Journal of Applied Botany and Food Quality 91, 126 - 134 (2018), DOI:10.5073/JABFQ.2018.091.018
1University
of Belgrade, Faculty of Biology, Institute of Botany and Botanical Garden “Jevremovac”, Belgrade, Serbia
2Institute for Medicinal Plant Research “Dr. Josif Pančić”, Belgrade, Serbia
Antineurodegenerative, antioxidant and antibacterial activities and
phenolic components of Origanum majorana L. (Lamiaceae) extracts
Sonja Duletić-Laušević1, Ana Alimpić Aradski1*, Stoimir Kolarević1, Branka Vuković-Gačić1,
Mariana Oalđe1, Jelena Živković2, Katarina Šavikin2, Petar D. Marin1
(Submitted: July 4, 2017; Accepted: March 7, 2018)
Summary
The aim of this study was to examine chemical composition, as well
as antineurodegenerative, antioxidant and antibacterial activity of
aqueous and ethanolic extracts of Origanum majorana L. (Lamiaceae) originating from Serbia, Greece, Egypt and Libya. Total phenolics and lavonoids, antioxidant activities, and acetylcholinesterase
and tyrosinase inhibitory activities were measured spectrophotometrically. Determination of phenolic compounds in extracts was done
using HPLC-DAD technique. Antibacterial activity included determination of minimum inhibitory concentration (MIC) and minimum
bactericidal concentration (MBC) using the microdilution method.
The highest phenolic and lavonoid contents were recorded in the
ethanolic extract of the Egyptian sample and in aqueous extract of
Serbian sample. The HPLC analysis showed high content of rosmarinic acid, with the highest amount found in the ethanolic extract
of the plants from Egypt. Water extracts showed prevalently better
antioxidant and antineurodegenerative activity in applied tests than
the ethanolic extracts. Gram-positive bacterial strains showed higher
sensitivity to tested extracts. According to the obtained results, sweet
marjoram samples from Serbia and Egypt can be marked as more
promising, due to the highest content of total phenolics and lavonoids and the best antioxidant, antibacterial and tyrosinase inhibitory
activity.
Keywords: Origanum majorana, extracts, chemical composition,
antioxidant activity, antibacterial activity, antineurodegenerative activity
List of abbreviations
AAE - ascorbic acid equivalent; ABTS - 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt; AChE - acetylcholinesterase; AD - Alzheimer disease; BHA - 2(3)-t-butyl-4-hydroxyanisole; BHI - Brain-Heart infusion; BHT - 3,5-di-tert-butyl4-hydroxytoluene; DMSO - dimethylsulfoxide; DPPH - 2,2-dyphenyl-1-pikrylhydrazyl (DPPH); DTNB - 5,5’-dithiobis(2-nitrobenzoic
acid); FRAP - ferric reducing ability of plasma; GAE - gallic acid
equivalent; L-DOPA - 3,4-dihydroxy-L-phenylalanine; MBC minimal bactericidal concentration; MHB - Mueller-Hinton broth;
MIC - minimal inhibitory concentration; QE - quercetin equivalent;
TFC - total lavonoid content; TPC - total phenolic content; TPTZ 2,4,6-tripyridil-s-triazin; TYR - tyrosinase; β-CB assay - β-Carotene
Bleaching assay
Introduction
The mint family (Lamiaceae) includes aromatic plants widely used
for culinary, medicinal, cosmetic and ornamental purposes, such
as basil, rosemary, sage, oregano, lavender, thyme and mint (Raja,
2012). The genus Origanum is composed of 42 species and 18 hybrids widely distributed in Eurasia and North Africa (IetswaaRt,
*
Corresponding author
1980), being native to the mountainous areas of Mediterranean and
Asia (ChIshtI et al., 2013). Species belonging to the genus Origanum are used since the ancient times as spices, medicinal, aromatic
and ornamental plants (MeyeRs, 2005). In vitro pharmacological investigations showed their antibacterial, antifungal, antioxidant, antispasmodic, antimutagenic, antitumoral, analgesic, antithrombin and
antihyperglycaemic activities (ChIshtI et al., 2013).
Origanum majorana L. (sweet marjoram) is herbaceous perennial
shrub, inhabiting dry slopes and rocky places, native to Cyprus and
Eastern Mediterranean area (IetswaaRt, 1980). It is used as a medicinal plant, against cold, as a spasmolytic, antirheumatic, diuretic,
and antiasthmatic drug, and as a culinary herb, for lavouring sausages, sauces, soups and condiments (ChIshtI et al., 2013).
The essential oil of marjoram of different origin was previously
analyzed for the composition and biological activities (teIxeIRa
et al., 2013; hajlaouI et al., 2016). Various extracts were studied for antioxidant (jun et al., 2001; VágI et al., 2005a; el-MaatI
et al., 2012; Roby et al., 2013; ayaRI et al., 2013; benChIkha et al.,
2013; VasudeVa et al., 2014; afIfI et al., 2014; eRenleR et al., 2015)
and antimicrobial activities (VágI et al., 2005b; leeja and thoppIl,
2007; abdel-MassIh et al., 2010).
In the presented manuscript for the irst time the marjoram extracts
were tested for the antineurodegenerative effects, which is of growing scientiic and public interest. The marjoram spices used in the
analyses were produced by respectable companies for culinary herbs
and spices, while Libyan marjoram was purchased at the local market. In several papers the commercially purchased marjoram material was subjected to various analyses (Mossa et al., 2015; wahby
et al., 2015; f eRnandes et al., 2016), because of importance of
testing the commercial, widely used spices in daily diet for possible
beneits for consumers health. Considering the insuficiency of data
on medicinal properties of O. majorana extracts, the goals of this
study were chemical analysis as well as investigation of antibacterial,
antioxidant and antineurodegenerative activity of marjoram aqueous
and ethanolic extracts obtained from plants cultivated in Serbia and
three Mediterranean countries (Greece, Egypt, Libya).
Material and methods
Chemicals and reagents
Methanol, ethanol, distilled water, glacial acetic acid, hydrochloric
acid and chloroform were purchased from Zorka Pharma, Šabac
(Serbia). Gallic acid, quercetin, ascorbic acid, 2(3)-t-butyl-4-hydroxyanisole (BHA), 3,5-di-tert-butyl-4-hydroxytoluene (BHT), 2,2-dyphenyl-1-pikrylhydrazyl (DPPH), 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 2,4,6-tripyridil-striazin (TPTZ), potassium acetate (C2H3KO2), potassium-persulfate
(K2S2O8), dimethylsulfoxide (DMSO), sodium carbonate anhydrous
(Na2CO3), aluminium nitrate nonahydrate (Al(NO3)39H2O), sodium
acetate (C2H3NaO2), iron(III) chloride (FeCl3), iron(II)-sulfate heptahydrate (FeSO4 ∙7H2O), β-carotene, Folin-Ciocalteu phenol reagent, sodium phosphate monobasic (NaH2PO4), sodium phosphate
Bioactivities of Origanum majorana extracts
dibasic (Na2HPO4), 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB),
acetylcholinesterase from Electrophorus electricus (electric eel)
(AChE), acetylcholine iodide, galanthamine hydrobromide from Lycoris sp., kojic acid, tyrosinase from mushroom and 3,4-dihydroxyL-phenylalanine (L-DOPA), streptomycin, rifampicin and resazurin
sodium salt (>90% (LC) were purchased from Sigma Chemicals Co.,
St. Louis, MO (USA), while Tween 40 and linoleic acid were purchased from Acros Organics (Belgium). Brain-Heart infusion (BHI)
and Mueller-Hinton broth (MHB) were purchased from Himedia
(India).
Plant material
Dried and crushed plant material of O. majorana, which was used in
the experimental procedure, originating from Serbia, Greece, Egypt
and Libya, was commercially purchased. Marjoram is cultivated at
appropriate ield and collected, stored and controlled by the respectable companies. The plant material from Serbia is a product of Institute for Medicinal Plant Research “Dr Josif Pančić“, the sample from
Greece is produced by “Kagia“ company, from Egypt is a product of
“Kotanyi“ company, while the plant material from Libya was purchased from the local market.
Preparation of extracts
Grinded plant material (50 g) was extracted during 24 h at room
temperature (5% w/v) using two solvents, hot water and 96% ethanol. The mixture was exposed to ultrasound 1 h before and after
24 h to improve extraction process. Subsequently, extracts were iltered through ilter paper (Whatman No.1) and evaporated under
reduced pressure (Buchi rotavapor R-114). During the process of
concentration of ethanolic extracts high temperatures were not used
in order to prevent the loss of analytes and artifact formation. The
obtained crude extracts were stored in a refrigerator at +4 °C for
further experiments.
Determination of total phenolic and lavonoid contents
The total phenolic (TPC) and lavonoid contents (TFC) were measured using JENWAY 6305UV/Vis spectrophotometer as described
before (Alimpić et al., 2015, 2017). Phenolic content of extracts was
calculated from gallic acid curve equation and expressed as gallic
acid equivalents (mg GAE/g dry extract). Flavonoid content of extracts was calculated from quercetin curve equation and expressed as
quercetin equivalents (mg QE/g dry extract). Values were presented
as mean ± standard deviation averaged from three measurements.
HPLC analysis
Phenolic compounds in the tested extracts were determined by comparing the retention times and absorption spectra (200-400 nm) of
unknown peaks with the reference standards. HPLC-DAD analysis
was performed on an Agilent 1200 Series HPLC (Agilent Technologies, Palo Alto, CA, USA) equipped with Lichrospher® 100 RP 18e
column (5 μm, 250 × 4 mm). Mobile phase A was formic acid in
water (0.17%), while mobile phase B was acetonitrile. The injection
volume was 10 μL, and low rate 0.8 mL/min with gradient program
(0-53 min 0-100% B). Stop time of the analysis was 55 min. The
investigated samples were analyzed in triplicate.
Evaluation of antioxidant activity
DPPH free radical scavenging assay was performed according to
previously described experimental protocol (Alimpić et al., 2015,
2017). BHA, BHT and ascorbic acid were used as antioxidant standards. Results were expressed as IC50 values (μg/mL) averaged from
three measurements.
127
ABTS free radical scavenging assay was performed according
to Alimpić et al. (2015, 2017). The extracts and antioxidant standards BHA and BHT were tested in concentration of 1 mg/mL and
0.1 mg/mL, respectively. ABTS activity was calculated from ascorbic acid calibration curve and expressed as ascorbic acid equivalents
per gram of dry extract (mg AAE/g), averaged from three measurements.
FRAP assay (ferric reducing ability of plasma), evaluating total antioxidant power of the sample, was performed according to Alimpić
et al. (2015, 2017). The extracts were tested in concentration of
0.5 mg/mL. BHA, BHT and ascorbic acid (0.1 mg/mL) were used
as reference antioxidants. FRAP values of samples were calculated from standard curve equation and expressed as μmol FeSO4 ×
7 H2O/g dry extract and presented as mean ± standard deviation
averaged from three measurements.
β-carotene bleaching (β-CB) assay was performed according to
Alimpić et al. (2017). Crude extracts and standards BHA, BHT and
ascorbic acid were tested in concentration of 0.5 mg/mL. The absorbances were measured using Tecan Sunrise SN microplate reader.
The antioxidant activity of the extracts was evaluated in term of
β-carotene bleaching using the following formula: [(A120-C120)/(C0C120)] × 100%, where A120 and C120 were the absorbance values measured at 120 minutes for sample and control, respectively, while C0 is
absorbance of control at 0 minutes.
Antibacterial assay
The antibacterial activity of extracts and standard antibiotics streptomycin and rifampicin was tested against four gram-negative
(Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC
15442, Salmonella enteritidis ATCC 12076, Shigella lexneri ATCC
9199) and four Gram-positive bacterial strains (Enterococcus fecalis
ATCC 29212, Staphylococcus aureus ATCC 25923, Bacillus subtilis
ATCC 6633 and Listeria innocua ATCC 33090). The lowest concentrations without visible growth, i.e. minimal inhibitory concentrations (MICs) were determined according to KolArević et al. (2016).
Antineurodegenerative activities
Acethylcholinesterase (AChE) and tyrosinase (TYR) inhibitory activity assays were performed according to spectrophotometric method using 96-well plates as described before (Alimpić et al., 2017).
The applied concentrations of extracts and standards (galanthamine
and kojic acid) were 25, 50 and 100 μg/mL. The absorbances were
measured using Tecan Sunrise SN microplate reader equipped by
XFluor4 software. The results are expressed as percents of inhibition
of samples comparing to controls.
Statistical analysis
Differences between the group means and their signiicance were
veriied by one-way ANOVA using the Software package STATISTICA v.7.0. The signiicance of differences was evaluated using Bonferroni’s test and statistical signiicance was set at p<0.05. Pearson’s
correlation coeficients were calculated between content of phenolic
components and antioxidant assays and interpreted according to
tayloR (1990). Calculations and constructing of the charts were
performed using MS Ofice Excel (2013).
Results and discussion
Yield of extracts, total phenolic and lavonoid contents
The yields of extracts varied depending on the plant origin and applied solvent (Tab. 1), with higher yields in aqueous extracts. Among
aqueous extracts, the extract of the Egyptian O. majorana had the
highest yield (29.51%), while the lowest yield was obtained for the
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S. Duletić-Laušević, A. Alimpić Aradski, S. Kolarević, B. Vuković-Gačić, M. Oalđe, J. Živković, K. Šavikin, P.D. Marin
Greek sample (16.47%). The highest yield among ethanolic extracts
was recorded for the Libyan sample (13.49%), and the lowest for the
Serbian plants (8.51%). In the study of VágI et al. (2005a) the yield
of ethanolic extract of plants from Egypt was higher compared to our
results (28.99% and 11.91%, respectively). tRIantaphyllou et al.
(2001), applied different extraction methods and obtained a yield
as low as 0.88%, in the water extract of Greek O. majorana. In the
study of benChIkha et al. (2013) ethanolic extract of Algerian O.
majorana yielded 8.16%, which is close to the Serbian sample in this
study.
Total phenolic and lavonoid contents of extracts were measured
spectrophotometrically and presented as gallic acid (mg GAE/g of
dry extract) and quercetin (mg QE/g of dry extract) equivalents, respectively (Tab.1). All tested aqueous extracts of O. majorana, except for the one originating from Egypt, showed higher phenolic
content comparing to ethanolic extracts (Tab. 1). The highest content
of phenolics was found in aqueous extracts of the Serbian O. majorana (122.71 mg GAE/g dry extract), and in ethanolic extracts of the
Egyptian plant (139.98 mg GAE/g dry extract). VágI et al. (2005a)
obtained higher percentage of phenolics in ethanolic extracts of O.
majorana from Egypt compared to the plants from Hungary. They
explained the results by inluence of different climate conditions,
while many more sunny days in Egypt could cause higher production
of self-defense compounds against radiation and microbes. Ethanolic
extract of Egyptian marjoram showed higher amount of total phenolic and lavonoid compounds, similar to the results of el-MaatI
et al. (2012), who analyzed extracts of ive Egyptian medicinal plants
and obtained higher amounts of phenolics and lavonoids in ethanolic
extracts of O. majorana compared to aqueous extracts. benChIkha
et al. (2013) studied ethanolic extracts of Algerian oregano species
and obtained higher content of phenolics in O. majorana than in O.
vulgare extract.
Flavonoids were detected in higher amounts in all tested ethanolic
extracts compared to the aqueous extracts (Tab. 1). Ethanolic extract
of the plant originating from Egypt was the most abundant in lavonoids (20.05 mg QE/g dry extract) compared to other samples. elMaatI et al. (2012) also obtained signiicantly higher amounts of
lavonoids in ethanolic extracts of the Egyptian O. majorana than
in the aqueous extract. When comparing the percentage of lavonoids in ethanolic extracts of Hungarian and Egyptian O. majorana
VágI et al. (2005a) obtained lower values in ethanolic extracts of O.
majorana from Egypt. benChIkha et al. (2013) found a high phenolic content in the ethanolic extract of O. majorana from Algeria
(266.86 mg GAE /g), but the extract was poor in lavonoids.
Tab. 1: Yields, total phenolic and lavonoid content of O. majorana extracts
HPLC analysis of the extracts
Chemical composition of O. majorana extracts was determined
using HPLC-DAD (Tab. 2) in order to determine components responsible for certain types of bioactivities. Among phenolic acids rosmarinic acid was present in a signiicant amount, especially in ethanolic
extracts of all tested samples. The ethanolic extract of the Egyptian
plant contained the highest amount of rosmarinic acid (41.54 mg/g).
Rosmarinic acid is restricted to the subfamily Nepetoideae of the
Lamiaceae family (peteRsen and sIMMonds, 2003). This acid was
previously reported as a dominant phenolic acid of the Lamiaceae
(wang et al., 2004; lee, 2010; eMbusCado, 2015), that has a range
of biological activities (al-dhabI et al., 2014). In the comparative
study of rosmarinic and caffeic acids contents of 76 Lamiaceae species using TLC-densitometric method, janICsak et al. (1999) have
found low content of rosmarinic acid (0.87-2.40 mg/g) and caffeic
acid (0.09-0.27 mg/g) in tested Origanum species.
eRenleR et al. (2015) studied the phytochemical content of the
Turkish O. majorana water-soluble ethyl acetate extract and also
detected rosmarinic acid and arbutin. In our samples, arbutin was,
generally, more abundant in ethanolic extracts with the higest con-
Sample origin
Yield (%)
TPC (mg GAE/g)
TFC (mg QE/g)
22.54
122.71±2.68a
15.64±0.52ad
Greece
16.47
111.10±1.32b
15.13±0.55abd
Egypt
29.51
95.33±4.61c
13.51±0.42b
24.81
84.76±1.84d
11.03±0.46c
Serbia
8.51
84.38±2.23d
16.33±0.44d
Greece
10.36
83.25±0.85d
18.85±0.74e
Egypt
11.91
139.98±1.14e
20.05±1.11e
Libya
13.49
58.62±0.51f
14.87±0.29abd
Aqueous extracts
Serbia
Libya
Ethanol extracts
Means followed with different letters in the same column are signiicantly
different at p<0.05.
Tab. 2: Phenolic components identiied in O. majorana extracts
Sample origin
Content of phenolic components (mg/g dry extract)
Rosmarinic acid
Caffeic acid
Chlorogenic acid
Arbutin
Luteolin-7-O-glucoside
Serbia
20.09±0.81a
1.02±0.03a
4.52±0.15a
14.73±0.52a
0.99±0.02a
Greece
9.37±0.19b
0.54±0.02e
3.91±0.10c
2.22±0.06e
3.82±0.13d
Egypt
23.98±0.79e
0.90±0.02f
4.42±0.17a
7.47±0.21f
5.57±0.19e
Libya
8.06±0.30b
1.88±0.07c
3.82±0.14c
3.33±0.09c
1.66±0.07b
Serbia
22.47±0.75ae
0.13±0.00b
0.04±0.00bd
10.02±0.38b
1.10±0.03a
Greece
27.71±1.02d
0.49±0.01e
0.34±0.01be
15.46±0.49a
0.65±0.02c
Egypt
41.54±1.42f
0.26±0.01d
0.62±0.02e
25.67±1.03g
1.90±0.06b
Libya
13.11±0.26c
0.32±0.01d
0.03±0.00d
13.73±0.42d
0.40±0.01c
Aqueous extracts
Ethanol extracts
Means followed with different letters in the same column are signiicantly different at p<0.05.
Bioactivities of Origanum majorana extracts
tent in Egyptian sample. On the contrary, Serbian sample contained
greater amount of arbutin in water extract. Arbutin has been shown
to have antityrosinase (ye et al., 2010), antimicrobial (KundAKović
et al., 2014), antioxidant, antihyperglycaemic and antihyperlipidemic
activities (shahaboddIn et al., 2011).
Water extracts of analyzed samples contained much more chlorogenic acid compared to ethanolic extracts. Chlorogenic acid showed
several beneicial health effects, such as antioxidant, neuroprotective,
antiinlamatory, hypoglycemic, antifungal, etc. (upadhyay and Rao,
2013). This acid is an ester of caffeic and quinic acids, but caffeic
acid showed stronger antioxidant activity when compared to chlorogenic acid in in vitro and in vivo experiments (sato et al., 2011).
Flavone luteolin and its glycosides have been reported to possess a
wide range of bioactivities involved in prevention and treatment of
various diseases (lopez-lazaRo, 2009). Among lavonoids, luteolin-7-O-glucoside was detected in all extracts with the highest content in aqueous extract of Egyptian sample.
After comparison of HPLC-DAD chromatograms of water and ethanolic extracts at 280 and 330 nm we have not detected any unusual
peak that might suggest formation of artifacts as a result of using
ethanol as extraction solvent.
129
the Hungarian one, exhibiting antioxidant power comparable to that
of BHT, probably caused by differences in the amounts of phenolic compounds. RaMadan et al. (2014) obtained a higher percentage of inhibition of DPPH radicals for ethanol than methanol extract
of Egyptian O. majorana at analyzed concentrations. Our results
showed the weakest activity of Libyan extracts, while benChIkha
et al. (2013) obtained signiicant results for the geographically close
Algerian O. majorana and O. vulgare ethanolic extracts, where all
samples had better antioxidant activity than used controls (BHA and
α-tocopherol), with better results for the extract of O. majorana.
Antioxidant activity of tested O. majorana extracts was also determined using ABTS radical decolorization assay and expressed as
ascorbic acid equivalents (Tab. 3). The tested aqueous extracts in
ABTS assay, with exception of the Egyptian sample, demonstrated
better activity than the ethanolic ones (Tab. 3). The aqueous extract
of the Serbian sample showed the best activity (2.06 mg AAE/g), and
the ethanolic extract of the Egyptian plant was the most powerful
against ABTS radicals (2.25 mg AAE/g), while the lowest results
for both extracts were obtained for the Libyan sample. el-MaatI
et al. (2012) obtained better ABTS activity using ethanolic extracts
compared to aqueous extracts of O. majorana from Egypt (81.1 and
33.3%, respectively), which is in accordance with our results for the
Egyptian plant. RaMadan et al. (2014) also studied Egyptian marjoram and obtained high ABTS scavenging capacity of ethanolic extract (854 μm trolox/g). palanIswaMy and padMa (2011) obtained
high percentage of inhibition of ABTS radicals for aqueous leaf
extract, but lower than the methanolic extract (78.31% and 88.96%,
respectively).
In the FRAP assay tested aqueous extracts showed much better activity than the ethanolic ones, except for the Egyptian plant extract
(Tab. 3). Among aqueous extracts, the Serbian sample showed the
strongest activity (826.45 μmol Fe (II)/g), while the Egyptian plants
showed the lowest activity (554.66 μmol Fe (II)/g). However, similarly to the results recorded in DPPH and ABTS assays, among ethanolic extracts, the strongest activity was obtained for the Egyptian
plants (796.25 μmol Fe (II)/g), while extract of the Libyan plants
showed the lowest activity (260.32 μmol Fe (II)/g). This assay was
previously applied to O. vulgare, which revealed various results,
better activity of the aqueous extracts of O. vulgare compared to
the ethanolic ones in Portuguese samples (teIxeIRa et al., 2013), or
Evaluation of antioxidant activity of extracts
Considering the facts that oxidative stress has been involved in the
development of different human diseases and occurrence of various
side effects of synthetic antioxidants, researchers have focused on
the beneicial health effects of phytochemicals (eMbusCado, 2015),
including those isolated from Origanum species.
Antioxidant activity of O. majorana extracts was measured using
four assays (Tab. 3). Tested aqueous extracts showed better activity against DPPH radicals compared to ethanolic extracts. Among
aqueous extracts, eficiency varied from the Serbian sample which
showed the best activity (28.25 μg/mL) to the Libyan sample with
low activity (51.84 μg/mL). Among ethanolic extracts the best activity was obtained for extract of the Egyptian plants (54.15 μg/mL)
which was also the most abundant in phenolics content, while the
Libyan extract showed the lowest eficiency (114.47 μg/mL).
In the study of VágI et al. (2005a) the ethanolic extract of the Egyptian herb showed stronger antioxidant properties when compared to
Tab. 3: Antioxidant activities of O. majorana extracts
DPPH (IC50, μg/mL)
ABTS (mg AAE/g)
FRAP (μmol Fe(II)/g)
β-CB assay (% inhibition)
Serbia
28.25±0.56a
2.06±0.03a
826.45±8.14a
83.24±2.40a
Greece
35.27±0.81a
1.88±0.09b
754.59±6.32b
46.54±0.81b
Egypt
50.90±1.54b
1.52±0.06c
554.66±3.50c
83.24±2.48a
Libya
51.84±1.33bc
1.31±0.04d
602.06±9.55d
71.28±2.97c
Serbia
59.07±0.60c
1.52±0.07c
362.00±7.64e
61.70±2.98df
Greece
74.27±0.77d
1.52±0.06c
288.23±6.39f
63.30±1.60df
Egypt
54.15±3.60cb
2.25±0.02e
796.25±7.52g
60.90±1.99def
Libya
114.47±6.15e
1.30±0.04d
260.32±7.64h
66.49±2.79dc
BHT
17.94±0.17f
2.78±0.02f
445.34±5.77i
53.72±2.26ebf
BHA
13.37±0.43f
2.82±0.01f
583.72±5.26d
57.71±3.39f
Ascorbic acid
5.11±0.14g
-
180.81±8.61j
17.82±1.13g
Sample origin
Aqueous extracts
Ethanol extracts
Standards
Means followed with different letters in the same column are signiicantly different at p<0.05.
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S. Duletić-Laušević, A. Alimpić Aradski, S. Kolarević, B. Vuković-Gačić, M. Oalđe, J. Živković, K. Šavikin, P.D. Marin
on the contrary, lower activity of the aqueous extracts of O. vulgare
from Tunisia than the ethanolic ones (béjaouI et al., 2013).
Similar to the results obtained in the other applied tests, in the βcarotene bleaching assay most of the aqueous extracts showed a
higher activity than ethanolic ones, except for the extracts of the
plants originating from Greece (Tab. 3). Among aqueous extracts, the
strongest activity was shown by the Egyptian and Serbian samples
(83.24%), while the lowest activity was recorded for the Greek plants
(46.54%). The strongest activity among ethanolic extracts showed
the Libyan plants (66.49%), while the etanolic extract of marjoram
originating from Egypt showed the lowest activity (60.90%). During the analysis of Egyptian O. majorana extracts, el-MaatI et al.
(2012) found a relatively low percentage of β-carotene inhibition,
obtaining a better result for the ethanolic extract.
The ethanolic extract of the Egyptian plants showed considerable antioxidant activity in applied tests, probably due to the high content of
rosmarinic acid, which is considered as a component with signiicant
antioxidant activity (tepe, 2008; eRenleR et al., 2015).
Correlation between antioxidant activity and phenolic components content
Correlation between results obtained for antioxidant activity and
phenolics content is presented as correlation coeficient (Tab. 4).
Antioxidant activity was more strongly correlated to total phenolic
than total lavonoid content which is in accordance to Wojdyło et al.
(2007). Chlorogenic acid content was strongly correlated to DPPH
and FRAP values, rosmarinic acid and arbutin to ABTS values,
while caffeic and chlorogenic acids were correlated to β-carroten
bleaching assay. Chlorogenic, caffeic and rosmarinic acids could be
responsible for the antioxidant activity of tested extracts. Antioxidant
properties of these phenolic acids were also proved by RaudonIs
et al. (2008), sato et al. (2011) and upadhyay and Rao (2013).
Strong correlation is established between DPPH and FRAP as well as
ABTS and FRAP assays, while correlations between other assays are
assessed as moderate to weak. Results obtained in DPPH and ABTS
assays showed moderate negative correlation between each other as
it was previously found for Brasilian spices by baRRos MaRIuttI
et al. (2008). In this study, ABTS and FRAP assays showed strong
correlation which is consistent with hossaIn et al. (2008).
Antibacterial activity of extracts
Search for natural antimicrobials is growing in current studies because of the undesirable health impact of synthetic antimicrobial
food preservatives and the occurrence of pathogenic microorganisms
resistant to pharmaceuticals (tajkaRIMI et al., 2010). In addition to
the lavoring effect, Origanum species are proved to have antimicrobial activity on human and plant pathogens (VágI et al., 2005b; leeja
and thoppIl, 2007; ashRaf et al., 2011; jabeR et al., 2012; ChIshtI
et al., 2013; teIxeIRa et al., 2013).
The results of antibacterial activity of extracts against Gram-positive
bacteria are presented in Tab. 5. The most sensitive strains among
Gram-positive bacteria were B. subtilis and L. innocua, both for
aqueous and ethanolic extracts, especially for O. majorana originating from Serbia and Egypt (Tab. 5). Marjoram extracts showed
almost no activity on tested concentrations against Gram-negative
bacterial strains, except aqueous extract of Libyan sample which produced bacteriostatic and bactericidal effect on S. lexneri.
Gram-positive bacterial strains showed higher sensitivity against
tested extracts compared to the Gram-negative ones, which has also
been proved by ayaRI et al. (2013). They found that some of the
tested bacterial strains showed higher sensitivity towards methanolic
extracts at lower values than antibiotics used as positive controls, as
in the case of B. subtilis, L. innocua and S. aureus. Antibacterial
effect of extracts of other Origanum species was analyzed by several authors. ashRaf et al. (2011) conducted the comparative analysis of antibacterial activity of O. vulgare chloroform, methanol and
aqueous extracts, using agar well diffusion method and obtained
promising results for chloroform and methanol extracts, but aqueous
extract was not active against most of the studied strains. jabeR
et al. (2012) compared the antibacterial activity of aqueous, ethanolic
and methanolic extracts of O. vulgare against three Gram-positive
and three Gram-negative bacterial strains, using the well diffusion
method. The ethanolic extract had higher activity than the aqueous
one. In determination of the minimum inhibitory concentration the
best activity against B. subtilis was obtained using the ethanolic extract, followed by the methanolic and aqueous extracts. teIxeIRa
et al. (2013) also reported on higher ability of the O. vulgare ethanolic extract for the inhibition of growth of seven bacterial strains
compared to the hot and cold water extracts, which showed almost
no inhibition.
Antineurodegenerative activities of extracts
Cholinesterase inhibition is a commonly used approach for treating
the symptoms of Alzheimer disease (AD), which is a widely distributed neurological disorder. Various plants could be employed as a
new strategy in the treatment of neurodegenerative diseases, inclu-
Tab. 4: Correlation between antioxidant assays and content of phenolic components in O. majorana extracts
DPPH assay
ABTS assay
FRAP assay
β-CB assay
Rosmarinic acid
-0.03a
0.56b
0.12a
0.07a
Caffeic acid
-0.36b
-0.26a
0.31a
0.48b
Chlorogenic acid
-0.72c
0.16a
0.65b
0.41b
Arbutin
0.23a
0.53b
0.05a
0.07a
Luteolin-7-O-glucoside
-0.44b
0.10a
0.37b
0.09a
Total phenolic content
-0,74c
0.95c
0.89c
-0.03a
Flavonoid content
0,08a
0.61b
0.03a
-0.35a
DPPH assay
1.00
-0.59b
-0.82c
-0.10a
1.00
0.79c
-0.13a
100
0.06a
ABTS assay
FRAP assay
β-CB assay
According to Taylor (1990): ar≤0.35 weak correlation; b0.36<r<0,67 moderate correlation; c0.68<r<1 strong correlation.
1.00
7.812
≤ 3.906
* MIC and MBC of antibiotics were presented as μg/mL.
50
Rifampicin*
-
≤ 3.125
3.125
>5
>5
≤ 3.125
1.562
≥5
5
≤ 1.562
50
>5
>5
25
12.5
>5
>5
≤ 12.5
3.125
2.5
1.25
≤ 1.532
25
2.5
1.25
≤ 25
200
>5
>5
≤ 200
2.5
3.125
≤ 2.5
≤ 3.125
Ethanolic
Streptomycin*
>5
5
≤5
>5
≥5
2.5
1.25
5
>5
>5
>5
>5
>5
>5
>5
>5
1.25
2.5
1.25
0.625
2.5
1.25
0.625
1.25
>5
2.5
≤ 2.5
>5
2.5
Aqueous
1.25
≤ 2.5
≤ 1.25
Ethanolic
Libya
>5
5
≤5
>5
≥5
≥ 2.5
2.5
5
>5
>5
>5
>5
>5
>5
>5
>5
2.5
5
≤5
1.25
2.5
1.25
0.625
1.25
>5
-
>5
2.5
Aqueous
1.25
≤ 2.5
≤ 1.25
Ethanolic
Egypt
>5
5
≤5
>5
>5
2.5
≤ 2.5
>5
>5
>5
>5
>5
>5
>5
>5
>5
1.25
5
≤5
0.625
1.25
>5
>5
0.625
5
≥5
5
≤5
≤ 1.25
1.25
≤ 0.625
Ethanolic
Greece
Aqueous
0.625
MBC
5
≤5
MIC
MBC
2.5
≤ 2.5
MIC
MBC
>5
>5
MIC
MBC
>5
>5
MIC
MBC
-
MIC
MBC
2.5
≤ 2.5
MIC
MBC
5
≤5
MIC
MIC
≤ 0.625
Aqueous
Serbia
MBC
S. aureus
L. innocua
Gram-positive bacteria
E. faecalis
B. subtilis
Extracts
Sample
origin
Tab. 5: Antibacterial activity of O. majorana extracts presented as MIC (mg/mL) and MBC (mg/mL)
0.625
S. lexneri
P. aeruginosa
E. coli
Gram-negative bacteria
S. enteritidis
Bioactivities of Origanum majorana extracts
131
ding several Lamiaceae species (peRRy et al., 2000; oRhan et al.,
2010, 2012).
O. majorana extracts were tested for the inhibition of acetylcholinesterase and tyrosinase (Tab. 6 and 7). Analyzed aqueous extracts
showed higher percentage of acethylcholinesterase inhibition than
the ethanolic ones, with the exception of aqueous and ethanolic extracts of Serbian marjoram (Tab. 6). Aqueous and ethanolic extracts
of the Greek plant showed the highest percentage of AChE inhibition
(22.80-37.29%), but lower than galanthamine (42.38-57.11%). The
obtained results were not concentration dependent, contrary to used
standard. The Egyptian (Mossa and nawwaR, 2010) and Tunisian
(hajlaouI et al., 2016) marjoram essential oils showed signiicant
antiacethylcholinesterase activity, but extracts were rarely examined. Chung et al. (2001) in screening of 139 spices have obtained
the highest inhibitory effect of marjoram ethanol extract, inding
the ursolic acid as an active component and potent AChE inhibitor.
alI-shtayeh et al. (2014) detected much higher inhibition of AChE
for ethanolic extract of Palestinian marjoram, while methanolic extract of Iranian O. majorana showed signiicantly lower inhibition
of AChE (gholaMhoseInIan and RazMI, 2012), when compared to
our results.
Several studies presented results for antiacethylcholinesterase activity of O. vulgare extracts. vlAdimir-Knežević et al. (2014) tested
ethanolic extracts of 26 Croatian medicinal plants for their inhibitory
activity against AChE, including O. vulgare which showed around
30% inhibition of enzyme activity at the concentration of 1 mg/mL.
oRhan et al. (2010) found 35.84% inhibition at 100 μg/mL for the
acetone extract of Algerian O. vulgare var. glandulosum, while at
25 and 50 μg/mL no activity was recorded.
Tyrosinase inhibitors are of great concern due to the role of tyrosinase in mammalian melanogenesis, fruit browning, and also in
production of dopamine quinone derivatives involved in the degeneration of nigrostriatal dopaminergic neurons in Parkinson’s disease
(hasegawa, 2010).
The ethanolic extracts of marjoram had higher activity than the aqueous ones in testing tyrosinase inhibitory activity (Tab. 7). Among
aqueous extracts, sample from Serbia showed the highest percentage of tyrosinase inhibition (7.77-16.38%), while among ethanolic
extracts the highest activity was exibited by the Egyptian herb (15.1123.09%). Mentioned extracts with the highest activities contained the
highest level of arbutin, which is used as strong tyrosinase inhibitor
and whitening agent in cosmetic products (lIM et al., 2009; saRIkuRkCu et al., 2015). The obtained results showed dependence on
the concentration of extracts. The used standard, kojic acid, showed
stronger activity than the examined extracts (33.93-51.81%).
There are very few literature data on the tyrosinase inhibitory activity of Origanum extracts and essential oils. lIn et al. (2011) found
that antityrosinase activities were 21.8% for O. majorana and 4.7%
for O. vulgare from China. Signiicant tyrosinase inhibition activity
of the methanolic extract and origanol A of the Indian O. vulgare was
recorded by Rao et al. (2011).
saRIkuRkCu et al. (2015) examined essential oils of two O. vulgare
subspecies and obtained signiicantly better results for O. vulgare
subsp. hirtum when compared to O. vulgare subsp. vulgare, probably
due to high linalool content. Essential oil of Italian O. vulgare signiicantly inhibited mushroom tyrosinase in a dose-depended manner
(fIoCCo et al., 2016).
Conclusion
Literature data related to O. majorana bioactivities are mostly limited to essential oils, but this study showed that water and ethanolic
extracts could also be promising natural sources of bioactive compounds. The aqueous extracts generally showed better antioxidant,
antimicrobial and antineurodegenerative activity than the ethano-
132
S. Duletić-Laušević, A. Alimpić Aradski, S. Kolarević, B. Vuković-Gačić, M. Oalđe, J. Živković, K. Šavikin, P.D. Marin
Tab. 6: Acetylcholinesterase inhibitory activity of O. majorana extracts and galanthamine (standard) presented as percentage of AChE inhibition (%)
Sample origin
Aqueous extracts
Ethanolic extracts
25 μg/mL
50 μg/mL
100 μg/mL
25 μg/mL
50 μg/mL
100 μg/mL
Serbia
6.32±0.38a
30.33±2.83a
8.60±0.78a
31.26±1.60a
18.70±3.30a
5.96±0.35a
Greece
34.18±2.45b
35.22±0.79a
33.12±2.20b
22.80±3.20a
27.67±0.93b
37.29±1.79b
Egypt
6.43±0.09a
33.33±2.17a
7.64±0.41a
30.07±4.91a
5.31±0.33c
6.44±0.18a
Libya
29.03±4.13b
32.02±4.66a
30.77±3.57b
6.53±0.48b
30.18±2.15b
34.88±2.84b
Galanthamine
42.38±0.74c
50.56±0.51b
57.11±1.68c
Means followed with different letters in the same column are signiicantly different at p<0.05.
Tab. 7: Tyrosinase inhibitory activity of O. majorana extracts and kojic acid (standard) presented as percentage of tyrosinase inhibition (%)
Sample origin
Aqueous extracts
Ethanolic extracts
25 μg/mL
50 μg/mL
100 μg/mL
25 μg/mL
50 μg/mL
100 μg/mL
Serbia
16.38±2.23a
15.53±0.80a
7.77±1.15a
16.28±3.21a
15.74±0.55a
7.87±0.66a
Greece
15.53±1.76a
13.19±3.04a
7.66±1.21a
17.45±3.70a
15.96±2.56a
9.89±1.21a
Egypt
14.04±2.44a
11.17±1.44a
7.87±1.21a
23.09±1.99a
18.83±2.71a
15.11±1.69b
Libya
15.64±2.17a
8.94±1.29a
6.17±0.96a
21.38±1.81a
14.79±2.78a
11.38±1.95a
Kojic acid
35.73±5.46b
33.93±3.78b
51.81±2.55b
Means followed with different letters in the same column are signiicantly different at p<0.05.
lic ones, with the exception of the Egyptian sample in some tests.
Among tested marjoram samples originated from Serbia, Greece,
Egypt and Libya, the most promising were plants from Serbia and
Egypt, which contained high level of rosmarinic acid. This acid was
found to be the predominant constituent in investigated extracts
and presumably had a substantial inluence on their antioxidant and
antineurodegenerative activities.
Acknowledgements
The authors are gratefull to the Ministry of Education, Science and
Technological Development of Serbia [grant number 173029], [grant
number 172058], [grant number 46013].
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