidin derivatives and quercetin was
performed using quercetin and cyanidin pure standards. The obtained results were: quercetin 9.5
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ppm, cyanidin-3-O(-6 -malonyl-glycoside) 5.2 ppm, cyanidin malonyl glycoside isomer 1.7 ppm and
cyanidin glycoside 2.1 ppm.
Figure 2. (A) Chromatographic separation of OLE constituents monitored in full mass ion positive mode
and (B) chromatographic separation of OLE constituents monitored in full mass ion negative mode.
All these substances and the other flavonoids present in smaller amounts exhibit anti-oxidative
activities combined with the anti-bacterial properties of benzoic acid derivatives.
2.2.2. Antibacterial Activity Assay

Preliminary experiments demonstrated that Staphylococcus epidermidis, Staphylococcus aureus,
Listeria innocua and Enterococcus faecalis are sensitive to OLE, for this reason Minimum inhibitory (MIC)
and minimum bactericidal (MBC) values were measured. The obtained results show the lowest MIC
and MBC values for both S. epidermidis and S. aureus therefore the most sensitive to OLE among the
investigated strains (Table 2). The values measured for S. epidermidis are the closest to the positive
control ampicillin suggesting its greater susceptibility to OLE in comparison to the other ones.
Table 2. Minimum inhibitory (MIC) and minimum bactericidal (MBC) values of OLE and the reference
antibiotic ampicillin expressed as mg/mL ± SD (n = 3).
S. epidermidis S. aureus L. innocua E. faecalis

OLE MIC 0.47 ± 0.00 0.94 ± 0.00 3.75 ± 0.00 3.75 ± 0.00
OLE MBC 0.94 ± 0.00 1.88 ± 0.00 7.50 ± 0.00 7.50 ± 0.00
Ampicillin MIC 0.13 ± 0.00 0.13 ± 0.00 0.50 ± 0.00 0.50 ± 0.00
Ampicillin MBC 0.50 ± 0.00 0.25 ± 0.00 1.00 ± 0.00 4.00 ± 0.00
2.2.3. Cytotoxicity Studies on RAW 264.7 and HaCaT Cell Lines

As macrophages play a key role in all phases of wound healing (inflammation, proliferation and
remodeling) [28], RAW 264.7 cell model was used to evaluate OLE anti-inflammatory activity. Firstly,
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in order to exclude false positives, OLE cytotoxicity on the macrophage cell line RAW 264.7 stimulated
with lipopolysaccharide (LPS, 50 ng/mL for 24 h) was investigated. By using eight two-fold dilutions
of OLE in the 0.015–2.0 mg/mL concentration range, after 24 h of incubation, it was observed that the
cell viability decreased below 75% at the concentration of 0.5 mg/mL. However, it was maintained
higher than 65% up to 1 mg/mL and dropped around 50%, with an increased fraction of dead/apoptotic
cells, at the concentration of 2.0 mg/mL (Figure 3A,B). For this reason, the concentration 2 mg/mL was
excluded from the study focusing the attention on the concentrations range of 0.015–1.0 mg/mL for
testing a potential OLE anti-inflammatory effect in LPS-treated RAW 264.7 cells.
Figure 3. (A) LPS-activated cells were treated for 24 h with OLE at the indicated concentrations. Cells
were stained using the PerCP-Annexin V and FVD 780 and analyzed by flow cytometry. Annexin
V/FVD—double negative cells (lower left quadrant) represented live cells, annexin V/FVD—double
positive cells (upper right quadrant) represented apoptotic cells and annexin V-negative/FVD-positive
cells (lower right quadrant) indicated dead cells. A representative dot plot is shown. The percentage of
viable, apoptotic and dead cells was reported in (B) for each OLE concentration. Data are the mean
percentage of two different experiments. Evaluation of OLE cytotoxicity and safety on HaCaT cell
line by (C) Trypan Blue exclusion and (D) MTT assays. Dotted lines indicate the 50% and 75% of cell
viability. ns, not significant OLE-treated versus untreated group (one-way ANOVA test).
Simultaneously, Trypan Blue exclusion assay and MTT test were carried out on human
immortalized keratinocyte cell line (HaCaT), in vitro model of stratum corneum, to evaluate OLE
safety in the same concentration range used for RAW 264.7 cells (0.015–2.0 mg/mL). The dose-response
curve obtained from Trypan Blue exclusion assay revealed that the number of cells was comparable to
the negative control until 0.5 mg/mL and slightly decreased at 1 mg/mL, reaching the 50% of viability
at 2.0 mg/mL (Figure 3C). Similar results were obtained by MTT assay. By using the same OLE
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concentration range (0.015–2.0 mg/mL), after 24 h of incubation HaCaT cells viability was around 75%
up to 1 mg/mL, decreasing below 50% at 2.0 mg/mL (Figure 3D). Overall, the cytotoxicity analyses
revealed that OLE is safe for both cell lines in the concentration range 0.015–0.5 mg/mL.
2.2.4. Anti-Inflammatory Activity

RAW 264.7 stimulation by LPS for 24 h induces NO synthase (iNOS) activation and thus NO release
in the culture supernatant [29], detectable by using Griess reaction. The incubation of LPS stimulated
RAW 264.7 with OLE for 24 h induces a significant decrease of NO release in the concentration range
0.125–1.0 mg/mL (Figure 4A). The obtained concentration curve provided an IC50 = 0.230 ± 0.022
mg/mL for the down-regulation of NO release by OLE (Figure 4B). The release of pro-inflammatory
cytokines, IL-6 and IL-1β in the same cell system was analyzed as reported in the literature [30,31].
It was observed a similar inhibitory effect exerted by OLE on the production of both cytokines in
LPS-treated RAW 264.7 macrophages. Specifically, both IL-6 and IL-1β were significantly inhibited
by OLE starting from the concentration of 0.030 mg/mL (Figure 4C,D). The inhibitory effect on IL-6
production was concentration-dependent, with an IC50 = 0.090 ± 0.008 mg/mL (Figure 4E). Differently,
the inhibitory effect on IL-1β production showed a concentration-dependence in the range 0.030–0.25
mg/mL, with an IC50 = 0.054 ± 0.002 mg/mL (Figure 4F). For higher concentrations (0.5–1.0 mg/mL) a
decreased inhibitory effect on IL-1β production was observed, probably due to the activation of the
inflammasome in increased apoptotic RAW 264.7 cells [32,33].
Figure 4. (A) LPS-activated RAW 264.7 cells were in vitro stimulated using different OLE concentrations
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for 24 h. NO release in the supernatant culture was quantified by using Griess reagent. Results are
reported as mean ± SD of three independent experiments, each conducted in triplicate. (B) *** p <
0.0001, OLE–treated versus LPS-treated group (one-way ANOVA test). Concentration-response curve
was obtained for the determination of the IC50 . Results are reported as mean of two independent
experiments, each conducted in triplicate. LPS-activated RAW 264.7 cells were in vitro stimulated with
different concentrations of OLE for 24 h. Supernatants were collected and the concentrations of IL-6 and
IL-1β were determined by ELISA test (C,D). ** p < 0.001, *** p < 0.0001, OLE-treated versus LPS-treated
group (one-way ANOVA test). Concentration-response curves were obtained for the determination of
the IC50 . For each curve results are reported as mean of two independent experiments, each conducted
in triplicate (E,F).
Overall, OLE inhibited the production of inflammatory mediators in LPS-treated RAW264.7 cell
line with IC50 lower than the highest cytotoxic concentration (i.e., 0.5 mg/mL).
2.3. Hydrogel Film Preparation

After these preliminary studies on pure OLE, a suitable dosage form was developed and
characterized. The antioxidant, anti-inflammatory and antibacterial activities observed for pure OLE
suggested that it could be a suitable active ingredient for wounds treatment. The wound healing
process is a natural post-trauma repairing course rather complex and sensitive; its interruption can
lead to the formation of non-healing chronic wounds. A chronic wound, in fact, can be defined as
a lesion in which the normal healing process has been interrupted at one or more points during the
phases of hemostasis, inflammation, proliferation and remodeling of the wound [34]. Diabetes, venous
or arterial disease, infections, metabolic deficiencies, oxidative phenomena and inflammation are the
main factors contributing to non-healing chronic wounds [35].
In order to exploit all the OLE biological activities a formulation suitable to be applied on severely
injured skin was developed. This formulation should (i) auto-adhere to skin (without the aid of glue),
(ii) protect the wound from mechanical solicitations, (iii) avoid occlusion and pain and (iv) be easily
removable and able to promote a sustained OLE release.
In order to reach this objective, OLE was formulated in a hydrogel film for potential dermal
applications. The formulation was projected and developed based on the following requirements:
(i) biocompatibility, (ii) autoadhesivity to skin/wound, (iii) easy and pain-free removal (atraumatic
removal) and (iv) an easy and scalable manufacturing method. The hydrogel films were prepared
starting from hydrogels whose composition was optimized referring to a previous study [36] by
using NaCMC (2%) and PVP K90 (0.1%) as bioadhesive polymers [37,38]. Many modifications in
the composition were tested in order to find the most suitable for the hydrogel film preparation
(Table S1). Differently from the previously developed films [36], bentonite nanoclay was introduced
in the composition as filler [39] to improve the hydrogel film mechanical properties. A preliminary
selection was made based on the following criteria: (i) hydrogel aspect (homogeneity and consistency)
and physical stability, (ii) easy casting (difficult for very viscous gels) and (iii) final film appearance
(detection of visible imperfection under visual inspection). Hydrogel 1 showed low consistency and
instability due to bentonite particles sedimentation after 24 h from the preparation.
This phenomenon was ascribed to the low hydrogel viscosity and for this reason three new
hydrogels (2, 3 and 4) were prepared with an increased amount of bentonite (Table S1) well known as
rheological modifier agent [40]. The idea was that the increase of bentonite content could improve
the hydrogel viscosity and thus its stability. Since, the sedimentation phenomenon was still observed,
the amount of bentonite was fixed at 4.0% and NaCMC content increased until 3.0% (hydrogel 5,
Table S1). These modifications allowed us to obtain a stable hydrogel, but very viscous and thick
making difficult the successive casting procedure. To solve this problem hydrogel 6 was then prepared,
having the same composition of hydrogel 1, with an increased NaCMC content (3.0%). The obtained
hydrogel showed stability and suitable consistency to allow an easy casting.
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2.4. Hydrogel Film Characterization
2.4.1. Hydrogel Film Antibacterial Activity

Starting from hydrogel 6 composition (Table S1), three different hydrogel films were prepared
loaded with 1.0, 3.0 or 5.0% w/w of OLE (Table S2). The corresponding hydrogel films were submitted
to antibacterial activity studies in order to evaluate their ability to reach OLE concentrations necessary
active against bacteria. Thus, the hydrogel films were evaluated against the same bacterial strains
resulted sensitive to the unformulated OLE: S. epidermidis, S. aureus, L. innocua and E. faecalis. Inhibition
halos of different sizes were generated by the three different films on the analyzed bacterial strains as
reported in Table 3. Specifically, the hydrogel film B1 produced inhibition zone just for S. epidermidis
and S. aureus, while B2 and B3 hydrogel films, containing OLE in higher amount, produced similar
inhibition halos for all the tested bacterial strains (Table 3). Analogously to the results obtained for
the unformulated OLE, S. epidermidis resulted in the most sensitive strain to the loaded hydrogel film.
Overall, these data suggested that the hydrogel film B2, containing 10.92 mg/cm2 of OLE, could be
the most suitable formulation for wounds application. For this reason further characterized in the
next studies.
Table 3. Inhibition halos measured for the hydrogel films B1–B3. Results are expressed as mm ± SD (n
= 3, n.i. = no inhibition).
Hydrogel Film
S. epidermidis S. aureus L. innocua E. faecalis
(OLE mg)
B1 (3.64) 23.00 ± 0.00 16.33 ± 0.58 n.i. n.i.
B2 (10.92) 28.67 ± 0.58 20.33 ± 0.58 21.67 ± 0.58 21.00 ± 0.00
B3 (18.21) 25.67 ± 0.58 21.00 ± 0.00 21.67 ± 0.58 21.33 ± 0.58
n.i. no inhibition.
The preliminary thermal characterization of the hydrogel film B2 showed a water content of 13.6%
(Figure S1) after drying and a high glass transition temperature (>200 ◦ C; Figure S2).
2.4.2. Hydrogel Film Thickness, Swelling Behavior and Matrix Erosion Capacity
The hydrogel film B2 (circle 3.14 cm2 ) in dry conditions showed a thickness of 0.43 mm (±0.05),
resulting very thin. In general, the low thickness of a film for skin use represents a suitable property for
ensuring imperceptibility after the application. After hydration the film thickness increased to 2.5 mm
(±0.04), suggesting that its swelling after the contact with SWF should not cause an excessive increase
of the dimensions that could compromise patient’s acceptability during the use. The swelling ability of
the hydrogel film B2 was an important parameter to be evaluated as this property is responsible for the
ability to absorb exudate from the wound. Moreover, it influences the bioadhesion capacity and OLE
release rate from the formulation.
The hydrogel film B2 exhibited a high capacity to absorb fluids, testified by the swelling %
measured. It swelled reaching two fold its original weight after 15 min and nine fold after 8 h
(Figure S3). This behavior can be attributed to the hydrophilic properties of the hydrogel film with a
marked water affinity. During the experiment the hydrogel film showed also weight loss, expressed
as erosion matrix % (Figure S3) and due to the gradual dissolution in the medium. The erosion %
was 40% after 15 min, then reached 60% after 60 min, maintaining this extent until the 8th h. Both
swelling and erosion % cannot be measured after 8 h as after this time the polymeric network resulted
completely relaxed due to the very high amount of the absorbed SWF. Thus, the complete hydrogel
film dissolution in SWF was observed. Based on these results, it is reasonable to think that the hydrogel
film can be easily removed by washing ensuring an atraumatic and pain free removal.
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2.4.3. Ex Vivo Adhesion Studies

The adhesion capability of the hydrogel film B2 was evaluated ex vivo by using pig skin samples
in which a wound was simulated as reported in Figure 5. The obtained results showed a detachment
force of 0.4 N ± 0.06 and detachment time of 13.00 sec ± 0.57. The measured adhesion capacity of the
hydrogel film B2 can be ascribed to the combination of both hydrophobic and hydrophilic interactions.
The hydrophobic interactions predominate in the case of the binding between hydrogel film and
stratum corneum of the peri-wound area. The hydrophilic interactions become more important in the
binding of the damaged area characterized by the presence of exudate.
Figure 5. Skin used for the assay (A) and simulation of an open wound (B).
The obtained results suggested that the developed hydrogel film B2 possesses the suitable balance
of hydrophobic and hydrophilic groups to bind wounded skin. In fact, the high swelling capacity
measured (Section 3.5.2) evidenced that it is able to interact with the exudate and to swell. This event
allowed the distension of NaCMC and PVP K90 polymeric chains exposing the hydrophilic groups
(-OH and carboxyl groups for NaCMC and carbonyl for PVP K90) to establish interactions (mainly
hydrogen bonds) with the subcutaneous tissues surrounding the wound.
The binding to the peri-wound area could mainly be attributed to hydrophobic groups (-CH3
for NaCMC and alkyl group for PVP K90) exposed to the outer side of the film and thus available to
interact with skin. This aspect is very important as the hydrogel film composition is suitable to adhere
to the skin surface avoiding the use of adhesives, not recommended in the open wounds management
because they are painful and discomfortable.
2.4.4. OLE In Vitro Release and Correlation with the Anti-Inflammatory Activity
The release capability of hydrogel film B2 loaded with OLE was evaluated in vitro by the Franz
diffusion cell. The obtained profile (Figure 6A) shows that OLE is released just after application
reaching a concentration of 0.060 mg/mL in 15 min and 0.62 mg/mL within 24 h. This suggests that the
hydrogel film B2 is able to release an amount of OLE necessary to obtain the anti-inflammatory activity
(Section 2.2.4) from the first minutes. At the same time OLE level remained below 1.0 mg/mL, a viable
concentration for both RAW 264.7 and HaCaT cell lines (Figure 3).
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Figure 6. (A) OLE In vitro release profile of from the hydrogel film B2 represented as mg/mL vs. time
and (B) mg/cm2 vs. time.
Despite the hydrogel film B2 has been projected for one daily application, OLE release monitoring
was performed until 48th h in order to evaluate if the produced concentrations remain in the safety
range. As reported in Figure 6A the amount of OLE released within 48 h increased slightly (0.69 mg/mL)
compared to 24th h. The concentration remained below the cytotoxic value (1.0 mg/mL) suggesting
that a prolonged application time did not impair skin cells viability.
It is interesting to evaluate the amount of OLE released per unit area (mg/cm2 ) as reported in
Figure 6B. The hydrogel film B2 was able to produce effective concentrations per cm2 for the antibacterial
and anti-inflammatory activities. Thus, wounds of different sizes could be treated modulating the
hydrogel film and is a suitable delivery system for OLE dermal applications for wounds treatment.
2.4.5. In Vitro Safety Studies of Hydrogel Film on HaCaT Cell Line

As the hydrogel film was projected for one daily application the safety studies on HaCaT cells
was evaluated within 24 h. A circular film (3.14 cm2 ) was incubated for 24 h at 37 ◦ C in DMEM
complete medium. Subsequently, the medium was used to treat HaCaT cells for 24 h. From the in vitro
release studies it was possible to know the exact OLE amount released from the hydrogel film B2.
Different concentrations comparable to OLE values (0.25, 0.5, 1, 1.5 and 2.0 mg/mL) were assayed.
The obtained results from MTT assay showed very similar results compared to unformulated OLE
(Figure 7) suggesting that both OLE alone and formulated in the hydrogel film B2 is cytotoxic at the
concentration of 2.0 mg/mL (above the maximum concentration obtained from the hydrogel film).
Thus, it is possible to conclude that both OLE and hydrogel film B2 are safe on an in vitro skin model.
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Figure 7. Evaluation of hydrogel film B2 cytotoxicity and safety on HaCaT cell line by an MTT assay.
Dotted lines indicate the 50% and 75% of cell viability. ns, not significant OLE-treated versus untreated
group (one-way ANOVA test).
3.1. Materials
Rojo Duro onion samples were provided by the farm “Azienda Agraria Turrioni Fiorella”
(Cannara-Perugia, Italy). The extract from OL was prepared about five months after the
onion harvesting.
Folin–Ciocalteu reagent, 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ),
6-hydroxy-2,5,7,8-tetramethyl-2-carboxylic acid (Trolox), 2,2-diphenyl-1-picrylhydrazyl (DPPH),
hydrochloric acid (HCl), ferric chloride (FeCl3 ), sodium acetate (NaOAc), sodium carbonate (Na2 CO3 ),
acetic acid (AcOH), gallic acid (GA) and ethanol (EtOH) and bentonite nanoclay were purchased
from Sigma-Aldrich (Milano, Italy). Polyvinylpyrrolidone K90 (PVP K90) was furnished by ISP
(Baar, Switzerland). Sodium carboxymethylcellulose (NaCMC) was purchased from Caelo (Hilden,
Germany). Ultrapure water was obtained from a reverse osmosis based Milli Q System (Millipore,
Milano, Italy). Other reagent grade chemicals and solvents were used without further purification.
Ultrapure water was sterilized in a steam autoclave (121 ◦ C, 2118 millibar absolute pressure and
relative pressure 1360 millibar).
The simulated wound fluid (SWF) pH 6.5 was prepared by dissolving 8.30 g of NaCl and 0.28 g of
CaCl2 in 1000 mL of ultrapure water [36].
3.2. Extraction Procedure

OL, usually produced during the manipulation and braiding of fresh onions, were collected, then
quickly washed with ultrapure water and finally dried by a cotton towel. OL (4 g) were suspended in
160 mL of absolute (abs) or 70% EtOH and the obtained suspension kept under magnetic stirring for
90 min at room temperature (RT) or at 60 ◦ C. The supernatant was recovered by decantation (or by the
use of a Pasteur pipette) and the residue resuspended in the same starting solvent (100 mL), kept under
magnetic stirring for 90 min at the same previously used temperature. In this case, the supernatant was
also recovered and the second extraction step repeated once again. The collected supernatants were
combined and the solvent removed by a rotary evaporator (water bath temperature 37.0 ± 0.1 ◦ C, Buchi
Italia s.r.l., Cornaredo, Italy). The obtained solid was suspended in ultrapure water (28 mL) and the
resulting suspension centrifuged at 4000 rpm, 20 ◦ C for 20 min. The supernatant solution was recovered
and the pellet twice submitted to the centrifugation step using 10 mL and 6 mL of ultrapure water for
suspending the second and the third pellet, respectively. The combined supernatants were freeze-dried
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and the obtained dry material stored at −20 ◦ C. The procedures were performed in duplicate for each
extraction method.
Folin–Ciocalteu assay [18] was performed using the sample (extract) dissolved in ultrapure
water, whereas in the case of FRAP (ferric ion reducing antioxidant power) and DPPH (2,2-diphenyl
-1-picrylhydrazyl) assays [18], the sample was dissolved in EtOH 75%. In any case, the solution was
prepared dissolving 20 mg of freeze-dried extract in 50 mL of solvent (0.4 mg/mL).
3.3. Extract Characterization
3.3.1. Fingerprint Analysis of OLE Constituents

A qualitative evaluation of OLE constituents was performed by LC-UV-high resolution mass
spectrometry. Freeze-dried OLE (100.0 mg) was solubilized in 10.0 mL of an EtOH/water (70:30)
solution. The obtained solution was centrifuged and 20.0 μL of the clear supernatant were injected in
a LC-UV-Vis-HRMS (High resolution mass spectrometry) system. The LC module was an Ultimate
3000 (Thermo Scientific, Rodano, Milan, Italy), the UV-Diode Array module was a Surveyor PDA Plus
Detector (Thermo Scientific, Rodano, Milan, Italy), and High Resolution Mass Spectrometer was a
linear ion trap linked to an Orbitrap System (LTQ XL, Thermo Scientific, Rodano, Milan, Italy).
OLE molecular species were separated by using a Luna Phenomenex RP-18 column (150 mm ×
2.1 i.d., 3 μm particles). Mobile phase was a binary mixture of 0.01% formic acid aqueous solution
(solvent A) and acetonitrile (solvent B) pumped through the column at a flow rate of 0.200 mL/min.
Two different chromatographic conditions were used aimed to suitably separate components with very
different polarity properties and mass spectrometric response. More polar compounds were separated
in isocratic conditions (A/B = 72/28) whereas the medium polarity constituents were separated by
a gradient elution program consisting of a linear gradient from A/B = 95/5 to A/B = 50/50. UV-Vis
detector was programmed in the wavelength range 220–650 nm.
High resolution and LC-tandem mass spectra were acquired either in negative and in positive
ion mode using a linear ion trap-Orbitrap detector equipped with an ESI source with the following
parameter settings: spray voltage 4.5 kV; sheath gas flow rate 35 (arbitrary units); drying gas flow rate
25 (arbitrary units) and capillary temperature of 250 ◦ C.
Full scan mass spectra were recorded in the range m/z 220–1200. MS/MS spectra were acquired in
the range between ion trap cut-off and precursor ion m/z values. High resolution mass accuracy of
recorded ions (vs. calculated) was ±5 millimass units (without internal calibration). High-resolution
spectra were acquired with the resolution R = 30,000 (FWHM).
3.3.2. Antibacterial Activity Assay

OLE antibacterial activity was evaluated for four bacterial species S. epidermidis WDCM (world
data centre for microorganisms) 00036, S. aureus WDCM 00034, E. L. innocua WDCM 00017 and faecalis
WDCM 00087. MIC and MBC concentrations were measured using a standard microdilution technique
according to Clinical Laboratory Standards Institute Guidelines, adapting the protocol as suggested
by J.M. Silván et al. [41]. The bacterial suspension used for the assay was prepared using bacteria
to approximately 1 × 105 CFU/mL in Muller Hilton Broth (MHB; Biolife Italiana s.r.l, Cod. 4017412).
The stored strains were revitalized on Brain Heart Infusion Broth (BHI, Biolife Italiana s.r.l, Cod.
4012302) and incubated according to their own growth conditions (Table S3). At the time of use, OLE
was dissolved in sterile ultrapure water to obtain the concentration required by the protocol for the
evaluation of the antibacterial effect. The assayed concentrations were: 30, 15, 7.50, 3.75, 1.88, 0.94 and
0.47 mg/mL. Moreover, for each bacterial strain three controls were set up. These included antibiotic
control (ampicillin), organism control (MHB and the bacterial suspension), negative control (MHB and
the solution of the extract at the same concentration tested). For each bacterial species the test was
performed in triplicate. The microplates were incubated according to the growth conditions of each
bacterial strain tested (Table S3). After incubation, serial decimal dilutions of each well were prepared
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in sterile saline solution (0.9% w/v NaCl) and plated onto agar with 5% sheep blood (Biolife Italiana
s.r.l, Cod. 4011552). Results are expressed as log CFU/mL.
3.3.3. Cell Lines and Cytotoxicity Studies

The mouse macrophage cell line RAW 264.7, obtained from the American Type Culture Collection
(ATCC, Manassas, VA, USA), was used to investigate OLE anti-inflammatory activity. RAW 264.7 cells
were cultured according to standard procedures in Roswell Park Memorial Institute 1640 medium
(RPMI-1640), whereas HaCaT cells were cultured according to standard procedures in Dulbecco’s
modified Eagle’s medium (DMEM). Both medium were supplemented with 10% heat-inactivated
Fetal Bovine Serum (FBS), 2 mM of L-glutamine and antibiotics (100 U/mL penicillin, 100 μg/mL
streptomycin; Gibco, Invitrogen, Carlsbad, CA, USA). Both cell lines were cultured at 37.0 ◦ C in a
5% CO2 atmosphere and the medium replaced every 3 days. RAW 264.7 and (HaCaT) were tested
for mycoplasma contamination before use. RAW 264.7 cells were activated with lipopolysaccharides
(LPS), serotype 055:B5 (Sigma-Aldrich, Saint Louis, MO, USA) at 50 ng/mL for 24 h. LPS-activated
RAW 264.7 cells were cultured at the concentration of 1 × 106 cells/700 μL in a 24-well plate and
co-treated with serial two-fold dilutions of OLE for 24 h. The percentage of live, apoptotic and dead
cells, was determined by using Annexin V Apoptosis Detection Kit PerCP-eFluor™ 710 and FVD
(Fixable Viability Dye eFluor™ 780; eBioscience, San Diego, CA, USA), according to the manufacturer’s
instructions. Specifically, each cell sample was washed and suspended in 100 μL of phosphate-buffered
saline before the staining. A dilution 1:1000 of FVD was then added to the sample and incubated at
4 ◦ C for 30 min in the dark. Annexin V-PerCP, diluted 1:20, was added and incubated for 15 min at RT
in the dark. Flow cytometry analysis was performed within 4 h.
HaCaT cells were purchased from I.Z.S.L.E.R. (Istituto Zooprofilattico Sperimentale della
Lombardia e dell’Emilia Romagna, Brescia, Italy) and used as model for assessing the epidermal
homeostasis during wound treatment. Cells viability was assessed after treatment for 24 h with OLE
both by Trypan Blue exclusion and MTT assays [42]. Cells were seeded at the density of 2 × 105
cells/well into 6-well culture plates in a final volume of 2 mL and 5 × 103 cells/well into 96-well flat
bottom culture plates in a final volume of 200 μL, for Trypan Blue exclusion and MTT assay respectively.
Cell viability was measured and expressed as a percentage relative to that of the control cells as
previously described [43].
3.3.4. Anti-Inflammatory Activity

For the quantification of nitric oxide (NO) release, LPS-activated RAW 264.7 cells were cultured at
the concentration of 0.15 × 106 cells/200 μL in a 96-well plate and co-treated with serial two-fold dilutions
of OLE for 24 h. The total production of NO was detected by incubating at RT for 7 min each culture
supernatant (50 μL) with 100 μL of Griess reagent (1% naphthylethylenediamine dihydrochloride in
distilled water and 1% sulfanilamide in 5% concentrate H3 PO4 , mixed 1:1). Nitrite concentration was
quantified by comparison with a sodium nitrite standard curve. The absorbance of samples was read
at λ= 530.0 nm, by using a spectrophotometer (TECAN). The assay was conducted in triplicate.
For the quantification of IL-6 and IL-1β, LPS-activated RAW 264.7 cells were cultured at the
concentration of 0.5 × 106 cells/500 μL in a 48-well plate and co-treated with serial two-fold dilutions of
OLE for 24 h. IL-6 and IL-1β were determined in the culture supernatants by Mouse IL-6 Uncoated
ELISA kit (Invitrogen, Carlsbad, CA, USA) and Mouse IL-1 β ELISA Ready-SET-Go!™ kit (eBioscience,
San Diego, CA, USA), respectively, as described by the manufacturer’s instructions. The experiments
were conducted in triplicate and repeated two times.
3.4. Hydrogel Film Preparation

The hydrogel films were prepared by the solvent casting method [36] starting from a hydrogel.
An OLE solution (1.0%, 3.0% or 5.0% w/w) was initially prepared in ultrapure water then, bentonite
(1%) was dispersed in this solution. Finally, glycerol (10%), NaCMC (3.0%) and PVP K90 (0.1%) were
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added. The mixing was performed at 600 rpm, for 20 min, at RT using a mechanical stirrer equipped
with a three blade helical impellers (DLS VELP® Scientifica, Usmate MB, Italy). In order to remove the
air incorporated during the mixing, each hydrogel was degassed in a conditioning planetary mixer
(Thinky mixer ARE-250) at 2000 rpm for 10 min. Afterwards, 3.5 g of the prepared mixture was casted
into silicon molds (ø = 3.5 cm) and left to dry in ventilated oven at 37.0 ◦ C ± 0.1 for 24 h. After drying,
the hydrogel films were stored at RT and 40% relative humidity (RH) until use.
3.5. Hydrogel Film Characterization
3.5.1. Hydrogel Film Antibacterial Activity

The test medium and bacterial suspension used for the assay were prepared as previously reported
(Section 3.3.2). The experiment was carried out on S. epidermidis WDCM 00036, S. aureus WDCM 00034,
E. faecalis WDCM 00087 and L. innocua WDCM 00017. Different inoculated media were used for each
bacterial strains. Sterile Petri dishes (diameter 90 mm) were filled with 20 mL of the prepared and
seeded media. After medium solidification, a small square (1 cm × 1 cm) of the hydrogel film was
placed in each series of plates and incubated according to their own growth conditions (Table S3).
The test was conducted in triplicate for each bacterial strain. Negative control was set up using a
hydrogel film OLE free. At the end of the incubation time the presence and the diameter of the
inhibition halo was evaluated by a gauge.
3.5.2. Hydrogel Film Thickness, Swelling Behavior and Matrix Erosion

Hydrogel film thickness was measured in both dry and wet state by a manual micrometer (Borletti,
Cremona, Italy). The thickness of the hydrogel film after hydration was measured after incubation in
10 mL of SWF thermostated at 32.0 ± 0.5 ◦ C for 8 h. After this period, the excess of SWF was removed
by filter paper and the thickness measured. The hydrogel film swelling capacity and erosion [44] were
calculated using Equations (1) and (2) respectively:
W2 − W1
swelling % = × 100. (1)
W1
W1 − W3
erosion % = × 100. (2)
W1
Each hydrogel film (circles of 3.14 cm2 ) was weighted (W1), immersed in 10 mL of SWF into a
Petri plate (ø = 5 cm) and thermostated at 32.0 ± 0.1◦ C for established times (15, 30, 60, 90, 120, 180, 300
and 1440 min). After immersion, the hydrogel films were wiped off from the excess of SWF using filter
paper and weighted (W2).
The erosion % was measured as follows. The swollen films were dried at 60 ◦ C for 24 h and kept
in desiccator over CaCl2 (40% RH) for 48 h and after drying the weighting was repeated (W3). The
obtained results represent the average of three measurements (n = 3).
3.5.3. Thermal Properties Measurement

Thermogravimetric (TGA) analysis was performed by a Netzsch STA 449C apparatus (Mettler
Toledo, Milano, Italy), in air flow and heating rate of 10 ◦ C/min, to determine the weight loss as a
function of increasing temperature. Differential scanning calorimetry (DSC) analysis was performed
using an automatic thermal analyzer (DSC821e, Mettler Toledo, Milano, Italy) and indium standard for
temperature calibrations. Holed aluminum pans were employed in the experiments for all samples and
an empty pan, prepared in the same way, was used as a reference. Samples of 3–6 mg were weighted
directly into aluminum pans and thermal analyses of the samples were conducted, at a heating rate of
5 ◦ C/min from 25 to 300 ◦ C.
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3.5.4. Ex Vivo Adhesion Studies

The hydrogel film adhesion force and time were assessed using pig skin samples (from shoulder
region), obtained from large white pigs weighing 165–175 kg, furnished by the Veterinary Service
of ASL N.1 Città di Castello (Perugia, Italy) and used within 12 h from pig death [36]. The ex vivo
adhesion force was measured by a dynamometer (Didatronic, Terni, Italy). By means of cyanoacrylate
glue the film was attached to the bottom of a cylindrical support and the porcine skin tissue (2 cm ×
2 cm) on the surface of a glass support thermostated at 32.0 ± 0.5 ◦ C. An incision of 2 cm was made
by a scalpel on the skin sample in order to simulate a wound. The latter was filled with 500 μL of
SWF and the hydrogel film was put in contact with the simulated wound by applying a light force
(0.5 N) for 60 sec. After this time, the force was removed and the hydrogel film kept in contact with the
skin for further 60 sec and then put in traction. The force necessary for film detachment to skin was
measured and expressed as average of three measurements (n = 3).
3.5.5. In Vitro Release of Extract from The Hydrogel Film

OLE In vitro release from the hydrogel film was evaluated by vertical Franz diffusion cell (USP
<1724>; PermeGear, Inc., Bethlehem, PA, USA, diameter 20 mm). A cellulose membrane (Whatman 41,
Whatman GmbH, Dassel, Germany) was placed between the two chambers. The receptor chamber
was filled with SWF as receptor medium (15 mL), thermostated at 32.0 ◦ C ± 0.5 and magnetically
stirred (600 rpm). The hydrogel film (circle of 3.14 cm2 ) was placed on top of the cellulose membrane.
The donor phase was represented by 2 mL of a 0.025-N K2 CO3 solution (simulating air CO2 ) [43].
All openings including donor top and receptor arm were occluded with parafilm® to prevent solvent
evaporation. At regular time intervals (5, 10, 15, 30, 45, 60, 120, 240, 300, 360, 420, 480, 1440 and 2880
min) samples of the receiving phase were withdrawn and OLE content was measured by a UV-vis
spectrophotometer (UV-Visible Agilent model 8453, Agilent Technologies, Cernusco sul Naviglio MI,
Italy) accomplished using a standard curve in SWF (λmax = 280.0 nm, r = 0.9998). All experiments
were performed in triplicate, each result represents an average of three measurements and the error
was expressed as standard deviation (±SD).
4. Conclusions
The manuscript described the use of the non-edible outside layers of the onion cultivar Rojo Duro
for the preparation of a hydrogel film for dermal application. This study proved that medical devices
could be developed by recycling a waste material from agricultural and food processing industries.
The use of the non-edible outside layers as a source of active ingredients is an efficient and smart
approach for avoiding the expensive disposal procedures while producing a high-value added product.
The extract (OLE) deriving from the non-edible outside layers was obtained by using eco-friendly
solvents thus without the production of further waste. OLE showed interesting antioxidant, radical
scavenging, anti-inflammatory and antibacterial activities. As these properties were suitable for
wounds treatment, OLE was formulated as autoadhesive biocompatible hydrogel film for application
on injured skin with the aim to promote the healing process through synergic mechanisms. This could
represent an effective alternative to conventional antibacterial therapies, limiting the use of antibiotics
and thus the resistance problem.
OLE (10.92 mg/cm2 ) was released from the hydrogel film by a sustained release reaching effective
concentrations to (i) exert the antibacterial activity against S. epidermidis, S. aureus and E. faecalis
(common strains responsible for wounds infections); (ii) obtain the anti-inflammatory activity and at
the same time and iii) maintain cells viability.
The hydrogel film is biocompatible, its composition is simple (PVP K90, NaCMC, bentonite),
allows a rapid adhesion to skin, without the use of adhesives, a mechanical protection of the wound
and an easy/pain-free removal by washing. Moreover, the hydrogel film is self-applicable. The last
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property, together to that described above, fulfills the compliance of the patient. The hydrogel film is
green, scalable and cheap, thus resulting advantageous for the industry.
The obtained results suggest that the developed formulation represents an innovative and effective
strategy useful for wounds treatment.

Figure S1: The TGA curve shows three steps: the first one until 200 ◦ C corresponding to the vaporization of
residual water (13.6%), the second step until 500 ◦ C due to the decomposition of the organic components, the
third one above 500 ◦ C characterized by a mineral residue (22.78%) due to bentonite content. Figure S2. PVP K90
shows a broad endothermic peak between 45 and 120 ◦ C due to the evaporation of bound water or moisture. For
bentonite it is detectable a broad band between 60 and 100 ◦ C attributable to adsorbed water loss. NaCMC shows
a glass transition temperature (Tg) at 75 ◦ C, corresponding and an exothermic peak at 284 ◦ C due to the melting
and the crystallization transition. Pre OLE thermogram shows that thermal process occurs at 80 ◦ C probably
related to the loss of volatile constituent of the sample (ethanol). No thermal decomposition was observed in
the temperature range investigated. For the hydrogel film B2 a glass transition temperature (Tg) at 228 ◦ C was
observed. The spectra are shown in arbitrary units (a.u.) and shifted for clarity. Figure S3. Swelling % and
erosion % of the hydrogel film B2 in SWF thermostated at 32 ◦ C for 480 min. Table S1. Hydrogels compositions.
Table S2. Compositions of hydrogels loaded with different OLE percentages. Table S3. Bacterial strains and
growth conditions.
Author Contributions: Conceptualization, M.M. and L.P.; Data curation, C.B., M.M., M.R.C., C.O., R.O. and L.P.;
Formal analysis, C.P., P.C., C.B., M.R.C., M.C., E.O., R.O. and M.R.; Funding acquisition, M.M., S.S., M.C.T. and
L.P.; Investigation, C.P., M.M., T.B., C.O., C.B., S.S. and L.P.; Methodology, C.P., M.M., C.B., T.B., C.O. and L.P.;
Resources, M.M., T.B., C.O., S.S., C.B. and L.P.; Supervision, C.P., M.M., M.R., M.C.T. and L.P.; Validation, P.C.,
C.B., M.R.C., M.C., E.O. and R.O.; Visualization, C.P., M.M., T.B., C.O., S.S. and L.P.; Writing—original draft, C.P.,
M.M., T.B., C.O., M.R., S.S. and L.P.; Writing—review and editing, C.P., M.M., M.R.C., C.O., R.O., M.C.T. and L.P.
All authors have read and agreed to the published version of the manuscript.
Funding: This research was financed by MIUR-Fondo d’Ateneo per la Ricerca di Base 2015.
Acknowledgments: Authors sincerely acknowledge Marco Marani (Department of Pharmaceutical Sciences)
for technical assistance, Fiorello Turrioni and “Azienda Agraria Turrioni Fiorella” (Cannara, Perugia, Italy) for
providing Rojo Duro onion skins, Simonetta De Angelis from ASL N. 1 (Città di Castello, Perugia, Italy), for
providing pig skin samples.
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29. Kwon, H.K.; Song, M.J.; Lee, H.J.; Park, T.S.; Kim, M.; Park, H.J. Pediococcus pentosaceus-Fermented
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in murine macrophages in pharmacological-induced apoptosis. Br. J. Pharmacol. 2010, 160, 1844–1856.
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Sample Availability: Samples of OLE and films are available from the authors.
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Review
Bee Products in Dermatology and Skin Care
Anna Kurek-Górecka 1, *, Michał Górecki 2 , Anna Rzepecka-Stojko 2 , Radosław Balwierz 1 and
Jerzy Stojko 3
1 Silesian Academy of Medical Sciences in Katowice, Mickiewicza 29, 40-085 Katowice, Poland;
radoslaw.balwierz@gmail.com
2 Department of Drug Technology, Faculty of Pharmaceutical Sciences in Sosnowiec,
Medical University of Silesia, Jedności 8, 41-200 Sosnowiec, Poland; mgorecki@sum.edu.pl (M.G.);
annastojko@sum.edu.pl (A.R.-S.)
3 Department of Toxycology and Bioanalysis, Faculty of Pharmaceutical Sciences in Sosnowiec,
Medical University of Silesia, Ostrogórska 30, 41-200 Sosnowiec, Poland; jstojko@sum.edu.pl
* Correspondence: akurekgorecka@interia.pl

Abstract: Honey, propolis, bee pollen, bee bread, royal jelly, beeswax and bee venom are natural
products which have been used in medicine since ancient times. Nowadays, studies indicate that
natural bee products can be used for skin treatment and care. Biological properties of these products
are related to flavonoids they contain like: chrysin, apigenin, kaempferol, quercetin, galangin,
pinocembrin or naringenin. Several pharmacological activities of phenolic acids and flavonoids, and
also 10-hydroxy-trans-2-decenoic acid, which is present in royal jelly, have been reported. Royal jelly
has multitude of pharmacological activities: antibiotic, antiinflammatory, antiallergenic, tonic and
antiaging. Honey, propolis and pollen are used to heal burn wounds, and they possess numerous
functional properties such as: antibacterial, anti-inflammatory, antioxidant, disinfectant, antifungal
and antiviral. Beeswax is used for production of cosmetics and ointments in pharmacy. Due to a
large number of biological activities, bee products could be considered as important ingredients in
medicines and cosmetics applied to skin.
Keywords: bee products; flavonoids, phenolic acids; skin care; therapeutic properties
1. Introduction
Nowadays, alternative medicine, which employs natural biologically active substances obtained
from bee products, is getting more and more attention. Bee products have been used not only in
treatment, but also for skin care as ingredients of cosmetics. The effect of bee products on the skin
has also been proved by numerous studies, and the use of honey, propolis, bee pollen and bee
venom in wound healing highlights their curative value [1–4]. Each bee product possesses specific
active substances which determine its use for various skin problems. Honey, propolis, bee pollen,
bee bread, beeswax and bee venom are the bee products which are used for medicinal purposes and
cosmetic production.
Honey is a natural product which is made by bees from nectar and honeydew. Honey is a
supersaturated solution of carbohydrates with numerous properties and wide use. Propolis, also
called bee glue, is a resinous substance collected by bees from buds of trees, shrubs, and green plants.
Both, propolis and honey were used in antiquity for embalming bodies, whereas folk medicine used
honey for wound healing and pain relief [5]. Bee pollen is collected from plants and transported to
the hive in form of pollen loads. The formation of loads involves moisturizing pollen with nectar
or honey. Pollen for winter supplies, which is deposited in the honeycomb cells, undergoes lactic
fermentation and produces bee bread. Bee bread and bee pollen are bactericidal and bacteriostatic

Molecules 2020, 25, 556
agents [6,7]. Beeswax is a substance produced by glands located in the bee abdomen. Wax obtained
from honeycombs constitutes a valuable ingredient used in cosmetology and pharmacy. Bee venom
also called apitoxin produced by honeybee. It consists a complex mixture of different peptides and
mast cell degranulating peptide, which therapeutic and cosmetic properties are used in many areas [8].
2. Selected Compounds of Bee Products

The chemical composition of bee products is quite diversified, and depends on the botanical
composition, geographical origin, time of collection and environmental conditions [9–11]. However,
each product made by bees has a specified composition and content of biologically active substances,
which give specific properties to each bee product. The chemical composition determines the curative
and properties of these products.
Honey contains at least 181 ingredients [12]. Honey is a supersaturated carbohydrates solution
containing mainly glucose and fructose [13]. Moreover, honey can have in its composition of sucrose,
rhamnose, trehalose, nigerobiose, isomaltose, maltose, maltotetraose, maltotriose, maltulose, melezitose,
melibiose, nigerose, palatinose, raffinose, and erlose [14,15]. It also contains enzymes, namely, glucose
oxidase, amylase, catalase, peroxidase, invertase, and lysozyme. Glucose oxidase produces hydrogen
peroxide which is one of responsible substances for the bactericidal activity of honey [16]. Honey
contains also organic acids: gluconic acid, citric acid, malic acid, lactic acid, succinic acid, oxalic acid,
tartaric acid, formic acid, acetic acid, benzoic acid, and pyromucic acid. The acids originate from
bee bodies and enzymatic conversions which occur during honey production. The content of these
acids is higher in mature honeys. Phenolic acids and flavonoids, which are responsible for many
biological properties and have antioxidative activity, are also important ingredients of honey. The
group of phenolic acids includes derivatives of hydroxycinnamic acid and hydroxybenzoic acid. The
derivatives of hydroxycinnamic acid are p-coumaric acid, caffeic acid, ferulic acid, and sinapic acid.
Whereas, the derivatives of hydroxybenzoic acid include p-hydroxybenzoic, vanillic, syringic, salicylic
and gallic acids and ellagic acid as a dimer of gallic acid [15]. In honey, flavonoids are represented
by naringenin, hesperetin, pinocembrin, chrysin, galangin, quercetin and kaempferol. However, a
significant decrease in the concentration of galangin, kaempherol, and myricetin is observed after honey
has been heated, while pasteurization causes a substantial decrease in myricetin concentration [17].
Honey contains also essential oils, whose composition includes terpenes (thymol, bisabolol, farnesol,
and cineol). Other components of honey comprise water, amino acids and proteins. Proline (50–80%)
dominates among amino acids, and its increased presence indicates honey maturity [14]. Vitamins
constitute a small group of compounds present in honey, and they are mainly: thiamine, riboflavin,
pyridoxine, p-aminobenzoic acid, folic acid, pantothenic acid, and vitamins A, C, E. Honey contains
also minerals: phosphorus, potassium, calcium, magnesium, sulfur, iron, copper, manganese, and zinc.
Although there is only a small amount of trace-elements in honey, they are highly bioavailable. It was
reported that copper, calcium, zinc, iron, manganese and magnesium from honey are characterized a
bioavailability of 80–90% [18].
In terms of chemical composition, propolis is a very diverse product. At present, at least 300 active
compounds have been identified in it [19]. Phenolic acids (caffeic, ferulic, chlorogenic, p-coumaric),
benzoic acid, cinnamic acid and flavonoids are the most important biologically active compounds.
Among flavonoids, we can enumerate chrysin, luteolin, apigenin, galangin, kaempherol, quercetin,
pinostrobin, pinocembrin, and terpene compounds, whose content is 0.5% (bisabolol), and alcohols
(cetyl, myricyl, mannitol and inositol) [20–22]. Propolis contains also minerals (calcium, magnesium,
manganese, zinc, copper, iron, cobalt and selenium), vitamins (B1, B2, B6, C and E) and enzymes
(succinate dehydrogenase, glucose-6-phosphatase, adenosine triphosphatase, acid phosphatase) [22,23].
Bee pollen comprises at least 200 biologically active substances. Proteins constitute about 22.7% of
bee pollen composition, including 10.4% essential amino acids: methionine, lysine, threonine, histidine,
leucine, isoleucine, valine, phenylalanine, tryptophan. Digestible carbohydrates constitute 30.8%,
while the percentage of reducing sugars is 25.7%. Among the fatty acids present in bee pollen, we can
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list acids such as gamma-linolenic acid, arachidonic acid, and linoleic acid (0.4%). Additionally, nucleic
acids and nucleosides are valuable components of bee pollen [2,24]. It contains also vitamins (B1, B2,
B3, B5, B6, C, H, E) and minerals (potassium, calcium, phosphorus, iron, zinc, copper, manganese) [9].
Protein content in bee bread is 12% lower than its content in bee pollen. The content of reducing
sugars increases by 40–50%, whereas the content of lactic acid rises to 3.1%. Bee bread contains vitamin
K and enzymes which cannot be found in bee pollen [25,26]. Bee bread is also a good source of phenolic
components. Among bee bread from different parts of the Baltic Region the p-coumaric acid, ferulic
acid, caffeic acid, kaempherol, isorhamnetin, naringenin and quercetin were identified [27].
Royal jelly contains peptides: jelleines I, II, III, IV, proteins, carbohydrates, lipids, vitamins
and minerals [28]. Among proteins we can list royalisin and enzymes: amylase, invertase, catalase,
acid phosphatase, and lysozyme. Proteins of royal jelly are rich in exogenous amino acids. The
carbohydrates in royal jelly are mainly monosaccharides: fructose, glucose and oligosaccharides.
Lipids play an important role in royal jelly composition [29]. 10-hydroxy-trans-2-decenoic acid,
3-hydroxydodecanoic acid, and 11-oxododecanoic acid can be included into the most valuable ones [28].
10-hydroxy-trans-2-decenoic acid (10H2DA) is the main and specific lipid component of this product.
10H2DA is used as a marker to validate the quality of royal jelly [28,30]. Royal jelly contains also volatile
compounds such as phenol, guaiacol and methyl salicylate. In royal jelly, there are also present trace
amounts of such bio-elements as potassium, sodium, magnesium, phosphorus, sulfur, calcium, zinc,
iron, and copper. Royal jelly contains mainly vitamins from group B: thiamine, riboflavin, pyridoxine,
pantothenic acid, nicotinic acid and biotin and it is also contains phenolic compounds: ferulic acid,
quercetin, kaempherol, galangin and fisetin, pinocembrin, naringin and hesperidin, apigenin, acacetin,
and chrysin [31,32].
Esters of acids and fatty alcohols are main constituents of beeswax and subsequent components,
in respect of amount, are free fatty acids [33]. Among the latter, 10-hydroxy-trans-2-decenoic acid
(10H2DA) exhibits antibacterial effect, which is important. Beeswax is composed of hydrocarbons
and free fatty alcohols [34,35]. Free fatty alcohols such as triacontanol, octacosanol, hexacosanol, and
tetracosanol are antioxidative and anti-inflammatory. Other substances are triterpenes, β-carotene,
volatile compounds and phenolic compounds. Among flavonoids, the main role is played by chrysin,
which relieves inflammation, has antimicrobial and regenerative effects. Sterols have a regenerative
effect, whereas an antiseptic effect is provided by three components: 10-hydroxy-trans-2-decenoic acid,
chrysin, and squalene [34,36].
Bee venom contains different peptides including melittin, apamine, adolapin, sekapin, prokamin
and mast cell degranulating peptide [37]. Peptides are main components of bee venom. Among
peptides especially melittin plays important role in inducing reactions associated with bee stings.
Melittin induces membrane permeabilization and lyses cells. It possesses also biologically active
amines like histamine, epinephrine, dopamine, norepinephrine and enzymes like phospholipase A2,
hyaluronidase, acid phosphomonoesterase, lysophospholipase. Bee venom has another components
than peptides including lipids, carbohydrates and free amino acids [8,38,39].
3. Bee Products as Raw Material for Medicines and Cosmetics Production

Honey in cosmetics is named “Honey” or “Mel” according to the International Nomenclature
of Cosmetic Ingredient (INCI), it is an emollient or humectant, and exhibits moisturizing properties.
Some cosmetics contain derivatives of honey, defined in the INCI as “Mel Extract” with moisturizing
properties, “Hydrogenated Honey” which is humectant, and antistatic “Hydroxypropyltrimonium
Honey”. Hydroxypropyltrimonium honey is used in shampoos and hair conditioners. More often the
concentration of honey in cosmetics is up to 10%. Higher concentrations (up to 70%) are obtained by
dispersing in oils, gels or polymer entrapment [40].
Most frequently, propolis has a form of aqueous or ethanol extracts. According to the INCI
nomenclature, in cosmetics we can find it under the following names: propolis and propolis extract.
Ethanol extracts of propolis are most frequently used. To obtain them, propolis is extracted with 70%
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ethanol, and then the extract is concentrated in reduced pressure conditions [41]. An aqueous extract
of propolis is used in antifungal cosmetics, while propolis dissolved in fats is used to produce lipsticks.
Royal jelly can most frequently be found in cosmetics in a lyophilized form, and the higher
percentage content of lyophilized royal jelly is, the less viscous cream becomes. However, royal jelly
content does not affect emulsion stability. Preparations with a higher content of royal jelly are well
absorbed, and do not leave greasy film. Creams with royal jelly have moisturizing properties especially
in concentration of 0.5% and 1% [42].
In cosmetic manufacturing, bee pollen is used in a form of aqueous, lyophilized and lipid extracts.
Active substances can be extracted with water, propylene glycols, glycerin and oils. Bee pollen
extacts are used in cosmetic in concentrations 0.5–5% [43]. In natural cosmetics, dried grains of bee
pollen—micronized and added to cosmetics—are also used.
Beeswax is used in cosmetics after honey has been removed from honeycombs, wax has been
melted, and impurities have been separated. To do this, various types of wax extractors are used: solar,
electric or steam ones. Yellow wax (Cera flava) or white wax (Cera alba) is used to produce cosmetics [34].
According to INCI, bee venom or apitoxin are defined as bee venom powder. It is yellow light
powder obtained by collecting a large amount of bee venom by electric stunning with using a bee
venom collector without harming the honey bee. Then bee venom has to be purified under strict
laboratory conditions. In next step purified bee venom is diluted in water, centrifuged, lyophilized and
refrigerated for use as cosmetic ingredient [44]. It is used as a cosmetic ingredients which possesses
antiaging, anti-inflammatory and antibacterial, antifungal and antiviral effects. Bee venom is used
to produce antiphotoaging and anti-acne products [8,44]. Bee venom is used in treatment psoriasis,
atopic dermatitis and alopecia [39].
4. The Effect of Bee Products on the Skin
4.1. Honey
Honey is used in medicine including due to its antimicrobial effect, which results from the
following factors: hydrogen peroxide, high osmotic pressure, high acidity, the presence of phenolic
acids, flavonoids and lysozyme [45]. Honey inhibits the growth of bacteria and fungi by reducing
their development on the skin surface. Honey is particularly suitable as a dressing for wounds and
burns, and has also been included in treatments against pityriasis, tinea, seborrhea, dandruff, diaper
dermatitis, psoriasis, hemorrhoids, and anal fissure [40]. Pinocembrin and lysozyme are responsible
for antifungal properties. Lysozyme inhibits growth of yeast-like fungi [46]. The effect of honey on
healing postsurgical wounds was documented [1]. Among 52 patients incisions on skin were covered
with honey dressing. The aesthetic outcome after third and six months was rated. The width of the
scars was smaller in compare to conventional dressing. After 5-day application of honey dressing,
an analgesic effect was obtained and wound healing was accelerated in women after plastic surgeries.
Honey induced extracellular Ca2+ entry results in wound healing. It is similar to role plays by Ca2+
signaling in tissue regeneration [47]. Moreover honey regulates the process of epithelial mesenchymal
transition (EMT) and it has a positive impact on wound healing. The effect on EMT depends on the
floral and origin of the honey [48]. Honey is the apitherapeutic agent in topical wounds treatment
due to killing bacteria, ability to bacterial biofilm penetration, lowing wounds pH, Reducing pain and
inflammation, promoting fibroblast migration and keratinocyte closure, promoting collagen deposition
so honey has a potential role in the area of tissue engineering and regeneration. Honey should be
considered to incorporate it to the biomaterial tissue templates for tissue regeneration. Honey was
used in electrospun templates, cryogels or hydrogels [49]. The main problem of use honey in tissue
engineering are: cytotoxicity of high concentrations of honey, the lack of prolonged release rates of
the honey over time. So future research should focus on these aspects. Among different types of
honey, a strong antibacterial effect was observed in manuka honey which contains larger amount of
methylglyoxal than European honeys [50]. The antibiotic activity of manuka honey is estimated by
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Unique Manuka Factor (UMF) and methylglyoxal (MGO) markers [46]. Due to an increased content of
glucose oxidase, a higher level of hydrogen peroxide than in European honeys can be observed [51].
Hydrogen peroxide is responsible for produce free radicals, which cause oxidative damage to bacterial
cell walls. The antimicrobial effect of honey from New Zealand is also evident in undiluted honeys
and it is not abolished by catalases, which differentiates manuka honey from other types of honey.
This type of honey is used in the treatment of various wounds, including burns. The inhibition value
against Staphylococcus aureus FDA 209P of manuka honey in dilutions from 1:2 to 1:128 is determined
in the range of 2.0–4.5 [50]. Manuka honey is used in medicine to heal burns, ulcers and wounds
difficult to heal, and brings satisfactory results. Manuka honey also soothes gum inflammation, and
inhibits the formation of dental plaque, fights thrush, and prevents periodontitis [52]. Another variety
of honey with antibacterial activity is Revamil from The Netherlands. The antibiotic factor in Revamil
is the peptide defensin-1 [46]. Bee defensin-1 permeabilizes bacteria and inhibits their RNA, DNA
and protein synthesis [49]. However in other varieties of honey also the phenolic compounds are
responsible for antibacterial effect.
Honey is a bee product with a high nutritional value and regenerative properties that is why it
is used in skin care products. A high content of carbohydrates, the presence of fruit acids and trace
elements are responsible for its nutritional and regenerative effects. Thanks to osmosis, microcirculation
in the dermal tissue is stimulated, which results in its better nutrition and oxygenation. In this way,
metabolic processes are also stimulated, which leads to eliminating harmful metabolites, and increasing
regenerative processes. Additionally, honey has hygroscopic properties, absorbing metabolites, and
causing detoxification of the dermal tissue. This results in an increase in the skin tension, improvement
of its elasticity, revitalizing its color, and smoothing out wrinkles [52]. Fruit acids, as honey components,
provide an exfoliating effect for dead skin cells. Honey can be used as peeling agent in a sugared
form [53]. As a result, many valuable nutritional components, including vitamins, can diffuse through
the skin more easily. Xerosis is relieved by fatty acids and mineral salts in honey. Honey soothes skin
irritations, it is a good cosmetic for chapped lips, rough, cracked hands, and frost bites. Honey is used
in balms and bath products because of its toning, relaxing, conditioning effects related to the high
content of simple sugars, the presence of essential oils, and bioelements [53]. Due to the presence of
flavonoids, honey can also play an important role in sun protection by preventing skin irritation [40].
4.2. Propolis
Propolis is widely used in medicine. Thanks to its antiseptic properties it is used in dermatology
to treat staphylococcal, streptococcal and fungal infections. Purulent skin infections, hidradenitis,
intertrigo, cheilosis, and thrush, among other things, are treated with propolis. As reported the
Propol T, which is a propolis preparation, is highly effective in treatment of skin burns [54]. There are
comparable therapeutic effects when propolis and sulfathiazole are used, however, bee glue is safer,
and has fewer adverse effects. Propolis is not only antimicrobial and anti-inflammatory but also it
increases cicatrization and reduces pain. Chrysin, which is a flavonoid, provides an analgesic effect.
Propolis used to treat burn wounds in pigs increased fibrolast proliferation, activation and growth
capacity. Propolis stimulates glycosaminoglycan accumulation what is needed for granulation, tissue
growth and wound closure. Propolis as apitherapeutic agent is more effectively than silver sulfadiazine.
Accumulation of collagen type I in matrix of an injury stimulates the repair process because collagen
type I is indispensable for the keratinocyte migration and reepithelization. Moreover, propolis increased
accumulation of collagen type III what accelerates healthy process. The usage of propolis ointment
to treat burns as a topical apitherapeutic product could contribute to reepithelization [3]. Topically
applied propolis decreased persistent inflammatory in diabetic wounds by normalizing neutrophil
and neutrophil elastase. Caffeic acid is responsible for anti-inflammatory effect of propolis [55].
Genistein from propolis accelerated wound healing and stimulated wound angiogenesis in mice with
diabetes type-1 [56]. Furthermore propolis may be effective in healing in different animal models
including animals with burns and diabetic wounds [3,55,56]. Moreover propolis is highly effective in
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the treatment of Acne vulgaris. Researchers confirmed the limitation of occurrence of Cutibacterium
acnes, i.e., a bacterium which plays a key role in acne vulgaris pathogenesis, after ethanol extract
of propolis was applied to the skin [57]. The ethanol extract of propolis inhibits also Staphylococcus
epidermidis. Propolis is used to manufacture cosmetics for the skin with acne, and to produce drugs
against bacterial and fungal infections [58]. Propolis in the concentration of 5–20% has regenerative,
repair effects and protects against external factors. It can be used to produce anti-bedsores preparations,
since it firms the dermal tissue and protects it against pathogenic microbes [59]. Propolis protects also
from ultraviolet radiation, since it can absorb UV light due to the presence of caffeic acid, coumaric
acid, and ferulic acid. Propolis is a good additive to sun blockers (creams, lotions, sticks, and lipsticks)
due to its properties of a natural filter, as well as antioxidative, anti-inflammatory and regenerative
effects [60]. Other researchers showed that Romanian propolis had photoprotective effects against
UVB after topical application to 30 Swiss mice [61]. Propolis is also used to produce protective lipsticks.
It is regenerative and antiviral in cold sores caused by herpex simplex virus. Flavones and flavonols
from propolis, especially galangin, kaempferol, quercetin, have a high antiviral activity against herpes
simplex virus type 1 in vitro [62]. Nolkemper et al. observed that both, aqueous and ethanol extracts
of propolis were strongly antiviral against herpes simplex type 2 (HSV-2) [63]. Skin care with products
based on propolis is helpful against fungal problems of the skin due to the presence of flavonoids
(pinocembrin and pinobanksin), phenolic acids (caffeic acid) and terpenes [59]. Pinocembrin isolated
from propolis inhibits the mycelial growth of Penicillium italicum by interfering energy homeostasis
and cell membrane damage of the pathogen [64]. Shampoos with bee glue can be a natural alternative
in treatment of dandruff and prevention of its recurrence due to its antifungal and anti-seborrheic
properties. Propolis has also been used for manufacturing toothpastes. Bee glue inhibits the formation
of dental plaque and is antimicrobial, thereby it reduces dental caries development. Propolis ethanol
extracts inhibit the growth of cariogenic bacteria, which include mainly Staphylococcus mutant and
Staphylococcus sobrinus. Glucosyltransferase makes bacteria produce glucan they feed on, which is
insoluble in water. Propolis eliminates cariogenic bacteria, inhibits the activity of glucosyltransferase,
and reduces adherent abilities of bacteria [65]. The conducted studies showed that the use of toothpaste
with propolis reduced dental plaque by 34.3% annually, whereas normal paste reduced the plaque
by 31.9%. After two-year use of the paste with propolis a further reduction of plaque by 12.4% was
observed, while normal paste managed to reduce it only by 5%. Rinsing the mouth with water with
0.5% propolis content complements the oral cavity care. After 21 days, this solution was able to reduce
dental plaque by 18.1% [66]. Propolis smoothes out wrinkles and has antiaging properties. A huge
role is played here by antioxidants such as phenolic compounds and flavonoids which neutralize an
unfavorable effect of free radicals on the skin. Bee glue lightens and smoothens the skin, reduces signs
of fatigue and moisturizes it [59].
4.3. Royal Jelly

Royal jelly has a broad spectrum of biological activities which determine the effect of royal jelly
on the skin, namely, antibacterial, anti-inflammatory, immunomodulatory, anti-allergic, antioxidant,
toning, moisturizing, and antiaging [67]. Royal jelly is a bee product with strong antimicrobial activity
within skin tissue, which is already evident in 20% concentration. Due to its anti-inflammatory
activity, royal jelly relieves periodontal diseases, inflammation of the oral cavity, tongue and throat.
Anti-inflammatory activity and wound healing results from its ability to inhibit the production of
pro-inflammatory cytokines (TNF-α, IL-6, IL-1). Royal jelly has a protective effect on blood vessels and
relieves hemorrhoids, and varicose veins of the lower extremities. It is used to treat lichen, ulcers, burns,
bed sores, shingles, in all cases where the regeneration of epidermis is expected, wound epithelialization,
nutritional effect, healing and antimicrobial activity. The effect of 5% royal jelly on ulcers on the diabetic
foot has been studied. The treatment lasted 3 months and involved dressing the wound with 5%
sterile royal jelly 3 times a week. Among eight treated ulcers, seven were cured, and in one case an
improvement was observed [68]. Royal jelly promotes wound reepithelization. The keratinocytes are
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responsible for the elevated production of MMP-9 (matrix metalloproteinase-9) after incubation with a
water extract of royal jelly. After applying water extract of royal jelly increased keratinocyte migration
and wound closure rates. The component of royal jelly responsible for stimulating MMP-9 production
is defensin-1. Moreover defensin-1 promotes reepithelization and wound closure. Similarly as in
honey, defensin-1 is responsible for cutaneous wound closure by enhancing keratinocyte and MMP-9
secretion [69]. Royal jelly is effective in the treatment of wounds, and is successfully used in cosmetics
for problem skin care. Royal jelly is an ingredient of preparations normalizing sebum secretion, for
seborrheic skin, acne-prone skin where frequently skin lesions and small wounds occur [31]. Due to
stimulating metabolism in tissues, royal jelly improves regenerative processes of tissues. Regenerative,
nutritional and healing properties are used in balms, creams, and lotions. Immunomodulatory and
antiallergenic activities of royal jelly are related to the properties of fatty acids, isolated from it. Both,
10HDA and 3-10-dihydroxydecanoic acid modulate immune response and lower the concentration
of IL-2 and IL-10. Anti-inflammatory and immunomodulatory activities of royal jelly were used
to treat atopic dermatitis, hypertrophy, hyperkeratosis and epidermis and dermis inflammation,
possibly through a blend of TNF-specific low adjustment of IFN-gamma specific production and high
adjustment of nitric-oxide synthase (NOS) expression [70]. 10-hydroxy-trans-2-decenoic acid, which
is present in royal jelly, stimulates fibroblast production of collagen by inducting the production of
transforming growth factor. As a result, royal jelly affects the production of collagen, which is an
important factor that supports the skin [28]. Royal jelly is highly moisturizing, and affects hydration of
the stratum corneum by retaining water in it. In consequence, the skin become more elastic and better
moisturized [42].
4.4. Bee Pollen

Bee pollen, another bee product, can also affect the skin. Bee pollen is a potent antifungal,
antimicrobial, antiviral, anti-inflammatory, immunostimulating agent, and it also facilitates the
granulation process of burn healing [71]. Pollen ethanol extract is antimicrobial against Staphylococcus
aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and has an antifungal activity
against Candida albicans. Flavonoids and phenolic acids provide antifungal and antibacterial properties
of bee pollen. Anti-inflammatory activity of bee pollen is due to inhibiting the activity of enzymes
participating in the development of inflammation, i.e., cyclooxygenase II and lipoxygenase. Phenolic
acids, fatty acids and phytosterols are responsible for anti-inflammatory characteristics. Additionally,
kaempferol inhibits hyaluronidase and elastase, which suppresses inflammatory response. Besides,
topical application of ointment with pollen extract to treat burns has been studied, since bee pollen can
regenerate damaged tissues [2].
Bee pollen is an active ingredient in cosmetics, usually in the concentration of 0.5–5% [43]. Its
significant effect on the skin tissue is due to a high content of flavonoids. Their presence allows bee
pollen to strengthen and seal capillaries, which is also increased by high vitamin C content, and that
is why bee pollen is used in creams for couperose skin. Bee pollen affects cell metabolism, boosts
regeneration and stimulates mitotic division. Bee pollen is used to produce shampoos and conditioners.
Its sebo-balancing activity, which involves reducing sebum secretion, is used in preparations for oily
hair. Bee pollen normalizes the activity of sebaceous glands due to presence of zinc, methionine and
phospholipids. Moreover, sulphur containing amino acids, mainly cysteine, present in bee pollen
strengthen hair shaft. Bee pollen is also added to anti-dandruff shampoos, since it limits fungal growth
and stops itching of the scalp, but it still has moisturizing, conditioning and regenerating properties.
Other researchers inform that a good solution would be to mix ethyl esters of essential unsaturated
fatty acids from flaxseeds with bee pollen. Essential fatty acids (EFA) would play the role of lipid
fraction solvent. Preparations with omega-3 and omega-6 acids enriched with diverse properties of bee
pollen could help in the care of atopic skin, sensitive skin, and the skin more vulnerable to scarring [43].
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4.5. Beeswax
When compared to other bee products, beeswax has the smallest range of biological activities.
K˛edzia [34] wrote that beeswax was added to ointments, liniments and creams used in treatment
of various dermatoses, e.g., boils, wounds, atopic dermatitis, psoriasis, diaper dermatitis caused by
Candida albicans. Beeswax is mainly used as an emulsifying agent. In cosmetics, beeswax is used as a
stiffener, a substance providing elasticity, plasticity and increasing skin adhesiveness. Beeswax is the
base for lipsticks, sticks and creams [72]. Beeswax has lubricating, softening activities and reduces
transepidermal water loss from skin. Sterols, which are also components of intercellular space, provide
these characteristics of beeswax. Squalene, 10-hydroxy-trans-2-decenoic acid and flavonoids (chrysin)
provide antiseptic properties to this product, and protect the skin against pathogenic microorganisms.
Beeswax constitutes a protective barrier against many external factors by forming a film on the skin
surface. β-carotene present in beeswax is a valuable source of vitamin A, into which it is converted.
Vitamin A delays collagen degradation, stimulates mitotic division in the epidermis, thus leads to
sooner regeneration of the skin after damage [34,36].
The main effects of flavonoids and phenolic acids present in above bee products on the skin are
presented in Table 1.
Table 1. Main effects of selected flavonoids and phenolic acids on skin.
Group Representative Structure Effect
+2 2 anti-inflammatory [73],
Chrysin antibacterial & antiviral [74],
antioxidant [22]
+2 2
Flavones
2+
+2 2 antiviral & antifungal [74],

Apigenin anti-allergic [75],
antioxidant [22]
+2 2
+2 2 antiviral [62],
Galangin antifungal [76],
2+ antioxidant [22]
+2 2
2+
anti-inflammatory [77],
+2 2
Flavonols antifungal & antiviral [74],
Kaempferol
antioxidant [22],
2+
+2 2
UV photoprotective [78]
2+
anti-allergic [2],
2+
antiviral & antifungal [74],
+2 2 antibacterial [12],
Quercetin
antioxidant [79],
2+ UV photoprotective [78],
+2 2 anti-inflammatory [77]
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Table 1. Cont.
Group Representative Structure Effect
+2 2
antifungal [76],
Pinocembrin
antioxidant [22]
+2 2
Flavanones
2+
UV photoprotective [80],
+2 2
antioxidant [79],
Naringenin
anti-inflammatory [77],
+2 2
antiviral [74]
2+ antiviral [20],
p-Coumaric
antibacterial [46]
+2
2
Phenolic acids anti-inflammatory [55],
2+ antiviral [20],
Caffeic
antibacterial [46],
+2
antifungal [59]
2+
2+ antibacterial [46],
Ferulic
photoprotective [60]
+2
2 &+
4.6. Bee Venom

Bee venom has been used in medicine in treatment but also as a cosmetic ingredient. Bee venom
has a wide spectrum of biological activity. It exhibit antibacterial and anti-inflammatory effects so it
can be used as a ingredient of anti-acne products. Bee venom shows inhibitory effects on Cutibacterium
acnes. Cutibacterium acnes is the main factor inducing the inflammation in acne. An et al. [81] showed
that topical application bee venom on mice skin, which previous obtained intradermally injected
Cutibacterium acnes into ears, limited number of inflammatory cells and also reduced level of tumor
necrosis factor (TNF)-α and interleukin IL-1β. Moreover, bee venom inhibited Toll like receptor
(TLR2) and CD14 expression in tissue which has been injected C. acnes. These results indicate that
bee venom can be used as anti-acne agent. Another researchers [82] also showed positive effects of
cosmetics containing bee venom on acne vulgaris. Purified bee venom reduced number of C. acnes
at concentration of 0.5 mg. Bee venom possesses bactericidal and bacteriostatic effects thanks to
melittin [38]. It has a significant antibacterial effect against Staphylococcus aureus, Staphylococcus
epidermidis and Staphylococcus pyrogenes [39,83]. Melittin is a toxic peptide that causes destruction of the
bacterial cell wall [38]. Bee venom can be used in fungi and viral skin infections. The antifungal effect
of bee venom against Trichophyton mentagrophytes, Trichophyton rubrum, Candida albicans and Malassezia
furfur was proved [84–86]. Antiviral effect of bee venom on herpes simplex virus has been studied.
Bee venom suppressed the replication this virus [87]. Moreover bee venom is a potential inhibitor
of 5 α-reductase, which is responsible for converse testosterone into dihydrotestosterone and plays
important role as hair growth promoter, what was confirmed in study on alopecia. Bee venom in
different concentrations 0.001%, 0.005% and 0.01% was applied in compare 2% minoxidil. Researchers
showed that bee venom promoted hair growth and inhibited transition from the anagen to catagen
phase. Additionally bee venom inhibited the expression of SRD5A2 which encodes a 5-α-reductase [88].
Bee venom can play role as a new therapy in localized plaque psoriasis. Intradermal bee venom and
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intradermal bee venom combined with oral propolis constitute effective treatment of localized plaque
psoriasis. Bee venom reduces level of IL-1β, TNF-α, and IL-6. Bee venom contains melittin, which
blocks the expression of inflammatory genes. Additionally bee venom inhibits the COX-2 expression,
so decrease production of prostaglandins which take part in inflammatory process [89]. Bee venom
compounds possess various, sometime opposing immune-related effects. Some components of bee
venom like apamin, histamine, mast cell degranulating (MCD) peptide and phospholipase A2 (PLA2)
increase inflammatory response, while polypeptide adolapin inhibits prostaglandins synthesis and
inhibit the activity of bee venom PLA2 and human lipoxygenase [90]. Anti-inflammatory effect of bee
venom is used also in treatment atopic dermatitis. Patients who applied emollient with bee venom
had lower eczema area, severity index and visual analogue scale value than patients who applied
emollient without bee venom [84]. The biological activities of bee venom have been used in wounds
healing. The mechanism of wound healing is associated with expressions of TGF-β1, fibronectin,
vascular endothelial growth factor (VEGF) and collagen-I. The research, which was conducted in mice
showed decreasing of wound size and increasing epithelial proliferation. Topical use of bee venom is
effective especially in reducing size of wounds in animal model [83]. The bee venom is using in wound
dressing combined with polyvinyl alcohol and chitosan. 4% bee venom in wound dressing in diabetic
rats accelerated healing and limited inflammatory process [91]. Another study showed that 6% bee
venom with chitosan supported wound healing [92]. Researchers indicated that bee venom stimulated
human epidermal keratinocyte proliferation and migration. Bee venom joined with hydrogel increased
collagen formation. Bee venom supports wound healing due to its anti-inflammatory, anti-microbial
and also antioxidant activity. Effective action of bee venom is very important in human melanoma
A2058 cells. Tu et al. exhibited that bee venom leads to apoptosis cell death by induction hydroxyl
radicals [93]. Recently bee venom also has been used as antiwrinkle agent. As a cosmetic ingredient
bee venom serum at a concentration of 0.006% was applied at amount 4 mL twice a day for 12 weeks
among twenty-two women from South Korea. It caused decreasing total wrinkle area, total wrinkle
count and wrinkle depth. Moreover bee venom possesses antimelanogenic activity by inhibiting
tyrosinase-related proteins [94]. The study conducted by Han et al. [44] reported that bee venom
exhibits photoprotective activity by reducting of the protein levels of matrix metalloproteinases.
Bee venom effectively inhibits photoaging processes so it can be used for photodamaged skin. Gel
containing 0.06% bee venom did not lead to photosensitive dermatitis what has been confirmed on
animal model [8,44].
5. Allergic Adverse Effects of Bee Products

The use of bee products for cosmetic as well as medicine production can involve the occurrence
of allergic reactions. An allergy to honey is seldom, and the most frequent allergen from honey that
causes hypersensitivity reactions is bee pollen. Additionally, bee protein in honey can cause an allergy.
Honey used to treat dermatoses undergoes thorough filtration to eliminate particles of bee pollen,
which are the main cause of honey allergic reactions. Honey allergy is very rare but sometimes causes
IgE-mediated hypersensitivity reaction [95]. In 2010 Basista conducted studies on beekeepers. None
of them was hypersensitive to honey [96]. More than 26 allergenic substances were determined in
propolis composition. Most frequently, an allergic reaction is caused by esters of caffeic acid and
cinnamic acid derived from poplar buds. In hypersensitive people, they cause a contact allergic
reaction. Due to the presence of these esters in other materials, cross allergic reaction can occur. The
most potent alergens are: LB-1, i.e., the compound consisting of 3-methyl-2-butyl-caffeate (54.2%),
3-methyl-3-butyl-caffeate (28.3%), 2-methyl-2-butylcaffeate (4.3%), caffeic acid (1.3%), benzyl caffeate
(1.0%), caffeic acid phenethyl ester (CAPE, 7.9%) and benzyl salicylate. An allergy to propolis is rare,
and an allergic response was more frequently reported after topical application than an oral one. In
the years 1989–2006, the World Health Organization registered only 26 notifications about side effects
after the contact with bee glue, of which just six were considered certain, and the remaining ones
were not fully credible. In healthy individuals, an allergy to propolis is rarely observed (0.64–1.3%),
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however, it occurs more frequently in people treated for allergies (1.2–6.7%). This hypersensitivity
is manifested by atopic eczema after the application of ethanol extract of propolis [97]. Moreover,
topical application of royal jelly in the form of ointments can cause skin rashes and eczemas [67].
Allergic and irritation reaction of bee venom have been associated with presence components likes:
phospholipase A2, melittin, hyaluronidase. Phospholipase A2 is a major allergen which is responsible
for inducing immuno-globulin E (IgE) [98]. Melittin causes cell lysis and fusion in addition to activation
of phospholipase A2. Hyaluronidase is a next allergen in venom, which is responsible for changes
in cell membranes. It caused spread of venom toxin through the gaps between cells. However, bee
venom can be toxic when large amount of venom is inoculated into body [98]. However, Han et al.
indicates that long term topically treatment with bee venom is safe what confimed their study [94].
6. Conclusions
Bee products constitute an important component of medicines and cosmetics. Honey is
regenerative and antimicrobial due to its high osmolarity, the presence of hydrogen peroxide and
lysozyme. Manuka honey thanks to the presence of methylglyoxal is a potent antiseptic agent. Propolis
is a bee product rich in phenolic compounds, which determine antimicrobial, UV protective, analgesic,
antioxidative and regenerative activities. Royal jelly is characterized by the presence of royalisin and
jelleines peptides. It also contains 10-hydroxy-trans-2-decenoic acid which improves the production
of collagen and is antiseptic. Bee pollen is rich in unsaturated fatty acids, vitamins, flavonoids and
hydroxy acids. Beeswax plays the most important role as emulsifier of the cosmetic forms. Moreover,
bee venom is an attractive and effective natural toxin rich in peptides. It plays an important role
in treatment and care skin especially in photodamage, acne, atopic dermatitis, alopecia or psoriasis.
Bee venom exhibits anti-inflammatory, antimicrobial, antifungal and antiviral action. Each of the bee
products is characterized by the content of certain active substances, which differentiates one bee
product from another, and causes that each of them is worth using for a different skin problem. The
effect of bee products on the skin has been proved by numerous studies, whose results are satisfactory,
and the use of these product in wound healing highlights their curative value. The advantage of
medicines and cosmetics based on bee products is their effectiveness with minimal side effects. Table 2
summarizes skin diseases where the therapeutic application of bee products has been studied.
Table 2. The summary of the skin diseases where the therapeutic application of bee products has
been studied.
Bee Product Components Effect Disease Reference

pinocembrin, lysozyme antifungal tinea [13,46,64]
methylglyoxal, defensin-1 peptide,
antibacterial wounds, burns, ulcers [13,16,45,46,50]
lysozyme, glucose oxidase, phenolic acids
Honey
fruits acids, sugars exfoliating wrinkles [13]
quercetin, naryngenin, kaempferol, chrysin anti-inflammatory wounds, gum inflammation [49,52]
carbohydrates, fruit acids, trace elements regenerative wounds [40,49,52,53]
chrysin analgesic wounds [3]
caffeic acid, quercetin anti-inflammatory wounds [19,55]
pinocembrin, galangin, caffeic acid antibacterial acne, wounds [19,57,65,66]
pinocembrin, pinobanksin, quercetin,
kaempherol, caffeic acid, p-coumaric acid, antifungal tinea, fungal infections [19,59]
terpenes,
Propolis infection of Herpes simplex
galangin, kaempferol, quercetin antiviral [62,63]
virus
caffeic acid, p-coumaric acid, ferulic acid,
photoprotective photoaging [60]
quercetin, kaempferol
phenolic acids, flavonoids antiaging wrinkles [59]
stimulates
genistein diabetic wound [56]
angiogenesis
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Table 2. Cont.
Bee Product Components Effect Disease Reference

wounds, diabetic foot ulcers,
defensin-1 peptid, ferulic acid antibacterial [32,67,68]
acne
10-hydroxydecanoic acid,
atopic dermatitis, wounds,
3-10-dihydroxydecanoic acid, amino, antiinflammatory [67]
Royal jelly hypertrophy, hyperkeratosis
gamma globulin
10-hydroxy-trans-2-decenoic acid,
antiaging wrinkles [67]
10-hydroxydecanoic acid
10-hydroxydecanoic acid, immunomodulatory autoimmune and
[70]
3-10-dihydroxydecanoic acid and antiallergenic inflammatory diseases
pinocembrin, apigenin, quercetin,
antifungal tinea [2,24,43]
kaempferol, ferulic acid, p-coumaric acid
kaempferol, phenolic acids antimicrobial burns [2,6,24,43]
Bee pollen
phenolic acids, fatty acids, phytosterols,
antiinflammatory atopic dermatitis, burns [2,43]
kaempferol, quercetin
methionine, zinc, phospholipids sebo-balancing acne [43]
squalene, 10-hydroxy-trans-2-decenoic acid, wounds, atopic dermatitis,
antibacterial [34,36]
chrysin psoriasis
Beeswax reduce
sterols transepidermal atopic dermatitis [34]
water loss
melittin antimicrobial wounds, acne [39,81–83,92]
melittin, apamin antifungal tinea [84–86]
melittin antiviral herpes simplex infections [84–87]
photoprotective,
not reported hiperpigmentation [44,94]
Bee venom antimelanogenic
plaque psoriasis, wounds,
melittin, adolapin antiinflammatory [83,84,89,91,92]
atopic dermatitis
phospholipase A2 pigmentation effect vitiligo [39]
promote hair
not reported alopecia [88]
growth
not reported antiwrinkle wrinkles [44,94]
Author Contributions: A.K.-G. designed the review; coordinated and participated in the writing of all sections
and wrote Abstract, Sections 1–3, Sections 4.1, 4.6 and 6 and collaborated in the creation of Table 1; M.G. wrote
Section 4.3 and created Tables 1 and 2 and collaborated in the writing of Section 4.2; A.R.-S. wrote Section 4.2 and
collaborated in the writing of Section 5; R.B. wrote Sections 4.4 and 4.5; J.S. wrote the Section 5. All authors were
involved in the editing process. All authors have read and agreed to the published version of the manuscript.
Funding: This work was funded by medical University of Silesia, Katowice, Poland grant number
KNW-1-163/N/9/O.
Acknowledgments: The authors thanks to medical University of Silesia, Katowice, Poland and Silesian Academy
of Medical Sciences in Katowice for their financial support.
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341
molecules
Article
Ulva intestinalis Protein Extracts Promote In Vitro
Collagen and Hyaluronic Acid Production by Human
Dermal Fibroblasts
Justine Bodin 1,2 , Amandine Adrien 2 , Pierre-Edouard Bodet 1 , Delphine Dufour 2 ,
Stanislas Baudouin 2 , Thierry Maugard 1 and Nicolas Bridiau 1, *
1 Equipe BCBS (Biotechnologies et Chimie des Bioressources pour la Santé), La Rochelle Université, UMR
CNRS 7266 LIENSs, Avenue Michel Crépeau, 17042 La Rochelle, France; justine.bodin1@univ-lr.fr (J.B.);
pierreedouard.bodet@univ-lr.fr (P.-E.B.); thierry.maugard@univ-lr.fr (T.M.)
2 SEPROSYS, Séparations, Procédés, Systèmes, 12 Rue Marie-Aline Dusseau, 17000 La Rochelle, France;
amandine.adrien@seprosys.com (A.A.); delphine.dufour@seprosys.com (D.D.);
stanislas.baudouin@seprosys.com (S.B.)
* Correspondence: nicolas.bridiau@univ-lr.fr; Tel.: +33-5-46-45-87-91; Fax: +33-5-46-45-82-65

Received: 1 April 2020; Accepted: 21 April 2020; Published: 30 April 2020
Abstract: With the increase in life expectancy, reducing the visible signs of skin aging has become a
major issue. A reduction in collagen and hyaluronic acid synthesis by fibroblasts is a feature of skin
aging. The green seaweed, Ulva intestinalis, is an abundant and rich source of nutrients, especially
proteins and peptides. The aim of this study was to assess the potential cosmetic properties of a
protein fraction from Ulva intestinalis (PROT-1) containing 51% of proteins and 22% of polysaccharides,
and its enzymatic peptide hydrolysates on human dermal fibroblasts. PROT-1 was extracted using
a patented acid- and solvent-free process (FR2998894 (B1)). The biochemical characterization and
chromatographic analysis showed a main set of proteins (25 kDa). To demonstrate the anti-aging
potential of PROT-1, fibroblast proliferation and collagen and hyaluronic acid production were
assessed on fibroblast cell lines from donors aged 20 years (CCD-1059Sk) and 46 years (CCD-1090Sk).
PROT-1 induced a significant increase in collagen and hyaluronic acid production per cell, and a
reduction in cell proliferation without increasing cell mortality. These effects were reversed after
protein hydrolysis of PROT-1, showing the central role of proteins in this promising anti-aging property.
Keywords: Ulva intestinalis; seaweed proteins; human dermal fibroblast; collagen; hyaluronic acid;
anti-aging
1. Introduction
In recent years, people have started to pay more attention to skin health and beauty. The cosmetic
industry is growing and many studies have focused on skin aging inhibition and delay. Wrinkles,
laxity and dryness are features of aging [1]. Skin aging is a complex biological process that can be
divided into two basic processes, intrinsic and extrinsic aging. Intrinsic aging is genetically inherited
(genetic, hormonal and metabolic processes, cellular metabolism); extrinsic aging is due to external
factors such as air pollution, toxins, nicotine consumption, lifestyle influence, chemicals and chronic
light exposures, which contribute to accelerate its consequences [2]. One of the major consequences
of skin aging is a reduction and alteration of extracellular matrix (ECM) components such as elastin,
collagen and hyaluronic acid (HA) [3]. For this reason, maintaining collagen and HA levels in the
dermis is essential to maintain a healthy skin.
HA is an ECM anionic glycosaminoglycan composed of d-glucuronic acid and N-acetyl-d-
glucosamine, synthesized by fibroblasts and keratinocytes [4]. It plays an important role in normal

Molecules 2020, 25, 2091
dermis, of water retention and activation of tissue/fibroblast proliferation. A decrease in HA level

results in impaired hydration and elastic properties of the skin [5].
Collagen is the main structural protein in the extracellular space. There are several types of
collagen: in the dermis, type 1 collagen represents most of the total collagen and is involved in
skin tension, elasticity and flexibility. The type 1 collagen subunit is a fibril-forming heterotrimeric
protein composed of two alpha-1 chains and one alpha-2 chain which fold into a stable and highly
ordered triple helix [6]. Collagen degradation is related to the expression of fibroblast-secreted matrix
metalloproteinases (MMP). Indeed, during the aging process, the MMP expression increases, causing
collagen degradation and reducing skin firmness [7,8].
The cosmetic industry is constantly looking for novelty, especially atypical molecules. From this
point of view, the marine environment is likely to open up many new possibilities [9]. Marine macroalgae
are abundant and sustainable sources of compounds for nutraceutical and cosmetic applications [10].
Among these interesting biomolecules, seaweed proteins have a high potential. The protein content
of seaweeds depends on and differs according to the species, season and environmental growth
conditions. Some seaweeds such as Rhodophyta or Chlorophyta contain high protein levels and are a
potential renewable source to isolate biomolecules with a nutritional interest [11,12]. Green seaweeds
of the Ulva genus can contain up to 44% of proteins [13]. Ulva species also contain a high level of
carbohydrates and are rich sources of vitamins and minerals [14]. Green seaweeds have various
biological activities, including anticoagulant, anti-aging, antimicrobial and antifungal, antioxidant,
mitogenic, immunomodulatory, anti-cholesterol and many other potential activities [15–17]. Among
the Ulva genus, the edible macroalga Ulva intestinalis contains 19.5% of proteins during summer [18].
In addition, it has been shown that a protein hydrolysate derived from Ulva lactuca exhibited an
anti-aging activity. This protein hydrolysate, which was composed of numerous amino acids, including
lysine, histidine, arginine, aspartic acid, proline, glycine, serine, glutamic acid, alanine, threonine,
tyrosine, isoleucine, leucine, and phenylalanine, optimized cellular respiration, stimulated collagen
production in the skin and inhibited elastase activity [19].
In this study, we used a previously developed acid-free and solvent-free procedure to extract and
purify proteins from Ulva intestinalis (Scheme 1) [20]. Then, we developed a hydrolysis procedure
of the obtained protein fraction (PROT-1) to produce a protein fraction of reduced molecular weight
(MW), H-PROT-1, in order to assess the influence of MW on collagen and hyaluronic acid production
by normal human dermal fibroblast (NHDF) cell lines from donors aged 20 and 46 years.
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Molecules 2020, 25, 2091
Scheme 1. Different steps and yield (η) of the extraction procedure developed by SEPROSYS [20].
2.1. Production and Characterization of Protein Fraction (PROT-1) and Protein Hydrolysates (H-PROT-1)
Ulva intestinalis material was shown to contain 26 ± 2.8% (w/wdry material ) of proteins, as determined
by the Kjeldahl method, which was within the high values reported for green seaweeds of the Ulva
genus (7–33% (w/wdry material )) [21].
The protein fraction PROT-1 was extracted by following the patented Seprosys process illustrated
on Scheme 1 [20]. The extracted protein fraction represented 4% (w/wdry material ) (21 g) of the desalinated
dry macroalgae (on 500 g of algae). The efficiency of the aqueous and solvent-free protein extraction
differs depending on the starting sample and protein accessibility. Indeed, some components such as
polysaccharides may interfere with algae protein extract. The yield obtained here was in the average of
the yields of protein aqueous extraction found in the literature [22].
The total amino acid composition of PROT-1 was determined after acid hydrolysis under strong
acidic conditions at 100 ◦ C for 2 h, using UHPLC-HRMS analysis of the amino acid mixture obtained
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Molecules 2020, 25, 2091
after neutralization. Figure 1 shows that the fraction was mainly composed of leucine (27.2%), isoleucine
(23.3%) and valine (28.4%). According to Lewis et al., major amino acids composing Ulva intestinalis
seaweeds are proline, methionine and aspartic acid [23], which is different from what we found.
However, amino acid composition of the alga can drastically vary with respect to the season, period or
geographic place of harvest, but also to its growth stage [24], which could explain these differences.
Arg Tyr Gly

Met
0,1% 2,9% <0.08% Ala
2,6% <0.7%
His
Glu <0.08%
1,1% Pro
Lys Phe 5,2%
0,5% 6,9%
Asp
<0.5% Val
28,4%
Leu
27,2%
Thr
<0.5%
Ile
23,3%
Figure 1. Total amino acids composition of the PROT-1 fraction determined after acid hydrolysis and
UHPLC-HRMS analysis.
The PROT-1 fraction was also enzymatically-hydrolyzed by using the protease preparation
Flavourzyme® , in order to obtain a fraction containing low MW peptides, thereafter referred as
PROT-1 hydrolysates (H-PROT-1 (t2h), (t4h), (t6h), (t24h)). The enzymatic hydrolysis procedure was
optimized to determine the optimal conditions (data not shown). In order to follow the time-course of
hydrolysis, a monitoring method involving a separation by high-performance liquid size exclusion
chromatography (HPL-SEC) was developed, and the MW of protein and peptide sets was estimated
using standards of known MW. Figure 2 shows the enzymatic hydrolysis time-course of the PROT-1
fraction. The analyses showed that this fraction contained a major set of proteins with a MW of about
25 kDa, eluted at 12 min, and a set of proteins with a MW higher than 400 kDa, eluted at 7 min.
During the hydrolysis, a shifting of the main peak towards higher retention times was observed, which
reflected a decrease in molecular weight. For example, at t1h, two major peaks at 13.4 and 13.9 min
corresponding to sets of proteins of 7.2 and 5.1 kDa, respectively, were observed. Furthermore, the
profile observed after 6 h of depolymerization was probably the consequence of a profound enzymatic
hydrolysis of peptides, causing the liberation of amino acids that were not or not much detected on the
chromatograms, due to their very low molar extinction coefficient compared to polypeptides.
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Molecules 2020, 25, 2091
Figure 2. Standard curve and time-course HPLC-SEC profiles of PROT-1 enzymatic hydrolysis catalyzed
by the protease preparation Flavourzyme® .
The PROT-1 fraction contained 51% of proteins and 22% of polysaccharides (Table 1). The presence
of polysaccharides may be explained by two reasons: the presence of a cell wall mucilage containing
polysaccharides [25] and the well-known presence of glycoproteins in algae of the Ulva genus [26].
The protein purity of the fraction could be improved by controlling the presence and composition
of anionic and neutral polysaccharides composing the mucilage. To characterize PROT-1 and its
hydrolysates obtained after enzymatic hydrolysis, all samples were analyzed using SDS-PAGE (Figure 3).
Lane 1 contains the size markers and lane 2 contains PROT-1. The visible trail in lane 2 indicates
a polydisperse set of proteins, very likely due to the partial hydrolysis of proteins that could occur
during the extraction step under harsh conditions, especially during flocculation. This protein trail
disappeared in accordance with the time-course of the enzymatic hydrolysis, showing its efficiency.
The presence of a band at 25 kDa at t2h is in line with the results reported by Rouxel et al. on the
existence of a water-soluble protein with a MW of 25 kDa in Ulva intestinalis [27]. The intensity of
this band at 25 kDa and others corresponding to low MW (around 10 kDa) decreased throughout
hydrolysis, which is consistent with the HPLC-SEC analyses.
Table 1. Biochemical composition of the PROT-1 fraction.
Ashes Proteins Neutral Sugars Uronic Acids Lipids Polyphenols

Fraction
% (w/wdry extract )
PROT-1 20 51 22 6 <1 <1
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Molecules 2020, 25, 2091
Figure 3. SDS-PAGE analysis of the PROT-1 fraction followed during enzymatic hydrolysis catalyzed
by the protease preparation Flavourzyme® .
Table 2 lists the peptides that were identified in the H-PROT-1 (t1h) fraction using UHPLC-HRMS.
88 peptides were identified, more precisely 14 dipeptides, 4 tripeptides, 17 tetrapeptides, 14 pentapeptides,
and 39 peptides of 6 to 15 amino acids. The amino acid composition of these 88 peptides was then
determined according to their sequences and showed that these peptides were mostly composed of
leucine or isoleucine (17.1%), alanine (9.7%), and glycine, glutamic acid, aspartic acid, tyrosine, threonine
and serine in similar proportions, between 7.1% and 7.9% (Figure 4).
Trp
0,5%
Tyr Ala
7,4% 9,7%
Val
5,5% Asp
7,9%
Thr
7,9%
Glu
7,6%
Ser
7,1% Phe
3,7%
Gln
3,7% Gly
Pro 7,6%
5,3%
Asn
Met 6,1% Leu/Ile
0,3% 17,1% His
Lys
0,8%
1,8%
Figure 4. Amino acid composition of the 88 peptides identified in the H-PROT-1 (t1h) fraction
by UHPLC-HRMS.
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Molecules 2020, 25, 2091
Table 2. Peptide sequences identified by UHPLC-HRMS in the H-PROT-1 (t1h) fraction.
Retention Experimental Mass Accuracy

Entry Ion Peptide or Amino Acid
Time (min) Monoisotopic Mass (Da) (ppm)
1 2.58 [M + H]+ 233.1130 0.86 VD
2 3.20 [M + H]+ 508.1899 2.56 EGESS
3 3.81 [M + H]+ 247.1284 2.02 VE
4 4.16 [M + H]+ 246.1456 2.84 I/LN
5 5.65 [M + H]+ 247.1289 2.02 I/LD
6 5.65 [M + H]+ 247.1294 2.02 EV
7 6.67 [M + H]+ 294.1545 2.72 I/L-hexose
8 8.21 [M + H]+ 261.1452 0.77 I/LE
9 8.81 [M + H]+ 261.1452 0.77 I/LE
10 9.57 [M + H]+ 590.2798 2.88 ETVNQ
11 9.80 [M + H]+ 404.2132 1.98 I/LSAN
12 10.40 [M + H]+ 419.2135 0.00 TVAE
13 10.70 [M + H]+ 449.2243 1.56 TEVT
14 10.93 [M + H]+ 328.1394 0.61 F-hexose
15 11.32 [M + H]+ 418.2293 0.96 I/LSAQ
16 11.52 [M + H]+ 555.2410 0.18 TGYTN
17 11.52 [M + H]+ 203.14 4.43 AI/L
18 11.83 [M + H]+ 546.2520 0.18 DAI/LNN
19 12.46 [M + H]+ 502.2625 1.00 AI/LNGQ
20 12.66 [M + H]+ 295.1294 1.69 FE
21 12.87 [M + H]+ 475.2515 0.84 AI/LSNA
22 13.41 [M + H]+ 367.1506 0.27 W-hexose
23 14.27 [M + H]+ 350.1740 1.43 I/LMS
24 14.48 [M + H]+ 526.2163 4.18 SAYDA
25 14.48 [M + H]+ 526.2163 4.18 * DADA
26 15.29 [M + H]+ 440.1667 0.68 EEY
27 16.18 [M + H]+ 403.2188 0.07 DVVA
28 16.65 [M + H]+ 237.1241 3.08 AF
29 17.22 [M + H]+ 205.0982 2.44 W
30 17.60 [M + H]+ 229.1552 2.18 I/LP
31 18.01 [M + H]+ 492.2662 0.61 SI/LTTA
32 19.09 [M + H]+ 458.2607 0.59 I/LPQT
33 19.09 [M + H]+ 458.2607 0.59 QPI/LT
34 19.98 [M + H]+ 295.1659 2.03 I/LY
35 21.39 [M + H]+ 302.2077 0.66 I/LI/LG
36 21.76 [M + H]+ 416.2517 1.20 * GSI/LA
37 21.76 [M + H]+ 416.2517 1.20 NVI/LA
38 22.01 [M + H]+ 409.2078 1.00 VGAY
39 22.01 [M + H]+ 409.2078 1.00 I/LNY
40 22.25 [M + H]+ 552.2316 1.81 EAEFG
41 22.64 [M + H]+ 411.7227 6.07 YTI/LDPP *
42 22.64 [M + H]+ 295.1654 0.34 YI/L
43 23.03 [M + 2H]2+ 720.3186 0.99 QAAEAAESGDFKSE
44 23.25 [M + 2H]2+ 841.8622 - * QSDWSEAEAAHS
45 23.45 [M + H]+ 777.3397 0.40 NQAAEAAESGDESKF
46 23.65 [M + H]+ 459.2179 3.64 EEYTFTK
47 23.65 [M + H]+ 459.2179 3.64 KFTTYEE
48 23.65 [M + H]+ 459.2179 3.64 KFYTTEE
49 23.85 [M + H]+ 550.2878 1.13 (I/L)GTYP
50 24.12 [M + H]+ 790.4323 2.21 TI/LATI/LSNA
51 24.32 [M + H]+ 329.1500 1.28 YF
52 24.53 [M + H]+ 373.2276 3.22 KDTVI/LI/LG
53 24.53 [M + 2H]2+ 766.8573 - * KDI/LI/LI/L *
54 25.18 [M + H]+ 401.2756 0.72 VVAI/L
55 25.18 [M + H]+ 600.3360 1.33 SPGGI/LGI/L
56 25.18 [M + H]+ 600.3360 1.33 SNI/LPGI/L
57 25.58 [M + H]+ 873.3639 1.57 (YG)YGDI/LDA
58 25.58 [M + H]+ 873.3639 1.57 (YG)GI/LDYDA
59 25.58 [M + H]+ 873.3639 1.57 (YG)DI/LGYDA
60 25.58 [M + H]+ 873.3639 1.57 (YG)DYGI/LDA
61 25.58 [M + H]+ 873.3639 1.57 (DI/L)GYGYDA
62 25.86 [M + H]+ 687.2986 0.17 DI/LGYGY
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Table 2. Cont.
Retention Experimental Mass Accuracy

Entry Ion Peptide or Amino Acid
Time (min) Monoisotopic Mass (Da) (ppm)
63 26.07 [M + H]+ 579.3143 0.98 NVI/LAY
64 26.07 [M + 2H]2+ 590.7799 3.71 QEYENI/LI/LGSQ
65 26.07 [M + H]+ 555.2821 1.31 YI/LYP
66 26.73 [M + H]+ 431.2497 0.86 DI/LAI/L
67 27.04 [M + H]+ 401.2756 0.72 I/LVGI/L
68 24.69 [M + 2H]2+ 604.7886 7.87 DSTWI/LTTAI/LSN
69 28.26 [M + 2H]2+ 566.2830 5.14 SG(Q/AG)QEI/LI/LDI/LE
70 28.26 [M + 2H]2+ 566.2830 5.14 QSGQEI/LI/LDI/LE
71 28.26 [M + 2H]2+ 566.2830 5.14 TNGQEI/LI/LDI/LE
72 28.46 [M + H]+ 726.4041 1.13 EI/LSPPI/LA
73 28.46 [M + H]+ 726.4041 1.13 ESI/LPPI/LA
74 28.46 [M + H]+ 726.4041 1.13 DTI/LPAI/LP
75 30.26 [M + H]+ 577.3329 2.72 AFI/LI/LN
76 30.47 [M + H]+ 645.4180 4.34 TI/LI/LVI/LS
77 30.47 [M + H]+ 645.4180 4.34 I/LI/LTVI/LS
78 30.47 [M + H]+ 645.4180 4.34 VI/LTI/LI/LS
79 30.88 [M + H]+ 938.4491 2.66 EI/LTDTVNF
80 30.88 [M + H]+ 938.4491 2.66 ETI/LDTVNF
81 31.35 [M + 2H]2+ 640.3069 4.08 EGESSI/LTTAI/LSNA
82 31.35 [M + 2H]2+ 640.3069 4.08 E(SG/DG/TG)ESI/LTTAI/LSNA
83 31.89 [M + 2H]2+ 616.3179 6.44 (AP)EI/LI/LTVDYNP
84 31.89 [M + 2H]2+ 616.3179 6.44 PVVDI/LTVDYNP
85 32.13 [M + H]+ 558.3867 0.95 I/LTVI/LI/L
86 32.50 [M + H]+ 746.4453 0.74 PI/LI/LGI/LYA
87 32.70 [M + H]+ 890.4997 - * I/LTI/LP
88 32.70 [M + H]+ 890.4997 - * YEI/LP
89 32.70 [M + H]+ 675.4078 0.25 I/LPGI/LI/LY
90 32.70 [M + H]+ 675.4078 0.25 I/LPI/LGI/LY
91 33.17 [M + 2H]2+ 634.3411 4.10 SI/LDI/LPHI/LPTQF
92 33.37 [M + 2H]2+ 691.8536 3.45 SI/LDI/LDPHI/LPTQF
* undetermined amino acids; () undetermined sequence.
2.2. Assessment of PROT-1 and H-PROT-1 Cytotoxicity on NHDF

The aim of this part was to assess the cytotoxicity of the PROT-1 and H-PROT-1 fractions (t2h,
t4h, t6h and t24h) on NHDF at 10, 100 and 500 μg/mL. Two fibroblast cell lines were used: one from a
20-year old woman (line 1059) and one from a 46-year old woman (line 1090). These two cell lines
were used to determine whether the effects could vary according to the age of the donor skin.
Figure 5 shows the effect of PROT-1 and H-PROT-1 on fibroblast viability. PROT-1 decreased
fibroblast proliferation, reducing by 18%, 28% and 42% the proliferation of the 1059-cell line at 10,
100 and 500 μg/mL, respectively. The effect was less marked for the 1090-cell line as there was no effect
on cell proliferation at 10 and 100 μg/mL but a decrease of 53% was observed at 500 μg/mL. Thus,
a slight difference was observed between both cell lines. The 1059-cell line was rapidly affected by the
extract, at a lower concentration than the 1090 cell line. The effect of the low MW peptide fractions
H-PROT-1 (t2h, t4h, t6h and t24h) on fibroblast proliferation was lower compared to that of PROT-1 on
both cell lines. Indeed, in the wells treated with H-PROT-1 (t24h), the cell viability reached 79% for the
1059-cell line and 78% for the 1090-cell line. These results showed that decreasing the MW of PROT-1
proteins led to a reduced effect on cell proliferation for both cell lines.
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Figure 5. Effect of PROT-1 and H-PROT-1 on fibroblast proliferation in vitro (MTT assay). Results
are expressed as the mean relative percentage of viable fibroblasts compared to the negative control
(100%). Significant differences between values obtained with samples and negative control (n = 20) are
indicated by * (p < 0.05), ** (p < 0.01) and *** (p < 0.001).
Figure 6 shows the effect of the PROT-1 fraction and H-PROT-1 hydrolysates on the cell mortality
by necrosis, compared to the negative control. PROT-1 had no effect on cell mortality while a slight
decrease in cell mortality was observed with H-PROT-1 at t2h, t4h and t6h: −8% (cell line 1059) and
−7% (cell line 1090) for example at t4h. Except for the highest concentration of PROT-1, 500 μg/mL,
which led to a very slight increase in cell mortality (+7% for the 1059-cell line and +9.5% for the
1090-cell line), no deleterious effect was observed on fibroblasts.
Overall, PROT-1 decreased cell proliferation without increasing cell mortality by necrosis. Besides,
we did not observe any alteration of the fibroblast morphology when checking the cells with optical
microscopy. PROT-1 thus appeared to have a cytostatic effect on both cell lines in a similar way, but
this effect was reduced after enzymatic protein hydrolysis. This would therefore indicate that the
molecular weight or the distinctive structure of some PROT-1 proteins is related to this cytostatic effect.
Figure 6. Effect of PROT-1 and H-PROT-1 on fibroblast mortality in vitro (LDH assay). Results are
expressed as the mean relative percentage of living fibroblasts compared to the negative control (0%).
Significant differences between values obtained with samples and negative control (n = 8) are indicated
by * (p < 0.05).
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2.3. Effect of PROT-1 and H-PROT-1 on Collagen Production

In this part of the study, the effect of the PROT-1 and H-PROT-1 fractions on collagen production
was assessed. Hyaluronic acid (HA) was used as a positive control. Indeed, HA is recognized by
CD44 receptors on the cell surface, which activates signaling pathways involved in the stimulation of
fibroblast proliferation and collagen production [28,29].
The protein fractions showed a higher activity than the positive control (Figures 7 and 8).
More specifically, at a HA concentration of 1000 μg/mL, the collagen production per well was increased
by 30.5% for the 1059 cell line (Figure 7) and by 28.5% for the 1090 cell line (Figure 8), while PROT-1
induced a significant dose-dependent rise in collagen production per well by 51.9% and 151.8% at
100 and 500 μg/mL, respectively, for the 1059 cell line, and by 52.5% and 79.5% at 100 and 500 μg/mL,
respectively, for the 1090 cell line. However, the reduction of PROT-1 proteins MW led to a strong
decrease in collagen production. Indeed, after 24 h of hydrolysis, the collagen production per well did
not significantly differ from the control conditions.
Figure 7. Collagen production per well and per cell by the human dermal fibroblast cell line 1059.
Results are expressed as the mean relative percentage of production, compared to the negative control
Figure 8. Collagen production per well and per cell by the human dermal fibroblast cell line 1090.
Results are expressed as the mean relative percentage of production, compared to the negative control
We also studied the collagen production per cell. In the presence of HA at a concentration of
1000 μg/mL, the collagen production per cell increased in a similar way with both cell lines, by about
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80%. The results obtained with the PROT-1 fraction were very interesting as they showed a very high
dose-dependent increase in collagen production per cell, compared to the negative control: +55%,
+128% and +331% for the 1059 cell line (Figure 7) and +13%, +76% and +371% for the 1090 cell line
(Figure 8), at 10, 100 and 500 μg/mL, respectively. It is noteworthy, however, that this pro-collagen
activity was lost when hydrolyzing proteins in the fraction PROT-1, particularly for the 1059 cell line.
Indeed, the production of collagen per cell did not significantly increase in presence of H-PROT-1 at
t24h. This effect seemed even closely related to the degradation of proteins in the H-PROT-1 fraction as
the pro-collagen activity of the PROT-1 fraction decreased in accordance with the time-course of the
enzymatic hydrolysis reaction. These results suggest that the native structure of the proteins that are
responsible for the pro-collagen activity of the PROT-1 fraction is essential. The results obtained with
the 1090 cell line were very similar, except that this cell line remained slightly more stimulated by the
H-PROT-1 fractions.
These results are of particular interest as the pro-collagen activity of the PROT-1 and even
H-PROT-1 fractions appeared to be superior to that of hyaluronic acid, which is a pro-collagen active
compound usually used in anti-aging skin care cosmetic formulations [30,31]. Furthermore, this activity
could be explained by the induction of a cell metabolic redirection, which would also be in accordance
with the cytostatic effect of the fractions that we previously observed. It means that fibroblast moved
towards collagen biosynthesis pathway(s) rather than cell growth. Indeed, fibroblasts underwent a
rapid decrease in cell viability but no significant change in cell mortality and morphology. Kmail et al.
showed a very similar behavior of hepatic macrophages in contact with extracts from Asparagus
aphyllus, Crataegus aronia, and Ephedra alata, which induced a significant cytostatic effect on macrophage
cultures, highlighted by MTT and LDH tests. These authors observed a decrease in cell viability but no
cytotoxicity of these antidiabetic extracts [32].
The very significant decrease in the pro-collagen activity that was observed with the hydrolyzed
protein fractions confirmed that the proteins were the molecules responsible for the collagen synthesis
stimulation in PROT-1. This result was consistent with what Ko et al. highlighted when assessing
the effect of a protein extract from Ulva pertusa on the proliferation and type I collagen synthesis of
replicative senescent fibroblasts [33], as well as the pro-collagen activity of a protein extract from Ulva
lactuca, AOSAINE® , developed by BiotechMarine. It was also consistent with the work of Montanari and
Guglielmo, who reported a tripeptide of sequence Lys-Val-Lys, which was shown to promote collagen
synthesis by human fibroblasts by 75% when associated to an Ulva lactuca aqueous extract [34]. On the
contrary, our results differed from what the same authors found when they extracted water-soluble
proteins from Ulva lactuca and produced a polypeptide containing the sequence Arg-Gly-Asp, after
depolymerization of these proteins. This polypeptide was shown to promote collagen I production
by stimulating the proliferation of fibroblasts and not collagen biosynthesis. Similarly, Honma et al.
demonstrated that peptide sequences Leu-Glu-His-Ala, Leu-Asp-His-Ala or Leu-Glu-His-Ala-Phe,
could promote extra-cellular collagen production by NHDF [35]. Nevertheless, the mechanisms of
actions involved in the activities of these peptides or proteins remain unclear. The protein extract from
Ulva pertusa was proved to directly inhibit MMP-1, which could explain its activity by preventing
type I collagen degradation. Joe et al. reported that an extract from Ecklonia stolonifera, containing
phlorotannins, could inhibit NF-kB or Ap-1 reporter gene expression and therefore suppress the
expression of MMP-1 in NHDP, which led to the increase in collagen production [36]. A very similar
activity was also shown with a peptide from Chlorella vulgaris [37]. At last, a pentadecapeptide from
Pyropia yezoensis (Asp-Pro-Lys-Gly-Lys-Gln-Gln-Ala-Ile-His-Val-Ala-Pro-Ser-Phe), was shown to be
able to activate the TGF-β/Smad signaling pathway, leading to increased type 1 collagen expression and
upregulated transcription factor specificity protein 1 (Sp1) expression, which is reportedly involved in
type 1 collagen expression [38].
None of these peptide sequences was found in the 88 peptide sequences identified in the H-PROT-1
(t1h) fraction, obtained after 1 h of PROT-1 hydrolysis by the enzyme preparation Flavourzyme® .
Nevertheless, one tripeptide sequence Thr-Val-Asn, including a central valine and two external polar
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amino acids, was found in the peptides eluted at 9.57 and 30.88 min (entries 10, 79 and 80 in Table 2).
This sequence is similar to that of the tripeptide Lys-Val-Lys, which was proved to exhibit a pro-collagen
activity by Montanari and Guglielmo [34]. Besides, two tripeptide sequences very close to tripeptide
sequences included in the pentadecapeptide from Pyropia yezoensis ( . . . -Asp-Pro-Lys- . . . and . . .
-Gln-Ala-Ile- . . . ) were found in the peptides eluted at 23.03, 23.45 and 33.37 min (entries 43, 45 and 92
in Table 2): Asp-Pro-His and Gln-Ala-Ala, respectively. Indeed, the only variation in the sequences
is located on the third amino acid, which is very similar in terms of property: another basic amino
acid for the first one and another hydrophobic amino acid for the second one. However, despite these
similarities, this does not mean that polypeptides or proteins exhibiting these sequences in the PROT-1
fraction are responsible for its pro-collagen activity, as biological functions of tripeptide sequences
occurring in large peptide structures would not be necessarily the same as those of smaller peptides.
Further studies involving the assessment of these particular peptides or proteins after purification are
needed to understand their structure–function relationship.
The MMP-1 inhibition potential of the PROT-1 fraction was also evaluated but showed no
significant inhibition (data not shown). In conclusion, the increase in collagen production induced
by this fraction was probably not due to a decrease in the extracellular fibrillar collagen degradation
directly associated with MMP-1 inhibition, contrary to what was shown by Ko et al. in their study
of an Ulva pertusa protein extract [33]. The increase in collagen production might therefore be linked
to the effect of the PROT-1 fraction on an intra-cellular mechanism, e.g., a biosynthesis pathway
such as NF-kB or TGF-β/Smad signaling pathways, chaperone synthesis pathway (maintenance of
conformation) or inhibition of MMP synthesis.
2.4. Effects of PROT-1 and H-PROT-1 on Hyaluronic Acid Production

Moisturization is the first step to fight against skin aging, by improving elastic properties of the
skin [5]. A major component of the skin ECM, hyaluronic acid, plays a key role in skin moisturization
by retaining water in the dermis, and thus lubricating the skin [39].
In this part of the study, the effects of PROT-1 and H-PROT-1 on the HA production by both the 1059
and 1090 fibroblast cell lines were investigated by an ELISA-like assay. TGF-β is known to stimulate
HA production by dermal fibroblasts [40] and was then used as a positive control. Figures 9 and 10
show that the HA production of both cell lines treated with the PROT-1 fraction was very significantly
increased, compared to the negative control, and even to the positive control, TGF-β. More precisely,
a TGF-β concentration of 10 μg/mL increased HA production per well and per cell of both cell lines, by
about 45%, as PROT-1 induced a dose-dependent increase in hyaluronan production per well, especially
with the 1059 cell line, reaching +46% and +87.3% at 100 and 500 μg/mL, respectively. However, unlike
TGF-β, the PROT-1 fraction strongly and dose-dependently stimulated the HA production per cell,
again mainly with the 1059 cell line; strong increases of 115%, 206% and 323% were indeed observed at
10, 100 and 500 μg/mL, respectively (Figure 9). The enzymatic hydrolysis of the PROT-1 fraction led to
a loss of its pro-hyaluronic acid activity, which was very similar to what was previously observed for
its pro-collagen activity. Indeed, the H-PROT-1 hydrolysates obtained after 2, 6 and 24 h induced a
significant drop of HA production per cell of both cell lines at 500 μg/mL, more specifically a maximal
reduction close to 245% after 24 h. This effect was particularly substantial for the 1059 cell line, which
showed a strong and quick decrease in HA production. Indeed, the activity was 30% lower than that
of the negative control after 6 h of hydrolysis. This loss of activity was slightly less significant with
the 1090 cell line, as the HA production per cell was stimulated by the H-PROT-1 (t6h) fraction by
about 42%. However, no stimulation could be observed at t24h. These results indicate that the native
structure of the active proteins, which seems to be essential for their pro-collagen activity, is necessary
for their pro-hyaluronic acid activity as well. They also confirm that fibroblast cell lines 1059 and 1090
are not equally stimulated by PROT-1 or H-PROT-1 fractions, pointing out that the physiological state
of dermal fibroblasts might affect their stimulatory capacity.
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Figure 9. Hyaluronic acid production per well and per cell by the human dermal fibroblast cell line
1059. Results are expressed as the mean relative percentage of production, compared to the negative
control (100%). Significant differences between values obtained with samples and negative control
(n = 9) by * (p < 0.05), ** (p < 0.01) and *** (p < 0.001).
Figure 10. Hyaluronic acid production per well and per cell by the human dermal fibroblast cell line
1090. Results are expressed as the mean relative percentage of production, compared to the negative
control (100%). Significant differences between values obtained with samples and negative control
(n = 9) by * (p < 0.05), ** (p < 0.01) and *** (p < 0.001).
Finally, TGF-β seemed to mainly enhance HA production by stimulating cell growth while the
activity of PROT-1 fractions completely differed. The fact that these fractions highly promoted HA
production per cell, compared to their effect on HA production per well, suggests that they would
be able to directly induce HA biosynthesis by the fibroblasts rather than their growth. Fayat et al.
showed that a brown seaweed aqueous extract from Padina pavonica inhibited the hyaluronidase [41].
This could be a way to further explore to better understand the mechanism of action involved in the
pro-hyaluronic acid activity of the PROT-1 fraction.
3. Material and Methods
3.1. Material
All chemicals and reagents were purchased from Merck (Darmstadt, Germany). Fetal bovine
serum, penicillin–streptomycin, trypsin–EDTA, and Eagle’s Minimum Essential Medium (EMEM)
were purchased from Eurobio Ingen (Les Ulis, France). ELISA-like hyaluronic acid assay microplates
were purchased from R&D systems (Wiesbaden, Germany). Human dermal fibroblasts (CCD-1059Sk,
lot number 62062292, and CCD-1090Sk, lot number 204756) were obtained from ATCC Cell (Manassas,
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VA, USA). They were derived from the skin of a 20- and 46-year old woman, according to the
provider’s information.
The green macroalga Ulva intestinalis, was cultivated and purchased from the aquaculture farm
Algorythme on the Island of Ré (Ars-en-Ré, France). They were collected in summer 2017.
3.2. Methods
3.2.1. Protein Extraction & SEPROSYS Extraction Procedure

First, 500 g of dehydrated Ulva intestinalis material was washed in 15 L of deionized water
(1/30 (w/w)) for 10 min at room temperature (RT) and wrung with a fabric cone to remove as much
water as possible (Scheme 1). The washed algae were then ground in 5 L of deionized water at 80 ◦ C
until obtaining 2-mm particles. The 5 L suspension of minced algae in water was transferred in a
thermostated tank at 80 ◦ C containing 2.5 L of deionized water. Extraction was processed under
constant agitation with a bladed stirrer at a rotation speed of 10 spins/min for 2 h. The pulps were
then removed from the tank and filtered with the fabric cone to collect the aqueous extract. Then,
13 L of extract was recovered and filtered with an ultrafiltration unit equipped with a 15 kDa Kerasep
KBW membrane (Novasep Process, Pompey, France). Filtration was carried out at 80 ◦ C at a pressure
of 5 bars and a circulation flow of 450 L/h (circulation speed of 5 m/s) until obtaining a retentate
around 4◦ B. Then, 1.5 L of retentate was demineralized by passage on a column containing 100 mL of
Amberlite FPA 98, a strong anionic resin in the OH− form, in series with a column containing 200 mL
of Amberlite IR 120, a strong cationic resin in the H+ form. Circulation was processed with a peristaltic
pump at a flow rate of 2 BV/h for the cationic resin. The deionized product was finally decanted in a
water bath at 80 ◦ C for 2 h (flocculation step). After centrifugation at 5000× g for 15 min at RT, the
fraction, referred to as PROT-1, was neutralized to pH 7 with 1 M NaOH and lyophilized [22].
3.2.2. Enzymatic Hydrolysis of PROT-1 Proteins

Five hundred milliliters of a 10 mg/mL solution of PROT-1 fraction was prepared in deionized water
and heated at 50 ◦ C, under magnetic agitation at 500 rpm. The protease preparation Flavourzyme®
was added at a ratio of 4% (v/v) and 10-mL aliquots were collected every hour for 24 h, and immediately
heated at 90 ◦ C for 20 min to inactivate the proteolytic enzymes. A control was also prepared without
adding the protease preparation and similarly treated. Hydrolyzed samples, referred to as H-PROT-1
(t1h), (t2h), (t3h), (t4h), (t6h) and (t24h), were subsequently freeze-dried.
3.2.3. Biochemical Composition

The neutral sugar content was determined according to the phenol-sulfuric method [42], using
rhamnose as a standard; rhamnose was chosen as it is the main moiety of the parietal water-soluble
polysaccharides of seaweeds from the genus Ulva, the so-called ulvans, which are the major
polysaccharides extracted by water maceration of these seaweeds. The protein content of the dehydrated
seaweed starting material was determined by the Kjeldahl method (N× 6.25) [43] while the protein
content of the PROT-1 fraction was determined by the Lowry assay [44], using bovine serum albumin
as a standard. The quantification method of polyphenols was adapted from the original one [45] using
gallic acid as a standard; briefly, 50 μL of Folin–Ciocalteu reagent and 200 μL of 20% sodium carbonate
were successively added to 100 μL of sample and the mixture was incubated in the dark for 45 min at
RT, prior to absorbance reading at 730 nm. The ash content was determined by measuring the mass
loss of samples heated for 15 h at 550 ◦ C. The lipid content was measured by the method of Chabrol
and Charonnat [46] using vegetal oil as a standard.
3.2.4. High-Performance Liquid Size Exclusion Chromatography

The structural analysis and the molecular mass distribution of the proteins and peptides were
assessed by high-performance liquid size exclusion chromatography (HPL-SEC), using a 1200 series
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HPLC system (Agilent Technologies), equipped with two size-exclusion chromatography columns in
series: TSK gel 3000 SW and TSK gel 2500 PW (TOSOH Biosciences). The temperature of analysis was
stabilized at 30 ◦ C and 30 μL of extract or standard at 1 mg/mL was injected. The products were eluted
with 20 mM phosphate buffer with 0.1% NaCl (pH 7) at a flow rate of 0.8 mL/min and detected at 210 nm.
The standard curve was prepared using protein and peptide standards ranging between 1000 and
669,000 Da (thyroglobulin, 669 kDa; apoferritin, 443 kDa; β-amylase, 200 kDa; alcohol dehydrogenase,
150 kDa; albumin, 66 kDa, carbonic anhydrase, 29 kDa; α-lactalbumin, 14.2 kDa; cytochrome c, 12 kDa;
insulin, 3 kDa; α-casomorphin 1-4, 1 kDa).
3.2.5. Ultra-High-Performance Liquid Chromatography Coupled to High Resolution Mass

Spectrometry Analysis (UHPLC-HRMS)
The total amino acid composition of the PROT-1 fraction was determined using an UHPLC system,
“Acquity UPLC H-class”, (Waters, Milford, MA, USA) coupled to a HRMS system, “XEVO G2 S Q-TOF”,
equipped with an electrospray ionization source (Waters, Manchester, England). The UHPLC system
was formed by a quaternary pump (Quaternary Solvent Manager, Waters) and an automatic injector
(Sample Manager-FTN, Waters) equipped with a 10 μL injection loop. Analyses were performed
according to the UHPLC and MS parameters given in Table 3. The standard curve for each of the 20
essential amino acids was prepared using a standard solution at a concentration within the range 1–25
mg/L. The detection and quantification of total amino acids was performed after total acid hydrolysis
of the sample. Briefly, 10 mg of the sample was solubilized in 1 mL of 1 M HCl and heated at 100
◦ C for two hours. After cooling at room temperature, the solution was neutralized by adding 1 M
NaOH and filtered through a 0.22 μm filter prior to analysis, starting from a 2.5 mg/mL sample solution
solubilized in water/methanol/formic acid 95:5:0.5 (v/v/v).
Table 3. Ultra-high-pressure liquid chromatography (UHPLC) and mass spectrometry (MS) parameters
used to determine the total amino acid composition of the PROT-1 fraction and to elucidate the peptide
sequences obtained in the H-PROT-1 (t1h) fraction.
Total amino Acid Composition

Elucidation of Peptide Sequences
Determination
UHPLC Parameters
Column Acquity UPLC HSST3 (150 mm × 2.1 mm × 1.7 μm) maintained at 25 ◦ C
Flow rate (μL.min−1 ) 0.3 mL·min−1
Gradient used for the total amino acid
0 min: 100% A; 2 min: 100% A; 2.5 min: 99%
composition determination: water/methanol
A; 3 min: 99% A; 3.5 min: 98% A; 4 min: 97%
95:5 (v/v) (A)/water/methanol 50:50 (v/v) (B)
0 min: 100% A; 2 min: 100% A; A; 5.5 min: 97% A; 8.5 min: 95% A; 10 min:
+ 0.5% (v/v) formic acid
7 min: 30% A; 8 min: 100% A; 95% A; 15 min: 90% A; 18 min: 90% A; 22 min:
13 min: 100% A. 80% A; 27 min: 80% A; 30 min: 70% A; 34 min:
Gradient used for the elucidation of peptide
70% A; 36 min: 0% A; 38 min: 0% A; 40 min:
sequences: water (A)/acetonitrile (B) + 0.1%
100% A; 45 min: 100% A.
(v/v) formic acid
Injection 5 μL (4 ◦ C)
MS Parameters
ESI+ MS/MS DDA, centroid:
ESI+ MSE , centroid: - Transition from MS to MS/MS and MS/MS to
- Function 1, low energy, 5 eV MS when ion intensity becomes higher than
Mode
- Function 2, high energy, 500,000/s and lower than 10,000/s (or after
ramping from 10 to 30 eV 10 s), respectively
- Collision energy ramping from 15 to 35 eV
Source temperature ◦
120 C
Desolvation temperature 500 ◦ C
Gas flow rate of the cone 50 L/h
Desolvation gas flow rate 300 L/h
Capillary voltage 3 kV
Sampling cone voltage 35 V
Source offset 80 V
Acquisition mass range 50–1200 m/z (0.5 scans/s) 50–2500 m/z (0.5 scans/s)
Lock-mass Leucine Enkephaline (MW = 555.62 Da,1 ng/μL) with a flow-rate of 5 μL/min
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Molecules 2020, 25, 2091
The sequence elucidation of peptides obtained in the H-PROT-1 (t1h) fraction was carried
out after filtration of a 10 mg/mL sample aqueous solution on a 50 kDa membrane (Amicon Ultra
0.5 mL centrifugal filters, Merck) and analyzed using the same UHPLC-HRMS system. Analyses were
performed according to the UHPLC and MS parameters given in Table 3.
The acquisitions and data processing were carried out using the Waters “Mass Lynx 4.1 version”
software. The peptide sequences were checked using “Fragment ion calculator” software online
(Institute for Systems Biology, Seatle, WA, USA), after the MS/MS fragmentation analysis.
3.2.6. SDS-PAGE
SDS-PAGE was carried out using a Protean II xi cell electrophoresis unit from BIORAD (Hercules,
CA, USA) with a stacking gel of 5% (w/v) and a separating gel of 17% (w/v) acrylamide in Tris-HCl
25 mM, pH 8.3, glycine 0.18 M and SDS 0.1% (w/v). The separation was performed at 75 mA for 2 h.
The protein bands were stained by Coomasie brilliant blue. The size markers (10–250 kDa) were
purchased from Precision Plus ProteinTM Standard, BIORAD (Hercules, CA, USA).
3.2.7. Cell Culture

Cells were cultured in EMEM supplemented with 10% (v/v) fetal bovine serum and 1% (v/v)
antibiotic solution (10,000 U/mL penicillin, 10 mg/mL streptomycin), used as the complete culture
medium. Cell lines 1059 and 1090 were cultured in a temperature-controlled humidified incubator
with 5% CO2 at 37 ◦ C. The cells were grown in 75 cm2 ventilated Falcon culture flasks (BD Biosciences,
Franklin Lakes, NJ, USA) and subcultured by trypsinization (0.05% (w/v) trypsin, PAN Biotech,
Aidenbach, Germany). The culture medium was changed every two or three days. Cells were used
between the third and ninth passages for the experiments.
3.2.8. Cell Viability

The MTT assay was used according to the method described by Mosmann [47]. This is a
colorimetric assay allowing assessing cell viability, based on the reduction of a yellow tetrazolium salt,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), by viable cells. In the presence
of mitochondrial succinate dehydrogenase found in active living cells, the tetrazolium ring of MTT is
reduced and forms a violet product, formazan. The yellow solution becomes purple and the intensity
of the purple coloration is proportional to the number of viable cells. The MTT test is used to determine
cell growth and cell viability.
Briefly, cells were seeded in 96-well Falcon microplates (BD Biosciences, Franklin Lakes, NJ, USA)
at 5 × 104 cells/mL in 100 μL of complete culture medium and incubated for 24 h. The medium was
then removed and 100 μL of PROT-1 or H-PROT-1 (at 10, 100 or 500 μg/mL) prepared in complete
culture medium was added in the wells. After 48 h, 25 μL of MTT (5% (w/v) in PBS) was added in
each well and the microplates were incubated for 4 h at 37 ◦ C. The medium was then removed and
200 μL of dimethyl sulfoxide was added in each well. The microplates were then incubated for 10 min
prior to absorbance reading at 550 nm using a Fluostar Omega microplate reader (BMG LABTECH,
Ortenberg, Germany).
3.2.9. LDH Assay

This method is based on the fact that, during cell death, the loss of the cell membrane integrity
leads to the release of cytoplasmic enzymes, such as lactate dehydrogenase (LDH), into the extra-cellular
medium. It measures the activity of LDH released by damaged cells in the cell supernatant, good
marker of cell death [48].
LDH release was measured with a commercially available LDH assay kit (Cytotoxicity Detection
Kit, Roche, France), following the supplier’s instructions. Briefly, LDH that is released in the cell
environment reduces NAD+ into NADH and H+ through the oxidation of lactate into pyruvate.
Thereafter, a catalyst (diaphorase) transfers H/H+ from NADH and H+ to a tetrazolium salt
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Molecules 2020, 25, 2091
(iodonitrotetrazolium, INT), to form a red colored formazan salt. The absorbance of the red colored
formazan salt produced was measured at 492 nm using a Fluostar Omega microplate reader (BMG
LABTECH, Ortenberg, Germany).
3.2.10. Collagen Quantification

NHDF were seeded at a density of 5 × 104 cells/well in 24-well culture microplates in complete
culture medium. After 24 h, the medium was removed and 500 μL of PROT-1 or H-PROT-1 (at 10,
100 or 500 μg/mL) or a negative or positive control in EMEM containing 1% (v/v) antibiotic solution
was added in each well. The microplates were incubated for 48 h at 37 ◦ C and the collagen production
was then measured using the Sirius Red staining procedure [49]. Briefly, the medium was removed
and the cells were washed twice with PBS and then fixed for 1 h with 1 mL of Bouin’s solution at RT.
Following fixation, the Bouin’s solution was removed and cells were washed twice with deionized
water. The cells were then stained with 1 mL of Sirius Red solution (0.5 g of Sirius Red 80 in 500 mL
of saturated aqueous solution of picric acid) for 1 h under stirring at RT. Samples were then washed
successively with deionized water and 0.01 M HCl to remove unbound dye. The bound dye was finally
solubilized in 500 μL of 0.1 M NaOH for 1 h under stirring and absorbance was read at 550 nm using a
Fluostar Omega microplate reader (BMG LABTECH, Ortenberg, Germany).
Results were then expressed as the mean relative percentage of collagen production, compared to
the negative control, using the following equations:
[collagen]sample
collagen production per well (%) = × 100 (1)
[collagen]negative control
collagen production per well (%)

collagen production per cell (%) = × 100 (2)
cell viability (%)
3.2.11. Hyaluronic Acid Quantification

Hyaluronic acid produced and secreted by fibroblasts in the extra-cellular medium was quantified
using an ELISA-like hyaluronic acid assay plate (R&D Systems, Minneapolis, MN, USA). Briefly, cells
were seeded at 5 × 104 cells/mL in 100 μL of complete culture medium in 96-well Falcon microplates
(BD Biosciences, Franklin Lakes, NJ, USA ) and cultured for 24 h. The medium was then removed and
150 μL of positive (10 μg/mL of TGF-β) or negative control prepared in incomplete culture medium,
was added in the wells. After 48 h, the hyaluronic acid concentration in the supernatants was measured
according to the manufacturer’s instructions.
Results were then expressed as the mean relative percentage of hyaluronic acid production,
compared to the negative control, using the following equations:
[hyaluronic acid]sample
hyaluronic acid production per well (%) = × 100 (3)
[hyaluronic acid]negative control
hyaluronic acid production per well (%)

hyaluronic acid production per cell (%) = × 100 (4)
cell viability (%)
3.2.12. Statistical Analysis

All data are presented as means ± standard deviations of at least triplicates. The student’s t-test
(independent, two-sided) was used to determine significant differences between experimental and
control samples, using Sigma Plot 12.5 (Systat Software Inc., San Jose, CA, USA).
4. Conclusions
In this study, a protein-rich fraction extracted from Ulva intestinalis (PROT-1) was produced
using an acid- and solvent-free procedure. PROT-1 contained various sets of proteins. The major
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Molecules 2020, 25, 2091
one exhibited a MW close to 25 kDa. We showed that the PROT-1 fraction significantly increased
in vitro collagen and hyaluronic acid production by normal human dermal fibroblasts and that these
increased productions were not due to an increase in cell number but rather to an activation of cell
metabolism related to collagen and hyaluronic acid biosynthesis. We also concluded that the molecular
weight significantly influenced this bioactivity. Indeed, H-PROT-1 fractions, which were the enzymatic
hydrolysates of the PROT-1 fraction, had no effect on fibroblast proliferation and did not stimulate
collagen and hyaluronic acid biosynthesis, compared to PROT-1. Moreover, the collagen and hyaluronic
acid productions were decreased in presence of some H-PROT-1 fractions, proving the protein or
polypeptide origin of the pro-collagen and pro-hyaluronic acid activities of the PROT-1 fraction.
This fraction could thus be of significant interest for skin care prevention or treatment as it
significantly promotes in vitro collagen and hyaluronic acid biosynthesis by dermal fibroblasts without
activating cell proliferation. In vitro assessment of these pro-collagen and pro-hyaluronic acid activities,
as well as cytotoxicity, are a fundamental and essential step to develop ingredients with anti-aging
potential. However, according to European Cosmetic guidelines, the evaluation of cosmetics should
mix instrumental measurements from both in vitro and ex vivo/in vivo model systems [50]. To further
envisage the commercial human application of the PROT-1 fraction, it would consequently have to
undergo ex vivo and in vivo experiments. Firstly, noninvasive ex vivo assays on reconstructed human
skin models could be performed, to measure percutaneous absorption, cell renewal and/or synthesis
and degradation of the MEC and its constituents, collagens and hyaluronic acid in particular. Secondly,
in vivo tests might be carried out on a panel of representative volunteers, starting from a neutral and
stable formulation, to determine its tolerability in terms of cutaneous (patch test) or ocular irritation,
the sensation felt after applying, and its effectiveness (measurement of mechanical properties of the
skin such as density, firmness, elasticity and moisture; assessment of depth and volume of wrinkles;
analysis of the epidermis microrelief).
Author Contributions: Conceptualization, J.B., A.A., D.D., S.B., T.M. and N.B.; Methodology, J.B., A.A., P.-E.B.
and N.B.; Software, J.B., A.A., P.-E.B. and N.B.; Validation, A.A., D.D., S.B., T.M. and N.B.; Formal Analysis, A.A.,
T.M. and N.B.; Investigation, J.B., A.A. and P.-E.B.; Resources, J.B., A.A., D.D., S.B., T.M. and N.B.; Data Curation,
J.B., A.A., P.-E.B. and N.B.; Writing—Original Draft Preparation, J.B., A.A., T.M. and N.B.; Writing—Review &
Editing, A.A. and N.B.; Visualization, J.B., A.A., D.D., S.B., T.M. and N.B.; Supervision, A.A., D.D., S.B., T.M. and
N.B.; Project Administration, D.D., S.B., T.M. and N.B.; Funding Acquisition, D.D., S.B., T.M. and N.B. All authors
have read and agreed to the published version of the manuscript.
Funding: This research was supported by the “Association nationale de la recherche et de la technologie”, by
providing a part of a PhD funding (Grant number 2016/1032).
Acknowledgments: We are very grateful to the “Association nationale de la recherche et de la technologie”, for
contributing to this work by providing a part of a PhD funding.
Abbreviations
RT room temperature
ECM extracellular matric
HA hyaluronic acid
MMP matrix metalloproteinase
TIMP tissue inhibitor of metalloproteinase
SM size markers
HPL-SEC high-performance liquid size-exclusion chromatography
MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
LDH lactate dehydrogenase
TGF-β transforming growth factor beta
AP-1 activator protein 1
NF-κB nuclear factor-kappa B
EDTA ethylenediaminetetraacetic acid
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Molecules 2020, 25, 2091
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Sample Availability: Samples of the PROT-1 and H-PROT-1 fractions are available from the authors.
363
molecules
Article
Potential Photoprotective Effect of Dietary Corn Silk
Extract on Ultraviolet B-Induced Skin Damage
Yeon-hee Kim 1,† , Amy Cho 1,† , Sang-Ah Kwon 1 , Minju Kim 1 , Mina Song 1 , Hye won Han 1 ,
Eun-Ji Shin 2 , Eunju Park 2 and Seung-Min Lee 1, *
1 Department of Food and Nutrition, BK21 PLUS Project, College of Human Ecology, Yonsei University,
Seoul 03722, Korea
2 Department of Food and Nutritional Science, Kyungnam University, Changwon 51767, Korea
* Correspondence: leeseungmin@yonsei.ac.kr; Tel.: +82-2-2123-3118
Received: 27 May 2019; Accepted: 15 July 2019; Published: 16 July 2019
Abstract: Ultraviolet B (UVB) irradiation causes adverse effects on the skin. Corn silk contains
flavonoids and other bioactive compounds and antioxidants, which may prevent skin photoaging
through antioxidant and anti-inflammatory effects. We aimed to investigate the potential
photoprotective effects of dietary corn silk on UVB-induced skin damage in mice and the mechanisms
behind these effects on human skin cells. Oral administration of corn silk water extract (CS) (2 or
4 g/kg/day) for 19 weeks decreased epidermal thickness, wrinkle formation, and positive staining
for PCNA, Ki67, and 8-OHdG, and increased collagen staining in UVB-irradiated SKH-1 hairless
mice compared with controls. The pro-inflammatory NF-κB target genes (IL-1β, iNOS, and COX-2)
and MMP-9 expressions were lower in the CS groups, and TGF-β/Smad signaling increased. Low
skin lipid peroxidation and blood DNA oxidation levels and high blood glutathione were detected.
Antioxidant transcription factor Nrf2-related catalase and SOD1 proteins and glutaredoxin mRNA
levels increased. The results of CS extract treatment and UVB irradiation in HaCaT cells showed
the same results in Nrf2 and NF-κB target genes. An LC-MS/MS analysis showed that the CS
extract contained potential antioxidants, which might have contributed to its anti-photoaging effects
in tissues and cells. CS extract may reduce UVB-induced skin damage through antioxidant and
anti-inflammatory mechanisms.
Keywords: corn silk extract; photoaging; ultraviolet B; NF-κB; antioxidant; inflammation
1. Introduction
Skin, the largest organ in the human body, acts as a barrier against external pollutants [1].
Skin tissue is constantly exposed to solar ultraviolet (UV) radiation [2]. When the antioxidant defense
system is damaged due to ultraviolet B (UVB) exposure of the skin, excessive reactive oxygen species
(ROS) are generated at the irradiated sites [3], which results in various changes, including the oxidation
of cellular components, DNA mutation, inflammation, and the activation of specific enzymes that
degrade the extracellular matrix [4]. These changes can lead to unrestrained cell proliferation [5]
and carcinogenesis [3]. Chronic exposure to UV leads to photoaging and even skin cancer [2].
Skin UVB-induced photoaging is associated with distinct clinical features, such as wrinkling and
thickening of the epidermis, which is a protective layer of the skin [5,6]. There are also related
histological characteristics such as disorganization of collagen in the dermis, which lies below the
epidermis, fragmentation, and dispersion [7].
The antioxidant system is thought to protect against cellular damage from UV-induced oxidative
stress through the scavenging of ROS or their byproducts [5]. Carotenoids, polyphenols, and vitamins E
and C have been shown to act as photo-protective barriers by quenching ROS [8]. The oral administration

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of natural product extracts, such as French maritime pine (Pinus pinaster) bark, Ginkgo biloba [6],
green tea (Camellia sinensis), and grape seed extracts [8], has been shown to prevent UV-induced skin
damage. Thus, the consumption of herbal substances that are rich in antioxidant components, such as
polyphenols, might offer protective effects against UVB damage [1].
Corn silk (CS, Zea mays L.) has been consumed as a herbal medicine in Korea [9], China, United
States, and France for millennia [10]. In folk medicine, it is used for the treatment of cystitis, edema,
diabetes mellitus, and prostatitis [10]. CS contains an abundance of phenolic compounds, such as
flavonoids (maysin, apigmaysin, luteolin) [11,12], anthocyanins (cyanidin, peonidin) [13], chlorogenic
acid, and other biologically active substances, such saponins and allantoin [11,14]. The antioxidant
capacity of CS was first reported by Zoran et al., who showed a positive correlation between
the polyphenol content and antioxidant activity in aqueous acetone CS extract in vitro [15]. Radical
scavenging activity and iron chelating activity of CS extracts were proven in test tube experiments [16,17].
The antioxidant capacities of the CS extract have been implicated in human breast cancer cells [18],
human neuroblastoma cells [19], clonal rat pancreatic β-cells [20], and CSP2, a polysaccharide isolated
from the CS extract [21]. In animal experiments, ad libitum drinking of CS extract for 28 days in healthy
albino mice elevated antioxidant enzyme activities and increased the content of reduced glutathione
in the kidney [22]. In another animal study, dietary CS extract rescued a high salt diet-induced
reduction in glutathione peroxidase [23] and alleviated radiation-induced oxidative stress in mice [24].
In addition, oral ingestion of CS-extracted flavonoids showed antioxidant effects against oxidative
stress under exhaustive exercise [14] and under streptozotocin-induced diabetes [25] in mice.
The effects of CS extracts on UVB-induced damage have not been extensively studied. There were
only several cell line experiments using CS constituents [26,27]. Trans-zeatin, purified from corn silk,
inhibited UVB-induced MMP-1 expression in skin fibroblasts [26]. Luteolin, another flavonoid found in
CS, exerted anticancer effects on UVB-irradiated mouse epidermal cells by suppressing cyclooxygenase
(COX) expression and NF-κB activity [27]. The role of dietary CS extract on UVB-damaged skin and its
antioxidant mechanism are still not clear. In our study, we aimed to demonstrate the effect of CS extract
on preventing UVB-induced skin damage in mice and further confirm the molecular mechanisms
underlying this effect in human skin cells. We hypothesized that the CS extract might contribute to
its photoprotective effect through antioxidant and anti-inflammatory pathways. We investigated the
effects of the oral administration or treatment of CS extract on UVB-induced damage in SKH-1 hairless
mice and human skin HaCaT cells.
2. Results
2.1. DPPH and ABTS Antioxidant Capacities of CS Extract

DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) radical scavenging activities of CS extract varied
from 18.99 to 83.92% (Figure 1A). ABTS (2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) assay
values ranged from 4.74 to 42.75% (Figure 1B). The half maximal inhibitory concentration (IC50 ) of CS
extract and the standard compound, L-ascorbic acid, in relation to DPPH and ABTS radical scavenging
activities (Table 1).
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Figure 1. DPPH and ABTS antioxidative assay of corn silk (CS) extracts and the general details of
SKH-1 mice during the experiment. Antioxidative effects of CS extracts were determined by DPPH
(A) and ABTS radical scavenging activity (B). For the assays, CS extracts at concentrations of 1.25,
2.5, 5, 10 mg/mL and DPPH or ABTS solution were mixed at a ratio of 1:4 and 1:99. Body weight (C),
food intake (D), liver weight (E), and spleen weight (F) of the mice were not significantly different
across all groups (n = 8~10 per group) including normal control group (NOR), UVB-irradiated group
(UVB), UVB-irradiated and low CS- (2 g/kg/day) treated group (CSL), and UVB-irradiated and high
CS- (4 g/kg/day) treated group (CSH). Values are mean ± SD. The results were analyzed by one-way
analysis of variance (ANOVA) followed by Duncan’s post-hoc test. Different lowercase letters over
bars (a, b, c, d, e) represent significant statistical differences (p < 0.05).
Table 1. IC50 values of DPPH and ABTS radical scavenging activities of corn silk (CS) extract and
ascorbic acid.
IC50 (mg/mL)
Sample
DPPH ABTS
Corn silk extract 3.60 ± 0.1 11.61 ± 0.2
L-ascorbic acid 0.08 ± 0.0 0.38 ± 0.1
Values are represented as mean ± SD of three replicates. Lower values represent higher radical scavenging activity
of the corresponding sample.
2.2. Food Intake, Body Weight, and Organ Weights of Animals

Body weights of the mice were not significantly different between groups during the study period
(Figure 1C). Mean daily food intake was also similar between groups (Figure 1D). Mouse liver and
spleen weights were not significantly different between the groups at sacrifice (Figure 1E,F).
2.3. Effects of CS Extract on Skinfold, Epidermal Thickness, and Wrinkle Formation in UVB-Irradiated Mice
The ANTERA 3D® images of skin replicas of animal groups are presented in Figure 2A. UVB
irradiation increased the epidermal thickness of the dorsal skin and the thickness of the skinfold
compared with the NOR group (Figure 2B–D). However, administration of the CS extract significantly
reduced the epidermal and skinfold thickness in the CSL and CSH groups compared with the UVB
group (Figure 2B–D). UVB irradiation increased the values of all parameters related to wrinkle
formation, including the volume of depression, the affected area of depression, the maximum valley
depth, and the average wrinkle length in comparison to the NOR (Figure 2E–H). These values were
significantly decreased in the CSL and CSH groups in comparison to the UVB group (Figure 2E–H).
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Figure 2. Effects of corn silk (CS) extract on UVB-induced wrinkle formation in the dorsal skin of SKH-1
mice at the end of the study (week 19). Photographs of the replica, replica analysis, and the backs of the
mice (A), Hematoxylin and eosin-stained sections (original magnification 100×) (B), epidermal thickness
(C), skinfold thickness (D), mean of skin wrinkle depression volume (E), mean depression area (F),
maximum depth (G), and wrinkle length (H) are presented for normal group (NOR), UVB-irradiated
group (UVB), UVB-irradiated and low CS- (2 g/kg/day) treated group (CSL), UVB-irradiated and high
(4 g/kg/day) CS-treated group (CSH). Values are mean ± SD. The results were analyzed by one-way
analysis of variance (ANOVA) followed by Duncan’s post-hoc test. Means with different lowercase
letters (a, b, c, d) represent statistically significant differences (p < 0.05). Bars with the same letters are
not significantly different.
2.4. Effect of CS Extract on Epidermal Expression Levels of PCNA and Ki67 in UVB-Irradiated Mice
The cell proliferation levels in the dorsal skin sections of the animal groups were examined by
the expression levels of proliferation marker genes PCNA (proliferating cell nuclear antigen) and
Ki67 (Figure 3A–D). PCNA- and Ki67-positive cells were localized to the stratum basale (basal layer)
between the epidermis and the dermis in the NOR whereas positive staining for PCNA and Ki67 were
detected throughout several layers of the epidermis in the UVB group (Figure 3A,B). The positive
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staining levels of PCNA and Ki67 were significantly decreased in CSL and CSH groups compared
with the UVB group (Figure 3C,D). Greatest reductions in the staining levels were detected in the
CSH group (Figure 3C,D). The CS extract significantly rescued the UVB-induced increase in PCNA
protein levels in mouse dorsal skin tissue (Figure 3E). These data indicated that the CS lessened the
UVB-mediated epidermal cell proliferation.
Figure 3. Effect of corn silk (CS) extract on the expression of PCNA and Ki67 in UVB-irradiated hairless
mice skin. Representative images of immunohistochemical staining of PCNA (A) and Ki-67 (B) are
shown. Immunostaining of each gene is depicted as brown areas at the original magnification of ×200.
PCNA (C) and Ki-67 (D) were quantified using ImageJ software. Representative Western blot image
and protein levels of PCNA (E) in mouse skin tissue are shown (repeated five times). Values are mean
± SE of the percentages of positive nuclear staining in the skin tissue or protein levels. The results were
analyzed by one-way ANOVA with Duncan’s post-hoc test. Bars accompanying different lowercase
letters (a, b, c, d) represent statistically significant differences (p < 0.05), whereas the same letters
represent no significant difference. Group abbreviations: Normal group (NOR), UVB-irradiated group
(UVB), UVB-irradiated and low CS- (2 g/kg/day) treated group (CSL), UVB-irradiated and high CS-
(4 g/kg/day) treated group (CSH).
2.5. Effect of CS Extract on Skin Collagen Fiber Content in UVB-Irradiated Mice

Skin connective tissue levels were assessed by MT and VVG staining, which detect collagen fibers
(Figure 4A,B). Compared with the NOR group, the UVB group showed lower staining levels in both
MT and VVG (Figure 4C,D). The CSL and CSH groups partly reverted the UVB-induced loss of the
staining density and the CSH showed the strongest staining compared to other groups in both MT
and VVG (Figure 4C,D). Next, protein expression levels of matrix metalloproteinase-9 (MMP-9) and
Tissue inhibitors of metalloproteinases (TIMP) which regulate extracellular matrix, were examined
(Figure 4E,F). Protein levels of MMP-9 and TIMP-1 in the skin were increased by UVB irradiation
compared with the NOR group, but CS treatment decreased MMP-9 at both low and high doses and
reduced TIMP-1 levels at the high dose (Figure 4E,F). Activation of TGF-β and Smad2/3 signaling
pathway was investigated in terms of collagen production [7]. The protein levels of TGF-β were not
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different between the UVB and the NOR groups, but there were modest elevations of TGF-β only in
the CSL and CSH groups compared with the UVB group (Figure 4G). The UVB irradiation lowered
phosphorylation levels of Smad2/3 compared with the NOR group, but this reduction was significantly
reverted in the CSH while not in the CSL group (Figure 4H). Procollagen type 1 levels significantly
increased in the CSH than in the UVB group (Figure 4I). These results suggested that UVB-diminished
TGF-β and Smad2/3 signaling pathway were re-activated in the CSH, and possibly in the CSL.
Figure 4. Effect of corn silk (CS) extract on collagen fiber content in UVB-photoaged mouse skin.
Collagen fibers were stained with Masson’s trichrome (MT) (A) and Verhoeffe Van Gieson (VVG)
(B). Representative histological images of collagen in mouse skin tissue are presented at an original
magnification of ×200. Collagen staining by MT and VVG appears blue and red, respectively. Staining
density of MT (C) and VVG (D) are shown, respectively. Western blotting detected MMP-9 (E), TIMP-1
(F), TGF-β (G), p-SMAD2/SMAD2 (H), and α-tubulin expression levels in the UVB-irradiated dorsal
skin protein extract of hairless mouse by using specific antibodies for each protein. Blot image is a
representation of three individual experiments. The blots were quantified using ImageJ software and
the signal intensities were normalized to the value of α-tubulin, except for p-SMAD2, which was
normalized to the expression of SMAD2. The mRNA expression of procollagen type 1 was assessed
by q-PCR analysis and was normalized to the intensity value of GAPDH, quantified using ImageJ
(I). Values are mean ± SE. Mean values not assigned with the same letter (a, b, c, d) are significantly
different, analyzed by ANOVA (p < 0.05). Group abbreviations: Normal group (NOR), UVB-irradiated
group (UVB), UVB-irradiated and low (2 g/kg/day) CS-treated group (CSL), UVB-irradiated and high
(4 g/kg/day) CS-treated group (CSH).
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2.6. Effect of CS Extract on Oxidative Stress and Skin Antioxidation Genes

To investigate the effects of CS on UVB-induced oxidative stress, skin and blood oxidative
stress-related markers were examined. The UVB group showed significant positive staining of
8-OHdG, an oxidative DNA damage marker, in the skin epidermis compared to the NOR group
(Figure 5A,B). However, CS treatment reduced 8-OHdG levels, with a greater reduction in the CSH
group (Figure 5A,B). In addition, lipid peroxidation products measured by Thiobarbituric acid reactive
substances (TBARS) in the skin were significantly decreased in the CSH group (Figure 5C). UVB also
increased the percentage of tail DNA and tail length of PBMC cells, indicating more DNA damage
compared with NOR (Figure 5D,E). However, the tail DNA and tail length of PBMC cells were reduced
in the CSL and CSH groups (Figure 5D,E). Plasma GSH, an antioxidant molecule, was slightly decreased
in the UVB group compared with that in the NOR group (p = 0.041), but significantly increased in
CSL (Figure 5F). These results indicated that the CS extract decreased UVB-induced skin and blood
oxidative damage.
Figure 5. Effect of Corn Silk (CS) extract on antioxidant gene expressions in response to UVB-induced
oxidative stress in mouse skin and liver. The UVB-induced formation of DNA/RNA damage marker,
8OHdG, was detected by immunohistochemistry. Representative images (A) and quantification (B)
of the staining are shown. MDA in the skin tissue was measured by the TBARS assay (C). Oxidative
stress in the blood was determined by the leukocyte comet assay and plasma glutathione concentration.
DNA damage was detected by the tail DNA (%) (D) and tail length (μm) (E). Plasma glutathione
(GSH) concentrations were compared (F). Nrf2 protein levels were assessed by Western blot and a
representative image of the blot is shown (G). The mRNA expression of catalase (H) and SOD1 (I) are
shown as assessed by q-PCR. Values are mean ± SE. Values with different letters (a, b, c, d) indicate
statistical significance (p < 0.05), as analyzed by one-way ANOVA. * p < 0.05, ** p < 0.01, student
t-test. Abbreviations: Normal group (NOR), UVB-irradiated group (UVB), UVB-irradiated and low
(2 g/kg/day) CS-treated group (CSL), UVB-irradiated and high (4 g/kg/day) CS-treated group (CSH).
UVB irradiation decreased Nrf2 protein expression in liver tissue, but CSL significantly recovered
the UVB-induced loss of Nrf2 levels (Figure 5H). In addition, catalase and SOD1 expression significantly
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increased in skin tissue CSH compared with the UVB group (Figure 5H,I). As a result, CS reduced
UVB-induced oxidative stress in the mouse skin and liver tissues.
2.7. Effect of CS Extract on Skin Inflammatory Gene Expression in UVB-Irradiated Mice

Expression levels of NF-κB target inflammatory genes including iNOS, IL-1β, and COX-2 in
UVB-irradiated mouse skin and liver tissue were examined (Figure 6A–D). In mouse skin, iNOS, IL-1β,
and COX-2 levels increased in the UVB group compared with the NOR group and decreased in the CS
groups compared with the UVB group (Figure 6A–C). In mouse liver, iNOS was decreased in CSL and
CSH compared with the UVB group (Figure 6D). Therefore, CS decreased levels of inflammatory gene
expression in UVB-irradiated skin and liver tissues.
Figure 6. Effect of corn silk (CS) extract on inflammatory gene expressions in UVB-irradiated mice
skin and liver. Protein expressions of IL-1β (A), COX-2 (B), and iNOS (C) in mouse skin, and iNOS in
mouse liver (D) were assessed by Western blot analysis and their representative images of multiple
independent experiments (n = 3) are presented. Protein results of iNOS in mouse liver is shown in (F)
and its representative blot image is shown in (E). Results are expressed as mean ± SE. Mean values
sharing different letters (a, b, c, d) over bars are significantly different (p < 0.05), as analyzed by ANOVA.
* p < 0.05, student t-test.
2.8. Effect of CS Extract on UVB-Irradiated Human Keratinocytes

Additional experiments were carried out on the human HaCaT keratinocyte cell line to further
examine the mechanism of the CS extract’s photoprotective effect on skin. First, MTT assay was
performed to investigate the cytotoxic effect of UVB and CS extract on HaCaT cells. A 24-h treatment
of CS extract did not show cytotoxicity at concentrations up to 5 μg/mL (Figure 7A). UVB irradiation
above 30 mJ/cm2 significantly inhibited cell growth in a concentration-dependent manner (Figure 7B).
Therefore, the UVB dose for cell experiments was set as 30 mJ/cm2 . In accordance with these results,
CS treatment on 30 mJ/cm2 UVB-irradiated cells did not show cytotoxicity up to 5 μg/mL (Figure 7C).
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Based on this data, 5 μg/mL was defined as the higher concentration (CSH), and a ten-fold dilution of
0.5 μg/mL was determined as the lower concentration (CSL).
Figure 7. Viability of CS- or UVB treated HaCaT cells and CS effect on antioxidant and anti-inflammatory
gene expressions in HaCaT cells. MTT assays showed the viability of HaCaT cells after exposure to
either CS extract (A), only UVB (B), or both UVB and CS extract treatment (C). Protein levels of Nrf2 (D)
were analyzed by Western blot and the representative images of the blot (three repetitions) are shown.
Protein levels of COX-2 (F) and iNOS (G) were analyzed by Western blot and the blot images represent
three repetitions. mRNA levels of Glutaredoxin (E), a target of Nrf2, and procollagen type 1 (H) was
analyzed by qPCR and normalized to GAPDH. Results are mean ± SE. Different lowercase letters (a, b)
represent statistical difference, as analyzed by ANOVA (p < 0.05). * p < 0.05, ** p < 0.01, student t-test.
Expression levels of Nrf2 were confirmed in human skin cells, which showed similar positive
effects of CS extract as did in mice. Nrf2 protein level was significantly reduced by UVB irradiation but
recovered by CS treatment at 5 μg/mL (Figure 7D). Nrf2 target gene, glutaredoxin, showed similar
regulation by CS treatment at both 0.5 and 5 μg/mL (Figure 7E). NF-κB target inflammatory genes,
including COX-2 and iNOS, were also examined in UVB-irradiated epithelial HaCaT cells. COX-2
and iNOS were significantly decreased in the UVB group treated with CS 5 ug/mL compared with the
non-CS treated UVB group (Figure 7F,G). Procollagen type 1 mRNA level was significantly improved in
both doses of CS extract compared with the UVB group (Figure 7H). Overall, the CS extract improved
Nrf2 signaling and alleviated inflammatory gene expression levels in UVB-irradiated HaCaT cells.
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2.9. Metabolite Identification in CS Using LC-MS/MS

LC-MS/MS analysis revealed putative detection of 6083 peaks in the CS extract, and the top
100 peaks in the order of peak intensities were listed in Supplementary Materials (Table S1). The peak
numbers below in parentheses indicate the order of the metabolites detected in the CS extract from
highest to lowest peak intensities. Out of the top 100 peaks in the CS extract, the top 30 metabolites were
further identified whether they exhibit antioxidant and/or anti-inflammatory effects based on previous
studies. Indole (1st peak), 3-(3-hydroxyphenyl)propanoic acid (2nd peak), and proline betaine (7th peak)
were revealed as the three most abundant metabolites in the CS extract that exhibit antioxidant and/or
anti-inflammatory effects (Table S1). This suggested that the CS contained antioxidant compounds
might have contributed to the antioxidant effect in skin protection. In mice, skin metabolites were
compared between NOR, UVB, CSL and CSH groups (Figure S1A). Five hundred eight metabolites
were altered in the CS groups compared to the UVB group (Figure S1B). Metabolites found in the
CS extract were also detected in among the 161 up-regulated metabolites in the skin of CS groups
compared with the UVB group. These included proline betaine (7th peak), L-proline (in the form of
L-phenylalanyl-L-proline, 11th peak), L-phenylalanine (in the form of L-Aspartyl-L-phenylalanine,
13th peak), phytosphingosine (14th peak), nicotinic acid (in the form of 6-Hydroxynicotinic acid,
23rd peak), ascorbic acid (in the form of dehydroascorbic acid, 28th peak), and vitamin A (29th peak) in
the order of highest to lowest peak intensities. These seven metabolites were previously implicated in
the antioxidant and/or anti-inflammatory roles.
3. Discussion
In the present study, we demonstrated that the CS water extract ameliorated the hyperproliferation
of UVB-induced skin epithelial tissues and wrinkle formation in addition to preserving epidermal
collagen content in UVB-irradiated SKH-1 hairless mice. CS extract was also effective in the alteration
of Nrf2 and NF-κB target inflammatory genes, which are influenced by oxidative stress, in mouse
skin and human skin cells. These anti-UVB effects appeared to be mediated by the antioxidant and
anti-inflammatory effects of CS, as shown in mice and in HaCaT cells.
The UVB-induced skin changes, including skinfold thickness, wrinkle depression volume
and epidermal thickness, were ameliorated conspicuously in the CS-treated groups. Reduction
in skin photoaging in the CS groups appeared to be due to inhibition of aberrant UVB-induced
hyper-proliferation because significant reductions in proliferation markers were detected in the skin of
the CS groups. Moreover, prolonged UV exposure in the skin is known to trigger cell proliferation with
damaged DNA [1]. However, oral administration of the CS markedly decreased hyperproliferation
and DNA damage.
The collagen content in the dermis, which lies below the epidermis, confers resilience and strength
to the skin [28]. Continuous UV exposure can lead to the loss of collagen through the reduction in
the production of type 1 collagen and increased activities of MMP [28]. In our study, MT and VVG
staining showed that collagen fiber was greatly reduced in mice exposed to UVB, but a significant
recovery was observed after treatment with CS or its components. The impairment of collagen
synthesis by UV irradiation occurs via interference in the TGF-β and Smad2/3 signaling pathway
in the skin [3,29]. These aberrations result in a reduction in the phosphorylation of Smad2/3, which
consequently decreases the transcription of type 1 procollagen [29]. Remarkable reactivation of TGF-β
and Smad2/3 signaling pathway was achieved by the CS in UVB-irradiated mice, suggesting that the
synthesis of type 1 procollagen might have been recovered by the CS. Similar results were shown in
HaCaT cells, where the CS extract significantly increased the low mRNA levels of procollagen type
1 in UVB-irradiated cells. In addition, expression of MMP-9, which displays proteolytic activities
and degrades the extracellular matrix containing collagen and elastin [4], was inhibited by the CS
groups. As seen in the CSH group, reduction in TIMP-1, a major inhibitor of MMP-9 [30], might have
reflected the condition of lowered MMP-9 to balance the activities of MMPs and TIMPs. Overall the CS
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prevented the UVB-induced loss of collagen fibers possibly by activating TGF-β and Smad2/3 signaling
and inhibiting MMP-9 expression.
Chronic UVB radiation on skin causes accumulation of ROS, adjacent and tumoral oxidative
stress, and oxidative damage [31]. As previously investigated by analytical methods, CS itself or its
components exert antioxidant capacities [15–17,32]. Antioxidative effects of our CS water extract were
in accordance with these studies, showing a dose-dependent increase in radical scavenging capacity.
The effects were far lower than that of ascorbic acid, therefore the CS extract may not be considered as
a direct antioxidant, but a potential material exerting positive effects on skin protection perhaps with
the synergistic effects of various compounds identified in the literature [11–14]. The radical scavenging
activities detected in the CS extract were in agreement with an increase in the murine blood levels of
GSH, an antioxidant molecule. GSH acts as a direct scavenger of free radicals [33]. Oxidative stress
levels in both blood and skin were lessened by the CS, as shown by the results of the skin 8OHdG,
TBARS, and blood comet assays. These antioxidant effects seen in the skin were especially exciting
considering that the CS extract was orally administered instead of being applied topically. The UV
exposure is known to disturb the antioxidant systems in the body other than the skin and increase
oxidative stress markers in the liver and blood [34]. The antioxidative effects observed in the skin tissue
and blood might have indicated that the CS reached the skin and blood circulation system and played
protective roles in the UVB-induced oxidative stress conditions. Similarly, other studies reported that
the oral administration of polyphenol-rich plant extracts prevented UV-induced lipid peroxidation in
skin and DNA damage in peripheral blood [35]. The administration of natural food extracts was able
to restore the blood GSH concentration in a diabetic animal model [36].
Nrf2 is a major regulator of antioxidant responses in cellular level through antioxidant response
element (ARE)-mediated transcriptional regulation [37]. Nrf2-deficient mice have shown accelerated
oxidative skin damage and photoaging in response to UVB radiation but no difference in carcinogenesis,
suggesting that Nrf2 system may play an essential role in relieving UVB-induced oxidative stress in
skin [38,39]. Catalase and SOD1 are well known antioxidant enzymes that neutralize excess reactive
oxygen species in cells [40]. SOD1 is a direct ROS quencher and its promoter is known to contain
Nrf2-ARE binding site [41]. No ARE site has been found in catalase promoter region so direct binding
of Nrf2 remains controversial [42], but Nrf2-dependent expression of catalase has been shown in
mouse-derived cells [43,44]. We showed that CS extract increased Nrf2 protein in liver tissue and
in HaCaT cells, suggesting a positive role of CS in the antioxidant pathway. In accordance with the
activation of Nrf2 protein, catalase and SOD1 increased in skin tissue compared with the UVB group.
Another Nrf2-regulated antioxidant, glutaredoxin, was reported to alleviate oxidative stress in human
retinal pigment epithelial cells [45,46]. We showed that the CS extract significantly improved the mRNA
levels compared with that of the UVB group in HaCaT cells. The Nrf2-mediated antioxidant enzyme
regulation could have contributed to the photoprotective effect of the CS extract on UVB-irradiated
skin. Although we expected CS dose-dependent increases in Nrf-2 protein levels in both animal and
cell experiments, we only observed these effects in the cells. The reasons we observed no further
increase in protein level of Nrf2 in CSH group could include possible toxic effects of a high dose of
CSH itself or effects of negative feedback mechanism after CS intake. According to Heo et al., Nrf2
protein is degraded after antioxidant enzymes [47]. In another study, with increased UVB irradiation,
HaCaT cells have been reported to exclude Nrf2 from the nucleus, compared with lower doses of UVB
where nuclear translocation was increased [48]. Thus, the Nrf2 protein in the CSH group may have
shown no increase due to tight regulation of this protein and possible negative feedback mechanism.
ROS induced by UVB radiation triggers signaling molecules such as NF-κB, a major regulator
of pro-inflammatory genes including iNOS [49]. Inflammatory response caused by UVB-irradiated
skin activates the transcription of MMPs, which degrade the dermal collagen and connective tissue in
skin [3]. UVB irradiation significantly activated NF-κB signaling, which in turn was blocked only by
the higher dose of CS. In contrast, IL-1β and iNOS, which are well-known NF-κB targets [50,51], were
successfully downregulated by the CS regardless of the doses in skin and liver tissues. On the other
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hand COX-2, another NF-κB target [51], was only lowered by the higher dose of CS. In HaCaT cells
iNOS was only decreased in the higher dose of CS extract. Collectively, the photoprotective effects of
CS might have been involved in the inhibition of UVB-activated NF-κB signaling pathway, leading to
the reduction in the expression of proinflammatory genes and MMP-9.
Metabolites both up-regulated in the skin of the CS group and found in the top 100 peaks in
CS extract included proline betaine, L-proline, L-phenylalanine, phytosphingosine, nicotinic acid,
ascorbic acid, and vitamin A, in the highest to lowest peak intensity order. Proline and glycine
betaine are antioxidants that also protect plants from dehydration [52], salt stress, and cell death [53].
L-phenylalanine exerts lipophilic antioxidant capacity as tested by DPPH and ABTS assays [54] and
anti-inflammatory effects on carrageenan-induced edema [55]. Phytosphingosine, an active lipid
abundant in both plants and animals, constitutes the stratum corneum (outer layer of skin) and exhibits
anti-inflammatory effect and defense against microbes [56]. Phytosphingosine-1-phosphate has been
reported to promote epidermal growth factor in human dermal fibroblasts, and promotes anti-aging
effects in human skin [57]. Nicotinic acid, known as vitamin B3 , stimulates keratinocyte differentiation,
stabilizes epidermal barrier function, and benefits aging skin by reducing wrinkles and exerting
anti-photocarcinogenesis effects [58]. In addition, nicotinic acid has shown anti-inflammatory effects
in TNF-α-exposed mouse adipocytes ascorbic acid by suppressing inflammatory chemokines [59].
Ascorbic acid, known as vitamin C, also benefits the skin by promoting collagen formation, scavenging
free radicals, and protecting from photoaging and UVB-induced lipid peroxidation [60]. Oral ingestion
of vitamin C has been suggested to be more effective on the skin than topical administration [61].
Vitamin A has been effective in alleviating inflammation in skin disorders, broncho-pulmonary
dysplasia, and pneumonia [62]. These substances in the CS might have attributed to the UVB protective
effects on the skin.
There are some limitations regarding the measurements of candidate antioxidants in the skin of
mice and its molecular relationship with CS’s antioxidant effects. The inclusion of groups for bioactive
constituents with the equivalent dose to their content in the CS extract would have provided further
support to our study. Previous studies suggested that allantoin [63] and luteolin [64] are present
in CS, and may exhibit anti-inflammatory or anti-oxidative effects [65–67]. However, allantoin and
luteolin in our CS extract were present as the 58th and 3244th most abundant chemical according
to our LC-MS/MS analysis and might have not significantly contributed to the CS effects. However,
when a 15-fold lower dose of allantoin and a 15-fold higher dose of luteolin were provided to the
UVB-irradiated animals, we observed significant improvements in UVB-induced skin damages along
with oxidative stress and inflammatory markers (unpublished data). In addition, our CS metabolite
analysis suggested other potential antioxidant and/or anti-inflammatory components. Further studies
are warranted to identify bioactive constituents for the UVB protective effects of the CS.
In conclusion, our data demonstrated that the oral administration of the CS extract ameliorated
UVB-induced skin photoaging by the prevention of aberrant cell proliferation and DNA damage, and
that these effects might be mediated by antioxidant and anti-inflammatory gene pathways. Histological
results of skin tissue showed that the CS extract effectively reduced UVB-induced wrinkle formation
and cell proliferation, and increased collagen synthesis. Mediators of the antioxidant defense system
such as Nrf2, catalase, SOD1, and glutaredoxin were elevated, and inflammation-related genes in
the NF-κB signaling pathway, such as IL-1β, COX-2, and iNOS were reduced upon oral ingestion
of dietary CS extract. In human cells, similar results were shown in the Nrf2 and NF-κB pathways.
As revealed by the LC-MS/MS results, the chemical composition of the CS extract included potential
antioxidants, which might have contributed to its anti-photoaging effects in animal tissue and in cells.
The results indicate that the CS extract was effective in the prevention of UVB-induced skin damage
through different signaling pathways. Further studies on the molecular level of the photoprotective
effect of CS water extract on the skin are required.
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4.1. Materials and Reagents

2,2-Diphenyl-1-picrylhydrazyl (DPPH) was purchased from Alfa Aesar (Haverhill, MA, USA).
2,2-azinobis(3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS), dimethyl sulfoxide (DMSO), and sodium
bicarbonate were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Potassium persulfate
was purchased from Duksan (Ansan-si, Gyeonggi-do, Korea). The assay kits for GSH analysis were
purchased from BioVision Research Products (Mountain View, CA, USA). Other chemicals were
commercially available and in analytical grade.
4.2. Preparation of CS Extract

Water extract of corn silk was prepared and supplied by Kwang Dong Pharmaceutical Co. Ltd.
(Seoul, Korea), and stored at 4 ◦ C until use. The corn silk was harvested from Jilin Province in
China, sterilized at 125 ◦ C for 30 min, extracted with water at 98 ◦ C–100 ◦ C for 1 h, filtered through
microfilter paper, and concentrated under reduced pressure at 50 ◦ C or lower (concentrated brix:
44–48 brix). After final sterilization at 95 ◦ C–98 ◦ C for 20 min, 1 L of corn silk extract was obtained
from 4 kg of corn silk. The moisture content of corn silk extract was 64.98%. The extract consisted
of 16.21% carbohydrate, 9.78% protein, 0.44% lipid, 5.54% dietary fiber, 0.06% crude fiber, and 8.59%.
The nutritional composition of the corn silk extract was determined using the Association of Official
Agricultural Chemists (AOAC) method (1996) established by the Korea Health Supplement Institute
(Seongnam, Korea) [68]. Finally, lyophilization using a freeze-dryer (FD8512, ilShin BioBase Co. Ltd.,
Yangju, Korea) for a minimum of 72 h removed water from the extract. In total, 125 g of the freeze-dried
corn silk extract (CS) was collected.
4.3. Measurement of Antioxidant Effects of CS Extract

To measure the DPPH radical scavenging activity of the CS water extract, 80 μL of DPPH solution
(0.4 mM DPPH in ethanol) was vigorously mixed with 20 μL of CS solution (1.25, 2.5, 5, 10 mg/mL
in water). For the control, 20 μL water was added to 80 μL DPPH After incubation for 10 min,
the absorbance of the solution was measured at 492 nm. The standard content was calculated using the
absorbance of L-Ascorbic acid (100 μg/mL) that were treated in the same way as the samples. The ABTS
radical scavenging activity was determined by the modified method of Re et al. [69]. In brief, 7 mM
ABTS in water was reacted with 2.45 mM potassium persulfate, allowed to stand in dark for 12–16 h to
make ABTS•+. This was diluted with methanol (50%, v/v), to absorbance of 0.70 ± 0.02 at 734 nm,
30 ◦ C, and 990 μL of this solution was added to 10 μL CS or water. Absorbance was measured at
734 nm after 1 min incubation. The free radical scavenging ability of DPPH and ABTS was calculated
by the following equation: Percentage of inhibition of DPPH or ABTS (%) = (1 – O.D. of sample/O.D.
of control) × 100. IC50 values of CS extract in relation to ABTS and DPPH free radicals was calculated
and compared using L-ascorbic acid (0, 12.5, 25, 50, 100 μg/mL) as a positive control.

Fifty-six 6-week-old female SKH-1 hairless mice were obtained from Orient Bio Inc. (Seongnam,
Korea). The animals were housed at 23 ± 2 ◦ C, with a relative humidity of 55 ± 10%, in a 12 h light/dark
cycle with free access to food (5L79, Orient Bio Inc., Seongnam, Korea) and water. After a one-week
acclimatization, the mice were randomly allocated into six groups: (i) Vehicle (saline)-treated normal
group (NOR, n = 10), (ii) UVB-irradiated group (UVB, n = 9), (iii) UVB-irradiated and 2 g/kg/day
CS-treated group (CSL, n = 9), and (iv) UVB-irradiated and 4 g/kg/day CS-treated group (CSH, n = 10).
The oral dose of each sample was adapted from Guo et al. and Wang et al. at which CS
administration did not induce weight loss, histopathological changes, or death [70,71]. The oral dose
of the group was set at 4.0 g/kg/day. The oral dose of the low dose group was set at 2.0 g/kg/day.
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Each sample was dissolved in saline and orally administered at a volume of 0.2 mL each. In the normal
and control groups, saline was administered at a dose of 0.2 mL/day.
To observe the protective effects of the extract on mice before tumorigenesis, we defined 19 weeks
as the endpoint of our study in reference to a report that UVB-induced tumor development time in 50%
untreated hairless mice was 20 weeks [28]. The animals were monitored daily and weighed weekly.
All experimental protocols were approved by the Institutional Animal Care and Use Committee
(IACUC) of Yonsei University, Korea (Permit number: 201608-495-02).
Mouse dorsal skin was exposed to UVB three times per week using the Biolink crosslinker BLX-312
(Vilbert Lourmat; Marne-La Vallee, France). Fifteen-centimeter distance was maintained between
the light source and mouse. The UVB source was 5 UVB lamps (5 × 8 W [8 J/s]) with a 312 nm peak
emission. The minimal erythematous dose was 180 mJ/cm2 . The UVB radiation was 180 mJ/cm2 in
weeks 2–11 and increased to 360 mJ/cm2 in weeks 12–19 by modified methods of Mantena et al. [72]
and Record and Dreosti [73]. No UVB was radiated in the first week.
4.5. Assessment of Skin Thickness and Wrinkle Formation in Mice

Hairless mice were anesthetized, and their dorsal skin was photographed at the end of the study
(19 weeks). The UV-induced skin fold thickness was measured at a point mid-way between the neck
and hips by using an electronic digital micrometer caliper (Marathon Watch Company Ltd., Ontario,
Canada). A replica of the mouse dorsal skin was obtained by using silicon rubber (Repliflo, CuDerm
Corp., TX, USA). The skin impressions were photographed using an Antera 3D® camera (Miravex,
Dublin, Ireland) and analyzed by Antera CS program (Miravex). The volume of the depression,
the affected area of depression, maximum depth, and wrinkle length were measured on the surface of
the replica (a circle with a 16.8 mm-diameter).
4.6. Histological and Immunohistochemical Analysis in Mice

The dorsal skin samples (1 × 1 cm2 ) were fixed in 10% neutralized formalin for at least 24 h and
embedded in paraffin. Prior to staining, 4 μm sections were deparaffinized in xylene, and rehydrated
through a graded ethanol series (100%–70%). The sections were stained with hematoxylin and
eosin (H&E), Masson’s trichrome (MT), and Verhoeffe–Van Gieson (VVG). The immunohistochemical
detection of proliferating cell nuclear antigen (PCNA), 8-hydroxy-2 -deoxyguanosine (8-OHdG),
and Ki67 was performed in 4 μm thick deparaffinized sections. For antigen retrieval, the sections
were incubated with Tris-EDTA buffer solution (pH 9.0) for PCNA or citrate buffer solution (pH 6.0)
for 8-OHdG and Ki67 for 15 min in a decloaking chamber (Biocare Medical). The sections were
treated with 3% H2O2 for 10 min to inhibit the endogenous peroxidase activity and rinsed in 0.1 M
Tris-buffered saline (TBS) solution. Non-specific binding sites were blocked by the incubation of
the sections with 5% BSA for 1 h. The sections were incubated with 1:100 diluted anti-8OHdG or
anti-Ki67 primary antibody (Abcam, Cambridge, UK) overnight at 4 ◦ C, followed by incubation with
the secondary antibody (DAKO Envision+ System-HRP Labelled Polymer: Anti-mouse for PCNA
and 8-OHdG, and anti-rabbit for Ki-67) for 1 h. The sections were washed in 0.1 M TBS, incubated
with 3,3 -diaminobenzidine tetrahydrochloride peroxidase substrate solution (k-3468, DAKO Corp.,
CA, USA) for 1–3 min, counterstained with Mayer’s hematoxylin solution, and dehydrated through
a graded series of 70%–100% alcohol in xylene. Representative photomicrographs were taken by a
light microscope (Eclipse Ti microscope; Nikon, Tokyo, Japan). The epidermal thickness and staining
density of collagen were quantified using ImageJ software (National Institutes of Health, MD, USA).
4.7. Measurement of Reduced GSH Content in Mouse Plasma

The plasma GSH level was determined by using the Glutathione Fluorometric Assay Kit (BioVision,
CA, USA) in accordance with the manufacturer’s instructions. Each sample was added to a 96-well
plate. Each well had a total volume of 100 μL containing 20 μL of the plasma from each mouse.
Subsequently, 2 μL of GST reagent and 2 μL of monochlorobimane (MCB) was added. After incubation
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for 1 h at 37 ◦ C, fluorescence was measured by using a GENios fluorescence plate reader at an excitation
wavelength of 335 nm and an emission wavelength of 460 nm.
4.8. Comet Assay (Alkaline Single-Cell Gel Electrophoresis)

The alkaline comet assay was conducted in accordance with the method of Singh et al., with minor
modifications [74]. Leukocytes were isolated from whole blood using Histopaque 1077 (Sigma Aldrich
Co.), mixed with 0.7% low melting-point agarose and added to slides. The slides were immersed in cold
lysing solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 1% sodium laurylsarcosine, pH 10, 1% Triton
X-100, and 10% DMSO) at 4 ◦ C for 1 h. After lysis, the slides were transferred to electrophoresis buffer
(300 mM NaOH and 10 mM Na2EDTA, pH 13.0) at 4 ◦ C for 40 min. For electrophoresis, an electric
current of 25 V/300 ± 3 mA was applied at 4 ◦ C for 20 min. The slides were washed three times with
neutralizing buffer (0.4 M Tris, pH 7.5) at 4 ◦ C and treated with ethanol for 5 min. All steps were
conducted in the dark to prevent additional DNA damage. Fifty cells from two replicate slides each
were analyzed using a fluorescence microscope (LEICA DM LB, Bensheim, Germany) and image
analysis software (Komet 4.0; Kinetic Imaging, UK) to compute the tail intensity (equivalent to the
percentage of DNA in the tail), tail length, and tail moment (tail length × tail intensity).
4.9. Determination of Lipid Peroxidation in Skin Tissue

Mouse skin tissue was homogenized in 1.15% KCl solution (30 mg/mL). After centrifugation
(1,500 g, 15 min, 4 ◦ C), 100 μL supernatant was reacted with 400 μL of TBARS reagent (0.8% TBA)
and incubated at 95 ◦ C for 1 h. To terminate the reaction, 250 μL of distilled water and 1.25 mL of
n-butanol/pyridine (15:1, v/v) were added, and the solution was centrifuged again (1,500 g, 15 min).
The absorbance of the supernatant was measured at 540 nm and the concentration of lipid peroxides
was calculated based on the malondialdehyde (MDA) standard curve and normalized to the protein
content of each sample by the bicinchoninic acid assay (BCA; Pierce, Rockford, USA).
4.10. Cell Culture

HaCaT cells (Item no. 300493, Cell Lines Service, Eppelheim, Germany) were cultured in Dulbecco’s
modified Eagle medium nutrient mixture F-12 (Ham) 1:1 (D-MEM/F-12, GibcoBRL, Braunschweig,
Germany) powder medium supplemented with 10% fetal calf serum (FCS) (GibcoBRL, Grand Island,
NY, USA), 1% antibiotic/antimycotic solution (Corning, NY, USA) and 1.2 g/L sodium bicarbonate
(NaHCO3; Sigma-Aldrich, cat. No. S6014). Cells were incubated in 5% CO2 at 37 ◦ C.
Cells were irradiated with UVB in Chambres Noires darkrooms (Vilber, France) under a UVB
light lamp (VL-6 MC; Vilber, France) emitting 315 nm. The UVB emission was calculated as 30 mJ/cm2
= 580 μW/cm2 × 52 s.
4.11. Cell Viability (MTT) Assay

Cytotoxicity of CS and UVB on HaCaT cells was measured by colorimetric MTT assay. Cells were
seeded in 96-well plates to make 2 × 104 cells/well. After 24 h incubation in complete medium, water
(control) or 0.1~15 μg/mL CS extract was added to each well. After 24 h incubation, cells were washed
and either replaced with fresh complete medium or UVB-irradiated at 30, 45, and 70 mJ/cm2 (52, 77,
120 s) in 200 μL PBS and then replaced with fresh complete medium with the same concentration of
CS extract or water (control). After 5 h incubation, 1 mg/mL MTT solution was added to each well
and allowed to react for 4 h. The supernatant was removed and 100 μL of isopropyl alcohol (Merck,
Darmstadt, Germany) was added to dissolve the generated formazan crystals. The absorbance was
measured at 570 nm using a microplate reader (Infinite® 200 PRO, Tecan, Switzerland).
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4.12. Western Blot Analysis

The UVB-irradiated total dorsal skin was homogenized (Polytron System PT 1200 E,
Luzernerstrasse, Switzerland) in RIPA lysis buffer with freshly added protease and phosphatase
inhibitor cocktails. After centrifugation (4 ◦ C, 12,500 g, 20 min), the supernatant protein concentration
was determined by using the Bradford reagent (BioRad, CA, USA). Equal amounts of total protein
were separated by SDS-PAGE and transferred to PVDF membranes (Millipore Corporation). Next,
the membranes were blocked by incubation in 5% fat-free dry milk in 1× phosphate-buffered saline (PBS)
for 1 h at room temperature. The membranes were then incubated overnight 4 ◦ C with 1:1000 dilutions
of the primary antibodies specific for α-tubulin, IL-1β, PCNA, MMP9, TIMP-1, Smad2, p-Smad2 (Santa
Cruz Biotechnology, Dallas, TX, USA), iNOS, COX-2 (BD Biosciences, San Jose, CA, USA), TGF-β (R&D
Systems Inc., Minneapolis, MN, USA), 8OHdG (Abcam, Cambridge, UK), Ki67 (Millipore Corporation,
Billerica, MA, USA), and Nrf2 (Invitrogen, MA, USA). The membranes were rinsed with PBST and
incubated with peroxidase-conjugated 1:5000 dilutions of the anti-mouse and anti-rabbit secondary
antibodies (Millipore Corporation, Billerica, MA, USA). The antibody signals were visualized by a
chemiluminescence detection system (GE Healthcare Life Sciences, Buckinghamshire, England) and
photographed by an AE-9300 Ez-Capture system (ATTO, Tokyo, Japan). Band density was quantified
using ImageJ software.
HaCaT cells were seeded in a 60 mm culture dish to make 8 x 105 cells/well and incubated in
complete media for 24 h. Then, water (NOR, UVB), 0.5 μg/mL CS extract or 5 μg/mL CS extract was
treated and incubated for 24 h. Then, cells were washed with PBS and replaced with fresh complete
medium (NOR) or UVB-irradiated at 315 nm for 52 s at 30 mJ/cm2 in 2 mL PBS, and then replaced
with fresh complete medium with the same concentration of CS extract or water (control). After 5 h
incubation, protein was extracted from the cells in RIPA lysis buffer for Western blot analysis.
4.13. Reverse Transcriptase (RT) and Quantitative Polymerase Chain Reaction (qPCR)
Skin tissue total RNA was extracted by homogenization in TRIzol reagent (MRC, Cincinnati,
OH, USA. Cell total RNA was extracted by scrapping in TRIzol reagent (MRC, Cincinnati, OH,
USA). The purity of the total RNA was measured with a spectrophotometer and reverse transcribed
using ImProm II Reverse Transcriptase kit (Promega, Madison, Wis., USA). cDNA was synthesized
according to the manufacturer’s protocol. qPCR was performed in a Mic real-time PCR system (BMS,
biomolecular systems, Australia) using 5x HOT FIREPOL® EvaGreen® qPCR Supermix (Solis biodyne,
Tartu, Estonia) in a volume of 18 uL depending on the manufacturer’s cycling conditions. Relative
gene expression was measured using the comparative 2-(ΔΔCq) method. Expression of housekeeping
GAPDH mRNA was used for qPCR data standardization. The primers used are shown in Table 2.
Table 2. Primers used for quantitative polymerase chain reaction (qPCR).
Gene Forward Sequence Reverse Sequence Product Size

m.Catalase AACGCTGGATGGATTCTCCC GCCCTAACCTTTCATTTCCCTTCAG 133
m.Procollagen type 1 CCCTAGCCTTTTCTCCGCC TGGCAACTCCAAGTCCATCAT 238
m.Nrf2 GTGAGACGTGGAAACCCGAG GCCATAGGACATCTGGGAAGC 347
m.TXN GAGCAAGGAAGCTTTTCAGGAG GTCCCGTTTTGGATCCGAGT 252
m.SOD1 ATGGCGACGAAGGCCGTGTG GACCACCAGTGTGCGGCCAA 360
m.GAPDH AAGGTCGGTGTGAACGGATTT CAGAAGGGGCGGAGATGATG 364
h.Glutaredoxin CATCGGCATGGCTCAAGAG AATCTGCTTTAGCCGCGTCA 313
h.Procollagen type 1 AGGACAAGAGGCATGTCTGGTT TTGCAGTGTAGGTGATGTTCTG 156
h.GAPDH AAGGTCGGTGTGAACGGATTT CAGAAGGGGCGGAGATGATG 364
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Molecules 2019, 24, 2587
4.14. Sample Preparation for Liquid Chromatography-Tandem Mass Spectrometer (LC-MS/MS) Analysis
Non-targeted LC-MS/MS was used to analyze the components present in the CS extract. The CS
extract was diluted 10,000 times with deionized water and filtered 0.45 μm. CS extract samples were
stored at −20 ◦ C until LC-MS/MS analysis.
4.15. LC-MS/MS Analysis and Identification of Metabolites

Ultimate 3000 UHPLC and Q-Extractive Orbitrap Plus was equipped with an ACQUITY C18
column (10 cm × 2.1 mm, particle size 1.7 μm, Waters, USA). The injection volume for each sample was
5 μL. The column was eluted using the following binary gradient solutions: A (deionized water with
0.1% formic acid) and B (methanol, UPLC graded) with a flow rate of 0.4 mL/min, A: B = 100: 0 at 1 min,
0: 100 at 16 min, 0: 100 until 20 min, and 100: 0 at 22 min. The full scan/dd-MS2 system parameters
were: FTMS, ESI-positive mode with mass resolution of 70,000, full scan range: 80~1000 m/z, dd-MS2
(Top 10) resolution of 17,500 with collision energy 30, flow rate of nitrogen sheath gas and auxiliary
gas: 40 (arbitrary units) and 10 (arbitrary units), spray voltage: 3.5 kV, capillary temperature: 320 ◦ C,
S-lensRF level: 50, auxiliary gas heater temperature: 300 ◦ C. Metabolomics analysis for mouse skin
was carried out as outlined in the supplementary methods.
The original data from the UPLC-Orbitrap-MS2 analysis were extracted from the XCMS online
platform (http://xcmsonline.scripps.edu/) for data alignment and peak detection. The parameters of
the XCMS online were 10-sec bandwidth, 15 ppm tolerance for database research, and the rest were
UPLC-orbitrap default values. The MS2 data for peak identification was extracted from the LC-raw file
using Xcalibur 2.2 (Thermo Fisher Scientific, San Jose, CA, USA). The LC-MS/MS search function of the
online database (MycompoundID (www.mycompoundid.org) and HMDB (www.hmdb.ca)) was used
to perform metabolite identification using retention time, exact mass, and MS/MS peak intensities.

The data are expressed as mean ± standard error (SE) or standard deviation (SD). The results were
analyzed by one-way analysis of variance (ANOVA) and one tail t-test using SPSS software (SPSS,
Version 23.0 IBM Inc., USA). The criterion for statistical significance was p < 0.05.

Table S1: Compounds identified in corn silk (CS) extract and mouse skin using LC-MS/MS; Figure S1: Heatmaps
of UVB-irradiated and CS extract-administered mouse skin metabolites.
Author Contributions: Conceptualization, S.-M.L.; methodology, Y.-h.K., A.C., S.-A.K., H.H.; validation, E.-J.S.,
E.P.; formal analysis and investigation, Y.-h.K., A.C., S.-A.K., H.w.H, E.-J.S., E.P., S.-M.L.; data curation, Y.-h.K.,
A.C., S.A.K., M.K., H.w.H.; writing—original draft preparation, Y.-h.K., A.C., M.S.; writing—revising and editing,
M.S., S.-M.L.; visualization, Y.-h.K., M.K.; supervision, S.-M.L.; project administration, S.-M.L., E.P.
Funding: This work was supported by the BK21 Plus Project for Bioactive Nutrition, Yonsei University, Seoul 03722,
Republic of Korea.
Acknowledgments: The corn silk extract was provided by Kwang Dong Pharmaceutical, Seoul, Republic of Korea.
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385
molecules
Article
Investigation of the Biological Activities and
Characterization of Bioactive Constituents of
Ophiorrhiza rugosa var. prostrata (D.Don) & Mondal
Leaves through In Vivo, In Vitro, and In
Silico Approaches
Md. Adnan 1,2,† , Md. Nazim Uddin Chy 3,4,† , A.T.M. Mostafa Kamal 3, *,
Md Obyedul Kalam Azad 1,5 , Arkajyoti Paul 4,6 , Shaikh Bokhtear Uddin 7 , James W. Barlow 8 ,
Mohammad Omar Faruque 7 , Cheol Ho Park 1 and Dong Ha Cho 1, *
1 Department of Bio-Health Technology, Kangwon National University, Chuncheon 24341, Korea;
mdadnan1991.pharma@gmail.com (M.A.); azadokalam@gmail.com (M.O.K.A.);
chpark@kangwon.ac.kr (C.H.P.)
2 Senior Scientist, Rentia Plant Factory, Chuncheon 24341, Korea
3 Department of Pharmacy, International Islamic University Chittagong, Chittagong 4318, Bangladesh;
nazim107282@gmail.com
4 Drug Discovery, GUSTO A Research Group, Chittagong 4000, Bangladesh; arka.bgctub@gmail.com
5 Head of Research and Technology, Rentia Plant Factory, Chuncheon 24341, Korea
6 Department of Microbiology, Jagannath University, Dhaka 1100, Bangladesh
7 Ethnobotany and Pharmacognosy Lab, Department of Botany, University of Chittagong, Chittagong 4331,
Bangladesh; bokhtear@cu.ac.bd (S.B.U.); omf@cu.ac.bd (M.O.F.)
8 Department of Chemistry, Royal College of Surgeons in Ireland, D02YN77 Dublin, Ireland;
jambarlow@rcsi.ie
* Correspondence: mostafa@pharm.iiuc.ac.bd (A.T.M.M.K.); chodh@kangwon.ac.kr (D.H.C.)

Abstract: Ophiorrhiza rugosa var. prostrata is one of the most frequently used ethnomedicinal
plants by the indigenous communities of Bangladesh. This study was designed to investigate
the antidiarrheal, anti-inflammatory, anthelmintic and antibacterial activities of the ethanol extract of
O. rugosa leaves (EEOR). The leaves were extracted with ethanol and subjected to in vivo antidiarrheal
screening using the castor oil-induced diarrhea, enteropooling, and gastrointestinal transit models.
Anti-inflammatory efficacy was evaluated using the histamine-induced paw edema test. In parallel,
in vitro anthelmintic and antibacterial activities were evaluated using the aquatic worm and disc
diffusion assays respectively. In all three diarrheal models, EEOR (100, 200 and 400 mg/kg) showed
obvious inhibition of diarrheal stool frequency, reduction of the volume and weight of the intestinal
contents, and significant inhibition of intestinal motility. Also, EEOR manifested dose-dependent
anti-inflammatory activity. Anthelmintic action was deemed significant (P < 0.001) with respect
to the onset of paralysis and helminth death. EEOR also resulted in strong zones of inhibition
when tested against both Gram-positive and Gram-negative bacteria. GC-MS analysis identified
30 compounds within EEOR, and of these, 13 compounds documented as bioactive showed good
binding affinities to M3 muscarinic acetylcholine, 5-HT3, tubulin and GlcN-6-P synthase protein
targets in molecular docking experiments. Additionally, ADME/T and PASS analyses revealed their
drug-likeness, likely safety upon consumption and possible pharmacological activities. In conclusion,
our findings scientifically support the ethnomedicinal use and value of this plant, which may provide
a potential source for future development of medicines.

Molecules 2019, 24, 1367
Keywords: Ophiorrhiza rugosa; Rubiaceae; antidiarrheal; anti-inflammatory; anthelmintic;

antibacterial; in silico molecular docking; ADME/T and PASS
1. Introduction
Due to ongoing reports of antibiotic resistance by various pathogens, researchers have refocused
their interest in the use of natural antimicrobial agents to treat infections instead of established
antibiotics [1]. Although some conventional antibiotics may be bactericidal, they remain unable to
inhibit the release of bacterial toxins which complicates the clinical picture [2]. Analogous to bacterial
infections, helminths can persistently infect both humans and animals throughout their lifespan.
Helminths exhibit greater complexity than other pathogens and are capable of producing chronic
disease, yet these diseases are often neglected in developing regions [3]. The association of bacteria and
parasites with gastrointestinal disorders is a common situation in developed and developing countries.
Various etiological (Salmonella, Campylobacter, Escherichia, Shigella, Yersinia enterocolitica, parasites, and
viruses) agents responsible for enteric infections may lead to dysentery-like chronic diarrhea [4].
Such infectious diseases cannot be cured easily at present, due to rapid resistance to available drugs;
therefore screening of new therapeutic avenues such as plants may provide an alternate and effective
approach for the development of novel agents.
Throughout the ages, plants have served humans for innumerable therapeutic interventions,
ranging from the common cold to life-threatening conditions. The value of phytomedicinal approaches
still resonates with the R&D departments of modern pharmaceutical giants [5]. The development
of modern medicines has, in many instances, stemmed from ethnic medicinal uses, and meticulous
investigation of naturally occurring bioactive compounds derived from plant screening programs
assists the development of new synthetic drugs [6]. As plant-derived drugs contain a pool of
metabolites with potential complementary pharmacological actions, their use in mitigating chronic
diseases through synergism is an area of intense interest [7]. In this light, indigenous knowledge
can help to contribute to the rational drug discovery and development of new drugs from medicinal
plants [8]. While indigenous communities typically have a rich knowledge of ethnic medicines,
these uses are based on empirical evidence, and proper mechanistic knowledge of biological
or pharmacological properties necessitates a scientifically sound investigation, followed by the
documentation and characterization of bioactive components of the studied species [9,10]. Hence,
proper research on medicinal plants is invaluable in the search for novel bioactive agents for the
management of the disease. Cognizant of these principles, we selected the ethnomedicinal plant
Ophiorrhiza rugosa var. prostrata for the present study.
Ophiorrhiza rugosa var. prostrata (D.Don) Deb & Mondal (syn: Ophiorrhiza harrisiana B.Heyne
ex Hook.f, Ophiorrhiza prostrata D.Don) is an annual herb belonging to the Rubiaceae family, which
naturally grows in Chittagong and both the Chittagong Hill Tract and Sylhet regions of Bangladesh,
where it is variously known as ‘Jari’ or ‘kalashona’ (Chakma), ‘Jariphul’ (Tanchangya) or ‘Pahari
mehedi’ (Marma). O. rugosa var. prostrata is used by the Tanchangya, Marma, and Chakma indigenous
communities for the treatment of different diseases. For example, a paste of the leaves is used for
the treatment of skin infections (boils) by the Tanchangya people. The Marma community prepares
a tea from the leaves, which is drunk daily for the treatment of body aches and chest pain, while
the Chakma community applies sun-dried crushed leaves to the ears for the treatment of earache
(personal communication). In addition, the crushed roots of the plant are used for the treatment of
dysentery [11,12]. Juice from the leaves is drunk in the treatment of diarrhea within the Marma
community (personal communication). However, despite such widespread use, there has been
no scientific investigation to date on either pharmacological or phytochemical aspects of the plant
to validate its traditional uses. Therefore, we aimed to investigate the bioactive components of
O. rugosa var. prostrata leaves using gas chromatography-mass spectrometry (GC-MS). As plants
388
Molecules 2019, 24, 1367
contain a mixture of phytochemicals, robust separation and identification methods are important to
elucidate potential bioactive and toxic constituents [13]. GC-MS, coupled with appropriate detection
systems is an invaluable tool for the separation and identification of the components of complex,
volatile mixtures [14]. Many plant secondary metabolites are sufficiently small, adequately volatile,
and thermostable in the GC environment to be easily analyzed by GC-MS [15]. In addition to the
phytochemical investigation, we aimed to investigate the known therapeutic applications of the plant
through a combination of in vivo (antidiarrheal and anti-inflammatory), in vitro (anthelmintic and
antibacterial) and in silico (molecular docking, ADME/T and PASS) analyses.
2. Results
2.1. GC-MS Analysis

The GC-MS analysis of EEOR revealed 30 compounds, which are listed in Table 1, along with their
chemical composition, while the total ionic chromatogram (TIC) is shown in Figure 1. The most
abundant component by peak area was shown to be phytol (15.50%), followed by γ-sitosterol
(14.94%), stigmasterol (7.92%), erucamide (5.39%), squalene (4.83%), methyl palmitate (3.95%), methyl
linoleate (2.96%), vitamin E (2.51%), methyl stearate (1.21%), ethyl linolenate (1.17%), loliolide (1.10%),
2-palmitoylglycerol (0.91%), and neophytadiene (0.86%). The structures of these compounds are
presented in Figure 2.
7,&

PLQ
Figure 1. Total ionic chromatogram (TIC) of EEOR (GC-MS, 70eV).
389
Molecules 2019, 24, 1367
Table 1. List of compounds identified in EEOR by GC-MS analysis.
S.N. RT (min) PA (%) Name of Compound Molecular Formula

1 7.624 0.92 Carbonic acid, hexadecyl methyl ester C18 H36 O3
2 10.130 1.77 1-Nonadecene C19 H38
Succinic acid, tridec-2-yn-1-yl
3 12.235 1.29 C24 H40 O4
trans-4-methylcyclohexyl ester
6-Hydroxy-4,4,7α-trimethyl-5,6,7,7α
4 12.388 1.10 C11 H16 O3
-tetrahydrobenzofuran-2(4H)-one, or Loliolide
5 12.471 1.97 1-Nonadecene C19 H38
2-Cyclohexen-1-one,
6 12.585 1.38 C13 H18 O3
4-hydroxy-3,5,6-trimethyl-4-(3-oxo-1-butenyl)-
7 13.003 0.86 Neophytadiene C20 H38
Hexadecanoic acid, methyl ester or Methyl
8 13.918 3.95 C17 H34 O2
Palmitate
9 14.588 5.47 9H-Pyrido[3,4-b]indole, 1-methyl- C12 H10 N2
9,12-Octadecadienoic acid (Z,Z)-, methyl ester
10 15.659 2.96 C19 H34 O2
or Methyl linoleate
11 15.716 4.33 8,11,14-Docosatrienoic acid, methyl ester C23 H40 O2
12 15.843 15.50 Phytol C20 H40 O
13 15.939 1.21 Methyl stearate C19 H38 O2
9,12,15-Octadecatrienoic acid, ethyl ester,
14 16.366 1.17 C20 H34 O2
(Z,Z,Z)- or Ethyl linolenate
15 17.203 1.92 Octadecanoic acid, 3-hydroxypropyl ester C21 H42 O3
16 17.825 1.61 1-Heptatriacotanol C37 H76 O
17 18.823 2.21 6,9-Octadecadienoic acid, methyl ester C19 H34 O2
Hexadecanoic acid, 2-hydroxy-1-(hydroxy-
18 19.377 0.91 C19 H38 O4
methyl)ethyl ester, or 2-Palmitoylglycerol
19 19.726 0.95 Diisooctyl phthalate C24 H38 O4
20 20.844 2.87 E,E,Z-1,3,12-Nonadecatriene-5,14-diol C19 H34 O2
21 20.914 1.10 Ethyl 9,12,15-octadecatrienoate C20 H34 O2
22 21.522 5.39 13-Docosenamide, (Z)- or Erucamide C22 H43 NO
23 21.885 4.83 Squalene C30 H50
24 22.372 1.14 α-Tocospiro B C29 H50 O4
1,6,10,14,18,22-Tetracosahexaen-3-ol,
25 22.765 0.92 C30 H50 O
2,6,10,15,19,23-hexamethyl-, (all-E)-
26 24.931 2.51 Vitamin E C29 H50 O2
27 26.431 5.00 Campesterol C28 H48 O
28 26.876 7.92 Stigmasterol C29 H48 O
29 27.768 14.94 γ-Sitosterol C29 H50 O
30 31.229 1.91 Lup-20(29)-en-3-ol, acetate, (3β)- C32 H52 O2
RT: Retention time; PA: Peak area.
390
Molecules 2019, 24, 1367
2+ 2
2
2
2
+2
3DOPLWR\OJO\FHURO 0HWK\O SDOPLWDWH
0HWK\O VWHDUDWH 1HRSK\WDGLHQH
+2
3K\WRO
6TXDOHQH
+ + 2
+2
+2
6WLJPDVWHURO
9LWDPLQ (
Figure 2. Chemical structures of the major bioactive compounds identified in the EEOR.
2.2. Acute Toxicity Test

The acute oral toxicity testing of EEOR did not show any particular evidence of toxicity or
behavioral abnormalities at doses of 5, 50, 100, 200, 400, 1000 or 2000 mg/kg. During the 72 h
inspection period, no mortality or physical changes such as allergic reactions, loss of body weight, etc.
were observed at the specified doses (data not shown).
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Molecules 2019, 24, 1367
2.3. Qualitative Phytochemical Screening

Preliminary phytochemical screening of EEOR suggested the presence of alkaloids, carbohydrates,
flavonoids, phenols, tannins, saponins, steroids, sterols, quinones, oxalate, coumarins, and terpenoids
(data not shown).
2.4. Effects of EEOR on Castor Oil-Induced Diarrhea in Mice

The effects of EEOR administration on castor oil-induced diarrhea are summarized in Table 2.
In this model, EEOR caused significant inhibition of diarrhea, in a dose-dependent manner.
The maximum inhibitory effect was observed at a dose of 400 mg/kg (62.50%, P < 0.001), which
is similar to the reference drug loperamide (65.62%, P < 0.001). In addition, EEOR caused a noticeable
reduction in defecation numbers at doses of 100 mg/kg (45.20%, P < 0.01), 200 mg/kg (52.05%, P < 0.001)
and 400 mg/kg (60.27%, P < 0.001) respectively, compared to the negative control. The reduction of
diarrheal feces was also exhibited dose-dependently, with the best antidiarrheal effect observed at the
higher dose of 400 mg/kg, compared to the standard drug.
Table 2. The effect of Ophiorrhiza rugosa extract on feces count in castor oil-induced diarrhea in mice.
% Inhibition of Total Number of % Inhibition of

Treatment (mg/kg) Total Number of Feces
Defecation Diarrheal Feces Diarrhea
Control (0.1
14.60 ± 0.74 6.40 ± 0.81
mL/mouse)
Loperamide (5) 5.40 ± 0.24 *** 63.01 2.20 ± 0.20 *** 65.62
EEOR (100) 8.00 ± 0.44 ** 45.20 5.00 ± 0.31 *** 21.87
EEOR (200) 7.00 ± 0.83 *** 52.05 3.80 ± 0.48 ** 40.62
EEOR (400) 5.80 ± 0.20 *** 60.27 2.40 ± 0.24 *** 62.50
Significantly different when compared with that of the control group at ** P < 0.01, *** P < 0.001. Results are
presented as mean ± SEM (n = 6).
2.4.1. Effects of EEOR on Castor Oil-Induced Enteropooling in Mice

The effect of EEOR on castor oil-induced enteropooling (Table 3) was a significant reduction in
the volume and weight of the intestinal contents. In comparison to the negative control (0.51 ± 0.025),
the mean volume of intestinal fluids decreased dose-dependently (0.440 ± 0.014, 0.40 ± 0.017 and
0.34 ± 0.063 at doses of 100, 200 and 400 mg/kg EEOR respectively). In addition, compared to the
standard drug loperamide, the dose of 400 mg/kg showed a maximal inhibitory effect on both volume
(32.29%, P < 0.05) and weight (49.57%, P < 0.001) of intestinal contents.
Table 3. The effect of Ophiorrhiza rugosa extract on castor oil-induced enteropooling in mice.
Volume of Intestinal Weight of Intestinal

Treatment (mg/kg) % Inhibition % Inhibition
Content (mL) Content (gm)
Control (0.1
0.51 ± 0.025 0.71±0.022
mL/mouse)
Loperamide (5) 0.26 ± 0.013 *** 49.42 0.29±0.012 *** 58.87
EEOR (100) 0.44 ± 0.014 ** 13.22 0.55±0.030 ** 22.25
EEOR (200) 0.40 ± 0.017 *** 21.78 0.44±0.090 ** 38.02
EEOR (400) 0.34 ± 0.063 * 32.29 0.35±0.047 *** 49.57
Significantly different when compared with that of the control group at * P < 0.05, ** P < 0.01, *** P < 0.001. Results
are presented as mean ± SEM (n = 6).
2.4.2. Effects of EEOR on Charcoal-Induced Intestinal Transit in Mice

The outcomes following different doses of EEOR on intestinal transit are shown in Table 4.
In contrast to the negative control, EEOR significantly (P < 0.001) reduced the peristalsis index at three
different doses. Of these, the 400 mg/kg dose produced the most significant inhibition (58.33%) of
intestinal motility, comparable to the standard drug loperamide (57.73%).
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Table 4. The effect of Ophiorrhiza rugosa extracts on intestinal transit in mice using a charcoal meal as
a marker.
Total Length of Distance Travelled Peristalsis Index % Inhibition Relative

Treatment (mg/kg)
Intestine (cm) by Marker (cm) (%) to Control
Control (0.1 mL/mouse) 48.60 ± 0.51 41.40 ± 0.93 85.19 ± 1.74
Loperamide (5) 49.20 ± 0.58 20.80 ± 0.73 42.26 ± 1.32 *** 57.73
EEOR (100) 44.20 ± 0.37 ** 29.20 ± 0.58 ** 66.07 ± 1.32 *** 33.92
EEOR (200) 43.00 ± 0.44 ** 24.80 ± 0.86 *** 57.69 ± 2.08 *** 42.30
EEOR (400) 48.30 ± 0.25 *** 20.10 ± 1.36 *** 41.66 ± 3.02 *** 58.33
Significantly different when compared with that of the control group at ** P < 0.01, *** P < 0.001. Results are
presented as mean ± SEM (n = 6).
2.5. Effects of EEOR on Histamine-Induced Mouse Paw Edema

The anti-inflammatory activity of EEOR and diclofenac sodium against histamine-induced edema
is shown in Table 5. The results show that the standard drug significantly (P < 0.001) inhibited the
inflammatory response (42.42%, 60.29%, 66.66% and 78.57%, respectively, at 1 h intervals for 4 h)
after sub-plantar injection of histamine, compared to the control group. On the other hand, oral
administration of EEOR (100–400 mg/kg) significantly blocked the inflammatory response induced by
histamine in a dose-dependent manner, with a dose of 400 mg/kg displaying statistically significant
38.38%, 42.64%, 54.76% and 57.14% (P < 0.001) reductions in paw edema at all hourly intervals over 4 h.
Table 5. Anti-inflammatory activity of Ophiorrhiza rugosa extract on histamine-induced paw edema.
Paw Volume (mm) (% Inhibition)

Treatment (mg/kg)
1h 2h 3h 4h
Control (0.1mL/mouse) 0.454 ± 0.010 0.392 ± 0.012 0.340 ± 0.007 0.312 ± 0.008
0.350 ± 0.004 *** 0.290 ± 0.007 *** 0.264 ± 0.010 *** 0.248 ± 0.012 ***
Diclofenac-Na (10)
(42.42) (60.29) (66.66) (78.57)
0.422 ± 0.005 ** 0.358 ± 0.015 *** 0.310 ± 0.010 *** 0.290 ± 0.004 **
EEOR (100)
(11.11) (17.64) (23.8) (21.42)
0.398 ± 0.007 *** 0.334 ± 0.009 *** 0.294 ± 0.006 *** 0.278 ± 0.006 ***
EEOR (200)
(20.20) (30.88) (35.71) (32.14)
0.344 ± 0.012 *** 0.300 ± 0.006 *** 0.260 ± 0.005 *** 0.246 ± 0.005 ***
EEOR (400)
(38.38) (42.64) (54.76) (57.14)
Each value is expressed as mean ± SEM (n = 6). ** P < 0.01, *** P < 0.001 compared with the control group
(Dunnett’s test).
2.6. Anthelmintic Activity

Figure 3 represents the anthelmintic activity of EEOR. The degree of anthelmintic activity shown
by the extract was found to be directly proportional to the concentration employed, ranging from the
lowest to highest concentration (5, 8, and 10 mg/mL). At concentrations of 5, 8 and 10 mg/mL, EEOR
showed significant (P < 0.001) paralysis times of (23.28 ± 1.07), (15.30 ± 0.72) and (10.67 ± 0.31) min,
while times to death were (57.63 ± 4.42), (32.83 ± 1.95) and (24.59 ± 1.43) min respectively. In the
experiment, the positive control (levamisole, 1 mg/mL) showed a paralysis time of (3.22 ± 0.08) min
and time to death of (6.19 ± 0.61) min.
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Figure 3. Anthelmintic activity of the ethanol extract of Ophiorrhiza rugosa leaves (EEOR). Each value in
the table is represented as mean ± SEM (n = 3); NC: Negative control; PC: Positive control, Levamisole
(1 mg/mL). *** P < 0.001 compared with PC (Dunnett’s test).
2.7. Antibacterial Activity

The antibacterial activity of EEOR is presented in Table 6. The most potent inhibitory effects
were exhibited against one Gram-positive (Bacillus subtilis), and two Gram-negative (Salmonella typhi
and Escherichia coli) bacteria. The broadest zone of inhibition (16.23 ± 0.68 mm) was found against
Escherichia coli at a concentration of 1000 μg/disc, followed by Bacillus subtilis (14.80 ± 0.72 mm) and
Salmonella typhi (12.80 ± 0.34 mm). On the other hand, the extract showed no inhibitory effect against
four bacteria, namely Staphylococcus aureus, Bacillus cereus, Salmonella paratyphi, and Pseudomonas
aeruginosa.
Table 6. Antibacterial effects of the ethanol extract of Ophiorrhiza rugosa leaves.
Zone of Inhibition (mm)

Bacterial Strain Name of the Bacteria Concentration (μg/disc) Kanamycin
(30 μg/disc)
EEOR 500 EEOR 800 EEOR 1000
Staphylococcus aureus (ATCC 6538) - - - 29.30 ± 0.60
Gram-positive Bacillus subtilis (ATCC 6633) 7.33 ± 0.57 11.70 ± 0.75 14.80 ± 0.72 32.81 ± 0.67
Bacillus cereus (ATCC 14579) - - - 27.50 ± 0.58
Salmonella typhi (ATCC 29629) - 7.33 ± 0.57 12.80 ± 0.34 28.218±0.81
Salmonella paratyphi (ATCC 9150) - - - 30.51 ± 0.50
Gram-negative
Escherichia coli (ATCC 8739) 8.20 ± 0.72 11.26 ± 1.16 16.23 ± 0.68 31.20 ± 0.82
Pseudomonas aeruginosa (ATCC 9027) - - - 26.28 ± 0.36
Values are presented as mean inhibition zone (mm) ± SD of three replicates; -: no activity.
2.8. Molecular Docking Study for Antidiarrheal Activity

Results of docking analyses for antidiarrheal activity are shown in Table 7, and the docking figures
are shown in Figures S1–S5. In this study, two major receptors (M3 muscarinic acetylcholine receptor,
PDB: 4U14; and 5-HT3 receptor, PDB: 5AIN) involved in intestinal motility were used to explore
the possible antidiarrheal activity of EEOR. In the case of the M3 muscarinic acetylcholine receptor
(PDB: 4U14), Vitamin E showed the highest docking score (−8.80 kcal/mol), better than the standard
drug loperamide (−7.32 kcal/mol). On the other hand, for the 5-HT3 receptor (PDB: 5AIN), loliolide
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(−5.47 kcal/mol) exhibited the highest docking score, followed by ethyl linolenate, phytol, methyl
linoleate, neophytadiene, methyl palmitate, and methyl stearate.
Analysis of the docking fits of each compound suggested various interactions between the
ligands and the target enzymes. Loliolide interacts with the M3 muscarinic receptor through
one H-bond to Asn507 and two π-π stacking interactions with Tyr529 and Tyr533 (docking score
−6.63 Kcal/mol). Ethyl linolenate interacts with the same enzyme through the formation of
two H-bonds with Ile222 and Leu225 residues (docking score −6.76 kcal/mol), while methyl
linoleate interacted with the enzymatic pocket by establishing one H-bond with Ile222 (docking
score −3.26 kcal/mol). 2-Palmitoylglycerol interacted through two H-bonds with Asn152 and
Ser151 (docking score −3.55 kcal/mol). Methyl palmitate (score: −2.00 kcal/mol), phytol (score:
−3.62 kcal/mol), and vitamin E (score: −8.80 kcal/mol) each form one H-bond, with Tyr148, Ile222
and Ser151 residues respectively.
Table 7. Docking scores of the major bioactive compounds.
Docking Score 1
Compound Name
4U14 5AIN 1SA0 1XFF
Loliolide −6.63 −5.47 −4.49 −4.88
Ethyl linolenate −6.76 −3.47 −5.36 −3.10
Methyl linoleate −3.26 −1.65 −1.87 0.25
Erucamide − − −2.35 −1.21
γ-Sitosterol − − −7.00 −
2-Palmitoylglycerol −3.55 − − −1.16
Methyl palmitate −2.00 −0.25 −1.10 +1.81
Methyl stearate − +1.62 − +2.76
Neophytadiene −2.55 −0.69 −0.59 +1.18
Phytol −3.62 −2.08 −2.30 −0.12
Squalene − − − −
Stigmasterol − − −7.13 −
Vitamin E −8.80 − −6.65 −
Reference drugs
−7.32 − −6.26 −2.73
(Loperamide/Levamisole/Kanamycin)
1 Docking scores in kcal/mol; Bold text indicates the highest score.
On the other hand, loliolide binds to the enzymatic pocket of the 5-HT3 receptor (PDB ID: 5AIN)
by forming one hydrogen bond with Ile116 (docking score −5.47 kcal/mol). Ethyl linolenate (score:
−3.47 kcal/mol) and methyl linoleate (score: −1.65 kcal/mol) interact with the same enzymatic
pocket, via one H-bond with Glu191 and Arg57 respectively. Methyl stearate interacts with this
same enzymatic pocket, by forming one H-bond with Arg57, with a docking score +1.62 kcal/mol.
Phytol interacts with the same enzymatic pocket by stabilizing one H-bond with Thr34 (docking score
−2.08 kcal/mol). However, methyl palmitate and neophytadiene did not show any interactions with
5AIN. The standard drug loperamide interacts with 4U14 by forming two π-π stacking interactions
with Trp525, with a docking score of −7.32 kcal/mol Figure S10C,F.
2.9. Molecular Docking Study for Anthelmintic Activity

Results of docking analysis for anthelmintic activity are presented in Table 7. From the results, it
is clear that stigmasterol showed the highest docking score against tubulin (−7.13 kcal/mol), followed
by γ-sitosterol (−7.00 kcal/mol), vitamin E (−6.65 kcal/mol), ethyl linolenate (−5.36 kcal/mol),
loliolide (−4.49 kcal/mol), erucamide (−2.35 kcal/mol), phytol (−2.30 kcal/mol), methyl linoleate
(−1.87 kcal/mol), methyl palmitate (−1.10 kcal/mol), and neophytadiene (−0.59 kcal/mol). Among
all compounds, three, namely stigmasterol (−7.13 kcal/mol), γ-sitosterol (−7.00 kcal/mol), and
vitamin E (−6.65 kcal/mol) showed better docking scores in comparison to the standard levamisole
(−6.26 kcal/mol). However, 2-palmitoylglycerol and squalene did not dock with tubulin (PDB: 1XFF).
In this study, the best fits found for illustrating the interactions with tubulin are shown in Figures
S6 and S7. Ethyl linolenate and methyl linoleate interact with tubulin by forming one H-bond with
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Lys254, whereas erucamide interacted with the same pocket by establishing one H-bond with Asn101.
Methyl palmitate and phytol instead form one H-bond with Lys254. Five compounds, namely loliolide,
γ-sitosterol, neophytadiene, stigmasterol, and vitamin E did not show any interactions with tubulin.
The docking figures of standard drugs are shown in Figure S10B,E.
2.10. Molecular Docking Study for Antibacterial Activity

Thirteen compounds of EEOR were docked with the GlcN-6-P synthase enzyme to assess possible
antibacterial activity. Our results indicated that loliolide had the highest binding affinity with the
GlcN-6-P synthase enzyme, with a docking score of −4.88 kcal/mol, followed by ethyl linolenate
(−3.10), erucamide (−1.21), 2-palmitoylglycerol (−1.16), phytol (−0.12), methyl linoleate (0.25),
neophytadiene (+1.18), methyl palmitate (+1.81) and methyl stearate (+2.76). Among all compounds,
loliolide and ethyl linolenate showed the best binding affinity against 1XFF, with docking scores of
−4.88 and −3.10 kcal/mol respectively, which also ranked better than the standard drug kanamycin
(−2.73 kcal/mol). In the antibacterial docking study, the best fit found for loliolide in the enzymatic
pocket of GlcN-6-P synthase involved stabilization through the formation of four H-bonds with Hie86,
Cyt1, Trp74 and Gly99. The best-ranked fit of ethyl linolenate to the same enzyme was to the binding
pocket of 1XFF through two H-bonds to Thr76, and one H-bond with each of Arg73, Asp123, and
His77. Methyl linoleate interacts with 1XFF by forming three H-bonds with Arg73, Hie86, and Thr76.
The best fit for erucamide in the same enzymatic pocket involved stabilization through the formation
of four H-bonds, with Hie86, Arg73, His77, and Asp123. 2-Palmitoylglycerol interacts with the enzyme
by forming two H-bonds with Thr76, and two H-bonds with Asp123 and His77. Methyl palmitate
interacts with 1XFF via only one H-bond with Arg73, and methyl stearate by forming two H-bonds,
with Hie86 and Arg73. Finally, phytol binds to the enzymatic pocket of 1XFF by forming two H-bonds
with Asp123 and Thr76. On the other hand, neophytadiene did not show any interactions with 1XFF.
The reference drug kanamycin interacts with GlcN-6-P synthase by forming three H-bonds with Trp74,
Cyt1, and Gly99, with a docking score of −2.73 kcal/mol. The docking scores obtained for each
compound are shown in Table 7, and the docking figures are shown in Figures S8 and S9. The docking
figures of standard drugs are shown in Figure S10A,D.
2.11. ADME Analysis

According to Lipinski’s rule of five, the compounds γ-sitosterol, squalene, stigmasterol, and
vitamin E violated rules of lipophilicity and molecular refractivity. Conversely, loliolide and
2-palmitoylglycerol met Lipinski’s conditions, which are considered to predict optimal drug-like
character. All other compounds contravened no more than one rule (Table 8).
Table 8. ADME property prediction for the major compounds of EEOR, obtained using Swiss ADME.
Molar
Compound Name MW 1 (g/mol) HB Acceptor 2 HB Donor 3 Log Po/w 4 Rule of Five 6
Refractivity 5
Loliolide 196.24 3 1 1.53 52.51 0
Ethyl linolenate 306.48 2 0 5.82 98.12 1
Methyl linoleate 297.47 2 0 5.69 98.78 1
Erucamide 337.58 1 1 6.77 110.30 1
γ-Sitosterol 414.71 1 1 7.19 133.1 2
2-Palmitoylglycerol 330.50 4 2 4.72 97.06 0
Methyl palmitate 270.45 2 0 5.54 85.12 1
Methyl stearate 298.50 2 0 6.24 94.73 1
Neophytadiene 278.52 0 0 7.07 97.31 1
Phytol 296.53 1 1 6.22 98.94 1
Squalene 410.72 0 0 9.38 143.48 2
Stigmasterol 412.69 1 1 6.96 132.75 2
Vitamin E 430.71 2 1 8.27 139.27 2
1 MW, Molecular weight (acceptable range: <500). 2 HB, Hydrogen bond acceptor (acceptable range: ≤10). 3 HB,
Hydrogen bond donor (acceptable range: ≤5). 4 Lipophilicity (expressed as Log Po/w , acceptable range: <5).
5 Molar refractivity should be between 40 and 130. 6 Rule of five: Number of violations of Lipinski’s rule of five;
recommended range: 0–4.
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2.12. PASS Prediction

PASS analysis indicated possible targets and likely pharmacological activities of each of the major
compounds within EEOR. We evaluated six biological properties for each compound, based on the
values of Pa > Pi and Pa > 7. This prediction approach suggested several important activities of
the compounds studied, including antibacterial, anthelmintic, anti-inflammatory, spasmolytic and
antiprotozoal actions, which are relevant to our present study. The predicted pharmacological activity
profiles of all major compounds are presented in Table 9.
Table 9. Biological activities predicted for Ophiorrhiza rugosa major compounds by PASS online.
Compound Name Biological Properties Predicted by Pass Online Pa Pi

Sugar-phosphatase inhibitor 0.727 0.028
Antibacterial 0.418 0.026
Spasmolytic, urinary 0.454 0.062
Loliolide
Anti-inflammatory 0.416 0.088
Antiperistaltic 0.345 0.018
Antihelmintic 0.345 0.071
Lipid metabolism regulator 0.951 0.003
Histamine release inhibitor 0.523 0.028
Ethyl linolenate
Antiparasitic 0.489 0.017
Anti-inflammatory, intestinal 0.438 0.015
Antisecretoric 0.781 0.005
Methyl linoleate
Reductant 0.637 0.009
Antihelmintic (Nematodes) 0.500 0.017
Anti-infective 0.424 0.038
Prostaglandin E1 antagonist 0.470 0.005
Erucamide
Albendazole monooxygenase inhibitor 0.450 0.026
Antitoxic 0.387 0.025
Antihypercholesterolemic 0.977 0.001
Antiviral (Influenza) 0.686 0.006
Antiinflammatory 0.572 0.038
γ-Sitosterol
Antiacne 0.529 0.005
Antiprotozoal (Leishmania) 0.316 0.091
Antibacterial 0.282 0.067
Antiinfective 0.757 0.005
2-Palmitoylglycerol
Calcium channel (voltage-sensitive) activator 0.637 0.014
Methyl palmitate
Reductant 0.523 0.020
Antimutagenic 0.513 0.014
GABA aminotransferase inhibitor 0.820 0.003
Methyl stearate
Gastrin inhibitor 0.716 0.004
Antinociceptive 0.538 0.019
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Table 9. Cont.
Compound Name Biological Properties Predicted by Pass Online Pa Pi

Carminative 0.691 0.007
Antiulcerative 0.585 0.012
Neophytadiene
Phytol
Spasmolytic 0.506 0.027
Squalene
Dermatologic 0.809 0.004
Antiacne 0.552 0.004
Antiinflammatory 0.541 0.045
Stigmasterol
Antisecretoric 0.367 0.068
Bone formation stimulant 0.306 0.020
Lipid peroxidase inhibitor 0.978 0.002
Free radical scavenger 0.783 0.003
Vitamin E
Spasmolytic 0.525 0.024
Pa = Probable activity; Pi = Probable inactivity.
3. Discussion
Infectious and parasitic diseases continue to represent intimidating issues for developing countries,
due to the lack of useful and safe drugs and the increasing resistance of pathogens to available
antibiotics or anti-parasitic agents. A common manifestation of these issues is infectious diarrhea,
attributable to both enteric bacterial pathogens and parasites [16]. Such infectious agents may evoke
not only adverse effects on intestinal functions but also increase systemic risk via compromising
host immunity, leading to increased morbidity and mortality [17]. To treat such infectious diseases,
different plant parts, plant extracts, and plant-derived products have been used in traditional medicine.
However, many of these traditional medicines have not been formally reported in the literature
to date. Recent comprehensive reports on plants used for the treatment of infectious diseases,
including diarrhea and dysentery have indicated their possible applications as alternative therapies [18].
In Ethiopia for example, a range of medicinal plants including Calpurnia aurea, Croton marcostachyus,
and Echinops kebercho have been scientifically validated as anti-infective agents [19]. In addition,
combined screening of anti-diarrheal and anti-infective properties of medicinal plants could prove a
valid strategy to identify novel therapeutics. A study conducted by Taylor et al. 2013 suggested that
plants demonstrating significant anti-bacterial activity against entero-pathogens could be considered
as potential diarrheal treatments [20]. In vitro and in vivo investigation of Rhus plants including Rhus
semialata, Rhus javanica, and Rhus tripartitum produced significant anti-bacterial and antidiarrheal
effects and the authors concluded that the presence of antibacterial agents might mediate the diarrhea
prevention [21,22]. However, to recognize the intrinsic value of plant extracts, the involvement of both
in vitro and in vivo approaches is important in the clinically search for effective anti-infective agents.
Studies of plants with established ethnomedicinal uses must consider ethnomedicinal preparation
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practices when evaluating materials scientifically in the laboratory environment. Thorough extraction
protocols are important to completely evaluate both therapeutic and toxicological potential of medicinal
plants. Typically, plant phytochemicals possess diverse chemical functionalities, yet most are readily
soluble in methanol or ethanol, due to their high extractability and high polarity. Many nonpolar
compounds are also soluble in this solvent [23,24]. Therefore methanol and ethanol are frequently used
for extraction of medicinal plants prior to evaluation of their therapeutic potential, and we selected
ethanol for our extraction of O. rugosa leaves, the most commonly used part of the plant. Our study
identified potential novel active components from the ethanol extract of Ophiorrhiza rugosa leaves
(EEOR), having antidiarrheal, anti-inflammatory, anthelmintic and antibacterial properties.
To verify the ethnomedicinal uses of Ophiorrhiza rugosa, we examined its antidiarrheal activity,
as well as its possible mechanism(s) of action in different animal diarrheal models. In all diarrheal
experiments, a high dose of the natural laxative castor oil (0.5 mL) was administered to each mouse.
The active metabolite of the oil (ricinoleic acid) is liberated via the action of small intestinal lipases,
thus altering the motility of gastrointestinal smooth muscle [25,26]. Upon binding of the metabolite
with EP3 prostanoid receptors on smooth muscle cells, it inhibits water and electrolyte absorption
from the intestine, resulting in accumulation of fluid and interruption of secretory functions, which in
turn generates a deleterious effect in the intestine [27,28]. Apart from its laxative effect, ricinoleic acid
causes intestinal dysfunction via local inflammation and stimulation of prostaglandin biosynthesis,
which also inhibits reabsorption of ions and water [29]. In all antidiarrheal assays, loperamide was
used as a standard drug, which enhances the rate of absorption by reducing the volume and movement
of intestinal contents [30].
In castor oil-induced diarrhea, the ethanol extract of O. rugosa produced a remarkable inhibitory
effect, in terms of both defecation rate and diarrhea. The extract, at all doses (100, 200, 400 mg/kg)
decreased the total number of feces at 1h intervals over 4h, while diarrheal feces were reduced,
indicating an alteration of defecation frequency and consistency. Among all three doses of EEOR,
200 and 400 mg/kg significantly (P < 0.001) reduced defecation numbers by 52.05% and 60.27%
respectively, which indicates a dose-dependent antidiarrheal action. A dose of the extract with
400 mg/kg EEOR exhibited inhibition (62.50%) of diarrhea that was comparable to the standard drug
loperamide (65.62%). This demonstrates that a relatively high dose of EEOR is required to evoke the
desired response, and a similar phenomenon has been observed by similar studies on different plant
species [31].
The anti-enteropooling potential of EEOR was investigated to explore its antidiarrheal efficacy
further and to aid mechanistic interpretation. Our results show that the extract markedly inhibited
castor oil-induced enteropooling into the small intestine, likely through suppressing castor oil
stimulated prostaglandin biosynthesis. All tested doses significantly decreased intraluminal fluid
compared to the control, with the highest dose of 400 mg/kg decreasing both the volume by 32.29%
(P < 0.05) and weight of intestinal contents by 49.57% (P < 0.001). These results confirm the antidiarrheal
efficiency of our extract and are comparable with an analogous study conducted by Agbon et al. [32].
To further characterize the effect of EEOR in reducing intestinal hypermotility, we investigated
gastrointestinal motility using a charcoal meal tracer. We observed that the administration of the
extract delayed the transit of the charcoal marker through the entire intestine. This inhibitory effect
was seen with all doses employed and implies that an anti-motility action underlies the mechanism
of action of the extract. Maximal inhibition of the peristaltic index was exhibited following a dose
of 400 mg/kg (41.66%, P < 0.001), and was equipotent with the standard drug loperamide (42.26%,
P < 0.001). Our findings suggest that the extract both decreases hypermotility and increases the transit
time through the suppression of intestinal muscle spasm, thus extending the time for absorptive
processes [33].
As aforementioned, castor oil promotes prostaglandin biosynthesis, which leads to the release
of various pro-inflammatory mediators, leading to inflammation and irritation. Non-steroidal
anti-inflammatory drugs (NSAIDs) may prevent diarrhea through inhibition of castor oil stimulated
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prostaglandin synthesis [34]. In this study, we assessed the anti-inflammatory activity of EEOR
following histamine challenge. Histamine causes contraction of the smooth muscle of small intestine,
uterus, bronchi, and bronchioles through activation of H1-receptors [35]. The mechanism of
the local inflammatory response induced by histamine is through the activation of vasodilation,
edema formation, vascular permeability, and cytokine release. [36]. Our results showed that EEOR
significantly (P < 0.001) suppressed histamine-induced paw edema, which provides evidence of a
potential anti-inflammatory effect. EEOR may thus ameliorate an acute inflammatory response via
inhibition of prostaglandins or other inflammatory mediators.
In the anthelmintic study, we utilized the aquatic worm Tubifex tubifex, a species of aquatic
oligochaete that is a suitable host for the Myxobolus cerebralis parasite, responsible for whirling disease
in salmonid fish [37]. Our data revealed that exposure to EEOR dose-dependently reduced (P < 0.001)
both paralysis and death times of the worm, indicating the presence of a potential anthelmintic
compound(s). The reference drug levamisole (a nicotinic receptor agonist) activates excitatory nicotinic
acetylcholine (nACh) receptors on the muscle of the worm, causing paralysis and death [38], and a
similar mechanism may account for the anthelmintic action of EEOR.
We investigated the antimicrobial activity of EEOR through the disc diffusion method, and the
extract induced a significant zone of inhibition against both Bacillus subtilis (a model Gram-positive
microorganism) and Escherichia coli (Gram-negative microorganism) at concentrations of 500, 800 and
1000 μg/disc. The lowest concentration (500 μg/disc) failed to show activity against Salmonella typhi
(Gram-negative microorganism), but the other two concentrations exhibited significant antibacterial
activity. These results indicate the existence of a broad-spectrum antibiotic effect of the plant extract
and represent the first such data on the extract. On the other hand, we did not find any noticeable effect
of our extracts on the Gram-positive Staphylococcus aureus or Bacillus cereus, or on the Gram-negative
organisms Salmonella paratyphi or Pseudomonas aeruginosa, even at 1000 μg/disc. Broadly, our results
suggest that EEOR constituents may interrupt general cellular functions or disrupt bacterial membrane
potential [39,40].
Generally, plants are rich in secondary metabolites with diverse biological actions, acting as natural
defense mechanisms against bacteria, insects, viruses, and fungi. Our preliminary phytochemical
evaluation suggested a distinct phytoconstituent profile in EEOR. Among these, alkaloids, flavonoids,
phenols, tannins, terpenoids, and saponins are commonly reported to possess both antibacterial
and anthelmintic activities [41,42]. Reports on various plant extracts suggest that antidiarrheal
effects may also be mediated through the action of saponins, tannins, steroids flavonoids and
alkaloids [43], whereas tannins and flavonoids are well known to aid reabsorption of intestinal
fluids and electrolytes [44]. Additionally, tannins reduce intestinal motility by inhibiting bowel
irritation, thereby exhibiting an antidiarrheal effect [45]. Various phytochemicals including flavonoids,
steroids, and phenols have been ascribed anti-inflammatory actions [46]. As EEOR showed significant
anthelmintic and antibacterial activity, especially on certain entero-pathogenic (Bacillus subtilis,
Salmonella typhi and Escherichia coli) organisms, coupled with its observed effects on gut motility,
this supports its possible utility in infectious diarrhea.
GC-MS analysis of EEOR identified a total of thirty different compounds. Based on the
literature, thirteen of these have already been documented to be bioactive. Loliolide [47], ethyl
linoleate [48], 2-palmitoylglycerol, and erucamide [49] have been shown to possess antibacterial
activity, while γ-sitosterol, stigmasterol, vitamin E, and squalene [47] have both antibacterial and
anti-inflammatory activities. Phytol and methyl palmitate have nematicidal, pesticidal, antibacterial,
and anti-inflammatory activities. Notably, phytol is very active against Salmonella typhi [49]. Finally,
neophytadiene [50] and methyl linoleate [47] have demonstrated anti-inflammatory activity.
Molecular docking studies have been widely used for the prediction of ligand-target interactions
and to obtain better insights into the biological activity of natural products. It also gives additional
clues about possible mechanisms of action and binding modes inside the binding pocket of various
enzymes [51]. In order to obtain better insight into the observed biological activity (antidiarrheal,
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anthelmintic, and antibacterial) of EEOR constituents, thirteen representative compounds within EEOR
were selected for docking analyses. These compounds were then docked against four targets, namely
the M3 muscarinic acetylcholine receptor (PDB ID: 4U14), the 5-HT3 receptor (PDB ID: 5AIN), tubulin
(PDB ID: 1SA0) and GlcN-6-P synthase (PDB: 1XFF).
Molecular docking studies with the M3 muscarinic acetylcholine receptor (PDB ID: 4U14) revealed
that, among the thirteen compounds, seven interacted with several amino acid residues through
hydrogen bonds and π-π stacking interactions (Tyr529, Tyr533, Ile222, Leu225, Asn152, Ser151, Tyr148),
with docking scores ranging between −2.00 and −8.80 kcal/mol. On the other hand, five compounds
interacted with a number of amino acid residues (Ile116, Glu191, Arg57, Arg57, and Thr34) within
the 5-HT3 receptor (PDB ID: 5AIN) with docking scores ranging from −0.69 to −5.47 kcal/mol. From
these results, we can conclude that the studied phytoconstituents may in part be responsible for the
antidiarrheal activity of EEOR through interaction with these target proteins.
In the anthelmintic docking study, the thirteen compounds were docked with tubulin (PDB ID:
1SA0) and showed docking scores ranging from −0.59 to −7.13 kcal/mol. From the results, it is
clear that the phytoconstituent stigmasterol displayed the highest score against tubulin, followed
by γ-sitosterol, vitamin E, ethyl linolenate, loliolide, erucamide, phytol, methyl linoleate, methyl
palmitate, and neophytadiene. It has been previously reported that phytol and methyl palmitate
possess nematicidal and pesticidal activities [49], and the anthelmintic activity of EEOR may be related
to these phytoconstituents. In the case of the antibacterial docking study, loliolide had the highest
binding affinity towards the GlcN-6-P synthase enzyme (PDB: 1XFF), followed by ethyl linolenate,
erucamide, 2-palmitoylglycerol, phytol, methyl linoleate, neophytadiene, methyl palmitate, and methyl
stearate. The antibacterial activity of the EEOR may thus be explained by the presence of loliolide,
ethyl linolenate, erucamide, 2-palmitoylglycerol, and phytol, which have good docking scores and for
which bioactivity has previously been reported [47,48].
All bioactive compounds were further characterized using the online-based prediction program
ADME analysis to explore their drug-likeness, pharmacokinetics and physiochemical characteristics.
Almost all compounds, except for γ -sitosterol, squalene, stigmasterol and vitamin E exhibited orally
active drug-likeness properties, according to Lipinski’s rule. It is reported that compounds with
lower molecular weight, lipophilicity, and hydrogen bond capacity have high permeability [52], good
absorption and bioavailability [53,54]. However, this analysis does not assess if a compound has any
particular pharmacological effect.
To predict a likely pharmacological profile of the compounds, we utilized the structure-based
biological activity prediction program Prediction of Activity Spectra for Substances (PASS). The results
suggested several activities, among these, we established probable activity values (Pa range 0.235–0.826)
for all 13 compounds for anthelmintic, antibacterial, anti-inflammatory, spasmolytic and antiprotozoal
actions, supporting our laboratory investigations of EEOR. Moreover, other activities were predicted,
suggesting the broader potential of this species. In summary, our comprehensive analyses, utilizing
complementary tools, support the traditional uses of EEOR. The observed effects may be due to the
combined actions of several phytoconstituents, both those documented herein and potentially other as
yet uncharacterized compounds.
4.1. Drugs and Chemicals

All drugs and chemicals used in this research were of analytical grade. Loperamide was obtained
from Square Pharmaceuticals Ltd. (Dhaka, Bangladesh), levamisole from ACI Limited (Dhaka,
Bangladesh), and castor oil from WELL’s Health Care (Madrid, Spain). Ethanol (Merck, Darmstadt,
Germany), Kanamycin (Sigma Chemical Co., St. Louis, MO, USA) and histamine (BDH Chemicals Ltd.
Poole, UK) were procured from the mentioned sources.
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4.2. Chemical Compounds Studied in This Article

Loliolide (PubChem CID: 100332); Ethyl linolenate (PubChem CID: 6371716); Methyl linoleate
(PubChem CID: 5284421); Erucamide (PubChem CID: 5365371); γ-Sitosterol (PubChem CID: 457801);
2-Palmitoylglycerol (PubChem CID: 123409); Methyl Palmitate (PubChem CID: 8181); Methyl stearate
(PubChem CID: 8201); Neophytadiene (PubChem CID: 10446); Phytol (PubChem CID: 5280435);
Squalene (PubChem CID: 638072); Stigmasterol (PubChem CID: 5280794); Vitamin E (PubChem CID:
14985).
4.3. Plant Collection, Identification, and Extraction

The leaves of Ophiorrhiza rugosa var. prostrata (D.Don) Deb & Mondal were collected in September
2017 from Kaptai National Park (22◦ 30 08”N 92◦ 12 04”E), Rangamati District, Chittagong Division,
Bangladesh. The plant was certified and authenticated by Dr. Shaikh Bokhtear Uddin, Professor,
Ethno-botany and Pharmacognosy Lab, Department of Botany, University of Chittagong, Bangladesh,
with a voucher specimen (accession no: 7609 CTGUH) deposited in the Herbarium of the University
of Chittagong (CTGUH). After subsequent washing with normal and distilled water, the collected
leaves were cut and oven-dried for a week at constant temperature (50 ◦ C), before milling into a coarse
powder using an automatic grinder. Then, the fine powder (350 g) was soaked in 850 mL of ethanol
for seven days at room temperature, with regular shaking and stirring on a shaker machine (model
VTRS-1, Nunes Instruments, Tamil Nadu, India). After 7 days, the macerate was filtered through a
sterilized cotton plug followed by Whatman filter paper No. 1, and the eluting solvent evaporated
on a rotary evaporator (RE 200, Sterling, Norman Way Industrial Estate, Cambridge, UK) at room
temperature to afford a semisolid extract (EEOR: 10 g), which was kept in a refrigerator (−4 ◦ C) until
further use.
4.4. Animals and Ethical Statements

Adult Swiss albino mice (20–25 g) of both sexes were obtained from Jahangir Nagar University,
Savar, Dhaka, Bangladesh. The animals were housed in polypropylene cages for adaptation, under
standard laboratory conditions (room temperature 25 ± 2 ◦ C; relative humidity 55–60%, 12 h light/dark
cycle), with food pellets and water ad libitum. All animals were acclimatized for 2 weeks and fasted
overnight before starting all experiments. This experiment was designed based on the Ethical Principles
and Guidelines guided by The Swiss Academy of Medical Sciences and the Swiss Academy of
Sciences. All tests were run in a remote and noiseless ambiance, between 9.00 a.m. and 5.00 p.m.
The study protocol was approved by both the Ethical review committee and the P&D committee of the
Department of Pharmacy, International Islamic University Chittagong, Bangladesh under the code
Pharm-P&D-61/08 16-122.
4.5. GC-MS (Gas Chromatography-Mass Spectroscopy) Analysis of EEOR

GC-MS analysis of EEOR was evaluated using a model 7890A capillary gas chromatograph along
with a mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). The column was a fused silica
capillary column of 95% dimethyl-poly-siloxane and 5% phenyl (HP-5MSI; length: 90 m, diameter:
0.250 mm and film: 0.25 μm). Parameters for GC-MS detection were an injector temperature of 250 ◦ C,
an initial oven temperature of 90 ◦ C gradually raised to 200 ◦ C at a speed of 3 ◦ C/min for 2 min
and with a final increase to 280 ◦ C at 15 ◦ C/min for 2 min. The total GC-MS run time was 36 min,
using 99.999% helium as a carrier gas, at a column flow rate of 1 mL/min. The GC to MS interface
temperature was fixed at 280 ◦ C, and an electron ionization system was set on the MS in scan mode.
The mass range evaluated was 50–550 m/z, where MS quad and source temperatures were maintained
at 150 ◦ C and 230 ◦ C respectively. The NIST-MS Library 2009 was used to search and identify each
component, and to measure the relative percentage of each compound, relative peak areas of the TIC
(total ionic chromatogram) were used, with calculations performed automatically.
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4.6. Acute Toxicity Testing of EEOR

Acute toxicity testing was assessed under standard laboratory conditions following OECD
guidelines [55]. Animals (n = 6) of the control and test groups were each administered 1% Tween-80 or
a single oral dose (5, 50, 100, 200, 400, 1000 and 2000 mg/kg body weight) of the test extract (EEOR).
Before administration of extract, mice were kept fasting overnight, and food was also delayed for 3 to
4 h after receiving the extract. All experimental animals were observed individually, paying particular
attention to any unexpected responses, including behavioral changes, allergic syndromes (itching, skin
rash), and mortality over the next 72 h.
4.7. Qualitative Phytochemical Screening of EEOR

Qualitative phytochemical analysis of EEOR was carried out following standard procedures, as
previously reported by Tiwari et al. [56].
4.8. Antidiarrheal Activity Evaluation of EEOR (In Vivo)
4.8.1. Castor Oil-Induced Diarrhea

The conditions of Awouters et al. [34] were followed with slight modifications. Mice were fasted
overnight prior to the experiment. Experimental animals were separated randomly into control and
test groups consisting of 6 mice in each category. Group-I served as a negative control, and received 1%
Tween-80 in distilled water; group-II (positive control) received loperamide (5 mg/kg BW; p.o), while
test groups III-V were treated with EEOR (100, 200 and 400mg/kg BW; p.o) respectively. After 1 h,
each mouse was put into an individual cage and diarrhea induced (0.5 mL castor oil, p.o). Blotting
paper on the floor of each cage was monitored to observe both the number and consistency of fecal
droppings. Blotting papers were replaced every 60 min during the 4h observation period. The total
numbers of both dry and wet feces excreted by the animals were counted. The following equation was
used to calculate percent inhibition of diarrhea:
Total number of diarrheal faces×(control−test groups)
percentage of inhibition of diarrhea = Total number of diarrheal faces of the control × 100, (1)
4.8.2. Castor Oil-Induced Enteropooling

Intraluminal fluid accumulation was evaluated by the method described by Robert et al. [57].
Dosing treatments were as for castor oil-induced diarrheal testing, again with six animals per group.
One hour post administrations of each test dose, animals were treated with castor oil (0.5 mL) to induce
diarrhea. Two hours later, the mice were sacrificed, and the small intestine was isolated from pyloric
sphincter to caecum. The small intestine was weighed (g) and the volume of intestinal contents (ml)
was measured by milking into a graduated tube. The intestine was reweighed, and the differences
between full and empty intestines were calculated. To calculate, the percentage volume and weight of
intestinal contents were determined using the following formula:
Mean of intestinal content × (control − test groups)

percentage of inhibition = × 100, (2)
Mean of intestinal content of the control
4.8.3. Gastrointestinal Motility

This experiment was performed based on the method of Mascolo et al. [58], with the treatment of
animals of each group (n = 6) as described in the castor oil-induced diarrhea test. In brief, 0.5 mL of
castor oil was administered to each animal to induce diarrhea. One hour after administration of each
test dose, animals were treated orally with 1 mL of a charcoal meal (10% charcoal suspension in 5%
gum acacia). After 1 h, animals were sacrificed and the distance traveled by the charcoal meal from
the pylorus to caecum was measured (cm) and expressed as a percentage of the total distance of the
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intestine. The following formulae were used to express the percentage of inhibition and Peristalsis
index:
Distance (cm) travel by the charcoal × (control − test groups)

inhibition (%) = × 100, (3)
Distance travel by the charcoal in the control group
Distance travel by the charcoal meal

Peristalsis Index = × 100, (4)
Total length of the small intestine
4.9. Histamine-Induced Paw Edema

The anti-inflammatory activity of EEOR was evaluated following injection of histamine into the
plantar surface of the mouse hind paw [59]. Animals were divided into four groups (n = 6); Group I
(negative control) received 1% Tween-80 (2 mL/kg); Group II (Positive control) received diclofenac
sodium (10 mg/kg BW; p.o); and Groups III and IV received EEOR (200 and 400 mg/kg BW; p.o)
respectively. 30 min following treatment, 0.05 mL histamine (1 mg/kg, in 1% Tween-80 with D.W) was
injected in the sub-plantar area of the right paw of each mouse to induce acute inflammation, and
micrometer slide calipers were used to measure the paw volume at 1, 2, 3 and 4 h. The percentage
inhibition of the inflammatory effect of the extract was calculated using the following expression:
Mean degree of inflammation (control − test groups)

% inhibition of inflammation = × 100 (5)
Mean degree of inflammation of control
4.10. Anthelmintic Activity of EEOR (In Vitro)

Anthelmintic activity was assessed following the method of Ajaiyeoba et al. with slight
modifications [60,61]. In this experiment, the sludge worm, or sewage worm (Tubifex tubifex, size:
2 to 2.5 cm in length), was used for its physiological and anatomical relevance to intestinal worms,
e.g., Annelida. Testing was performed in triplicate. In brief, 5 to 10 worms were randomly placed in
each Petri dish, divided into four groups (I, II, III and IV). To each, 3 mL of either EEOR at a specified
concentration (5, 8 or 10 mg/mL) or the standard drug levamisole (1 mg/mL) added. Anthelmintic
activity was calculated at two different stages, namely ‘time of paralysis’ and ‘time of death’ of the
worms. Time to paralysis was counted as the time when worms lost their natural movement. The time
of death was recorded after confirming that the worms moved neither when vigorously shaken nor
when dipped in slightly warm water.
4.11. Antibacterial Activity of EEOR (In Vitro)

The antibacterial effect of EEOR was evaluated by the disc diffusion technique [62]. Prepared
Nutrient agar was placed into Petri dishes under laminar airflow for solidification. Overnight cultures
of Gram-positive Bacillus subtilis (ATCC 6633), Staphylococcus aureus (ATCC 6538) and Bacillus cereus
(ATCC 14579) and Gram-negative Salmonella typhi (ATCC 29629), Salmonella paratyphi (ATCC 9150),
Escherichia coli (ATCC 8739) and Pseudomonas aeruginosa (ATCC 9027) organisms were each prepared
with 100 μL bacteria (bacterial inocula were adjusted to 108 CFU/mL), spread smoothly on the agar
surface. Dry sterile discs (6mm diameter) were laid upon the seeded agar plate using a sterile forceps.
Each desired concentration of EEOR (500, 800 or 1000 μg) was loaded on these discs and then incubated
(at 37 ◦ C for 24 h). The diameter of each zone of inhibition was recorded and measured in mm. As a
positive control, kanamycin (30 μg/disc) was used.
4.12. In silico Molecular Docking

The major bioactive compounds of EEOR, as detected by GC-MS, were selected for molecular
docking studies, to understand better possible molecular interactions based on their affinity to interact
with different target proteins. Docking studies were performed using the Schrödinger suite-Maestro
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v10.1, LLC, New York, NY, USA, and Accelrys Discovery Studio 4.0 software (BIOVIA, San Diego, CA,
USA) was used for visualization of 3D structures.
4.12.1. Ligand Preparation

The structures of thirteen major compounds were obtained from the PubChem compound
repository, and the ligands prepared using the LigPrep tool embedded in Maestro v 10.1 (Schrödinger
suite, LLC New York, NY, USA), neutralized at pH 7.0 ± 2.0 using Epik 2.2, and minimized by force
field OPLS_2005.
4.12.2. Receptor Preparation

3D crystal structures of the proteins used for the test were downloaded from the Protein Data Bank;
RCSB PDB [63], GlcN-6-P synthase (PDB ID: 1XFF) [64], tubulin (PDB ID: 1SA0) [65] 5-HT3 receptor
(PDB ID: 5AIN) [66] and M3 muscarinic acetylcholine receptor (PDB ID: 4U14) [67]. The Protein
Preparation Wizard of the Schrödinger suite-Maestro version 10.1 was used to prepare and refine
the crystal structures. Charges and bond orders were assigned, hydrogens added to heavy atoms
and selenomethionines and selenocysteines converted into methionines and cysteines respectively,
followed by removing all water molecules. Using force field OPLS_2005, minimization was performed
to set a maximum heavy atom RMSD to 0.30 Å.
4.12.3. Grid Generation and Molecular Docking

Receptor grid generation and molecular docking experiments were performed using Glide
(Schrödinger suite-Maestro version 10.1) [68,69] For each protein, a grid was produced using the
following default parameters: van der Waals scaling factor 1.00 and charge cut-off value 0.25, subjected
to the OPLS_2005 force field. A cubic box of definite dimensions centered on the centroid of the active
site residues was generated for the receptor, and the box size was set to 14 Å × 14 Å × 14 Å for docking.
Docking experiments were carried out using the Standard Precision (SP) scoring function of Glide,
and only the best scoring fit with docking score was noted for each ligand.
4.13. In Silico ADME Analysis

The pharmacokinetic properties of all major identified compounds were evaluated using Lipinski’s
rule of five [70]. Lipinski stated that a compound could show drug-like behavior if it does not fail
more than one of the following criteria: (i) molecular weight not more than 500; (ii) H-bond donors
≤5; (iii) H-bond acceptors ≤10; (iv) Lipophilicity <5; and (v) molar refractivity between 40 and
130. The web tool Swiss ADME [71] was used to assess the ADME parameters of all compounds.
Compounds which obey Lipinski rule are considered as ideal drug candidates.
4.14. In Silico PASS Prediction

Possible biological activities of identified major compounds were evaluated using the online
computer program PASS (Prediction of Activity Spectra for Substances) [72]. This tool predicts up
to 3750 biological properties of a compound, associated with an analysis of its chemical structure.
The outcomes of this analysis were denoted as Pa (probable activity) and Pi (probable inactivity),
where the values of both Pa and Pi may differ from 0.000 to 1.000. We considered values of Pa > Pi and
Pa > 0.700 to indicate biological activity for a compound [73].
5. Statistical Analysis
Data were analyzed using SPSS 20.0 statistical software (SPSS, IBM Corporation, Armonk, NY,
USA). Results were presented as mean ± SEM (standard error of the mean), and one-way ANOVA
followed by Dunnett’s test was applied. A p-value of less than 0.05 was considered significant.
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6. Conclusions
In summary, our study demonstrates that EEOR possesses significant and dose-dependent
antidiarrheal activity in different models, which supports the traditional use of this plant in folk
medicine. The study also provides further evidence of inhibition of inflammatory mediators,
which rationalise the anti-inflammatory activity of the plant extract. The positive results regarding
anthelmintic and antibacterial activities increase the value of this plant. Collectively, these outcomes
support the ethnomedicinal use of O. rugosa for the management of various infectious diseases.
Furthermore, various potential bioactive constituents identified by GC-MS analysis showed promising
binding affinity toward different proteins in molecular docking experiments, and their drug-like
characteristics were demonstrated through ADME/T analysis. PASS predictions of bioactive
constituents were in agreement with our laboratory findings. Therefore, O. rugosa may represent
a viable candidate for the treatment of infectious diseases. However, further studies are needed
to identify and isolate the pure compounds responsible for the observed biological effects, and to
characterize its toxicity profile and longer-term safety.
Supplementary Materials: The following are available online, Figure S1. 2D interactions of the best fit found
for (A) Loliolide, (B) Ethyl linolenate, (C) Methyl linoleate, (D) 2-Palmitoylglycerol, (E) Methyl palmitate,
(F) Neophytadiene, (G) Phytol, and (H) Vitamin E docked to the M3 muscarinic acetylcholine receptor (PDB ID:
4U14); Figure S2. Best ranked fit of (A) Loliolide, (B) Ethyl linolenate, (C) Methyl linoleate, (D) 2-Palmitoylglycerol,
(E) Methyl palmitate, (F) Neophytadiene, (G) Phytol and (H) Vitamin E in the binding pocket of the M3 muscarinic
acetylcholine receptor (PDB ID: 4U14); Figure S3. 2D interactions of the best fit found for (A) Loliolide, (B) Ethyl
linolenate, (C) Methyl linoleate, (D) Methyl palmitate, (E) Methyl stearate and (F) Neophytadiene docked to
the 5-HT3 receptor (PDB ID: 5AIN); Figure S4. Best ranked fit of (A) Loliolide, (B) Ethyl linolenate, (C) Methyl
linoleate, (D) Methyl palmitate, (E) Methyl stearate and (F) Neophytadiene in the binding pocket of the 5-HT3
receptor (PDB ID: 5AIN); Figure S5. Best ranked fit of Phytol (A) in the binding pocket of 5-HT3 (PDB ID: 5AIN)
and 2D representation of key interactions in the binding pocket for Phytol (B); Figure S6. 2D interactions of the
best fit found for (A) Loliolide, (B) Ethyl linolenate, (C) Methyl linoleate, (D) Erucamide, (E) γ-Sitosterol, (F)
Methyl palmitate, (G) Neophytadiene, (H) Phytol, (I) Stigmasterol and (J) Vitamin E docked to tubulin (PDB ID:
1SA0); Figure S7. Best ranked fit of (A) Loliolide, (B) Ethyl linolenate, (C) Methyl linoleate, (D) Erucamide, (E)
γ-Sitosterol, (F) Methyl palmitate, (G) Neophytadiene, (H) Phytol, (I) Stigmasterol and (J) Vitamin E in the binding
pocket of tubulin (PDB ID: 1SA0); Figure S8. 2D interactions of the best fit found for (A) Loliolide, (B) Ethyl
linolenate, (C) Methyl linoleate, (D) Erucamide, (E) 2-Palmitoylglycerol, (F) Methyl palmitate, (G) Methyl stearate,
(H) Neophytadiene and (I) Phytol docked to GlcN-6-P synthase (PDB ID: 1XFF); Figure S9. Best ranked fit of (A)
Loliolide, (B) Ethyl linolenate, (C) Methyl linoleate, (D) Erucamide, (E) 2-Palmitoylglycerol, (F) Methyl palmitate,
(G) Methyl stearate, (H) Neophytadiene and (I) Phytol in the binding pocket of GlcN-6-P synthase (PDB ID: 1XFF);
Figure S10. (A) Best fit and (D) 2D interaction diagram of Kanamycin docked at the binding pocket of GlcN-6-P
synthase (PDB ID: 1XFF). (B) Best fit and (E) 2D interaction diagram of Levamisole docked at the binding pocket
of tubulin (PDB ID: 1SA0). (C) Best fit and (F) 2D interaction diagram of Loperamide docked at the binding pocket
of M3 muscarinic acetylcholine receptor (PDB ID: 4U14).
Author Contributions: M.A. and M.N.U.C. conceived and designed the experiments, prepared the plant extract,
carried out all the experimental works and collected and analyzed the data. M.A. drafted the final manuscript.
M.O.K.A. helped to perform in vivo antidiarrheal experiments. M.N.U.C. and A.P. performed the in silico study.
D.H.C., A.T.M.M.K., S.B.U., J.W.B., M.O.F., and C.H.P. supervised the study, evaluated the experimental data and
reviewed the final manuscript. All authors read and approved the final manuscript.
Funding: This research received no external funding.
Acknowledgments: This research was supported by the Department of Pharmacy, International Islamic University
Chittagong, Bangladesh, Kangwon National University, and Central Laboratory of Kangwon National University,
Chuncheon, 24341, Korea.
Conflicts of Interest: The authors declare that they have no conflict of interest.
Abbreviations
EEOR Ethanol extract of Ophiorrhiza rugosa leaves
p.o. per oral
i.p. Intraperitoneal
ANOVA Analysis of variance
BW body weight
SEM standard error of mean
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SPSS statistical package for social science

ADME/T Absorption, Distribution, Metabolism, Excretion, and Toxicity
PASS Prediction of Activity Spectra for Substances
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molecules
Review
Genus Ophiorrhiza: A Review of Its Distribution,
Traditional Uses, Phytochemistry, Biological
Activities and Propagation
Muhammad Taher 1, *, Siti Syazwani Shaari 1 , Deny Susanti 2, *, Dayar Arbain 3, *
and Zainul Amiruddin Zakaria 4, *
1 Department of Pharmaceutical Technology, Kulliyyah of Pharmacy, International Islamic
University Malaysia, Kuantan 25200, Malaysia; syazwani1897@gmail.com
2 Department of Chemistry, Kulliyyah of Science, International Islamic University Malaysia,
Kuantan 25200, Malaysia
3 Faculty of Pharmacy, Universitas 17 Agustus 1945, Jakarta 14350, Indonesia
4 Department of Biomedical Science, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia,
Serdang 43400, Selangor, Malaysia
* Correspondence: mtaher@iium.edu.my (M.T.); deny@iium.edu.my (D.S.); dayararbain@gmail.com (D.A.);
zaz@upm.edu.my (Z.A.Z.); Tel.: +60-(09)-5704842 (M.T.)

Received: 25 March 2020; Accepted: 15 April 2020; Published: 4 June 2020
Abstract: Almost 50 species of Ophiorrhiza plants were reviewed in this work and the main objective
is to critically analyse their distribution, phytochemical content, biological activity, and propagation.
Moreover, the information would be useful in promoting the relevant uses of the plant, especially in
the medicinal fields based on in vitro and in vivo studies. To this end, scientific sources, including
theses, PubMed, Google Scholar, International Islamic University Malaysia IIUM EBSCO, PubChem,
and Elsevier, were accessed for publications regarding the Ophiorrhiza genus in this review. Scientific
literature regarding the Ophiorrhiza plants revealed their wide distribution across Asia and the
neighbouring countries, whereby they were utilised as traditional medicine to treat various diseases.
In particular, various active compounds, such as alkaloids, flavonoids, and terpenoids, were reported
in the plant. Furthermore, the Ophiorrhiza species showed highly diverse biological activities, such
as anti-cancer, antiviral, antimicrobial, and more. The genus propagation reported could produce a
high quality and quantity of potent anticancer compound, namely camptothecin (CPT). Hence, it is
believed that the relevant uses of natural compounds present in the plants can replace the existing
crop of synthetic anticancer drugs associated with a multitude of unbearable side effects. Additionally,
more future studies on the Ophiorrhiza species should be undertaken to establish the links between its
traditional uses, active compounds, and pharmacological activities reported.
Keywords: genus Ophiorrhiza; distribution; traditional uses; bioactive molecules; biological

activity; propagation
1. Introduction
Genus Ophiorrhiza belongs to the Rubiaceae family, which is one of the Indo-Malaysian genera
widely spread in the wet forests across tropical and subtropical Asia, Australia, New Guinea, and
the Pacific Islands [1,2]. Most of the genera members are perennial herbs capable of growing from
approximately 10 cm to 1 metre of height [2]. Normally, the Ophiorrhiza genus can be characterised
by its succulent stems, five-petal flowers with slightly unequal opposite leaves, laterally compressed
fruits, and numerous capsular seeds of small rhomboid shapes [3]. Currently, the Ophiorrhiza L. genus
consists of 321 species, five varieties, and one subspecies. In particular, 46 species and five varieties are

Molecules 2020, 25, 2611
mainly distributed in the north-eastern states and Western Ghats of India [2], whereas 16 species and
three varieties can be found in the state of Kerala, India [4].
2. Botany
For the past period of years, herbal medicines have been served as therapeutic agents and continue
to be important lifesaving drugs for mankind. It is undeniable that plants can treat various forms of
diseases including cancer. The demand for herbal drugs increases due to the increased awareness of
the side effects and toxicity linked to synthetic drugs [5]. Plants from family Rubiaceae are known to
contain several secondary metabolites like alkaloids, flavonoids, steroids, terpenoids, and fatty acids.
Some of the compounds from this family, such as caffeine, quinine, emetine, and camptothecin, are of
major pharmaceutical importance [6]. These metabolites can be utilized as natural medicines as they
can inhibit the activity of DNA topoisomerase, which are the clinical targets for anticancer drugs [7].
Camptothecin (CPT) is a potent anti-cancer compound that has been widely isolated from Ophiorrhiza
species (O. mungos, O. mungos var. angustifolia, O. rugosa var. decumbens and many others), after
being first isolated from a Chinese tree, Camptotheca acuminate, in 1966 [6,8]. Later, many researchers
extensively developed several other CPT analogues, and among them are topotecan and irinotecan,
which showed better DNA-topoisomerase-1 inhibitor activity and are well-tolerated compared to
natural CPT [9].
3. Methodology
In-depth information on the Ophiorrhiza genus was obtained via a literature search conducted
for publications using various electronic databases, such as Google Scholar, International Islamic
University Malaysia (IIUM), EBSCO, PubMed, PubChem, and Elsevier. Accordingly, ChemDraw
software was employed to draw the bioactive molecules found in the Ophiorrhiza plants. In terms
of the publications selected for review, no limitations for the range of years were subjected in this
paper. The keyword ‘genus Ophiorrhiza’ was used for the primary searches, while the following
terms were employed for secondary searches: ‘Plant name’, ‘Phytochemicals’, ‘Biological activity’,
‘Pharmacological activity’, and ‘Propagation’. To highlight the therapeutic uses of genus throughout
the medical field, not all of the identified plants were included in this paper. Only those subjected to
in vitro and in vivo studies were presented in this review. Therefore, this review was not exhaustive
for all Ophiorrhiza species typically utilised in traditional or modern medicines. The layout of the
searching methodology is presented in Figure 1.
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Molecules 2020, 25, 2611
Figure 1. Methodology conducted.
4. Distribution
The Western Ghats are known as one of the diversity centres of Ophiorrhiza species following the
Western Himalayas, which comprise of about 22 distributed taxa for the species [10]. Some of them that
can be found in Andaman and the Nicobar islands of India include O. infundibularis Balakr, O. mungos
L, O. nicobarica Balakr, and O. trichocarpa BL [11]. Meanwhile, about 30–35 species are documented
in the shady and moist-to-wet areas in Thailand’s lowland and mountain forests both [3], whereas
Peninsular Malaysia is home to 21 species [6]. In contrast, 70 species of the Ophiorrhiza are distributed
in China in which most of them are found in the southern area of Yangtze River, which traverses the
provinces of Yunnan and Guangxi [12]. Figure 2 shows some of the Ophiorrhiza collections found in
West Sumatra, while Table 1 shows the distribution of this species worldwide.
In general, most of the Ophiorrhiza species are widely discovered in the Asian regions, particularly
India, Indonesia, and Malaysia. This suggests the highly suitable nature of the regional tropical and
forest environments for the growth and distribution of these plants. Therefore, it is believed that newer
Ophiorrhiza species can be discovered via the undertaking of extensive research in Asian countries.
(a) (b)
Figure 2. Cont.
413
Molecules 2020, 25, 2611
(c) (d)
(e) (f)
(g) (h)
(i) (j)
Figure 2. Photo collections of Ophiorrhiza species. (a) Ophiorrhiza communis [13]. (b) Ophiorrhiza sp.
(ex. Gunung Singgalang) [14]. (c) Ophiorrhiza “Siberida DA-RT 6030” [15]. (d) Ophiorrhiza sp. (ex. Sako,
TNKS) [16]. (e) Ophiorrhiza longiflora BI. [17]. (f) Ophiorrhiza cf. kunstlery King. [18]. (g) Ophiorrhiza ex.
Padang Panjang [19,20]. (h) Ophiorrhiza “DA-RT 7895” [21]. (i) Ophiorrhiza “Air Sirah DA-RT 6604” [22].
(j) Ophiorrhiza “Sako DA-RT 7577” [16].
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Molecules 2020, 25, 2611
Table 1. Distribution of Ophiorrhiza species around the world.
Species Location References

O. marginata Mt Keronsong, Jambi, Indonesia [23]
O. cf. rosacea Solok, West Sumatra [24]
Ulu tembeling Malaysia
O. communis [6]
Bungus, Padang, Indonesia
O. cf. communis Sijunjung, West Sumatra [13]
O. tomentosa Cameron Highland, Malaysia [6]
O. bracteata Lubuk Alung West Sumatra [25]
O. major Anai Reserved Forest, West Sumatra [26]
O. cf ferruginea Anai forest, West Sumatra [27]
O. rosacea The Seven Mountain, Jambi [28]
O. kunstlery Pariaman, West Sumatra [28]
Mount Letter W Kabupaten Padang
O. cf. kunstlery King [29]
Pariaman, Indonesia
O. filistipula Pangian Lintau, West Sumatra [30]
O. blumeana Mount Tandikat, West Sumatra [31]
O. trichocarpon Blume Kerala, South India [4]
O. pumila Japan [32]
O. pectinata Tamil Nadu Kerala, India [33]
O. radicans Western Ghats Kerala, India [34]
O. shendurunii Kerala Western Ghast, India [35]
Agastyamala Hills of Western Ghats
O. prostrata [1]
Thiruvananthapuram Kerala, India
O. rugosa The northern part of Thailand [3]
O. rugosa var. prostrata (D.Don) Chittagong Hill Tract and Sylhet regions of
[36]
Deb & Mondal Bangladesh
Ishigaki Island of Okinawa, Japan, Taiwan,
O. liukiunseis [37]
Philipines
Ishigaki and other south-west islands of
O. kuroiwai [38]
Okinawa
Ishigaki and other south-west islands of
O. japonica [38]
Okinawa
O. alata Craib Chantaburi, Thailand [39]
O. nicobarica Balakr. Andaman and Nicobar Islands, India [11]
O. infundibularis Great Nicobar Island, India [2]
O. jojui Andaman Islands, India [2]
O. shiqianensis Guizhou Province, South-western China [12]
O. neglecta BI. ex DC Kabupaten 50 Kota, Indonesia [40]
O. longiflora BL. Tarok Lubuk Bonta, Indonesia [17]
O. palidulla Ridl. Tilatang Kamang, Indonesia [41]
O. sp. Kotamadya Padang Panjang, Indonesia [19,20]
O. klosii Ridl. Kabupaten Solok, Indonesia [42]
O. ex. Simanau DA-RT 61,
Kabupaten Solok, Indonesia [43]
Solok
O. neglecta BI. Ex DC Kabupaten 50 Kota, Indonesia [40]
O. anonyma Val. Silayang Kabupaten Agam, Indonesia [44]
Mount Singgalang Kabupaten Agam,
O. ex. Gunung Singgalang [14]
Indonesia
Air Sirah Kabupaten Solok Sumatera Barat,
O. “Air Sirah DA-RT 6604” [22]
Indonesia
O. “Sako DA-RT 7577” Sako Kabupaten Sumatera Barat, Indonesia [16]
National Park Siberida Rengat Riau,
O. “Siberida DA-RT 6030” [15]
Indonesia
O. DA-RT 82 AT Tes Lake Bengkulu, Indonesia [45]
Kerinci Seblat National Park (TSNK) Jambi,
O. DA-RT 7895 [21]
Indonesia
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Molecules 2020, 25, 2611
5. Traditional Uses
The plants species of Ophiorrhiza (Rubiaceae) genus have been associated to claims of various
medicinal properties and wide-ranging applications in traditional and modern medicine alike [4,10,33].
Traditionally, the plants are used to treat inflammation, pain, cancer, and bacterial and viral-based
infections. Furthermore, the Ophiorrhiza species are capable of healing snakebite, stomatitis, ulcers, and
wounds [5,10,33], while also acting as an antioxidant [46], antitussive, and analgesic alternative [4].
They are also applied to tend to cases of gastropathy, leprosy, and amenorrhea, besides possessing
sedative and laxative properties obtained from the extract of their root barks [33]. In fact, it interesting
to note that O. mungos is specifically known as ‘snakeroot’ due to its known use as a treatment for
snakebite [6].
In modern medicine, Ophiorrhiza plants are popular as a result of their constituent camptothecin’s
anti-cancer properties, which are attributable to its ability in inhibiting deoxyribonucleic acid (DNA)
topoisomerase-1 [10]. However, their usage in treating different diseases may be dissimilar between
varying tribes. For example, the Tanchangya people in Bangladesh uses the paste of O. rugosa var.
prostrata (D.Don) Deb & Mondal in order to treat boils, those of the Mama tribe make tea from its leaves
in tending to body aches or juices them for diarrhoea, whereas the Chakma tribe treats earache by
applying sun-dried crushed leaves on the site of pain [36]. Regardless, various alternative traditional
uses of this particular species have been detailed, which are shown in Table 2. The Ophiorrhiza species
are evidently rich with bioactive molecules, offering superior pharmacological effects as they can be
used to treat a multitude of diseases throughout mild to chronic stages.
In this review, all available information about Ophiorrhiza genus and its bioactive molecules linked
with significant pharmacological properties are collected. This act is particularly crucial to promote the
plants and their relevant applications in the medicinal fields according to pharmacological evidence
obtained via in vitro and in vivo studies.
Table 2. Traditional uses of Ophiorrhiza species.
Hippocratic
Ophiorrhiza Species Traditional Uses References
Screening
O. discolor Br. Skin infections [47]
O. filistipula Miq. Skin infections and inflammation [47]
O. cf. rosacea Ridl. Vasodilator effect [47]
Analgesic and
Ophiorrhiza DART 6526 [47]
muscle relaxant
O. major Ridl. Skin disorders like eczema [25]
Herpetic lesions, skin infections,
O. nicobarica [11]
and irritation
O. mungos Cancer and snakebite,
[48]
(root) Sedative and laxative properties
O. communis Poultice and treating cough [6]
O. tomentosa Poultice [6]
Skin infections such as boils, body
O. rugosa var. prostrata
aches, and chest pain, earache, [36]
(D. Don) & Mondal
dysentery, diarrhea
O. singaporiensis Snakebite [17]
6. Phytochemistry
The Rubiaceae family is known with its capability of producing bioactive metabolites such as
iridoids, indole alkaloids, anthraquinones, terpenoids (i.e., diterpenes and triterpenes), flavonoids,
and many other derivatives of phenolic compounds, which result in their respective significant
pharmacological activities [46]. The phytochemical analyses done for several species of Ophiorrhiza
(Rubiaceae) have revealed the positive presence of alkaloids, flavonoids, and triterpenes, specifically
in O. radicans [10], O. mungos [5], O. liukiuensis [37], and O. nicobarica [11]. Furthermore, some of the
416
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important phytochemicals found in most Ophiorrhiza species include camptothecin and its derivatives,
namely pumiloside, luteolin, harman, tetrahydroalastonine, bracteatine, blumeanine, strictosidinic
acid, and lyalosidic acid [49]. According to many studies, the therapeutic uses of these compounds can
described by their anti-inflammatory, anticancer, antiviral, and antibacterial activities exhibited [46].
Therefore, the compounds derived from Ophiorrhiza species are perceived as useful as one of the
natural alternative sources in drug development in order to replace chemically-synthesised medications
associated with undesirable side effects such as dizziness, nausea, and vomiting.
6.1. Alkaloids
The Ophiorrhiza (Rubiaceae) species are highly prevalent among plant-rich indole alkaloids [24],
which are characterised by a low molecular weight and contain at least one nitrogen atom in an
amine-type structure. In their pure form, most of the alkaloids are colourless, non-volatile, and exist
as crystalline solids [47]. They are typically useful due to their biological activities and defensive
properties observed in plants, as well as for medicinal treatment purposes as they have excellent
pharmacological properties [50].
One of the common alkaloids found in Ophiorrhiza species is harman (1). In particular,
harman-2-oxide (2) generally found in O. rosacea is a crystalline alkaloid that can be reduced to
harman (1) by reduction with zinc in acetic acid. Furthermore, strictosidinic acid (3) is another major
bioactive molecule in Ophiorrhiza plants besides harman (1) [28]. Through Carbon-13 nuclear magnetic
resonance (C-NMR), harman (1) can be characterised as a compound containing six non-substituted
aromatics and one primary and three substituted aromatic carbons, whereas strictosidinic acid (3)
consists of six quaternary, five methane, and 16 methylene carbons [6].
Next, palicoside (4) or N-methylstrictosidinic acid depicts a quite similar structure compared to
strictosidine (5). However, the positioning of additional methyl groups at R1 and R2 differentiates them
into two different structures. In particular, the formation of normalindine (6) and isonormalindine
(7) can be derived from strictosidine (5) by introducing the β and α-configurations of a methyl group
between N4 and C19, respectively [28]. Similarly, isomalindine-16-carboxylate (8) and malindine
(10) display close spectroscopic data with isomalindine (9) at the C3,18 carbons and the N-Me group.
However the difference between them is that there is a carboxylate group placed at C6 carbon of
isomalindine-16-carboxylate (8), while in the case of malindine (10), there is a different orientation
of the methyl group at C19 as compared to the other two compounds, namely isomalindine (9) and
isomalindine-16-carboxylate (8) [13].
On top of this, camptothecin (11) is a modified monoterpene indole alkaloid having potent
anti-cancer property has been reported to be found in abundance in O. mungos and O. mungos
var. angustifolia, rendering them the main sources for its biomass production [8]. It consists of a
pentacyclic ring structure, which includes a pyrrole (3,4β) quinoline moiety and an asymmetric centre
within the α-hydroxyl lactone ring with 20S configuration [7]. Additionally, the pumiloside (12) and
deoxypumiloside (13) found in O. pumila are further considered as the precursors for the biosynthesis of
camptothecin (11). This is attributable towards their formation from the half structure of camptothecin
(11) and half structure of strictosamide (14) accordingly [32].
Meanwhile, the two glucoalkaloids of lyalosidic acid (15) and 10-hydroxylyalosidic acid
(16) have been found to coexist with their respective β-carbolines alkaloid counterparts, namely
6-hydroxyharman (17) and harman (1) in O. japonica. This suggests that these two constituents are
closely correlated, whereby the hydrolysis of harman (1) at C6 will produce 6-hydroxyharman (17).
Similarly, the diazomethane methylation of lyalosidic acid (15) and 10-hydroxylyalosidic (16) acid
accordingly yields the two products of lyaloside (18) and 10-methoxylyaloside (19), respectively [51].
Moreover, the chemical structures of ophiorrhizine (20) isolated from O. major Ridl [25], ophiorrine
A (21), and ophiorrine B (22) extracted from O. japonica [38], and six ophiorrhisides, namely ophiorrhisides
A (23), B (24), C (25), D (26), E (27), and F (28) isolated from O. trichocarpan Blume [52] are quite
similar. However, they are dissimilar regardless due to the differences in the additional groups and
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glucose orientation at a certain position of carbon atom. For example, the difference in stereochemistry
configuration at C16 will thus differentiate ophiorrine A (21) and ophiorrine B (22) into two different
betaine-type of indole alkaloids [38]. Accordingly, the chemical structures of the alkaloids under the
umbrella of the Ophiorrhiza species are displayed in Figure 3.
1+
1
++ +
1 2*OF
1 2
+
0H 2
&2 +
+
Harman (1) Harman-2-oxide (2) Strictosidinic acid (3)
+1
10H 1+ +
1 1
+ +
2*OF 2*OF
1 1
2 2
+2 & 0H2 &
Palicoside (4) Strictosidine (5) Normalindine (6)
1 1
1 1
+ + + +
1 1+
Isomalindine-16-carboxylate (8) Isomalindine (9) Malindine (10)
2
2
2 1 1
1 + +
+
+ +
2 +
2
+ 2*OF
2*OF
Deoxypumiloside (13)
Camptothecin (11) 3H-΅: (3S)-Deoxypumiloside, Strictosamide (14)
3H-Ά: (3R)-Deoxypumiloside
+ + +2 +
1 1
1 1
+ +
2*OF 2*OF
2 2
+2 & +2 &
Lyalosidic acid (15) 10-hydroxylyalosidic acid (16) 6-hydroxyharman (17)
Figure 3. Cont.
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Molecules 2020, 25, 2611
&22
+
+ + 1 +
0H2 +
+
1 1
1 1
+ + 1+ 2
2*OF + + +
2*OF +
+ +
2 2 2 2
0H2 & 0H2 & +2+ &
2+
+2
+2
Lyaloside (18) 10-methoxylyaloside (19) Ophiorrine A (21)
+
+
1 &22
2
+ 2+
10H +2 2+
2 1 2
1+ + + + 2+
+ 2+
+2 2+
2 2
+2+ & 2 2 2
2
2+ 0H2 &
+2
+2
Ophiorrine B (22) Ophiorrhiside A (23)

2
10H
1
+ + 2+
+2 2+ 2+
2 2 +2 2 2+
2
2 2+
0H2 &
Ophiorrhiside B (24)
2
10H
1
+ 2+ 2
+2 2+
2 2 20H
2
2
0H2 & 2+
Ophiorrhiside C (25)
2
2
10H 2
1 10H
+ 1 10H
2*OF + 1
2*OF +
2 +
0H2 & 2
0H2 & +22& 2*OF
20H
2
Ophiorrhiside D (26) Ophiorrhiside E (27) Ophiorrhiside F (28)
Figure 3. Cont.
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Harmaline (29) d-1,2,3,4-tetrahydroharmine

Harmine (30)
(31)
1 0H
0H
&2 0H
1 + 1
+
1
&+2
+ 1
+
0H
Vallesiaschotamine (32) Didehydro-19-

Tetrahydroalstonine (33)
methylnormalindine (34)
20H
β ' *OF 2
2
1
1
2
2+ 2
Decarbomethoxydihydrogambirtannine
Chaboside (36) Isovallesiachotamine (37)
(35)
+2 2
&O
1 1+
1 + + 2*OF
+ 1
2*OF +
&20H 2
Bracteatine (39) 3,4,5,6-tetrahydrodolichantoside (46)
Figure 3. Alkaloids from the genus of Ophiorrhiza. *Glc = glucose molecule.
Furthermore, prior reports have detailed the pharmacological properties of some alkaloids, one of
which is harmaline (29), also known as harmidine, which is found in O. nicobarica. When subjected
to oxidation, it will be converted to a psychoactive substance known as harmine (30), whereas a
reduction process will give rise to d-1,2,3,4-tetrahydroharmine (31) [11]. Similarly, compounds such as
vallesiaschotamine (32), strictosidinic acid (3), and tetrahydroalstonine (33) have been proven to be
capable of depressing the central nervous system, relaxing the muscles, and offering relief pain (i.e.,
tetrahydroalstonine only) when administered to mice, whereas lyalosidic acid (15) yields a vasodilator
effect [24].
Various different alkaloids that can be found in Ophiorrhiza plants are summarised accordingly
in Table 3. The most common types for this particular species consist of harman, ophiorrhine, and
camptothecin as well as their respective derivatives. Most of these compounds display a pentacyclic
amine-ring structure, which is an indication that they are of the alkaloid group. Therefore, it is
undeniable that the Ophiorrhiza species is rife and abundant with alkaloid compounds, rendering them
plants with a high potential of excellent therapeutic effects.
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Table 3. Alkaloids contents in Ophiorrhiza species.
Species Alkaloids Contents Parts of Plant References

O. rosacea Harman (1), Harman-2-oxide (2) Aerial part [28]
Palicoside (4), isomalindine (9),
O. kunstlery King Aerial part [18,28,29]
3,14-didehydro-19-methylnormalindine (34)
O. communis Harman (1) Leaves [53]
O. tomentosa Harman (1), strictosidinic acid (3) Leaves [53]
Lyalosidic acid (15), 10-hydroxylyalosidic acid (16),
O. japonica Aerial part [51]
6-hydroxyharman (17)
O. japonica Ophiorrine A (21), Ophiorrine B (22) Leaves [38]
O. kuroiwai Mak Ophiorrine A (21), Ophiorrine B (22), lyasoside (18) Aerial part [38]
Decarbomethoxydihydrogambirtannine (35),
O. marginata Aerial part [23]
strictosidinic acid (3)
O. cf. communis Isomalindine-16-carboxylate (8) Aerial part [13]
O. liukiuensis Camptothecin (11), 6-hydroxyharman (17) Whole plant [37]
Camptothecin (11), chaboside (36), pumiloside (12),
O. pumila Whole plant [54]
deoxypumilosides (13)
O. nicobarica Harmaline (29) Whole plant [11]
Tetrahydroalstonine (33), Vallesiaschotamine (32),
O. DA-RT 6526 Aerial part [47,55]
Isovallesiachotamine (37)
Ophiorrhiza DA-RT 6526b Strictosidinic acid (3) Aerial part [24,56]
Ophiorrhiza DA-RT 82 A Tetrahydroalstonine (33) Aerial part [45]
O. longiflora BL. Tetrahydroalstonine (33) Aerial part [17]
O. cf. rosacea Harman (1), lyalosidic acid (15) Aerial part [24]
O. mungos Camptothecin (11) Leaves [57]
Ophiorrhizine-12-carboxylate (38), bracteatine (39),
O. blumeana Korth Aerial part [47]
blumeanine (40)
O. bracteata Korth Ophiorrhizine (20), bracteatine (39) Aerial part [47]
O. discolor Br. Tetrahydroalstonine (33) Aerial part [47]
7-methoxy-camptothecin (41), normalindine (6),
O. filistipula Miq. Aerial part [47]
strictosidinic acid (3)
Dihydrocycloakagerine (42), mostuenein (43),
O. cf. ferruginea Valeton Aerial part [47]
tetrahydroakagerine (44), isomalindine (9)
O. major BI. Ophiorrhizine (20) Aerial part [47]
O. teysmaniana Miq. Tetrahydroalstonine (33) Aerial part [47]
Ophiorrhiside A (23), Ophiorrhiside B (24),
Ophiorrhiside C (25), Ophiorrhiside D (26),
O. trichocarpon Blume Ophiorrhiside E (27), Ophiorrhiside F (28), Whole part [52]
dolichantoside (45), 5-carboxystrictosidine (46),
lyaloside (18), 3,4,5,6- Tetrahydrodolichantoside (47)
6.2. Flavonoids
Flavonoids are the subgroups of phenolic compounds, which are characterised with at least one
aromatic ring and one or more hydroxyl substituents [50]. Their basic structures consist of two benzene
rings linked through a heterocyclic pyrane ring [58], whereupon the compounds can be classified
into several subclasses, such as isoflavonoids, chalcones, flavanones, flavones, dihydroflavonols,
flavonols, anthocyanidins, and catechins [7]. The presence of flavonoids in plants is responsible for
providing an attractive colour in order to attract plant pollinators, protecting the leaves from fungal
pathogens and UV-B radiation, and controlling normal physiological activities such as respiration and
photosynthesis [58]. Furthermore, the compounds are linked to several pharmacological properties,
such as anti-inflammatory, anti-platelet, anti-proliferative, anti-carcinogenic, and many others, as well
as a minimal toxicity effect [59]. In fact, some of the flavonoids also possess anti-snake venom activity:
they occur by inhibiting phospholipase A2, which is an essential constituent in snake venoms [5].
Various plants of the Ophiorrhiza species contain flavonoids, whereby O. mungos Linn. and
O. liukiuensis have been specifically reported to have luteolin-7-O-glucosides (48) and hyperin (also
known as quercetin-3-O-galactoside) (49) respectively (Figure 4). In particular, luteolin-7-O-glucosides
(48) possess three aromatic ring structures with the characteristics of 5,7,3 ,4 oxygenated
flavones. Hydrolysis of luteolin-7-glycosides with acid will generate luteolin-7-O-β-glucosides [60].
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Luteolin-7-O-glucosides (48) typically yield potent antioxidant, anti-inflammatory, and anti-cancer

properties [60]. Additionally, it is newly reported that it can protect the gastrointestinal tract from
ethanol and indomethacin-induced gastric ulcer upon its modelling in rats [59].
2+
2+
2+
*OF2 2 +2 2
2+
2
+
2*OF
2+ 2 2+
Luteolin-7-O-glucoside (48) Hyperin (49)
Figure 4. Flavonoids from the genus of Ophiorrhiza. *Glc = glucose molecule.
Meanwhile, hyperin (49) is a tetrahydroxyflavone compound composed of quercetin and a

beta-D-galactosyl residue attached at position 3 [61]. The butanol extract of O. liukiuensis whole
plant has revealed the presence of hyperin (49) and other alkaloid compounds concomitantly [46].
Previously discovered in Ericaceae, Guttifera, and Celastraceae, the compound has been reported to
produce anti-inflammatory effects. For example, it can the inhibit lipopolysaccharide (LPS)-induced
inflammatory response by terminating the TLR4 and NLRP3 signalling pathways, which are generally
associated with acute kidney injury [61]. This review thus reveals that flavonoid compounds are less
abundant and present as much as alkaloids are in the Ophiorrhiza species. Regardless, several previous
studies have shown that they are capable of providing a significant therapeutic effect to certain diseases
in comparison with their alkaloid counterparts.
6.3. Terpenoids
Terpenoids, which are also known as isoprenoids, are derived from two precursors, namely
isopentenyl diphosphate (IPP) and its isomer dimethyl allydiphosphate (DMAPP). Accordingly, they
can be classified into monoterpenes (C10 ), sesquiterpenes (C15 ), diterpenes (C20 ), sesterterpenes (C25 ),
triterpenes (C30 ), tetraterpenes (C40 , carotenoids), and steroids (C18–30 ) [50]. All terpenoids are derived
from a repetitive fusion of branched five-carbon units based on the isopentane skeleton [62] and offer
varying beneficial uses as flavourings and medicines alike [50].
In particular, ursolic acid (50) has been found together with camptothecin (11),
10-methoxy-camptothecin, and harman (1) from the chloroform extract of O. liukuensis whole plant [46].
It is a pentacyclic amphiphilic triterpene compound with a hydroxylated polycyclic structure, which is
capable of exhibiting many functions. For example, it has selective induction of cell death properties with
the help of caspase-3, may prevent the stimulation of lipoxygenase and cyclooxygenase, and selectively
inhibits the cyclic AMP phosphotransferase and AMP-dependent protein kinase. Consequently, the
metabolic activity of a cell, as well as its division, gene expression, and development, can be maintained
accordingly [11], thereby collectively preventing the cell overgrowth that potentially leads to cancer.
In recent years, new triterpenoid fatty acid esters named lupan-20-ol-3(β)-yl hexadecanoate (51),
lupan-20-ol-3(β)-yl acetate (52), and olean-18-en-3(β)-yl hexadecanoate (53) have been isolated from
the hexane extract of O. shendurunii, which present as a white and waxy solid. These compounds
manifest certain antimicrobial, antifungal, and anti-yeast activities through the diffusion disc method.
In contrast, other compounds isolated from the hexane extract include doctriacontanoic acid (54) and
stigmasterol (55), whereas rubiadin (56), nonadecanoic acid (57), hexadecanoic acid (58) (i.e., palmitic
acid), and camptothecin (11) are obtained from the chloroform extract of O. shendurunii whole plant [35].
Figure 5 and Table 4 below show the terpenoid content in some of the Ophiorrhiza species accordingly.
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Ursolic acid (50) Lupan-20-ol-3 (Ά)-yl hexadecanoate (51)
Lupan-20-ol-3(Ά)-yl acetate (52) Olean-18-en-3(Ά)-yl hexadecanoate (53)

&22+
2 +
+2
&22+ 2
+ 2
+
2*OF
2 2
+ 2
2*OF
2*OF
Demethylsecologanol (59) 3-o-glucosylsenburiside II (60)
2 2 2
+
2
2*OF
Epivogeloside (61)
Figure 5. Terpenoids from the genus of Ophiorrhiza. *Glc = glucose molecule.
Table 4. Terpenoids content in Ophiorrhiza species.
Species Triterpenes Parts of Plants References

Ursolic acid (50), demethylsecologanol
O. liukiuensis (59), 3 -o-glucosylsenburiside II (60) Whole plant [37,46]
(monoterpene), epivogeloside (61),
O. nicobarica Ursolic acid (50) Whole plant [11]
Lupan-20-ol-3 (β)-yl hexadecanoate
O. shendurunii (51), lupan-20-ol-3(β)-yl acetate (52), Whole plant [8]
olean-18-en-3(β)-yl hexadecanoate (53)
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7. Biological Activity
7.1. Anticancer Activity

In general, normal cells will grow and divide themselves for some time before their growth
stops, whereas reproduction will only occur in order to replace any damaged or dead cells. Therefore,
cancer occurs in the presence of abnormal cell growth due to the altered gene expression of DNA in
the affected cells, consequently resulting in their resistance to apoptosis and continuous division [63].
Moreover, the existence of reactive oxygen species (ROS) may further promote the development of
these tumour cells as they directly alternate the cellular metabolic process. If the natural antioxidants
found in the body cannot control the production of ROS or destroy them, severe damage to body
tissues may be observed while the cancer cells continue to actively proliferate [60].
The conventional synthetic anticancer drugs have unbearable side effects such as fatigue,
sleep disturbances, and hair loss, thereby rendering their substitution slowly with nature-derived
alternatives [63]. In particular, the therapeutic effects of secondary metabolites possessed by alkaloids,
flavonoids, terpenoids, polyphenols, and quinones may potentially inhibit the activity of DNA
topoisomerase enzyme [62] and suppress the ROS activity, thus leading to fewer side effects [60].
The actions of the DNA topoisomerase enzyme causes DNA strands to be separated and re-joined,
resulting in changes of the structure [62]. Accordingly, camptothecin (11) is one of the powerful alkaloids
found in Ophiorrhiza plants in possession of excellent anticancer activity and acts as a potent inhibitor
of DNA topoisomerase I [10,64]. The component is capable of preventing the enzymatic activity of
DNA topoisomerase I specifically by trapping the reaction intermediate (i.e., the cleavable complex)
during the breaking and re-joining process, causing disrupted cancer cell division [62]. Furthermore, it
can bind to tubulin and inhibit microtubule formation in the dividing cells, subsequently enhancing the
inhibition of the DNA topoisomerase [64]. Therefore, this review offers the notion that the development
of novel anticancer drugs extracted from natural sources is possible due to the pharmacological effects
of unique compounds present in the Ophiorrhiza plants.
Besides camptothecin (11), a multitude of phytochemicals show great anticancer properties as
well, such as harmane (1) and harmaline (29)’s reported roles in scavenging the ROS and inhibiting
thiol oxidation [65]. Accordingly, a comparative study has noted luteolin and quercetin’s ability to
inhibit topoisomerase II catalytic activity in Chinese hamster ovary AA8 cells, underlining them as a
pioneering lead for cancer treatment [7]. Meanwhile, the UV filtering property of 11 natural plants
including O. mungos has also been assessed in a preliminary study. Following this, the results suggest
that secondary metabolites such as polyphenols and flavonoids can absorb UV radiation and scavenge
the ROS produced from body cells [66]. This is an essential outcome as the free reactive atoms or
molecules are harmful due to their ability to damage normal and key biological macromolecules in
the body, such as lipid, protein, and DNA. In contrast, the anthraquinone fraction of O. rugosa van
decumbens functions by causing cytotoxicity in cancer cells and increasing the intracellular ROS, thus
leading to cell apoptosis [67].
Several works assessing the anti-inflammatory activities of the extracts obtained from Ophiorrhiza
species have been conducted as they are highly beneficial in determining their respective anticancer
properties. Inflammation is typically complicated, beyond one’s control, and a continuous process that
results in the progression of diseases such as atherosclerosis and cancer. Therefore, carrageen-induced
inflammation is a commonly employed method in order to investigate the anti-inflammatory activity
of certain compounds. To date, no exact mechanism of action for the anti-inflammatory activity of
anthraquinone fraction from O. rugosa has been underlined. However, it is believed that inflammation
reduction occurs via the inhibition of the lipid peroxidation and scavenging of hydroxyl radicals [67].
Table 5 shows the anticancer activities of certain Ophiorrhiza species based on previous studies.
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Table 5. Anticancer activity.
Extraction and Method (Dose/

Species Phytochemicals Biological Activity References
Isolation Concentration)
Ophiorrhiza mungos Luteolin-7-O- Gastroprotective effect against ethanol-induced
25 mg/kg b.w p.o [59]
Linn. glucosides (48) and indomethacin-induced gastric injury in rats
Molecules 2020, 25, 2611
Alcohol extract: 400 mg/kg b.w

Alcohol and
Anti-cancer property on Dalton’s Ascites p.o (14 days)
aqueous extracts of [68]
Lymphoma (DAL) in mice Aqueous extract: 800 mg/kg b.w.
O. mungos leaves
p.o (14 days)
Anti-cancer activity against COLO 320 DM, AGS,
MCF-7, and A549 cancer cell lines in rats
Luteolin-7-O- Methanolic extract Notes: COLO 320 DM (human colon 20 mg/kg b. w subcutaneously for
glucoside (48), of leaves and roots adenocarcinoma), AGS (human gastric cancer cell 16 weeks and 120μM (Incubated [60]
camptothecin (11) of the plant line), MCF-7 (human breast cancer cell line), A549 for 24 h)
(human lung cancer cell lines)
Suppressed β-catenin in COLO 320 DM
Methanolic extract 1 mg/mL (The extract was exposed
UV filtering potential [66]
of leaves to direct sunlight for 21 days)
425
Anti-cancer property in mice with: 130 μg/mL b.w
Ophiorrhiza rugosa Ehrlich ascites carcinoma (EAC) 160 μg/mL b.w
Anthraquinones [67]
van decumbens Dalton’s lymphoma ascites (DAL) 200 mg/kg b.w
Solid tumors (p.o for 10 days)
Major chemicals:
Ophiorrhoza ursolic acid (50), Analgesic and anti-inflammatory activity in rats 200 and 300 mg/kg b.w p.o
Alcoholic extract [11]
nicobarica β-sitosterol, and mice Fractions: 50 mg/kg b.w p.o
harmaline (29)
Presence of 10 μg/μL (Incubation of the
Prevention of hemorrhagic lesion induced by
Ophiorrhiza flavonoids, cardiac venom with the extract before
snake venom on the yolk sac membrane of the [5]
mungos glycosides and applying to the embryo for 30
chick embryo.
phenolics min)
b.w = body weight; p.o = oral route.
Molecules 2020, 25, 2611
7.2. Camptothecin Interest

Camptothecin (11) was first isolated via the extraction of the bark and stem of the Chinese
tree Camptotheca acuminata in 1958 [8,69]. It was later found in C. lowreyana, C. yunnanensis,
Nothapodytes nimmoniana, Pyrenacantha klaineana, Merrilliodendron megacarpum, Ervatamia heyneana,
Mostuea brunonis, O. mungos, O. pumila, and O. filistipula as well [4,10,34,70]. Even though camptothecin
is one of the potent topoisomerase-1 inhibitors, its usage and application in clinical trials and global
market has been withdrawn as a result of its low aqueous solubility and severe toxicity during the
S-phase of the cell cycle, which can lead to normal cell death [9]. In fact, the early 1970s yielded various
camptothecin clinical trials in which the respective researchers had failed across several attempts in
developing its sodium salt form to overcome its poor solubility characteristics. Following this, they
had found that for camptothecin (11) to show its activity, the lactone ring moiety must remain intact.
However, the ring was opened during the sodium salt preparation, thus resulting in its failure to show
the anticipated activity [71].
Another earlier study has also suggested that a complete pentacyclic ring structure with the D
ring pyrridone and E ring lactone formed via a 20S configuration is crucial towards the anticancer
property of camptothecin (11) [7]. Scholarly interest in camptothecin molecules steadily increased
in the mid-1980s in which two first-generation water-soluble camptothecin analogues with an intact
lactone ring were developed, namely camptosar (irinotecan or CPT-11) (62) and hycamtin (topotecan)
(63) (Figure 6). These two structures were obtained by modifying the A and B rings of camptothecin
accordingly [7]. In 1996, the United State Food and Drug Administration (FDA) approved the use
of these two analogues in the pharmaceutical industry, whereby they were marketed by Pharmacia
(Pfizer) and GlaxoSmithKline for the treatment of metastatic colorectal, primary colon, and metastatic
ovarian cancers [71].
Regardless, recent studies undertaken have shown that the isolated natural camptothecin
derivatives such as 9-methoxycamptothecin and 10-hydroxy-camptothecin are water-soluble and
possess antitumor activity [70]. Following this, more advanced therapeutic strategies by using
target-oriented drug delivery systems have crowned camptothecin (11) as a major candidate for the
treatment of multi-drug resistant cancers [8]. In fact, newer drug delivery via liposomal and copolymer
vehicle-mediated systems are capable of significantly improving the safety and efficacy of CPT [9]. For
example, its release mediated by pH-sensitive block copolymer is believed to enhance its subsequent
delivery in the body, alongside better safety considerations [72]. Additionally, another new alternative
strategy of stem cell-based research in the apoptogenic signalling of CPT is still in progress [73].
From these findings, clearly several approaches have been discovered by researchers in order to
stimulate the application of CPT in regenerative medicine by using its chemical analogues. Among
them, methods such as manipulating its ring structure, conjugating it with other molecules, developing
its prodrug, and using specific protein-based drug delivery systems have been suggested to establish a
better efficacy and safety of anticancer drugs. However, most of the analogues are still in the phase of
clinical trials and their safety issues are attributed as the major reason why these compounds have yet
to enter the market. Table 6 shows the camptothecin analogues that are still in the clinical trial phase
in detail.
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Table 6. Clinical trials of camptothecin analogues. Adapted from [9].
Camptothecin Year Clinical

Indication Company
Analogues Started Trials/Progression
Ovarian and malignant
9-aminocamptothecin 1993 Phase I/II
lymphoma
Karenitecin Malignant melanoma and brain
Phase II
(BNP-1350) tumor
Diflomotecan Solid tumors and small cell lung
2007 Phase II Ipsen
(BN-80915) cancer (SCLC)
Advanced solid tumors and
Gimatecan (ST-1481) Phase II recurrent epithelial ovarian and
fallopian tube cancers
Elomotecan
Phase I Advanced solid tumors Ipsen and Roche
(BN-80927)
Dr. Reddys
DRF-1042 Phase I
Laboratories
Gastric cancer and relapsed
Exatecan mesylate Phase II
rhabdomyosarcoma 9 in children)
Phase II/III but
already withdrawn
Rubitecan Pancreatic cancer
due to unfavorable
results
CZ-48 * Phase I Solid tumors
TP-300 * Phase I Advanced solid tumors
EZN-2208 * Phase I Advanced malignancy
Discontinue after Pharmacia and
MAG-CPT **
Phase I Upjohn
XMT-1001 ** Phase II Lung cancer
CRLX-101 ** Phase I/II Advanced solid tumor
* Conjugated form of camptothecin analogue; ** Prodrug form of camptothecin.
Irinotecan (62) Topotecan (63)
Figure 6. Campthotechin analogues.
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Molecules 2020, 25, 2611
7.3. Antiviral Activity

Generally, flavonoids are one of the phytochemicals responsible for the activities against multiple
viruses. For example, catechin can inhibit the property of viral infectivity of the respiratory syncytial
virus (RSV) and herpes simplex virus-1 (HSV-1) but not their intracellular replication, whereas quercetin
is capable of effectively reducing the infectivity of majority of viruses [74] and partially inhibiting
DNA gyrase [58]. Meanwhile, coumarins may possess antiviral effects by stimulating the macrophages
towards preventing infections and recurrence of cold sores caused by HSV-1 in humans, while
terpenoids are active against yeasts such as Candida albicans. Similarly, harmane offers intercalating
properties with the DNA of viruses [74] and has a strong activity against the intracellular amastigote
of Leishmania infantum, which is partly due to its ability to inhibit the leishmanial protein kinase C
(PKC) activity [75].
Furthermore, harmaline (29) or 7-methoxy-1-methyl-4,9-dihydro-3H-pyrido[3,4-β] indole alkaloid
isolated from O. nicobarica is known for its potent antiviral activity against herpes simplex virus 1 (HSV-1.
Rather than interfering with viral entry, it affects the recruitment of lysine-specific demethylase-1 (LSD1)
and immediate-early (IE) complex binding on the promoter of ICP0. This will, in turn, contribute
to the suppression of the viral IE gene synthesis, thereby causing a reduction of ICP4 and ICP27
expression [76]. Similarly, camptothecin (11) and 10-methoxycamptothecin previously isolated from
the leaves of O. mungos have been noted to yield an active activity against the herpes virus [77]. Table 7
shows the antifungal, anti-yeast, and antiviral activities of five Ophiorrhiza species according to prior
studies conducted on these plants.
428
Table 7. Antiviral activity.

Antifungal activity was determined by the
Ophiorrhiza trichocarpon Alkaloid, coumarin, agar-well diffusion method.
Methanolic 50 mg/mL
Bl., Ophiorrhiza rugosa, anthraquinone Inhibition zone:
Molecules 2020, 25, 2611
extract of the 30 mg/mL [3]

and Ophiorrhiza aff. glycoside, scopoletin, Candida albicans (10 mm)
whole plant 75 mg/mL
nutans Cl. ex Hk. f. saponins Trichophyton mentagophyte (12 mm)
Aspergillus flavus (12 mm)
Anti-yeast/antifungal activity of:
The three compounds against:
C. albicans (inhibition zone; 15, 10 and
16 mm respectively)
Lupan-20-ol-3(β)-yl Fusarium oxysporum (inhibition zone; 10,
hexadecanoate (51), Hexane and 8 and 8 mm respectively).
lupan-20-ol-3(β)-yl chloroform
Ophiorrhiza shendurunii 100 μL [35]
acetate (52), extract of the Hexane extract:
olean-18-en-3(β)-yl whole plant Candida albican (28/28 mm)
429
hexadecanoate (53) Fusarium oxysporum (18/26 mm)
Chloroform extract:
Candida albican (28/28 mm)
Fusarium oxysporum (24/26 mm)
0.25 and 0.5
mg/kg b.w p.o
Ophiorrhiza nicobarica harmaline (29) Anti-HSV-1 activity in mice [76]
once daily
(8 days)
Molecules 2020, 25, 2611
7.4. Antimicrobial Activity

The components of alkaloids, carbohydrates, tannins, anthraquinones, hormones, terpenoids,
saponins, flavonoids, essential oils, glycosides, and potassium are undeniably considered to have
a curative effect against several pathogens [78]. Flavonoids, for example, can form a complex with
extracellular bacterial soluble proteins and cell walls; those that are lipid-soluble may also disrupt the
microbial membranes as they are lipophilic in nature. Meanwhile, coumarins and terpenoids are active
against positive bacteria such as Bacillus subtilis and Staphylococcus aureus, despite the latter’s lesser
effect against gram-negative bacteria [74].
A majority of works assessing the antimicrobial activities of extracts from Ophiorrhiza species
have been conducted using the diffusion assay method, namely by observing the inhibition zone of
these extracts on certain microorganism growth. Different plant extracts will typically display distinct
antibacterial activities, which are attributable to their varying phytochemical contents and properties.
For instance, the ethyl acetate and methanol extracts have been underlined as the best options amongst
other solvent extracts tested. Here, the lesser activity of other extracts is due to their inability to either
extract the bioactive molecules with antimicrobial constituents or produce an effective concentration of
antibacterial constituents [79]. Table 8 shows the antimicrobial activities of five Ophiorrhiza species,
whereas their biological activities are summarised in Figure 7.
430
Table 8. Antimicrobial activity.

Presence of Ethyl acetate and methanol extract showed potent
Hexane, chloroform,
carbohydrates, antimicrobial activity against nine microorganisms (B.
ethyl acetate,
proteins, phenolics, subtilis, L. lactis, S. aureus, M. luteus, P. aeruginosa, S.
Molecules 2020, 25, 2611
Ophiorrhiza methanol and

terpenoids, typhimurium, K. pneumonia, P. vulgaris, E. coli.). 500 μg/mL [79]
mungos ethanol extracts of
glycosides, reducing Inhibition zone:
leaves and stem of
compounds and Ethyl acetate extract: 19–28 mm
the O. mungos.
saponins Methanol extract: 11–26 mm
Carbohydrate, Antimicrobial activity against six pathogens with
tannin, terpenoids, inhibition zone:
Ethanolic extract of
saponins, flavonoids, B. subtilis (20 mm), K. pneumonia (19 mm), P. aeruginosa (14 0.02 mL [78]
fresh flower
alkaloids and mm), S. aureus (14 mm), S. mutans (11 mm)
glycosides C. albicans (16 mm)
Antibacterial activities of:
Ophiorrhiza trichocarpon BI. 75 mg/mL
E.coli (10 mm) 10 mg/mL
Ophiorrhiza S.aureus (10.5 mm) 10 mg/mL
431
trichocarpon BI., Alkaloids, Pseudomonas aeruginosa (10 mm)
Ophiorrhza coumarins, Ophiorrhza rugosa 100 mg/mL
Methanolic extract of
rugosa and anthraquinone E.coli (9.5 mm) 10 mg/mL [3]
the whole plant
Ophiorrhiza aff. glycosides, S.aureus (10 mm) 50 mg/mL
Nutans Cl. Ex scopoletin, saponins Pseudomonas aeruginosa (10 mm)
Hk. f. Ophiorrhiza aff. Nutans Cl. Ex Hk. f. 100 mg/mL
E.coli (10 mm) 10 mg/mL
S.aureus (10.5 mm) 10 mg/ml
Pseudomonas aeruginosa (10 mm)
Lupan-20-ol-3(β)-yl Antibacterial activities of the chloroform extract using agar
hexadecanoate (51), well diffusion assay are significant against:
Hexane and
Ophiorrhiza lupan-20-ol-3(β)-yl B. subtilis (22/18 mm)
chloroform extracts 100 μL [35]
shendurunii acetate (52), E. coli (19/23 mm).
of the whole plant
olean-18-en-3(β)-yl The hexane extract showed no antibacterial activity against
hexadecanoate (53) Escherichia coli and Bacillus subtilis.
Molecules 2020, 25, 2611
Figure 7. Summary of biological activity of phytochemicals in Ophiorrhiza species.
8. Propagation
Over time, several researchers have conducted biotechnology research on certain species of
Ophiorrhiza in order to develop an efficient method for the development of camptothecin (11) and
recognition of novel secondary metabolites from cultivated plants [32]. The high demand for
camptothecin and other active pharmaceutical ingredients, as well as the overexploitation of C. accumata
and Nothapodytes foetida as the main sources of this component have resulted in the development of
various protocols for its in vitro cultivation and production by using Ophiorrhiza plants [1]. In fact,
camptothecin is a highly valuable drug with market prices ranging from US$3500 to US$350,000 per
kilogram. Therefore, it is considerably interesting for scholars to identify alternative ways of producing
the component, such as by using plant cells and tissue cultures, as a result of its commercial value [64].
The successful regeneration of O. pumila from the callus tissue of its leaves and shoot has
been reported, whereby a new glucosyloxy camptothecin (i.e., 9-β-glucosyloxycamptothecin) is
isolated along with 15 metabolites, including six alkaloids linked to camptothecin (11). However,
(3S)-deoxyppumiloside as one of camptothecin’s possible biogenetic precursors has not been detected,
whereas the (3R) epimer is isolated from regenerated plants. Accordingly, these well-developed callus
cultures can also generate the anthraquinones which are not producible by non- culture O. pumila
plants [54]. Therefore, a method for the micropropagation and generation of camptothecin from
O. mungos via in vitro plants has been documented, whereas a protocol is also developed for rapid
root and organogenesis proliferation. As a result, the high performance liquid chromatography
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Molecules 2020, 25, 2611
(HPLC) analysis of the method has proven that its in vitro production generates higher yields than
natural-grown plants [80].
Moreover, a study on the effect of jasmonic acid in the cell suspension culture of O. mungos species
has revealed the suspension’s ability to increase the production of camptothecin significantly [81].
Similarly, another study has substantiated the effect of silver nitrate and yeast extract on the cell
growth, camptothecin (11) accumulation, and cell viability, thereby leading to a significant increment
in the production of biomass and camptothecin (11) alike [82]. In fact, a prolonged subculture of
O. trichocarpose Blume by alternating the medium intensity in each subculture has resulted in a notable
increment in the development of the plant’s shoots and biomass [83].
In line with this, different concentrations of various auxins in O. mungos var. angustifolia culture
have also been reported to influence the biomass camptothecin (11) production, whereby the result
shows that both explants will not induce callus growth in the absence of exogenous hormones.
In contrast, they will induce the growth of calli following their culture in the Murashige and Skoog
(MS) medium at different strengths together with butyric acid (BA) and naphthalene acetic acid (NAA)
solutions. The study underlined the ideal combination of solutions as half-strength MS solid medium
with 10.74 μM NAA + 4.44 μM BA. Further, the scholars have discovered that the in vitro leaves are
the best explants that can help to produce a higher content of camptothecin compared to their in vivo
counterparts [69].
In contrast, an efficient plant regeneration system has been introduced in a study through somatic
embryogenesis. Here, the embryogenic callus tissues extracted from aseptic leaf explants were used to
induce the somatic embryos under callus tissues incubated across the different periods of 10 to 60 days.
The outcomes showed that 40-day-long incubation in 0.5 mg/L 2,4-dichloro phenoxy acetic acid media
was the optimum environment for the somatic embryogenesis in O. pectinata. Therefore, the study
successfully substantiated the influence of callus tissue incubation period towards the differentiation
of embryos in suspension cultures [33].
Moreover, a high production of camptothecin (11) has been obtained using the root cultures of both
transformed and untransformed O. mungos [64] and O. alata Craib [39], respectively, thus promoting
an alternative novel system in sustaining its bio-production as raw material for the pharmaceutical
industry. The transformed hairy roots were obtained by infecting the node explants with Agrobacterium
rhizogenes TISTR 1450 [39,64]: it was found that the CPT (11) concentration was consequently double
the amount in the soil-growing plant [39].
In general, culture media containing ammonium as the nitrogen source is preferable, whereby
nitrate is essential in regulating the pH value and promoting the growth of root and synthesis of
camptothecin (11) [64]. The addition of polystyrene resin or Diaion HP-20 capable of absorbing
camptothecin (11) has further led to its increased concentration in culture media, namely seven-fold
higher compared to the control media [39]. Moreover, camptothecin (11) can be extracted more in root
and hairy root cultures as they consist of differentiated tissues. Here, the secondary plant metabolite
biosynthesis is still active and has not decreased in contrast to the cultures of undifferentiated callus or
cell suspension [39,64].
Nevertheless, the accumulation of camptothecin (11) in the different parts of plants is identical [84],
rendering it crucial for one to consider the specific selection and isolation of cells or organ lines in order
to induce in vitro calli in biomass metabolite production. For example, undifferentiated callus and
suspension cultures do not always produce the desired amount of compound of interest. In contrast,
the shoot, root, and hairy root cultures will often generate the same compounds as in the appropriate
organs [67]. Hence, the type of medium culture, suitable salt strength, nitrate level, phosphate level,
and growth regulator level should be considered beforehand to obtain the desired metabolites at a
high quantity and quality [84].
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Molecules 2020, 25, 2611
9. Conclusions
To conclude, compounds derived from Ophiorrhiza plants play an important role in treating
diseases although they may not directly consider drugs. The compounds can be served as lead
compounds that are beneficial for the development of potential anticancer drugs, particularly. This
may help new researchers in understanding the diseases better, providing more efficient therapies
with a new mechanism of action, increasing the patient compliance, reducing the synthetic anticancer
drugs-related adverse effects as well as encouraging the development of future and novel anticancer
drugs. The Ophiorrhiza species represent an enormous diversity throughout the world, yet not many
have been explored. With the development of technology of apparatus and equipment nowadays,
newly discovered bioactive compounds showing inhibition effects on cancer cells may rapidly be
recognized. Thus, further extensive research and studies should be continued to discover more bioactive
molecules in this species and their therapeutic effects to highlight the use of natural source-derived
drugs in the medicinal field which are believed to be more well-tolerated and cost-effective.
Funding: This research was funded by INTERNATIONAL ISLAMIC UNIVERSITY MALAYSIA, grant number
P-RIGS18-028-0028. The APC was funded by the Universiti Putra Malaysia.
Acknowledgments: The authors are thankful to the International Islamic University Malaysia for funding this
work via Grant No. P-RIGS18-028-0028.
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molecules
Review
Lamium Plants—A Comprehensive Review on Health
Benefits and Biological Activities
Bahare Salehi 1 , Lorene Armstrong 2 , Antonio Rescigno 3, *, Balakyz Yeskaliyeva 4 ,
Gulnaz Seitimova 4 , Ahmet Beyatli 5 , Jugreet Sharmeen 6 , Mohamad Fawzi Mahomoodally 6, *,
Farukh Sharopov 7, *, Alessandra Durazzo 8 , Massimo Lucarini 8 , Antonello Santini 9, *,
Ludovico Abenavoli 10, * and Javad Sharifi-Rad 12, *
1 Student Research Committee, School of Medicine, Bam University of Medical Sciences, Bam 44340847, Iran;
bahar.salehi007@gmail.com
2 Department of Pharmaceutical Sciences, State University of Ponta Grossa, Ponta Grossa,
Paraná 84030900, Brasil; lorenearmstrong@hotmail.com
3 Department of Biomedical Sciences, University of Cagliari, s.s. 554 bivio Sestu, I-09042 Monserrato, CA, Italy
4 Faculty of Chemistry and Chemical Technology, Al-Farabi Kazakh National University,
Almaty 480012, Kazakhstan; balakyz@mail.ru (B.Y.); sitigulnaz@mail.ru (G.S.)
5 Department of Medicinal and Aromatic Plants, University of Health Sciences, 34668 Istanbul, Turkey;
ahmet.beyatli@sbu.edu.tr
6 Department of Health Sciences; Faculty of Science, University of Mauritius, Réduit 80837, Mauritius;
sharmeenjugs@gmail.com
7 Department of Pharmaceutical Technology, Avicenna Tajik State Medical University, Rudaki 139,
Dushanbe 734003, Tajikistan
8 CREA-Research Centre for Food and Nutrition, Via Ardeatina 546, 00178 Rome, Italy;
alessandra.durazzo@crea.gov.it (A.D.); massimo.lucarini@crea.gov.it (M.L.)
9 Department of Pharmacy, University of Napoli Federico II, Via D. Montesano, 49-80131 Napoli, Italy
10 Department of Health Sciences, University Magna Graecia, viale Europa-Germaneto, 88100 Catanzaro, Italy
11 Department of Agricultural Sciences, University of Naples Federico II, 80055 Portici, Italy; rafcapas@unina.it
12 Zabol Medicinal Plants Research Center, Zabol University of Medical Sciences, Zabol 61615-585, Iran
* Correspondence: rescigno@unica.it (A.R.); f.mahomoodally@uom.ac.mu (M.F.M.); shfarukh@mail.ru (F.S.);
asantini@unina.it (A.S.); l.abenavoli@unicz.it (L.A.); javad.sharifirad@gmail.com (J.S.-R.);
Tel.: +39-0706754516 (A.R.); +23-057327341 (M.F.M.); +992-93-995-0370 (F.S.); +39-0812539317 (A.S.);
+39-0961-3694387 (L.A.); +98-21-88200104 (J.S.-R.)
Academic Editor: Raphaël E. Duval

Received: 16 April 2019; Accepted: 14 May 2019; Published: 17 May 2019
Abstract: This work is an updated snapshot of Lamium plants and their biological activities.
The main features of the plant are described and the components of its essential oils are summarized.
The traditional medicinal uses of Lamium plants has been reported. The presence of these chemicals
i.e., hydroxycinnamic acids, iridoids, secoiridoids, flavonoids, anthocyanins, phenylpropanoids,
phytoecdysteroids, benzoxazinoids, betaine can provide biological activities. After the discussion
of antioxidant properties documented for Lamium plants, the biological activities, studied using
in vitro models, antimicrobial, antiviral, anti-inflammatory, anti-nociceptive activity, and pain therapy
and cytotoxicity and cytoprotective activity are here described and discussed. Finally, targeted
examples of in vivo studies are reported.
Keywords: Lamium plants; antiviral; antioxidant; anti-inflammatory; cytotoxicity
1. Introduction
Medicinal plants have been used since antiquity to treat illness and discomfort; their knowledge
has been propagated from generation to generation by indigenous and local populations instigating

Molecules 2019, 24, 1913
the ethnobotanical study. Considering their global use, traditional, therapeutic and industrial value,
many natural products are being investigated to lead the production of new drugs, wherefore, families
and genera of plants with great potential are being researched for this purpose [1–3]. The recent review
of Durazzo et al. [4] gives a current picture of main features of botanicals, by describing the strict
relationship between the main plant biologically activity compounds and the nutraceutical role of
botanicals [4–10].
Lamiaceae is a widespread family of flowering plants, also known as the mint family [11]. It is well
distributed around the continents and, has about 250 genera described, which have 7,852 accepted
species names [12]. The largest genera are Salvia, Scutellaria, Stachys, Plectranthus, Hyptis, Teucrium,
Vitex, Thymus and Nepeta [3,11,13]. The plants of this family are normally shrubs or herbs with aromatic
compounds in their leaves or flowers, such as essential oils. Many species are cultivated for their
medicinal properties like antiseptic, antispasmodic, calmative, antimicrobials and, it is also used for
culinary, fragrance, flavor and aromatherapy [11,14–16].
Within the family Lamiaceae, the genus Lamium is herbaceous and possess annuals or perennials
forms, comprising around 40 species found in temperate and subtropical regions of Africa, Asia
and Europe [16–19]. Regarding the botanical aspects, the leaves are cordate or reniform, ovate to
lanceolate with an acute apex and cordate base, being petiolate on the lower nodes and sessile or
uncommon amplexicaule at the upper nodes. It presents inflorescences with verticillasters in the axils
of the floral leaves (2-12 flowered). The calyx is campanulate or tubular with subequal teeth. The corolla
is bilabiate and shows a dark purple, yellowish green, white, yellow, etc. [17,19]. Species well studied
of the genus are: L. album L., L. purpureum L., and L. maculatum. The popular name of “dead nettle”
is due to the superficial similarity of the stinging nettles and, they do not have trichomes which can
release toxic compounds [20].
The genus is used in folk medicine as antispasmodic, astringent, anti-proliferative,
anti-inflammatory, antiviral, regulatory for sebaceous secretions and, also for the treatment of
hypertension, scrofula, paralysis, prostate, menorrhagia, uterine hemorrhage, leucorrhea, trauma,
fracture [19–22].
Ecologically this genus shows important characteristics such as self-pollination, being hosting
for different insects species and they attract bumblebee queens and honeybees, which represents
the entomophilous pollination [19]. This focused review wants to give an updated snapshot of main
beneficial properties of Lamium plants, with a focus on antimicrobial, antiviral, anti-inflammatory,
anti-nociceptive activity, and pain therapy and cytotoxicity and cytoprotective activity, in order to
better address nutraceutical uses, formulations and applications; therefore, this review aims to bring
information found in the literature data about different aspects of the Lamium genus and to demonstrate
its importance regarding traditional, medicinal, chemical composition, biological and pharmacological
activities, as well as industrial potential and economic use. The search was carried out using search
engines Scopus, Science Direct, and PubMed and the following keywords were typed: Lamium
and health benefits; Lamium and biological activities; Lamium and antioxidant properties; Lamium
and antiviral activity; Lamium and anti-inflammatory activity; Lamium and antimicrobial activity;
Lamium and antischistosomal activity; Lamium and antinociceptive activity; Lamium and pain therapy;
Lamium and cytotoxicity; Lamium and cytoprotective activity; Lamium and anti-tyrosinase activity;
Lamium and anticancer activity; Lamium and in vitro studies; Lamium and in vivo studies; Lamium
and clinical studies.
2. A Shot on the Biologically Active Compounds in Lamium Plants

Chemically, Lamium is distinguished by the presence of different classes of chemical constituents,
which we can mention hydroxycinnamic acids, terpenoids, among them iridoids and secoiridoids,
flavonoids, anthocyanins, phenylpropanoids, phytoecdysteroids, benzoxazinoids, betaine [20–22].
Thus, the presence of these chemicals can provide biological activities tested in vitro and in vivo
assays, such as antioxidant, anti-inflammatory, antimicrobial, antischistosomal, for pain relief in
440
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rheumatism and arthritis, a tonic for constipation, antinociceptive, anticancer [16,20–22]. Most of
the bioactivities of Lamium species are linked to their principal constituents, that is, phenolics
and essential oils [23–26]. Polyphenols, flavonoids, terpenes, steroidal derivatives, and ecdysteroids
account for various biological activities of this species of Lamium [27–31]. In this regard, it is worth
mentioning the review of Carović-Stanko et al. [32] focused on the Lamiaceae species and their secondary
metabolites encompassing a wide array of beneficial functions and their applicability as sources of
functional foods.
2.1. The Phenolic and Terpenoid Compounds of Lamium Plants

Here some studies showing the isolation of phenolic and terpenoid compounds are reported.
For instance, Deng et al. [33], by studying constituents in the herb of Lamium maculatum L. var Kansuense,
isolated and characterized ten compounds were obtained and they were identified as d-mannitol,
beta-sitosterol, stigmasterol, rutin, 3 -methylquercetin-3-O-rutinoside, n-butyl-beta-D-fructopyranoside,
daucosterol, acteoside, 20-hydroxyecdysone, allantoin. Nugroho et al. [34] isolated and characterized
two new flavanol glycosides, together with other three just known using column chromatography from
aerial parts of L. amplexicaule. The Czerwinska et al. [22] reported that the main constituents of the aerial
part of L. Album were phenylpropanoids, iridoids, flavonoids and phenolic acids. Czerwinska et al. [35]
in the herb of L. album underlined phenylpropanoid glycosides along with iridoids and flavonoids might
be the valuable bioactive compounds present in these species. The peculiar constituents of Lamium plants
are phenlypropanoids and iridoids. The phenylpropanoids consist in a group of compounds with a great
diversity of structures including simple phenylpropanoids (i.e., derivatives of cinnamyl alcohols, cinnamic
acids, phenylpropanes) and complex phenylpropanoids [36]. Generally they can occur rarely in the free
form and are often present in bound form, linked to carbohydrates, as examples of main forms found
in Lamium plants. Beside phenylpropanoids, another main constituent and potential chemotaxonomic
marker of Lamium plants are iridoids: they are monoterpenes consisting of a cyclopenta[c]pyranoid
skeleton (iridane skeleton) and mainly occurring as glycosides [37]. Molecules such as verbascoside,
cis-acteoside, and lamalboside (lamiusides A), lamiusides B, C, and E, lamiridoside, lamiol, caryoptoside,
albosides A and B are examples of phenylpropanoid glycosides, described Lamium album L. [21,27,35,38,39].
For instance, in the aerial parts of the L. album plant two phenylpropanoids glycosides, verbascoside
and isoverbascoside (Figure 1), can make up 55% of the total phenolic compounds measured [40].
The iridoids found in the genus can work as markers of L. album, L. amplexicaule, L. garganicum,
L. maculatum, and L. purpureum [20].
Figure 1. The chemical structure of some biologically active molecules from Lamium species.
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Molecules 2019, 24, 1913
2.2. Essential Oil Constituents of Lamium Plants

Considering how the therapeutic importance of Lamiaceae species is mostly based on their volatile
oils [41], here essential oil constituents of Lamium plants are described. Even though plants belonging
to the Lamiaceae family are recognized to contain an eminent amount of essential oils, the plants from
the genus Lamium of the subfamily Lamioideae possess a small number of essential oils. The yields of
the essential oils extracted from the fresh flowers of Lamium plants differ between 0.01–0.31% [42].
Numerous authors have studied the flavor composition from Lamium species [42–44].
However, the volatile components of only some species have been well studied so far, out of the 25 species
of Lamium that are accepted [12]. Various studies have demonstrated that the biological and chemical
activities of essentials oils from Lamiaceae plants, not merely varied within the species and its
varieties but even amid the different agro-climatic and geographical regions [45–49]. Such variations
in the composition of Lamiaceae plant essential oils can be essentially related to genetic as well
as environmental factors that also define the genetic expressions and thereby influence the chemical
constituents of the oils [50]. A study by Alipieva et al. [43] on the essential oils of four Lamium species
from Bulgaria (namely, L. album, L. purpureum, L. garganicum, and L. maculatum flowers) collected
from nine natural populations were analyzed by using GC-MS. Although a similarity of the volatile
profiles of all samples was observed, quantitative and qualitative variations in oil composition
of the plants obtained from different locations were also seen. For instance, hexahydrofarnesyl
acetone was observed in L. maculatum (at Losen mountain: 1.9% while at Ljaskovetz: 1.2%),
L. purpureum (at Vlado Trichkov: 1%) and L. album (at Vlado Trichkov: 0.2%) though not from
all location sites. Besides, predominantly in L. maculatum and L. garganicum were identified other
terpenoids, such as β-caryophyllene and α-humulene, found in highest concentrations in L. maculatum
(at Losen mountain: 1.6% and 0.8%). Table 1 depicts more such examples where variations in
the composition and percentages of main constituents of essential oils of Lamium species grown in
different regions and/or countries.
Furthermore, the study of Flamini and workers [42] showed that the composition of essential oils
varied, in different organs of Lamium species investigated. For instance, in L. purpureum, the content
of α- and β-pinene was 35.7% in the leaves, 7.5% in the bracts while it was 75.3% in the flowers.
In addition, in L. bifidum, bracts and leaves displayed a profile characterized by the common presence of
germacrene D, β-caryophyllene, α-humulene, and β-elemene, while contrastingly, the flowers emitted
profiles revealing high percentages of myrcene (47.2%), β-caryophyllene (11.8%) and sabinene (11.0%).
The previous work of Kapchina-Toteva et al. [51] also investigated the effect of micropropagation
on the essential oil content in leaf extracts of Lamium album L. plants grown under in vitro and ex
vitro conditions in contrast to in situ-grown plants. The in situ-grown plants were found to consist
of 45 hydrocarbons, and as a result of micropropagation, the content of these compounds reduced
about two-fold, reaching 24 during in vitro cultivation, and finally 19 after ex vitro acclimation.
Moreover, while the in situ plants were characterized by the accumulation of long-chain alkanes
(nonadecane; heneicosane; heptadecane; tricosane), both in vitro and ex vitro plants contained shorter
hydrocarbons such as octane (3.9% and 1.1%, respectively) and undecane (3.0% in in vitro samples).
Besides, certain alcohols such as phytol; octen-3-ol; 3-hexene-1-ol and acetate were observed to rise
in micropropagated plants compared to in situ-grown ones. As for terpenes, 60 compounds were
detected in the oil of in situ plants while it decreased in both in vitro and ex vitro ones—44 and 35
compounds. A variation in the terpene composition was also detected. The major sesquiterpenes
in the in situ-grown plants were germacrene D (6.9%) and β-caryophyllene E (1.1%). In the course
of micropropagation, elevated accumulation of these compounds was observed in both, in vitro-
and ex vitro-grown plants [germacrene D (44.1% and 46.7%, correspondingly) and β-caryophyllene E
(13.0% and 6.5%, correspondingly)]. Therefore, these results demonstrated the significance of growing
conditions which were accountable for the variability in the level and composition of plant metabolites.
The chemical structure of the main volatile secondary metabolites from Lamium species are represented
in Figure 2.
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Molecules 2019, 24, 1913
Figure 2. The chemical structure of the main volatile secondary metabolites from Lamium species.
443
Table 1. Main components of essential oil from Lamium species studied from the year 1976–2018.
Plant Name Place/Country of Collection Parts Used Extraction Method Used % Yield Main Components References
aerial parts (flowers Trans-phytol (44.8%), octadecanol (12.0%), hexadecanoic acid
(1) Lamium amplexicaule L. Khorassan-e Razavi province in northeastern Iran Hydro-distillation 0.1 (w/w) [52]
and leaves) * (11.8%) and hexahydrofarnesyl acetone (10.6%)
Germacrene D (18.5–34.9%), (E)-caryophyllene (2.5–11.9%),
Huntsville, Alabama, USA aerial parts ** Hydro-distillation NA α-pinene (2.2–16.2%), β-pinene (2.0–10.6%) and 1-octen-3-ol [53]
(3.5–8.0%)
Trans-chrysanthenyl acetate (41.1%), germacrene D (28.9%)
Pergole, Arcidosso Municipality, South Tuscany, Italy Flowering aerial parts Hydro-distillation NA [42]
Molecules 2019, 24, 1913
and α-pinene (6.8%), ocimene (0.8%)

Northeast of Tehran, Iran aerial parts * Hydro-distillation 0.1(v/w) Germacrene-D (22.3%) and camphor (18.1%) [54]
Isophytol (14.8%), 9,12,15-ocadecanoic acid methyl ester
(19.2%), 6,10,14-trimethyl-2-pentadecanone (8.0%), dibutyl
El Dakahlyia governorate, Egypt Leaves NA NA [55]
phathalate (6.1%), nonacosane (5.5%), hexadecanoic acid
(3.4%) and nonyl phenol (3.2%)
Syria NA Dry evaporation NA Imedazol and pyrimidene [56]
NA NA NA 0.09 NA [57]
Germacrene D (15.0–46.3%), α-pinene (4.1–15.3%), β-pinene
(2) Lamium purpureum L. Huntsville, Alabama, USA aerial parts ** Hydro-distillation NA (6.3–16.3%), and 1-octen-3-ol (4.2–15.3%), β-elemene [53]
(3.7–16.0%)
Germacrene D (35.4%), β-pinene (26.8%) and α-pinene
Pergole (Arcidosso Municipality, South Tuscany, Italy Flowering aerial parts Hydro-distillation NA [42]
(13.4%), ocimene (2.9%)
1-Octen-3-ol, cis-3-hexen-1-ol, phenethyl alcohol, benzyl
Japan Aerial parts Steam distillation NA [44]
alcohol, phenol, 0-, m-, and p-cresols, guaiacol, eugenol
β-caryophyllene (14.8%), caryophyllene oxide (13.8%),
Experimental station of Faculty of Pharmacy, Z,E-α-franesene (10.1%), dihydroedulan I (9.13%),
(3) Lamium maculatum L. aerial parts ** Hydro-distillaion 0.35 (v/w) [58]
Zagazig University, Egypt α-humulene (6.1%), bornyl formate (6.0%) and α-bisabolene
444
(5.3%)
NA Leaves NA NA Hexahydrofarnesylacetone (22%) [59]
Germacrene D (39.0%), (Z)-ocimene (8.7%), methyl salicylate
(4) Lamium hybridum Vill Pergole (Arcidosso Municipality, South Tuscany, Italy Flowering aerial parts Hydro-distillation NA [42]
(7.5%) and β-caryophyllene (6.1%), ocimene (11.6%)
Germacrene D (34.9%), sabinene (12.4%), β-caryophyllene
(5) Lamium bifidum Cyr. Pergole (Arcidosso Municipality, South Tuscany, Italy Flowering aerial parts Hydro-distillation NA [42]
(11.5%), α-humulene (6.8%)
(6) Lamium garganicum L.
Athens, Greece aerial parts *** Hydro-distillation 0.31 1,8-cineole (47.5%), citronellal (25.1%) and isoeugenol (11.8%) [60]
subsp. laevigatum Arcangeli
6,10,14-trimethyl-2-pentadecanone (10.2%)
(7) Lamium album L. Behshahr, Mazandaran Province, North of Iran Flowering aerial parts Hydro-distillation 0.2 (w/w) [61]
and 4-hydroxy-4-methyl-2-pentanone (9.1%)
Prenol, farnesene-beta- E, tridecanol n, dodecanoic acid n,
Supercritical carbon
Experimental field of the Kaunas Botanical Garden of Plants in the vegetation hexadecane-n, squalene, tetradecanol-n, undecane–n,
dioxide extraction NA [62]
Vytautas Magnus University, Lithuania period benzoate-isopentyl, dodecanoate -butyl, phytone,
method
neophytadiene
NA Aerial part NA 0.04–0.46 NA [57]
NA Flowers NA 0.05 NA [57]
Kharkiv region, Ukraine Leaves NA NA α-Terpeniol, linalool, squalene, spatulenol, α-Bisabolol [63]
(8) Lamium moschatum Mill. NA Flowers Steam-washed NA Caryophyllene [64]
(9) Lamium striatum
Flowers Steam-washed NA Carboxylic acids [64]
Sibth. et. Smith
NA—Not Available/applicable; * Air-dried; ** fresh; *** fresh air-dried.

Molecules 2019, 24, 1913
3. Traditional Medicinal Uses of Lamium Plants

Plants have been used by a human as food and medicine since ancient times. Examples of
the traditional medicinal uses of Lamium plants are here described. White dead-nettle (Lamium album
L.) used for decades in Europe, China, and Japan during times of famine [65]. Different aerial parts of
this plant are edible and traditionally used as raw or cooked food in some countries. Especially in some
dishes in Mediterranean and neighboring areas [66]. In addition, non-stinging nettle is considered
a base component of some well-known vegetarian dishes and salads [40]. L. album, when added to food
supplements, can prevent menstrual, musculoskeletal disorders and ameliorate fat metabolism [67,68].
L. amplexicaule is used in the preparation of Japanese traditional rice porridge which called “seven
spring herbs” [69].
In traditional and folk medicine worldwide some Lamium species used in the treatment of
fracture, hypertension, leucorrhoea, paralysis, putrescence, trauma and some gynecological diseases
like menorrhagia, uterine hemorrhage, vaginal and cervical inflammation, bleeding after childbirth
and believed to be a contraceptive, etc. [70–72]. Ethnobotanical studies indicate the use of aerial parts
and floral branches of L. album for different kidney problems like the exertion of stones [73,74]. Leaves
decoction and infusion for respiratory tract problems [75].
Moreover, there are many types of activities, e.g., antipyretic, astringent, bronchitis,
diuretic, emollient, expectorant, insomnia, pains, sciatica, vasodilator, hemostatic, wound healing,
antihypertensive, anti-inflammatory which recorded by literature [76–80]. L. amplexicaule used
as anti-rheumatic, laxative and diaphoretic [81]. Sometimes fresh leaves of L. amplexicaule are crushed
a paste formed used topically to joints swelling [82]. Aerial parts decoction of L. galeobdolon traditionally
used for fever, malaria, warts, constipation, hair loss, rheumatism, dandruff, hemorrhage [83,84]
depression, nerve tonic [85].
4. Biological Activities Lamium Plants

Our mental and physical well-being is directly related to what we introduce with the diet. In fact,
the nutritional content of what we eat affects the composition of our cell membranes, blood, tissues,
organs, skin, and so on. It has long been a common opinion, widely shared also by the scientific world,
that diet and nutrition are also important factors that can, depending on the case, protect us or promote
pathological conditions and chronic diseases. From this perspective, the great variety of biologically
active molecules present in plants can offer a general condition of ‘protection’ and play an important
role in maintaining a state of well-being of the individual.
In the years 1970–1980, scholars of popular culture and history of medicine began to record
the traditional health habits of the peoples in the various countries, with an approach that tried
to capture the full breadth without expressing value judgments. In this context, the notion of folk
medicine has extended its meaning since the boundaries between popular traditions and scientific
evidence present undeniable differences. Over time, therefore, the research has tried to find scientific
evidence to validate or refute practices in the profane medical culture.
Folk medicine has made extensive use of plants of the Lamium genus over the centuries. The most
common uses are described in the countries of the Mediterranean basin (Europe and North Africa)
and in Western Asia. Buds, leaves, and flowers are also widely used in the kitchen for the preparation
of various recipes of local tradition in many countries of the Mediterranean basin [21]. From health
benefits, plants of the Lamium genus have found widespread applications in folk medicine thanks to
the large presence of chemical compounds that constitute effective active ingredients in many situations
of therapeutic interest.
4.1. In Vitro Studies

Most of the scientific evidence on the Lamium genus has been conducted through in vitro studies
over the past 15–20 years. Extracts from the shoots, leaves, and flowers, have shown many biological
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activities. In this section, particularly, we will review the species of the Lamium genus used about
various medicinal uses, highlighting those most promising studies given a possible use of the active
ingredients extracted from these plants.
4.1.1. Antioxidant Activity

As is known, antioxidant molecules can be technically defined as agents that prevent or slow
down the phenomenon of oxidation. Reactive oxygen species (ROS) are unavoidable sub-products of
cellular aerobic metabolism [86]. However, free radicals can also originate from prolonged exposure to
UV rays, cigarette smoke, and air pollution. The reactive oxygen molecules are capable of damaging
the structures of the cell through the establishment of so-called oxidative stress, a situation that, if not
kept under control, can lead or exacerbate many pathological states [87]. A large number of molecules,
so-called antioxidants, can interrupt the chain of radical reactions and thus prevent damage to cells [88].
Our body can counteract the activity of free radicals through endogenous antioxidant mechanisms
and the introduction through the diet of exogenous substances [89]. Among the endogenous factors
are enzymes such as superoxide dismutase (SOD; EC 1.15.1.1) which catalyzes the dismutation
of the superoxide radical (O2 − ) into either molecular oxygen (O2 ) or hydrogen peroxide (H2 O2 ),
catalase (CAT; EC 1.11.1.6), which removes intracellular H2 O2 , and reduced glutathione (GSH) [90].
Among the non-exogenous non-enzymatic substances with antioxidant properties, we recall Vitamin E,
Vitamin C, carotenoids, polyphenols, and anthocyanins instead.
While many foods have a protective effect on free radicals, on the other, improper eating habits
can increase their activity, for example, a diet too rich in animal fats, excessive consumption of some
vegetable oils and fatty fish, excess of iron, food intolerances.
The antioxidant properties are determined in vitro by common assays: the trolox equivalent
antioxidant capacity (TEAC), oxygen radical absorbance capacity (ORAC), total radical-trapping antioxidant
parameter (TRAP), ferric-reducing antioxidant power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH)
radical scavenging activity assay [91,92]. They vary in their principles, mechanisms, and experimental
condition, in the reference compounds used, i.e., trolox, gallic acid, or catechins and in how endpoints are
measured [93–95].
These methods are based on the transfer of electrons (ET) or the transfer of hydrogen atoms
(HAT) [96,97]. Another antioxidant mechanism is the transition metal chelation, (TMC): transition metals
ions may be chelated by polyphenols, leading to stable complexed compounds [98]. The determination
of the antioxidant power should be measured with at least two or three assays [99,100]. A more in-depth
and correct investigation must also include the determination of the phenolic content and that of
the flavonoids.
As marked by several authors [101–103] in the procedure of determination of antioxidant properties
three critical elements should be taken into account: the extraction procedure, the antioxidant capacity
measurements and the expression of results.
In this regard it is worth mentioning the point of view of Durazzo and Lucarini, [7] that well
summarized the actual main strategies of research for evaluating antioxidant properties: the evaluation
of bioactivities of pure compounds and/or their mixtures; the study of different biologically active
compound-rich extracts and how these fractions contribute to the activity of total food extract;
the isolation of extractable and non-extractable compounds.
The assessment of the interaction of bioactive compounds as antioxidant properties represents
the first step for the evaluation of the health properties of medicinal plants [4]. Many species belonging
to the Lamium genus have been studied to identify their antioxidant properties. Carović-Stanko et al. [32]
pointed out that most of the Lamiaceae sources of antioxidants belong to the subfamily Nepetoideae,
such as basil, lemon balm, marjoram, mint, oregano, rosemary, sage, etc [104]. Lamium album L. is
absolutely the most studied species. Trouillas et al. [105] have studied and compared the water-soluble
fraction of 16 plants, including L. album, typical of Limousin, a central-southern region of France.
Presumably, the whole plant was used, whose main constituents are acid phenols, flavonoids,
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mucilage, iridoids. Water-soluble extracts have been tested for the ability to inhibit the DPPH radical,
the superoxide radical generated by the xanthine/xanthine oxidase (X/XO) system, and the inhibition
of the hydroxyl radical generated by the Fenton reaction. It is interesting to note that in the first two
cases (DPPH and X/XO) the extracts of L. album (non-stinging nettle) showed to be very effective when
compared with the other species studied; particularly, it was 12-16 times more effective than extracts of
Urtica dioica (stinging nettle). This result diverged, significantly, from that obtained with a methanol
extract of L. album that showed the same ability to inhibit the DPPH radical at an extract concentration
about 140 times lower [106]. This discrepancy could be due to the better ability of methanol to extract
flavonoids (≈193 mg of gallic acid equivalents per g of extract (mg GAE/g)). This higher extraction
capacity was corroborated by the greater amounts of phenolic compounds extracted when compared to
extraction in water. The content of phenolic compounds of the methanol extract was almost five times
greater than the aqueous extract. This result was in agreement with the content of phenolic compounds
found in methanol extracts of specimens of another species of the Lamium genus, L. amplexicaule
L. collected during the flowering period in a region of south-eastern Anatolia, Turkey (≈184 mg
GAE/g) [107]. The presence of polyphenols could, moreover, be the determining factor in explaining
why the methanol extract and hexane of L. amplexicaule showed a significant reduction in the formation
of nicked DNA and increased the native form of plasmid DNA pBR322.
Hydroxyl radicals generated by the Fenton reaction are known to cause oxidatively induced breaks
in DNA strands via the subsequent free radical-induced reaction on plasmid DNA. Hydroxyl radicals
can react with nitrogenous bases of DNA producing base radicals and sugar radicals. Polyphenols are
potential protecting agents against the lethal effects of oxidative stress and offer protection to DNA
by chelating redox-active transition metal ions [108,109]. For instance, Yumrutas et al. [107] showed
that L. amplexicaule hexane extract seemed to possess a greater ability to protect DNA than the methanol
extract. Hence, it might be said that available non-polar compounds in the hexane extracts might be
contributing to phenolic compounds for protecting DNA.
As another example, the antioxidant effect of butanol extracts from wild specimens
L. album and L. purpureum L. (red dead nettle) collected in Romania were compared for DPPH
and chemiluminescence activity. A possible correlation between the chemical composition, especially
for the amount of total phenols, and the antioxidant activity of the extracts was found [110]. In both
cases, the extracts possessed dose-dependent scavenger activity evaluated after 30 min. Incubation
with extracts, in all dose levels tested, whereas, the L. purpureum extract (1% concentration) exhibited
the highest scavenging activity compared with L. album extract.
Vladimir-Knežević et al. [111], by studying different medicinal plants of the Lamiaceae family
such as Salvia officinalis, Mentha longifolia, Melissa officinalis, Lavandula angustifolia, Satureja montana
concluded that Lamiaceae species are a rich source of various natural AChE inhibitors and antioxidants.
Danila et al. [112] aimed at assessing the phenolic content of L. album and L. maculatum methanolic
extracts, and their antioxidant capacity: for the DPPH assay the EC50 (μg/mL) values were 32.3 ± 0.1
for L. maculatum extract and 63.5 ± 0.7 for L. album extract, while in the ABTS assay EC50 (μg/mL)
values were 13.2 ± 0.1 for L. maculatum extract and 19.9 ± 0.5 for L. album extract.
On the other hand, many studies have proven that some natural antioxidants are a double-edged
sword. They can, under certain conditions, act as pro-oxidants in vitro, triggering lipid peroxidation,
DNA damage and apoptotic phenomena [113]. Phenolics and carotenoids can also exhibit prooxidant
activities, mainly in the presence of redox-active transition metal ions [114–116].
4.1.2. Antiviral Activity

As is known, viruses can replicate only within a host cell, exploiting their metabolic apparatus
and using their own genetic information; however, multiplication occurs only in cells susceptible to
the virus, that is, provided with specific superficial receptors and able to perform the replicative phases
of its genome.
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The search for an antiviral compound must be based on the interaction of the drug with specific
stages of viral replication; for example, it can act on cellular penetration of the virus, on the replication
of its genome, on protein synthesis or on the release of new viruses from the host cell. Herbal medicines
and purified natural products provide a rich resource for novel antiviral drugs [117].
Some chemical compounds have been isolated in plants of the Lamium genus having interesting
antiviral activities that are here reported. It is worth mentioning a phytochemical study of the aqueous
extract of the flowering tops of L. album, a component herb in a commercial liver health herbal formula,
that led to the identification of the antiviral activity of some iridoids [118]. Isomers lamiridosins
A and B were found to significantly inhibit hepatitis C virus entry in vitro showing an IC50 2.31 μM.
Interestingly, the parent iridoid glucosides demonstrated no anti-HCV entry activity.
4.1.3. Antimicrobial Activity

Generally, the term ‘natural antibiotics’ refers to those substances endowed with antibacterial
activity deriving from plants. In fact, antibiotics of natural origin do not derive only from plants, but also
from fungi, bacteria, and animals. Antibiotics are substances used to fight bacterial infections and may
have bacteriostatic action (i.e., inhibit bacterial growth) or bactericidal (i.e., they can kill bacteria).
A similar argument can be made for the antimycotics that are used to counteract the development of
pathogenic fungi.
Some types of plants can produce antibacterial and antifungal substances, even if they present
an activity, usually, much lower than that possessed by antibiotics deriving from fungi and bacteria [119].
Furthermore, it is good to remember that the antibacterial or antifungal substances contained in
these plants can interfere with possible pharmacological treatments already in place. Plants also
contain other compounds that could potentially be hazardous to health. However, in the popular
medicine of emerging countries, the use of plant preparations to counteract the growth of pathogenic
microorganisms is often described.
Antifungal activity of L. tenuiflorum Fisch and Mey against some medical yeast species
was described by Dulger et al. [120]. The ethanol extracts obtained from the leaves, rootstock,
and the combined formulation of Turkish endemic specimens have been investigated for their
antifungal activities against medical yeast Candida and Cryptococcus species. The extract (in the form of
sticky black substances) was dissolved in DMSO before testing. The combination of plant extracts
(1:1 ratio) was used in this test. Comparing the obtained result with those of the antifungal drug
ketoconazole used as a reference, it was noted that the combination of plant extracts (both leaves
and rootstock) exhibited greater antifungal effect against Candida albicans and that Candida spp. was
more susceptible than Cryptococcus spp. Unfortunately, in that study, there is no correlation between
quantity and type of polyphenols present in the extract of L. tenuiflorum and the antimycotic activity
found. Conversely, none of the extracts obtained from L. galactophyllum Boiss and Reuter, L. macrodon
Boiss and Huet and L. amplexicaule displayed activity towards Candida albicans ATCC 10231 [121].
Lacking such an antimycotic power could be due to the different quantity of polyphenols contained in
these species, ranging between 94 and 112 mg GAE/g in respect to higher values found for L. album
and L. amplexicaule which extracts ranged between 184 and 193 mg GAE/g [106,107].
The great variety of species of the Lamium genus present in Turkey [121] has meant that traditional
folk medicine made extensive use of Lamium herbs. On the contrary, L. album extracts possess
little antifungal activity as found by Chipeva et al. [122] who evaluate the antimicrobial activity of
L. album plants. The extracts, obtained from leaves and flowers, were harvested either in the wild or
from in vitro propagated plants. Four solvents (chloroform, methanol, ethanol, and water) and two
methods of extraction (Sоxhlet, thermostat) were used. The different combinations of extraction
solvent and extraction method led to results that also varied due to the origin of the plant, i.e., wild
or in vitro propagated. In conclusion, L. album extracts possessed a broad spectrum of antibacterial
activity with greater efficacy towards Gram-positive bacteria. However, as the extracts have not been
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chemically characterized it is impossible to determine which molecule may be more responsible for
such an antibacterial activity.
4.1.4. Anti-Inflammatory, Anti-Nociceptive Activity, and Pain Therapy

Arachidonic acid is the main precursor of eicosanoids, substances involved in the body’s
inflammatory response. In the presence of tissue damage, enzymes belonging to the phospholipase class
A2 release the arachidonic acid from the membrane phospholipids. From this, two different molecular
types can be obtained: the series 2 of prostaglandins and thromboxanes (from the cyclooxygenase
pathway) and the series of leukotrienes (from the lipoxygenase pathway). The synthesis of the series
2 of prostaglandins (PG2) and thromboxanes is mediated by the enzyme cyclooxygenase, which is
present in the human organism in the form of COX1 and COX2. The synthesis of leukotrienes is linked
to the activity of the enzyme 5-lipoxygenase. From these observations, pharmacological research on
molecules able to counteract the effects of arachidonic acid derivatives by inhibiting the enzymes
involved in the inflammatory cascade has started. To this end, enzymes extracted from both animal
and vegetable sources used as enzymatic model systems to evaluate the anti-inflammatory effect of
plant extracts and active ingredients are also used.
Aqueous extracts of L. album have been shown to inhibit lipoxygenase activity in vitro [105],
at relatively low concentrations of extract (IC50 ≈1.5 mg/mL). As these extracts have not been further
characterized, it is not possible to attribute this inhibition activity of lipoxygenase to a specific molecule.
It is very likely that this effect is due to more than one molecule and through several mechanisms.
In fact, many studies have established that free oxygen radicals are implicated in inflammatory
processes [123] and that phenolic compounds can block lipoxygenase activity, or they can function
as scavengers of free radicals which are released during the inflammatory cascade of arachidonic
acid [124]. Anti-inflammatory bioactivity of compounds was also proved with aqueous-methanolic
extract of L. album herb in human neutrophils as recently showed by Czerwinska [35]. This effect
appears to be due to the inhibition of the release of some inflammation mediators, such as the IL-8
and 3 TNF cytokines, by neutrophil granulocytes.
Rheumatoid arthritis is a multifactorial chronic, systemic and disabling inflammatory disease
with an undefined etiology, but probably of autoimmune origin. It mainly affects the symmetrical joints,
but also tendons, synovium, muscles, bags and other tissues of the organism. Rheumatoid arthritis
develops because, in a genetically predisposed subject, an environmental triggering event activates
an auto-immune response; there is thus an abnormal activation of the immune system, which affects
the joints causing chronic inflammation and consequent joint damage.
Rheumatoid arthritis can also be fought with plants [125] whose extracts can be used at least to
reduce the quantities of methotrexate, one of the elective drugs used in the treatment of this pathology.
As for the Lamium genus, the evidence that some species can provide benefits in the treatment of
rheumatoid arthritis is quite weak [126] and are mostly speculative and based on the antioxidant
and anti-inflammatory properties of L. album extracts.
About the inflammatory activity of L. album extracts, cannot be ignored a study in which
the leaves of this herb have been used as a placebo to test the anti-inflammatory activity of U. dioica
(stinging nettle) [127]. That study prompted from the observation that the sting of the common stinging
nettle has long been used for self-treatment of arthritic pain. A randomized controlled double-blind
crossover study in twenty-seven patients with osteoarthritic pain at the base of the thumb or index
finger was performed to ascertain if the daily application of nettle leaves in the painful area brought
relief. L. album leaves were chosen as a placebo since leaves are almost indistinguishable from stinging
nettle leaves. It was remarkable that after a week of treatment the score reduction with U. dioica leaves
was significantly greater than that with placebo (L. album leaves).
Pain therapy aims to recognize, evaluate and treat chronic pain in the most appropriate way.
There are several classes of drugs that can be used for the treatment of pain. The type of drug to be used
can vary depending on the origin, the nature and the intensity of the painful stimulus that is intended
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to be treated. Depending on the circumstances can be used: non-steroidal anti-inflammatory drugs

(e.g., ketoprofen, diclofenac, naproxen and nimesulide), opioid analgesics (e.g., codeine, tramadol,
buprenorphine, fentanyl, oxycodone, methadone, hydromorphone and morphine), antidepressants,
very useful in the treatment of neuropathic pain (e.g., amitriptyline, clomipramine, duloxetine);
anticonvulsants, also useful in the treatment of neuropathic pain (e.g., gabapentin, pregabalin);
local anesthetics (e.g., lidocaine). Unfortunately, the use of such drugs is often accompanied
by sometimes serious side effects, which may sometimes outweigh the benefits of the drug. For this
reason, over the past few years, general attention has shifted to non-pharmacological therapies
(e.g., radiotherapy, cryotherapy, thermotherapy, massages, physiotherapy, relaxation techniques).
Increasing attention has also concerned the active ingredients from medicinal plants. Although widely
used in traditional folk medicine, there are only a few studies investigating the potential analgesic
effects of the Lamium genus. Most of the studies have been carried out concerning different genera of
the Lamiaceae family, but very few are those concerning the Lamium genus [128].
4.1.5. Cytotoxicity and Cytoprotective Activity

Cytotoxicity is the measurement of how much a substance can damage or kill cells.
This measurement can be performed both in vitro and in vivo, and this difference is significant, since it
is one thing to measure the cytotoxic activity of a chemical agent on a cell culture in a homogeneous
medium (e.g., a fibroblast layer on a medium of culture) and the measurement of cell viability in vivo
is very different, where many biochemical and other factors are involved. Most of the cytotoxicity
studies of plant extracts have been conducted in vitro because it is the simplest method. In vitro
studies, in turn, offer several advantages; e.g., they are highly reproducible simplified systems, they
allow to analyze the cellular and molecular mechanisms of toxicity, the identification of early damages
and allow to contain costs and get rapid responses concerning animal experimentation. The main
criticism that can be found in systems for measuring cytotoxicity in vitro concerns the excessive
simplification of such methods concerning a multicellular organism. Nevertheless, in vitro tests are
widely used; the cytotoxicity can be carried out and evaluated with different essays. The most used is
the MTT assay, the SRB assay (with Sulforhodamine B), the TB assay (with Trypan blue). In the study
of cytotoxicity mechanism, the choice of the cellular model should respond to the need to study in
detail the organ-specific effects of certain compounds or mechanisms of action in specific cell types.
Unfortunately, most often the cellular model is chosen independently of the needs mentioned above;
for this reason, the results obtained in the different studies are hardly comparable.
As instance, Veleva et al. [129] studied the changes in the functional characteristics of tumor
and normal cells after treatment with extracts of white dead-nettle, by adhesion test, MTT
(3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyl tetrazolium bromide), transepithelial resistance (TER),
immunofluorescence staining and trypan blue exclusion test: extracts from L. album L. change TER
and actin filaments, and somehow may block cell mechanisms, leading to the polarization of MDCK II
cells (Madin-Darby canine kidney cells II).
From another work [105], aqueous extracts of the whole L. album plant have been shown to induce
cytotoxicity in the mouse tumor cell line, the B16 mouse melanoma cells at relatively low concentrations.
It is difficult to determine what may be the causes of this cytotoxicity although it could be related to
the content of phenolic compounds of the extract [105].
Polyphenols can act as antioxidants through various mechanisms, including hydrogen donating
reactions, metal chelation, inhibition of cytochrome P450 isoforms and up-regulation or protection of
antioxidant defenses (e.g., intracellular glutathione levels) [130]. In particular, the possible cytotoxicity
of these phenylpropanoids has long been debated. For instance, verbascoside has shown increased
chromosome aberrations and in vitro sister chromatid exchanges in human lymphocyte cultures [131].
However, the results may be due to instability and degradation of verbascoside in caffeic acid
and 3,4-dihydroxyphenyl ethanol [131]. The genotoxicity of verbascoside seems to have been ruled
out entirely by a recent study [132]. This study clearly demonstrates that diets rich in verbascoside
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do not give rise to any mutagenic activity, resulting in non-cytotoxic to animals and suggesting its
possible use in both animal and human diets.
In fact, products based on dried leaves and flowers of L. album are already on the market. L. album
is numbered among dermatological plants with anti-inflammatory activity, and this plant is also used
in wound healing [21]. Skin fibroblasts proliferation is considered as the most important initial stage of
tissue repair. Thus, Paduch et al. [133] analyzed the plant extracts activity on human skin fibroblasts
(HSF) proliferation and viability in order to add information on the effectiveness of these products.
The sensitivity of HSF cells in culture to methanol, ethyl acetate, and heptane extracts of Lamii albi flos
were investigated. Extracts with methanol, ethyl acetate and heptane of Lamii albi flos were prepared
by heating 20 g of plant material with 300 mL of the appropriate solvent for 5 h at a temperature
of 60 ◦ C at reflux. Each of these extracts was subsequently concentrated under reduced pressure
at 30 ◦ C up to a volume of 100 mL. The extracts thus obtained were used for the determination of
flavonoids, pentacyclic triterpenes, and iridoids. The triterpene component seemed to be responsible for
the absence of cytotoxicity of the heptane extracts even at high concentrations and, indeed, triterpenes
can exert stimulatory effects on the proliferative capacity of HSF cells.
When a model of chemical stress induced by potassium dichromate in human hepatoblastoma
HepG2 cells was used, ethanolic extracts of L. album showed a cytoprotective effect in vitro [40].
Purified extract counteracted ROS formation in oxidative stress conditions in tested cells.
The cytoprotective effect of 50 μg/mL L. album purified ethanolic extract seems related to the presence
of verbascoside, which exhibited the highest cytoprotective action from all the polyphenols identified
in the ethanolic extract.
It is interesting to reiterate that solvent extracts of the same species, L. album, can exert cytoprotective
or cytotoxic effects according to the methods of extraction, application of the extract and the tested cell
lines. Moscova-Doumanova et al. [134] investigated the effect of methanol, and chloroform extracts,
obtained from in vivo and in vitro cultivated plants of L. album, on the cell viability, adhesion, and cell
cycle of the type A549 human lung cell line. Different combinations of methanol and chloroform
extracts were tested. Preliminary results showed that both the extracts have a cytotoxic effect on lung
cancer cells. They caused a reduction in the adhesion properties of the cells with a stronger effect
by extracts from in vivo plants. After 48 h of incubation time, all extracts cause retention in the G2
phase while a mixture of them leads to the apoptosis. However, without characterization of the extracts,
it is not possible to hypothesize the molecular mechanism of the observed phenomena.
Other applications on corneal disease are described as follows. The cornea is the membrane that
covers the front of the eye, through which it is possible to glimpse the iris and the pupil. Transparent
and avascular, this structure represents the first ‘lens’ that the light encounters in its path to the brain.
The cornea is, in fact, an essential element of the ocular dioptric system: it allows the passage of
light rays towards the internal structures of the eye and helps to focus the images on the retina.
The cornea’s optical function is carried out thanks to its perfect transparency and the regularity of
the contact surface with the air. Therefore, any inflammation and damage to the corneal epithelial
should be quickly eliminated to maintain corneal transparency. In this context, the powerful antioxidant
and anti-inflammatory properties of L. album extracts above described could make, in perspective,
this medical herb a promising candidate in the formulation of natural remedies for topical use in
corneal diseases.
This prompted Paduch et al. [135] to evaluate the effect of L. album extract on human corneal
epithelial cells (10.014 pRSV-T cell line) cultured in vitro. In that study, the first goal achieved
was to ascertain the ethanol extract of L. album was non-toxic to human corneal epithelial cells
at concentrations up to 125 μg/mL. Ethanol extract contained polar compounds which contribute to
maintaining cells intact, or even, stimulate cellular mitochondrial metabolism as verified by MTT
assay. Besides, flavonoids and polyphenolic compounds, better represented in ethanol extracts, also
contributed to the reduction of inflammatory phenomena and ROS scavenging. Therefore, it cannot
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be excluded that soon, after further in vivo experiments, supplements for the treatment of mild eye
diseases based on extracts of L. album herb may be used.
At this point, it is worth mention L. galeobdolon L., commonly known as the ‘yellow archangel, that
has good potential as an ingredient for the preparation of functional foods. This species is a wildflower
widespread in Europe and has been introduced elsewhere as a garden plant. An ethnobotanical study
carried out on 49 edible wild plants traditionally harvested and consumed in a region of the Basque
Country, Northern Spain, described the recreational use by children of nectar sucked from the base
of L. galeobdolon flowers. Different benzoxazinoids (BXs), present as glucosides (Figure 3), have been
identified in the yellow archangel.
Figure 3. Benzoxazinone glucoside skeleton.
These compounds represent a class of indole-derived plant metabolites that work in defense
against numerous parasites and pathogens [136]. Many recent studies have reported antimicrobial,
anticancer, reproductive stimulatory effects, system stimulators central nervous system, and reduction
of appetite and weight of BXs derivatives and their derivatives [137].
4.1.6. Antityrosinase Activity

Tyrosinase (EC 1.14.18.1) is a ubiquitous enzyme containing two copper ions. The dinuclear copper
center of tyrosinase catalyzes the o-hydroxylation of monophenols, oxidation of catechols [138,139],
quinonization of dihydroxycoumarins [140], o-aminophenols and aromatic o-diamines [141]. Tyrosinase is
also one of the key enzymes in melanin biosynthesis. In animals, as the enzyme catalyzes the first two main
steps of the melanogenesis. Overproduction of melanin can result in various hyperpigmentation disorders
including melasma and age spots. Thus the discovery of new tyrosinase inhibitors has been since many
years the main goal of numerous investigations [142,143]. Many studies deal with chemical compounds
extracted from plants. Even though the presence of phenolic compounds in plants of the genus Lamium is
abundant, and potentially there are many phenolic compounds able to influence the activity of tyrosinase,
there are very few studies that describe such an influence on the tyrosinase enzyme.
Nugroho et al. [34] reported how two flavanol glycosides in methanol extracts of L. amplexicaule
showed in vitro inhibitory activity against the mushroom tyrosinase. The tyrosinase inhibition
mechanism and related kinetics have not been studied in detail. In the study, it is hypothesized that
the high group of hydroxyl groups present on the isolated flavonoid molecule may be responsible
for binding to the catalytic site of tyrosinase, and result in an inhibition phenomenon according to
previous studies [144,145].
The current study of Etsassala et al. [146] applied a fast screening method using a cyclic voltammetry
technique for evaluating anti-tyrosinase activity of twenty-five species of plants from the Lamiaceae
family: among these, those that showed a fast current inhibition rate at a minimum concentration
when compared to a kojic acid standard were classified as having the greatest anti-tyrosinase activity
such as Salvia chamelaeagnea, S. dolomitica, Plectranthus ecklonii, P. namaensis, and P. zuluensis.
4.2. In Vivo Studies

The observation of in vivo phenomena is often considered more relevant to factual reality than
in vitro reproductions, although the latter is equally useful because they allow analyzing a single
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phenomenon, isolating it from the context that could create a background noise that is too high to be
able to distinguish the phenomenon clearly.
Unfortunately, in vivo studies using plants of the Lamium genus are very few compared to
in vitro studies.
An in-depth study evaluated the anti-inflammatory and antinociceptive activities of various
extracts prepared with methanol, dichloromethane, n-butanol, and water from the aerial parts of some
species of the Lamium genus [25]. In this study, conducted on male Swiss albino mice, extracts of
L. eriocephalum subsp. eriocephalum, L. garganicum subsp. laevigatum, L. garganicum subsp. pulchrum
and L. purpureum var. purpureum were administered to the animals to alleviate inflammatory pain in
a model of ear edema and in carrageenan-induced and Prostaglandin E2 -induced hind paw edema.
The experimental data demonstrated that L. garganicum subsp. laevigatum and L. garganicum subsp.
pulchrum displayed remarkable anti-inflammatory and antinociceptive activities in mice at 200 mg/kg
dose without inducing any gastric damage.
The biological activities of the herbal extracts of the genus Lamium described up to now have
concerned organic solvent extracts or water extracts. These extracts allowed to obtain concentrated
solutions of phenolic compounds, flavonoids, iridoids, terpenes, steroidal derivatives, enriched
with the various components depending on the polarity of the extracting mixture.
An unusual extraction procedure has been described in a study of the biological activity of
an L. album oil extract [147] by using a biphasic solvent system consisting of 70% ethanol and sunflower
oil at a ratio of 1:1, after the maceration of above-ground of L. album in water. The oil extract
(OE) was then studied in models of hemolytic anemia (HA) induced with intramuscular (IM)
administration of phenylhydrazide chloride to white mongrel male rats. Three groups of animals were
formed: (i) intact animals, (ii) negative control group (with induced hemolytic anemia but untreated,
(iii) OE treated (with induced hemolytic anemia and treated). The results showed that the administration
of extract had anti-anemic effects since all blood parameters (e.g., number of erythrocytes, hemoglobin
levels, hematocrit, and red blood cell indices) were significantly better throughout the twelve weeks of
the experiment. The authors suggested that the anti-anemic effect of OE may be due to the antioxidant
action of chlorophyll preparation. This explanation is not fully convincing since the sunflower oil used
to obtain OE contains on average 59% of linoleic acid [148]. Recently, linoleic acid has been proved to
induce red blood cells and hemoglobin damage via an oxidative mechanism, eventually leading to
partial acute anemia [149]. In other words, if chlorophyll had effectively carried out anti-anemic action,
such an action would be even stronger as it would have contrasted not only the anemic effect due to
the administration of phenylhydrazide chloride, but also that induced by the linoleic acid contained in
OE. It would have been interesting to know the effect of the administration of the extractor oil only in
a further control group, but this eventuality has not been taken into consideration by the authors of
this study.
An interesting in vivo use of L. amplexicaule has been described in Punjab, a region of
southern Pakistan. In this region, the leaves of L. amplexicaule are administered orally to ruminants
affected by helminth infections, at a rate of 250 g at a time [150]. The duration of treatment is not
standardized and is not performed by veterinary staff but by local pastors. The efficacy of the treatment,
therefore, is not certified by veterinarians and needs further investigation carried out with criteria
and procedures of the veterinary medicine. However, the wide use of L. amplexicaule as an anthelmintic
in ruminants suggests that there are concrete possibilities for effective action by substances contained
in the plant.
4.3. Clinical Studies

Randomized controlled trial (RCT) is a study in which people are allocated at random (by chance
alone) to receive one of several clinical interventions. If the in vivo studies conducted with herbs
belonging to the Lamium genus are few, the RCTs studies are even less. Below we give an account of
the few RCTs we have come to know.
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Molecules 2019, 24, 1913
Atopic dermatitis (AD) is a pruritic, chronic and inflammatory skin disease, the onset of which
often coincides with the pediatric age. The ‘atopic’ appellation, attributed to dermatitis, underlines
the absence of a skin location. Shapira et al. [151] describe a brief report about forty-nine patients
who were recruited for a two weeks treatment to test the efficacy of tri-herbal combination on AD in
a randomized, placebo-controlled trial. L. album was one of these three herbs. The medication was taken
orally three times daily for two weeks. That study found that tri-herbal combination induced a highly
significant improvement in both objective and subjective parameters of AD. However, placebo treatment
induced equally positive results in all measured aspects, so it is not possible to attribute any therapeutic
effect to the medicament L. album containing.
5. Conclusions and Future Perspectives

Beside specific aspects of plant biochemistry, this review underlined the use of Lamiun plants
(some species) in the formulation of natural remedies for topical use, preparation of functional
foods, possible action as tyrosinase inhibitors, among others. Lamiaceae is one of the most extensive
and diverse plant families about their ethnomedicinal properties. Besides the great representativity of
species, chemotaxonomic markers are individuated as well as targeted biological functions.
Generally, the ancient science of phytotherapy study represents a great challenge for future research
in pharmacological and medical fields: from the exploitation of chemistry of plants to intervention studies
until clinical trials in humans. New frontiers should be based on an integrated and multidisciplinary
approach of research, in terms of health benefits and sustainable health applications and addressed
towards advanced technologies such as the nanotechnologies and chemometrics.
Author Contributions: All authors contributed equally to this work. B.S., A.R., M.F.M., F.S., A.S., L.A. and J.S.-R.
critically reviewed the manuscript. All the Authors read and approved the final manuscript.
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molecules
Review
Cucurbits Plants: A Key Emphasis to Its
Pharmacological Potential
Bahare Salehi 1 , Esra Capanoglu 2 , Nabil Adrar 3 , Gizem Catalkaya 2 , Shabnum Shaheen 4 ,
Mehwish Jaffer 4 , Lalit Giri 5 , Renu Suyal 5 , Arun K Jugran 6 , Daniela Calina 7 ,
Anca Oana Docea 8 , Senem Kamiloglu 9 , Dorota Kregiel 10 , Hubert Antolak 10 ,
Ewelina Pawlikowska 10 , Surjit Sen 11,12 , Krishnendu Acharya 11 , Zeliha Selamoglu 13 ,
Javad Sharifi-Rad 14, *, Miquel Martorell 15, *, Célia F. Rodrigues 16 , Farukh Sharopov 17 ,
Natália Martins 18,19, * and Raffaele Capasso 20, *
1 Student Research Committee, School of Medicine, Bam University of Medical Sciences, Bam 44340847, Iran;
bahar.salehi007@gmail.com
2 Faculty of Chemical & Metallurgical Engineering, Food Engineering Department, Istanbul Technical
University, 34469 Maslak, Turkey; capanogl@itu.edu.tr (E.C.); catalkaya.gizem@gmail.com (G.C.)
3 Laboratoire de Biotechnologie Végétale et d’Ethnobotanique, Faculté des Sciences de la Nature et de la Vie,
Université de Bejaia, Bejaia 06000, Algérie; n.adrar@hotmail.fr
4 Department of Plant Sciences, LCWU, Lahore 54000, Pakistan; shabnum_shaheen78@hotmail.com (S.S.);
meh.jaffer@gmail.com (M.J.)
5 G.B. Pant National Institute of Himalayan Environment & Sustainable Development Kosi-Katarmal,
Almora 263 643, India; lalitorchid@gmail.com (L.G.); renusuyal04@gmail.com (R.S.)
6 G.B. Pant National Institute of Himalayan Environment & Sustainable Development Garhwal Regional
Centre, Srinagar 246174, India; arunjugran@gbpihed.nic.in
7 Department of Clinical Pharmacy, University of Medicine and Pharmacy of Craiova, 200349 Craiova,
Romania; calinadaniela@gmail.com
8 Department of Toxicology, University of Medicine and Pharmacy of Craiova, 200349 Craiova, Romania;
daoana00@gmail.com
9 Mevsim Gida Sanayi ve Soguk Depo Ticaret A.S. (MVSM Foods), Turankoy, Kestel, 16540 Bursa, Turkey;
senemkamiloglu87@gmail.com
10 Institute of Fermentation Technology and Microbiology, Lodz University of Technology, Wolczanska 171/173,
90-924 Lodz, Poland; dorota.kregiel@p.lodz.pl (D.K.); hubert.antolak@p.lodz.pl (H.A.);
ewelina.pawlikowska@edu.p.lodz.pl (E.P.)
11 Molecular and Applied Mycology and Plant Pathology Laboratory, Department of Botany, University of
Calcutta, Kolkata 700019, India; surjitsen09@gmail.com (S.S.); krish_paper@yahoo.com (K.A.)
12 Department of Botany, Fakir Chand College, Diamond Harbour, West Bengal 743331, India
13 Department of Medical Biology, Faculty of Medicine, Nigde Ömer Halisdemir University, Campus,
51240 Nigde, Turkey; zselamoglu@ohu.edu.tr
14 Zabol Medicinal Plants Research Center, Zabol University of Medical Sciences, Zabol 61615-585, Iran
15 Department of Pharmacy, Faculty of Pharmacy, University of Concepcion, Concepcion 4070386, Chile
16 LEPABE, Department of Chemical Engineering, Faculty of Engineering, University of Porto, Rua Dr.
Roberto Frias, s/n, 4200-465 Porto, Portugal; c.fortunae@gmail.com
17 Department of Pharmaceutical Technology, Avicenna Tajik State Medical University, Rudaki 139,
Dushanbe 734003, Tajikistan; shfarukh@mail.ru
18 Faculty of Medicine, University of Porto, Alameda Prof. Hernâni Monteiro, 4200-319 Porto, Portugal
19 Institute for Research and Innovation in Health (i3S), University of Porto, 4200-135 Porto, Portugal
20 Department of Agricultural Sciences, University of Naples Federico II, 80055 Portici, Italy
* Correspondence: javad.sharifirad@gmail.com (J.S.-R.); martorellpons@gmail.com (M.M.);
ncmartins@med.up.pt (N.M.); rafcapas@unina.it (R.C.); Tel.: +98-21-88200104 (J.S.-R.);
+56-41-266-1671 (M.M.); +351-22-5512100 (N.M.); +39-081-678664 (R.C.)
Received: 2 April 2019; Accepted: 13 May 2019; Published: 14 May 2019
Abstract: Cucurbita genus has received a renowned interest in the last years. This plant species,
native to the Americas, has served worldwide folk medicine for treating gastrointestinal diseases

Molecules 2019, 24, 1854
and intestinal parasites, among other clinical conditions. These pharmacological effects have
been increasingly correlated with their nutritional and phytochemical composition. Among those
chemical constituents, carotenoids, tocopherols, phenols, terpenoids, saponins, sterols, fatty acids,
and functional carbohydrates and polysaccharides are those occurring in higher abundance. However,
more recently, a huge interest in a class of triterpenoids, cucurbitacins, has been stated, given its
renowned biological attributes. In this sense, the present review aims to provide a detailed overview
to the folk medicinal uses of Cucurbita plants, and even an in-depth insight on the latest advances
with regards to its antimicrobial, antioxidant and anticancer effects. A special emphasis was also
given to its clinical effectiveness in humans, specifically in blood glucose levels control in diabetic
patients and pharmacotherapeutic effects in low urinary tract diseases.
Keywords: cucurbits; pumpkin; squash; antimicrobial; antioxidant; anticancer; traditional medicine
1. Introduction
Cucurbita plants have been applied in different cultures as traditional medication. For instance,
Native Americans have used pumpkins for the treatment of intestinal worms and urinary ailments,
this therapeutic strategy being approved by American doctors in the early nineteenth century as
an anthelmintic for worms annihilating [1]. Seeds are used as an anthelmintic, to treat issues of
the urinary framework, high blood pressure, to prevent the development of kidney stones, to ease
prostate disorders and even to improve the erysipelas skin contamination [2]. In southeastern Europe,
Cucurbita pepo L. (pumpkin) seeds have been applied to heal irritable bladder and prostate enlargement.
Specifically, in Germany, the use of pumpkin seeds was adopted for application by the authority
for irritated bladder conditions and micturition problems of prostate enlargement, although the
monograph written in 1985 noted a lack of pharmacological studies that could confirm its effective
clinical effects. On the other hand, in the USA, the purchase of all such non-prescription medications
for the therapy of prostate enlargement was banned in 1990. In traditional Chinese medicine, Cucurbita
moschata Duchesne seeds were also applied for handling the parasitic diseases caused by worms,
while Mexican herbalists have used Cucurbita ficifolia Bouché as a remedy for reducing blood sugar
levels [3–7].
Indeed, increasing evidence has shown that cucurbits’ medicinal properties depend upon the
chemical compounds present, which produce a specific physiological effect in the human body [8–10].
Specifically, cucurbits fruits are found to be beneficial in blood cleansing, purification of toxic substances
and good for digestion, besides giving the required energy to improve human health. These species
possess a higher amount of proteins, phytosterols [11,12], unsaturated fatty acids [13,14], vitamins
(like carotenoids, tocopherols) [15] and microelements (e.g., zinc) [16]. Fruits, seeds and leaves
from various Cucurbita members (pumpkin, watermelon, melon, cucumber squash, gourds, etc.)
possess different pharmacological effects [17,18], such as antidiabetic [19–21], antiulcer, analgesic,
nephroprotective [22] and anticancer activities [18]. In this sense, this review provides a detailed
overview to the folk medicinal uses of Cucurbita plants, an in-depth insight on the latest advances
regarding its antimicrobial, antioxidant and anticancer effects, and lastly, a special emphasis to its
clinical effectiveness in humans, specifically in blood glucose levels control and low urinary tract
diseases (Figure 1).
464
Molecules 2019, 24, 1854
Figure 1. Most pronounced and investigated biological effects of Cucurbita spp.
2. Cucurbita Plants: A Brief Overview to Its Ethnopharmacological Uses

Recent ethnopharmacological studies showed that C. pepo and Cucurbita maxima Duchesne are
among the most commonly used Cucurbita plants for traditional medicinal treatments. As shown in
Table 1, many different components of Cucurbita plants are applied in diverse regions of the globe for
handling different diseases.
Table 1. Cucurbita plants traditionally applied in the cures of different diseases in diverse regions of
the world.
Scientific Name
Disease(s)
(Common Location Local Name Parts Used Administration References
Treatment
Name)
Digestive
Basque Country,
Kalabazea Seeds Oral (Intestinal worms, [23]
Iberian Peninsula
Constipation)
Mkuranga District,
Maboga Leaves Oral Anemia [24]
Tanzania
Polish people in Intestinal
Zapallo Seeds Oral [25]
Misiones, Argentina parasites
Vomiting blood,
Cucurbita Nelliyampathy hills Seeds Oral
Parangi Blood bile [26]
maxima of Kerala, India
Fruits Oral Urinal disorders
Duchesne Flowers Dermal Cataract
(Squash) Mauritius Giromon Seeds Oral Renal failure [27]
Fruits Dermal Wound
Agro Nocerino
Sarnese, Campania, Cocozza Seeds Oral Prostatitis [28]
Southern Italy
India UNSP Flowers UNSP Osteosarcoma [29]
Pakistani descent in
Blood pressure,
Copenhagen, Kadoo Fruits Oral [30]
constipation
Denmark
Ashanti region, Cancer (lung,
UNSP Leaves Oral [31]
Ghana head)
465
Molecules 2019, 24, 1854
Table 1. Cont.
Scientific Name
Disease(s)
(Common Location Local Name Parts Used Administration References
Treatment
Name)
Cucurbita pepo L. Ghimbi District,
Buqqee Seeds Oral Gonorrhea [32]
(Pumpkin) Southwest Ethiopia
Mexico, Central
Calabaza Whole plant Oral Obesity [33]
America, Caribbean
Ripollès district, Acne, Dermatitis,
Pyrenees, Catalonia, Carbassa Flowers Dermal Ecchymosis, Fever, [34]
Iberian Peninsula Toxicity, Wound
Fruits Dermal Infection
Nkonkobe
Municipality, Arthritis, Blood
Imithwane Leaves Oral [35]
Eastern Cape, booster
South Africa
West Bank, Palestine Kare’a Seeds Oral Breast cancer [36]
Delanta,
Gastritis,
Northwestern Wello, UNSP Fruits Oral [37]
Stomachache
Northern Ethiopia
Leaves Dermal Dandruff
Local Government
Area,
Okeugu Leaves Oral Malaria [38]
south-eastern
Nigeria
Cucurbita
galeottii Cogn. Mauritius Giraumon Seeds Oral Mucous discharge [39]
(Pumpkin)
UNSP: Unspecified.
In particular, the positive health effects of C. maxima seeds are well-documented [23,25–28]. Raw
C. maxima seeds are orally administered for the treatment of digestive disorders, such as intestinal
worms [23,25], constipation [23] and vomiting blood and blood bile [26] by the local people in the
Iberian Peninsula, Argentina and India, respectively. Also, sun-dried seeds of C. maxima are ingested
in Mauritius for the treatment of renal failure [27], whereas raw seeds are consumed to treat prostatitis
in the Agro Nocerino Sarnese in Campania, Southern Italy [28]. C. maxima seeds, fruits, flowers and
leaves are also used as traditional medicine [24,26,27,29–31], where the treatment of urinary disorders,
blood pressure regulation and prevention of constipation can be achieved with oral consumption of
C. maxima fruits, and the wound healing with dermal application [26,27,30]. In Mkuranga district in
Tanzania, C. maxima leaves are used for healing anemia [24], and in the Ashanti region in Ghana, this
plant part is orally consumed for lung and head cancer treatment [31]. Furthermore, in Mauritius,
C. maxima fruits are compressed externally on eyes against cataract [27], while in India the same petals
are used to treat osteosarcoma [29]. Nonetheless, and to the authors knowledge, much is needed to
support both the in vitro and in vivo biological effects of this plant, since most of the efforts has been
made towards its agro-industrial applications.
With regards to C. pepo seeds, they are mainly regarded as agro-industrial wastes, while in some
parts of the globe they are used raw, roasted or cooked, at a domestic scale [40]. Accordingly, in a study
carried out in Ghimbi District in Southwest Ethiopia [32], it was reported that oral administration of
cultivated seed of C. pepo is used as a gonorrhea therapy. Moreover, C. pepo seeds are also used as an
herbal remedy by breast cancer patients in West Bank in Palestine [36]. In another study, conducted in
Nkonkobe municipality in Eastern Cape, South Africa [35], it was indicated that arthritis and blood
booster are treated with orally taken C. pepo leaves. C. pepo leaves are also used for the treatment of
malaria and dandruff in the local government area in south-eastern Nigeria and Ghimbi District in
Southwest Ethiopia, respectively [37,38]. In the latter study, it was also pointed out that the fruits of
C. pepo are consumed to treat gastritis and stomachache [37]. Topical use of C. pepo fruit as an external
antiseptic was reported in Ripollès district, the Pyrenees in Catalonia and Iberian Peninsula, whereas
in the same location the flowers of this plant are used for antigenic, antidermatitic, antiecchymotic,
antiophidian, antipyretic and anti-toxic purposes [34]. C. pepo, as the whole plant, is also applied in the
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folk medicine of Mesoamerica and Caribbean for the therapy of fitness due to its pancreatic lipase
inhibition activity [33]. In addition to the above, the decoction prepared from the Cucurbita galeottii
Cogn. seeds is used against mucous discharge in Mauritius [39].
3. Cucurbita Plants Phytochemical Composition

Carotenoids are highly present in the fruit of these plants, namely α-carotene, β-carotene,
ζ-carotene, neoxanthin, violaxanthin, lutein, zeaxanthin, taraxanthin, luteoxanthin, auroxanthine,
neurosporene, flavoxanthin, 5,6,5 ,6 -diepoxy-β-carotene, phytofluene, α-cryptoxanthin and
β-cryptoxanthin [41]. Total carotenoid content varied between 234.21 μg/g to 404.98 μg/g in C. moschata
fruit [42], and 171.9 μg/g to 461.9 μg/g in C. pepo fruit [43]. There are also several publications on the
carotenoid content of a number of Cucurbita plants such as C. moschata, C. pepo [42] and C. maxima [44].
Edible Cucurbita seeds are also rich in vitamin E (49.49 μg/g to 92.59 μg/g), γ-tocopherol is more
abundant than α-tocopherol and the fruit contains less [45].
The study of Yang et al. [46] showed no flavonoid content (below detection limit: 0.05 mg/100 g)
in either the immature or the mature fruit of C. maxima. Only the shoots and buds showed positive
results. Sreeramulu and Raghunath [47] reported that average total phenolic content of C. maxima
was 46.43 mg gallic acid equivalent (GAE)/100 g. In another study, C. maxima was analyzed for its
flavonoid content and kaempferol was found to be the only flavonoid in this species at a concentration
of 371.0 mg/kg of dry weight [48].
C. pepo was found to be very weak in polyphenol content. Only 0.02 mg GAE/100 mg sample
has been found in its fresh fruit by Mongkolsilp et al. [49]. However, Iswaldi et al. [50] have reported
for the first time a list of 34 polyphenols including a variety of flavonoids in the fruit of C. pepo., in
addition to other unknown polar compounds. Besides, the flowers of C. pepo may contain considerable
amount of phenolic compounds. Andjelkovic et al. [51] studied the phenolic content of six pumpkin
(C. pepo) seed oils and identified the following compounds: Tyrosol, vanillic acid, vanillin, ferulic acid
and luteolin. Among them, tyrosol was the most abundant compound ranging from 1.6 mg/kg to
17.7 mg/kg.
Peričin et al. [52] studied the phenolic acid content of C. pepo seeds. p-Hydroxybenzoic acid was
found to be the prevailing phenolic acid, with 34.72%, 67.38% and 51.80% of the total phenolic acid
content in whole dehulled seed, kernels and hulls, respectively. Aside from p-hydroxybenzoic acid,
the most dominant phenolic compounds can be listed in a decreasing order of quantity as follows:
Caffeic, ferulic and vanillic acids in whole dehulled seeds. Trans-synapic and protocatechuic acids,
and p-hydroxybenzaldehyde were the abundant phenolic acids presented in the kernels of hulled
pumpkin variety; the hulls comprised p-hydroxybenzaldehyde, vanillic and protocatechuic acids with
considerable amounts. Table 2 presents the main phenolic compounds found in the Cucurbita spp. and
their structures.
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Table 2. Main chemical structures of the phenolic compounds found in the Cucurbita spp.*.
Compound Name Synonym(s) Empirical Formula Structure References
[52]
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Protocatechuic acid 3,4-Dihydroxybenzoic acid C7 H6 O4 http://phenol-explorer.eu/

compounds/412
[52]
p-Hydroxybenzoic acid 4-Hydroxybenzoic acid C7 H6 O3 http://phenol-explorer.eu/
compounds/418
468
[52]
p-Hydroxybenzaldehyde 4-Hydroxybenzaldehyde C7 H6 O2 http://phenol-explorer.eu/
compounds/725
[52]
4-Hydroxy-3-methoxybenzoic
Vanillic acid C8 H8 O4 http://phenol-explorer.eu/
acid; p-Vanillic acid
compounds/414
Table 2. Cont.
[52]
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3,4-Dihydroxycinnamic
Caffeic acid C9 H8 O4 http://phenol-explorer.eu/
acid
compounds/457
[52]
3,5-Dimethoxy-4-hydroxybenzoic
Syringic acid C9 H10 O5 http://phenol-explorer.eu/
acid
metabolites/420
469
[52]
trans-4-Hydroxycinnamic
trans-p-coumaric acid C9 H8 O3 http://phenol-explorer.eu/
acid
compounds/454
3-Methoxy-4-Hydroxycinnamic [52]
Ferulic acid acid; 3-Methylcaffeic acid; C10 H10 O4 http://phenol-explorer.eu/
Coniferic acid compounds/459
Table 2. Cont.
[52]
trans-4-Hydroxy-3,5-dimethoxy-cinnamic
trans-sinapic acid C11 H12 O5 http://phenol-explorer.eu/
acid; trans-Sinapinic acid
Molecules 2019, 24, 1854
compounds/464
p-HPEA;
4-(2-Hydroxyethyl)phenol; [52]
Tyrosol 2-(4-Hydroxyphenyl)ethanol; C8 H10 O2 http://phenol-explorer.eu/
2,4-Hydroxyphenyl-ethyl-alcohol; compounds/673
4-Hydroxyphenylethanol
4-Hydroxy-3-methoxy-benzoic
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aldehyde;
[52]
Methylprotocatechuic
Vanillin C8 H8 O3 http://phenol-explorer.eu/
aldehyde;
compounds/724
Vanillic aldehyde;
p-Vanillin
Table 2. Cont.
[52]
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Luteolin 5,7,3 ,4 -Tetrahydroxyflavone C15 H10 O6 http://phenol-explorer.eu/

compounds/229
OH
OH O [52]
Kaempferol 3,5,7,4 -Tetrahydroxyflavone C15 H10 O6 http://phenol-explorer.eu/
compounds/290
OH
OH O
* The data were collected from the Phenol-Explorer database, which is an online comprehensive database on polyphenol contents in foods, http://phenol-explorer.eu/ (Accessed on
09.12.2018).
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4. Looking at Cucurbita Plants Biological Activity
4.1. Antimicrobial Activity of Cucurbita Plants
4.1.1. In Vitro Studies

Pumpkin extracts showed a positive activity towards bacterial and fungal infections. They
were effective against gram-positive: Staphylococcus aureus, Bacillus subtilis, as well as gram-negative
bacterium: Escherichia coli, Proteus vulgaris, Pseudomonas aeruginosa, Salmonella spp. or Klebsiella spp.
Pumpkin extracts also showed antibacterial activity against water borne bacteria Vibrio cholerae as
well as intestinal flagellated parasite Giardia lamblia, often isolated from surface water. Other studies
documented that pumpkin extracts showed a wide range of antifungal activity against species from the
Fusarium, Trichoderma, Aspergillus, Verticillium, Phytophora, Botrytis, Candida and Saccharomyces genera
(Table 3). However, the mechanisms of antimicrobial activity of pumpkin extracts are still unknown,
although it seems to exist a synergistic action between all extracted bioactive substances. It is well
known that plant extracts exert biological effects more prominent than their isolated compounds. In
fact, recent evidence has established that, in whole matrices, the major compounds interact with those
in trace amounts to potentiate their own potential, to provide additional properties other than those
often recommended and even to help counterbalance the side effects of these isolated compounds. In
addition, and not the least important to emphasize, is that such minor compounds by strengthening
the biological effects of a specific bioactive also reduces the dose required to achieve a similar effect.
Table 3. Antimicrobial activity of Cucurbita spp. extracts evaluated in vitro.
Cucurbita spp./Plant Part Extract Microbial References

Cucurbita pepo L.
Water Escherichia coli [53]
fruits
Bacillus cereus
Bacillus subtilis
Escherichia coli
Enterobacter aerogenes
Enterobacter agglomerans
Salmonella enteritidis
Salmonella choleraesuis
Cucurbita pepo L.
Methanol Staphylococcus aureus [54]
fruits
Pseudomonas aeruginosa
Enterobacter faecalis
Klebsiella pneumoniae
Bacillus sphericus
Bacillus thruengenesis
Cryptococcus meningitis
Penicillium chrysogenum
Serratia marcescens
Escherichia coli
Phosphate buffered Streptococcus thermophilous
Cucurbita pepo L. [55]
saline (PBS) Fusarium oxysporium
Trichoderma reesei
Aspergillus niger
Cucurbita pepo L.
Ethanol extract Heligmosoides bakeri (worm) [56]
fruits
Staphylococcus aureus
Cucurbita pepo L. Escherichia coli
Water, methanol [57]
cortex Proteus mirabilis
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Table 3. Cont.
Cucurbita spp./Plant Part Extract Microbial References

Cucurbita pepo L. Methanol, Staphylococcus aureus
[58]
seeds, backpeel ethanol Salmonella typhi
Serratia sp.
Cucurbita pepo L. Escherichia coli
Ethanol [59]
leaves Klebsiella pneumoniae
Bacillus subtilis
Providencia stuartii
Cucurbita pepo L. Klebsiella pneumoniae
Methanol [60]
leaves Escherichia coli
Enterobacter aerogenes
Enterobacter cloacae
Bacillus subtilis
Cucurbita pepo L. Ethyl acetate, n-butanol,
Pseudomonas aeruginosa [61]
leaves water
Candida albicans
Rhodotorula rubra
Cucurbita moschata Duchesne
Methanol Trichoderma viride [62]
seeds oil extract
Penicillium chrysogenum
Rhizopus oligosporus
Aspergillus fumigatus
Aspergillus parasiticus
Aspergillus niger
Cucurbita moschata Duchesne
crude protein from rinds, seeds Acetone [62]
Bacillus subtilis
and pulp
Escherichia coli
Cucurbita maxima Duchesne Petroleum ether and
Giardia lamblia [63]
fruit methanol
Salmonella typhi,
Escherichia coli
Cucurbita maxima Duchesne Enterobacter faecalis,
Alcohol [64]
flowers Bacillus cereus
Curvularia lunata
Candida albicans
Escherichia coli
Cucurbita maxima Duchesne
Water Pseudomonas sp. [65]
peels
Vibrio cholerae
Entamoeba histolytica
Bacillus subtilis
Cucurbita maxima Duchesne
Ethanol Pseudomonas aeruginosa [66]
seeds
Escherichia coli
Candida albicans
Aspergillus niger
Bacillus subtilis
Staphylococcus werneri
Cucurbita maxima Duchesne Pseudomonas putida
Ethanol [67]
seeds Pseudomonas aeruginosa
Proteus mirabilis
Escherichia coli
Kleibsella pneumoniae
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4.1.2. In Vivo Studies

Not only pumpkin extracts, but also proteins and peptides isolated from Cucurbita spp. were
identified and characterized in terms of antimicrobial activity. Three pumpkin proteins inhibited
the growth of fungi Fusarium oxysporum, Verticillium dahliae and Saccharomyces cerevisiae [68]. The
antifungal peptide—cucurmoschin—isolated from black pumpkin seeds also demonstrated inhibitory
activity against mold growth: Botrytis cinerea, F. oxysporum and Mycosphaerella oxysporum [69]. The
ribosome-inactivating protein extracted from C. moschata showed an antimicrobial effect towards
phytopathogenic fungi Phytophora infestans as well as against bacteria Pseudomonas solanacearum and
Erwinia amylovora [70]. Additionally, PR-5 protein isolated from leaves of pumpkin, demonstrated
synergism with combination of nikkomycin, a chitin synthase inhibitor, towards to Candida albicans [71].
Protein Pr-1 isolated from pumpkin rind inhibited the growth of plant pathogenic fungi, namely
B. cinerea, F. oxysporum, F. solani and Rhizoctonia solani, as well as the opportunistic pathogenic yeast
C. albicans [72]. These results demonstrate that the proteins from pumpkin may be of importance to
clinical microbiology with a wide range of therapeutic applications (Table 4). As the most prominent
ones, and given the current evidence, namely regarding its ability to trigger fungal membranes
damages and to improve the plasma membranes permeability, they can be effectively used to combat
fungal infections and even to use in combination with current antifungal agents, both to improve its
effectiveness and even to reduce its side effects.
Table 4. In vitro antimicrobial activity of Cucurbita spp. proteins.
Cucurbita spp. Proteins Microbial References

Fusarium oxysporum,
Cucurbita maxima Duchesne seeds proteins Verticillium dahliae [68]
Saccharomyces cerevisiae
Phytophora infestans,
Cucurbita maxima Duchesne seeds protein RIP Erwinia amylovora, [70]
Pseudomonas solanacearum
Pumpkin leaves protein PR-5 Candida albicans [71]
Botrytis cinerea,
Fusarium oxysporum,
Pumpkin rind protein Pr-1 Fusarium solani [72]
Rhizoctonia solani,
Candida albicans
Botrytis cinerea,
Black pumpkin seeds protein cucurmoschin Fusarium oxysporum [69]
Mycosphaerella oxysporum
Pumpkin pulp, due to its antimicrobial properties, is widely used to relieve intestinal inflammation
or stomach disorders [73] (Table 5). Pumpkin and its seeds, in the traditional world medicine, are
often employed as an anti-helminthic remedy and for supportive therapy in functional diseases of
the bladder as well as in the case of digestion problems. The usage of an extract of C. pepo cortex
towards urinary tract infections may correspond to a new source of antibiotics against bacterial urinary
tract infections [57]. Other studies represented the importance of oil from seeds of a pumpkin as
a hopeful drug for treating wounds in vivo [74]. The researchers demonstrated a premium quality
of pumpkin oil with a high quantity of polyunsaturated fatty acids, tocopherols that were able to
perform efficient wound healing [74]. Morphometric evaluation and histological evidence in rats
showed healed biopsies from pumpkin oil and a complete re-epithelialization with a recurrence of skin
appendages and well re-growing collagen fibers out of cells inflammation.
Pumpkin-based foodstuff is well recognized as a source of anti-inflammatory remedies, which
can be useful in arthritis treatment [75]. Pumpkin seed oil notably prevent adjuvant-induced arthritis
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in rats, similar to indomethacin, a well-known anti-inflammatory substance. Its clinical applicability as

an antioxidant was also assessed on rheumatoid arthritis [76] and recently confirmed by Dixon [77].
Table 5. Antimicrobial property of Cucurbita spp. and its importance in vivo.
Antimicrobial Property References

Cucurbita pepo L. seeds Wounds healing [74]
Anthelmintic, treatment of bladder
Pumpkin seeds [78]
functional disorders
Pumpkin seed oil Arthritis prevention [75]
Control of gastrointestinal
Pumpkin fruits [56]
nematode infections
Effective treatment of bacterial
Cucurbita pepo L. cortex extract [57]
urinary tract infections
4.2. Anticancer Activities of Cucurbita Plants

Cucurbitacins are a unique gathering of triterpenoids that containing a cucurbitane basic structure
skeleton, and an extensive variety of natural exercises is related with their substance basic multifaceted
nature. Cucurbitacins have been accounted for the most secondary metabolites in the Cucurbitaceae.
They have the biogenetically 10α-cucurbit-5-ene [19(10→19β) abeo-10α-lanostane skeleton, which
is related to their cytotoxicity. A few reports credited in vitro and in vivo cytotoxic exercises to the
cucurbitacins [79,80]. Jayaprakasam et al. [81] exhibited the anticancer properties of cucurbitacins B, D,
E and I, confined from the products of Cucurbita andreana Naudin, towards colon, bosom, lung and focal
sensory system disease cell lines. Cucurbitacin B is the most considered and keeping in mind that few
investigations exhibited its viability in various models of malignant growth, incorporating into vivo
tumor xenografts [82–85], there is a contradiction concerning the components hidden its anticancer
action. Concealment of the oncogene STAT 3 (signal transducer and activator of transcription 3) has all
the earmarks of being identified with tumor restraint [86], which does not prohibit elective. A malignant
growth is in charge of 12% of the global impermanence. Medications incorporate chemotherapy
medical procedure and radiation therapy. Be that as it may, chemotherapy endures constraints of
medication obstruction, poisonous quality, reactions and lacking explicitness toward tumor cells [87].
Consequently, there is a solid enthusiasm for the utilization of plants as a promising wellspring of
increasingly productive anticancer medications.
In Vitro Anticancer/Antitumor Effects

Presently over forty cucurbitacins have been isolated from the Cucurbitaceae family and different
types of the herbs. The apoptotic impacts of cucurbitacins are expected of their capacity to change the
qualities, transcriptional exercises using atomic components and mitochondrial trans-film potential
and their ability to initiate or hinder ace or hostile to apoptotic proteins. Cucurbitacins are specific
inhibitors of the JAK/STAT pathways; likewise, different instruments are involved in their apoptotic
impacts, for example, PARP cleavage, MAPK pathway, articulation of dynamic caspase-3, diminished
JAK3 and pSTAT3 levels and also diminishes in different downstream STAT3 targets, for example,
Bcl-2, Mcl-1, cyclin D3 and BclxL, which are all embroiled in the cell cycle control [88].
C. pepo alcohol extract demonstrated cytotoxicity towards HepG2 and CT26 cell lines with IC50
values 132.6 μg/mL and 167.2 μg/mL, individually. The ethanol extract of C. pepo was displayed a huge
portion subordinate inhibitory impact towards HeLa cell development [89].
Cucurbita glycosides A and B isolated from C. pepo ethanol extract demonstrated cytotoxic action
in vitro towards HeLa cells with IC50 17.2 μg/mL and 28.5 μg/mL, individually [90]. Cucurbitacin B
and E isolated from C. pepo cv dayangua demonstrated an antiproliferative in MCF-7, HCT-116, SF-268,
A549 and NCI-H460 cell lines [81].
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The antiproliferative impact of 23,24-dihydrocucurbitacin F, on human PCa cells may jump out at
the enlistment of the cofilin-actin pole development and actin collection delivering to cell cytokinesis
disappointment, hindered cell development cycle capture at G2/M stage and apoptosis [91]. Likewise,
23,24-dihydrocucurbitacin F has an inhibitory effect on Epstein-Barr infection actuation initiated by
the tumor advertiser, 12-O-tetradecanoyl-phorbol-13-acetic acid derivation and furthermore, shows
altogether hostile to tumor-advancement action on mouse skin tumor advancement [88].
Treatment with cucurbitacins B and E resulted in apoptosis and cell cycle capture of MDAMB-231
and MCF-7 breast cancer cell lines. Additionally, they tweaked the outflow of proteins associated
with cell-cycle control in both of the estrogen-autonomous (MDA-MB-231) and estrogen-subordinate
(MCF-7) in human bosom malignant growth cell lines. Growth hindrance and cytotoxic impact of
cucurbitacin B on bosom disease cell lines SKBR-3 and MCF-7 were credited to G2/M stage capture
and apoptosis. Cucurbitacin B treatment repressed Cyclin D1, c-Myc and β-catenin articulation levels,
translocation to the core of β-catenin and galectin-3. Western smear investigation demonstrated
expanded PARP cleavage proposing actuated caspase action and diminished mitogenic Wnt-related
flagging particles galectin-3, β-catenin, c-Myc and cyclin D1 with changes in phosphorylated GSK-3β
levels [92].
Cucurbitacin E caused disturbance of the cytoskeleton structure of actin and vimentin inhibiting
the multiplication of prostate disease cells. Cucurbitacins also additionally hindered the expansion of
endothelial cells joined by an interruption of the F-actin and tubulin microfilaments cytoskeleton, typical
mitogen-prompted T-lymphocytes and lessened cell motility recommend an enemy of angiogenesis
and hostile to the metastasis job for cucurbitacins. It is also fit for instigating and keeping up high
multiplication rates in lymphocytes [93].
The literature has indicated that secondary metabolites of C. pepo have the potential anticancer
activity, which represents great interest for the development of new chemotherapeutic agents for
preventive growth of the tumor.
5. Clinical Effectiveness of Cucurbita Plants in Humans
5.1. Control of Blood Glucose Level in Diabetic Patients

Diabetes mellitus is a chronic disease characterized by changes in saccharide, lipid and protein
metabolism resulting from a deficiency in insulin secretion from the pancreas, insulin resistance or
both. The main clinical symptom is represented by increased blood sugar levels (hyperglycemia) that
uncontrolled lead in time to a wide spectrum of complications [94]. Natural therapeutic alternatives to
allopathic treatment always attracted the researchers to the intention of finding new drugs with fewer
side effects [95–100]. Thus, the hypoglycemic effect of Cucurbita species (Table 6) is known and used
for long traditional medicine in many countries, like China, India, Iran and Mexico [101–104].
Table 6. Pharmacotherapeutic effects of Cucurbita plants in human clinical studies.
Part of the Plant with Active Compounds Cucurbita spp. References

Cucurbita maxima
Polysaccharides from pulp fruit Duchesne [105,106]
Hypoglycemic Cucurbita ficifolia Bouché
Non-pectines polysaccharides and pectines
Cucurbita ficifolia Bouché [107–109]
from pulp; proteins and oil from seeds
Reduced clinical symptoms of Δ5-Δ7-Δ8-Phytosterols, unsaturated fatty
Cucurbita pepo L. [110–113]
benign prostatic hyperplasia acids from seeds extracts, lignans
Positive effects in stress urinary
Oil, sterols from seeds Cucurbita pepo L. [114,115]
incontinence in female
Cucurbita pepo L. [116]
Improved urinary symptoms in Seeds oil (sterols) blended with soy germ
human overactive bladder extract (phenols, isoflavones) Cucurbita maxima
[117]
Duchesne
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Mahmoodpoor et al. [106] in a recent study performed on patients with severe diabetes from the
Intensive Care Unit showed the hypoglycemic effect of C. maxima pulp. The subjects received five
grams of C. maxima powder per 12 h for three consecutive days. After the treatment, it was observed
a decrease of serum glucose levels from 214.9 mg/dL to 214.9 mg/dL associated with a reduction of
insulin doses from 48.05 IU to 39.5 IU [106]. C. ficifolia also showed a good hypoglycemic effect when
the extract was administered in doses of 4 mL/kg to patients with type 2 diabetes and moderately
elevated blood glucose level [105]. Five hours after administration, the mean of serum glucose level
decreased from 217.2 mg/dL to 150.8 mg/dL [105].
The most important hypoglycemic active substances in pumpkin are non-pectines polysaccharides
and pectines from pulp, proteins and oil obtained from seeds [107–109]. Alenazi et al. [118] reported a
clinical case of a 12-year-old Asian diabetic patient that ate every day for four months 200 g of pumpkin.
After two months of daily pumpkin consumption, a decrease of glycosylated hemoglobin (HbA1C)
from 10.8% to 8.5% was observed [118]. The same positive hypoglycemic effect was also revealed in
another study by Jain et al. [119]. Fourteen patients diagnosed with type 2 diabetes received C. ficifolia
juice for 40 days, and glycosylated hemoglobin decreased with 22.5% [119]. Shi et al. [120] investigated
the antidiabetic activity of pumpkin carbohydrate granules in patients with type 2 diabetes compared
to a control placebo group. After one month of treatment, both blood and urine glucose levels were
significantly decreased compared with the placebo control group [120]. The results of a randomized,
placebo-controlled trial conducted showed that a rich diet in pumpkin (C. maxima) seeds significantly
reduced postprandial blood glucose of adults with normal glycaemia [121]. This study included 25
normoglycemic adults who consumed daily 65 g of pumpkin seeds [121]. Possible mechanisms of
antihyperglycemic action of Cucurbita species are not fully understood but several studies investigated
this subject in the last decades. Zhang et al. [122] demonstrated that C. moschata heteropolysaccharides
regenerate pancreatic islets by stimulating proliferation of pancreatic β-cells. Quanhong et al. [123]
showed that polysaccharides bounded by protein (polysaccharide 41.21% and protein 10.13%) increase
glucose tolerance level and reduce hyperglycemia. In the light of these results, supplements with
natural extracts from Cucurbita plants can be considered as alternative hypoglycemic products and
further multicenter randomized studies can confirm these results.
5.2. Pharmacotherapeutic Effects in Low Urinary Tract Diseases

Benign prostatic hyperplasia (BPH) represents an increase in the volume of the prostate under the
influence of androgenic hormones, and 70% of aging men suffer from this condition. Since clinical
evolution of urinary signs is slow, prevention of BPH is useful, phytotherapy being an alternative
way [124]. For example, oil obtained from C. pepo seeds is traditionally used to treat urinary symptoms
in BPH as the daily frequency of urination, nycturia, time of the bladder emptying and residual
volume [110,111]. The main mechanism through which these effects are obtained is represented by the
inhibition of 5-α-reductase. This enzyme is required to convert testosterone to dihydrotestosterone,
which has a higher affinity than testosterone for androgen receptors. As a result, protein synthesis
increases the volume of the prostate implicitly [113].
In a multicenter clinical trial, thousands of patients diagnosed with BPH were treated with capsules
containing 500 mg of C. pepo seeds extracts. Their quality of life has been significantly improved by
reducing the urinary symptoms of BPH [125].
Other modern studies have shown pharmacotherapeutic synergism in BPH when C. Pepo is
administrated simultaneously with other plants. Thus, the combination with Serenoa repens (W. Bartram)
Small significantly improved the urinary symptoms of BPH and decreased blood dihydrotestosterone
levels [111]. Hong et al. [112] obtained similar results on urinary symptoms in Korean men with BPH
treated with 320 mg of C. pepo plus 320 mg of S. repens. They also observed a decrease in prostatic
antigen levels after the treatment, but without changes in prostate volume [112]. In a randomized
Phase II clinical trial carried out by Coulson et al. [126] the efficacy of the ProstateEZE Max formulation
obtained from a mixture of plants traditionally used in treating BPH was evaluated. ProstateEZE is a
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natural formulation containing C. pepo, S. repens, Pygeum africanum Hook.f., E. parviflorum Schreb. and
lycopene. Fifty-seven male patients diagnosed with BPH were selected in the study. Thirty-two of
them received a capsule of ProstateEZE Max daily for three consecutive months, and 25 patients were
treated with a placebo. In patients treated with Prostate EZE, the clinical symptoms of BPH decreased
by 35.9% compared with only 8.3% for the placebo. The frequency of nocturnal urination was reduced
with 39.3% in subjects treated for three months with ProstateEZE compared to the placebo group [126].
Due to these beneficial therapeutic effects of Cucurbita plants in BPH, the European Medicines
Agency approved the use of C. pepo for both BPH and other bladder disorders, such as urinary stress
incontinence in women [127].
Urinary stress incontinence occurs when pelvic muscles that support the bladder and the sphincter
muscle, which controls the urinary flow, are weakened. This disorder is associated with aging in women.
The main symptom is urinary incontinence [115]. The seeds extract of C. pepo have a therapeutic effect
in this condition through a double mechanism. Directly by relaxing the bladder muscles leading to
a decrease in nycturia and indirectly through a hormonal mechanism by inhibiting 5-α reductase.
This inhibition determines the anabolic effects that strengthen the bladder sphincter muscles [115,127].
The main chemical compounds in the pumpkin seeds that explain these effects are sterols (sitosterol,
spinasterol) and fatty oil, which contain oleic, linoleic, palmitic acids and tocopherol) [114]. Gažová et
al. [128] demonstrated these effects in a study of 86 women with urinary incontinence stress who were
treated for twelve weeks with the preparation of a plant mix: C. pepo, Equisetum arvense L. and Linum
usitatissimum L. Episodes of urinary incontinence during the day were reduced to 35% and nocturnal
urinary frequency to 54% [128].
Overactive bladder syndrome (OAB) is characterized by the frequent urge to urinate during the
day and night, followed by an involuntary loss of urine [116]. A human clinical trial conducted by
Shim et al. (2014) investigated the efficacy and utility of Cucuflavone (tablets with a mixture of plant
extracts 87.5% C. pepo seeds and 12.5% soy) in reducing OAB symptoms [116]. The active compounds
of Cucuflavone are phenols (pyrogallol) and isoflavones (genistein, daidzin). One hundred and twenty
patients were included in the study, divided into two groups: The Cucuflavone group and the placebo
group. Patients from Cucuflavone group received two tablets twice a day (a total of 875 mg of C. pepo
seed extract and 125 mg of soy extract daily) for twelve weeks. The final results of the investigation
showed that urinary incontinence, the frequency of daily and nocturnal urination was statistically
significantly reduced compared to the initial parameters [116]. In a recent investigation, Nishimura et
al. obtained similar results. They confirmed the efficacy of C. maxima seeds oil on urinary disorders in
OAB. Forty-five subjects with OAB were included and treated daily with 10 g of C. maxima seed oil for
twelve weeks. At the end of the investigation, the frequency of average daily urination was reduced
from 10.96 to 8.00 [117].
6. Conclusions and Future Perspectives

In short, the use of Cucurbita species and their active constituents in various clinical and
pharmacological studies revealed the presence of multiple, effective and useful compounds, which
provide the opportunity for further production of antidiabetic, analgesic, anti-inflammatory and
cardioprotective drugs and foods. Indeed, the use of Cucurbita plants in the treatment of several
diseases, including gastrointestinal disorders, intestinal parasites and hypertension, dates from a long
time ago. The antimicrobial and antioxidant properties of these species have triggered a huge interest
for multiple applications. First of all, free radicals are generated through various metabolic activities
in the body, ultimately resulting in various deleterious diseases [99]. These diseases can be treated
by supplementation of cucurbits as activities of some cucurbits are comparable with commercially
available antibiotics. The present review markedly highlights that Cucurbita species have preventive
and therapeutic abilities for treatment of different diseases. The presence of active phytochemicals in
Cucurbita species further strengthens the opportunity for their application as an upcoming anticancer,
antidiabetic, analgesic, anti-inflammatory and cardioprotective drugs, as well as foods. Finally, and
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Molecules 2019, 24, 1854
not the least important, the application of Cucurbitaceae members in public health, as nutraceuticals is
associated with great availability and a good safety profile.
Author Contributions: All authors contributed equally to this work. J.S.-R., M.M., N.M., and R.C., critically
reviewed the manuscript. All the authors read and approved the final manuscript.
Acknowledgments: This work was supported by CONICYT PIA/APOYO CCTE AFB170007. N. Martins
would like to thank the Portuguese Foundation for Science and Technology (FCT-Portugal) for the
Strategic project ref. UID/BIM/04293/2013 and “NORTE2020—Northern Regional Operational Program”
(NORTE-01-0145-FEDER-000012) and C.F.R. for the project UID/EQU/00511/2019—Laboratory for Process
Engineering, Environment, Biotechnology, and Energy—LEPABE funded by national funds through
FCT/MCTES (PIDDAC).
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485
molecules
Review
Fragaria Genus: Chemical Composition and
Biological Activities
Radu Claudiu Fierascu 1,2 , Georgeta Temocico 1, *, Irina Fierascu 1,2, *, Alina Ortan 1 and
Narcisa Elena Babeanu 1
1 University of Agronomic Sciences and Veterinary Medicine of Bucharest, 59 Mărăs, ti Blvd., 011464 Bucharest,
Romania; radu_claudiu_fierascu@yahoo.com (R.C.F.); alina_ortan@hotmail.com (A.O.);
narcisa.babeanu@gmail.com (N.E.B.)
2 Emerging Nanotechnologies Group, National Institute for Research & Development in Chemistry and
Petrochemistry—ICECHIM Bucharest, 202 Spl. Independentei, 060021 Bucharest, Romania
* Correspondence: gtemocico@gmail.com (G.T.); dumitriu.irina@yahoo.com (I.F.)
Academic Editors: Raffaele Capasso, Lorenzo Di Cesare Mannelli and Nicola Volpi
Abstract: The strawberries represent in our days one of the main fresh fruits consumed globally,
inevitably leading to large amounts of by-products and wastes. Usually appreciated because of their
specific flavor, the strawberries also possess biological properties, including antioxidant, antimicrobial,
or anti-inflammatory effects. In spite of the wide spread of the Fragaria genus, few species represent
the subject of the last decade scientific research. The main components identified in the Fragaria
species are presented, as well as several biological properties, as emerging from the scientific papers
published in the last decade.
Keywords: Fragaria genus; chemical composition; biological properties
1. Introduction
The production of different fruits all around the world exceeds millions of tons, depending on
geographical zones, consumption, and growing traditions, inevitably leading to large amounts of
by-products and wastes. Fragaria genus (Rosaceae), commonly known as strawberry, represents one of
the most important food plants all over the world, with a double global production compared with all
other fruit berries combined [1]. Their widespread use, primarily because of their flavor, can also lead
to considerable benefits to human health. Among other characteristics, nonvisual properties like taste,
nutritional values, or aroma make these fruits to be in the top of consumer preferences [2].
Known and consumed for thousands of years, Fragaria species are encountered throughout the
northern hemisphere, as well as in some areas of South America [1]. Several authors present the
historical consumption of strawberries in pre-Columbian sites, Picunche and Mapuche people (Chile),
Romans or ancient China. [1,3–5] The exact number of accepted species of the genus remains a subject
of debate, ranging from 22 [6] to 16 [7]. In addition, there are many hybrids and cultivars representing
ploidy levels ranging from diploid (2n = 2x = 14) to decaploid (2n = 10x = 70), influencing the size
of the fruits. Most of the research regarding the genus can be traced to the extraordinary work
of Antoine Nicolas Duchesne, that offered botanical description, details on the history, cultivation,
sex, and polyploidy of different species [5]. Generally speaking, all Fragaria species share some
common characteristics: are low-growing perennials, with usually evergreen and trifoliolate leaves,
insect-pollinated, with white actinomorphic flowers (usually 5-petalled). The main difference between
species is represented by the animal-dispersed accessory aggregate fruits, in terms of color, shape,
and achene (the 1-seeded simple fruits) and calyx positions at maturity. From the different composition
and other characteristics of those fruits, also arise the potential commercial value of the species. Many

Molecules 2020, 25, 498
cultivars are perennials that vary in their photoperiod needs, leading to varying harvesting times
(June-bearers, ever-bearers, day-neutral) [8].
Among the 247 varieties known and listed, only few present commercial interest:
Fragaria x ananassa Duchesne (octoploid hybrid-containing 56 chromosomes, known as garden
strawberry, native to northern America, cultivated all over the world), and, to a lesser extent,
Fragaria vesca L. (diploid species, known as wild strawberry, native to Northern hemisphere) and
Fragaria chiloensis (L.) Mill. (octoploid species, known as Chilean strawberry, native to northern, pacific
and southern America) [1].
As previously mentioned, the strawberries represent one of the most important fruit plants.
Their production reached 9.22 million tones (world level) in 2017, the major producers being China
(40.3% of total world production), United States (15.7%), Mexico (7.14%), Egypt (4.42%), Turkey
(4.34%), Spain (3.9%), Republic of Korea (2.28%), Poland, Russian Federation, Morocco, Japan,
Germany, United Kingdom, and Italy (between 1 and 2%) [9]. This mass-production invariably
leads to large amounts of wastes, that can be further exploited in multiple areas, including medicine,
cosmetics, and food industry [10]. The wastes are generated throughout the growth cycle (maturation,
multiplication, and expansion), while the large-scale methods of cultivation (e.g., in fields, plastic
tunnels., etc.) leads to large amounts of wastes from leaves, stolons, fruits, etc.
The present review paper aims to present the identified components in different species of the
Fragaria genus, as well as their potential biological activities, as emerging from the scientific papers
published in the past decade. The selection of the articles to be included in the present review was
performed using the well-known data-bases (Scopus, Web of Science, ScienceDirect, and PubMed),
using specific keywords (“composition”, “therapeutic”, “cytotoxic”, “anti*”—returning results for
“antimicrobial”, “antifungal”, “anti-inflammatory”, etc.). The validation of the articles was performed
manually (by reading the entire article) and in the present review were inserted only articles with
significant contribution to the field of research.
2. Composition of Fragaria L. Genus

Giampieri et al. [11] reviewed the composition of the strawberry (Fragaria x ananassa),
while Morales-Quintana and Ramos [12] reviewed the composition and potential applications of
the Chilean strawberry (Fragaria chiloensis (L.) Mill.), while the functional properties of the berries,
in general, and of the strawberries, in particular, were reviewed by Jimenez-Garcia et al. [13]. As
resulting from various literature studies [11–15], the general composition of the strawberries (in terms
of major components) can be summarized in Table 1 (with a general image provided in Figure 1).
Table 1. Major (common) components in Fragaria L. aggregate fruits (adapted from [11–15]).
Class Compound Ref.

Pelargonidin 3-glucoside, cyanidin 3-glucoside, cyanidin 3-rutinoside,
Anthocyanins pelargonidin 3-galactoside, pelargonidin 3-rutinoside, pelargonidin [11–13]
3-arabinoside, pelargonidin 3-malylglucoside
Flavonols Quercetin, kaempferol, fisetin, their glucuronides, and glycosides [11–13,16]
Flavanols Catechin, proanthocyanidin B1, proanthocyanidin trimer, proanthocyanidin B3 [11]
Sanguiin H-6, ellagitannin, ellagic acid, lambertianin C,
Ellagitannins [11]
galloylbis-hexahydroxydiphenoyl-glucose
Phenolic acids 4-coumaric acid, p-hydroxybenzoic acid, ferulic acid, vanillic acid, sinapic acid [15]
Vitamins Vitamin C, vitamin B9 [14]
Minerals Mn, K, mg, P, Ca [11]
Others Sugars (glucose, fructose, and sucrose), fibers [11]
488
Molecules 2020, 25, 498
Figure 1. Main components Fragaria species identified according literature data.
The presented composition varies with a series of factors, including the value of the cultivar,
seasonal variation, and the degree of fruit ripeness. In the reviewed time period, several studies
presented the evaluation of species belonging to Fragaria genus. Their main findings are presented in
Table 2, while relevant studies are presented in the following paragraphs.
As the major bioactive constituents of Fragaria fruits are represented by anthocyanins, most of the
literature studies are focused on their identification/quantification. Cerezo et al. [17] identified multiple
anthocyanins and other phenolic compounds present in Fragaria x ananassa (Camarosa variety) puree,
among which three of them (delphinidin-3-glucoside, peonidin-3-glucoside, cyanidin-3-galactoside)
were proposed for the first time in the literature. A correlation between the cultivation system
(classic/organic) and the composition of the strawberries was established by Crecente-Campo et al. [18].
The authors observed higher values of the identified anthocyanins and ascorbic acid (accompanied by
a darker, redder color and a superior nutritional value) in the case of organic cultivated strawberries.
The differences in terms of volatile esters composition between wild and cultivated strawberries were
presented by Dong et al. [19]. The authors suggested that the composition in volatile esters (dominated
in the case of F. vesca by acetate esters, and by ethyl hexanoate, in the case of Fragaria x ananassa) is the key
factor in the differences in terms of aroma patterns between the two species. Yang et al. [20] evaluated
the phenolic compounds present in Fragaria x ananassa Duch. cv. Falandi fruits, identifying flavone
glucuronides, lignan glycosides, and other compounds. The authors also isolated, for the first time in
the literature, three phenolic glucosides (2,3 -epoxy-4-(butan-2-one-3-yl)-5,7,40-trihydroxy flavane
3-glucoside, kaempferol 3-(6-butylglucuronide), benzyl 2-glucosyl-6-rhamnosylbenzoate), offering their
spectroscopic characteristics in support of the suggested structures. Roy et al. [21] studied comparatively
the polyphenolic composition of Fragaria x ananassa and Fragaria vesca mutant fruits (white-colored)
with regular fruits. Their study revealed the presence of 22 compounds belonging to different
groups, as anthocyanins (cyanidin-3-glucoside, pelargonidin-3-glucoside, peonidin-3-glucoside,
pelargonidin malonyl-glucoside, peonidin-malonyl-glucoside, and cyanidin-malonyl-glucoside)
flavonols (quercetin, quercetin-3-glucoside, kaempferol-3-glucoside, kaempferol-acetyl glucoside,
and kaempferol-coumaroyl hexoside), flavan-3-ols (proanthocyanidin dimers, catechin and epicatechin),
489
Molecules 2020, 25, 498
hydroxycinnamic acids (caffeic acid, chlorogenic acid, and p-coumaroyl hexose), and ellagic
acid-derived compounds (ellagic acid deoxyhexoside, methyl ellagic acid pentoside, and dimethyl
ellagic acid pentoside). The major difference recorded between the white and red fruits (for both
species) was the anthocyanins content. The white fruits had much lower total anthocyanin levels
(0.11–0.35 for F. vesca and 0.89 mg/100 g fresh fruits for Fragaria x ananassa), compared with the red
fruits (8.36, respectively 15.20 mg/100 g fresh fruits). Another major difference was recorded in terms
of free ellagic acid and its derivatives (higher for F. vesca white fruits compared with the red fruits).
Although the study was focused on the identification of specific mutations in different white-fruited
genotypes, the article offers a very good insight on the variation of the phenolic composition, both with
species and genotype. This could be further useful for the selection of the phenotype for separation of
bioactive compounds for targeted applications. Two different compounds (a protease enzyme with
molecular weight 65.8 kDa, stable at high temperatures and over a wide pH range, with specificity
toward hemoglobin, respectively a cysteine protease inhibitor cystatin FchCYS1) were isolated in 2018
from Fragaria x ananassa [22], respectively Fragaria chiloensis [23], while a new ellagitannin (a galloylated
derivative of agrimoniin, with molecular weight 2038) named fragariin A was isolated in 2019 by
Karlińska et al. from strawberry fruits (Fragaria x ananassa Duch.) [24]. The distribution of the active
compounds found in Fragaria x ananassa Duch. fruits was elucidated in 2019 by Nizioł et al. [25],
by applying mass spectrometry imaging with 109 Ag nanoparticle enhanced target. The authors studied
thirty-two known metabolites and reached the conclusion that γ-aminobutyric acid, quinic acid,
vitamin C, catechin, xylose, 4-hydroxy-2,5-dimethyl-3(2H)-furanone and nonanal are located under
the fruit’s skin, aldehydes (hexanal, benzaldehyde) and ketones (1-penten-3-one, geranylacetone)
are distributed throughout the fruits (in the inner core and in the cortex layer), while asparagine,
lysine, gambriin C, oxalic acid and 2-methylbutanoic acid are found on/around the surface of the
achenes. The authors suggested that their distribution is strongly connected with both the sites of their
biosynthesis and to their function.
490
Table 2. Composition of Fragaria species (as presented by original works published in the reviewed period; references presented in chronological order).
Species Plant Part, Other Variables Identified Compounds and Main Findings Identification Method Ref.
Anthocyanins (cyanidin 3-O-glucoside, pelargonidin 3-O-glucoside cyanidin-malonyl-glucoside and
F. chiloensis Ripe fruits HPLC-DAD, LC-ESI-MS [26]
pelargonidin-malonyl-glucoside); procyanidins, ellagitannins, ellagic acid and flavonol derivatives
F. chiloensis Leaves Procyanidins, ellagitannins, ellagic acid and flavonol derivatives HPLC-DAD, LC-ESI-MS [26]
F. chiloensis Rhizomes Procyanidins, ellagitannins, ellagic acid and flavonol derivatives HPLC-DAD, LC-ESI-MS [26]
Anthocyanins (pelargonidin-3-glucoside, pelargonidin-3-rutinoside, cyanidin-3-rutinoside, pelargonidin-3,5-diglucoside,
Molecules 2020, 25, 498
Fragaria × ananassa Fruits pelargonidin-3-(6-acetyl)-glucoside, 5-carboxypyranopelargonidin-3-glucoside, delphinidin-3-glucoside, peonidin-3-glucoside, LC-MS/MS, HPLC-UV/Vis [17]
cyanidin-3-galactoside), p-hydroxybenzoic acid, (+)-catechin, ellagic acid, p-coumaric acid, quercetin glucoside
Fruits, cultivar and seasonal
Fragaria × ananassa Vitamin C, β-carotene, total phenolics, total anthocyanins; genotype influence is stronger than the environmental influence Colorimetric [27]
variations
Fruits, different cultivars on Total vitamin C, total phenolics, total anthocyanins, total ellagic acid/pelargonidin-3-glucoside and cyanidin-3-glucoside;
Fragaria × ananassa Colorimetric/HPLC-DAD [28]
different ripeness stage higher amounts in pink fruits compared with fully ripped fruits
Fruits, different farming
Fragaria × ananassa Total phenolics/pelargonidin-3-glucoside and cyanidin-3-glucoside, vitamin C, higher in organic farming fruits Colorimetric/HPLC-DAD [18]
methods
Fruits, different cultivars (27) Phenolic compounds (multiple classes, including anthocyanins, flavanols and ellagitannins); composition dependent on
Fragaria × ananassa HPLC-DAD-MS [29]
and ripening stages cultivar, cinnamic acid conjugates and anthocyanins levels increased with the ripening stage
Fragaria × ananassa,
Fruits Quercetin and isorhamnetin glycosides (higher levels in wild strawberry) HPLC-DAD, LC-ESI-MS [30]
F. vesca
Fragaria × ananassa, Volatile esters (including ethyl acetate, hexyl acetate, methyl butanoate, ethyl butanoate, hexyl butanoate, methyl hexanoate,
Fruits, different cultivars GC-MS [19]
F. vesca ethyl hexanoate, hexyl hexanoate); higher levels in cultivated strawberries.
Anthocyanins (cyanidin 3-O-glucoside, pelargonidin 3-O-glucoside, peonidin 3-O-glucoside, cyanidin 3-O-malonylglucoside,
pelargonidin 3-O-malonylglucoside, peonidin 3-O-malonylglucoside), dihydroflavonol and flavonols (taxifolin
3-O-arabinoside, kaempferol 3-O-glucoside, quercetin 3-O-glucoside, quercetin-acetylhexoside, kaempferol
F. vesca Fruits, two different cultivars HPLC-DAD [31]
3-O-acetylhexosides), flavan-3-ols and proanthocyanidins (catechin, B type proanthocyanidin dimers, trimers, and tetramers),
ellagic acid and derivatives (glycosylated, methyl pentoside, methylellagic acid methyl pentoside, ellagitannins), other
compounds (benzoic acid, ferulic acid hexose derivative, citric acid, furaneol glucoside)
491
Anthocyanins (cyanidin, pelargonidin), cyanidin glycosides (cyanidin 3-glucoside, cyanidin 3-arabinoside, cyanidin
Fragaria × ananassa,
Fruits 3-sambubioside, delphinidin 3-galactoside, delphinidin 3-glucoside, delphinidin 3-malonylglucoside); higher levels of cyanidin HPLC-DAD [32]
F. vesca
glycosides in wild species
Ellagitannins (sanguiin H-2 isomer, sanguiin H-10 isomer, sanguiin H-6/agrimoniin/lambertianin A isomer,
F. vesca Leaves LC-PDA-ESI-MS [33]
castalagin/vescalagin isomer, sanguiin H-10 isomer, sanguiin H-2 isomer, casuarictin/potentillin isomer)
Fruits, different cultivars and Vitamin C, anthocyanins (pelargonidin 3-glucoside, cyanidin 3-glucoside, pelargonidin 3-rutinoside), ellagic acid; strongly
Fragaria × ananassa HPLC-UV/Vis [34]
production years dependent on the cultivar and production year
Vitamin C, pelargonidin-3-rutinoside, ellagic acid, cyanidin-3-glucoside, quercetin (red fruits), neochlorogenic,
Fragaria × ananassa Fruits, at different ripening stage LC-ESI-TOF [35]
pelargonidin-3-glucoside, pelargonidin-3-rutinoside, epicatechin, quercetin-3-β-d-glucoside, ellagic acid (green fruits)
Quercetin-3-β-d-glucoside, ellagic acid, kaempferol-3-O-glucoside, vitamin C (red), catechin, quercetin-3-β-d-glucoside, ellagic
Fragaria × ananassa Calyx (red and green) LC-ESI-TOF [35]
acid (green)
Fragaria × ananassa Flower Catechin, quercetin-3-β-d-glucoside, ellagic acid, kaempferol-3-O-glucoside, vitamin C LC-ESI-TOF [35]
Fragaria × ananassa Leaf Procyanidin dimer and trimer, catechin, quercetin-3-β-d-glucoside, vitamin C, ellagic acid LC-ESI-TOF [35]
Fragaria × ananassa Stolon Neochlorogenic, procyanidin dimer, catechin, quercetin-3-β-d-glucoside, ellagic acid, vitamin C, kaempferol-3-O-glucoside LC-ESI-TOF [35]
Fragaria × ananassa Stem Procyanidin dimer, catechin, ferulic acid, quercetin-3-β-d-glucoside, ellagic acid LC-ESI-TOF [35]
Fragaria × ananassa Crown Procyanidin dimer and trimer, catechin, propelargonidin dimer, ellagic acid LC-ESI-TOF [35]
Fragaria × ananassa Root Procyanidin dimer and trimer, catechin, neochlorogenic, propelargonidin dimer LC-ESI-TOF [35]
Phenolic acids (p-coumaric acid, ellagic acid, ferulic acid derivative, p-coumaric acid derivatives), monomeric flavanols
((+)-catechin), flavonols (quercetin 3-O-glucoside, fisetin, quercetin 3-O-glucoside derivative), anthocyanins (cyanidin
Fragaria × ananassa Fruits, different novel cultivars 3-glucoside, cyanidin 3-rutinoside, cyanidin pentoside, pelargonidin 3-galactoside, pelargonidin 3,5-diglucoside, pelargonidin HPLC-DAD, LC-ESI-QTOF [36]
3-glucoside, pelargonidin 3-rutinoside, cyanidin 3-Oacetylglucoside, cyanidin hexoside, pelargonidin 3-O-monoglucuronide,
pelargonidin derivatives)
Table 2. Cont.
Species Plant Part, Other Variables Identified Compounds and Main Findings Identification Method Ref.
Hydroxybenzoic acid, p-coumaric acid, other hydroxycinnamic acids, (+)-catechin, (−)-epicatechin, procyanidins, flavonols,
Fruits, grown on different
Fragaria × ananassa anthocyanins (cyanidin 3-glucoside, pelargonidin 3-glucoside, pelargonidin derivative); higher levels recorded at lower HPLC-DAD [37]
altitudes, on consecutive years
altitudes.
Kaempferol 3-(6-methylglucuronide), quercetin 3-(6-methylglucuronide), isorhamnetin 3-(6-methylglucuronide), trichocarpin,
2-p-hydroxybenzoyl-2,4,6-tri hydroxyphenylacetate, 2-p-hydroxyphene thyl-6-caffeoylglucoside, zingerone 4-glucoside, 1H NMR, 13 C NMR, HMBC,
b-hydroxypropiovanillone 3-glucoside, (+)-isolariciresinol 90-glucoside, (−)-isolariciresinol 90-glucoside, aviculin,
Molecules 2020, 25, 498
Fragaria × ananassa Fruits HPLC-UV/Vis, LC-MS/MS, [20]

(−)-secoisolariciresinol 4-glucoside, cupressoside A, cedrusin, icariside E4, dihydrodehydrodiconiferyl alcohol 90-glucoside,
HR-ESI-MS,
massonianoside A, urolignoside, (−)-pinoresinol 4-glucoside, 2,3”-epoxy-4-(butan-2-one-3-yl)-5,7,40-trihydroxy flavane
3-glucoside, kaempferol 3-(6-butylglucuronide), benzyl 2-glucosyl-6-rhamnosylbenzoate
39 phenolic compounds (including cyanidin 3-O-glucoside, delphinidin-3-O-glucoside, pelargonidin-3-O-glucoside, LC-ESI-Orbitrap-MS,
Fruits, wild and cultivated, from
F. vesca pelargonidin-3-O-rutinoside, (+) catechin, (−) epicatechin, procyanidin B1 and B2, isoquercetin, gallic acid, p-coumaric acid, LC-ESI-QTrap-MS, [38]
different geographical areas
phloridzin); composition dependent on the geographical area LC-ESI-QTrap-MS/MS
Cyanidin 3-O-glucoside, pelargonidin-3-O-glucoside, pelargonidin-O-rutinoside, total anthocyanins content, dependent on the
Fragaria × ananassa Fruits, different cultivars UPLC-PDA-ESI-MS, HPLC-DAD [39]
cultivar
Volatile composition—one hundred compounds (including esters, aldehydes, ketones, alcohols, terpenoids, furans and
F. vesca Fruits GS-MS [40]
lactones).
27 metabolites (organic acids, flavonoids, catechin and its oligomers, ellagitannins), including quinic acid, chelidonic acid,
F. vesca Leaves LC-ESI-Orbitrap-MS [41]
quercetin derivatives, catechin and procyanidins, phloridzin, pedunculagin, methyl ellagic acid glucuronide.
Fragaria × ananassa, White-fruited mutants, different
Anthocyanins, flavonols, flavan-3-ols, hydroxycinnamic acids, and ellagic acid—derived compounds, dependent on genotype LC-ESI-MS/MS [21]
F. vesca genotypes
Anthocyanins (cyanidin-3-O-glucoside, pelargonidin hexoside, cyanidin manlonyl hexoside, pelargonidin-malonyl hexoside),
F. chiloensis Fruits ellagitannins (ellagic acid hexoside, pentoside, rhamnoside), proanthocyanidin dimers, epicatechin, flavonols (quercetin HPLC-DAD, LC-ESI-MS [42]
pentoside, glucuronide)
Anthocyanins, flavonoids, cinnamic acid derivatives, tannins and related compounds, triterpenoids; concentration dependent UPLC-ESI-QTOF-MS/MS,
Fragaria × ananassa Fruits, different cultivars [43]
on the cultivar HPLC-DAD
492
where: 13 C NMR—Carbon-13 nuclear magnetic resonance; GC-MS—gas chromatography–mass spectrometry; 1 H NMR—proton nuclear magnetic resonance; HMBC —heteronuclear
multiple bond correlation; HPLC-DAD—high-performance liquid chromatography with diode array detector; HPLC-UV/Vis—high-performance liquid chromatography equipped
with UV/vis detector; HR-ESI-MS—high-resolution electrospray ionization mass spectrometry analysis; LC-ESI-MS(/MS)—liquid chromatography electrospray ionization (tandem)
mass spectrometry analysis; LC-ESI-Orbitrap-MS—liquid chromatography electrospray ionization Orbitrap mass spectrometry; LC-ESI-QTrap-MS(/MS)—liquid chromatography
electrospray ionization quadrupole ion trap mass spectrometry; LC–ESI–(Q)TOF—liquid chromatography electrospray ionization with (quadrupole) time-of-flight; LC-MS/MS—liquid
chromatography–tandem mass spectrometry; LC-PDA-ESI-MS—liquid chromatography equipped with photodiode array detector coupled to mass spectrometry using the electrospray
ionization interface; UPLC-ESI-QTOF-MS/MS—ultra-performance liquid chromatography equipped quadrupole time of flight coupled to tandem mass spectrometry using the
electrospray ionization interface; UPLC-PDA-ESI-MS—ultra-performance liquid chromatography equipped with photodiode array detector coupled to mass spectrometry using the
electrospray ionization interface.
Molecules 2020, 25, 498
The composition of the fruits, although representing a characteristic of each species,

can be influenced by a number of factors (as presented in Table 2), including the
characteristics of the cultivar [21,27,29,31,34,39,43–46], cultivation factors, and the environmental
conditions [18,27,37,38,47–50], ripening stage [28,29,35,51], or by biotechnological approaches [52–54].
For example, the variation in composition of a large number of Fragaria × ananassa
Duch. cultivars was presented in 2019 by Nowicka et al. [43]. The authors identified as
main components in the strawberry fruits the anthocyanins (pelargonidin-3-O-β-glucoside,
cyanidin-3-O-β-glucoside, pelargonidin-3-O-rutinoside, cyaniding-3-O-(6”malonyl)glucoside,
pelargonidin-3-O-(6”malonyl)glucoside)), flavonoids (derivatives of quercetin, kaempferol and
isorhamnetin), cinnamic acid derivatives (isomers of 1-O-p-coumaroylhexose, 1-O-feruloylhexose,
1-O-p-coumaroyl-β-glucose, 1-O-trans-cinnamoyl-β-glucose), tannins and related compounds
(gallotannins, glycosides of ellagic acid and methylellagic acid, free ellagic acid, flavan-3-ol derivatives),
and triterpenoids (methyl or hydroxyl derivatives of tormentic or dihydrotormentic acid). More
importantly, using the variation of the analyzed compounds allowed the authors to propose some of
the cultivars for future studies and biomedical uses. Another interesting study was presented in 2011
by Pineli et al. who studied the compositional differences between two Fragaria x ananassa cultivars
(Osogrande and Camino Real) in different ripening stages. Interestingly, the authors observed higher
amounts of total phenolics, total ellagic acid, and vitamin C in the pink strawberries (3/4 ripe, as
defined by the authors), compared with the green and fully ripe ones [28]. The differences recorded
between the cited literature studies can be thus explained by the influence of those factors, as well as by
the extraction procedure followed (which also have a strong influence on the final composition of the
extracts), as demonstrated by Pawlaczyk-Graja et al. on the structure of polyphenolic-polysaccharide
conjugates obtained from the leaves of Fragaria vesca using different classical and modern methods [55].
As the primary economic importance of the Fragaria genus is related to their fruits, it must
be stated that, besides the previously presented factors, their composition (and finally, their health
benefits) is correlated with the applied processing methods. The most encountered forms of products
(besides fresh fruits) are represented by the dried fruits and puree. Méndez-Lagunas et al. [56]
studied the influence of the thermal drying on the anthocyanins and total phenolic content of
Fragaria × ananassa Duch. fruits. The study revealed a 26% loss of anthocyanins content when
drying at 50 ◦ C, respectively a 45% loss upon drying at 60 ◦ C. The total phenolic content was even
more seriously affected by the thermal process, with 60.9% loss associated with 50 ◦ C, and 78.1% for
the 60 ◦ C treatment, respectively. Thus, the importance of fresh fruits consumption and when not
possible, application of appropriate drying methods was emphasized; the authors propose the 50 ◦ C
treatment as suitable for preserving the bioactive compounds found in the strawberries (particularly
anthocyanins). Álvarez-Fernández et al. [57] evaluated the variation in non-anthocyanin phenolic
composition (hydrolyzed tannins, flavanols and condensed tannins, ellagic acid and derivatives,
hydroxycinnamic acids, flavonol glycosides and stilbenes) of strawberry puree in different production
stages. If mashing process affected the content of some phenolics (gallic acid, monogalloylglucoside
and ellagic acid), the pasteurization process induced the decrease of all the compounds’ concentration.
However, the non-anthocyanin phenolic profile was not significantly affected, suggesting that the
strawberry puree represents a good source of phenolic compounds.
The strawberry “seeds” (achenes) were proven to be a valuable source of unsaturated fatty acids.
Thus, several studies revealed that commercially available achenes oil contained high amounts of
linoleic and α-linoleic acids (over 70%), with a total content of over 90% unsaturated fatty acids (also
including oleic and traces of palmitoleic acid), while the saturated fatty acids were mainly represented
by palmitic and stearic acids [58,59]. In the same time, the seeds could also be considered a source of
dietary fibers, proteins, polyphenols (mainly ellagitannins), and vitamins [60]. Figure 1 summarizes
the main classes of constituents found in Fragaria genus.
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Molecules 2020, 25, 498
3. Biological Activities of Fragaria Genus
3.1. Antioxidant Properties

Traditionally consumed in the form of fruits (as previously presented), Fragaria species have
also found application in traditional medicine. For example, Fragaria vesca leaves and fruits were
traditionally used for the treatment of external rashes, as well as internally, as blood purification and
roborontarium, for the treatment of diarrhea [61], as macerate for renal stones, or as tea (together with
other medicinal plants) for treating stomach inflammations, sedation, or regulation of digestion [62].
The following paragraphs presents the main biological properties of different Fragaria species, as
emerging from the literature data published in the past decade. Particularly, the anthocyanins
family represent the subject of several review papers published in the last years, dealing with their
bioavailability and potential health benefits [63–65]. The following chapters includes only the studies
regarding the biological activity of compounds or extracts obtained from Fragaria species (not studies
presenting the activity of compounds that are found in those plants).
The major classes of compounds found in the Fragaria species (anthocyanins and non-anthocyanin
phenolic compounds) are known for their antioxidant properties [65,66]. As can be expected, the vast
majority of the literature presenting the biological activities of species belonging to Fragaria genus
present their antioxidant activity. However, the studies that will be presented should be carefully
considered, as many are performed using assays predisposed to positive results in the presence of
oxygenated functions on aromatic rings (thus being considered more “class-related” than as specific
molecular targeting) [67]. Thus, those studies, although useful as screening tools, should be confirmed
by more specific assays, such as in vivo or cell-based models [66,67].
Pineli et al. [28] performed a study on two Fragaria × ananassa Duch. cultivars (Osogrande
and Camino Real), regarding the correlation between composition and the antioxidant activity
(established using 2,2-Diphenyl-1-picrylhydrazyl radical-scavenging activity—DPPH and ferric
reducing antioxidant power—FRAP assays) of acetone extracts obtained from fruits at different
ripening stages. Very interestingly, although, as expected, the anthocyanins levels were higher in the
red fruits (full ripe), the best antioxidant activities, for both cultivars and both assays were obtained for
the pink fruits. The only exception is represented by the DPPH assay results for Osogrande cultivar
(for which the best results were obtained for the red fruits); however, the differences between the
results of the DPPH assay for the cultivar at the three ripening stages were not statistically significant.
The differences between the results can be explained by the different mechanisms of the assays (as
previously described by our group) [66]. A much better correlation was observed by the authors
with the total phenolic and vitamin C content. Finally, the Osogrande cultivar presented superior
antioxidant properties (associated with higher levels of the total phenolic and total ellagic acid content,
especially in pink and red fruits). Zhu et al. [35] evaluated the influence of the solvent used for
room-temperature extraction (water/ethanol) and of the extracted Fragaria × ananassa var. Amaou
parts (red fruit, green fruit, red calyx, green calyx, flower, leaf, stolon, stolon leaf, stem, crown, and
root) on the antioxidant properties of the extract. Their conclusion was that the ethanol was the solvent
of choice (because of different polarities of the phenolic compounds found in the plant) and, among
the plant parts, best results (presented as Trolox equivalents—TE per gram or extract and per 100 g of
plant fresh weight—FW) were obtained for flower extracts (1460.1 μMol TE/g extract), respectively for
crown (6212.3 μMol TE/100g FW), when reported to a fresh weight basis. Stolon leaves (1456.7 μMol
TE/g extract, respectively 5244.2 μMol TE/100g FW) also exhibited a very good antioxidant activity.
Individual compounds (including phenolic glucosides, flavone glucuronides, and lignan
glycosides) were isolated from the Fragaria x ananassa Duch. cv. Falandi fruits by Yang et al. [20] and
tested for antioxidant properties. The best results were obtained for flavone glucuronides (in the ABTS
and DPPH assays), and a lignan glycoside (in the FRAP assay), respectively. Considering the results
obtained for the positive control used in the antioxidant assays (ascorbic acid), the authors suggested
that the investigated phenolic compounds play an important role in the overall antioxidant property of
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the plant. Contrary to other studies, Chaves et al. [39] demonstrated a correlation between the total
anthocyanin content and the antioxidant potential of strawberry fruits, in a study over seven cultivars.
The antioxidant potential of plants, as it results from literature studies, seems to be correlated with
total anthocyanins and not with total phenolic content.
In 2019, Nowicka et al. [43] published a study regarding the variation in composition and
antioxidant properties of 90 cultivars of Fragaria × ananassa Duch. fruits over two years of production.
The results (average values presented in Table 3) revealed not only that some cultivars can be considered
as having superior antioxidant properties (Roxana, Gigaline, Selvik, Thuriga ISK, Eratina, Siria, Dagol,
Plarionfre, Grenadier, and Kimberly), but also, considering the phytochemical profile, that the main
compounds responsible for the activity are the tannins, especially ellagitannins and procyanidins.
As previously stated, because of the increasing request, strawberries are often commercialized as
processed products. The effect of fruit drying on the antioxidant potential of the Fragaria × ananassa
Duch., Diamante var. fruits was presented by Méndez-Lagunas et al. [56]. The antioxidant assay
performed on the processed fruits (DPPH) revealed 74.7% loss of antioxidant activity for the thermal
treatment at 50 ◦ C, while the 60 ◦ C treatment led to a 66.2% loss of the activity. The results suggested
that, beyond temperature, heat treatment time has a stronger effect on the antioxidant activity (as
at higher temperature, shorter periods are necessary). Similar, several researches were performed
regarding the changes of antioxidant activity (determined using ORAC and DPPH assays) during
different stages of puree production [57]. Although slight reduction of the antioxidant properties
was recorded (statistically significant only for the pasteurization step), the authors recommended the
strawberry puree as an excellent source of antioxidants. The same group [68] observed no effect of the
gluconic fermentation of strawberry puree (applied for the production of beverages) on the antioxidant
activity (determined using the DPPH assay); the authors even reported an increase of the antioxidant
potential after the pasteurization step, which was correlated with an increase in the gallic acid and
hydroxycinnamic derivatives content. The results would suggest that the gluconic fermentation could
maintain the antioxidant potential of the fresh products upon processing.
The antioxidant potential of strawberries could rapidly find industrial applications, as was the
case for other plant-derived antioxidants [69,70] in, for example, meat industry, as recently reviewed
by Lorenzo et al. [71], for increasing the shelf-life of different products (as sausages or raw, cooked,
and cooked-chilled porcine patties).
Table 3 summarizes the main findings regarding the antioxidant potential of Fragaria species, as
well as the responsible classes of compounds (as presented by the authors).
As a general remark, it can be observed that most authors assign the antioxidant potential
to the total phenolic content in general, and in particular to some classes of compounds, such as
anthocyanins, flavan-3-ols or tannins. Considering the individual species, Fragaria x ananassa fruits
presented antioxidant properties in the DPPH assay (the assays with the widest application) between
76.73–100 mg/mL (IC50 ) for various cultivars (the best results being obtained for the Camarosa
cultivar) [39] or between 300 and 1300 μMol trolox/100 g fresh weight, for a larger survey (comprising
90 cultivars) [43]. Also, regarding the differences between the antioxidant potential of different plant
parts, for F. chiloensis methanolic extracts the best activity was observed for fruits [26], while for
Fragaria × ananassa for the crown ethanolic extract (6213.3 μMol trolox/100 g fresh weight). Fragaria
vesca was mainly evaluated in terms of leaves, roots, or vegetative parts antioxidant activity, with
antioxidant potential ranging from 13.46 mg/L to approx. 140 mg/L (IC50 ), strongly dependent on the
source of vegetal material and applied extraction technique [72,73].
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Table 3. Antioxidant properties of different extracts obtained from Fragaria species (references presented in chronological order).
Species Extraction Method Antioxidant Assay Antioxidant Potential Responsible Compounds Ref.
ORAC: 2.7–24.46 mmol Trolox/g; FRAP: 2.75–12.5 mmol Fe2+ /g (depending on
Fragaria × ananassa, Camarosa var. fruits Anthocyanins isolated using CCC ORAC, FRAP Anthocyanins [17]
the fraction)
Fragaria chiloensis spp. chiloensis form Aglycone and glycosylated
Methanol: formic acid (99:1 v/v) extraction DPPH, SAS DPPH assay: IC50 = 38.7 mg/L; SAS: 79.3%) [26]
chiloensis fruits ellagic acid and flavonoids
Methanol: formic acid (99:1 v/v) extraction DPPH, SAS DPPH assay: IC50 = 49.4 mg/L; SAS: 67.60% [26]
chiloensis leaves ellagic acid and flavonoids
Molecules 2020, 25, 498

Methanol: formic acid (99:1 v/v) extraction DPPH, SAS DPPH assay: IC50 = 64.8 mg/L; SAS: 55% [26]
chiloensis rhizomes ellagic acid and flavonoids
Fragaria x ananassa Osogrande var. DPPH: 11.91–12.83 μMol BHT eq./g FW; best results for ripe fruits FRAP: Total phenolic content,
Acetone (80%) extraction DPPH, FRAP [28]
frozen fruits 27.37–36.75 μMol FS eq./g FW; best results for green fruits vitamin C
Fragaria x ananassa Camino Real var. DPPH: 9.75–12.01 μMol BHT eq./g FW, FRAP: 24.13–28.49 μMol FS eq./g FW (best Total phenolic content,
Acetone (80%) extraction DPPH, FRAP [28]
frozen fruits results for pink fruits) vitamin C
F. vesca leaves Methanol, ultrasounds extraction DPPH, FRAP DPPH: IC50 = 13.46 mg/L; FRAP: 0.878 mmol Fe2+ /g DW Total phenols, total tannins [72]
DPPH, FRAP, Total phenolics, total
IC50 , mg/L: DPPH—50.03/50.56/50.62; FRAP—40.98/44.78/49.23; β-C
F. vesca roots, wild-growing Hydromethanolic extraction, infusion, decoction β-Carotene bleaching flavan-3-ols, total [73]
bleaching—116.26/44.88/66.10; TBARS—35.76/4.76/6.14
inhibition, TBARS dihydroflavonols,
DPPH, FRAP, Total phenolics, total
F. vesca roots, commercial Hydromethanolic extraction, infusion, decoction β-Carotene bleaching flavan-3-ols, total [73]
bleaching—68.34/23.44/114.67; TBARS—6.69/24.25/10.62
inhibition, TBARS dihydroflavonols,
Fragaria × ananassa var. Amaou, fruits, at
Ethanol or water room temperature extraction Modified ABTS assay Ethanol: 150.5/151.9; water: 227.2/189.4 (red/green fruits) μMol TE/100 g FW Total phenolic content [35]
different ripening stage
Fragaria × ananassa var. Amaou calyx (red
Ethanol or water room temperature extraction Modified ABTS assay Ethanol: 241.1/1239.9; water: 1716.6/577.7 μMol TE/100 g FW (red/green calyx) Total phenolic content [35]
and green)
Fragaria × ananassa var. Amaou flower Ethanol or water room temperature extraction Modified ABTS assay 4234.4/387.5 μMol TE/100 g FW (ethanol/water) Total phenolic content [35]
Fragaria × ananassa var. Amaou leaves Ethanol or water room temperature extraction Modified ABTS assay 2401.7/241.1 μMol TE/100 g FW (ethanol/water) Total phenolic content [35]
Fragaria × ananassa var. Amaou stolon Ethanol or water room temperature extraction Modified ABTS assay 1089.4/1856.7 μMol TE/100 g FW (ethanol/water) Total phenolic content [35]
Fragaria × ananassa var. Amaou stem Ethanol or water room temperature extraction Modified ABTS assay 1338.6/1123.1 μMol TE/100 g FW (ethanol/water) Total phenolic content [35]
496
Fragaria × ananassa var. Amaou crown Ethanol or water room temperature extraction Modified ABTS assay 6213.3/128.7 μMol TE/100 g FW (ethanol/water) Total phenolic content [35]
Fragaria × ananassa var. Amaou root Ethanol or water room temperature extraction Modified ABTS assay 253.1/69.2 μMol TE/100 g FW (ethanol/water) Total phenolic content [35]
Hydromethanolic and aqueous extracts; DPPH, FRAP, Total phenolics, total
F. vesca vegetative parts (leaves and stems), bleaching—56.71/12.34/13.40; TBARS—12.63/3.12/5.03
wild-growing infusion microencapsulated in β-Carotene bleaching flavan-3-ols, total [74]
wild-growing (hydromethanolic/infusion/decoction); Final formulation (mg/mL)—DPPH—2.74;
alginate and incorporated in k-carrageenan gelatine inhibition, TBARS dihydroflavonols,
FRAP = 1.23
DPPH, FRAP, IC50 , mg/L: DPPH—139.33/121.94/118.89; FRAP—324.49/91.88/88.20; β-C Total phenolics, total
F. vesca vegetative parts (leaves and stems),
Hydromethanolic and aqueous extracts β-Carotene bleaching bleaching—388.90/76.41/69.98; TBARS—24.36/23.07/17.52 flavan-3-ols, total [74]
commercial
inhibition, TBARS (hydromethanolic/infusion/decoction). dihydroflavonols,
Best results (IC50 ): ABTS—4.42 μM kaempferol 3-(6-methylglucuronide);
Fragaria x ananassa cv. Falandi fruit 22 compounds isolated from ethanolic extracts ABTS, DPPH, FRAP DPPH—32.12 μM quercetin 3-(6-methylglucuronide); Individual compounds [20]
FRAP—0.05 mmol/g—urolignoside.
Fragaria x ananassa cv. Albion, Aromas,
Camarosa, Camino Real, Monte Rey, Portola, Ultrasonic extraction with acidified methanol DPPH IC50 (mg/mL) ranging from 76.73 (Camarosa)—100 (Camino Real) Total anthocyanin content [39]
and San Andreas fruits
Ultrasonic extraction with ethanol: water solvent Condensed tannins and
F. vesca leaves native to Italy TEAC 0.34–0.35 mg/mL Trolox eq., compared with quercetin (0.40) [41]
(70:30, v/v) flavonoid derivatives
0.07 (simple supercritical extraction)—5.82 μMol BHT/g sample (with ethanol,
Fragaria x ananassa cv. Tochiotome leaves Supercritical CO2 extraction with different entrainers DPPH Phenolic compounds [10]
dried at 40 ◦ C)
Ultrasonic aqueous methanol (70%) acidified with
Fragaria × ananassa fruits (90 cultivars) DPPH, ABTS Average values (μmol Trolox/100 g):765.06 (DPPH), 1637.96 (ABTS) Tannin-based compounds. [43]
1.5% formic acid, at room temperature
where: ABTS—2,2 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) assay; BHT—butylated hydroxytoluene; DPPH—reduction of 2,2-diphenyl-1-picrylhydrazyl; DW—dry weight;
eq.—equivalents; FRAP—ferric reducing ability of plasma; FS—ferrous sulphate; FW—fresh weight; IC50 —half maximal inhibitory concentration; ORAC—oxygen radical absorbance
capacity; SAS—superoxide anion assay; TBARS—thiobarbituric acid reactive substances assay; TEAC—Trolox equivalent antioxidant capacity.
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3.2. Anti-Inflammatory Properties

As previously stated, one of the traditional uses of Fragaria is as an anti-inflammatory agent [61,62].
Most of the authors assign the anti-inflammatory properties to the presence of anthocyanins
(the most representative being pelargonidin and cyanidin derivates) [75], molecules with known
anti-inflammatory potential [76,77], demonstrated both in vitro and in vivo [78,79]. Similar to the
other biomedical potential, the anti-inflammatory action is also correlated with the composition of
different Fragaria species. The traditional use of F. vesca as an anti-inflammatory agent was supported
by the study of Liberal et al. [80]. The authors observed the decrease of a relevant mediator of
the inflammatory response (nitric oxide) produced by macrophages, cultured in the presence of a
NO-production inducing bacterial endotoxin (LPS). The ethanolic extract obtained from Fragaria vesca
leaves, used at non-cytotoxic concentrations (80 and 160 mg/L), induced a 31%, and 40% inhibition,
respectively. The authors assigned the NO decrease to a direct scavenging effect (as demonstrated by a
23% inhibition of the nitrite content in the culture media, correlated with the absence of a significant
effect when quantifying the inducible nitric oxide synthase—iNOS and the pro-inflammatory cytokine
IL-1β). The authors also observed a statistically insignificant increase in the phosphorylated IκBα
(nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha) content, suggesting
either an increase of its expression or a decrease in its degradation. More than that, the authors observed
an increased conversion of the microtubule-associated protein light chain LC3-I to LC3-II (a marker of
autophagy), suggesting further anti-cancer properties. Methanolic extracts of Fragaria x ananassa, var.
Alba fruits were also confirmed by Gasparrini et al. [81] to lower the intracellular levels of reactive
oxygen species (ROS), decrease apoptotic rate and improve antioxidant defenses and mitochondria
functionality in E. Coli induced inflammation in human dermal fibroblast cells. Their results showed
significant decrease of TNF-α (tumor necrosis factor alpha), IL-1β and IL-6 (interleukin 6) levels.
The authors proposed as responsible mechanism the action on AMPK (5 AMP-activated protein
kinase) related pathways (increment in phosphorylated AMPK expression). Between the two presented
studies there are several experimental design/inputs differences that can explain the different obtained
results (including the species and the solvent used for extraction). Thus, the higher levels of bioactive
molecules in the Fragaria x ananassa fruits (compared with the F. vesca leaves, as previously presented)
can explain the differences observed by the authors in terms of anti-inflammatory action. Similar
observations were made by Molinett et al. [82] who proved the anti-inflammatory and hepatoprotective
effect of aqueous F. chiloensis fruits extracts on LPS-inducted liver injury on rats. The anti-inflammatory
effect of the Fragaria x ananassa var. Camarosa fruits was also in vivo evaluated on female mice by
Duarte et al. [83], who assigned the activity to the presence of anthocyanins. Moreover, the authors
performed in vitro experiments using pelargonidin-3-O-glucoside (the major anthocyanin in Fragaria)
in order to establish its molecular mechanism of action. Regarding the in vivo experiments the authors
noticed the inhibition of the carrageenan-induced leukocyte influx to the pleural cavity upon crude
extract treatment, due to the reduction in neutrophil migration. The extract also induced a reduction
of myeloperoxidase activity and reduced the exudate concentration in the pleural cavity and NO
levels. The pure compound pelargonidin-3-O-glucoside produced similar results, inhibiting IkBα, also
reducing the phosphorylation of p65 NF-kB (nuclear factor kappa-light-chain-enhancer of activated
B cells) subunit. The authors proposed as mechanism of the anthocyanin the mitogen-activated
protein kinase (MAPK) pathways, leading to the decrease in NF-kB and activated protein 1 (AP-1)
translocation. In 2019, Van de Velde et al. [84] supported the previously reported anti-inflammation
properties of strawberry extracts, also proposing another possible application, by evaluating the
wound-healing effects. The extract and polyphenolics/anthocyanins-enriched fractions influenced
the skin fibroblast migration (45% of the migration registered for the positive control—fetal bovine
serum, for the crude extract, 50% of the positive control for the anthocyanins enriched fraction at
1 mg/L, 30% of the positive control for the polyphenolics enriched fraction), suggesting that the
wound-healing properties are strongly associated with the anthocyanins presence. The dietary use of
strawberry achenes commercial oil has also been proven to reduce the activity of superoxide dismutase
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(SOD) and glutathione peroxidase (cGPx) in rats (38.73, respectively 10.5 international units/gram of
hemoglobin—U/g Hb, compared with the control group—67.33, and 22.9 U/g Hb, respectively), thus
being qualified as a potential nutraceutical reducing oxidative stress [58].
3.3. Other Potential Applications

The anti-microbial properties were evaluated within the reviewed time period, especially for F. vesca.
Hydromethanolic extracts obtained from leaves and roots of Fragaria vesca L. were evaluated by
Gomes et al. [85] as antimicrobial agents a series of S. aureus strains. The results suggested a weak
antimicrobial potential of the extracts (5–9 mm inhibition halos in the qualitative assays), which did
not qualify the extracts for quantitative determinations. Superior results in terms of antimicrobial
properties were obtained by Cardoso et al. [86]. Using hydroalcoholic extracts, the authors observed
good antimicrobial properties of the crude extract against a series of Helicobacter pylori isolates (inhibition
zones ≥ 15 mm) at a 25 mg/mL concentration. The ellagitannin-enriched fraction was efficient against
all isolates at lower concentrations (7.5 mg/mL), which led the authors to assume that the ellagitannins
were the main class of compounds responsible for the anti-microbial properties. As the H. pylori
represents a pathogen involved in several gastric pathologies (including gastritis, gastroduodenal
ulcer disease, gastric adenocarcinoma and mucosa-associated lymphoid tissue lymphoma), the authors
proposed the wild strawberry extract as a potential candidate for human health applications.
The anti-allergenic potential of several compounds (linocinnamarin,
1-O-trans-cinnamoyl-b-d-glucopyranose, p-coumaric acid, cinnamic acid, chrysin, kaempferol,
catechin, and trans-tiliroside) isolated from Fragaria x ananassa var. Minomusume fruits were
evaluated by Ninomiya et al. [87], through the determination of their inhibitory effects on
antigen-stimulated degranulation in rat basophilic leukemia RBL-2H3 cells. Among the studied
compounds, linocinnamarin (95% inhibition of control at 100 μM) and cinnamic acid (approx. 80% of
control at 100 μM) were the most efficient in degranulation suppression (through direct inactivation of
spleen tyrosine kinase), being proposed as promising tools for alleviating symptoms of type I allergy.
The commercially-available strawberry freeze-dried powder was demonstrated by Abdulazeez [88]
to reverse alloxan-induced diabetes (results not presented in Table 4 as authors used commercial
powder product); in a similar study, Yang et al. [12] evaluated the potential anti-diabetic
application of new and known compounds isolated from strawberry fruits (as presented in
Section 2) by determining the α-glucosidase inhibitory activity. The best results were obtained
for cupressoside A (IC50 = 25.39 μM), kaempferol 3-(6-methylglucuronide) (IC50 = 65.22 μM), and
2-p-hydroxybenzoyl-2,4,6-tri hydroxyphenylacetate (IC50 = 97.81 μM), with very good results obtained
for a newly proposed structure (kaempferol 3-(6-butylglucuronide)-IC50 = 107.52 μM); results superior
to the positive control (acarbose-IC50 = 619.94 μM) were also obtained for five other compounds.
Another interesting study is represented by the one performed by Zhu et al. [35]. Besides
the phytochemical and anti-oxidant studies (previously presented), the authors also evaluated the
anti-obesity, anti-allergy, and skin-lightening effects of extracts obtained from different parts of
strawberry in different ripening stages. The extracts exhibited anti-obesity activity (the water extract
of unripe fruit and the ethanol extracts of the stem, stolon leaf, and crown ripe fruits exhibiting
anti-lipase activity, as well as inhibitory effect on adipocyte differentiation), anti-allergy function (the
ethanol extracts of flower, stolon leaf and red calyx showing strong suppression effect on the release of
β-hexosaminidase), and skin-lightening potential (ethanol extracts of ripe fruits, unripe fruit and the
crown exhibiting melanogenesis inhibitory action, correlated with the tyrosinase-inhibitory activities).
The cytotoxic potential of the Fragaria species was demonstrated in several studies.
Somasagara et al. [89] evaluated the potential application of methanolic strawberry extract in leukemia
(CEM) and breast cancer (T47D) cell lines ex vivo, as well as its therapeutic and chemopreventive
potential in vivo. The MTT, trypan blue and LDH assays revealed the cytotoxicity of the extract on cancer
cells, in a concentration-dependent manner, while the in vivo studies revealed the anti-proliferative
action on tumor cells. Forni et al. [90] investigated the antiproliferative and differentiation potential of an
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anthocyanin-rich strawberry fruit extract on B16-F10 murine melanoma cells. Their results showed the
reduction of cell proliferation (30% after 48 h), accompanied by the lowering of the intracellular levels of
polyamines (63.8% decrease of spermidine, 52.9% decrease of spermine, after 72 h), and the enhancement
of tissue transglutaminase (172% increase after 48 h). The used extract also down-regulated p53 and
p21 expression (47.2%, and 32.6%, respectively). Liberal et al. [33] presented the cytotoxic potential
of an ellagitannin-enriched fraction from Fragaria vesca leaves on human hepatic carcinoma cell line
(HepG2). Their results showed that the crude extract and, more pronounced, the ellagitannin-enriched
fraction, were able to interfere with cell cycle distribution. The ellagitannin-enriched fraction also
induced necrosis and apoptosis in the threated cells, decreased chymotrypsin-like activity of the 26S
proteasome, impaired autophagic flux, promoted the accumulation of ubiquitinated proteins, and
decreased the expression of several proteasome subunits. Lucioli et al. [91] evaluated the influence
of hydroalcoholic extracts (methanol, ethanol, isopropanol) from in vitro cell suspension on the
proliferation of several cancer cells (neuroblastoma, colon, and cervix carcinoma cell lines). The extracts
induced a statistically significant reduction of cell growth but did not affect the human fibroblasts
from healthy donors. The chemoprotective action of strawberries was also studied by Casto et al. [92]
(results not presented in Table 4 as authors used commercial strawberry powder). The chemoprotective
role of strawberries on colorectal cancer in inflammatory bowel disease was recently reviewed by
Chen et al. [93], who proposed a mechanism of action involving the suppression of cytokines release,
decrease of oxidative stress, reduction of genomic instability, and inhibition of NFκB (nuclear factor
kappa-light-chain-enhancer of activated B cells) and related signalling pathways. Table 4 summarizes
the main biological activities (except anti-oxidant properties, presented in Table 3), as emerging from
the literature survey, considering the main constituents of the tested extracts.
499
Table 4. Main biological activities presented in the literature (references listed in chronological order).
Action Plant Extraction Method Assay Results Responsible Compounds Ref.
Prevention of increase in colon weight and disease activity
Anti-inflammatory
index, decrease in macroscopic and microscopic lesion
on inflammatory Fragaria vesca leaves Eth. extraction MPO activity; GSH, SOD and CAT levels Phenolic acids, flavonoids [94]
score; significant improvement of MPO, CAT and SOD
bowel disease
levels at 500 mg/kg 5 days oral treatment
Nitric oxide production, western blot analysis
Inhibition of nitrite production on pre-treated cells (at 80
Eth. extraction at room (expression of pro-inflammatory proteins in
Molecules 2020, 25, 498
Anti-inflammatory Fragaria vesca leaves and 160 mg/L—31%/40%); 23% inhibition in culture media, Phenolic content [80]
temperature, infusion lipopolysaccharide-triggered macrophages);
at 160 mg/L
nitric oxide scavenger activity
Determination of ROS intracellular levels, Reduction of intracellular ROS levels (significant at
apoptosis detection, antioxidant enzyme 100 mg/L), decreased apoptotic rate (significant at 50 and
Fragaria x ananassa, Meth. extraction at room Vitamin C, anthocyanins,
Anti-inflammatory activities, immunoblotting analysis, 100 mg/L); Increased ARE-antioxidant enzymes expression, [81]
var. Alba fruits temperature, infusion flavonoids
determination of mitochondrial respiration and reduced NO and inflammatory cytokines production (at 50
extracellular acidification rate in cells and 100 mg/L) to control levels
maintained hepatocellular membrane, structural integrity,
Histological analyses, determination of
Anti-inflammatory, Fragaria chiloensisssp. attenuated hepatic oxidative stress, and inhibited
Aq. extracts transaminases, cytokines, F2-isoprostanes, and Phenolic content [82]
hepatoprotective Chiloensis fruits inflammatory response in LPS-induced liver injury;
glutathione assays
downregulation of cytokines (TNFa, IL-1β, and IL-6)
In vivo: quantification of the leukocyte content,
exudate concentration, MPO and ADA
Inhibition of the carrageenan-induced leukocyte influx to Phenolic compounds,
Fragaria x ananassa Ultrasonic-assisted, acidified activities, nitric oxide products, TNF-α and IL-6
Anti-inflammatory the pleural cavity; reduction of myeloperoxidase activity, anthocyanins (particularly [83]
var. Camarosa fruits meth. extraction, separation levels; in vitro: MTT assay, measurement of
exudate concentration, NO levels. pelargonidin-3-O-glucoside)
nitric oxide products, TNF-α and IL-6 levels,
western blot analysis
Ultrasound-assisted
Fragaria x ananassa MTT assay, ROS, NO levels, effects on
Anti-inflammatory, extraction, acidified meth.: ROS reduction, suppression of IL-1β, IL-6 and iNOS gene Polyphenolic compounds,
500
var. San Andreas inflammatory markers and on skin fibroblast [84]
wound healing aq. (80:20); separation of expressions; enhanced skin fibroblast migration especially anthocyanins
fruits migration
different fractions
Fragaria vesca leaves Centrifugation extraction 6–9 mm inhibition zones for leaves, 5–9 mm for roots
Anti-microbial Disc diffusion assay Phenolic compounds [85]
and roots with meth.: aq. (80:20) (depending on S. aureus strain)
Hydroalcoholic extraction, Good inhibition potential at 25 mg/mL, better effect for the
Anti-microbial Fragaria vesca leaves Disc diffusion assay Ellagitannins [86]
separation ellagitannin-enriched fraction
Fragaria x ananassa degranulation suppression (95–60% inhibition for
Methanol fraction of fruits Antigen-stimulated degranulation in RBL-2H3 Best results -
Anti-allergenic var. Minomusume linocinnamarin, cinnamic acid, chrysin, kaempferol, [87]
juice (obtained by squeezing) cells phenylpropanoid glycoside
fruits trans-tiliroside)
Fragaria x ananassa Compounds isolated from IC50 values better than the positive control (acarbose) for
Anti-diabetic α-glucosidase inhibitory activity Individual compounds [20]
var. Falandi fruits eth. extracts nine compounds (537.43 to 25.39 μM)
Fragaria ×ananassa
var. Amaou, entire
plant (red fruit, Anti-lipase assay, adipocyte differentiation
Anti-obesity,
green fruit, red Eth. or aq. room temperature inhibition assay, melanogenesis inhibition assay, Crown, stolon leaf and flowers extracts exhibited the
anti-allergy, Total phenolic content [35]
calyx, green calyx, extraction β-hexosaminidase inhibition assay, tyrosinase highest effects
skin-lightening
flower, leaf, stolon, inhibition assay
stolon leaf, stem,
crown and root)
Reduction of uric acid at 100 mg/mL
Fragaria x ananassa Supercritical CO2 extraction
Antihyperuricemic Uric acid production in AML12 hepatocytes (96 mmol/2 h/mg protein), compared with the control Kaempferol, quercetin [10]
cv. Tochiotome leaves with different entrainers
(16,096 mmol/2 h/mg protein)
Cytotoxic, Fragaria x ananassa Ex vivo: cell viability assay; in vivo: developing Cytotoxic on cancer cells, blocked the proliferation of
Meth. extraction Phenolic compounds [89]
anti-proliferative fruits tumor size determination tumor cells
Table 4. Cont.
Action Plant Extraction Method Assay Results Responsible Compounds Ref.
reduction of cell proliferation, lowering of the intracellular
Fragaria x ananassa Acidified hydro-eth. Transglutaminase assay and polyamine
Antineoplastic levels of polyamine, enhancement of tissue Anthocyanins [90]
var. Pajaro fruits extraction detection, immunoblot analysis
transglutaminase activity
Hydroalcoholic extract at Effects on HepG2 cells—cell viability
Inhibition of HepG2 cell viability IC50 = 690 mg/L
Fragaria vesca L. room temperature, assessment, cell proliferation, cell cycle and cell
Cytotoxic (extract)/113 mg/L (fraction); fraction induced necrosis and Ellagitannins [33]
leaves ellagitannins-enriched death analysis, Western blot analysis,
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apoptosis, influenced the cellular proteolytic mechanisms

fraction proteasome chymotrypsin-like activity
anthocyanins,
Ultrasound-assisted Histological studies, Western blot analysis, Decreased tumor incidence, decreased levels of TNF-α,
Lyophilized Fragaria ellagitannin/ellagic
Chemopreventive extraction with acidified PGE2 measurement, and nitrate/nitrite IL-1β, IL-6, COX-2 and iNOS, inhibition of the [95]
x ananassa fruits acid/ellagic acid derivatives
acetone colorimetric assay phosphorylation of PI3K, Akt, ERK, and NFκB
flavonols
Hydroalcoholic extracts Under 50% viable cells for colorectal adenocarcinoma and
Fragaria x ananassa
Cytotoxic (meth., eth., isopropanol) Cell proliferation, cell viability colon adenocarcinoma upon treatment with extracts Polyphenols [91]
leaves
from in vitro cell suspension containing 0.29 mM ethoxy-dihydrofuro-furan
where: ADA—adenosine-deaminase; Akt—Protein Kinase B; aq.—water (aqueous); CAT—catalase; COX-2—cyclooxygenase-2 enzyme; ERK—extracellular signal-regulated kinase;
eth—ethanol; GSH—glutathione; HepG2—human liver cancer cell line; IC50— half maximal inhibitory concentration; IL-1β—Interleukin 1 beta cytokine protein; IL-6—interleukin
6; iNOS—inducible nitric oxide synthase; meth.—methanol; MPO—myeloperoxidase; MTT—3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NFκB—nuclear factor
kappa-light-chain-enhancer of activated B cells; NO—nitric oxide; PGE2 —Prostaglandin E2 ; PI3K—phosphatidylinositol 3-kinase; RBL—rat basophilic leukemia cells; ROS—reactive
oxygen species; SOD—superoxide dismutase; TNF-α—tumor necrosis factor alpha;.
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4. Current Limitations and Future Perspectives

In spite of the wide spread of the Fragaria genus, few species represent the subject of the last decade
scientific research, with many works focused on the composition and bioactivities of wild and garden
species. Although those species, with certain commercial value, represent a very valuable source of
different classes of polyphenols (including proanthocyanidins, anthocyanins, ellagitannins, flavonoids,
phenylpropanoids, stilbenes, phenol glycosides, and dihydrochalcones) and thus possessing important
nutritional value [96], the researchers should also focus on less-studied species, native to different parts
of the world. This could represent an important opportunity for future studies. Also, the evaluation of
other possible application (such as cosmetic products) could represent an interesting area of research.
As an example, Sikora et al. [59] used supercritical CO2 extraction for obtaining strawberry seeds
(achenes) oil and applied it (in varying concentration from 0.5–2%) for the development of shower/bath
cosmetics with good skin-moisturizing properties, without influencing the stability of the products.
A drawback that limits the potential beneficial effects of the strawberries’ consumption is
represented by their processing. As previously mentioned, the thermal treatment reduces both the
bioactive compounds and their biological activities. In this area, new protective coatings obtained using
nanotechnological approaches were proposed for increasing their shelf-lives [97], or as post-harvest
treatments, alternatives to the classical thermal treatments currently applied [98,99].
As emerging from the literature study, most of the research is performed via classical extraction
methods in order to align with traditional uses of the species (infusion, decoction), and only few are
including modern extraction and separation techniques (such as countercurrent chromatography).
In this area, would be beneficial to evaluate the use of modern methods of extraction/separation of
biological active compounds [100].
The rich anthocyanin content of the genus seems to offer promising compounds for very important
applications, such as anti-cancer or chemoprotective agents. Another surprising aspect emerging from
the reviewed works is the relatively few studies concerning the potential of Fragaria species towards
anti-microbial applications. Although the literature abounds in examples regarding the anti-microbial
potential of different plants extracts, it appears that the Fragaria could still offer some surprising results
in this domain (considering its rich composition). Also, the application of Fragaria extracts in other areas,
such as nanotechnology, is only “surface-scratched” at this moment. For example, Demirbas et al. [101]
evaluated anthocyanins-rich berry extracts (including strawberry) for the phytosynthesis of silver
nanoparticles and evaluated their antioxidant and anti-microbial potential. Although the results of
the phytosynthesis process were promising (smallest dimensions, compared with the blackberry or
raspberry extracts (35 nm), and with less aggregation, the antimicrobial effect was relatively poor (and
only on B. cereus). The field of nanotechnology in general, and of nanoparticles phytosynthesis in
special, represents a continuously increasing domain, so the extracts obtained from different species of
the genus could demonstrate their usefulness in this area [102]. Other different nanoparticles could
be obtained using the extracts, as well with tuned properties, using enrichment of the extracts [103].
Correlated with the anti-microbial properties, the potential toward nanotechnology could also lead to
the development of other materials (such as polymeric encapsulated nanoparticles or even natural
extracts) for different anti-microbial applications or for wider applications in increasing the quality of
food products [104,105].
Finally, as a more general remark, the absence of standardized methods for the evaluation of
different potential applications represents a major draw-back in the comparison of the results presented
by different authors.
5. Conclusions
Fragaria represents a widely spread genus, with species encountered all over the world. The
current study aimed to present the progress made in the last decade in the study of the composition
and potential applications of the species belonging to the Fragaria genus. However, in spite the
wide spread of its species, only a few represents the subject of current research. The literature study
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revealed that three species represent the major subject of research, respectively the wild, garden and
beach strawberry.
Used in traditional medicine especially as an anti-inflammatory adjuvant, the scientific research
supports this application, as well as several other potentially important uses, for example as a
chemoprotective agent.
The composition of the genus, rich in polyphenolic compounds in general, and in anthocyanins
in particular, suggests its possible application in multiple other areas. The relatively under-study of
the genus (and the severe lack of literature for some of the species) offers in turn an opportunity for
future research. At the same time, elucidation of the composition and properties of the commercially
valuable products represents a very important aspect, as the characteristics of such a widely consumed
product should be thoroughly elucidated.
Author Contributions: R.C.F., A.O., N.E.B., G.T., and I.F. contributed to data collection and analysis, and
manuscript design. R.C.F., G.T., and I.F. prepared and revised the manuscript, and are the main authors of the
study. All authors have read and agreed to the published version of the manuscript.
Funding: The authors gratefully acknowledge the support obtained through the project SusMAPWaste, SMIS
104323, Contract No. 89/09.09.2016, from the Operational Program Competitiveness 2014-2020, project co-financed
from the European Regional Development Fund.
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Sample Availability: Not available..
508
molecules
Article
In Vitro and In Situ Characterization of the Intestinal
Absorption of Capilliposide B and Capilliposide C
from Lysimachia capillipes Hemsl
Xu Zhang 1 , Xiao Cheng 2 , Yali Wu 1 , Di Feng 1 , Yifan Qian 1 , Liping Chen 1 , Bo Yang 1 and
Mancang Gu 1, *
1 College of Pharmacy, Zhejiang Chinese Medical University, Hangzhou 311402, China;
zhangxu@zcmu.edu.cn (X.Z.); wuyali@zcmu.edu.cn (Y.W.); fengdi@zcmu.edu.cn (D.F.);
qianyifan@zcmu.edu.cn (Y.Q.); LipingChen@zcmu.edu.cn (L.C.); Yangbo@zcmu.edu.cn (B.Y.)
2 Huzhou Institute for Food and Drug Control, Huzhou 313000, China; cheng_xiao1981@163.com
* Correspondence: gmancang@zcmu.edu.cn; Tel.: +86-571-617-68158; Fax: +86-571-866-13606
Academic Editor: Raffaele Capasso

Abstract: The goal of this investigation was to determine the processes and mechanism of intestinal
absorption for capilliposide B (CAPB) and capilliposide C (CAPC) from the Chinese herb, Lysimachia
capillipes Hemsl. An analysis of basic parameters, such as drug concentrations, time, and behavior in
different intestinal segments was analyzed by liquid chromatography-tandem mass spectrometry
(LC-MS). The susceptibility of CAPB and CAPC to various inhibitors such as P-glycoprotein (P-gp)
inhibitor (verapamil); multidrug resistance-associated protein 2 (MRP2) inhibitor (indomethacin);
cytochrome P450 protein 3A4 (CYP3A4) inhibitor (ketoconazole); and the co-inhibitor of P-gp, MRP2
and CYP3A4 (cyclosporine A) were assessed using both caco-2 cell monolayer and single-pass
intestinal perfusion (SPIP) models. As a result, CAPB and CAPC are both poorly absorbed in the
intestines and exhibited segment-dependent permeability. The intestinal permeability of CAPB and
CAPC were significantly increased by the co-treatment of verapamil, indomethacin. In addition, the
intestinal permeability of CAPB was also enhanced by ketoconazole and cyclosporine A. It can be
concluded that the intestinal absorption mechanisms of CAPB and CAPC involve processes such
as facilitated passive diffusion, efflux transporters, and enzyme-mediated metabolism. Both CAPB
and CAPC are suggested to be substrates of P-gp and MRP2. However, CAPB may interact with the
CYP3A4 system.
Keywords: capilliposide B; capilliposide C; intestinal absorption; caco-2 cell; single-pass intestinal

perfusion; liquid chromatography-tandem mass spectrometry
1. Introduction
The oral delivery route for the therapeutic administration of drugs remains one of the most
desirable and important routes in drug delivery. With a combination of increased patient compliance,
safety and ease of administration, orally-delivered drugs often offer greater clinical efficacy than other
options, particularly in minimizing potential infections [1]. However, many therapeutics are unable to
utilize this sought-after delivery route due to the poor solubility and instability of many compounds
in gastrointestinal fluids. Furthermore, properties such as rapid metabolic elimination, low intestinal
permeability, and efflux by protein transporters are major obstacles to the oral delivery of various
compounds [2]. In recent years, there have been many attempts to overcome these hurdles described
by the aforementioned limitations on oral administration.
The efflux transporters, such as the adenosine triphosphate-binding cassette transporters (ABC
transporters), play a critical role in the absorption and distribution of drugs in the intestinal

Molecules 2019, 24, 1227
organs. These transporters are important factors dictating the bioavailability of the administered
drug in addition to the enzymes responsible for drug metabolism [3,4]. ABC transporters such as,
P-glycoprotein (P-gp), multidrug resistance-associated proteins (MRPs) and breast cancer resistance
protein (BCRP) are capable of excreting drugs in the cellular compartment into the interstitial
compartment, decreasing bioavailability [5]. Many active components of traditional Chinese
medicine (TCM), such as saponins (akebia saponin D, ginsenoside Rh2, araloside A, the total
saponins of Mao-Dong-Qing), flavones (apigenin, epimedins) and alkaloids (reserpine, vincristine,
vinblastine) [6–12] have been shown to be substrates of efflux transporters, which negatively affect the
absorption of drugs in the intestine and result in poor bioavailability.
Aside from ABC transporters, drug metabolism enzymes are also critical factors in determining
the bioavailability of drugs, among which the cytochrome P450 enzyme (CYP450) plays a major
role [13]. The CYP3A4 subset of CYP450 accounts for 30% of the total CYP450 enzyme system [14,15]
and is responsible for the bio-transformation of several drugs with a key role in pharmacological effects.
Several permeability models have been employed to understand mechanisms of intestinal
absorption, including an in vitro epithelial cell monolayer model such as caco-2 monolayer model and
MDCK-MDRI epithelial cell monolayer model [16,17], in vitro everted intestinal sac [18], in vivo
or in situ intestinal perfusion models [19], and in vivo pharmacokinetic models. Among these
permeability models, the caco-2 cell monolayer model and in situ intestinal perfusion (SPIP) technique
are considered as the gold standard in intestinal permeability research and have been wildly utilized
to predict intestinal permeability [20,21]. Caco-2 cells express active transport systems, such as P-gp
and MRPs [5,22]. However, because of its low expression levels of P450 enzymes and the absence of a
protective mucus layer, the cell lines cannot be used for studying the interaction of transporters with
underlying metabolic actions.
Lysimachia capillipes Hemsl, a Chinese herb and medicinal plant, is widely used as a remedy
for the treatment of colds and arthritis as observed for Cannabis sativa extracts [23,24]. Recently,
pharmacological investigations revealed that capilliposide B (CAPB) and capilliposide C (CAPC),
the major components of saponins isolated from the Lysimachia genus [25], exhibit an inhibitory
effect on cell proliferation in various cancers, such as esophageal cancer [26], human non-small cell
lung cancer [27], prostate cancer [28], and nasopharyngeal cancer [29]. Although CAPB and CAPC
possess anticancer activity, their poor intestinal absorption obstructs further applications. This poor
performance may be attributed to two key factors. On one hand, systemic exposure of CAPB and CAPC
was low with significant variation among individuals after oral administration. This is likely a result of
its extensive biotransformation in the gastrointestinal tract [30]. On the other hand, the low intestinal
permeability of CAPB and CAPC may also serve as a critical barrier, resulting in poor bioavailability
and low exposure in tissues after oral administration [31]. However, the specific mechanisms which
affect the permeability of CAPB and CAPC through the intestinal epithelium are unknown. Thus, a
systemic study is required to investigate the intestinal absorption of CAPB and CAPC.
Therefore, the primary aim of this study was to investigate the process and mechanisms by which
CAPB and CAPC are absorbed by intestinal cells using caco-2 cell monolayer and in situ intestinal
perfusion models. The effect of parameters such as drug concentration, transport time and behavior
in different intestinal segments were analyzed. Furthermore, the susceptibility of CAPB and CAPC
absorption to treatment with various inhibitors, such as P-gp inhibitor (verapamil), MRP2 inhibitor
(indomethacin); CYP3A4 inhibitor (ketoconazole); and the co-inhibitor of P-gp, MRP2 and CYP3A4
(cyclosporine A) were also assessed.
2. Results
2.1. Liquid Chromatography-Tandem Mass Spectrometry Analysis Method Validation

The concentration of CAPB and CAPC across caco-2 cell monolayer was detected by liquid
chromatography-tandem mass spectrometry (LC-MSn). The total ion chromatogram and product ion
510
Molecules 2019, 24, 1227
mass spectra of CAPB, CAPC and dioscin (IS) are shown in Figure 1, while the standard curves of
CAPB and CAPC over the concentrate range of 1.0–5000 ng/mL are shown in Figure S1. The equations
of the regression line were y = 40.84x − 8.1502 (r2 = 0.998) for CAPB (over 1–250 ng/mL), y = 64.736x
− 147.9 (r2 = 0.998) for CAPB (over 250–5000 ng/mL), y = 38.961x − 11.532 (r2 = 0.987) for CAPC (over
1–250 ng/mL), and y = 68.756x − 104.59 (r2 = 0.994) for CAPC (1–5000 ng/mL). Extraction recoveries
at concentrations of 10, 150 and 3000 ng/mL were determined to be 91.6%, 98.41% and 98.44% for
CAPB; and 82.05%, 96.65% and 81.65% for CAPC, respectively. The matrix effect of CAPB and CAPC
was between 1.01 and 1.11 (RSD < 3.3%) as evaluated internally by a standard-normalized matrix
factor. Intra-day and Inter-day variations were both less than 4%.
Figure 1. Total ion chromatogram (top panel), product ion mass spectra (middle panel) and the multiple
reaction monitoring (MRM) transitions of the deprotonated molecular ions mass spectrogram (bottom
panel) of capilliposide B (CAPB, A), capilliposide C (CAPC, B) and dioscin (IS, C). The chromatograms
monitoring of CAPB was at m/z 1197.0→1060.6, CAPC at m/z 1183.0→1022.7 and IS at m/z
869.5→725.0.
2.2. The Characterization of Caco-2 Cell Monolayer

The integrity of the monolayer was evaluated by measuring the trans-epithelial electrical
resistance (TEER) and phenol red permeability studies. In our data, the monolayer displayed a
TEER values >300 Ωcm2 andthe apparent permeability (Papp , cm/s) values of Phenol <105 cm/s after
growing for 20 days. These results indicate that caco-2 cell monolayer may be used for permeability
studies. In addition, cell viability was verified by MTT assay. As shown in Figure S2, CAPB and CAPC
less than 40 μg/mL did not inhibit cell growth significantly. However, a concentration of CAPB and
CAPC over 40 μg/mL resulted in significantly decreased cell viability. Hence, 10, 20 and 40 μg/mL of
CAPB and CAPC was selected as the testing concentrations for the drug transport study.
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2.3. The Characterization of the Intestinal Permeability Features of CAPB and CAPC In a Caco-2
Cell monolayer
A caco-2 cell monolayer model was used to explore the intestinal permeability features of CAPB
and CAPC. Firstly, the Papp values were measured at different drug concentrations (10, 20, 40 μg/mL),
and the Papp of CAPB and CAPC were all found to be less than 2 × 10−6 cm/s, as shown in Figure 2A,B.
Our data also showed that Papp values increased as the CAPB and CAPC concentration increased
to high and medium concentrations. Both CAPB and CAPC displayed a significant increase in
permeability compared to lower concentrations (p < 0.05). However, no significant difference was
found between high concentrations and medium concentrations of CAPB and CAPC (p > 0.05).
Furthermore, the Papp values obtained after incubation with CAPB and CAPC for 45, 60 and 90 min
across caco-2 cell monolayer in the AP-BL and BL-AP direction is presented in Figure 2C. It was found
that Papp values showed an upward trend over time. As shown in Figure 2D, the Papp (BA)/Papp (AB)
values (efflux ratio values, ER values) of CAPB and CAPC at different initial drug concentrations (10,
20, 40 μg/mL) were more than 1.0 and less than 1.5.
Figure 2. Bidirectional transport studies of capilliposide B (CAPB, A), capilliposide C (CAPC, B) at

different initial drug concentrations (10, 20 and 40 μg/mL). p < 0.05 (*), comparison with the 10 μg/mL
capilliposides (CAPs) group. The apparent permeability (Papp , cm/s) values of CAPB and CAPC at 0,
45, 60 and 90 min (C). The Papp (BA)/Papp (AB) values of CAPB and CAPC were at different initial
drug concentrations (10, 20 and 40 μg/mL) (D). All results are expressed as mean ± S.D. (n = 3).
2.4. The Role of P-gp, MRP2 and CYP3A4 on CAPB and CAPC Transport Across Caco-2 Cell Monolayer
It was also speculated that the efflux transporter and metabolism enzyme plays an important role
in the permeability of CAPB and CAPC across caco-2 cell monolayer as shown in Figure 3. Compared
with treatment using CAPs alone, for the co-treatment with either P-gp inhibitor (verapamil); MRP2
inhibitor (indomethacin); or the co-inhibitor of P-gp, MRP2 and CYP3A4 (cyclosporine A), the Papp
values of CAPB increased about 25 times (p < 0.01), 11 times (p < 0.05) and 10 times (p < 0.05),
respectively, on the AP-BL side and around 30 times (p < 0.01), 10 times (p < 0.05) and 10 times
(p < 0.05), respectively on the BL-AP side. The Papp values of CAPC on the AP-BL side were also
significantly increased up to 11 times (p < 0.05) and 16 times (p < 0.01) in the presence of verapamil
and indomethacin, respectively. However, in the presence of cyclosporin A, the permeability of CAPC
showed only a small increase (p > 0.05), while CAPB showed a 10-fold increase (p < 0.05). Therefore,
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the in vitro transport data indicates that CAPB and CAPC may be the substrate of the efflux protein
P-gp and MRP2; moreover CAPB may also be affected by CYP3A4.
Figure 3. The effect of different factors on the transportation of capilliposide B (CAPB) and capilliposide
C (CAPC) across caco-2 cell monolayer. The data are presented as the apparent permeability (Papp , cm/s).
Effect of P-glycoprotein (P-gp) inhibitor (verapamil hydrochioride); multidrug resistance-associated
protein 2 (MRP2) inhibitor (indomethacin); and the co-inhibitor of P-gp, MRP2 and cytochrome P450
protein 3A4 (CYP3A4) on caco-2 cell monolayer for CAPB (A) and CAPC (B). p < 0.05 (*), p < 0.01 (**),
comparison with control. All results are expressed as mean ± S.D. (n = 3).
2.5. The Characterization of the Intestinal Permeability of CAPB and CAPC In Rats
The SPIP model was used to further explore the intestinal permeability features of CAPB and
CAPC in rats. Firstly, we determined the stability of CAPB and CAPC in the Krebsringer buffer (K-R
buffer) across different pH values. As shown in Figure 4A, CAPB and CAPC were more stable at
pH 5.0 and pH 6.55 than pH 7.43. In other words, CAPB and CAPC were stable in a weakly acidic
environment. Because the pH of intestinal juice was close to 6.55, we used a K-R buffer at pH 6.55 as
the perfusion solution.
The effective permeability (Peff , cm/s) and absorption rate constants (ka, s−1 ) values of CAPB and
CAPC were measured at different drug concentrations (20, 50 and 80 μg/mL). As shown in Figure 4B,
the Peff and Ka values of CAPB showed a slight decline between 20 and 50 μg/mL. However, a
significant increase was seen at 80 μg/mL of drug concentration. However, the Peff and Ka values of
CAPC showed a slight increase between 20 to 50 μg/mL followed by a significant decrease (p < 0.05)
as drug concentration increased.
Subsequently, the Peff and Ka values of CAPB and CAPC in the different intestinal segments
including duodenum, jejunum and ileum were assessed. As shown in Figure 4C, the order of Peff and
Ka values of CAPB in three different intestinal segments was duodenum > jejunum > ileum. The order
of CAPC followed the same pattern as CAPB. The permeability in the duodenum was significantly
greater than that in jejunum and ileum, respectively (p < 0.05). Our data demonstrated that CAPB and
CAPC may exhibit segmental-dependent permeability and were best absorbed in the duodenum.
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Figure 4. The characterization of the intestinal permeability of capilliposide B (CAPB) and capilliposide
C (CAPC) in single-pass intestinal perfusion (SPIP) model. The stability of CAPB and CAPC were
measured at different pH values (pH 5, pH 6.55, and pH 7.34, respectively) (A). The absorption rate
constants (ka, s−1 ) values (B) and effective permeability (Peff , cm/s) values (C) for CAPB and CAPC
at different initial drug concentrations (20 μg/mL, 50 μg/mL, and 80 μg/mL, respectively). p < 0.05
(*) compared with with the group at 80 μg/mL. The Ka values (D) and Peff values (E) of CAPB and
CAPC obtained from the duodenum, jejunum, and ileum in SPIP models. p < 0.05 (*) compared with
the duodenum. All the results are expressed as mean ± S.D. (n = 3).
2.6. The Role of P-gp, MRP2, and CYP3A4 on Intestinal Permeability of CAPB and CAPC
To further confirm the role of P-gp, MRP2 and CYP3A4 on the intestinal permeability of CAPB and
CAPC, the Peff and Ka values were measured in the presence of verapamil, indomethacin, ketoconazole,
and cyclosporine A. As the findings in Figure 5 indicate, The P-gp inhibitor, verapamil, resulted in
a 1.5-fold increase (p < 0.05) on the intestinal transport of CAPB and CAPC. In the presence of
indomethacin, the Ka and Peff values increased significantly (p < 0.05). The Peff value of CAPB showed
an even more significant increase at 4.2 fold while the Peff of CAPC only increased 2 fold. It is worth
noting that the Peff values of CAPB and CAPC are equal in the absence of an inhibitor. Therefore it is
indicated that the affinity between CAPB and MRP2 is stronger than that of CAPC.
In the presence of the CYP3A4 inhibitor, ketoconazole, CAPB showed a two-fold higher
permeability than CAPB alone (p < 0.05). However, CAPC exhibited minimal alteration of permeability
in the presence of ketoconazole (p > 0.05). The substrate of CYP3A4 protein is therefore elucidated to
be CAPC over CAPB. In the presence of cyclosporin A, a co-inhibitor of P-gp, MRP2, and CYP3A4, the
permeability of CAPC increased slightly (p > 0.05), while CAPB increased around two-fold (p < 0.05).
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Figure 5. The effect of different factors on intestinal absorption of capilliposide B (CAPB) and
capilliposide C (CAPC). The data are presented as absorption rate constants (ka, s−1 ) values (B)
and effective permeability (Peff , cm/s). P-glycoprotein (P-gp) inhibitor (verapamil hydrochioride);
multidrug resistance-associated protein 2 (MRP2) inhibitor (indomethacin); and the co-inhibitor of
P-gp, MRP2 and cytochrome P450 protein 3A4 (CYP3A4) (cyclosporine A); and CYP3A4 inhibitor
(ketoconazole) on small intestinal absorption of CAPB (A) and CAPC (B). p < 0.05 (*), in comparison
with control. All the results are expressed as mean ± S.D. (n = 3).
3. Discussion
CAPB and CAPC, two major compounds of L. capillipes Hemsl, exhibit significant cytotoxicity
against many human cancer cell lines, including prostate cancer cell PC3 and DU145, along with
nasopharyngeal cancer CNE-2 cells; ovarian cancer SK-OV-3 and A2780 subtypes; and lung cancer
PC-9, A549, H1299, and H460 cells [25–30]. Moreover, CAPB and CAPC were found to inhibit tumor
growth without inducing significant toxicity to hepatic or renal tissues in a mouse model [26,27,32].
Previously, studies have evaluated the pharmacokinetics, tissue distribution, and excretion of CAPB
and CAPC systematically [31,33,34]. Both compounds showed poor bioavailability and low exposure
in tissues after oral administration [31]. However, the intestinal absorption mechanics and underline
transport systems of CAPB and CPAC were still unclear. In this study, we revealed that both CAPB and
CAPC showed low permeability across intestinal epithelial cells. However, the intestinal absorption of
CAPB and CAPC may involve facilitated passive diffusion, and may be affected by efflux transporters
and metabolic enzymes.
In a caco-2 cell model, our data showed that the Papp of CAPB and CAPC were both less than
2 × 10−6 cm/s, which represents a low permeability of compounds in vitro. The amount of CAPB
and CAPC across the cell monolayer accumulated as concentration and time increased. Meanwhile,
the ER values of CAPB and CAPC were between 1.00 and 1.50, indicating that facilitated diffusion
and efflux mechanisms may be involved in the intestinal epithelium transportation of CAPB and
CAPC [22,35]. On the other hand, Peff values of CAPB and CAPC were both much less than 0.2 ×
10−4 cm/s in a single-pass intestinal perfusion model, which confirmed the in vitro transportation
results. Interestingly, we found that the Peff of CAPB and CAPC fluctuated only slightly from 20
to 50 μg/mL but were drastically restrained at 80 μg/mL in an in situ intestinal infusion model.
A possible explanation was that the facilitated passive diffusion may involve intestinal transport,
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similar to the transport mechanism of sodium taurocholate [36]. However, we did not assess the
transportation features of CAPB and CAPC at 80 μg/mL in the caco-2 monolayers model, as the drug
showed cytotoxicity when the concentration of CAPB and CAPC was above 40 μg/mL. Because the
Papp of CAPB and CAPC increased linearly from 10μg/mL to 40 μg/mL in the caco-2 monolayer model,
we wanted to further characterize the intestinal permeability of compounds at higher concentrations
in the SPIP model. In addition, this data showed that Peff and Ka values of CAPB and CAPC in the
duodenum were significantly higher than that in the jejunum and ileum. This may be related to pKa of
the drug, the degree of dissociation, the pH in the four intestinal segments, the relative abundance of
microvilli and villi, and the distribution of efflux transporters and uptake transporters [37].
The P-gp and MRP2 proteins are two major efflux transporters which affect the absorption of drugs
in the intestines [38,39] and are shown to be highly expressed in intestinal epithelium as well as on the
membranes of caco-2 cells [4]. Numerous studies have shown that active components of traditional
Chinese medicine are the substrates of P-gp and MRP2 proteins, which are likely explanations for the
reduced intestinal absorption of oral ginsenoside Rh2, a member of the saponins [40]. As examples,
akebia saponin D demonstrates poor intestinal absorption as a result of MRPs in the intestine [41].
Ginsenoside Rh2 [42,43] and araloside A [22] have poor intestinal absorption, because they are both
substrates of P-gp.
This in vitro transport data and in situ intestinal infusion data showed that the inhibition of P-gp
and MRP2 activity could significantly enhance the permeability of CAPB and CAPC across intestinal
epithelia. These results indicated that CAPB and CAPC, also members of the saponins family, may be
substrates for the efflux protein P-gp and MRP2. The intestinal permeability of CAPB and CAPC may,
at least partly, be limited by P-gp and MRP2.
In addition to the ABC transport protein, metabolic enzymes play a critical role in the intestinal
absorption of drugs [44]. For example, CYP3A4 was shown to also influence ginsenoside Rh2 intestinal
absorption as it was the predominant enzyme responsible for the oxidation of ginsenoside Rh2 [43,45].
The best example perhaps, is with paclitaxel, whose poor bioavailability is caused by a combination of
poor water solubility, P-gp efflux, and CYP3A4 metabolism. In an excretion study of CAPB, it was
proven that CAPB experienced extensive metabolism prior to excretion [34]. Likewise, CAPC also
demonstrated extensive metabolism in rat intestinal microflora and a strong anticancer activity [46].
Prior studies also systematically characterized 19 metabolites of CAPB and CAPC in mice and proposed
a major metabolic pathway (deglycosylation and esterolysis) following oral dosing [32]. However,
caco-2 cells do not always express the appropriate amount of metabolic enzymes, such as P450
enzymes, which can affect the uptake of certain drugs that are transported through metabolic-specific
pathways [4,47]. Because of its low expression levels of P450 enzymes, the role of CYP3A4 was
investigated primarily by adding the co-inhibitor of P-gp, MRP2 and CYP3A4 (cyclosporin A) in the
caco-2 cells and SPIP model. Furthermore, the inhibitor of CYP3A4 (ketoconazole) was added to the
SPIP model only for the reasons mentioned above. Our in vitro transport data and in situ intestinal
infusion data showed that the inhibition of CYP3A4 could significantly enhance the permeability of
CAPB across the intestinal epithelia. It was found that CAPB may be the substrate of CYP3A4, but
CAPC may not.
While the results are promising, the current study still suffers from certain limitations.
The intestinal absorption of drugs was affected by numerous factors, such as transporters, and
intestinal microflora [37]. However, the scope of this study sought to focus on the permeability
features of CAPB and CAPC, along with the major transporters and metabolic enzymes in intestinal
epithelium including P-gp, MRP2 and CYP3A4 [5,13]. While previous studies have already evaluated
the pharmacokinetics, distribution, intestinal metabolism, and excretion of CAPB and CAPC [31–34],
the absorption characteristics of CAPB and CAPC in the intestinal tract were largely unknown.
Additionally, classical inhibitors were used to test the possible drug transport mechanisms instead of a
knockout cell or mouse model. This was chosen to make the study more straightforward. In future
studies, our group seeks to focus on the effects of intestinal microflora, along with other transporters
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and metabolic enzymes on the intestinal absorption mechanisms of CAPB and CAPC. A gene-editing
protocol may also be employed to further investigate the transport mechanisms of CAPB and CAPC.
4.1. Materials
Capilliposide B (C58 H96 O24 , CAPB), capilliposide C (C57 H94 O24 , CAPC) and Lysimachia capillipes
Hemsl API (more than 70% total of CAPB and CAPC) were obtained from Professor Tian Jingkui,
College of Biomedical Engineering and Instrument Science, Zhejiang University (Zhejiang, China).
Dioscin (Internal standard, IS, purity ≥ 98%) and Verapamil Hydrochloride were obtained from
National Institutes for Food and Drug Control (Beijing, China). Phenol Red, Ketoconazole, Novobiocin
and Cyclosporin (purity ≥ 98%) were purchased from Shanghai Yuanye Biotechnology Co., Ltd.
(Shanghai, China). The caco-2 human cell line was obtained from the Shanghai Cell Bank of The
Chinese Academy of Sciences. HPLC-grade methanol and acetonitrile and other chemicals were of
analytical grade.
4.2. Liquid Chromatography-Tandem Mass Spectrometry (LC-MS) Analysis

The liquid chromatography system used in this study was an Agilent Technologies model 1290
Infinity (Agilent Technologies, Santa Clara, CA, USA). Separations were carried out using a UItimate
XB-C18 column (250 × 4.6 mm, 5 μm, Phenomenex, Toran, CA, USA) at 40 ◦ C. The mobile phase was
composed of 0.3% formic acid in water as mobile phase A (MA) and acetonitrile as mobile phase B
(MB) using a gradient elution of 49% MB (0–10 min) and 49% to 90% MB from 10–25 min. Separation
was carried out at a flow rate of 1.0 mL/min. The sample injection volume was 10 μL.
An Agilent 6460 Triple Quad mass separometer (Agilent Technologies) equipped with a
Turboionspray source (TIS) was operated in the positive ionization mode with multiple reaction
monitoring (MRM) for LC-MS analysis. The MS parameter was optimized as follows: TIS temperature,
600 ◦ C; ionspray voltage, −4500 V; curtain gas, nitrogen, 30 psi; nebulizing gas, 50 psi; declustering
potential, 135 V for CAPB, 135 V for CAPC and 90 V for dioscin (IS); entrance potential, 10 V; collision
energy 10 eV for CAPB, CAPC and dioscin (IS); collision cell exit potential, 15 V. The following MRM
transition was used: m/z 1197→1060.6 for CAPB, 1182.8→1022.0 for CAPC, 869.5→725.6 for dioscin.
4.3. Caco-2 Cell Culture

Caco-2 cells were routinely cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM)
containing 10% fetal bovine serum (FBS), 1% nonessential amino acids, and 1% penicillin/streptomycin.
Cells were kept at 37 ◦ C in a 90% relative humidity atmosphere containing 5% CO2 . Cells were seeded
onto transwells purchased from Corning Costar Co. (New York, NY, USA) on 12-well plates for
transport studies and uptake studies at 5 × 104 cells per insert. Cells were grown for 20 days. The
integrity of monolayer was evaluated by measuring the trans-epithelial electrical resistance (TEER)
and by phenol red permeability studies.
The effects of CAPs on caco-2 cell viability were checked by 3-(4,5-dimethyl-2-thiazolyl)-
2,5-diphenyl-2-H-tetrazolium bromide (MTT) colorimetric assay [12], which is adapted to analyze cell
proliferation and drug cytotoxicity. Cells with decreased viability are considered to be less metabolically
active and hence will reduce less MTT. In brief, caco-2 cells were seeded at 4 × 103 cells/well into
96-well culture plates and were incubated at 37 ◦ C for 24 h before the assay. The cells were treated
with different concentrations (0, 10, 20, 40, 80, 120, 160 and 240 μg/mL) of CAPs. After 24 h of
incubation, 20 μL of MTT (5 mg/mL) was added to each well and the cells were incubated for another
4 h. After removing the culture medium, 100 μL of dimethyl sulfoxide was added to dissolve the
contents in the plate. Then, the absorbance was measured at 570 and 630 nm (reference wavelength)
using a microplate reader (Multiskan MK3; Thermo Fisher Scientific, Waltham, MA, USA).
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4.4. Permeability Studies Using Caco-2 Cells

DMEM was removed and the monolayer was washed with Hank’s Balanced Salt Solution (HBSS).
The blank HBSS was replaced by 0.4 mL of HBSS containing 40, 20 and 10 μg/mL of drug on the
apical (AP) side and 2.1 mL of HBSS on the basolateral (BL) side. In the AP-BL direction studies,
0.4 mL of HBSS containing 40 μg/mL drug and P-gp inhibitor (53 μg/mL verapamil); MRP2 inhibitor
(17.9 μg/mL indomethacin); or the co-inhibitor of P-gp, MRP2 and CYP3A4 (10 μg/mL cyclosporin A)
were added to the AP side; 2.1 mL of HBSS was added to the BL side. In the BL-AP direction studies,
0.4 mL of HBSS containing 40 μg/mL drug and different inhibitors were added to the BL side, and
2.1 mL of HBSS was added to AP side. Samples (0.4 mL) were taken from the BL or AP side after
45, 60 and 90 min incubation at 37 ◦ C. An additional 0.4 mL of HBSS was added at the same time.
The concentrations of samples were analyzed by LC-MS.
4.5. In Situ Single-Pass Intestinal Perfusion (SPIP) Studies In Rats

Male Sprague–Dawley (SD) rats (280 ± 30 g) were supplied by the Laboratory Animal Center,
Zhejiang Chinese Medical University (Zhejiang, China). All animals were fasted overnight (12–18 h)
with free access to water before experiments. All experiments were performed in accordance with the
guidelines for the care and use of animals as established by the Laboratory Animal Centre, Zhejiang
Chinese Medical University (Zhejiang, China) (approval number: ACXK20150016).
The perfusion experiment was performed as previously reported. In brief, rats were fasted
overnight but permitted to drink water freely the day before the experiments. After being anesthetized,
the rats were placed on the surface of a thermostatic device and maintained at 37 ◦ C. An incision of
approximately 3 cm was made along the midline of the abdominal cavity to expose the contents of the
abdomen. Perfusate entered the duodenum (1 cm below pylorus) and exited from the jejunum (15 cm
from the pylorus). Then the incision was made at both sides of the segment. The intestinal contents
were rinsed with saline preheated at 37 ◦ C, drained with air, and connected to the perfusion system
with the catheter.
One-hundred millitlers of pre-prepared perfusate was taken and preheated at 37 ◦ C. The perfusion
was started at a circulation speed of 1.0 mL/min for 10 min. The flow rate was subsequently increased
to 0.2 mL/min for 30 min to ensure steady-state conditions. Samples were collected in glass tubes
(2 mL/per) at 10-min intervals for 90 min. The samples were filtered through a membrane filter
(0.45 μm) and the concentration of CAPB, CAPC and phenol red were measured by LC-MS. The rest of
the samples were stored at −20 ◦ C for further studies.
4.6. Data Analysis

The apparent permeability (Papp , cm/s) across caco-2 cell monolayer was calculated from the
linear plot of drugs accumulated in the receiver side versus time using Equation (1):

1 dQ
Papp = (1)
C0 A dt
where dQ/dt represents the steady-state flux of the drug on the receiver (serosal in the case of AP-BL
studies or mucosal in the case of BL-AP studies) side, C0 is the initial concentration of the drug in the
donor side, and A is the monolayer growth surface area (4.67 cm2 ). Linear regression was carried out
to obtain the steady-state appearance rate of the drug on the receiver side.
Effective permeability (Peff ) and absorption rate constants (Ka) were calculated using the following
Equations (2), (3) and (4), respectively
C out Cout Cin,phenolred

= × , (2)
C in Cin Cout,phenolred
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−Qln C out /C in
Peff (m/s) = , (3)
2πRL
C out
Ka = Q(1− )πR2 L, (4)
C in
where Cin phenol red and Cout phenol red is equal to the concentrations of phenol red in the inlet
and outlet samples, respectively; C out /C in is the ratio of the outlet and inlet concentration of the
tested drug that has been adjusted for water transport, Q is the perfusion buffer flow rate (0.2 and
0.1 mL/min for rats and mice, respectively), R is the radius of the intestinal segment (set to 0.2 and
0.1 cm for rats and mice, respectively), and L is the length of the intestinal segment.

All experiments were performed in triplicate (minimum) and results were expressed as mean
values ± standard deviation (SD). Statistical comparisons were performed by Student’s t-tests or
one-way analysis of variance (ANOVA) using the SPSS version 22 software. Comparisons between two
groups were analyzed using Student’s t-tests. When a p-value was smaller than 0.05, it was considered
statistically significant. All data were represented for at least three independent experiments.
5. Conclusions
The present study has revealed that CAPB and CAPC are poorly absorbed in the intestines and
likely exhibited segmental-dependent permeability; it can also be found that the intestinal absorption
mechanism of CAPB and CAPC may involve facilitated passive diffusion associated with the efflux
transporters P-gp and MRP2, along with the metabolic enzyme CYP3A4. As a whole, CAPB may be
the substrate of the P-gp, MRP2 and CYP3A4, while CAPC may be the substrate of the P-gp and MRP2,
but not of the CYP3A4. In conjunction with results from previous studies along the direction of CAPB
and CAPC, these results provide updated information concerning the intestinal absorption process
and the possible mechanism of these two compounds.
Supplementary Materials: The following are available online at http://www.mdpi.com/1420-3049/24/7/1227/

s1, Figure S1: The standard curve of CAPB and CAPC across caco-2 cell monolayer was detected by LC-MS.
Figure S2: CAPs inhibited the proliferation of caco-2 cells.
Author Contributions: X.Z. and X.C. wrote and substantively revised the manuscript. X.Z., Y.W. and D.F.
performed the experiments. Y.Q., L.C. and B.Y. analyzed the data. M.G. acted as a supervisor.
Acknowledgments: The work was supported by National Natural Science Foundation of China grants (Grant
No. 81673607, 81303235, 81774011, 81473434); Natural Science Foundation of Zhejiang Province grants (Grant No.
Y19H280009, LY17A040010); as well as the Public Welfare Research Project of Huzhou Science and Technology
Grant (Grant No. 2018GZ24). We appreciate Tian Jinkui from Zhejiang University for giving the Capilliposide B,
capilliposide C and Lysimachia capillipes Hemsl API as kindly gifts.
Conflicts of Interest: The authors report no conflicts of interest.
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molecules
Article
Solvent Fractionation and Acetone Precipitation for
Crude Saponins from Eurycoma longifolia Extract
Lee Suan Chua 1,2, *, Cher Haan Lau 1 , Chee Yung Chew 2 and Dawood Ali Salim Dawood 1
1 Metabolites Profiling Laboratory, Institute of Bioproduct Development, Universiti Teknologi Malaysia,
Skudai, Johor Bahru 81310 UTM, Johor, Malaysia; cherhaan@gmail.com (C.H.L.);
alisalim883@yahoo.com (D.A.S.D.)
2 Department of Bioprocess and Polymer Engineering, School of Chemical and Energy Engineering, Faculty of
Engineering, Universiti Teknologi Malaysia, Skudai, Johor Bahru 81310 UTM, Johor, Malaysia;
chee_yung@live.com.my
* Correspondence: chualeesuan@utm.my; Tel.: +607-5531566

Abstract: Eurycoma longifolia is a popular folk medicine in South East Asia. This study was focused
on saccharide-containing compounds including saponins, mainly because of their medical potentials.
Different organic solvents such as ethyl acetate, butanol, and chloroform were used to fractionate the
phytochemical groups, which were consequently precipitated in cold acetone. Solvent fractionation
was found to increase the total saponin content based on colorimetric assay using vanillin and sulfuric
acid. Ethyl acetate fraction and its precipitate were showed to have the highest crude saponins after
acetone precipitation. The samples were shown to have anti-proliferative activity comparable with
tamoxifen (IC50 = 110.6 μg/mL) against human breast cancer cells. The anti-proliferative activities of
the samples were significantly improved from crude extract (IC50 = 616.3 μg/mL) to ethyl acetate
fraction (IC50 = 185.4 μg/mL) and its precipitate (IC50 = 153.4 μg/mL). LC-DAD-MS/MS analysis
revealed that the saccharide-containing compounds such as m/z 497, 610, 723, 836, and 949 were
abundant in the samples, and they could be ionized in negative ion mode. The compounds consisted
of 226 amu monomers with UV-absorbing property at 254 nm, and were tentatively identified as
formylated hexoses. To conclude, solvent fractionation and acetone precipitation could produce
saccharide-containing compounds including saponins with higher anti-proliferative activity than
crude extract against MCF-7 cells. This is the first study to use non-toxic solvents for fractionation of
bioactive compounds from highly complex plant extract of E. longifolia.
Keywords: Eurycoma longifolia; Simaroubaceae; solvent fractionation; acetone precipitation; saponins;

LC-DAD-MS/MS.
1. Introduction
Eurycoma longifolia has traditionally been used as ethnomedicine by indigenous people from
ASEAN (Association of Southeast Asian Nations) countries to treat many illnesses, such as tertian
malaria, ulcer, syphilis, gonorrhea, and dysentery, as well as to relieve headache, stomachache,
and insect bites [1]. The roots of the plant, which is locally known as Malaysian Ginseng, are popular
mainly because of its aphrodisiac effects [2–4]. Quassinoids are the most extensively studied
phytochemicals from the roots of the plant, mostly C18-20 quassinoids [5]. They are degraded
and highly oxygenated triterpenes, which mainly contribute to the bitter taste of plants of the
Simaroubaceae family. Saponins are another important class of bioactive phytochemicals, but they
are relatively limited in literature for this plant. This could be due to the difficulties in separating and
identifying saponins.

Molecules 2019, 24, 1416
Recently, spectrophotometric techniques have been widely applied to estimate total saponin content,
based on the method proposed by Hiai et al. [6] for plant samples. This method uses strong acids such as
sulfuric acid and perchloric acid to oxidize triterpene saponins and react with vanillin to give a distinctive
red-purple colored complex, which can be measured at the visible range of wavelengths ranging from
473 to 560 nm. The reaction is also dependent upon the structure of ring A in triterpenes [7]. Although
it is a simple assay to estimate total saponins, specific saponin compounds need to be identified by
chromatographic technique. Generally, saponins do not have a chromophore for ultraviolet absorbance
in liquid chromatography. Moreover, saponins exhibit low sensitivity in detectors like refractive index
and evaporative light scattering, partly due to the restriction in solvent of choice and gradient condition.
Although derivatization using 4-bromophenacyl bromide prior to HPLC analysis was proposed by
Slacanin et al. [8], Oleszek et al. [9] and Nowacka and Oleszek [10] found that the derivatized standards
decomposed in a short period of time (48 h) at room temperature. Therefore, liquid chromatography
coupled with mass spectrometry has been the method of choice in the detection and identification of
saponins in recent years. Previously, this combined technique has been used to chemically characterize
saponins from Pulsatilla chinensis [11], Paris polyphylla [12], Chenopodium quinoa [13], Tribulus terrestris,
and Panax ginseng [14], and Quillaja saponaria bark extract [15].
Saponin consists of triterpenoid or steroidal aglycones that are substituted with different number
of sugar moieties or organic acids. Saponins are sometimes called glycosaponins, and the unsubstituted
aglycones are classified as sapogenins, which are usually nonpolar. Sapogenins could be either
triterpenoid (C30) or steroid (C27) aglycones. The hydrophilic sugar moiety and hydrophobic aglycone
of saponins make them act as biological detergents. Sawai and Saito [16] reported that plants often
accumulate triterpenoids, including steroids in their glycosylated form, saponins. Indeed, glycosylation
could stabilize the compounds against thermal degradation during heat processing, and result in
greater bioactivities than their aglycones [17].
According to the Malaysian Standard (MS 2409: 2011) [18], the total glycosaponins of the plant
could make up more than 40% w/v in the freeze dried water extract. This yield is significantly higher
than that value recorded for Panax ginseng, which is only up to 15% of total saponins [19]. Saponins
have been associated with various biological activities, such as anti-inflammatory, cholesterol lowering
and anti-cancer properties [20–22]. Therefore, it is important to investigate saponins in E. longifolia
extract in order to explain its enthopharmacological properties scientifically.
The present study investigated total saponin content in E. longifolia extract, and in its fractions
and precipitates. Because of the complex phytochemicals present in the plant extract, liquid–liquid
extraction was used to partition the target compounds, using different solvents such as ethyl acetate,
butanol, and chloroform. Subsequently, the organic fractions were precipitated in cold acetone to
further recover the compounds. Saponins can be precipitated by lowering the dielectric constant
of medium using acetone. Therefore, this work compared the estimated total saponin in different
organic fractions and their precipitates, as well as highlighting the fragmentation patterns of the
identified saccharide-containing compounds from the fractions and precipitates. The anti-proliferative
activities of ethyl acetate fraction and precipitate were also examined, using human breast cancer
for comparison.
2.1. Effect of Ethanol Concentration on Saponin Content

Different concentrations of ethanol (0–100%) were used to extract phytochemicals from the roots
of E. longifolia. In the present study, aqueous ethanol was selected as the solvent of choice, mainly
due to the low toxicity and high solubility for terpenes and saponins [23]. The yield of crude extract
was around 3.5% using aqueous ethanol ranged from 0–70% (Figure 1). The yield was significantly
decreased when the concentration of ethanol was more than 80%. The reduction of solvent polarity
by increasing ethanol content showed most saponins in the plant extract to have low solubility.
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Molecules 2019, 24, 1416
Hence, E. longifolia roots might have a high content of polar to semi-polar saponins. The diverse
characteristics of saponins are attributed to different functional groups attached to the triterpenoid
(pentacyclic structure) or steroidal (tetracyclic structure) aglycones.
Figure 1. Yield of extraction (line) and total saponins using diosgenin (dot bar) and escin (line bar) as
standard chemicals.
The saponin assay showed that the total saponins of E. longifolia extracts increased proportionally
with the increase of ethanol concentration in the solvent system used for reflux extraction. This also
indicates higher solubility of saponins in ethanol than in water. The results expressed as escin
equivalent or diosgenin equivalent are almost similar. The ratio of both results is near 1 at different
ethanol concentrations. Although diosgenin is not a saponin, the hydroxyl group at C-3 and double
bond at C-5 could react with acidic vanillin under oxidization of sulfuric acid to form chromogen.
The assay involves the condensation reaction of the aldehyde group of vanillin with the hydroxyl
group of triterpenic acid to form red condensates for detection.
2.2. Fractionation by Liquid–Liquid Extraction

Organic solvents such as ethyl acetate, butanol, and chloroform were used to partition crude
extract using the technique of liquid–liquid extraction. The highly polar substances, such as organic
acid, polysaccharides, and proteins, may stay in the aqueous phase, while the other relatively less polar
compounds, including terpenoids and saponins, are partitioned into the organic phase. This explains
the increment of total saponin content after fractionation (Figure 2). Ethyl acetate fraction was found
to have the highest total saponin content among the organic fractions.
Figure 2. Total saponins of organic fractions (solid bar) and precipitates (line bar) expressed as mg
DE/g in different solvent systems.
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Molecules 2019, 24, 1416
The chromatograms shown in Figure 3 show the peaks detected at 254 nm after fractionation.
The chromatographic profiles of the organic fractions are almost similar, but their mass spectra
are significantly different. The chromatographic profiles display the compounds with UV-absorbing
property, whereas compounds like terpenoids and saponins mostly do not have such a property. On the
other hand, the mass analyzer will only detect ionizable compounds in the samples. Ethyl acetate and
butanol fractions were found to have less polar compounds, whereas chloroform seemed to partition
more polar compounds from crude extract. The difference between the organic fractions can also be
seen from the results of colorimetric assays. In line with the colorimetric assay, the butanol fraction
exhibited the lowest peak area.
Figure 3. Chromatograms (black line) and total ion chromatograms (blue line) of mass spectrometer
for organic fractions from ethyl acetate (a), butanol (b), and chloroform (c).
2.3. Cold Acetone Precipitation

Cold acetone precipitation appeared to slightly increase the total saponin content from the organic
fractions. The increment was more significant for butanol fraction. Again, ethyl acetate precipitate
could obtain the highest total saponin content. The fractions were added dropwise into cold acetone,
and precipitate was formed due to the sudden drop of dielectric constant of media. The dielectric
constant (ε) of acetone is 20.7, which is about half the dielectric constant of 70% ethanol (ε = 41.1).
Precipitation occurred, most probably because of the steric hindrance of lipophilic terpenoidal skeleton,
limiting its solubility in acetone. Usually, glycosylated terpenoids or steroids were precipitated in
acetone, thus contributing to higher saponin content.
The chromatograms of the precipitates show peak 1 (10.6 min), peak 2 (10.9 min), peak 3 (11.1 min),
and peak 4 (12.2 min), as illustrated in Figure 4. They are tentatively identified as m/z 427, m/z 239
(β-carboline-1-propionic acid), m/z 497, and m/z 723, respectively. The mass spectra at those retention
times revealed the presence of m/z 497 [2M + HCOOH − H]− , m/z 610 [2M + HCOOH + 113 −
H]− , m/z 723 [3M + HCOOH − H]− , m/z 836 [3M + HCOOH + 113 − H]− , and m/z 949 [4M
+ HCOOH − H]− . In the present study, M was found to be the monomer of 226 amu, which is
formylated hexose. Most probably, the neutral loss of 113 is acylglycerol. The formylated saccharides
have the same fragmentation patterns as the compounds detected in Rhamnus davurica Pall [24] and
Rubia cordifolia L. [25] extracts. Hence, cold acetone was likely to precipitate saccharide-containing
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Molecules 2019, 24, 1416
compounds, as presented in Table S1 (Supplementary Materials). The table lists the product ions and
neutral losses attributed to sugar moieties. The neutral loss of the peaks revealed that the precipitated
compounds were saccharide-containing compounds, including saponins. Since saponins do not have
chromophores for UV detection, their mass spectra are presented in Figure 5. The mass spectra of the
precipitates clearly show the fragment ions, which were mostly ionized saccharides, as intense peaks.
The common sugar fragment ions in the figure are m/z 179 (hexose − H), 225 (hexose + HCOOH − H),
341 (dihexose − H2 O − H), 377 (dihexose + H2 O − H), and 387 (dihexose − H2 O + HCOOH − H).
Negative ionization was also found to be more preferable for the precipitated compounds in this study.
Figure 4. Chromatograms of precipitates in acetone using different organic fractions.
Figure 5. Mass spectra of ethyl acetate (a), butanol (b), and chloroform (c) precipitates.
High performance unsupervised statistical techniques, namely heat mapping and principal
component analysis, were used to classify the huge datasets. The heat map explains that the number of
metabolites precipitated from butanol fraction was less than the other two organic fractions (Figure 6a).
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Molecules 2019, 24, 1416
The butanol precipitate contained higher masses of compounds, mostly higher than 600 Da, while the
ethyl acetate precipitate was found to have a wide range of compounds. The mass profile of the ethyl
acetate precipitate was close to the mass profile of the chloroform precipitate based on the dendogram.
In line with the dendrogram, ethyl acetate and chloroform precipitates showed closer metabolite
profiles, as explained by the first principal component (PC1) in Figure 6c. The first two principal
components explain 84.6% of the total variance for the precipitates. Hence, different organic solvents
extracted different metabolites from the crude extract of E. longifolia, subsequently contributing to
different profiles of metabolites in those precipitates.
Figure 6. Heat mapping (a) and principal component analysis with score (b) and loading (c) plots.
2.4. Phytopharmacological Significance of Bioactive Precipitate

Ethyl acetate fraction and its precipitate were found to exhibit the highest total saponin content.
Therefore, the anti-proliferative activities of ethyl acetate fraction and its precipitate were tested on
a human breast cancer cell line in the subsequent experiments. The results found that the inhibitory
action of the samples was improved from crude extract (IC50 = 616.3 μg/mL) to ethyl acetate fraction
(IC50 = 185.4 μg/mL) and its precipitate (IC50 = 153.4 μg/mL). The IC50 of ethyl acetate precipitate was
close to the value of tamoxifen, 110.6 μg/mL (Figure 7). Tamoxifen is the most common drug used
to treat breast cancer patients [26]. Previous studies also reported that saponins could be potential
anticancer agents [21,27]. Hence, the processing technology to concentrate terpenoids and their
glycosylated derivatives could increase the performance of herbal samples in suppressing MCF-7
cell proliferation. This can also be seen from the high IC50 (733.7 μg/mL) of ethyl acetate filtrate,
which means a high concentration of sample is required to exhibit its anti-proliferative activity. Direct
precipitation using crude extract seemed to be less cytotoxic against MCF-7 cells. The combination of
solvent fractionation and acetone precipitation could increase the biological activity of the plant extract.
This technique should be recommended, as previous researchers used toxic solvents such as methanol
and chloroform to get bioactive fraction in their pharmacological studies [28,29]. The presence of
solvent residues in the plant extracts make them unsuitable for product formulation, particularly
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Molecules 2019, 24, 1416
products for human consumption. Therefore, food grade solvents such as ethanol, acetone, and ethyl
acetate are the primary choice of consumers.
Figure 7. MCF-7 cell viability after treated with tamoxifen (Δ), crude extract (), crude precipitate (x),
crude filtrate (), ethyl acetate fraction (), ethyl acetate precipitate (•), and ethyl acetate filtrate ( ).
3.1. Chemicals and Plant Material

The roots of Eurycoma longifolia (SK 3317/18) were harvested from Bentong, Pahang, Malaysia.
The samples were then dried and shredded into chip form, about 1 cm in size. Human breast cancer cell
line (MCF-7) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA)
and maintained in Dulbecco’s Modified Eagle Medium. Dimethyl sulfoxide (DMSO, 99.9%), vanillin
(≥97%), oleanolic acid (≥97%), and escin (≥95%) were purchased from Sigma-Aldrich, St. Louis,
MO, USA. Ethanol, acetic acid, perchloric acid, sulfuric acid, formic acid, ethyl acetate, butanol,
chloroform, acetonitrile, and acetone were sourced from Merck, Darmstadt, Germany.
3.2. Heat Reflux Extraction

The dried E. longifolia chips were then finely ground into powder (~2 mm) by a grinder.
The samples (25 g) were extracted with different concentrations of ethanol (250 mL) in a heat reflux
system for 2 h. After extraction, the solution was cooled and filtered for drying using a rotary
evaporator. The extraction yield was recorded for each solvent system.
3.3. Liquid-Liquid Extraction

The crude extract of 70% ethanol was used in the fractionation process. The fractionation was
carried out using the technique of liquid–liquid extraction. Three types of organic solvents, namely
ethyl acetate, butanol, and chloroform were selected to partition crude extract into individual fractions.
Crude extract (0.5 g) was reconstituted in water (10 mL) and vigorously extracted by ethyl acetate
(20 mL) in a 100 mL separating funnel. The solution was left for phase separation after extraction.
The organic phase was withdrawn, and another 20 mL ethyl acetate was added into the remaining
aqueous solution for extraction again. This fractionation process was repeated in triplicate. The organic
fraction of ethyl acetate was combined and dried by a rotary evaporator. A similar fractionation
process was also carried out to prepare butanol and chloroform fractions.
3.4. Cold Acetone Precipitation

The fractions of ethyl acetate, butanol, and chloroform were then reconstituted in 70% ethanol
(5 mL) and added dropwise into cold acetone (20 mL). Phytochemicals with poor solubility in cold
acetone were precipitated. The precipitated phytochemicals were filtered and dissolved in 50%
methanol for LC-DAD-MS/MS analysis, and dissolved in 50% ethanol for total saponin assay.
529
Molecules 2019, 24, 1416
3.5. Total Saponin Content

The total saponin content was determined colorimetrically according to the procedures described
by Makkar et al. [30]. A 250 μL sample (1 mg/mL) was mixed with 250 μL vanillin (8g/100 mL ethanol)
and topped up with 2.5 mL sulfuric acid (72%). The mixture was heated for 10 min at 60 ◦ C, and then
cooled in an ice-water bath for 5 min. The absorbance of the mixture was recorded by a UV-vis
spectrophotometer (UV-1800, Shimadzu, Japan) at 544 nm. Escin (5.7–71.4 mg/L) was used as the
standard chemical to build a calibration curve. The results are expressed as mg escin equivalent
per g sample (mg EE/g), or mg diosgenin equivalent per g sample (mg DE/g).
3.6. Cell Proliferation Using MTT Assay

MTT assay was performed to determine the viability of MCF-7 cells treated with samples (crude
extract, ethyl acetate fraction, and its precipitate). Standard chemicals, namely escin and tamoxifen
were used as positive controls in the experiments, whereas DMSO was used as a negative control.
Tumor cells (1 × 105 cell/mL) were seeded in 96 flat well microtiter plates, with 200 μL culture medium
in each well. The microplate was covered by sterilized parafilm and shaken gently before incubation at
37 ◦ C, in 5% CO2 for 24 h. After incubation, the medium was removed and two-fold serial dilutions of
samples were added to the wells for 24 h treatment at 37 ◦ C with 5 % CO2 . A 10 μL MTT solution was
added to each well and further incubated at 37 ◦ C for 4 h. The media solution was carefully removed
and 100 μL of solubilization solution was added into each well. The absorbance was determined
using an ELISA reader at a wavelength of 575 nm. Each concentration of samples was assayed in
triplicate. The growth of MCF-7 cells treated with herbal samples was determined based on their
viability after treatment. The results are expressed in effective concentration required to inhibit 50% of
viable cells (IC50 ).
3.7. LC-DAD-MS/MS
A liquid chromatograph (Dionex Corporation Ultimate 3000; Sunnyvale, CA, USA) integrated
with a diode array detector (Dionex Ultimate 3000) and a quadrupole and time-of-flight (QTOF) mass
spectrometer (AB SCIEX QSTAR Elite; Foster City, CA, USA) was used to screen phytochemicals.
A C18 reversed phase XSelect HSS T3 column (2.1 × 100 mm, 2.5 μm) with a flow rate of 150 μL/min
was used for separation, and compound peaks were detected at 254 nm. A binary gradient system
consisting of solvent A (water with 0.1% formic acid) and solvent B (acetonitrile) was programmed as:
0–10 min, 10% B; 10–20 min, 10–85% B; 20–25 min, 85% B; 25–25.1 min, 85–10% B; 25.1–30 min, 10% B.
The injection volume was 5 μL. All samples were filtered with 0.2 mm nylon membrane filter prior
to injection.
The QTOF mass spectrometer was used for phytochemical screening from m/z 100–2000. A single
information dependent acquisition (IDA) method was created to acquire both TOF MS and two
dependent runs of product ion scan with rolling collision energy. Nitrogen gas was used for nebulizing
(40 psi) and curtain gas (20 psi). Collision gas was set at 3, the accumulation time was 1 s for TOF
MS and 2 s for each product ion scan. The voltage of ion spray was 4500 V for negative ion mode.
The declustering potential was 40 V and the focusing potential was set at 300 V.

Heat-map and cluster analysis were used to visually classify the detected ions in precipitates
using R version 2.11.1. Principal component analysis was carried out using Pareto scaling in the data
processing software MarkerView 1.1 (Applied Biosystems/MDSSciex, Foster City, CA, USA).
4. Conclusions
The technique of solvent fractionation, followed by acetone precipitation seems to be able to
recover saccharide-containing compounds from the highly complex crude extract of E. longifolia.
530
Molecules 2019, 24, 1416
Usually, large molecules like triterpenoids and saponins which have intermediate polarity would
be recovered and then further precipitated in a highly polar acetone. Ethyl acetate appears to be
more effective to recover saccharide-containing compounds. This is the first study to concentrate
saccharide-containing compounds for MCF-7 cell inhibition. Further investigation will be carried out
to identify the recovered saccharide-containing compounds.
Supplementary Materials: The following are available online, Table S1: Precipitated compounds in cold acetone
from organic fractions of Eurycoma longifolia.
Author Contributions: Conceptualization, L.S.C.; methodology, L.S.C.; data curation, C.H.L., C.Y.C. and D.A.S.D.;
writing, L.S.C.; supervision, L.S.C.; funding acquisition, L.S.C.
Funding: This research was funded by the Ministry of Higher Education, Malaysia, grant number HICoE 4J263
and The APC was funded by the Ministry of Higher Education, Malaysia.
Acknowledgments: The authors would like to thank the assistance of internship students in the laboratory.
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molecules
Article
Inhibitory Effect of Osthole from Cnidium monnieri
on Tobacco Mosaic Virus (TMV) Infection in
Nicotiana glutinosa
Ya-Han Chen 1,2 , Dong-Sheng Guo 2 , Mei-Huan Lu 2,3 , Jian-Ying Yue 1 , Yan Liu 4 ,
Chun-Ming Shang 4 , De-Rong An 2, * and Ming-Min Zhao 1, *
1 College of Horticulture and Plant Protection, Inner Mongolia Agricultural University, Hohhot 010019, China;
yhchen1018@nwafu.edu.cn (Y.-H.C.); yuejianying2018@163.com (J.-Y.Y.)
2 College of Plant Protection and State Key Laboratory of Crop Stress Biology for Arid Areas, Northwest A&F
University, Yangling 712100, China; gds1995908@163.com (D.-S.G.); lu_meihuan@sina.com (M.-H.L.)
3 Microbial Resources of Research Center, Microbiology Institute of Shaanxi, Xi’an 710043, China
4 Academy of Agriculture science in Baotou, Baotou 014010, China; liuyanww@126.com (Y.L.);
chunmingsh@163.com (C.-M.S.)
* Correspondence: anderong323@163.com (D.-R.A.); Mingminzh@163.com (M.-M.Z.);
Tel.: +86-158-2909-7529 (D.-R.A.); +86-157-7136-0659 (M.-M.Z.); Fax: +86-029-8708-2710 (D.-R.A.);
+86-0471-638-5801 (M.-M.Z.)
Academic Editor: Raffaele Capasso

Received: 3 December 2019; Accepted: 18 December 2019; Published: 24 December 2019
Abstract: The coumarin compound of osthole was extracted from Cnidium monnieri and identified
by LC-MS and 1 H- and 13 C-NMR. Osthole was tested for anti-virus activity against tobacco mosaic
virus (TMV) using the half-leaf method. The results showed that stronger antiviral activity on TMV
infection appeared in Nicotiana glutinosa than that of eugenol and ningnanmycin, with inhibitory,
protective, and curative effects of 72.57%, 70.26%, and 61.97%, respectively. Through observation of
the TMV particles, we found that osthole could directly affect the viral particles. Correspondingly,
the level of coat protein detected by Western blot was significantly reduced when the concentrations
of osthole increased in tested plants compared to that of the control. These results suggest that
osthole has anti-TMV activity and may be used as a biological reagent to control the plant virus in the
half-leaf method.
Keywords: Cnidium monnieri; osthole; tobacco mosaic virus; half-leaf method; inhibitory
1. Introduction
Tobacco mosaic virus (TMV) belongs to the genus Tobamovirus and is transmitted by mechanical
inoculation and insects with chewing mouthparts in a propagative manner [1,2]. TMV is an economically
and destructively important plant virus with a wide host range, infecting more than 400 plant species
from 36 families [3]. Recently, a survey of plant viruses was collected from 31 provinces in mainland
China over a period from 2013 to 2017, which included over 41,000 vegetable crop samples from
the Solanaceae, Cucurbitaceae, Leguminosae, and Cruciferae families. The results showed that TMV is
distributed in all the surveyed provinces and is one of the most dominant viruses among 63 virus
species detected in these four families [4]. TMV leads to one hundred million dollars losses in crops
around the world in a year [5]. TMV is dependent on the plant cell to replicate and infect, which causes
extreme difficulty for antiviral therapies to inhibit only the virus without damaging the host [6,7].
Therefore, the chemical method was not effective in controlling plant viruses in crop fields.
The use of pesticides has brought with it a host of issues, like the increase in drug resistance of plant
pathogens, environmental pollution, and health risks to animals and humans [8,9]. In recent years, more

Molecules 2020, 25, 65
and more people have begun to focus on the use of botanical pesticides, which display great development
potential for controlling plant viral diseases, because they have low reside and are environmentally safe,
biodegradable, and safe to non-target organisms [10,11]. Up until now, many kinds of plant compounds
have already been demonstrated to have anti-viral ability, such as Amaranthaceae, Nyctaginaceae,
Asteraceae, Chenopodiaceae, Asclepiadaceae, Polygonaceae, Simaroubaceae, Acanthaceae, Liliaceae, Cruciferae,
Leguminosae sp., Boraginaceae, Oleaceae, Taxaceae, Ranunculaceae, Juglandaceae, Saxifragaceae, Theaceae,
Schisandraceae, Cupressaceae, Labiatae, and Caryophyllaceae [12–15].
Among these plants, the effective antiviral compounds are mainly proteins, alkaloids, flavonoids,
phenols, essential oils, and polysaccharides. In China, four reported plant-derived ingredients have
been widely used in viral disease control, including oligosaccharides, rhyscion, matrine, and fatty
acids [10]. Many studies have reported the inhibitory effects of plant-derived antiviral pesticides on
TMV. Tagitinin C (Ses-2) and 1β-methoxydiversifolin-3-0-methyl ether (Ses-5), two sesquiterpenoids
isolated from Tithonia diversiflia, were found to have higher inhibitory activities than the control agent
ningnanmycin [16]. Wang et al. found that sulfated lentinan induced systemic and long-term protection
against TMV in tobacco [17].
Cnidium monnieri (L.) Cusson is a traditional Chinese medicine that is widely distributed throughout
China. Many studies have suggested that it has pharmacological functions, such as anti-allergic,
antipruritic, antibacterial, antidermatophytic, anti-osteoporotic, and antifungal activities [18–22].
C. monnieri was reported to contain a number of biologically active compounds such as osthole,
imperatorin, bergapten, isopimpinellin, xanthotoxol, xanthotoxin, cnidimonal and cnidimarin,
glucosides, sesquiterpenes, etc. [23–25]. The anti-viral activity of ethanol extracted from C. monnieri in
plants remains unknown.
In this study, we performed the osthole isolation from C. monnieri. We investigated whether
the exogenous application of osthole is able to induce anti-viral activity in the tobacco plant
when infected with TMV. The inhibitory, protective, and curative effects on TMV infection were
measured. Furthermore, we observed whether osthole could affect the TMV particles and coat protein
(CP) accumulation.
2. Results
2.1. Compound Structure of Osthole

Osthole (7-methoxy-8-isopentenylcoumarin): is a white solid with the molecular formula
C15 H17 O3 , as identified by high-performance liquid chromatography (HPLC) (Figure 1), proton
nuclear magnetic resonance (1 H-NMR), carbon-13 nuclear magnetic resonance (13 C-NMR) (Figure 2),
and high-resolution mass spectrometry (HR-MS) spectra (Figure 3). As indicated by Figure 1, the purity
was greater than 98%. The spectral data was identical to that previously reported in the literature [26].
Figure 1. The HPLC chromatogram of osthole.
534
+&2 2 2
RVWKROH
Figure 2. Samples and structure of the compound identified from osthole.
(A) (B)
7,&RI72)06IURP6DPSOH $'5*'6$3&,3 RI'5*'6$3&,3ZLII 'XR6SUD\ 0D[HFSV 72)06PLQIURP6DPSOH $'5*'6$3&,3 RI'5*'6$3&,3ZLII 0D[HFSV
D HW H 'XR6SUD\

H

H
H
H
H H
H H
H
H
H
H H
H H
H
H
H
H H
H
H
,Q WH Q V LW\ F S V
,Q WH Q V LW\ F S V
H
H
H
H H
H
H
H
H
H
H H
H
H
H
H
H
H H

H
H
H
H
H

7LPHPLQ P]'D
Figure 3. The high-performance liquid chromatography/mass spectrometry (HPLC/MS) chromatogram

of osthole. (A) The HPLC/MS of chromatogram of osthole. (B) The MS of chromatogram of osthole.
1H-NMR (500 MHz, DMSO-d6): δ (ppm) 1.61 (s, 3H), 1.71 (s, 3H), 3.40 (d, J = 7.2 Hz, 2 H), 3.89 (s,
3H), 5.11–5.14 (m, 1 H), 6.26 (d, J = 9.6 Hz, 1 H), 7.05 (d, J = 8.4 Hz, 1 H), 7.55 (d, J = 8.4 Hz, 1 H), 7.96
(d, J = 9.6 Hz, 1 H); 13 C-NMR (125 MHz, DMSO-d6): δ (ppm) 18.1, 21.9, 25.9, 56.7, 108.5, 112.7, 113.1,
116.6, 121.7, 127.6, 132.2, 145.1, 152.6, 160.2, 160.7; HR-MS (ESI): m/z calculated for C15 H17 O3 ([M +
H]+ ) 245.1170, found 245.1169. For the NMR data please see the Supplementary Figures S1 and S2.
2.2. Anti-TMV Activities of Osthole

The anti-TMV activity of osthole from C. monnieri (L.) Cusson was tested at a concentration of
5 mg/mL in N. glutinosa using the half-leaf method. Based on the inhibition rates of local lesions on the
leaves of N. glutinosa (Figure 4), the antiviral activity of osthole was shown to be superior to that of
eugenol and ningnanmycin (Table 1), with an inhibitory effect of 72.57%, protective effect of 70.26%,
and curative effect of 61.97%.
535
Figure 4. Anti-tobacco mosaic virus (TMV) activities of osthole in N. glutinosa. The half-leaf was
smeared with osthole extract mixed with TMV at the same volume, and the right half-leaf was smeared
with 40 μL of TMV. (A) Osthole extract (5 mg/mL) and water. (B) Osthole extract (5 mg/mL) and TMV.
(C) Ningnanmycin SL (8%; 1000-X dilution) and TMV.
Table 1. The anti-viral activity of osthole against TMV.
Drug Inhibitory Effect (%) Protective Effect (%) Curative Effect (%)
Osthole 72.57 ± 9.24 aA 70.26 ± 10.49 aA 61.97 ± 7.84 aA
Eugenol 60.39 ± 5.48 aA 56.04 ± 4.98 aA 60.83 ± 4.49 bB
8% Ningnanmycin SL
64.11 ± 2.43 aA 60.57 ± 7.24 aA 55.45 ± 10.96 aA
(1000-X dilution)
Values are presented as the mean ± SE. Different upper and lower letters in the same column indicate significant
difference at p < 0.01 or p < 0.05 level by Duncan’s new multiple range test.
2.3. The Effect of Osthole on Viral Particles

In order to determine whether osthole could directly affect the viral particle, TMV particles were
mixed with the osthole at 3 mg/mL and 5 mg/mL with an equal volume for 45 min at room temperature.
We found that non-treated TMV particles as observed by a Hitachi H-600 Electron Microscope appeared
normal and baculiform (Figure 5A). In contrast, those treated with osthole presented with a strong
detrimental effect on the virus particles (Figure 5B,C). The virus particles were gradually destroyed: as
the concentration of osthole increased, the more severely the virus particles were damaged.
Figure 5. Electron microscopic observation of TMV particles after treatment with osthole for 45 min.
The concentration of the purified TMV was 0.60 mg/mL. The sample was observed under 49,000×
magnifications using a Hitachi H-600 Electron Microscope. (A) Normal TMV particles. (B) TMV treated
with osthole at 3 mg/mL for 45 min. (C) TMV treated with osthole at 5 mg/mL for 45 min.
536
2.4. Kinetic Analysis of the Effect of Osthole Against TMV Infection

To examine which concentration of osthole could be most effective against TMV infection, a kinetic
analysis was performed. The level of CP was detected by Western blot. We found that the CP level
was significantly reduced when the concentration of osthole increased from 1 to 7 mg/mL in treated
plants, as compared to that of the control (Figure 6). Osthole at 7 mg/mL could completely inhibit
CP accumulation in TMV. This result indicates that osthole may inhibit the replication of TMV in
plants. As shown in Table 2, we found that there was a significant positive correlation between the
concentration of osthole and inhibitory effects on TMV infection.
Table 2. The anti-viral activity of osthole against TMV at varying concentrations.
Concentration (mg/mL) Inhibitory Effect (%)

1 34.46 ± 5.19 cC
3 53.23 ± 3.13 bB
5 72.57 ± 9.24 aA
7 83.22 ± 3.68 aA
Data in the table are mean ± SD. Different upper and lower letters in the same column indicate significant difference
at p < 0.01 or p < 0.05 level by Duncan’s new multiple range test.
Figure 6. TMV coat protein (CP) accumulation detected by Western blot analysis.
3. Discussion
In this study, osthole was isolated from C. monnieri with 98% purity. Osthole is a coumarin
compound, a kind of secondary metabolite in plants, which has been shown to play an important
role in plant defense responses [27]. Additional properties of osthole include antibacterial, antifungal,
and pesticidal functions [28]. It was reported that osthole exhibits a wide range of inhibition in
mycelial growth against many fungal diseases (Rhizoctonia solani, Macrophoma kawatsukai, and Fusarium
graminearum) [28]. However, its anti-viral activity against plant viruses has not been reported. In this
study, we found that osthole has stronger anti-viral activity than eugenol and ningnanmycin.
Furthermore, we evaluated whether osthole could directly inhibit viral particles. Through
observation of TMV particles using a Hitachi H-600 Electron Microscope, we found that the
compound could directly affect the particles; TMV particles were gradually destroyed. When
the osthole concentration increased, the more severely the viral particles were damaged. Many reports
have indicated that many changes occur in the morphology of TMV particles after treatment with
plant extracts. Wang et al. found that TMV particles treated with eugenol showed ruptures and
abnormality [29]. Particles were destroyed and shortened by treated with Eupatorium adenophorum leaf
extract as reported by Jin et al. (2014) [30]. These results suggest that the method underlying viral
particle destruction is a common mechanism by which plant-derived reagents act on viral infection.
CP is critical for systemic infection and viral replication, protecting nucleic acid from enzymatic
degradation, which is related to the long-distance movement of TMV and the expression of host
symptoms [31–33]. In this study, we found the level of CP was significantly reduced to varying degrees
when the concentration of osthole increased in treated plants, as compared to that of the control.
Osthole at a concentration of 7 mg/mL completely inhibited expression of the TMV CP. The present
results are in agreement with those reported by Li et al. (2007), Wang et al. (2014), and Chen et al.
537
(2018) [7,11,17]. However, it remains to be further studied whether the function of osthole is through
inhibiting CP synthesis or the stereoscopic assembly of the virus.
In conclusion, osthole was isolated and purified from C. monnieri, and identified by 1 H- and
13 C-NMR and HR-MS. Osthole showed potent inhibitory activity against TMV infection. However,
the antiviral mechanism of osthole on plant viruses remains unclear. In the future, we will examine
whether osthole exerts its effect on CP synthesis or the stereoscopic assembly of TMV. This is the first
published report on the anti-TMV activities of osthole.
4.1. Chemicals and Materials

Ningnanmycin AS (8%) was obtained from Deqiang Biology Co., Ltd. (Harbin, China.). Eugenol
was purchased from Mckuin biological Co. LTD (Shanghai, China)
C. monnieri (L.) Cusson was purchased from the Jihetang Pharmacy (Yangling, China), and was
identified by Professor Xiaoqian Mu at Northwest A&F University (Yangling, China).
TMV isolates were provided by the Laboratory of Molecular Plant Pathology, Southwest University
(Chongqing, China) in the form of virus infected plants of N. benthamiana.
The seeds of N. glutinosa were provided by the Laboratory of Plant Virus, Inner Mongolia
Agricultural University (Hohhot, China), and cultivated in an insect-free greenhouse at 24 ± 1 ◦ C. The
experiments were conducted when the plant had grown 5–6 leaves.
4.2. Virus Purification

The Gooding method [34] was used for the purification of TMV-inoculated N. benthamiana, and
the isolates were stored at −20 ◦ C and diluted to 50 μg/mL with 0.01 M PBS (phosphate-buffered saline)
before use. Absorbance values were estimated at 260 nm by using an ultraviolet spectrophotometer
Tu-1901 (Beijing General Instrument co. LTD, Beijing, China). To calculate the concentration of virus,
the following formula was used (Equation (1)):
Virus concentration = (A260 × dilution ratio)/E0.1%,260

1 cm
nm
(1)
4.3. Isolation and Purification of Active Compounds and Structure Analysis

The C. monnieri (100 g) was powdered and extracted with 500 mL 90% methanol by reflux
three times (1.5 h each). The combined methanol extract was concentrated (50 g) and incubated with
quicklime (100 g) for 24 h. Subsequently, it was washed three times using five times diluted hydrochloric
acid and concentrated, suspended in chloroform (90 mL), and isolated with alkaline water (0.5% NaOH
solution). The crystal was filtered from the solution by adjusting the pH value to 7. After drying at a
low temperature, the solution that was filtered from the crystal with petroleum for 1 h was naturally
cooled to room temperature, and the final product was analyzed by liquid chromatography-mass
spectrometry (LC-MS). The structure was identified by 1 H- and 13 C-NMR spectra.
4.4. Inhibitory Effect of Osthole on TMV Infection

The compounds of osthole and eugenol were dissolved in DMSO (dimethyl sulfoxide) (1000 mg/mL)
and diluted to the required concentration with Tween-20 and distilled water (1:1000 v/v). Ningnanmycin
(8%) was diluted with water to a concentration of 500 μg/mL and used for the following experiment.
The inhibitory, protective, and curative effects were examined using the half-leaf method.
Osthole, eugenol, and ningnanmycin were mixed with the virus (TMV at 6 × 10−3 mg/mL) at the
same volume or concentration for 10 min, and then were smeared with a cotton swab onto the left
leaves of tobacco (N. glutinosa) along the main vein, whereas the virus sap and the DMSO solvent in
the right half of the leaves were inoculated in each of the three treatment groups (total of 12 leaves).
Each half of the leaf was smeared with 40 μL of TMV extract, and each inoculated leaf was washed
538
with water after 10 min. The local lesion numbers were recorded for 3–4 days after inoculation and
each compound and control agent was repeated three times.
The inhibition rates of osthole, magnolol, honokiol, and ningnanmycin were recorded and
calculated according to the following formula (Equation (2)):
Inhibition rate (%) = [(C − T)/C] × 100% (2)
where C is average lesion number of the control halves and T is the average mean lesion number on
the drug-treated half-leaves.
4.5. Protective Effect of Osthole on TMV Infection

Osthole, eugenol, and ningnanmycin were gently smeared with cotton swabs on the left side of
the leaves. The DMSO solution was spread as a negative control in the right lobe of tobacco leaves of
the same ages. After 24 h, 40 μL of TMV (50 μL/mL) was inoculated onto whole leaves of N. glutinosa,
each dealing with three treatment groups repeated in triplicate (total of 12 leaves were recorded), and
each inoculated leaf was washed with water after 10 min. The number of lesions on tested leaves was
investigated for 3–4 days.
4.6. Curative Effect of Osthole on TMV Infection

TMV (6 × 10−3 mg/mL) was inoculated on the whole leaves of N. glutinosa by cotton swabs. Then,
the leaves were washed with water and dried. After 24 h, osthole, eugenol, and ningnanmycin were
smeared onto the left leaf side, while the DMSO solution was smeared onto the right side for the
control. The local lesion numbers were recorded for 3–4 days after viral inoculation. Each experiment
was repeated three times.
4.7. Viral Particle Observation by Transmission Electron Microscope (TEM)

TMV particles were mixed with the osthole at 3 and 5 mg/mL with an equal volume for 60 min at
room temperature [30]. Then, samples were placed on a carbon-coated grid, and negatively stained with
a few drops of 2% phosphotungstic acid for 1 min at room temperature. They were then washed and
the excess fluid was absorbed on filter paper. The samples were observed with an electron microscope
(H-500, Hitachi Co. Ltd., Tokyo, Japan). An untreated virus sample served as a negative control.
4.8. Western Blot Analysis to Detect the CP of TMV

The levels of the TMV CP were analyzed by Western blotting. Total protein was extracted from
leaves of N. glutinosa (0.1 g, fresh weight), that were treated by water, and 1, 3, 5, and 7 mg/mL of osthole.
Samples were ground in liquid nitrogen and dissolved in 200 μL extraction buffer (125 mM Tris-HCl,
pH 7.5, 2% SDS, 6 M UREA, 5% β-mercaptoethanol and bromophenol blue). The extracts were then
heated at 95 ◦ C for 10 min and centrifuged at 12,000× g at 4 ◦ C for 10 min. Equal sample volumes
(5 μL) were loaded on a 12% polyacrylamide gel, and proteins were separated by electrophoresis at
120 V for 70 min. After being transferred to a PVDF membrane, CP was detected using a primary
antibody (1:800) and was subsequently probed with AP-coupled goat anti-rabbit IgG (1:5000; Abcam,
Cambridge, UK). The signals on the membrane were visualized using Clarity Western ECL Substrate
(Bio-Rad Company, Hercules, CA, USA).

All data were expressed as the mean ± SD by measuring three independent replicates. The Data
Processing System 15.10 (Hefei, China) was used to perform the statistical analysis. The significance of
the statistical differences between three means was determined using Duncan’s new complex range
method at the 5% level.
539
Supplementary Materials: The following are available online, Figure S1: H-NMR of osthole, Figure S2: C-NMR
of osthole.
Author Contributions: Y.-H.C., D.-S.G. and M.-H.L. isolated and purified of active compounds and analyzed the
structure. Y.-H.C. tested anti-TMV activities of osthole. D.-S.G. tested Transmission Electron Microscope (TEM).
J.-Y.Y., C.-M.S. and Y.L. analyzed the kinetic analysis of the osthole effect against TMV infection. D.-R.A. and
M.-M.Z. designed the experiments and supervised the study. M.-M.Z. and Y.-H.C. wrote the manuscript. All
authors have read and agreed to the published version of the manuscript.
Funding: The study was supported by Start-up Funding of High-level Talent Researcher in Inner Mongolia
Agricultural University (No. NDGCC2016-23) to Mingmin Zhao. Science and Technology Major Project of Inner
Mongolia (2018) to Chunming Shang, and the Demonstration and Application of Control Technology of Plant
Pests and Diseases in Shaanxi Province (No. K4030218261) to Derong An.
Acknowledgments: We thank Xian-Chao Sun (Southwest University, the People’s Republic of China) for providing
the primary antibody of TMV. We thank the Life Science Research Core Services (LSRCS) of Northwest A&F
University, the People’s Republic of China, for their help collecting the LC-MS and NMR data.
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About the Editors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

Raffaele Capasso and Lorenzo Di Cesare Mannelli

Thilina L. Gunathilaka, Kalpa W. Samarakoon, P. Ranasinghe and L. Dinithi C. Peiris

Thania Alejandra Urrutia-Hernández, Jorge Arturo Santos-López, Juana Benedı́,

Chukwuemeka R. Nwokocha, Isheba Warren, Javier Palacios, Mario Simirgiotis,

Serena Silvestro, Santa Mammana, Eugenio Cavalli, Placido Bramanti and

Laura E. Ewing, Charles M. Skinner, Charles M. Quick, Stefanie Kennon-McGill, Mitchell

Mario A. Tan, Mark Wilson D. Lagamayo, Grecebio Jonathan D. Alejandro and

Uthaiwan Suttisansanee, Somsri Charoenkiatkul, Butsara Jongruaysup, Somying Tabtimsri,

Thais Biondino Sardella Giorno, Carlos Henrique Corrêa dos Santos,

Phyu Phyu Myint, Thien T. P. Dao and Yeong Shik Kim

Seham Abdel-Shaﬁ, Abdul-Raouf Al-Mohammadi, Mahmoud Sitohy, Basma Mosa,

Anna Kurek-Górecka, Michał Górecki, Anna Rzepecka-Stojko, Radosław Balwierz

Justine Bodin, Amandine Adrien, Pierre-Edouard Bodet, Delphine Dufour,

Muhammad Taher, Siti Syazwani Shaari, Deny Susanti, Dayar Arbain

Bahare Salehi, Lorene Armstrong, Antonio Rescigno, Balakyz Yeskaliyeva,

Radu Claudiu Fierascu, Georgeta Temocico, Irina Fierascu, Alina Ortan

Academic Editor: Derek J. McPhee

Molecules 2020, 25, 5131; doi:10.3390/molecules25215131 1 www.mdpi.com/journal/molecules

Academic Editors: Raﬀaele Capasso and Lorenzo Di Cesare Mannelli

Abstract: Rheum palmatum L. is a traditional Chinese medicine with various pharmacological

Molecules 2019, 24, 1454; doi:10.3390/molecules24081454 7 www.mdpi.com/journal/molecules

2.1. Anthraquinone-Glycosides Content of PAGR

Figure 1. The structure of the emodin-8-O-β-d-glucoside (I), aloe-emodin-8-O-β-d-glucoside (II),

2.2. Acute Toxicity Study

2.3. Changes inBody Weight and Fasting Blood Glucose in Rats

Figure 3. Eﬀects of puriﬁed anthraquinone-Glycoside from Rheum palmatum L. (PAGR) on total

2.5. The Eﬀect of PAGR on Histopathological Changes

2.5.1. Eﬀects of PAGR on Histopathological Changes in Liver

Figure 4. The eﬀects of puriﬁed anthraquinone-Glycoside from Rheum palmatum L. (PAGR) on

2.5.2. Eﬀects of PAGR on Histopathological Changes in Kidney

Figure 5. Eﬀects of puriﬁed anthraquinone-Glycoside from Rheum palmatum L. (PAGR) on

2.5.3. Eﬀects of PAGR on Histopathological Changes in Pancreas

Figure 6. Eﬀects of puriﬁed anthraquinone-Glycoside from Rheum palmatum L. (PAGR) on

4. Material and Methods

4.1. Drugs and Chemicals

4.2. The Preparation and Determination of PAGR

4.3. Determination of Three Anthraquinone-Glycosides in PAGR by HPLC

4.3.1. HPLC Chromatographic Conditions

4.3.2. Preparation of Working Solution and Standard Curve

4.3.3. Stability and Precision

aloe-emodin-8-O-β-d-glucoside, and chrysophanol-8-O-β-d-glucoside was 2.30%, 1.26%, and 1.20%,

4.4. Acute Toxicity Test

4.5. AnimalsTreatment and Sample Collection

4.6. Biochemical Measurements

4.7. Histopathological Examinations

4.8. Western Blot Analysis

4.9. Statistical Analysis

Academic Editor: Raffaele Capasso

Keywords: dihydrochalcone; advanced glycation end-product; Eysenhardtia polystachya; diabetic

Molecules 2019, 24, 1214; doi:10.3390/molecules24071214 23 www.mdpi.com/journal/molecules

2. Results and Discussion

2.1. Identiﬁcation of 3 -O-β-D-glucopyranosyl α,4,2 ,4 ,6 -pentahydroxy–dihydrochalcone (1)

Table 1. Effect of dihydrochalcone (1) on urine protein of diabetic-nephropathy mice.

Urine Protein (mg/dL)

2.4. Effect of Dihydrochalcone on MCP-1

2.5. Effect of Dihydrochalcone on Glycosylated Haemoglobin Concentration (HbA1c)

Groups Glycosylated Haemoglobin (%) AGEs (RFU/mg Protein)