Bioactive Extraction and Application in Food and Nutraceutical Industries

Methods and Protocols
in Food Science
Tanmay Sarkar · Siddhartha Pati Editors
Bioactive Extraction
and Application
in Food and
Nutraceutical
Industries
METHODS AND PROTOCOLS IN FOOD SCIENCE
Series Editor
Anderson S. Sant’Ana
University of Campinas
Campinas, Brazil
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Methods and Protocols in Food Science series is devoted to the publication of research
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Bioactive Extraction
and Application in Food
and Nutraceutical Industries
Edited by
Tanmay Sarkar
Dept. of Food Processing Tech, West Bengal State Council of Technical Education, Malda, India
Siddhartha Pati
NatNov Bioscience Private Limited, Odisha, India
Editors
Tanmay Sarkar Siddhartha Pati
Dept. of Food Processing Tech NatNov Bioscience Private Limited
West Bengal State Council of Technical Education Odisha, India
Malda, India
ISSN 2662-950X ISSN 2662-9518 (electronic)

Methods and Protocols in Food Science
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Preface to the Series
Methods and Protocols in Food Science series is devoted to the publication of research
protocols and methodologies in all fields of food science. The series is unique as it includes
protocols developed, validated and used by food and related scientists as well as theoretical
basis are provided for each protocol. Aspects related to improvements in the protocols,
adaptations and further developments in the protocols may also be approached.
Methods and Protocols in Food Science series aims to bring the most recent develop-
ments in research protocols in the field as well as very well established methods. As such the
series targets undergraduate, graduate and researchers in the field of food science and
correlated areas. The protocols documented in the series will be highly useful for scientific
inquiries in the field of food sciences, presented in such way that the readers will be able to
reproduce the experiments in a step-by-step style.
Each protocol will be characterized by a brief introductory section, followed by a short
aims section, in which the precise purpose of the protocol is clarified. Then, an in-depth list
of materials and reagents required for employing the protocol is presented, followed by a
comprehensive and step-by-step procedures on how to perform that experiment. The next
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The Methods and Protocols in Food Science series will fill an important gap, addressing
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detailed in scientific papers. With this, the series has a potential to become a reference
material in food science laboratories of research centers and universities throughout the
world.
University of Campinas Anderson S. Sant’Ana

Campinas, Brazil
v
Preface
The field of bioactive extraction and its application in the food and nutraceutical industries
has witnessed significant growth and innovation in recent years. Bioactive compounds,
which possess beneficial properties for human health, have garnered attention for their
potential in improving dietary quality and promoting overall well-being. This book, titled
Bioactive Extraction and Application in Food and Nutraceutical Industries, aims to provide
a comprehensive overview of the various aspects of bioactive compound extraction and its
industrial applications.
The chapters in this book cover a wide range of topics, encompassing both traditional
and novel extraction techniques, as well as exploring diverse sources of bioactive com-
pounds. The primary objective is to present a holistic view of the field, catering to the
needs of researchers, industry professionals, and students who are interested in this rapidly
evolving area.
Chapter 1 delves into traditional extraction techniques commonly employed in the
food, nutraceutical, and biotechnology industries. It provides a solid foundation by discuss-
ing the technological advancements and applications of these techniques in extracting
bioactive compounds, enabling readers to grasp the fundamentals of the field.
Chapters 2 and 3 specifically focus on the extraction of bioactive compounds and
nutraceuticals from plants and marine sources, respectively. These chapters shed light on
the wide array of bioresources available and explore the applications of plant-based and
marine-derived compounds in the food and nutraceutical sectors. Readers will gain insights
into the diverse range of bioactive compounds that can be extracted from these sources and
their potential benefits.
The subsequent chapters delve into the application of specific extraction techniques.
Chapter 4 discusses the utilization of microwave-assisted extraction for bioactive com-
pounds, highlighting its efficiency and effectiveness. Chapter 5 explores ultrasound-assisted
extraction, another powerful technique that has gained popularity in the food, pharmacy,
and biotech industries. Both of these chapters present practical insights into these innovative
extraction methods.
Chapters 6 and 7 introduce supercritical and subcritical fluid extraction and novel
solvent-based extraction, respectively. These advanced techniques offer unique advantages
in terms of efficiency, selectivity, and sustainability. The chapters provide comprehensive
coverage of their principles, applications, and potential in the extraction of bioactive
compounds.
Chapter 8 focuses on enzyme-assisted extraction, a promising method that harnesses the
power of enzymes to enhance extraction efficiency. This chapter delves into the enzymatic
hydrolysis of plant and microbial sources and its potential for extracting valuable bioactive
compounds.
Chapter 9 introduces pulsed electric fields as a green technology for the extraction of
bioactive compounds. This emerging technique utilizes electrical pulses to disrupt cell
membranes and facilitate the release of bioactive compounds, offering a sustainable alterna-
tive to traditional extraction methods.
vii
viii Preface
Chapter 10 explores pressurized liquid extraction, which utilizes high-pressure solvents

to extract bioactive compounds. This chapter discusses its applications and advantages,
providing valuable insights for researchers and industry professionals.
Chapter 11 presents case studies and applications of different novel extraction methods
discussed throughout the book. It showcases real-world examples to demonstrate the
practical implementation and effectiveness of these techniques. Chapter 12 explores pres-
surized liquid extraction for the isolation of bioactive compounds and the application of this
high-pressure solvent extraction technique in obtaining valuable bioactive compounds with
efficiency and precision.
The subsequent chapters continue to broaden the scope by exploring the extraction of
bioactive compounds from fruit waste (Chapter 13) and plant seeds (Chapter 14), providing
innovative approaches to utilize these underutilized bioresources. Chapter 15 focuses on
essential oils, discussing their sources, extraction techniques, and nutraceutical perspectives.
Chapter 16 addresses green and clean technologies for the production of novel nutra-
ceuticals, emphasizing sustainable practices and environmentally friendly approaches.
Chapters 17 and 18 delve into the optimization of nutraceutical extraction processes
and the computational approaches used in this field, respectively. These chapters provide
valuable insights into improving extraction efficiency and understanding the behavior of
bioactive compounds through advanced modeling and simulation techniques.
In conclusion, Bioactive Extraction and Application in Food and Nutraceutical Indus-
tries aims to serve as a comprehensive guide for researchers, industry professionals, and
students involved in the extraction and application of bioactive compounds. The diverse
range of topics covered in the chapters will provide readers with a solid foundation, practical
knowledge, and insights into the latest advancements in the field. It is our hope that this
book will contribute to the continued growth and development of this exciting area of
research and application.
Malda, India Tanmay Sarkar

Odisha, India Siddhartha Pati
Contents
Preface to the series. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi
1 Technologies for Extraction of Bioactive Compounds

and Its Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Rinku Sudarshan Agrawal and Nilesh Prakash Nirmal
2 Extraction of Bioactive and Nutraceuticals from Plants
and Their Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Hadia Hemmami, Bachir Ben Seghir, Soumeia Zeghoud,
Ilham Ben Amor, Abdelkrim Rebiai, and Imane Kouadri
3 Extraction of Bioactive and Nutraceuticals from Marine Sources
and Their Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Nikheel Rathod, Vijay Reddy, Martina Čagalj, Vida Šimat,
Merina Dahal, Nilesh Prakash Nirmal, and Siddhnath Kumar
4 Microwave-Assisted Extraction of Bioactive and Nutraceuticals . . . . . . . . . . . . . . . 79
Moufida Chaari, Sarra Akermi, Khaoula Elhadef,
Hussein A. H. Said-Al Ahl, Wafaa M. Hikal,
Lotfi Mellouli, and Slim Smaoui
5 Ultrasound-Assisted Extraction for Food, Pharmacy,
and Biotech Industries. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
Manab Jyoti Goswami, Utpal Dutta, and Dwipen Kakati
6 Super- and Subcritical Fluid Extraction of Nutraceuticals
and Novel Phytocompound . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
Pankaj Koirala, Saphal Ghimire, Sampurna Rai,
and Nilesh Prakash Nirmal
7 Novel Solvent Based Extraction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
Ratnnadeep C. Sawant, Shun-Yuan Luo, and Rahul B. Kamble
8 Enzyme-Assisted Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
Sadhana B. Maled, Ajay R. Bhat, Subrahmanya Hegde,
Yuvaraj Sivamani, Arunachalam Muthuraman,
and Sumitha Elayaperumal
9 Pulsed Electric Fields as a Green Technology for the Extraction
of Bioactive Compounds. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201
Radhika Theagarajan, Susindra Devi Balendran,
and Priyanka Sethupathy
10 Pulsed Electric Field Extraction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
Subrahmanya Hegde, Yuvaraj Sivamani, Arunachalam Muthuraman,
11 Case Studies and Application of Different Novel Extraction Methods . . . . . . . . . 255
Muskaan Sharma, Sakshi Vaishkiyar, and Sunidhi Kumari
ix
x Contents
12 Pressurized Liquid Extraction for the Isolation of Bioactive Compounds . . . . . . 275

Rakesh Barik, Sinoy Sugunan, and Mohd Affendi Bin Mohd Shafri
13 Fruit Waste: Potential Bio-Resource for Extraction of Nutraceuticals
and Bioactive Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299
Milan Dhakal, Saphal Ghimire, Geeta Karki,
Gitanjali Sambhajirao Deokar, Fahad Al-Asmari,
14 Plant Seeds: A Potential Bioresource for Isolation
of Nutraceutical and Bioactive Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333
Gitanjali Sambhajirao Deokar, Nilesh Prakash Nirmal,
and Sanjay Jayprakash Kshirsagar
15 Essential Oils: Sustainable Extraction Techniques
and Nutraceuticals Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373
Olusegun Abayomi Olalere, Chee-Yuen Gan, Abiola Ezekiel Taiwo,
Oladayo Adeyi, and Funmilayo Grace Olaiya
16 Green and Clean Extraction Technologies for Novel Nutraceuticals . . . . . . . . . . . 391
Insha Arshad, Gulden Gosken, Mujahid Farid, Mudassar Zafar,
and Muhammad Zubair
17 Optimization of Nutraceuticals Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 419
Shanza Malik, Ayesha Jabeen, Farooq Anwar,
Muhammad Adnan Ayub, Muhammad Nadeem Zafar,
and Muhammad Zubair
18 Computational Approach and Its Application in the Nutraceutical
Industry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 449
Prabina Bhattarai, Sampurna Rai, Pankaj Koirala,
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 469
Contributors
OLADAYO ADEYI • Department of Chemical Engineering, Michael Okpara University of

Agriculture, Umudike, Abia State, Nigeria
RINKU SUDARSHAN AGRAWAL • Department of Patronage of Traditional and Speciality Foods,
MIT School of Food Technology, MIT Art, Design and Technology University, Pune,
Maharashtra, India
SARRA AKERMI • Laboratory of Microbial Biotechnology and Engineering Enzymes (LMBEE),
Center of Biotechnology of Sfax (CBS), University of Sfax, Sfax, Tunisia
FAHAD AL-ASMARI • Department of Food Science and Nutrition, College of Agriculture and
Food Sciences, King Faisal University, Al-Hofuf, Saudi Arabia
ILHAM BEN AMOR • Department of Process Engineering and Petrochemical, Faculty of
Technology, University of El Oued, El Oued, Algeria; Renewable Energy Development Unit
in Arid Zones (UDERZA), University of El Oued, El Oued, Algeria; Laboratory of
Biotechnology Biomaterials and Condensed Materials, Faculte de la Technologie, University
of El Oued, El Oued, Algeria
FAROOQ ANWAR • Department of Food Science, Faculty of Food Science and Technology,
University Putra Malaysia, Serdang, Selangor, Malaysia; Institute of Chemistry,
University of Sargodha, Sargodha, Pakistan
INSHA ARSHAD • Department of Chemistry, University of Gujrat, Gujrat, Pakistan
MUHAMMAD ADNAN AYUB • Department of Chemistry, University of Sahiwal, Sahiwal,
Pakistan
SUSINDRA DEVI BALENDRAN • Department of Home Science and Research Centre, Faculty of
Food Processing and Management, Thassim Beevi Abdul Kader College for Women,
Kilakarai, Ramanathapuram, Tamil Nadu, India
RAKESH BARIK • GITAM School of Pharmacy, GITAM Deemed to be University, Hyderabad,
Rudraram, Patancheru Mandal, Sangareddy District, Telengana, India
AJAY R. BHAT • Department of Biotechnology and Bioinformatics, JSS Academy of Higher
Education and Research, Mysore, Karnataka, India
PRABINA BHATTARAI • Department of Health and Human Development, Montana State
University, Bozeman, MT, USA
MARTINA ČAGALJ • University Department of Marine Studies, University of Split, Split,
Croatia
MOUFIDA CHAARI • Laboratory of Microbial Biotechnology and Engineering Enzymes
(LMBEE), Center of Biotechnology of Sfax (CBS), University of Sfax, Sfax, Tunisia
MERINA DAHAL • Department of Nutrition and Dietetics, Central Campus of Technology,
Tribhuvan University, Kathmandu, Nepal
GITANJALI SAMBHAJIRAO DEOKAR • Department of Quality Assurance, MET’s Institute of
Pharmacy, Bhujbal Knowledge City, Nashik, Maharashtra, India
MILAN DHAKAL • Institute of Nutrition, Mahidol University, Salaya, Nakhon Pathom,
Thailand
UTPAL DUTTA • Department of Chemistry, Rajiv Gandhi University, Rono Hills, Doimukh,
Arunachal Pradesh, India
SUMITHA ELAYAPERUMAL • Department of Biotechnology and Bioinformatics, JSS Academy of
Higher Education and Research, Mysore, Karnataka, India
xi
xii Contributors
KHAOULA ELHADEF • Laboratory of Microbial Biotechnology and Engineering Enzymes

MUJAHID FARID • Department of Environmental Science, University of Gujrat, Gujrat,
Pakistan
CHEE-YUEN GAN • Analytical Biochemistry Research Centre (ABrC), Universiti Sains
Malaysia, Penang, Malaysia
SAPHAL GHIMIRE • Food Engineering and Bioprocess Technology, Asian Institute of
Technology, Khlong Luang, Pathum Thani, Thailand
GULDEN GOSKEN • Department of Food Technology, Vocational School of Technical Sciences,
Tarsus University, Mersin, Turkey
MANAB JYOTI GOSWAMI • Department of Chemistry, Rajiv Gandhi University, Rono Hills,
Doimukh, Arunachal Pradesh, India
SUBRAHMANYA HEGDE • Department of Biotechnology and Bioinformatics, JSS Academy of
HADIA HEMMAMI • Department of Process Engineering and Petrochemical, Faculty of
Applied Chemistry and Environment, Faculty of Exact Sciences, University of El Oued,
El Oued, Algeria
WAFAA M. HIKAL • Medicinal and Aromatic Plants Research Department, Pharmaceutical
and Drug Industries Research Institute, National Research Centre (NRC), Giza, Egypt;
Department of Biology, Faculty of Science, University of Tabuk, Tabuk, Saudi Arabia
AYESHA JABEEN • Department of Chemistry, University of Gujrat, Gujrat, Pakistan
DWIPEN KAKATI • Department of Chemistry, Rajiv Gandhi University, Rono Hills,
Doimukh, Arunachal Pradesh, India
RAHUL B. KAMBLE • Department of Botany, Dr. Ambedkar College, Deekshabhoomi, Nagpur,
Maharashtra, India
GEETA KARKI • Institute of Nutrition, Mahidol University, Salaya, Nakhon Pathom,
Thailand
PANKAJ KOIRALA • Food Science for Nutrition, Institute of Nutrition, Mahidol University,
Salaya, Nakhon Pathom, Thailand
IMANE KOUADRI • Department of Process Engineering, Faculty of Science and Technology,
University of 8 May 1945, Guelma, Guelma, Algeria
SANJAY JAYPRAKASH KSHIRSAGAR • MET’s Institute of Pharmacy, Nashik, Maharashtra, India
SIDDHNATH KUMAR • Department of Harvest and Post-harvest Technology, College of
Fisheries, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana,
Punjab, India
SUNIDHI KUMARI • Independent Researcher, Noida, Uttar Pradesh, India
SHUN-YUAN LUO • Department of Chemistry, National Chung Hsing University, Taichung,
Taiwan
SADHANA B. MALED • Department of Biotechnology and Bioinformatics, JSS Academy of
SHANZA MALIK • Department of Chemistry, University of Gujrat, Gujrat, Pakistan
LOTFI MELLOULI • Laboratory of Microbial Biotechnology and Engineering Enzymes
ARUNACHALAM MUTHURAMAN • Pharmacology Unit, Faculty of Pharmacy, AIMST
University, Bedong Kedah, Malaysia
Contributors xiii
NILESH PRAKASH NIRMAL • Institute of Nutrition, Mahidol University, Salaya, Nakhon

Pathom, Thailand
FUNMILAYO GRACE OLAIYA • School of Industrial Technology, Universiti Sains Malaysia,
Penang, Malaysia
OLUSEGUN ABAYOMI OLALERE • Department of Chemical Engineering, University of Bath,
Claverton Down, Bath BA2 3GB, Bath, United Kingdom
SAMPURNA RAI • Food Science for Nutrition, Institute of Nutrition, Mahidol University,
Salaya, Nakhon Pathom, Thailand
NIKHEEL RATHOD • Department of Post Harvest Management of Meat, Poultry and Fish, PG
Institute of Post Harvest Technology & Management (Balasaheb Sawant Konkan Krishi
Vidyapeeth, Dapoli), Killa-Roha, Raigad, Maharashtra, India
ABDELKRIM REBIAI • Renewable Energy Development Unit in Arid Zones (UDERZA),
University of El Oued, El Oued, Algeria; Laboratory of Applied Chemistry and
Environment, Faculty of Exact Sciences, University of El Oued, El Oued, Algeria;
Chemistry Department, Faculty of Exact Sciences, University of El Oued, El Oued, Algeria
VIJAY REDDY • Department of Harvest and Post-harvest Technology, College of Fisheries,
Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab, India
HUSSEIN A. H. SAID-AL AHL • Medicinal and Aromatic Plants Research Department,
Pharmaceutical and Drug Industries Research Institute, National Research Centre
(NRC), Giza, Egypt
RATNNADEEP C. SAWANT • Department of Chemistry, Dr. Ambedkar College, Deekshabhoomi,
Nagpur, Maharashtra, India
BACHIR BEN SEGHIR • Department of Process Engineering and Petrochemical, Faculty of
Technology, University of El Oued, El Oued, Algeria; Laboratory of Industrial Analysis and
Materials Engineering (LAGIM), University of 8 May 1945, Guelma, Guelma, Algeria
PRIYANKA SETHUPATHY • Riddet Institute, Massey University, Palmerston North,
New Zealand
MOHD AFFENDI BIN MOHD SHAFRI • Biomedical Science Department, Kulliyyah of Allied
Health Sciences, International Islamic University Malaysia, Kuantan, Pahang, Malaysia
MUSKAAN SHARMA • Independent Researcher, Delhi, India
VIDA ŠIMAT • University Department of Marine Studies, University of Split, Split, Croatia
YUVARAJ SIVAMANI • Department of Pharmaceutical Chemistry, Cauvery College of
Pharmacy, Mysore, Karnataka, India
SLIM SMAOUI • Laboratory of Microbial Biotechnology and Engineering Enzymes (LMBEE),
Center of Biotechnology of Sfax (CBS), University of Sfax, Sfax, Tunisia
SINOY SUGUNAN • School of Pharmaceutical Sciences, JSPM University Pune, Wagholi, Pune,
Maharashtra, India
ABIOLA EZEKIEL TAIWO • Faculty of Engineering, Mangosuthu University of Technology,
Durban, South Africa
RADHIKA THEAGARAJAN • Department of Home Science and Research Centre, Faculty of Food
Processing and Management, Thassim Beevi Abdul Kader College for Women, Kilakarai,
Ramanathapuram, Tamil Nadu, India
SAKSHI VAISHKIYAR • Independent Researcher, Patna, India
MUDASSAR ZAFAR • Department of Biochemistry, University of Gujrat, Gujrat, Pakistan
MUHAMMAD NADEEM ZAFAR • Department of Chemistry, University of Gujrat, Gujrat,
Pakistan
xiv Contributors
SOUMEIA ZEGHOUD • Department of Process Engineering and Petrochemical, Faculty of

Applied Chemistry and Environment, Faculty of Exact Sciences, University of El Oued, El
Oued, Algeria
MUHAMMAD ZUBAIR • Department of Chemistry, University of Gujrat, Gujrat, Pakistan
Chapter 1
Technologies for Extraction of Bioactive Compounds and Its

Applications
Rinku Sudarshan Agrawal and Nilesh Prakash Nirmal
Abstract
The successively growing consumer interest in natural bioactive compounds associated with human health
promotion and disease prevention has received huge attention in the food market toward effective
extraction techniques. Bioactive compounds can be extracted from natural sources and commercially
utilized in the development of nutraceuticals and functional foods. Various novel extraction technologies,
such as supercritical fluid extraction, ultrasound and microwave-assisted extraction, and accelerated solvent
extraction, have been considered effective for large-scale recovery, less extraction time, and superior extract
quality. The choice of an appropriate extraction technique could be based on the final applications or the
process optimization of bioactive compounds. This chapter aims to present conventional and emerging
techniques suitable for extraction of bioactive compounds from natural sources and its potential utilization
in food and nutraceutical industries.
Key words Bioactive, Health promotion, Extraction, Application
1 Introduction
Consumers’ attitude toward a healthy and well-balanced diet is

encouraging the utilization of natural products. Plant-derived nat-
ural products have gained extensive attention as a significant source
of active compounds with functional efficacy. Plants are found to be
an effective reservoir of numerous bioactive compounds and thus
have been hugely exploitated in various commercial sectors, espe-
cially in the food and pharmaceutical industries. These substances
are chemical structures that perform specialized functions at the
biological level. Bioactive compounds derived from plants, also
referred as phytonutrients, are produced as secondary metabolites
eliciting pharmacological effects in human health management [1].
Natural bioactive compounds influence the health of living
organisms as they are associated with some extra nutritional con-
stituents that typically occur in low quantities in foods. Different
Tanmay Sarkar and Siddhartha Pati (eds.), Bioactive Extraction and Application in Food and Nutraceutical Industries,
Methods and Protocols in Food Science, https://doi.org/10.1007/978-1-0716-3601-5_1,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024
1
2 Rinku Sudarshan Agrawal and Nilesh Prakash Nirmal
fruits, vegetables, whole grains, millets, etc. provide health benefits

beyond the basic nutritive function. A number of bioactive com-
pounds, such as polyphenols, phytosterols, dietary fiber, flavonoids,
phytoestrogens, carotenoids, and vitamins, are known to exert
beneficial physiological effects. The quest for bioactive compounds
from natural sources has been driven by scientific research of these
targeted molecules against a vast array of diseases, besides their use
in food science and technology. Bioactive compounds are thus
recognized as a vital ingredient in human health owing to their
multiple biological effects, such as reduction in risk factors of
various cardiovascular diseases, and they also act as antioxidant,
antifungal, antiviral, anti-carcinogenic, antiallergenic, anti-
inflammatory, and antimicrobial agents [2]. Phytochemicals being
a natural source of health-promoting compounds find greater
opportunity in the development of fortified foods with these func-
tional ingredients.
Extraction is one of the most important steps to analyze the
bioactive compounds present in plant material. Bioactive com-
pounds from green plants are currently the subject of considerable
research interest, but their extraction as part of phytochemical
and/or biological investigations presents a key challenge. The use
of bioactive compounds in different commercial sectors, such as
food and pharmaceutical industries, signifies the need for the most
appropriate and standard technique for the extraction of these
active components from plant materials. The main conventional
techniques related to extraction of bioactive compounds are macer-
ation, percolation, hydro distillation, and Soxhlet extraction. Along
with conventional methods, numerous advanced techniques have
emerged as green or clean extraction techniques due to less con-
sumption of solvent and energy, faster extraction rate, high-quality
products, better yield, and eco-friendliness [3]. No single method
is recommended as a standard method for extracting bioactive
compounds from plant materials as individual methods are asso-
ciated with some strengthes and weaknesses. Biological activities of
the extract show significant variation depending on the extraction
method, and this also opens a gateway for selecting a suitable
extraction method.
Thus, the extraction of active compounds from plants needs an
appropriate extraction technique that can provide bioactive
ingredients–rich extracts and fractions. The extraction procedures,
therefore, play a crucial role in the yield and nature of the phyto-
chemical content. Hence, this chapter aims to provide an overview
of bioactive extraction from natural sources with various conven-
tional and emerging technologies and their potential application in
the food and pharmaceutical industries (Fig. 1).
Technologies for Extraction of Bioactive Compounds and Its Applications 3
Plant derived bioactive

compounds (Fruits, Vegetables
and by-products)
Extraction of bioactive
compounds
Conventional extraction Advanced extraction
Purification
(TLC, Column chromatography, HPLC)
Application of bioactiove compounds
Fig. 1 Schematic representation of bioactive compounds and their applications
2 Extraction of Bioactive Compounds
Extraction is one of the most important unit operations followed in

the food industry that involves separation of medicinally active
compounds from plant materials using selective solvents through
standard procedures. Extraction of bioactive constituents has
always been a great challenge for scientists as the target compounds
may be non-polar to polar, thermally labile to resistant, and the
suitability of the extraction techniques must be considered. The
extraction of these active compounds requires a suitable extraction
technology that considers the plant parts used as starting material,
extraction time, nature of the solvent, particle size, and the stirring
during extraction [4]. The bioactive compounds can be extracted
by conventional and advanced technologies, which have their own
advantages and disadvantages (Table 1).
Conventional methods such as pressing, hydro-distillation,
steam distillation, and solvent extraction have been used since
ancient times in food processing industries (Fig. 2). The major
challenges of conventional extraction are longer extraction time,
requirement of costly and high-purity solvent, more quantity of
solvents, loss of volatile compounds, degradation of thermolabile
compounds, the possibility of leaving toxic solvent residues in the
extract, low yield and extraction efficiency. Consequently, there is a
Table 1
Advantages and disadvantages of extraction techniques
Conventional extraction techniques Advanced extraction techniques

Longer extraction time Shorter extraction time
Requirement of costly and high-purity solvent Reduced solvent usage and environmental friendly
Extraction efficiency is limited High extraction efficiency
Extraction yield is less Improved extraction yield
Labor intensive Less labor is required
Loss of volatile compounds Effective for volatile compounds
Degradation of thermolabile compounds Suitable for thermolabile compounds
Less initial capital investment Initial capital investment is more
Conventional Extraction Advanced Extraction

Techniques Techniques
Supercritical fluid
Maceration
extraction
Ultrasound assisted
Percolation extraction
Microwave assisted
Hydrodistillation extraction
Pulsed electric field

Soxhlet Extraction
assisted extraction
Enzyme assisted
extraction
Pressurized liquied
extraction
Fig. 2 Conventional and advanced extraction methods

growing demand for emerging extraction techniques that have

several advantages, such as less energy and solvent consumption
and reduced extraction times and can replace conventional solvents
with eco-friendly substitutes. The qualitative and quantitative stud-
ies of bioactive compounds from natural sources mostly rely on the
selection of proper extraction methods. The selection of extraction
technology is based on their advantages, disadvantages, operation
parameters, required degree of purity of the extract, physical and
chemical properties of the compound of interest, cost-effectiveness,
and value of the extracted product [5].
3 Conventional Extraction Techniques
Conventional or classical techniques such as maceration, Soxhlet

extraction, and hydro distillation are the most widely used methods
for extraction of bioactive compounds from natural sources. These
methods are primarily based on liquid–solid extraction [6]. The
efficiency of conventional extraction methods depends on the
choice of solvent and the polarity of the compound.
3.1 Maceration Maceration, since long, is one of the simplest and inexpensive
techniques widely used for the extraction of essential oil and bioac-
tive compounds from plant material. The whole or coarsely pow-
dered plant material is soaked with a solvent, such as ethanol,
acetone, or hexane, in a closed vessel. This is allowed to stand at
room temperature for 2–3 days with frequent stirring, which facil-
itates extraction. The process is intended to rupture the cell struc-
ture and help in the removal of different plant components. The
mixture is then pressed or strained by filtration or decantation after
a specific time [7]. The extraction efficiency was lowest in the
extracts of the maceration method, and it is a time-consuming
method. But it could be used for the extraction of thermolabile
components.
3.2 Percolation Percolation is a more efficient extraction method than maceration

as it is conducted by passing the boiled solvent through the plant
material at a controlled and moderate rate because it is a continuous
process in which the saturated solvent is constantly being replaced
by fresh solvent [8].
3.3 Hydro Distillation Hydro distillation is another conventional method that uses water
or steam for the extraction of bioactive compounds, especially
essential oils from plants. Hydro distillation is often carried out
using an equipment known as Clevenger apparatus or simple steam
distillation. In the Clevenger apparatus, sample mixed water is
boiled to evaporate volatile components, while in the steam distil-
lation approach, the steam is passed through a bed of the sample. In
both methods, two layers (aqueous and oil-rich) are obtained and
oil can be further separated via separating funnels [9]. Hydro dis-
tillation consumes more time, high levels of energy distillation rates
may vary if the heat source is not controlled, and the direct heat
source may cause charring of plant material at the base of the
chamber [10].
3.4 Soxhlet Soxhlet extraction is one of the most popular conventional techni-
Extraction ques for extracting valuable bioactive compounds from various
natural sources. This is an automatic continuous extraction tech-
nique having high extraction efficiency that requires less time and
solvent consumption than maceration or percolation. However, it is
widely applied to compounds with high thermal stability [8].
The finely ground material is placed in a thimble-holder made
from cellulose or filter paper and kept in a Soxhlet apparatus.
Extraction solvents are heated in a round bottom flask, vaporized
into the sample thimble, condensed in the condenser, and dripped
back. When the liquid reaches an overflow level, a siphon aspirates
the whole content of the thimble-holder and unloads it back into
the distillation flask, carrying the extracted analytes in the bulk
liquid. This process is continued until complete extraction is
achieved [6]. The efficiency of this process depends on parameters
such as solubility, mass transfer, and solid material characteristics.
The selection of a suitable solvent is one of the most important
factors for the extraction of bioactive compounds since it must be
based on its ability to extract the target compound [11]. This
technique has also been combined with microwave-assisted extrac-
tion and ultrasonic extraction in an attempt to improve extraction
efficiencies.
4 Advanced Extraction Techniques
The attention toward novel approaches for the extraction and

isolation of bioactive compounds from plant-based materials is
increasing in the field of research and development. Various new
and promising extraction techniques have become the recent area
of interest for extraction of bioactive compounds as they are able to
overcome the limitations of conventional methods. These advanced
extraction methods are being explored as clean, green, and environ-
mentally sustainable technologies that act as efficient alternatives to
conventional extraction technologies. Green extraction aims to
reduce the usage of solvent and energy, generates less waste, and
prevents environmental pollution while obtaining the highest
product yield with good quality [12].
Advanced techniques are also referred to as “assisted” extrac-
tion techniques where an additional physical phenomenon (ultra-
high pressure, ultrasound, electric fields, use of enzymes) is used for
identification of the process [13]. Owing to the high yield, reduced

processing time, high-quality products, and less generation of
waste, these emerging technologies have replaced the conventional
extraction methods. The effectiveness of these techniques varies
with the properties of the source matrix, its chemical structure,
and process parameters such as solvent, pressure, time, and temper-
ature. The food industry in the extraction sector should select a
suitable extraction method. The main objectives of advanced
extraction processes are related to achieving high yield, increased
heat and mass transfer, more effective use of energy with less time
consumption, reduction in the number of processing steps, low
environmental impact, and ensuring a desirable balance between
product quality and process efficiency [14].
4.1 Supercritical The supercritical fluid extraction (SFE) technique offers numerous
Fluid Extraction (SFE) operational advantages over conventional methods as it uses super-
critical solvents with various physico-chemical properties. The SFE
technology is extensively adapted for the extraction of thermolabile
biomolecules without any degradation of compounds. It involves
modulation of physical features, such as increasing the temperature
and pressure of a substance or solvent above its critical values. The
changes in fluid density in its supercritical state allow for variation in
solvency power, which results in selective extractions of compounds
of interest [15].
This technique makes use of supercritical fluids such as CO2,
ethanol, and water, which are generally recognized as safe (GRAS)
by the US Food and Drug Administration (FDA). Particularly,
supercritical carbon dioxide (SC-CO2) is extensively used as a
solvent as it is inert, nontoxic, economical, and easily separable
from the final product [8]. SC-CO2 is characterized by low viscos-
ity, high density, and diffusivity than conventional solvents, which
helps to have enhanced transport properties than liquids, diffuses
very easily through solid materials, and thus increases the extraction
rate of compounds [16]. SC-CO2 has unique solvent properties
that make it a desirable compound for separating antioxidants,
essential oils, pigments, flavors, fragrances, and fatty acids from
plant and animal materials. The productivity and profitability of a
supercritical fluid extraction (SFE) process largely depend on the
selection of process parameters.
4.2 Ultrasound- Ultrasound-assisted extraction (UAE) is extensively used in the

Assisted Extraction food industry as a viable alternative to the conventional extraction
(UAE) techniques that have been inclusively reported for achieving high
extraction rates of plant-derived bioactive compounds [17]. UAE is
also referred to as ultrasonic extraction or sonication, which uses
ultrasonic wave energy in the extraction. This technology involves
the use of acoustic vibrations or mechanical waves with ultrasound
to produce cavitation in which microbubbles form in the liquid
phase. Ultrasonic treatment causes mechanical impact on the cell

wall. The plant cell wall can get disrupted, which enhances solvent
penetration into the cell and thus helps to improve the mass trans-
fer. Ultrasound in the solvent-producing cavitation accelerates not
only dissolution but also diffusion of the solute and heat transfer,
which improves the extraction efficiency [18].
The factors affecting the efficiency of UAE are extraction time,
power, solvent, liquid/solid (L/S) ratio, plant material, frequency,
amplitude, and intensity. Ultrasound power directly affects the
cavitation and shear forces in the extraction medium. The fre-
quency used for the extraction of bioactive compounds from natu-
ral products usually ranges between 20 and 120 kHz [19].
UAE is considered a more promising choice than conventional
extraction methods due to lower consumption of solvent and
energy. It is explored as a more sustainable process as it is associated
with several advantages, such as: rapid heat and mass transfer effi-
ciency, greater solvent penetration into solid material, reduction of
extraction temperature and time, and lowest carbon emission
[6]. UAE also provides faster extractions, with high reproducibility
and higher purity of the final product when compared to conven-
tional extraction methods. UAE is widely applicable for the extrac-
tion of thermolabile and unstable compounds from natural
sources [8].
4.3 Microwave- Microwave-assisted extraction has been accepted as an effective

Assisted alternative technique to recover bioactive compounds from plant
Extraction (MAE) materials using microwave energy. Microwaves are electromagnetic
radiation that occur in frequencies ranging from 300 MHz to
300 GHz and wavelengths between 1 mm and 1 m. These waves
are composed of two mutually perpendicular oscillating fields, that
is, an electrical field and a magnetic field, which are used as energy
and information carriers [6].
The principle of microwave heating depends on its direct
impact on polar materials. Microwaves can penetrate biomaterials
and interact with polar molecules such as water to generate heat.
The heat is generated by ionic conduction and dipole rotation
mechanisms [20]. During microwave treatment, polar compounds
align themselves in the direction of electric field and rotate at high
speed which leads to disruption of the cell membrane. The cell wall
breakage enhances the release of the extract from the source
materials.
The important parameters that influence the extraction effi-
ciency of MAE techniques are microwave power and temperature,
microwave energy density, plant sample characteristics and its water
content, solvent-to-feed ratio, and irradiation time [21]. The com-
bination of ultra-sonication with microwave amplifies the efficiency
of the extraction process. MAE is a promising novel extraction
technique that offers many advantages, such as rapid heating
rates, increased extract yield, short processing time, energy saving,

and no further generation of secondary waste. Additionally, due to
less consumption of organic solvent, this technique gets a broad
recognition as a green extraction technique [22].
4.4 Pulsed Electric Pulsed electric field involves the application of high voltage pulses
Field (PEF)-Assisted for a brief time period (nanoseconds/milliseconds) through the
Extraction sample placed between two conducting electrodes [23]. Pulsed
electric fields (PEF)-assisted extraction is attracting great attention
as a nonthermal extraction technology due to being a cost-effective
technique that is energy efficient, time-saving, and eco-friendly
[24]. PEF has proven to be a promising technique to enhance the
extraction of valuable bioactive compounds such as anthocyanin,
polyphenols, and plant oil from plant tissues, as well as their bypro-
ducts and soluble intracellular matter from microorganisms.
The targeted material is placed between the electrodes, and a
high voltage electric field of 10–60 kV is applied through electro-
des. The applied high voltage pulse induces pores in the cell mem-
branes (electroporation), which enhances the permeability of the
cell membrane. The cell membrane loses its structural functionality,
leading to cell disintegration, which accounts for the leakage of
intracellular content, and the plant material is extracted [25]. PEF
has the ability to electroporate the cell membranes and is thus
commonly used as pretreatment to facilitate the extraction of bio-
active compounds, followed by subsequent conventional or
advanced extraction techniques [11]. This technique is usually
preferred for liquid foods as electrical current flows into the liquid
food more fast and effectively and the transfer of pulses from one
point to another in liquids is quite easy due to the presence of
charged molecules [26].
Numerous studies concluded PEF as a promising tool to
recover phytonutrients used in the food and pharmaceutical indus-
tries. PEF-assisted extraction leads to cost efficiency, higher extract
yield of bioactive compounds, lower energy consumption, and less
treatment time, providing the optimum process parameters. The
extraction efficiency of bio ingredients with PEF treatment depends
on electrical parameters such as electric field strength, energy input,
pulse polarity, and delay time between pulses of opposite polarity.
4.5 Enzyme-Assisted Enzyme-assisted extraction is a novel approach for extracting bio

Extraction (EAE) ingredients from plant materials. The application of enzymes for
extraction of bioactive compounds is a recent and emerging
approach from small-scale, laboratory optimization studies to
large-scale, industrial applications. Enzymes are ideal catalysts that
can assist in the efficient extraction of bio ingredients from natural
sources. The EAE technique has been applied for extraction of
macromolecules such as proteins, polysaccharides, and low
molecular compounds such as phenols and polyphenols, oils and

fatty acids, essential oil, sugars, di- and triterpenes, and
vitamins [27].
Enzyme-assisted extraction (EAE) technique makes use of spe-
cific enzymes to hydrolyze cell wall components. This disintegrates
the structural integrity of plant cell wall and increases the perme-
ability of the intracellular material for extraction thereby releasing
bioactive compounds. In EAE, the plant material is pretreated with
desirable enzymes such as protease, pectinase, pectin esterase,
cellulase, hemicellulase, cellobiase, and α-amylase to hydrolyze the
cell walls and release the phytochemicals bound to lipid and carbo-
hydrate chains inside the cell [28].
Enzymes are applicable to extract many phenolic compounds,
including flavonoids and anthocyanidins. EAE can also be applied
for the additional recovery of compounds such as pectins from
agricultural wastes and byproducts by increasing the permeability
of plant cell wall. During enzymatic treatment, the highest enzyme
efficiency mainly depends on the type, dosage, and required condi-
tion of the enzymes, treatment time and temperature combina-
tions, substrate ratio, and particle size [5]. This technique has also
been used in combination with other extraction techniques such as
UAE, SFE, and MAE to improve the overall recovery of bioactive
compounds from source materials [29]. The combination of ultra-
sonic waves with enzymes can improve the capability of enzymes.
Many research studies have shown highly positive effects of both
ultrasonic and enzymes in the improvement of extraction yield with
superior product quality for nutraceutical and pharmaceutical
applications.
EAE has been regarded as a valuable substitute for conventional
techniques to isolate various biologically active compounds more
rapidly and with better recovery. EAE has emerged as an
eco-friendly alternative to recover bioactive compounds with sev-
eral advantages, such as increased yield and quality of product,
reduction in extraction time, lower solvent consumption, in addi-
tion to the increased transparency of the system. However, EAE has
probable commercial and technical limitations, such as worldwide
regulations of enzyme usage and relatively high cost of enzymes for
large industrial production. An important area of research is inves-
tigating the stability of enzymes and their interaction with other
food and plant ingredients during processing and storage.
4.6 Pressurized Pressurized liquids are able to recover phytonutrients faster than
Liquid Extraction (PLE) conventional low pressure methods. PLE can be viewed as an
extension of supercritical fluid extraction, utilizing organic solvents
instead of carbon dioxide. PLE uses liquid solvents below their
critical point with controlled temperature and pressure [30]. PLE
was first introduced as accelerated solvent extraction (ASE) tech-
nology in 1995 by Dionex Corporation as an alternative to other
extraction techniques. It is also referred as pressurized solvent

extraction (PSE), enhanced solvent extraction (ESE) or pressurized
fluid extraction (PFE) [31]. Pressurized liquid extraction (PLE)
technology has gained remarkable research interest to achieve fast
and efficient extraction of the bioactive components from the food
matrix in short time using organic solvents at elevated temperature
and pressure.
The sample is placed in the extraction cell with an organic
solvent such as toluene or hexane/acetone at an elevated tempera-
ture (up to 200 °C) and relatively high pressure (500–3000 psi) to
increase the efficiency of the extraction process. The use of a closed
system allows extraction at elevated temperatures since the boiling
point of the solvent increases. The rise in temperature usually above
their boiling points causes dramatic changes in the physical and
chemical properties of water such as increase in the diffusion rate
and decrease in the viscosity of solvents. The PLE conditions
provides better solubility of target analytes with the solvent. The
elevated temperature can also break down analyte-matrix interac-
tions and increases mass transfer of the essential compounds present
in the plant matrix to the solvent, as well as the stability of the
process [32].
PLE has been consolidated as a high-throughput and green
extraction technique for sustainable extraction of bioactive com-
pounds from various natural sources. This technique requires less
amounts of solvents due to the combination of high pressure and
temperature, which dramatically decreases the time consumption of
extraction. It is a solid-liquid extraction technique that offers
improved extraction efficiency with less time and cost, is
eco-friendly, automates minimum production of waste, and elim-
inates post-extraction steps such as filtration and centrifugation
[33]. The main limitations of the process are high equipment cost
and the need for a thorough optimization of variables to avoid a
matrix-dependent efficiency [34].
4.7 Combination of The combination of different extraction techniques is a desirable

Modern Techniques for approach that presents advantages to overcome the limitations of
Effective Extraction of an individual extraction technique. Many research studies have
Bioactive Compounds shown that a combination of these novel extraction strategies can
be effective for rapid and efficient extraction with the aim of
enhancing the amount of the target molecules and reducing the
waste of solvents.
Ultrasound is a promising choice, particularly when it is com-
bined with other substitutes among the different available combi-
nations. Ultrasound can be used for rapid heat and mass transfer in
the extraction field when combined with other technologies
[35]. Ultrasound-assisted extraction technique combined with
SFE has been recently estimated to improve the flexibility in the
extraction process [36]. Different combinations, such as
ultrasound-assisted enzymatic extraction (UAEE), microwave-

assisted enzymatic extraction (MAEE), and ultrasonic microwave-
assisted extraction (UMAE), can provide a synergistic effect and
exhibit higher potential extraction ability [37].
5 Purification of the Bioactive Compounds
Plant materials are a multi-component mixture that contains

numerous types of bioactive compounds having different polarities;
however, their separation still remains a big challenge. The isolation
and purification of bioactive compounds is very important to eval-
uate its biological activity by in vitro and in vivo assays [38]. Chro-
matography is a separation technique immensely used for
identification and/or purification purposes in different research
and industrial sectors. The chromatographic technique is a widely
adopted technique that has a vital role in the chemistry of plant-
derived natural products, as well as a significant contribution to the
discovery of innovative bioactive compounds of pharmaceutical and
biomedical importance. Chromatographic techniques such as paper
chromatography, thin-layer chromatography, high-performance
liquid chromatography (HPLC), gas chromatography, column
chromatography, or combination of several chromatographic tech-
niques can be potentially used to obtain pure compounds [39].
Various scientific studies support that silica gel column chro-
matography and thin-layer chromatography (TLC) are still mostly
used due to their convenience, economy, and availability in various
stationary phases [40]. High-performance liquid chromatography
(HPLC) is an accurate and selective chromatographic technique
potentially used in pharmaceutical industries for the isolation and
purification of bioactive compounds from medicinal plants. Various
nonchromatographic techniques, such as immunoassay, phyto-
chemical screening assay, and Fourier-transform infrared spectros-
copy (FTIR), are recognized as valuable tools for the
characterization and identification of bioactive compounds present
in an unknown mixture of plant extracts [41].
6 Extraction of Bioactive Compounds from Agro-industrial Waste
The huge quantity of waste produced from agricultural and food

production remains a great challenge as well as an opportunity for
the food industry. Agro-industrial waste has a great potential to
generate food additives that can be beneficial in ensuringe global
food sustainability [42]. Agro-industrial waste is composed mainly
of seed, skin, rind, and pomace and is a promising source of
potentially valuable bioactive compound components such as
proteins, polysaccharides, fibers, lipids, carbohydrates, peptides,

carotenoids, phenolic and other compounds, which can be reused
as nutraceuticals and functional ingredients [43].
Due to their complex chemical composition, animal and vege-
table byproducts can be utilized as a low-cost raw material to obtain
bioactive compounds using suitable extraction technology
[44]. The recovery of these bioactive compounds present in agri-
cultural residues such as fruits, vegetables, and plants involves the
use of various conventional and modern extraction technologies.
The availability and suitability of extraction techniques provides an
opportunity for optimal use of any of these for recovery of specific
active compounds. The utilization of bioactive compounds isolated
from agricultural wastes can reduce the risk and cost of waste
treatment as well as potentially add more value to agricultural and
food production. These recovered biomolecules help to enhance
consumer health by developing nutraceuticals, functional foods,
dietary supplements, and active or smart agents for biodegradable
materials and packaging. Thus, valorization of bioactive com-
pounds from the byproducts of agricultural industry reduces the
waste disposal burden and mitigates the environmental problem
with sustainable use of natural resources [45].
Bioactive phytochemicals extracted from tomato byproducts
such as tocopherols, carotenes, polyphenols, and terpenes have
showed significant amounts of antioxidant activities. Therefore,
these value-adding components isolated from such waste can be
potentially utilized as a natural antioxidant source in the formula-
tion of functional foods or can serve as additives in food products to
extend their shelf-life [46]. Bioactive compounds recovered from
various fruit and vegetable processing waste with their bioactivity
are summarized in Table 2.
7 Nano Emulsion as Potential Delivery Systems for Bioactive Compounds
Nanotechnology has found enormous applications in different sec-

tors, and nano-emulsion of bioactive compounds and functional
food ingredients have a promising potential for application in food
industries. Nano-emulsions are small, droplet-sized in the range of
100 nm and are kinetically stable colloidal systems. As compared to
conventional emulsions, a nano-emulsion-based delivery system
plays an important role in improving the solubility, functionality,
and textural properties of bioactive compounds, especially lipophi-
lic active food ingredients. Nano-emulsions include encapsulation
of poorly soluble compounds (natural preserving agents, nutraceu-
ticals, colorants, flavors), texture modification, and enhancement in
bioavailability, digestibility, solubility, etc. [55].
Table 2
Bioactive compounds from fruit and vegetable processing waste and by-products
Source Residue Bioactive components Bioactivity References

Tomato Skin, Lycopene, carotenoids, Antioxidant, antimicrobial [47]
pomace Flavonones activity
Mango Peel, Phenolic compounds, Lower the risk of cancer, [48]
kernel, carotenoids, flavonoids, cataracts, Alzheimer’s
pomace, vitamin C, and dietary fiber disease, anti-oxidant
seed
Banana Peel Flavonols, phenolic acids, Antioxidant, antibacterial, [49]
catechins reducing blood sugar &
cholesterol
Citrus Peel, pulp, Pectin, hesperidin Immunomodulatory, anti- [50]
fruits seeds oxidant, anti-cancer
Apple Pomace Phenolic acids, flavonoids, Antioxidant, antimicrobial, [51]
anthocyanins anti-inflammatory,
Cardioprotective
Carrots Peel Phenols, Beta-carotene Provitamin A, dietary fiber, [52]
antioxidant,
Cauliflower Stem and Bioactive peptides, flavonoids, Antioxidant, anti-obesity, [53]
leaves quercetin, Kaempferol Chemopreventive
Beetroot Pomace Betalins, flavonoids, phenolic Antioxidant, [54]
compounds Hepatoprotective activity
Lipophilic bioactive compounds such as carotenoids, omega-3

fatty acids, polyphenols, flavonoids, phytosterols, and tocopherols
are susceptible to being incorporated in food products. The incor-
poration of highly lipophilic bioactive compounds in food is a great
challenge due to their poor water solubility, fast oxidation, and
instability in food formulations. Nano-sized structures such as
nano-emulsions of oil-in-water are regarded as useful tools with
great potential in the food sector to incorporate food ingredients.
The reduction in the size of bioactive compounds incorporated
within a solution would help to increase the surface area per mass
unit of nano-emulsions, thus enhancing the solubility and stability
in foods. The smaller droplet size of nano-emulsions will enable
higher bioaccessibility of bioactive compounds encapsulated [56].
The nano-emulsion formulations of active ingredients can be
used to develop biodegradable coating and packaging films to
enhance the quality, functional properties, nutritional value, and
shelf life of foods. However, food grade nano-emulsions can find
widespread application only if their production cost is commercially
feasible and meets the safety standards of the food industry [57].
8 Application of Bioactive Compounds
8.1 Functional Foods The successively growing demand for foods with beneficial effects
on human health, while contributing to the sustainable use of
natural resources, enhances the research interest in potential utili-
zation of bioactive compounds. Bioactive compounds are receiving
more popularity due to their diverse biological activities and huge
exploitation in various commercial sectors, such as food,
pharmaceutical, and cosmetic industries. These compounds exhibit
beneficial effects such as antioxidant activity, anti-diabetic, anti-
cancerous, antidiuretic, anti-atherosclerotic, and so on for human
beings. Bioactive compounds have multiple applications in food,
acting as antimicrobials, antioxidants, natural dyes, fortifying ingre-
dients, texture modifiers, and others [58]. Bioactive ingredients
such as anthocyanins, curcumins, tannins, and carotenoids are
commonly applied as natural colorings in food product prepara-
tions [59]. Moreover, they have also been used for the develop-
ment of active and smart biodegradable food packaging
materials [60].
The technological advancements made possible the extraction
of bioactive compounds not only from natural sources but also
from byproducts and their reintroduction into foods. Bioactive
compounds can be used to improve the quality of conventional
foods with respect to nutritional, sensorial, and technological prop-
erties (e.g., water and oil holding capacities, foaming, emulsion,
and gelatinization) [61]. Bioactive compounds are key factors in
the development of nutraceuticals and functional foods. The possi-
bility of applying bioactive components in food products and in
new technologies to enhance food product quality and safety is
enormous. Due to the diversity of compounds, their possible inter-
actions, and various physiological activities, each component must
be properly evaluated for the production of food, beverages, and
active and smart packaging applied to food to guarantee maximum
potential in the applications [45].
8.2 Food Phenolic compounds are well known for their health benefits
Preservatives related to antioxidant activity and thus have potential use as bio-
preservatives. These compounds are extensively studied for their
potential application in the food sector for improving the shelf life
of perishable food products. The biological activity of phenolic
compounds delays or inhibits the oxidation and growth of micro-
organisms; these compunds are thus considered as biopreservatives
for safe extension of perishable products [62].
8.3 Pharmaceuticals Phytochemicals are biologically active, naturally occurring chemical

compounds in plants with various therapeutic benefits beyond
those attributed to macronutrients and micronutrients. Nature-
derived bioactive components have been investigated to elucidate
their biological activity for conventional medicines in the preven-

tion and treatment of several chronic diseases. There is currently a
growing interest in the study of bioactive compounds, extracts, and
new ingredients from natural sources to produce pharmaceuticals,
nutraceuticals, and dietary supplements. In recent years, the
demand for herbal medicines and several natural products is consis-
tently increasing for a healthy and sustainable life.
Bioactive food ingredients have emerged as a key component
related to health-promoting and disease-preventing functions.
Plant-derived bioactive compounds can help to suppress inflamma-
tion by inhibiting oxidative damage and communicating with the
immune system. These bioactive ingredients control diet-related
medical conditions such as obesity, cancer, cardiovascular diseases,
osteoporosis, and other metabolic diseases [63]. Isoflavones can
decrease dietary cholesterol absorption and low-density plasma
lipoproteins via binding cholesterol in the intestinal tract.
Bioactive compounds are considered promising ingredients
used to meet the human body’s requirement and are usually con-
sumed in the form of pharmaceutical preparations, such as pills,
tablets, capsules, and powders [64]. The most commonly marketed
and used nutraceuticals are amino acids, vitamins (C and E), miner-
als (copper, selenium, and zinc), carotenoids (β-carotene, lutein,
zeaxanthin, and lycopene), fatty acids (omega-3 and omega-6),
polyphenols, and several others [65], which can be extracted from
agro-industrial commodities and its byproducts as well.
9 Conclusion and Future Perspective
Bioactive compounds are recognized due to their pharmacological

and nutraceutical value and can be potentially utilized in the devel-
opment of functional foods. The ever-growing demand for the
extraction of plant-derived bioactive compounds continuously
encourages the search for convenient extraction methods.
Researchers have been seeking to use these techniques alone or in
association with conventional techniques that result in better yield
with less extraction time, extraction of heat-labile compounds, and
less generation of toxic waste. The selection of these techniques
depends on factors such as time, yield, and cost of extraction.
Technological advancement and environmental awareness are two
important factors for the development of different advanced extrac-
tion techniques. Many studies are still being carried out to improve
these new green extraction techniques further, with the intention of
reducing the cost of extraction, the time consumed, the quality of
the extract, health, and environmental safety. These technologies
could provide an innovative approach in the upcoming years to
increase the production of specific compounds for use as nutraceu-
ticals or as ingredients in the design of functional foods.
References
1. Bernhoft A (2010) A brief review on bioactive 552–557. https://doi.org/10.1016/B978-0-
compounds in plants. In: Bernhoft A 12-384947-2.00261-0
(ed) Proceedings from symposium held at 11. Santos TRJ, de Aquino Santana LCL (2022)
The Norwegian Academy of Science and Let- Conventional and emerging techniques for
ters, Oslo, November 13–14, 2008: Bioactive extraction of bioactive compounds from fruit
compounds in plants – benefits and risks for waste. Braz J Food Sci Technol 25:e2021130,
man and animals. The Norwegian Academy of 1–18. https://doi.org/10.1590/1981-6723.
Science and Letters, Oslo, pp 11–17 13021
2. Aanchal W, Kumar GA, Vatsala S (2019) Role 12. Wei Z-F, Wang X-Q, Peng X, Wang W, Zhao
of bioactive compounds in human health. Acta C-J, Zu Y-G, Fu Y-J (2015) Fast and green
Sci Med Sci 3(9):25–33 extraction and separation of main bioactive fla-
3. Soquetta MB, de Marsillac Terra L, Bastos CP vonoids from Radix Scutellariae. Ind Crop
(2018) Green technologies for the extraction Prod 63:175–181. https://doi.org/10.1016/
of bioactive compounds in fruits and vegeta- j.indcrop.2014.10.013
bles. J Food 16(1):400–412 13. Huie CW (2002) A review of modern sample-
4. Jurinjak TA, Benkovic M, Valinger D, Jurina T, preparation techniques for the extraction and
Belscak-Cvitanovic A, Gajdos KJ (2018) Opti- analysis of medicinal plants. Anal Bioanal Chem
mizing bioactive compounds extraction from 373:23–30
different medicinal plants and prediction 14. Jacotet-Navarro M, Rombaut N, Deslis S,
through nonlinear and linear models. Ind Fabiano-Tixier A-S, Pierre F-X, Bily A, Chemat
Crop Prod 126:449–458. https://doi.org/ F (2016) Towards a “dry” bio-refinery without
10.1016/j.indcrop.2018.10.040 solvents or added water using microwaves and
5. Azmir J, Zaidul ISM, Rahman MM, Sharif KM, ultrasound for total valorization of fruit and
Mohamed A, Sahena F, Jahurul MHA, vegetable byproducts. Green Chem 18(10):
Ghafoor K, Norulaini NAN, Omar AKM 3106–3115. https://doi.org/10.1039/
(2013) Techniques for extraction of bioactive C5GC02542G
compounds from plant materials: a review. J 15. Silva RPFF, Rocha-Santos TAP, Duarte AC
Food Eng 117:426–436. https://doi.org/10. (2016) Supercritical fluid extraction of bioac-
1016/j.jfoodeng.2013.01.014 tive compounds. Trends Anal Chem 76:40–51.
6. Stéphane FFY, Jules BKJ, Batiha GE-S, Ali I, https://doi.org/10.1016/j.trac.2015.11.013
Bruno LN (2021) Extraction of bioactive com- 16. Diaz-Reinoso B, Moure A, Domı́nguez H,
pounds from medicinal plants and herbs. In: Parajó JC (2006) Supercritical CO2 extraction
Natural medicinal plants. IntechOpen, and purification of compounds with antioxi-
L o n d o n . h t t p s : // d o i . o r g / 1 0 . 5 7 7 2 / dant activity. J Agric Food Chem 54(7):
intechopen.98602 2441–2469
7. Azwanida NN (2015) A review on the extrac- 17. Williams OJ, Raghavan GSV, Orsat V, Dai J
tion methods use in medicinal plants, principle, (2004) Microwave-assisted extraction of cap-
strength and limitation. Med Aromat Plants saicinoids from capsicum fruit. J Food Biochem
4(3):1–10. https://doi.org/10.4172/ 28:113–122
2167-0412.1000196 18. Altemimi A, Lakhssassi N, Baharlouei A, Wat-
8. Zhang Q-W, Lin L-G, Ye W-C (2018) Techni- son DG, Lightfoot DA (2017) Phytochem-
ques for extraction and isolation of natural icals: extraction, isolation, and identification
products: a comprehensive review. Chin Med of bioactive compounds from plant extracts.
13(20):1–26. https://doi.org/10.1186/ Plan Theory 6(42):1–23. https://doi.org/10.
s13020-018-0177-x 3390/plants6040042
9. Ashraf R, Ghufran S, Akram S, Mushtaq M, 19. Machado APF, Sumere BR, Mekaru C,
Sultana B (2020) Cold pressed coriander (Cor- Martinez J, Bezerra RMN, Rostagno MA
iandrum sativum L.) seed oil. In: Cold (2019) Extraction of polyphenols and antiox-
pressed oils: green technology, bioactive com- idants from pomegranate peel using ultra-
pounds, functionality and applications. Aca- sound: influence of temperature, frequency
demic Press, London, pp 345–356 and operation mode. Int J Food Sci Technol
10. Cook CM, Lanaras T (2016) Essential oils: 54:2792–2801. https://doi.org/10.1111/
isolation, production and uses. In: Encyclope- ijfs.14194
dia of food and health. Academic Press, pp
20. Jain T (2009) Microwave assisted extraction 32. Ridgway K, Smith RM, Lalljie SPD (2012)
for phytoconstituents – an overview. Asian J Sample preparation for food contaminant
Res Chem 2(1):19–25 analysis. In: Comprehensive sampling and sam-
21. Nour AH, Oluwaseun AR, Nour AH, Omer ple preparation, vol 3. Academic Press, Amster-
MS, Ahmed N (2021) Microwave-assisted dam, pp 819–833
extraction of bioactive compounds 33. Khaw KY, Parat MO, Shaw PN, Falconer JR
(review). In: Microwave heating. IntechOpen, (2017) Solvent supercritical fluid technologies
L o n d o n . h t t p s : // d o i . o r g / 1 0 . 5 7 7 2 / to extract bioactive compounds from natural
intechopen.96092 sources: a review. Molecules 22:1186.
22. Alupului A (2012) Microwave extraction of h t t p s : // d o i . o r g / 1 0 . 3 3 9 0 /
active principles from medicinal plants. UPB molecules22071186
Sci Bull Ser B 74(2):129–142 34. Wijngaard H, Hossain MB, Rai DK, Brunton
23. Saulis G (2010) Electroporation of cell mem- N (2012) Techniques to extract bioactive com-
branes: the fundamental effects of pulsed elec- pounds from food by-products of plant origin.
tric fields in food processing. Food Eng Rev Food Res Int 46:505–513. https://doi.org/
2(2):52–73. https://doi.org/10.1007/ 10.1016/j.foodres.2011.09.027
s12393-010-9023-3 35. Usman I, Hussaina M, Afzaala M, Saeed F,
24. Pereira RN, Avelar Z, Pereira SG, Rocha CMR, Javed M, Afzal A, Ashfaq I, Al Jbawi E, Saewan
Teixeira JA (2022) Pulsed electric fields for the SA (2022) Traditional and innovative
extraction of proteins and carbohydrates from approaches for the extraction of bioactive com-
marine resources. In: Innovative and emerging pounds. Int J Food Prop 25(1):1215–1233
technologies in the bio-marine food sector. 36. Chemat F, Rombaut N, Sicaire A-G,
Academic Press, London, pp 173–195 Meullemiestre A, Fabiano-Tixier A-S, Abert-
25. Ranjha MMAN, Kanwal R, Shafique B, Arshad Vian M (2017) Ultrasound assisted extraction
RN, Irfan S, Kieliszek M, Kowalczewski PŁ, of food and natural products. Mechanisms,
Irfan M, Khalid MZ, Roobab U, Aadi RM techniques, combinations, protocols and appli-
(2021) A critical review on pulsed electric cations. A review. Ultrason Sonochem 34:540–
field: a novel technology for the extraction of 560. https://doi.org/10.1016/j.ultsonch.
phyto-constituents. Molecules 26:4893–4916 2016.06.035
26. Thulasida JS, Varadarajan GS, Sundararajan R 37. Le W, Zhang Z, Sun D-W, Sivagnanam SP,
(2019) Pulsed electric field for enhanced Tiwari BK (2020) Combination of emerging
extraction of intracellular bioactive compounds technologies for the extraction of bioactive
from plant products: an overview. Nov Appro compounds. Crit Rev Food Sci Nutr 60(11):
Drug Des Dev 5(2):1–7 1826–1841. https://doi.org/10.1080/
27. Łubek-Nguyen A, Ziemichód W, Olech M 10408398.2019.1602823
(2022) Application of enzyme-assisted extrac- 38. Lemes AC, Sala L, Ores JDC, Braga ARC,
tion for the recovery of natural bioactive com- Egea MB, Fernandes KF (2016) A review of
pounds for nutraceutical and pharmaceutical the latest advances in encrypted bioactive pep-
applications. Appl Sci 12(7):3232–3252 tides from protein-richwaste. Int J Mol Sci 17:
28. Shinwari KJ (2021) Emerging technologies for 9 5 0 – 9 6 0 . h t t p s : // d o i . o r g / 1 0 . 3 3 9 0 /
the recovery of bioactive compounds from saf- ijms17060950
fron species. In: Saffron. Academic Press, 39. Ingle KP, Deshmukh AG, Padole DA, Dudhare
London, pp 143–182 MS, Moharil MP, Khelurkar VC (2017) Phy-
29. Marathe SJ, Jadhav SB, Bankar SB, Singhal RS tochemicals: extraction methods, identification
(2017) Enzyme assisted extraction of and detection of bioactive compounds from
bioactives. In: Food bioactives. Springer, pp plant extracts. J Pharmacogn Phytochem 6(1):
171–201 32–36
30. Otero-Pareja MJ, Casas L, Fernández-Ponce 40. Zhang Z, Pang X, Xuewu D, Ji Z, Jiang Y
MT, Mantell C, Martı́nez de la Ossa EJ (2005) Role of peroxidase in anthocyanin deg-
(2015) Green extraction of antioxidants from radation in litchi fruit pericarp. Food Chem 90:
different varieties of red grape pomace. Mole- 4 7 – 5 2 . h t t p s : // d o i . o r g / 1 0 . 1 0 1 6 / j .
cules 20:9686–9702 foodchem.2004.03.023
31. Li J, Pettinato M, Campardelli R, De Marco I, 41. Sasidharan S, Chen Y, Saravanan D, Sundram
Perego P (2022) High-pressure technologies KM, Latha LY (2011) Extraction, isolation and
for the recovery of bioactive molecules from characterization of bioactive compounds from
agro-industrial waste. Appl Sci 12:3642–3667 plants’ extracts. Afr J Tradit Complement
Altern Med 8(1):1–10
42. Gorguc A, Gençdag E, Yılmaz FM (2020) Bio- in apple pomace from juice and cider
active peptides derived from plant origin manufacturing: potential use in dermal formu-
by-products: biological activities and techno- lations. Trends Food Sci Technol 90:76–87.
functional utilizations in food developments–a https://doi.org/10.1016/j.tifs.2019.05.014
review. Food Res Int 136:109504. https://doi. 52. De Andrade Lima M, Charalampopoulos D,
org/10.1016/j.foodres.2020.109504 Chatzifragkou A (2018) Optimisation and
43. Sagar NA, Pareek S, Sharma S, Yahia EM, Lobo modelling of supercritical CO2 extraction pro-
MG (2018) Fruit and vegetable waste: bioac- cess of carotenoids from carrot peels. J Super-
tive compounds, their extraction, and possible crit Fluids 133:94–102. https://doi.org/10.
utilization. Compr Rev Food Sci Food Saf 17: 1016/j.supflu.2017.09.028
5 1 2 – 5 3 1 . h t t p s : // d o i . o r g / 1 0 . 1 1 1 1 / 53. Gonzales GB, Raes K, Coelus S, Struijs K,
1541-4337.12330 Smagghe G, Van Camp J (2014) Ultra (high)-
44. Prado DMF, Almeida AB, Oliveira-Filho JG, pressure liquid chromatography-electrospray
Alves CCF, Egea MB, Lemes AC (2020) ionization-time-of-flight-ion mobility-high-
Extraction of bioactive proteins from seeds definition mass spectrometry for the rapid
(corn, sorghum, and sunflower) and sunflower identification and structural characterization
byproduct: enzymatic hydrolysis and antioxi- of flavonoid glycosides from cauliflower waste.
dant properties. Curr Nutr Food Sci 17(3): J Chromatogr A 1323:39–48. https://doi.
3 1 0 – 3 2 0 . h t t p s : // d o i . o r g / 1 0 . 2 1 7 4 / org/10.1016/j.chroma.2013.10.077
1573401316999200731005803 54. Vulic JJ, Cebovic TN, Canadanovic-Brunet
45. Lemes AC, Egea MB, de Oliveira Filho JG, JM, Cetković GS, Canadanovic VM, Djilas
Gautério GV, Ribeiro BD, Coelho MAZ SM, Tumbas Šaponjac VT (2014) In vivo and
(2022) Biological approaches for extraction of in vitro antioxidant effects of beetroot pomace
bioactive compounds from agro-industrial extracts. J Funct Foods 6:168–175. https://
by-products: a review. Front Bioeng Biotech- doi.org/10.1016/j.jff.2013.10.003
nol 27(9):1–18. https://doi.org/10.3389/ 55. Saini A, Panesar PS, Bera MB (2019) Valoriza-
fbioe.2021.802543 tion of fruits and vegetables waste through
46. Kalogeropoulos N, Chiou A, Pyriochou V, green extraction of bioactive compounds and
Peristeraki A, Karathanos VT (2012) Bioactive their nanoemulsions-based delivery system.
phytochemicals in industrial tomatoes and their Bioresour Bioprocess 6(26):1–12
processing byproducts. LWT Food Sci Technol 56. Odriozola-Serrano I, Oms-Oliu G, Martı́n-
49:213–216 Belloso O (2014) Nano-emulsion-based deliv-
47. Fermoso FG, Serrano A, Alonso-Fariñas B, ery systems to improve functionality of lipophi-
Fernández-Bolaños J, Borja R, Rodrı́guez- lic components. Front Nutr 1:1–4. https://
Gutiérrez G (2018) Valuable compound doi.org/10.3389/fnut.2014.00024
extraction, anaerobic digestion, and compost- 57. Aswathanarayan JB, Vittal RR (2019) Nanoe-
ing: a leading bio refinery approach for agricul- mulsions and their potential applications in
tural wastes. J Agric Food Chem 66:8451– food industry. Front Sustain Food Syst 13(3):
8468. https://doi.org/10.1021/acs.jafc. 1–21. https://doi.org/10.3389/fsufs.2019.
8b02667 00095
48. Ayala-Zavala JF, González-Aguilar GA (2011) 58. Veneziani G, Novelli E, Esposto S, Taticchi A,
Use of additives to preserve the quality of Servili M (2017) Chapter 11 – Applications of
fresh-cut fruits and vegetables. In: Advances recovered bioactive compounds in food
in fresh-cut fruits and vegetables processing. products. In: Galanakis CM (ed) Olive mill
CRC Press, Boca Raton, pp 231–254 waste. Academic Press, Cambridge, MA, pp
49. Vu HT, Scarlett CJ, Vuong QV (2018) Pheno- 231–253. https://doi.org/10.1016/b978-0-
lic compounds within banana peel and their 12-805314-0.00011-x
potential uses: a review. J Funct Foods 40: 59. Luzardo-Ocampo I, Ramı́rez-Jiménez AK,
238–248. https://doi.org/10.1016/j.jff. Yañez J, Mojica L, Luna-Vital DA (2021)
2017.11.006 Technological applications of natural colorants
50. Naqash F, Masoodi FA, Rather SA, Wani SM, in food systems: a review. Foods 10(3):
Gani A (2017) Emerging concepts in the 6 3 4 – 6 4 5 . h t t p s : // d o i . o r g / 1 0 . 3 3 9 0 /
nutraceutical and functional properties of foods10030634
pectin-a review. Carbohydr Polym 168:227– 60. Alizadeh-Sani M, Mohammadian E, Rhim J-W,
239. https://doi.org/10.1016/j.carbpol. Jafari SM (2020) pH-sensitive (halochromic)
2017.03.058 smart packaging films based on natural food
51. Barreira JCM, Arraibi AA, Ferreira ICFR colorants for the monitoring of food quality
(2019) Bioactive and functional compounds and safety. Trends Food Sci Technol 105:93–
144. https://doi.org/10.1016/j.tifs.2020.08. 63. Soumya NPP, Mini S, Sivan SK, Mondal S

014s (2021) Bioactive compounds in functional
61. Guimaraes RM, Pimentel TC, de Rezende food and their role as therapeutics. Bioact
TAM, Silva J d S, Falcão HG, Ida EI (2019) Compd Health Dis 4(3):24–39
Gluten-free bread: effect of soy and corn 64. Kumar K, Yadav AN, Kumar V, Vyas P, Dhali-
co-products on the quality parameters. Eur wal HS (2017) Food waste: a potential bio
Food Res Technol 245(7):1365–1376. resource for extraction of nutraceuticals and
https://doi.org/10.1007/s00217-019- bioactive compounds. Bioresour Bioprocess
03261-9 4(1):1–14. https://doi.org/10.1186/
62. Martillanes S, Rocha-Pimienta J, Cabrera- s40643-017-0148-6
Bañegil M, Martı́n-Vertedor D, Delgado-Adá- 65. Souyoul SA, Saussy KP, Lupo MP (2018)
mez J (2016) Application of phenolic com- Nutraceuticals: a review. Dermatol Ther (Hei-
pounds for food preservation: food additive delb) 8(1):5–16. https://doi.org/10.1007/
and active packaging. In: Phenolic compounds. s13555-018-0221-x
IntechOpen, pp 1–12
Chapter 2
Extraction of Bioactive and Nutraceuticals from Plants

and Their Application
Hadia Hemmami, Bachir Ben Seghir, Soumeia Zeghoud, Ilham Ben Amor,
Abdelkrim Rebiai, and Imane Kouadri
Abstract
Natural bioactive compounds are a useful source of molecules for the development of nutraceuticals, food
additives, and functional foods because they contain a wide variety of diverse structural and functional
properties. Although extracting them for use in biological and/or phytochemical investigations presents
some particular challenges, plant-derived bioactive and nutraceutical compounds are now the subject of a
lot of studies since they offer a variety of biological properties and therapeutic benefits. In order to cut costs
related to their synthesis and separation, the process of extracting active phytochemicals is also carefully
taken into account. Despite the fact that natural bioactive compounds and functional foods have been used
as traditional medicines to treat chronic diseases for decades, recent scientific studies emphasize the health
advantages of functional meals and reveal the underlying processes that underlie their activities. To cure and
prevent inflammatory and oxidative diseases, phytochemicals perform essential bioactive roles. Plant-
derived bioactive compounds that don’t cause oxidative damage and interact with the immune system
might lessen inflammation. The capacity to bind to poisons or carcinogens that impact the digestive system
exists in many bioactive compounds.
This chapter’s goal is to present, summarize, and assess the many approaches utilized to extract bioactive
and nutritional components from plants, besides their most recent applications.
Key words Extraction, Bioactive compounds, Nutraceuticals compounds, Plants
1 Introduction
Herbal items made from medicinal plants are useful sources that
come from all over the world and can include a wide range of
ingredients [1, 2]. Natural remedies have been used to cure both
acute and chronic illnesses [3] for as long as human civilization has
existed. One of the most significant sources of food and medicine
for people is natural products [4]. They often contain a range of
physiologically active chemicals, such as phenolic substances, vita-
mins, sulfur compounds, pigments, terpenoids, and other naturally
occurring antioxidants [5], which are efficient in enhancing defense
21
22 Hadia Hemmami et al.
and treating cancer and cardiovascular disorders [6]. The amount

of active components in natural products has a significant impact on
their efficacy. The habitat, age, harvest season, drying techniques,
and other factors can all affect the chemical makeup of natural
products [7]. Natural products are widely used; however due to
their accessibility, there are concerns about their precision, efficacy,
and safety. Many products are available over the counter and are
made up of a mixture labeled with various herbal ingredients [2, 8].
The choice of the best extraction technique is crucial to both
quantitative and qualitative analyses of bioactive chemicals derived
from plant materials [9, 10]. Despite the fact that extraction is the
fundamental initial step in both quantitative and qualitative analyses
of the components of medicinal plants. An extraction technique
should ideally be comprehensive in terms of the components to be
studied, quick, easy, affordable, and highly automated [11]. In the
field of natural products, chromatographic and spectrometric
methods have made major contributions, particularly in the identi-
fication, separation, and characterization of bioactive chemicals
from plant sources [12]. The most frequent variables influencing
extraction processes are matrix properties of the plant part, pres-
sure, temperature, time, and solvent. The advancement of bioactive
analysis over the past 10 years has been fuelled by a greater under-
standing of the dynamic chemical nature of the various bioactive
molecules. These strategies and techniques produced a significant
shift toward this “green Eldorado” between 1990 and 2000
[13]. The usage of natural chemicals from plant origin is attracting
a lot of attention from numerous industrial sectors and individuals
globally. The cosmetic, culinary, and pharmaceutical sectors are
using natural substances derived from plants more often, and they
may one day replace synthetic chemicals [14]. Different plant com-
ponents, including leaves, roots, stems, fruits, seeds, and flowers,
contain a variety of nutrients and bioactive substances. The identi-
fication and characterization of components and the study of bio-
active substances have become simpler because of the development
of advanced and sophisticated instrumentation techniques [15].
Different extraction techniques can be used to extract plant
components. Over the past 50 years, novel techniques have been
created that are more environmentally friendly because they utilize
less synthetic and organic chemicals, operate more quickly, and
provide extracts of higher yield and quality. Ultrasound, pulsed
electric field [16], enzyme digestion [17], extrusion [18], micro-
wave heating [19], ohmic heating [20], supercritical fluids [21–
26], and accelerated solvents [19, 27] have all been used to increase
the overall yield and selectivity of bioactive components from plant
materials. have being researched as alternative techniques. Tradi-
tional extraction techniques like Soxhlet are still used as a bench-
mark for evaluating the efficacy of newly developed methodologies.
There are several scientific publications, book chapters, and
Extraction of Bioactive and Nutraceuticals from Plants and Their Application 23
Fig. 1 Chapter summary
monograph where nontraditional methodologies have been thor-

oughly examined [9, 28–31]. These works emphasize the applica-
tion of extraction techniques for nutraceuticals, food additives, and
many other industries, but they do not discuss the extraction of
bioactive chemicals from herbal plants. The goal of this chapter is to
give a thorough overview of various techniques. Figure 1 sum-
marizes the main points of the chapter.
2 Primary and Secondary Metabolites
Small molecules called metabolites are intermediary biological pro-

cesses. Primary metabolites are recognized as crucial or necessary
substances and have a direct role in plants’ typical growth, repro-
duction, and development. Cell components such as carbohy-
drates, polysaccharides, sugars, amino acids, lipids, and proteins
and fermentation byproducts such as ethanol, acetic acid, citric
acid, and lactic acid are examples of primary metabolites that are
primarily used by organisms during their growth and development
stages [32–34].
Secondary metabolites typically have a function but are not very
vital for the organism because they are not directly involved in those
processes (e.g. phenolic, steroids, lignans, etc.). They express the
uniqueness of species and can only be found in certain organisms or
groups of organisms [10, 32, 34]. In many cases, the purpose of
these molecules and how they help the organism are not fully
understood, and they are not always generated under all circum-
stances. Plants contain many primary and secondary metabolites
(Fig. 2).
Fig. 2 Primary and secondary metabolites
3 Bioactive Compounds
A plant extract is a material or an active ingredient having useful

qualities that have been extracted from the tissues of a plant,
typically by subjecting it to a solvent treatment, for use in a specific
application. Although not yet recognized as essential nutrients,
“bioactive substances” are typically referred to as physiologically
significant chemicals [35]. Essential (e.g., vitamins) and nonessen-
tial (e.g., alkaloids and polyphenols) substances that naturally
occur, are fed to animals, and have the potential to have an impact
on human health are known as bioactive compounds [36]. They
come from a variety of natural sources, including animals, fungi,
plants, and marine organisms (like lichens). Natural sources often
only contain small amounts of bioactive substances [37, 38].
Plant composites typically contain plant-active chemicals. All
plant organs and parts, including leaves, barks, roots, woods,
tubers, gums or oleoresin exudations, figs, fruits, rhizomes, flowers,
twigs, berries, as well as the entire plant, are capable of synthesizing
active chemicals in minute amounts and at various concentrations.
After extraction, additional steps could be needed to separate or
purify the target chemicals.
4 Bioactive Compound Types “Natural Phenols”
According to their chemical makeup, bioactive compounds in

plants can be classified as antioxidant vitamins, polyphenols, ter-
pene derivatives, phytoestrogens, minerals, polyunsaturated fatty
acids, dietary fiber, and phytic acid [39]. Additionally, they can be
grouped according to their distribution in diverse plants (specific to
vegetable species or widespread), range of concentration found in
plant-based diets and the human body, possible sites of action, and
efficiency against various [40]. Compounds with an aromatic ring
and one or more hydroxyl groups are known as phenols. They are
secondary metabolites that originate from the phenyl propanoid,
shikimate, or pentose phosphate pathways in plants [41]. They are
widely spread across the plant world and range in complexity from
simple phenolic acids to tannins. The majority of them are now an
essential component of the human diet and may be found in all
plant components, including all vegetative organs, as well as in
flowers, vegetables, cereals, fruits, seeds, grains, and other foods.
Flavonoids make up half of these ubiquitously found phenolic
bioactive chemicals [42]. Phenolics are frequently used in a variety
of UV radiation protection systems or to fend off attacks from
viruses, parasites, predators, etc. Additionally, it incorporates a
number of additional defense-related biochemical processes that
work as antioxidants, antimutagenic, and anticarcinogens, but
most critically, they further alter gene expression [43]. Additionally,
it affects the coloring of rare plant species. Primarily divided into
various classes, phenolic substances include phenolic acids, tannins,
stilbenes, lignins, and flavonoids (Fig. 3).
5 Techniques for the Extraction, Isolation, and Purification of Bioactive Compounds
Phytochemicals are chemical compounds found in plant extracts

that have medicinal significance and have a particular physiological
effect on humans. These plant compounds have been employed in
homeopathic and ayurveda medicine to cure illnesses ever since
ancient times. These are nonnutritive compounds with defensive
or preventing qualities. Alkaloids, flavonoids, tannins, and phenolic
chemicals make up the majority of these bioactive substances
[44]. The primary raw materials used to produce new drugs are
these molecules in various combinations. Numerous bioactive sub-
stances with antibacterial characteristics are found in plants to
provide defense against aggressor agents, particularly microbes
[10]. For individuals who work on herbal medications, the main
issues and major hurdles in the separation, extraction, and
N
N
CH3 OH
N
Alkaloids Alkaloids Monoterpenes
OH O OH
OH
HO O
CH3 CH3
O O
O
OH
Polyacetylenes Sesqueterpenes
Polyketides
OH
C O HO O
OH
OH
OH
RO
OH O
Triterpenes, saponins, steroid Flavonoids
Fig. 3 Bioactive compounds
characterization of bioactive components in botanicals and herbal

formulations are covered. The extraction of components from raw
herbal preparations is the most crucial stage in the study of con-
stituents; the pros and downsides of various extraction methods are
covered in the following. These methods include chromatographic
procedures and phytochemical screening tests (high-pressure liquid
chromatography [HPLC], immunoassay, thin-layer chromatogra-
phy [TLC], and Fourier transform infrared [FTIR]).
The WHO estimates that there are over 20,000 medicinal
plants in 91 countries with massive biodiversity. Mexico, Peru,
Ecuador, Madagascar, India, Zaire, Colombia, Brazil, Australia,
Indonesia, Malaysia, and China were among the original 91 nations.
Later, the United States, South Africa, the Democratic Republic of
the Congo, the Philippines, and Venezuela were joined, bringing
the total to 18 nations. The initial steps in employing a bioactive
molecule from plant bodies include extraction, pharmacological
screening, isolation and characterization of bioactive compounds,
toxicological estimation, and clinical assessment.
An overview of the usual methods for extracting, separating,
and characterizing bioactive compounds from plant extract is given
in Fig. 4.
Fig. 4 Steps involved in extraction, isolation, and characterization of bioactive compounds from plant extract
5.1 Extraction The primary step in analyzing important plant components for
Methodology separation and characterization is extraction.
Prewashing, drying plant materials in the air, obtaining a
homogeneous sample by grinding, and often enhancing the kinet-
ics of extract by increasing sample contact with the solvent system
are the most crucial processes. The proper steps must be followed
to prevent possible active extract ingredients from being distorted
or destroyed during sample preparation. If the plants are chosen
based on their traditional usage [45], plant sample extracts are
made according to the traditional healer’s instructions in the same
sequence to mimic the traditional “herbal” medication as closely as
feasible. The clear-cut nature of the bioactive compounds being
targeted is a major factor in the solvent system selection. For
instance, the hydroalcoholic leaf extract of Aegle marmelos was
found to be effective against MNU-induced toxicity, which is
responsible for liver inflammation and hepatocarcinogenesis
[46]. There are several extraction solvent systems available to
extract the bioactive components from natural goods. Polar sol-
vents like methanol, ethanol, or ethyl acetate are used to isolate
hydrophilic chemicals, whereas dichloromethane or a 1:1 combina-
tion of dichloromethane and methanol is utilized to isolate more
hydrophobic compounds.
To get rid of chlorophyll, hexane extraction might be helpful

occasionally [47]. For the extraction of plant materials, a variety of
techniques, including sonification, heating under reflux, and a few
others, are frequently utilized [48]. Additionally, Soxhlet extrac-
tion, fresh plants, or dried, crushed plant materials can be macer-
ated or percolated in water and/or organic solvent systems to create
plant extracts. The most popular modern extraction methods
include solid-phase microextraction, supercritical-fluid extraction,
pressurized liquid extraction, solid-phase extraction, surfactant-
mediated, and microwave-assisted extraction techniques. These
methods have some advantages over chromatographic analysis,
including a reduction in the amount of organic solvent used and a
reduction in sample degradation.
5.2 Identification Modern methods allow for the concurrent creation of several com-
and Characterization plex bioassays and their accessibility, while also offering precise
methods for isolation, separation, and purification [49]. The objec-
tive of searching for bioactive chemicals is to find a method that is
suitable for monitoring the source material for bioactivity, such as
antibacterial, cytotoxicity, or antioxidant, with simplicity, specific-
ity, and speed (Fig. 5) [50]. Due to the expense, length of time, and
potential for ethical problems associated with animal research,
in vitro procedures are typically more favorable than in vivo trials.
Although diverse plant sections and/or many of them will create
different chemicals, in addition to their varying chemical structures
and physicochemical qualities, the isolation and characterization
techniques for bioactive compounds are not difficult [51]. The
Fig. 5 Methods used for bioactive compound extraction

Fig. 6 Plant extract as natural antioxidant
selection and gathering of plant materials are regarded as the first

steps in the isolation and characterization of bioactive phytochem-
icals. The subsequent step entails gathering ethnobotanical data in
order to identify potential bioactive compounds.
To separate and purify the active chemicals that are responsible
for the bioactivity, extracts can be created using a number of
solvents. Based on their characteristics, bioactive substances can
be isolated and purified using column chromatographic methods.
The purification of the bioactive compounds is sped up by tools like
HPLC. The purified chemicals may be identified using a variety of
spectroscopic methods, including UV-visible (UV-Vis), mass spec-
troscopy infrared (IR), and nuclear magnetic resonance (NMR)
[52–55] (Fig. 6).
5.3 Purification of Paper or TLC and column chromatography have been used to
the Bioactive identify and characterize a large number of bioactive chemicals.
Molecules Due to their accessibility, affordability, and ease in a variety of
stationary phases, TLC and column chromatography are still fre-
quently utilized. The most effective methods for separating the
phytochemicals may be found in silica, alumina, cellulose, and
polyamide.
High concentrations of diverse phytochemicals present in plant

materials make effective separation challenging. Therefore, for
highly valued separations, increasing the polarity of the various
mobile phases is beneficial. Column chromatography has long
been used to evaluate the chosen fractions of substances using
TLC. Bioactive compounds have been separated using TLC and
silica gel column chromatography using a variety of analytical tools
[56]. The needed bioactive chemicals’ structures are explained and
their identities are determined using information from a variety of
spectroscopic techniques, including UV-Vis, IR, NMR, and mass
spectroscopy. The fundamental principle of spectroscopy is that
electromagnetic radiation is transmitted by organic molecules,
some of which absorb radiation while others do not. A spectrum
may be created by figuring out how much electromagnetic energy is
absorbed. The bonds contained in a molecule are peculiar to certain
spectra. The organic molecule’s structure can be roughly identified
or determined based on these spectra. For structural explanation,
scientists mostly employ the IR, UV visible, radio frequency, and
electron beam spectra generated from three or four areas [52].
5.3.1 UV-Visible UV-visible spectroscopy may be used for qualitative analysis and the
Spectroscopy (UV-Vis) identification of certain types of compounds in both pure and
biological mixtures. Quantitative research on aromatic chemicals
uses UV-visible spectroscopy because they are potent UV chromo-
phores. Natural chemicals can be identified via UV-visible spectros-
copy [56]. Anthocyanidins, tannins, polymer dyes, and phenols are
phenolic chemicals that may be easily identified by UV-Vis spec-
troscopy [57]. It was discovered that UV-Vis methods are less
discriminating and provide complicated data on total polyphenol
content. The total phenolic extract (280 nm), phenolic acids
(360 nm), flavones (320 nm), and anthocyanidins are all quantified
using UV-Vis spectra (520 nm). Comparing this procedure to
others, it is both less time- and money-consuming [57].
5.3.2 Infrared When infrared light passes through a sample of an organic mole-
Spectroscopy (IR) cule, some of the frequencies will be absorbed while other frequen-
cies will pass through the sample undetected. When a molecule is
exposed to infrared light, it undergoes changes in vibration that are
related to infrared absorption. As a result, it is possible to think of
infrared spectroscopy as a type of vibrational spectroscopy. The
vibrational frequencies of various bonds (C-C, C=C, CC, C-O,
C=O, O-H, and N-H) vary [56]. By identifying the distinctive
frequency absorption band in the IR spectra, it is possible to deter-
mine if such bonds are present in an organic molecule [10]. A high-
resolution analytical technology called Fourier Transform Infrared
Spectroscopy (FTIR) is used to pinpoint the chemical components
and clarify the structural compounds. Herbal extracts or powders
can be quickly and nondestructively fingerprinted using FTIR.
5.3.3 Fourier Transforms Infrared spectroscopy using the Fourier transform is a useful tech-
Infrared Spectroscopy nique for locating functional groups in plant extracts. It aids in
(FTIR) molecular identification and structural determination. It is a high-
resolution analytical instrument for deciphering structural com-
pounds and identifying chemical ingredients. Herbal extracts or
powders may be quickly and nondestructively fingerprinted using
FTIR [58]. The spectrum of an unknown molecule can be identi-
fied by comparison with a known compound spectra for the major-
ity of typical plant compounds. There are several ways to prepare
samples for this method. The easiest method for liquid samples is to
sandwich one drop of the sample between two plates of sodium
chloride. Between the plates, the drop creates a thin layer. Potas-
sium bromide (KBr) may be used to crush solid materials, and the
resulting thin pellet can subsequently be examined. Another
method for solid samples is to first dissolve the preparation in a
solvent system, such as methylene chloride, and then transfer the
resulting solution to a single salt plate. A thin coating of the original
material is then formed on the plate once the solvent has evapo-
rated. FTIR is a high-resolution analytical method used to reveal
the structure of compounds and identify the chemical constituents.
Herbal extracts or powders can be quickly and nondestructively
analyzed using FTIR [59].
5.3.4 Nuclear Magnetic The magnetic properties of atomic nuclei, notably those of the
Resonance hydrogen atom, proton, carbon, and an isotope of carbon, are the
Spectroscopy (NMR) primary focus of NMR. By comparing the differences between a
variety of magnetic nuclei, which gives a clear image of where these
nuclei are positioned in the molecule, NMR spectroscopy has
enabled numerous researchers to investigate molecules. It also
shows which atoms are present in the clusters that are close
by. Finally, it can be argued that a lot of atoms are present in each
of these settings [57, 60].
5.3.5 Identification of In mass spectrometry (MS), molecules are attacked with a mixture
Chemical Compounds of electrons or lasers before being changed into charged ions, which
Using Mass Spectrometry have a high energy. A mass spectrum is a graph that shows how
many fragmented ions there are in relation to their mass/charge
ratio. By using MS, using information about the areas where the
molecule has been split apart, it is feasible to calculate the relative
molecular mass (molecular weight) with high precision and con-
struct a precise molecular formula [61]. The most significant and
popular advanced technology is tandem mass spectrometry
(MS/MS). In addition to increasing selectivity, MS/MS offers a
plethora of structural data, enabling the identification and measure-
ment of even co-eluting molecules [62]. For the first time, the
phenolic compounds quercetin, crysin, quinic acid, chlorogenic
acid, and kaempferol were successfully screened and identified in a
hydromethanolic extract of A. aspera by UPLCPDA (ultra-

performance liquid chromatography ephotodiode array) and
MALDI-TOF-MS (matrix-assisted laser desorption/ionizatione-
time-of-flight mass spectrometry) [63]. Nowadays, MALDI-LC/
MS is often utilized for phenolic compound chemical analysis.
However, because of its high ionization efficacy, particularly for
phenolic compounds, the electrospray ionization (ESI) approach
is a favored technique.
5.3.6 Nonchromato- Research on receptor binding analysis, enzyme assay, and quantita-
graphic Techniques tive and/or qualitative analytical procedures in animals or plants has
made monoclonal antibody (MAb) against drugs and tiny molecu-
lar weight bioactive chemicals an indispensable tool. The immuno-
blotting approach is based on the western blotting method, which
makes use of the antigen-antibody binding capabilities to identify
larger molecule analytes like peptides and proteins in a precise and
sensitive manner. ELISA, a highly sensitive, precise, and easy-to-use
technology, was created for individual competitive testing
[64]. Since their introduction, monoclonal antibodies (MAbs),
which have a wide range of applications, have grown in significance
as a tool in contemporary bioscience research. Recently, several
researchers have concentrated on the creation of MAbs against
the secondary metabolites, or natural compounds, obtained from
medicinal plants [65].
The procedures used to create monoclonal antibodies using
hybridoma technology against plant-based medications are as
follows:
(i) Adequate purification and characterization of the required
antigen.
(ii) Giving mice an immunity boost using the purified antigen.
(iii) The cultivation of myeloma cells that are unable to produce
the hypoxanthine-guanine-phosphoribosyl transferase
(HGPRT) enzyme required for the nucleic acid salvage
pathway.
(iv) The removal of mouse spleen cells and their fusion with mye-
loma cells.
(v) The hybridomas were raised in hypoxanthine aminopterin
thymidine (HAT) medium after fusion [66].
5.3.7 Phytochemical Phytochemicals are a diverse group of bioactive substances (sec-

Screening Assay ondary metabolic substances) derived from plants that have a
strong potential to improve human health [67].
Three fundamental stages are needed to undertake a natural
product analysis: an effective means to separate the different phy-
tochemicals in the extracts, a way to identify and quantify the
phytochemicals, and a mechanism to extract the phytochemicals of

interest from the raw plant material. Pure standards are necessary
for this.
In only a few minutes, analytical tools are now available that can
quickly isolate, clearly identify, and reliably quantify phytochemicals
from plant materials [68].
6 Bioactive Compounds: Their Role in the Prevention and Treatment of Diseases
Many bioactive substances seem to have positive impacts on health.

Before we can start making dietary recommendations that are
supported by science, there is still a lot of scientific study to be
done. Despite this, there is enough data to suggest ingesting foods
that are high in bioactive substances. This translates into advocating
a diet high in a range of vegetables, fruits, oils, nuts, legumes, whole
and grains from a practical standpoint [69].
6.1 Use of Natural Bioactive food components are essential in both the prevention and
Bioactive Compounds treatment of diseases since various chemicals are involved in the
in the Food and pathophysiology of many disease processes. Several bioactive che-
Pharmaceutical micals actively control the inflammatory process, which is the
Industries underlying cause of diabetes, cancer, and other inflammatory dis-
eases. Dietary practices, food components, and bioactive substances
with anti-inflammatory characteristics have all been found to be
protective. Utilizing bioactive food substances with antioxidant
and anti-inflammatory characteristics that are found in spices and
herbs may thus help avoid inflammation that can lead to carcino-
genesis or cardiovascular disorders [70]. For instance, the two most
successful nonpharmaceutical therapies for inflammatory bowel
disease are dietary changes and functional diets (IBD). IBD can
be treated with probiotics and nonstarchy polysaccharide dietary
supplements. Omega-3 fatty acids, vitamins, phytochemicals, and
plant extracts are a few examples of bioactive compounds. These
dietary peptides and functional foods have potent anti-
inflammatory effects in both human and animal studies [71]. In
order to reduce inflammation, functional foods can alter inflamma-
tory cytokines and work with the immune system. The manufacture
of nutraceuticals for inflammatory-related disorders is made possi-
ble by the anti-oxidative and anti-inflammatory action of cotonea-
ster’s polyphenolic components [72]. Angiotensin-converting
enzyme (ACE) inhibitors, which are bioactive components of Cor-
iandrum sativum, are thought to have anti-hypertensive
effects [73].
Plant-derived phytochemicals offer a promising new route for
the creation of diabetic mellitus therapies. The more significant
alkaloids include flavonoids, glycosides, terpenoids, and steroids
[74]. Numerous phytochemicals with possible antidiabetic
properties may be found in many fruits, vegetables, oils, legumes,

and nuts. Aloe vera, mango, banana, avocado, coffee, blueberries,
bitter gourd, cinnamon, black tea, ginger, garlic, guava, grape,
pomegranate, pumpkin, olive oil, jackfruit, papaya, onion, and
others are some of them [75]. Dietary flavonoids regulate insulin
sensitivity, beta-cell function, glucose metabolism, and the func-
tional availability of antioxidants [76].
The South Indian fruit crop known as jackfruit (Artocarpus
heterophyllus Lam) is prized for its therapeutic qualities. It includes
a variety of antioxidants that aid in the prevention of numerous
chronic illnesses, including diabetes and heart disease. The glyce-
mic index (GI) of jackfruit is said to be quite low, limiting a sharp
rise in blood sugar levels [77]. It also has plenty of vitamin C,
carotenoids, and flavones, all of which contribute to its anti-
inflammatory properties and lower the risk of chronic heart disease,
cancer, hypertension, and type 2 diabetes [78, 79]. Antioxidant,
antiulcer, antihypertensive, anticancer, and anti-aging activities may
be found in jack fruit phytonutrients [80, 81]. Banana inflorescence
(Musa paradisiaca) supplementation has also been shown to reduce
inflammation, hyperglycemia, and oxidative stress in diabetic rats
produced by streptozotocin. Contrarily, the bioflavonoid morin
exerts its antidiabetic effects via a mechanism that mimics the
actions of insulin [82].
Dietary phytochemicals have also been shown to include a
number of anticancer substances. Yang et al. examined the struc-
tural characteristics and anticancer activity [83] of water-soluble
polysaccharides from Kaempferia galanga (aromatic ginger). By
consuming a polyphenol-rich herbal congee with a combined
extract of Morus alba and Polygonum odoratum leaves, menopausal
women’s bone mineral density was enhanced, according to
Wattanathorn et al. [84].
There are also claims that certain bioactive substances have
neuroprotective effects. Several conditions that can affect the
brain include Parkinson’s disease (PD), Prion disease, experimental
autoimmune encephalomyelitis (EAE), Alzheimer’s disease (AD),
multiple sclerosis (MS), ischemic stroke, and neuropathic pain.
Numerous studies have demonstrated that altering lifestyle vari-
ables, such as adopting a suitable diet, might postpone or prevent
the onset of Alzheimer’s disease, an age-related form of dementia.
Promising substances include phenolic compounds, fat-soluble
vitamins, isothiocyanates, omega-3 fatty acids, and carotenoids.
These bioactive substances have antioxidant and anti-inflammatory
properties, and they actively contribute to the formation of tau
tangles and amyloid plaques [85]. A member of the Caryocaraceae
family commonly referred to as “pequi,” Caryocar Brasiliense
(Camb), is a potential neuroprotective phytomedicine with anti-
oxidant and anti-cholinesterase properties as well as neuroprotec-
tive benefits [86].
Bioactive substances such as omega-3 fatty acids, plant sterol

esters, and phenolic compounds may lower the risk of atheroscle-
rosis and cardiovascular diseases by decreasing inflammation, LDL
cholesterol levels, and oxidative stress [87]. Therefore, nutrition
and functional foods will be key in treating and preventing illnesses.
6.2 Use of Bio-Based The Product and Drug Administration (FDA) defines “natural” as
Compounds as Food a food that “does not include anything artificial or synthetic,
Additives including additives,” despite the fact that the phrase has no legal
definition. Due to studies indicating negative impacts of the usage
of synthetic ingredients, there has been an increase in research and
demand for natural foods during the past few years. Additionally,
the phrase “natural” adds value to the product because it is now
fashionable to consume goods made entirely of natural substances
[88, 89].
Because they include chemicals that are good for health, plants,
fruits, and spices are well known. The biologically active com-
pounds found in plants that are used as food additives can be
unofficially categorized as antioxidants, antimicrobials, flavorings,
colorants, and others. More research has been done as a result of
increased public awareness of the benefits of ingesting natural
goods, leading to potential sources of natural additives [90].
There are a ton of raw materials with a high concentration of
bioactive compounds in the by-products and biowaste from the
food sector. For instance, orange peels may be used as flavorings,
sweeteners, and antioxidants since they include essential oils, cellu-
lose, pectin, hemicellulose, and soluble sugars (galactose, sucrose,
fructose, and glucose) [91].
The utilization of vegetable byproducts and bio-residues is an
alternative to synthetic additives since consumers are more inter-
ested in additives derived from natural sources and have a sustain-
able mentality [92].
6.3 Neuroprotective A host defensive process known as neuroinflammation is linked to

Effects of Biological the neutralization of an injury and the restoration of the brain’s
Activity and Toxicity of normal structure and function. All significant CNS illnesses have
Plant Nutraceuticals the characteristic of neuroinflammation [93].
In the majority of neurological disorders, including Prion dis-
ease, Alzheimer’s disease (AD), multiple sclerosis (MS), Parkin-
son’s disease (PD), experimental autoimmune encephalomyelitis
(EAE), neuropathic pain, and ischemic stroke, neuroinflammation
is the primary mediator of secondary brain damage. Both aging-
dependent circumstances and aging-independent pathogenic
events can cause neuroinflammation because they both involve
related inflammatory cascades [69].
It makes sense to explore natural treatments for transient cere-
bral ischemia-reperfusion injury (TCI-RI), as well as to research
their mechanisms of action. AFF, an extract of the total flavonoids
from A. esculentus flowers, was studied by Y. Luo et al. [94] for its
potential protective effects on TCI-RI. The researchers demon-
strated that AFF had protective effects against TCI-RI by scaveng-
ing free radicals and indirectly boosting the neuronal Nrf2-ARE
pathway to reduce oxidative stress damage.
Alzheimer’s disease (AD) is a neurodegenerative illness of the
central nervous system that gradually impairs cognition and mem-
ory. The disease’s molecular characteristics include extracellular
amyloid peptide (A) deposition in senile plaques, the emergence
of intracellular neurofibrillary tangles (NFT), cholinergic deficit,
significant neuronal loss, and synaptic alterations in the cerebral
cortex, hippocampus, and other brain regions crucial for cognitive
and memory functions. A deposition kills neurons by a number of
different possible mechanisms, including oxidative stress, excito-
toxicity, energy depletion, inflammation, and apoptosis [95].
A member of the Caryocaraceae family known as “pequi,”
Caryocar brasiliense (Camb), is one of the promising neuroprotec-
tive phytomedicines. In their article, “Neuroprotective Effect of
Caryocar brasiliense Camb.,” T. S. de Oliveira et al. [86] investi-
gated the antioxidant and anticholinesterase activities as well as the
neuroprotective effects of C. brasiliense leaf extracts to provide new
information on the potential use of this plant against neurodegen-
erative disorders.
Memory loss that worsens over time, along with other cogni-
tive impairments, are common AD symptoms. The amyloid
hypothesis states that synaptic malfunction and consequent neuro-
degeneration in AD are primarily caused by amyloid- (A-) asso-
ciated toxicity and imbalance. A has been proposed as a possible
therapeutic target for the treatment of AD as a result. Procyanidins
extracted from Lotus seedpod ameliorate amyloid—induced
toxicity in rat Pheochromocytoma cells [96] study by H. Huang
et al. confirms the anti-A activities and protective mechanisms as a
potential natural product for AD therapy. The authors assessed the
LSPC’s ability to mitigate the harm caused by A-25-35 to rat
pheochromocytoma (PC12) cells.
In the case of infection, inflammation, trauma, ischemia, and
neurodegeneration in the central nervous system (CNS), microglia
cells act as scavenger cells and play a crucial function as resident
immunocompetent and phagocytic cells [97]. While prolonged
activation of microglia and astrocytes causes neuroinflammation,
which can start or accelerate dementia, it is a brain defense system
to combat dangerous infections and damaged tissues [98].
Numerous herbal plants and their active ingredients have sur-
faced in recent years and have been the focus of in-depth study.
When opposed to contemporary trendy supplements, these medi-
cines have been time-tested and verified by traditional usage.
Recent research has shown that traditional herbal remedies with
reliable ethnopharmacological qualities have neurotrophic and
neuroprotective effects. These features can be helpful in avoiding

different types of neuronal cell death in neurodegenerative and
neuroinflammatory illnesses. Over the last 20 years, a number of
natural compounds have been investigated for their potential to
reduce neuroinflammation, treat neurodegenerative disorders, or
have positive effects on the central nervous system. Many plants
have been found to contain anti-inflammatory and antioxidant
properties, which may shield the brain from the harm caused by
inflammation. The evidence for the neuroprotective effects of tra-
ditional herbal extracts, particularly their anti-inflammatory proper-
ties, is growing [99–104]. Natural substances that are especially
designed to prevent microglial activation may be more effective in
treating neurodegenerative and neuroinflammatory illnesses that
are linked to microglia. In the parts that follow, we’ll concentrate
on well-known and significant natural products, their active ingre-
dients, and their anti-inflammatory properties based on suppressing
microglial activation.
7 Future Perspectives
Due to the fact that there are still 500,000 plants in the world that
need to be found, examined, and investigated by the scientific
community for their potential therapeutic capabilities to treat a
variety of ailments, bioactive chemicals and herbal medications
have a bright future. Worldwide demand for items made from
plants has expanded, yet traditional indigenous medical methods
are still in their infancy. For the treatment and healing of a wide
range of ailments, herbal compositions and diverse formulations
have been used for many generations with careful selection and
application [105]. For their medical requirements, almost 85% of
the population in Asia, Africa, Latin America, and the Middle East
largely trust traditional herbal remedies. Skin conditions, jaundice,
cancer, TB, hypertension, diabetes, and many other infectious dis-
orders are successfully treated using bioactive chemicals and their
analogs at both the chronic and acute levels [106]. These bioactive
compounds do, however, have some disadvantages, including
changes in composition with climate, the concurrent occurrence
of synergistic effects of compounds, their mode of administration,
and stability in active form, which may have unfavorable or unex-
pectedly positive effects on bioactivity. These problems can be
resolved by utilizing state-of-the-art techniques for the isolation
of pure bioactive compounds, their synthesis as herbal nanoparti-
cles, and convenient examination of their therapeutic effects in
addition to toxicity analysis prior to medication after keeping the
extract for longer periods of time [107]. A. aspera leaf extract was
characterized using MALDI-TOF-MS, which reveals the presence
of chlorogenic acid (CGA). The extract was also utilized to create
herbal gold nanoparticles, and fresh splenocyte cell culture was
employed to test the extract’s toxicity [108, 109]. The anticancer-

ous effects of CGA were later observed separately in vitro, in vivo,
and in silico [110]. liquid chromatography mass spectrometry
(LC/MS), the development of HPLC/MS, magnetic field, and
nuclear magnetic resonance (NMR) has thus significantly lessened
the problem of identifying the structures of these compounds from
extracts, despite the fact that active compounds are present in plants
in very small concentrations [111].
Herbal remedies are becoming more and more popular because
of their inherent safety, low cost, and accessibility. However, when
compared to traditional allopathic medications, concerns are raised
about their pharmacognosy, clinical validation, and standardiza-
tion. Despite these concerns, attempts to scientifically fix them
have increased over the past 25 years in both wealthy and develop-
ing nations. Additionally, this prompts us to consider the necessity
of further research in creating herbal medicines as cutting-edge
therapeutic agents.
8 Conclusions
The world’s health systems’ greatest sources of therapeutic phar-

maceuticals and currently used natural remedies will be bioactive
chemicals produced from medicinal plants. These natural remedies
may be the most significant and necessary source of medications for
both illness treatment and health maintenance. Although these
traditional remedies have just recently gained attention, they have
been mentioned in ancient literature for thousands of years, and
local people from many civilizations have extensive knowledge and
firsthand experience with them. However, in-depth scientific inves-
tigation and the identification of bioactive chemicals are required to
develop the subsequent generation of medicines based on natural
formulations. Prioritizing specific aspects, such as the assessment of
the quality of raw extracts and their combinations, will enable
future therapies to compete with existing ones. New and improved
methods for their purification, efficient animal research, and palat-
able clinical studies are also required for the justified use of these
medicinal plant extracts with safety and efficacy.
References
1. Zeghoud S, Hemmami H, Rebiai A, Ben review. Comb Chem High Throughput
Seghir B (2022) Using FTIR spectroscopy Screen 25:945–972
and chemometrics for classifying of algerian 3. Rebiai A, Seghir BB, Hemmami H,
medicinal plant species. Vegetos 35:298–305 Zeghoud S, Amor IB, Kouadri I et al (2022)
2. Rebiai A, Hemmami H, Zeghoud S, Ben Quality assessment of medicinal plants via
Seghir B, Kouadri I, Eddine LS et al (2022) chemometric exploration of quantitative
Current application of chemometrics analysis NMR data: a review. Compounds 2:163–181
in authentication of natural products: a
4. Prabu SL, Suriyaprakash T, Kumar CD, 15. Muhamad II, Hassan ND, Mamat SN, Nawi
Sureshkumar S, Ragavendran T (2012) NM, Rashid WA, Tan NA (2017) Extraction
Nutraceuticals: a review. Elixir Pharm 46: technologies and solvents of phytocom-
8372–8377 pounds from plant materials: physicochemical
5. Donno D, Beccaro GL, Mellano MG, Cerutti characterization and identification of ingredi-
AK, Bounous G (2013) Medicinal plants, ents and bioactive compounds from plant
chemical composition and quality: may black- extract using various instrumentations. In:
currant buds and blackberry sprouts be a new Ingredients extraction by physicochemical
polyphenol source for herbal preparations? J methods in food. Elsevier, pp 523–560
Appl Bot Food Qual 86:79–89 16. Toepfl S, Mathys A, Heinz V, Knorr D (2006)
6. Canterino S, Donno D, Mellano MG, Bec- Potential of high hydrostatic pressure and
caro GL, Bounous G (2012) Nutritional and pulsed electric fields for energy efficient and
sensory survey of Citrus sinensis (L.) cultivars environmentally friendly food processing.
grown at the most northern limit of the Med- Food Rev Intl 22:405–423
iterranean latitude. J Food Qual 35:108–118 17. Gaur R, Sharma A, Khare S, Gupta MN
7. Rohman A, Nugroho A, Lukitaningsih E, (2007) A novel process for extraction of
Sudjadi (2014) Application of vibrational edible oils: enzyme assisted three phase parti-
spectroscopy in combination with chemo- tioning (EATPP). Bioresour Technol 98:
metrics techniques for authentication of 696–699
herbal medicine. Appl Spectrosc Rev 49: 18. Rijo P, Falé PL, Serralheiro ML, Simões MF,
603–613 Gomes A, Reis C (2014) Optimization of
8. Razmovski-Naumovski V, Tongkao-on W, medicinal plant extraction methods and their
Kimble B, Qiao VL, Beilun L, Li KM et al encapsulation through extrusion technology.
(2010) Multiple chromatographic and che- Measurement 58:249–255
mometric methods for quality standardisation 19. Kaufmann B, Christen P (2002) Recent
of Chinese herbal medicines. World Sci Tech- extraction techniques for natural products:
nol 12:99–106 microwave-assisted extraction and pressurised
9. Smith RM (2003) Before the injection— solvent extraction. Phytochem Anal 13:105–
modern methods of sample preparation for 113
separation techniques. J Chromatogr A 20. Lakkakula NR, Lima M, Walker T (2004)
1000:3–27 Rice bran stabilization and rice bran oil extrac-
10. Sasidharan S, Chen Y, Saravanan D, tion using ohmic heating. Bioresour Technol
Sundram K, Latha LY (2011) Extraction, iso- 92:157–161
lation and characterization of bioactive com- 21. Marr R, Gamse T (2000) Use of supercritical
pounds from plants’ extracts. Afr J Tradit fluids for different processes including new
Complement Altern Med 8:1–10 developments—a review. Chem Eng Process
11. Mandal V, Dewanjee S, Sahu R, Mandal SC Process Intensif 39:19–28
(2009) Design and optimization of ultra- 22. Lang Q, Wai CM (2001) Supercritical fluid
sound assisted extraction of curcumin as an extraction in herbal and natural product
effective alternative for conventional solid liq- studies—a practical review. Talanta 53:771–
uid extraction of natural products. Nat Prod 782
Commun 4:1934578X0900400121 23. Meireles MAA (2003) Supercritical extraction
12. Kumar BR (2017) Application of HPLC and from solid: process design data (2001–2003).
ESI-MS techniques in the analysis of phenolic Curr Opinion Solid State Mater Sci 7:321–
acids and flavonoids from green leafy vegeta- 330
bles (GLVs). J Pharm Anal 7:349–364 24. Wang L, Weller CL, Schlegel VL, Carr TP,
13. David B, Wolfender J-L, Dias DA (2015) The Cuppett SL (2008) Supercritical CO2 extrac-
pharmaceutical industry and natural products: tion of lipids from grain sorghum dried dis-
historical status and new trends. Phytochem tillers grains with solubles. Bioresour Technol
Rev 14:299–315 99:1373–1382
14. Ramawat K, Dass S, Mathur M (2009) The 25. Ghafoor K, Park J, Choi Y-H (2010) Optimi-
chemical diversity of bioactive molecules and zation of supercritical fluid extraction of bio-
therapeutic potential of medicinal plants. In: active compounds from grape (Vitis labrusca
Herbal drugs: ethnomedicine to modern B.) peel by using response surface methodol-
medicine. Springer, Berlin, Heidelberg, pp ogy. Innovative Food Sci Emerg Technol 11:
7–32 485–490
26. Ghafoor K, Al-Juhaimi FY, Choi YH (2012) 41. Duthie GG, Duthie SJ, Kyle JA (2000) Plant
Supercritical fluid extraction of phenolic com- polyphenols in cancer and heart disease:
pounds and antioxidants from grape (Vitis implications as nutritional antioxidants. Nutr
labrusca B.) seeds. Plant Foods Hum Nutr Res Rev 13:79–106
67:407–414 42. Tungmunnithum D, Thongboonyou A,
27. Smith RM (2002) Extractions with super- Pholboon A, Yangsabai A (2018) Flavonoids
heated water. J Chromatogr A 975:31–46 and other phenolic compounds from medici-
28. Moldoveanu SC, David V (2002) Sample nal plants for pharmaceutical and medical
preparation in chromatography. Elsevier aspects: an overview. Medicines 5:93
29. Szumski M, Buszewski B (2002) State of the 43. Ribarova F, Atanassova M, Marinova D,
art in miniaturized separation techniques. Crit Ribarova F, Atanassova M (2005) Total phe-
Rev Anal Chem 32:1–46 nolics and flavonoids in Bulgarian fruits and
30. Wang L, Weller CL (2006) Recent advances in vegetables. J Univ Chem Technol Metall 40:
extraction of nutraceuticals from plants. 255–260
Trends Food Sci Technol 17:300–312 44. Mallikharjuna P, Rajanna L, Seetharam Y,
31. Majors RE (2003) Advances in HPLC col- Sharanabasappa G (2007) Phytochemical
umn packing design. LC GC Eur 16:8–13 studies of Strychnos potatorum Lf-A medici-
nal plant. J Chem 4:510–518
32. Rungsung W, Ratha KK, Dutta S, Dixit AK,
Hazra J (2015) Secondary metabolites of 45. Cos P, Vlietinck AJ, Berghe DV, Maes L
plants in drugs discovery. World J Pharm Res (2006) Anti-infective potential of natural pro-
4:604–613 ducts: how to develop a stronger in vitro
“proof-of-concept”. J Ethnopharmacol 106:
33. Yolci Omeroglu P, Acoglu B, Özdal T, Tamer 290–302
CE, Çopur ÖU (2019) Extraction techniques
for plant-based bio-active compounds. In: 46. Verma S, Bahorun T, Singh RK, Aruoma OI,
Natural bio-active compounds. Springer, pp Kumar A (2013) Effect of Aegle marmelos
465–492 leaf extract on N-methyl N-nitrosourea-
induced hepatocarcinogensis in Balb/c mice.
34. Cooper R, Nicola G (2014) Natural products Pharm Biol 51:1272–1281
chemistry: sources, separations and struc-
tures. CRC Press 47. Huie CW (2002) A review of modern sample-
preparation techniques for the extraction and
35. Varma N (2016) Phytoconstituents and their analysis of medicinal plants. Anal Bioanal
mode of extractions: an overview. Res J Chem Chem 373:23–30
Environ Sci 4:8–15
48. Altemimi A, Watson DG, Kinsel M, Lightfoot
36. Biesalski H-K, Dragsted LO, Elmadfa I, DA (2015) Simultaneous extraction, optimi-
Grossklaus R, Müller M, Schrenk D et al zation, and analysis of flavonoids and poly-
(2009) Bioactive compounds: definition and phenols from peach and pumpkin extracts
assessment of activity. Nutrition 25:1202– using a TLC-densitometric method. Chem
1205 Cent J 9:1–15
37. Patel K, Panchal N, Ingle P (2019) Techni- 49. Mulinacci N, Prucher D, Peruzzi M,
ques adopted for extraction of natural pro- Romani A, Pinelli P, Giaccherini C et al
ducts extraction methods: maceration, (2004) Commercial and laboratory extracts
percolation, soxhlet extraction, turbo distilla- from artichoke leaves: estimation of caffeoyl
tion, supercritical fluid extraction. Int J Adv esters and flavonoidic compounds content. J
Res Chem Sci 6:1–12 Pharm Biomed Anal 34:349–357
38. Zhang Z, Li G (2010) A review of advances 50. SaraJliJa H, ČukelJ N, Mršić GND, Brnčić M,
and new developments in the analysis of Ćurić D (2012) Preparation of flaxseed for
biological volatile organic compounds. lignan determination by gas
Microchem J 95:127–139 chromatography-mass spectrometry method.
39. Barba FJ, Esteve MJ, Frı́gola A (2014) Bioac- Czech J Food Sci 30:45–52
tive components from leaf vegetable products. 51. Popova I, Hall C, Kubátová A (2009) Deter-
Stud Nat Prod Chem 41:321–346 mination of lignans in flaxseed using liquid
40. Carbonell-Capella JM, Buniowska M, Barba chromatography with time-of-flight mass
FJ, Esteve MJ, Frı́gola A (2014) Analytical spectrometry. J Chromatogr A 1216:217–
methods for determining bioavailability and 229
bioaccessibility of bioactive compounds from 52. Zhang Z, Pang X, Xuewu D, Ji Z, Jiang Y
fruits and vegetables: a review. Compr Rev (2005) Role of peroxidase in anthocyanin
Food Sci Food Saf 13:155–171
degradation in litchi fruit pericarp. Food 63. Narayan C, Kumar A (2013) Identification
Chem 90:47–52 and characterization of phenolic compounds
53. Hemmami H, Ben Seghir B, Ben Ali M, in hydro methanolic extract of Achyranthes
Rebiai A, Zeghoud S, Brahmia F (2020) Phe- aspera (HMEA) by UPLC and MALDI-
nolic profile and antioxidant activity of bee TOF-MS and in vivo antioxidant activity. Ori-
pollen extracts from different regions of ent Pharm Exp Med 13:51–59
Algeria. Ovidius Univ Ann Chem 31:93–98 64. Tanaka H, Morinaga O, Uto T, Fuji S, Aboa-
54. Zeghoud S, Rebiai A, Hemmami H, Ben gye FA, Tung NH et al (2012) Application of
Seghir B, Elboughdiri N, Ghareba S et al monoclonal antibodies against bioactive nat-
(2021) ATR–FTIR spectroscopy, HPLC ural products: eastern blotting and prepara-
chromatography, and multivariate analysis for tion of knockout extract. Int J Anal Chem
controlling bee pollen quality in some Alger- 2012:260425
ian regions. ACS Omega 6:4878–4887 65. Yan X, Zhao Y, Zhang Y, Qu H (2017)
55. Rebiai A, Ben Seghir B, Hemmami H, Monoclonal antibodies and immunoassay for
Zeghoud S, Belfar ML, Kouadri I (2021) medical plant-derived natural products: a
Determination of some phenolic acids in review. Molecules 22:355
Algerian propolis. Ovidius Univ Ann Chem 66. Ansar W, Ghosh S (2013) Monoclonal anti-
32:120–124 bodies: a tool in clinical research. Indian J Clin
56. Altemimi A, Lakhssassi N, Baharlouei A, Wat- Med 4:S11968
son DG, Lightfoot DA (2017) Phytochem- 67. Sharma M, Kaushik P (2021) Vegetable phy-
icals: extraction, isolation, and identification tochemicals: an update on extraction and
of bioactive compounds from plant extracts. analysis techniques. Biocatal Agric Biotechnol
Plan Theory 6:42 36:102149
57. Urbano M, De Castro MDL, Pérez PM, Gar- 68. Berhow MA (2002) Modern analytical tech-
cı́a-Olmo J, Gomez-Nieto MA (2006) niques for flavonoid determination. In: Flavo-
Ultraviolet–visible spectroscopy and pattern noids in cell function. Springer, Boston, pp
recognition methods for differentiation and 61–76
classification of wines. Food Chem 97:166– 69. Taylor AK, Cao W, Vora KP, Cruz JDL, Shieh
175 W-J, Zaki SR et al (2013) Protein energy
58. Rasul MG (2018) Extraction, isolation and malnutrition decreases immunity and
characterization of natural products from increases susceptibility to influenza infection
medicinal plants. Int J Basic Sci Appl Comput in mice. J Infect Dis 207:501–510
2:1–6 70. Teodoro AJ (2019) Bioactive compounds
59. Hazra K, Roy R, Sen S, Laskar S (2007) Iso- of food: their role in the prevention and treat-
lation of antibacterial pentahydroxy flavones ment of diseases. Oxid Med Cell Longev
from the seeds of Mimusops elengi Linn. Afr J 2019:3765986
Biotechnol 6:1446–1449 71. Al Mijan M, Lim BO (2018) Diets, functional
60. Ingle KP, Deshmukh AG, Padole DA, Dud- foods, and nutraceuticals as alternative thera-
hare MS, Moharil MP, Khelurkar VC (2017) pies for inflammatory bowel disease: present
Phytochemicals: extraction methods, identifi- status and future trends. World J Gastroen-
cation and detection of bioactive compounds terol 24:2673
from plant extracts. J Pharmacogn Phyto- 72. Kicel A, Kolodziejczyk-Czepas J, Owczarek A,
chem 6:32–36 Rutkowska M, Wajs-Bonikowska A, Granica S
61. Christophoridou S, Dais P, Tseng L-H, Spraul et al (2018) Multifunctional phytocom-
M (2005) Separation and identification of pounds in cotoneaster fruits: phytochemical
phenolic compounds in olive oil by coupling profiling, cellular safety, anti-inflammatory
high-performance liquid chromatography and antioxidant effects in chemical and
with postcolumn solid-phase extraction to human plasma models in vitro. Oxid Med
nuclear magnetic resonance spectroscopy Cell Longev 2018:3482521
(LC-SPE-NMR). J Agric Food Chem 53: 73. Hussain F, Jahan N, Rahman K-u, Sultana B,
4667–4679 Jamil S (2018) Identification of hypotensive
62. Di Stefano V, Avellone G, Bongiorno D, biofunctional compounds of Coriandrum
Cunsolo V, Muccilli V, Sforza S et al (2012) sativum and evaluation of their angiotensin-
Applications of liquid chromatography–mass converting enzyme (ACE) inhibition poten-
spectrometry for food analysis. J Chromatogr tial. Oxid Med Cell Longev 2018:4643736
A 1259:74–85
74. Gaikwad SB, Krishna Mohan G, Sandhya Rani (2018) Neuroprotective effect of Caryocar
M (2014) Phytochemicals for diabetes man- brasiliense Camb. leaves is associated with
agement. Pharm Crops 5:11–28 anticholinesterase and antioxidant properties.
75. Beidokhti MN, J€ager AK (2017) Review of Oxid Med Cell Longev 2018:9842908
antidiabetic fruits, vegetables, beverages, oils 87. Scolaro B, Soo Jin Kim H, de Castro IA
and spices commonly consumed in the diet. J (2018) Bioactive compounds as an alternative
Ethnopharmacol 201:26–41 for drug co-therapy: overcoming challenges in
76. Vinayagam R, Xu B (2015) Antidiabetic prop- cardiovascular disease prevention. Crit Rev
erties of dietary flavonoids: a cellular mecha- Food Sci Nutr 58:958–971
nism review. Nutr Metab 12:1–20 88. Baines D (2012) Defining the term “natural”
77. Hettiaratchi U, Ekanayake S, Welihinda J in the context of food products. In: Natural
(2011) Nutritional assessment of a jackfruit food additives, ingredients and flavourings.
(Artocarpus heterophyllus) meal. Ceylon Woodhead Publishing, pp 1–22
Med J 56:54–58 89. Kumar Y, Yadav DN, Ahmad T, Narsaiah K
78. De Faria A, De Rosso VV, Mercadante AZ (2015) Recent trends in the use of natural
(2009) Carotenoid composition of jackfruit antioxidants for meat and meat products.
(Artocarpus heterophyllus), determined by Compr Rev Food Sci Food Saf 14:796–812
HPLC-PDA-MS/MS. Plant Foods Hum 90. Gokoglu N (2019) Novel natural food pre-
Nutr 64:108–115 servatives and applications in seafood preser-
79. Jagtap UB, Panaskar SN, Bapat V (2010) vation: a review. J Sci Food Agric 99:2068–
Evaluation of antioxidant capacity and phenol 2077
content in jackfruit (Artocarpus heterophyllus 91. Ayala-Zavala JF, González-Aguilar G, Siddi-
Lam.) fruit pulp. Plant Foods Hum Nutr 65: qui MW (2018) Plant food by-products:
99–104 industrial relevance for food additives and
80. Lin J-A, Wu C-H, Fang S-C, Yen G-C (2012) nutraceuticals. CRC Press
Combining the observation of cell morphol- 92. Faustino M, Veiga M, Sousa P, Costa EM,
ogy with the evaluation of key inflammatory Silva S, Pintado M (2019) Agro-food bypro-
mediators to assess the anti-inflammatory ducts as a new source of natural food addi-
effects of geranyl flavonoid derivatives in tives. Molecules 24:1056
breadfruit. Food Chem 132:2118–2125 93. Farooqui AA, Horrocks LA, Farooqui T
81. Gupta A, Tandon N (2004) Reviews on (2007) Modulation of inflammation in brain:
Indian medicinal plants. Indian Council of a matter of fat. J Neurochem 101:577–599
Medical Research, New Delhi 94. Luo Y, Cui H-X, Jia A, Jia S-S, Yuan K (2018)
82. Paoli P, Cirri P, Caselli A, Ranaldi F, The protective effect of the total flavonoids of
Bruschi G, Santi A et al (2013) The insulin- Abelmoschus esculentus L. flowers on tran-
mimetic effect of Morin: a promising mole- sient cerebral ischemia-reperfusion injury is
cule in diabetes treatment. Biochim Biophys due to activation of the Nrf2-ARE pathway.
Acta 1830:3102–3111 Oxid Med Cell Longev 2018:8987173
83. Yang X, Ji H, Feng Y, Yu J, Liu A (2018) 95. Parihar M, Hemnani T (2004) Alzheimer’s
Structural characterization and antitumor disease pathogenesis and therapeutic inter-
activity of polysaccharides from Kaempferia ventions. J Clin Neurosci 11:456–467
galanga L. Oxid Med Cell Longev 2018: 96. Huang H, Yan P, Sun T, Mo X, Yin J, Li P et al
9579262 (2018) Procyanidins extracted from lotus
84. Thomas R, Williams M, Sharma H, seedpod ameliorate amyloid-β-induced toxic-
Chaudry A, Bellamy P (2014) A double- ity in rat pheochromocytoma cells. Oxid Med
blind, placebo-controlled randomised trial Cell Longev 2018:4572893
evaluating the effect of a polyphenol-rich 97. Kim SU, de Vellis J (2005) Microglia in health
whole food supplement on PSA progression and disease. J Neurosci Res 81:302–313
in men with prostate cancer—the UK NCRN 98. Barbalace MC, Malaguti M, Giusti L,
Pomi-T study. Prostate Cancer Prostatic Dis Lucacchini A, Hrelia S, Angeloni C (2019)
17:180–186 Anti-inflammatory activities of marine algae
85. Grodzicki W, Dziendzikowska K (2020) The in neurodegenerative diseases. Int J Mol Sci
role of selected bioactive compounds in the 20:3061
prevention of Alzheimer’s disease. Antioxi- 99. Chen L-W, Wang Y-Q, Wei L-C, Shi M, Chan
dants 9:229 Y-S (2007) Chinese herbs and herbal extracts
86. de Oliveira TS, Thomaz DV, da Silva Neri HF, for neuroprotection of dopaminergic neurons
Cerqueira LB, Garcia LF, Gil HPV et al and potential therapeutic treatment of
Parkinson’s disease. CNS Neurol Disord 105. Khan MSA, Ahmad I (2019) Herbal medi-
Drug Targets 6:273–281 cine: current trends and future prospects. In:
100. Ho Y-S, Yu M-S, Lai CS-W, So K-F, Yuen New look to phytomedicine. Elsevier, pp
W-H, Chang RC-C (2007) Characterizing 3–13
the neuroprotective effects of alkaline extract 106. Jamshidi-Kia F, Lorigooini Z, Amini-Khoei H
of Lycium barbarum on β-amyloid peptide (2018) Medicinal plants: past history and
neurotoxicity. Brain Res 1158:123–134 future perspective. J Herbmed Pharmacol 7:
101. Lee H, Kim YO, Kim H, Kim SY, Noh HS, 1–7
Kang SS et al (2003) Flavonoid wogonin 107. Colegate SM, Molyneux RJ (2007) Bioactive
from medicinal herb is neuroprotective by natural products: detection, isolation, and
inhibiting inflammatory activation of micro- structural determination. CRC Press
glia. FASEB J 17:1–21 108. Tripathi A, Kumari S, Kumar A (2016) Toxic-
102. Li F-Q, Wang T, Pei Z, Liu B, Hong J-S ity evaluation of pH dependent stable Achyr-
(2005) Inhibition of microglial activation by anthes aspera herbal gold nanoparticles. Appl
the herbal flavonoid baicalein attenuates Nanosci 6:61–69
inflammation-mediated degeneration of 109. Narayan C, Kumar A (2014) Antineoplastic
dopaminergic neurons. J Neural Transm and immunomodulatory effect of polypheno-
112:331–347 lic components of Achyranthes aspera (PCA)
103. Shimazawa M, Chikamatsu S, Morimoto N, extract on urethane induced lung cancer
Mishima S, Nagai H, Hara H (2005) Neuro- in vivo. Mol Biol Rep 41:179–191
protection by Brazilian green propolis against 110. Tripathi A (2018) Immunomodulatory com-
in vitro and in vivo ischemic neuronal dam- motion of HMGB 1 in urethane primed
age. Evid Based Complement Alternat Med 2: hepatocarcinogenesis. In: School of biotech-
201–207 nology. Banaras Hindu University, Varanasi
104. Suk K, Lee H, Kang SS, Cho GJ, Choi WS 111. Schroeder FC, Gronquist M (2006) Extend-
(2003) Flavonoid baicalein attenuates ing the scope of NMR spectroscopy with
activation-induced cell death of brain micro- microcoil probes. Angew Chem Int Ed 45:
glia. J Pharmacol Exp Ther 305:638–645 7122–7131
Chapter 3
Extraction of Bioactive and Nutraceuticals from Marine

Sources and Their Application
Nikheel Rathod, Vijay Reddy, Martina Čagalj, Vida Šimat, Merina Dahal,
Nilesh Prakash Nirmal , and Siddhnath Kumar
Abstract
The marine resource hosts wide biodiversity, regarded as a rich source of diverse bioactive compounds,
which are widely investigated for their bioactive composition and nutraceutical applications having several
health benefits. Several in vitro and in vivo studies have reported the impacts of marine bioactive com-
pounds in controlling several lifestyle disorders. Based on their high bioactive compounds and nutraceutical
value, their extraction techniques are known to influence their bioactivity widely. The present chapter
summarizes the knowledge on novel extraction technologies of bioactive compounds from marine sources;
further, based on their bioactivity and nutraceutical property, their ability to modulate development of
several disorders is addressed. Besides, the chapter covers food application of derived bioactive compounds
to develop nutraceutical foods.
Key words Bioactive, Nutraceuticals, Extraction, Functional food
1 Introduction
Oceans are habitat to the most diverse plants and creatures on the
planet. Marine organisms (planktons, seaweeds, microalgae,
microbes, invertebrates, fish, and so on) have been discovered to
be a potential source of numerous bioactive and nutraceutical
compounds [1, 2]. The compounds exhibit a wide range of func-
tional characteristics, including antioxidant, anti-inflammatory,
anti-microbial, anti-hypertensive, anti-cancer, neuroprotective,
and so on. In an animal model, consumption of seaweed potentially
ameliorates conditions associated with chronic diseases such as
cardiovascular diseases, hyperlipidaemia, diabetes, obesity, and
hypertension [3, 4] through modulation of various signaling
pathways. The major marine-sourced phytochemicals with health-
protective effects are phenolics, flavonoids, terpenoids,
phytosterols, and alkaloids [5]. In addition to phytochemicals,
45
46 Nikheel Rathod et al.
carbohydrate-based bioactive compounds from marine

sources such as laminarin, alginic acid, fucoidan, carrageenan, sul-
fated polymannuronate, heparin, dermatan sulfate, fucosylated
chondroitin sulfate, glycolipids, and glycoproteins possess potent
health benefits [2]. The peptides from marine animals, on the other
hand, have shown promising pharmaceutical and healing proper-
ties. Despite the fact that marine sources are a rich source of
bioactive and nutraceutical compounds with expanded functional
characteristics, extracting these bioactive compounds with pre-
served bioactivity remains a challenge.
Extraction is an operation where plant or animal tissues are
treated through a specific procedure to obtain targeted com-
pounds. For the separation, characterization, and significant health
applications of the bioactive and functional compounds, their opti-
mal extraction with excellent quality is most important. However,
no universal process has yet been developed for ideal extraction.
Maceration, Soxhlet extraction, and hydrodistillation are some
conventional extractions that are still in practice for extraction of
phytochemicals from marine weeds. These methods do, however,
have certain limitations. For example, conventional extraction
approach has a prolonged extraction time, high operating cost,
and poor compound selectivity. In addition to this, these processes
have high solvent consumption, low solvent recovery, and eventu-
ally different environmental hazards. Moreover, the heat-sensitive
bioactive compounds are thermally decomposed by high tempera-
tures during processing. Researchers and investigators are explor-
ing the best possible methods for optimal quantification of the
bioactive compounds [6]. The selection of the extraction process
and medium of extraction, along with other factors, is usually based
on the targeted compound, so one should consider the possible
influencing factors during extraction. Thus, considering the draw-
backs of conventional methods and limiting factors, a lot of novel
procedures have been explored and applied to the extraction of
bioactive compounds from marine resources. Ultrasonication,
microwave-assisted extraction (MAE), pulse electric field (PEF),
eutectic solvent-based extraction, and sub- and supercritical fluid-
based extraction are some of the major green extraction technolo-
gies developed in the last half-century [7]. These procedures are
claimed to be more environmentally friendly since fewer solvents
and other chemical substances are utilized during these processes.
Furthermore, these methods can significantly improve extraction
yields with better quality extracts, and the operational time is
noticeably less than conventional methods. Through this chapter,
we have attempted to provide insight into the current trend of
novel technology applications in the extraction of bioactive com-
pounds from marine sources. Also, the chapter has articulated the
bioactivity and nutraceutical properties of compounds from
marine-based sources, along with their commercial applications.
Extraction of Bioactive and Nutraceuticals from Marine Sources. . . 47
2 Novel Extraction Technologies
2.1 Ultrasound Ultrasound-assisted extraction employs application of high power

and low frequency (20 kHz to 100 MHz and 10 to 1000 W/cm2)
ultrasound waves that generate acoustic cavitation (production of
bubbles), which in turn expands and compress in size and finally
collapse due to the cavitation cycle (compression and expansion)
affecting the cellular structure responsible for improved extraction
of target compounds [8, 9]. The collapse of bubbles generates
extreme local conditions (high pressure, temperature), causing
destruction of cell walls; reducing particle size, resulting in higher
contact between solvent and medium; and causing improved
extraction of target compounds by diffusion from the cell. Ultra-
sound results in increased extraction of target compounds, lower
solvent requirements, shorter time requirements, extraction of
heat-labile compounds, and lesser energy requirement, which
make it an environment-friendly technique.
Ultrasound extraction is influenced by several parameters, such
as application of power and frequency, extraction time and temper-
ature, viscosity, solubility and stability of sample, and optimized
solvent-to-substrate ratio [8–11]. Several recent studies have
focused on the ability of ultrasound to extract bioactive and nutra-
ceutical compounds from marine sources [12–16].
A recent study by [15] optimized the ultrasonic-based extrac-
tion of astaxanthin from shrimp shell. Application of ultrasound
yielded significantly higher quantity of pigment. The astaxanthin
yield increased based on polarity of solvent (petroleum ether/ace-
tone/water: 15:75:10) used and extraction time for 6 h achieved
highest yield. Based on optimized conditions, extraction using
23.6% ultrasound amplitude for 13.9 min resulted in the highest
astaxanthin yield (51.5%), exhibiting the highest antioxidant
(1705 μmol of Fe2+/g and 73.9% of radical scavenging) activity.
Similarly, [17] reported the impacts of ultrasound-assisted extrac-
tion on qualities of oil from fish (Labeo rohita) head. The oil
extracted by using ultrasound had higher proportion of unsatu-
rated fatty acids and exhibited high thermal and oxidative stability.
The higher extraction was obtained using ultrasound treatment due
to disintegration of cell structures releasing lipid.
2.2 PEF Pulsed electric field is based on the principle of electroporation of

cell membranes enhancing mass transfer. However, for electropo-
ration (permanent), the electric field applied should be over the
threshold value (membrane potential) [8, 18–20]. Under these
conditions, the electric field compromises the membrane perme-
ability responsible for extraction of compounds. The nature of
electroporation varies based on strength of the field, exposure
time, pulse number, pulse frequency, properties of cell, and
treatment medium [21]. Application of PEF helps in extraction of

bioactive compounds without inactivation [22]. The low tempera-
ture or no external heating requires less solvent operation, which
gives it advantages for the recovery of bioactive compounds.
For valorizing fish discards from sea bream and sea bass (gills,
bones, and heads), pulsed electric field with water as medium was
evaluated by Franco et al. [23]. Discards were found to be rich
sources of minerals and amino acids. Furthermore, application of
PEF resulted in breaking the membrane and releasing the contents,
which increased the antioxidant activity. Similarly, valorizing fish
bones were evaluated for extraction of nutraceutical ingredients
[24]. Electric filed application at 15 kV/cm resulted in the highest
recovery of calcium (10.140 mg/mL), chondroitin sulfate
(5.899 mg/mL), and collagen (0.156 mg/mL). Further, the opti-
mization study suggested PEF application at 22.79 kV/cm at pulse
number 9 and liquid solid ratio of 11 resulted in higher yield of
calcium, chondroitin sulfate, and collagen as 19.8, 39.268, and
3.875 mg/mL, respectively. The higher extraction yield was related
to the PEF ability to rupture cell wall.
PEF for extraction of lipids from shrimp cephalothorax was
reported by Gulzar and Benjakul [25]. Higher level of cellular
disintegration by applying increase pulses was reported, which
directly resulted in increase of lipid extraction. Lipid extracted by
PEF followed by solvent extraction exhibited higher stability (per-
oxide value, TBARS, and free fatty acid content). Application of
PEF was found to exhibit positive impacts on lipids extracted form
cephalothorax containing higher unsaturated fatty acids and pig-
ments in monoester and diester forms.
High-intensity PEF (10–35 kV/cm) application for extraction
of protein from mussels was evaluated [26]. Optimized operating
conditions (20 kV/cm, pulse number of 8.12, and enzymolysis for
2 h) resulted in the highest protein yield of 77.08%. Hence, PEF
could be successfully used for speed and cleaner recovery of pro-
teins from mussels.
2.3 MAE Microwave-assisted extraction works on the principle of fast heat-

ing by electromagnetic radiations (300 MHz to 300 GHz; 915 and
2450 MHz) at high power for a short duration. The exposure to
microwaves increases the heat penetration in the matrix, increasing
temperature and pressure and resulting in improved mass transfer
by penetration of solvent in the matrix. The application of direct
heating results in rapid heating, lowering the time and solvent
required for extraction. Several factors such as microwave power,
frequency, solvent-to-matrix ratio, and exposure time affect the
extraction efficiency. Also, the characteristics (dielectric constant)
of the solvent used are regarded as important factors determining
the solvating capacity.
Microwave-assisted (600 W, 70 °C for 10 min) extraction of

lipids from fish (Upeneus moluccensis and Saurida undosquamis)
using ethanol solvent was reported by Ozogul and team
[27]. Application of microwave yielded highest lipids in compari-
son to other methods (bligh and dryer, Soxhlet extraction, and
ultrasound). Lipids extracted from Saurida undosquamis contained
higher proportion of unsaturated fatty acids, suggesting the
method of extraction (microwave-assisted extraction) had impacts
on the fatty acid profile of fish and lipid quality. Similarly, Afolabi
and others [28] reported microwave-assisted extraction (800 W)
yielded high quantity (16.13% w/w) and good quality lipid (free
fatty acid—1.35% and acid value- 2.69 mg KOH/g) using ethanol
solvent. The extracted lipid was rich in bioactive lipid constituents
(MUFA-11.11%, PUFA-55.56%, arachidonic acid, eicosapentae-
noic acid, and docosahexaenoic acid). Additionally, the FTIR spec-
tra predicted the presence of groups primarily responsible for
antimicrobial activities. Recently, [29] extracted lipids from salmon
processing waste using microwave-assisted extraction and com-
pared with results obtained from Soxhlet extraction. Microwave
extraction helped in the extraction of 69% of lipids from heads
(<11 min at 50 W using 80 g/L) and backbones (<15 min at
300 W using 80.1 g/L), while 92% recovery was obtained from
viscera (<15 min at 960.6 W using 99.5 g/L) using n-hexane
solvent. The lipid extracted using microwave technology was supe-
rior in terms of fatty acid profile and bioactivity in comparison to
Soxhlet extraction.
The ability of the microwave field to disrupt the cell wall was
demonstrated by Magnusson and others [30] for improving extrac-
tion of polyphenol (phloroglucinol) from brown seaweed in com-
parison to solid-liquid extraction. Results suggested water was the
best solvent for extraction of polyphenols, the biomass-to-water
ratio increased the extraction, and lower temperature conditions
exhibited higher phenolic extraction. Furthermore, the conditions
were optimized to increase (70%) the yield of bioactive phenolic
compounds using 1:30 water-to-solvent ratio, at 160 °C for 3 min.
2.4 Supercritical CO2 It uses solvents at supercritical temperature and pressure condi-
Extraction tions, exhibiting properties of between a liquid and a gas, helping
in improved extraction of bioactive compounds [31]. Considering
the low critical conditions and higher diffusivity of carbon dioxide
(30 °C and 7.38 MPa), it is used for recovery of target compounds.
The proper relation between the pressure and temperature can be
so managed to ensure extraction of heat-labile compounds
[32]. The major advantage of using this technique is reduced
usage of toxic organic solvents. The low polarity of carbon dioxide
limits the extraction of nonpolar compounds. Hence, compounds
are used to modify the polarity ensuring improved salvation
capacity.
Considering the high bioactivity possessed by lipids from

marine origin, several novel techniques for extracting marine lipids
have gained importance [33, 34]. Atlantic salmon, widely con-
sumed globally due to their health benefits, while processing dis-
cards, accounts a large proportion of waste; hence, [35] evaluated
supercritical carbon dioxide method for extraction of phospholipids
from frame bone. This method employed ethanol as co-solvent at
45 °C and 27.5 Mpa; the conditions yielded 6.9% lipids of 80%
purity and 5.6% of phospholipids. The extracted lipids were rich in
bioactive astaxanthin (27.6 μg/g), unsaturated fatty acids, and
phospholipids compared to extraction with organic solvents (etha-
nol). Furthermore, the lipids extracted at lower pressures exhibited
higher stability over extraction at higher pressures. Furthermore,
the extracted lipids exhibited high antioxidant activity, which
increased in dose dependent manner. Similarly, Mexxomo and
others [36] evaluated the hypolipidemic effects of extracts derived
from pink shrimp processing residue using supercritical fluid extrac-
tion method. Significant increase (more than 3 times) in PUFA
contents of fatty acid profile of residue extracts from pink shrimp
extract by supercritical carbon dioxide extraction (30 MPa using
CO2) was observed. Based on bioactive composition, the extracts
exhibited hypolipidemic (reducing cholesterol and triglyceride con-
tent) and anti-obesity effects (weight reduction). The abilities were
attributed to synergistic effects due to the presence of phenolic,
pigments, and fatty acid contents.
Similarly, phospholipids extracted from the viscera of Pacific
saury by supercritical carbon dioxide method employing ethanol
solvent were evaluated for their neuroprotective activity
[37]. PUFA was regarded as the main bioactive constituent present
in lipid fractions derived. The extract exhibited inhibition of amy-
loid beat (Aβ)1–42 by 69% responsible for neurodegenerative dis-
orders. Authors suggested the bioactivity possessed by omega-3
fatty acids plays a vital role in imparting neuroprotective capacity
to lipids from marine origin.
3 Bioactivities and Nutraceutical Application of Bioactive Compounds

from Marine Sources
3.1 Bioactivity Antioxidants are used to lessen oxidative damage to the human
body and to extend the shelf life of lipid-containing foods and
3.1.1 Antioxidant
maintain their nutritious content. They also work to prevent dam-
Property
age caused by free radicals. In contrast to naturally occurring anti-
oxidants such tocopherol, ascorbate, and carotenoids, synthetic
antioxidants like butylated hydroxy anisole (BHA), tertiary-butyl-
hydroquinone (TBHQ), and butylated hydroxytoluene (BHT) are
also available. Natural antioxidants from marine sources, such as
protein hydrolysates, peptides, and amino acids, are increasingly
being investigated due to worries about the potential for carcino-

genic effects. Table 1 represents the bioactive compounds isolated
from different marine sources and their bioactivities. Generally
speaking, a peptide’s ability to stabilize free radicals depends on
its power to provide or take electrons away from the radical in order
to reduce its reactivity. The hydrophobicity, chain length of pep-
tides, size (0.5–3 kDa), and amino acid composition and sequence
(hydrophobic and aromatic) have shown advantages over proteins
in terms of antioxidative activity. When compared to other natural
antioxidants like tocopherol, high antioxidant peptides isolated
from the visceral organs of horse mackerel utilizing gastrointestinal
digestion demonstrated superior antioxidative effect [38].
By-catch byproducts of crabs and shrimp can be inexpensive
sources of natural antioxidant raw materials [40]. Antioxidant pep-
tides of marine origin act in the scavenging of free radicals or in
preventing oxidative damage by interrupting the radical chain reac-
tion of lipid peroxidation, which compromises the cell’s viability
[59]. As a functional dietary ingredient or supplement, cephalotho-
rax hydrolysates of Pacific white shrimp were found to have antiox-
idant effects [39]. Shrimp hydrolysates from Acetes chinensis
demonstrated antioxidant peptides [40]. The crayfish Procambaru-
sclarkii hydrolysates showed significant antioxidant activity
[41]. Seaweeds contain significant amounts of polyphenols or phe-
nolic chemicals, especially those with strong antioxidants. Phloro-
tannins, a class of polyphenols, have drawn a lot of attention as
functional food additives. Phlorotannins are tannin compounds
created when phloroglucinol (1,3,5-trihydroxybenzene) units are
polymerized through the acetate-malonate route. They are primar-
ily found in brown algae [42] and are secreted in the cell wall, where
they combine with other compounds like alginic acid to form
complexes.
Significant levels of phenolic compounds with possible antioxi-
dant action have also been found in sea cucumbers. It’s possible
that the considerable hepatoprotective effect was facilitated by the
active phenolic compounds found in the extract of the body wall of
Holothuria atra. In comparison to solvent extracts, which only
contained slightly higher total phenolic contents (1.53–2.90 mg
GAE/g DW), aqueous extracts of the Holothurialeucospilota,
Holothuriascabra, and Stichopuschlorontus had significantly higher
total phenolic contents (4.85–9.70 mg GAE/g DW) and demon-
strated strong antioxidant properties [43].
Phycobilins (PC and PE), a type of protein covalently
connected to chromophores, make up phycobiliproteins. These
water-soluble proteins can be utilized as a natural food colorant
and are effective antioxidants. The cyanobacteria Lyngbya spp. and
Arthrospira spp. both generate PE, a pink-colored protein pig-
ment, and PC, a blue-colored phycobiliprotein [60]. Spirulina,
Botryococcus, Chlorella, Dunaliella, Haematococcus, and Nostoc are
Table 1
Bioactive compounds from marine sources and their activities
Active compound Activity Source References

Peptides Antioxidant activity Visceral organs of horse mackerel [38]
Cephalothorax Antioxidant activity Pacific white shrimp [39]
hydrolysates
Hydrolysates and Antioxidant activity Shrimp (Aceteschinensis) [40]
peptides
Hydrolysates Antioxidant activity Crayfish (Procambarus clarkia) [41]
Phlorotannins Antioxidant activity Brown algae [42]
Phenolic compounds Antioxidant activity Sea cucumber (Holothurialeucospilota, [43]
Holothuriascabra, and
Stichopuschlorontus)
Porphyrans Immunoregulatory, Porphyraspp [44]
antioxidant, and
anticancer properties
Fucoidans Antioxidant activity Seaweed (Sargassum thunbergi, [44]
Ascophyllum nodosum, Viz
fucusvesiculosus, Laminaria japonica,
Fucusevanescens, and Laminaria
cichorioides)
Ethyl acetate extract Antimicrobial activity European abalone (Haliotistuberculata [45]
coccinea)
Peptide fractions Antimicrobial activity Chlorella vulgaris [46]
Hydrolyzed secondary Antimicrobial activity Atlantic mackerel (Scomberscombrus) [47]
raw material and half-fin anchovy (Setipinnataty)
Short-chain fatty acids Antimicrobial activity Microalgae. Isochrysisgalbana, [48]
and unsaturated Scenedesmus sp., and Chlorella sp.
chain fatty acids
Peptides Antihypertensive Tuna frame protein hydrolysate [49]
activity
Tri-peptides Antihypertensive Northern shrimp (Pandalus borealis) [50]
activity
Peptides Antihypertensive Sardine muscle [51]
activity
Triterpene glycosides Anti-cancer properties Sea cucumber [43]
Fucoidans Anticoagulant Seaweed [52]
properties
Fucoidans Anticoagulant Seaweed (Saccharina japonica, [53]
properties Ochrophyta, Phaeophyceae)
(continued)
Table 1
(continued)
Active compound Activity Source References

Polysaccharide Anticoagulant Marine alga (Dictyopterisdelicatula, [54]
fractions properties Ochrophyta, Phaeophyceae)
Laminarin Would healing property Cystoseirabarbata [55]
Alginates Would healing property Cell wall of brown macroalgae [56]
Fucoxanthin Neuroprotective Brown algae [57]
property
Carotenoids Neuro protective Marine fungi [58]
property
just a few of the algae that have been identified as excellent sources
of phycobiliproteins. These pigments have been found to have
antioxidant capabilities, according to a recent study by Menaa and
others [44]. Carrageenans derived from Hypnea spp. exhibit anti-
oxidant characteristics and hypocholesterolemic effects by lowering
cholesterol and sodium absorption while increasing potassium
absorption [60]. The complex sulfated polysaccharide, known as
porphyran, which is derived from red Porphyra spp., possesses
immunoregulatory, antioxidant, and anticancer properties
[44]. Sargassum thunbergi, Ascophyllum nodosum, Viz fucusvesicu-
losus, Laminaria japonica, Fucusevanescens, and Laminaria cichor-
ioides are a few examples of brown algae that contain fucoidans,
which have antioxidant properties [44].
The antioxidant properties of sulfated polysaccharides isolated
from Monostromaangicava are correlated with the degree of sulfa-
tion and a moderate molecular weight [43]. A powerful antioxi-
dant, iodine is present in most edible seaweeds [44]. The water-
soluble vitamins thiamine, riboflavin, niacin, pantothenic acid, and
biotin, as well as the fat-soluble vitamins retinoic acid and toco-
pherols with antioxidant properties, are both abundant in
seaweeds [44].
3.1.2 Antimicrobial Food antimicrobials are used to stop the development of germs that
Property lead to food spoiling. The most widely used antimicrobial medica-
tions are organic acids (sorbic acid, acetic acid, citric acid, etc.),
which alter the permeability of cell membranes to substrates and
create pH conditions that are unfavorable to bacterial development.
Despite being a very effective preservative, organic acids like sorbic
acid are known to break down when exposed to water and generate
potentially dangerous compounds like acetaldehyde. Ethyl acetate
was used as the extraction solvent to produce an extract from the
edible abalone species Haliotistuberculata coccinea, sometimes
known as “European abalone,” by Tortorella et al., 2021. It

demonstrated antimicrobial action against Staphylococcus aureus
ATCC 6538P, the most susceptible strain, the developing multi-
drug-resistant Stenotrophomonas maltophilia D71, and the
methicillin-resistant Staphylococcus epidermidis strain RP62A. Addi-
tionally, it demonstrated anthelmintic action as measured by its
toxicity toward the target model helminth Caenorhabditis elegans.
The antibacterial efficacy of Chlorella vulgaris peptide fractions
that were pepsin-digested and demonstrated antimicrobial activity
against E. coli CECT 434 was assessed by Sedighi and others
[46]. Guzmán et al. [61] reported the discovery of antibacterial
peptides from Tetraselmissuecica, specifically the AQ-1766 peptide
(LWFYTMWH), which exhibited antimicrobial activity against
both gram-positive and -negative bacterial strains, including
methicillin-resistant S. aureus, B. cereus, M. luteus, and
P. aeruginosa. The majority of antimicrobial peptides have been
found to be cationic, meaning they have a net positive charge as a
result of positively charged amino acid groups such as lysine and
arginine, which also have hydrophobic and amphipathic properties
that help them bond with their hosts and become soluble in aque-
ous (lipid) membranes. Antimicrobial peptides are hypothesized to
function by opening pores in the membrane before entering the
cell, where they release biological components from the microbes
and kill the cell. Peptides with less than 50 amino acids and a low
molecular weight of less than 10 kDa are typically found to have
antimicrobial activity when released under the right hydrolysis
circumstances [62].
Atlantic mackerel (Scomber scombrus) and half-fin anchovy
(Setipinnataty) hydrolyzed secondary raw materials exhibited anti-
bacterial efficacy against gram-negative Escherichia coli and gram-
positive Listeria innocua [47]. Microalgae have tremendous appli-
cation potential as a natural antibiotic and have the ability to reduce
microbial infection in aquaculture [63]. Now, numerous researches
have demonstrated the antibacterial action of short-chain fatty acids
and unsaturated chain fatty acids isolated from microalgae. Isochry-
sisgalbana, Scenedesmus sp., and Chlorella sp. were the three micro-
algae whose major chemicals were identified by Alsenani et al. [48]
as DHA, EPA, linoleic acid, and oleic acid and whose extracts may
suppress the growth of gram-positive bacteria
3.1.3 Antihypertensive Different marine fish and fish body parts, such as those from tuna,
Property yellowfin sole, scad, Hoki, Pacific hake, yellow stripe trevally, and
conger eel, have been used to make peptides (fish bones, muscles,
skin, intestines, etc.). Peptides from fish have been found to have
antihypertensive effects [64]. The production of Angiotensin-
converting enzyme inhibitory peptides is influenced by the hydro-
lysis circumstances, substrate protein, and peptidase type.
Angiotensin-converting enzyme-inhibitory protein hydrolysates

were produced by the enzymatic digestion of a marine protein
substrate, according to studies [65]. Lee et al. [49] extracted the
peptide GDLGKTTTVSNWSPPKYKDTP from tuna frame protein
hydrolysate. This peptide significantly reduced the systolic blood
pressure of spontaneously hypertensive rats, and its antihyperten-
sive efficacy was comparable to that of captopril. Sardine cannery
byproducts are another potential source of bioactive peptides. In
the hydrolysate produced from Northern shrimp (Pandalus borea-
lis), two novel ACE inhibitory tri-peptides, FTY and FSY, were
revealed [50]. The two peptides are quite similar since both of the
terminal residues are the same and both of the intermediate resi-
dues (Serine and Threonine) have R-groups that contain hydroxyl.
The sole distinction is that FTY is noticeably bulkier than FSY due
to the presence of a free methyl in addition to the hydroxyl in the
R-group of Thr. The findings demonstrate that this modest change
has a large impact on ACE inhibitory activity since FSY is around
30 times more active than FTY. The peptides KW, RVY, and MY,
which are recognized as antihypertensive peptides, have been
reported to have ACE-inhibitory activity in sardine muscle hydro-
lysates with alkaline protease [51].
3.1.4 Anticancer The world’s population has been impacted by cancer as a significant
Property cause of mortality in both direct and indirect ways. Although cancer
cases are on the rise, some of them may be avoidable or even
treatable by using natural substances. The risk of chronic diseases
is said to be reduced and overall health is maintained by bioactive
peptides that can be found on land and in the water. Fish bypro-
ducts are a source of bioactive peptides and may be anticarcinogenic
[66]. Anticarcinogenic peptides inhibit the growth of cancer cells in
a number of different methods, such as (1) in the cytoplasm, (2) by
promoting membrane rupture through micellization, and (3) by
interacting with cells during apoptosis via gangliosides on the
surface. Anticancer properties were found in peptides made from
leftover fish processing raw material [62].
Cytotoxicity is the most frequent biological characteristic of sea
cucumber glycosides, making them one of the most researched
anticancer drugs. To present, a variety of sea cucumber species
have produced more than 300 triterpene glycosides with notable
pharmacological characteristics. Argusides A–E, triterpene glyco-
sides derived from Bohadschiaargus, have exhibited significant
in vitro cytotoxicity against a number of human carcinoma cell
lines. Against six different tumor cell lines (P-388, A-549,
MCF-7, MKN-28, HCT-116, and U87MG), the triterpene glyco-
sides, pentactasides I–III, as well as philinopsides A–B, isolated
from Pentactaquadrangularis elicited a remarkable in vitro cyto-
toxicity effect with an IC50 value ranging from 0.60 to 3.95 M. In
numerous types, including pancreatic ductal adenocarcinoma,

colon, prostate, cervical, and bladder cancer cells, frondoside A, a
triterpene glycoside derived from the orange-footed sea cucumber
Cucumariafrondosa, has shown anticancer properties. It has been
claimed that this glycoside has anti-cancer properties through a
variety of mechanisms of action, including the activation of cellular
apoptosis, suppression of cancer cell proliferation, migration,
metastases development, invasion, and angiogenesis. On human
leukemia HL-60 and human hepatoma BEL-7402 cells, it was
found that the triterpene glycosides fuscocinerosides A–C,
pervicoside C, and holothurin A, which were isolated from the sea
cucumber Holothuriafuscocinerea, had a strong cytotoxic effect.
Three sulfated triterpene glycosides from Pseudo colochirus violaceus
ides have demonstrated notable in vitro cytotoxicity action against
stomach cancer MKN-45 and CT-116 cells. Sea cucumber may be
utilized as a functional diet to prevent cancer, despite the fact that
the precise mechanism(s) behind the anticancer effects of numer-
ous triterpene glycosides are still completely unknown [43].
3.1.5 Anticoagulant A series of events take place during blood coagulation that, if
Property unchecked, might result in coronary artery blockage. During plate-
let activation, a large number of platelets gather. Next, prothrom-
bin is changed into thrombin, a serine protease that turns soluble
fibrinogen into its insoluble form, fibrin. As more thrombin is
produced, the conversion of fibrinogen to fibrin is enhanced. The
combined effects of vasoconstriction and obstruction of coronary
arteries by fibrin complex formation within the blood vessel result
in myocardial ischemia and heart attacks. Limiting platelet aggre-
gation will thereby reduce vasoconstriction and the risk of myocar-
dial ischemia in these circumstances. The king of physiologically
active chemicals, fucoidan is a highly branched, diverse monosac-
charide with a high molecular weight (10,000–100,000 Da). The
complex chemical structure promotes its anticoagulant
potential [52].
The molecular weight of fucoidans and the amount of galac-
tose they contain both affect the seaweeds’ ability to prevent clot-
ting. Four of the seven fucoidans isolated from the seaweed
Saccharina japonica (Ochrophyta, Phaeophyceae) differ in both
average molecular weight and the ratio of fucose to galactose.
These fucoidans’ results from the activated partial thromboplastin
time (APTT) assay demonstrate that larger molecular weight fucoi-
dans have strong anticoagulant activity, which further raises the
galactose concentration [53]. Sulfated polysaccharides from the
brown seaweed Sargassum fulvellum also showed substantial anti-
coagulant action in the APTT assay, as did several fucans isolated
from the brown algae Padina gymnospora. The APPT test demon-
strated that six families of sulfated polysaccharides from the marine
alga Dictyopterisdelicatula (Ochrophyta, Phaeophyceae) inhibit

both the intrinsic and common pathways of coagulation and that
some polysaccharide fractions have anticoagulant activity compara-
ble to that of clexane (a commercial anticoagulant drug). Three
seaweeds, Laurencia filiformis (Rhodophyta), Ulva compressa
(Chlorophyta), and Turbinariaconoides (Ochrophyta, Phaeophy-
ceae), contain polysaccharides that prolonged the coagulation of
human plasma tested by the APTT assay [54].
In the APTT assay, an extract of the green seaweed Udotea
flabellum, which is rich in sulfated polysaccharides, revealed a
plasma coagulation time that was twice as long, comparable to the
outcomes from 1 g of heparin [53]. The use of ulvans as an
anticoagulant is based on the polar interaction of sulfated polysac-
charide with proteins, such as heparin cofactor II, which is thought
to be the cause of the anticoagulant activity [67]. The ulvan’s
anticoagulant effect was the most anticipated biological character-
istic because of its molecular similarity to mammalian heparinoid
substances. There have been reports of substantial antithrombotic
and anticoagulant activity in sulfated polysaccharides isolated from
Ulvales spp. [68].
As a naturally occurring, highly sulfated mucopolysaccharide,
algal sulfated polysaccharide has an anticoagulant effect similar to
heparin. Due to the sulphate group’s anionic nature, sulfated poly-
saccharides are good candidates for bio-applications as antioxidant
and anticoagulant medicines. A fascinating prospect for use in
medicine is microalgal sulfated polysaccharide, a glycosaminogly-
can that contains sulfate ester in its different sugar units [69]. While
some seaweed polysaccharides directly decrease fibrin polymeriza-
tion and thrombin activity without interacting with antithrombin
III (AT III) and heparin cofactor II (HC II), others exhibit antico-
agulant effect via influencing AT III and boosting HC II, a key
endogenous inhibitor [53].
The anticoagulant properties of Chlorella sorokiniana could be
attributed to a variety of elements, such as the sulfate concentration
and their binding site, monosaccharide residues, and glycoside
bonds that are important for the bioactivity of
polysaccharides [70].
The anticoagulant and antiplatelet effects of bioactive peptides
made from fish muscle are also established [62]. Anticoagulants
from the hydrolysate of yellowfin sole protein were recognized by
Phadke and team [62] in 2021. It formed an inactive compound
that prevented activated coagulation factor XII regardless of Zn2+.
Peptides comprising the amino acids His-Cys-Phe, Cys-Leu-Arg,
Leu-Cys-Agr-Agr, and Leu-Cys-Arg have higher anticoagulant
activity. Protein hydrolysates from the adductor muscle of the sea
bivalve mollusk Mytilus edulis showed better anticoagulant ability at
1.49 mg/mL. Similar to this, blood coagulation factor was
suppressed by the hydrolysate of an anticoagulant peptide from an

oyster (Crassostrea gigas). The inhibition of thrombin time was
observed to be prolonged and partial thromboplastin time was
activated by oyster (Crassostrea gigas) hydrolysate and Scapharcab-
roughtonii protein (26 kDa) [71].
3.1.6 Wound Healing The process of healing a wound involves a number of steps, includ-
Property ing cell migration and proliferation as well as the production of new
extracellular matrix. Brown algal polysaccharide laminarin has a
small molecular weight (MW; 5 kDa). Laminaria and Saccharina
species as well as some Ascophyllum and Fucus species contain
it. Laminarin is made up of sβ-(1,6)-intrachain linkages and (1,3)-
β-D-glucan. Laminarin from Cystoseirabarbata (5% cream) greatly
accelerated reepithelization, increased wound contraction, and per-
mitted restitution of mice skin tissue during the in vivo healing
process [55]. Alginate is a linear polysaccharide made up of consec-
utive block structures of (1–4-)linked β-D-mannuronic acid
(M) and α -L-guluronic acid (G) monomers. The cell wall of
brown macroalgae contains it naturally. Alginates have use in mate-
rials for dental impressions and alginate fiber wound dressings [56].
Carrageenan is a sulfated polysaccharide with a high molecular
weight that is a structural component of the cell membranes of red
macroalgae. It is made up of alternating linear chains of
α-1,3-galactose and β-1,4,3,6-anhydrogalactose with ester sul-
phates (15–40%). Carrageenan is used in the transport of drugs,
the regeneration of bone and cartilage tissue, and wound healing
due to its physiochemical characteristics and gelling mechanism
[72]. Additionally, gelatin is produced into sterile sponges for use
in medical and dental operations, as well as a material for treating
wounds [73]. Oral administration of peptides from different fish
species and their byproducts, like collagen hydrolysates, shows
moisture retention across the face in addition to improved visco-
elastic characteristics and decreased sebum levels. Enzymatic pro-
tein hydrolysates made from the silver carp’s bones and isolated
peptides were more effective at promoting keratinocyte metabolism
and wound healing processes, highlighting the potential of bone
peptides for treating wounds in the cutaneous region [74].
3.1.7 Neuroprotective There has been a significant increase in research on marine micro-
Property bial pigments in recent years. Pigments are molecular structures
that can absorb specific wavelengths of light and reflect the rest of
the visible spectrum. Pigment production by marine bacteria is
thought to be mediated by a quorum-sensing mechanism [75]. Car-
otenes, which are pure hydrocarbon carotenoids without any sub-
stituents in their structures, and xanthophylls, which are molecules
containing oxygen, are the two main categories of carotenoids
[76]. Astaxanthin, a xanthophyll carotenoid compound, has

demonstrated its ability to provide neuroprotection through the
inhibition of lipopolysaccharide-induced neuroinflammation, amy-
loidogenesis, and oxidative activity in mouse models [77, 78]; the
prevention of hippocampal insulin resistance and complications of
Alzheimer’s disease in Wistar rats [79]; and the prevention of brain
damage in offspring exposed to prenatal epilepsy seizures [80]. Fur-
thermore, the neuroprotective potential of astaxanthin and fuco-
xanthin against amyloid-mediated toxicity in pheochromocytoma
neuronal cells has also been studied. Results showed many neuro-
protective effects but also pointed to fucoxanthin’s greater poten-
tial as a potential treatment approach [57].
Furthermore, research into the potential of β-carotene for the
treatment of acute spinal cord injury revealed that it could slow the
advancement of secondary damage events by blocking the nuclear
factor-κB pathway [58]. Due to its advantageous effects on the
central nervous system, lycopene is a biocompound that has been
extensively studied. According to one study, it has the ability to
lessen oxidative stress and the cell apoptosis caused by tert-butyl
hydroperoxide, two important elements in the pathogenesis of
Alzheimer’s disease. Following lycopene injection, cell survival
and neuronal morphology were enhanced, mitochondrial mem-
brane potential was restored, and reactive oxygen species were
reduced [81]. Additionally, giving lycopene to rats with hippocam-
pal lesions caused by aluminum chloride has improved cognition
impairment and reduced oxidative stress by lowering levels of mal-
ondialdehyde and 8-hydroxy-2′-deoxyguanosine and raising levels
of glutathione and superoxide dismutase activity. In turn, these
mechanisms have demonstrated that they can stop neuroinflamma-
tion and apoptosis [82]. Moreover, 1-methyl-4-phenyl-1,2,3,6-
tetrahydropyridine-treated Parkinson’s disease mouse models
showed that lycopene had neuroprotective effects by raising dopa-
mine levels and lowering oxidative stress levels [83]. In rat models
of spinal cord ischemia/reperfusion injury, lycopene has also shown
promise for improving neurological function recovery and inhibit-
ing neuronal death and neuroinflammation [84], including preven-
tion of cerebral vessel injury caused by hyperlipidemia by reducing
astrocyte activation and inflammatory cytokine production [85].
Through several research studies, marine biocompounds have
demonstrated their neuroprotective benefits, primarily targeting
the prevention of neurodegeneration and the decrease of oxidative
stress in the central nervous system. However, the study of chemi-
cals with neuroprotective properties derived from marine sources is
still in its early stages, necessitating additional research.
Fig. 1 Various bioactive compounds obtained from different marine sources
3.2 Nutraceutical The marine ecosystem is so diverse that it presents a great pool for
Property obtaining new compounds that are beneficial for health improve-
ment and can potentially be used in food, supplement, or therapeu-
tic industries [86]. Thus, newly identified or isolated compounds
from marine sources that carry nutraceutical properties have been
intensively researched in recent years. Marine-origin nutraceuticals
include polysaccharides, peptides and proteins, polyunsaturated
fatty acids (PUFAs), and other lipids, pigments, enzymes, phenolic
compounds, minerals, and vitamins (Fig. 1) [86–88]. The sources
from which these compounds have been isolated include seaweeds
[89], microalgae [90], fish, and a large group of marine inverte-
brates (arthropods, echinoderms, sponges, mollusks, cnidarians,
lophophorates, marine worms, and the hemichordates) [91, 92].
Sulfated polysaccharides found in seaweeds are the most stud-
ied group due to their biological activities. They have shown antic-
arcinogenic, anti-inflammatory, antioxidant, antiviral, and
anticoagulant properties [89, 93]. Polysaccharides make up more
than 80% of the seaweed weight; they serve as structural com-
pounds and energy reserves. The main sulfated polysaccharides
isolated from brown seaweeds are fucoidan, laminarin, and alginate;
from red seaweeds is carrageenan; and from green seaweeds ulvan
[86]. Microalgal polysaccharides also exhibit various biological
activities; also they are components of the microalgal cell wall,
energy reservoirs and serve as cell protection. They are mainly
composed of pentose and hexose monosaccharides with glycosidic
linkage [94]. Marine invertebrates, such as sea cucumbers, asci-
dians, sea urchins, and nudibranchs, are also the source of
polysaccharides, mainly belonging to the group of glycosaminogly-

cans. These can be used as nutraceuticals as they are known to have
anti-inflammatory effects, improve cartilage structure and function,
and relieve the osteoarthritis pain. Invertebrates that do not possess
glycosaminoglycan synthesize sulfated polysaccharides, which have
the structure and biological functions similar to glycosaminogly-
cans [95]. Sulfated polysaccharides that carry antimicrobial activity
and can be used as nutraceuticals can also be isolated from fish
skins [96].
Marine algae, fish, and invertebrates are considered a great
source of proteins and bioactive peptides. Seaweeds are natural
reservoirs of bioactive peptides with numerous health benefits,
and they are considered an alternative source of protein. However,
extraction of proteins and bioactive peptides from seaweeds is
challenging and yields are low. Further improvements in extraction
are needed to break down the covalently bounded polysaccharides
and protein complex structures present in seaweeds [97]. Further,
more than 50% of microalgal dry weight consists of proteins. Some
species belonging to the genera Dunaliella and Arthrospira are
currently marketed as nutraceuticals [98]. The amino acid profile
of fish proteins makes them a high-quality nutritive component
since they contain all essential amino acids that are easily digestible
and have high bioavailability. Besides, they are a rich source of
bioactive peptides, biomolecules derived from fish protein hydro-
lysates that have shown numerous biological activities (antioxidant,
antimicrobial, antihypertensive, antiproliferative, immunomodula-
tory, antidiabetic, etc.). For this reason, they are considered bio-
molecules with positive health effects and potential therapeutic
applications [91]. Proteins and peptides derived from marine inver-
tebrates, squids, snails, shellfish, and jellyfish have shown antioxi-
dant, anti-inflammatory, anticancer, antiviral, antibacterial,
immunomodulatory, and antihypertensive activities [99].
Marine sources have unique lipid composition containing large
amounts of PUFAs. The long-chain n - 3 PUFAs, eicosapentae-
noic acid (EPA) and docosahexaenoic acid (DHA), have numerous
health benefits. They reduce the risk of cardiovascular diseases,
arthritis, and Alzheimer’s and Parkinson’s diseases. Besides, they
have neuroprotective effect, and they stimulate neurodevelopment
and neurotransmission in humans. Fatty fish, microalgae, seaweeds,
krill, and fungi are significant sources of these fatty acids [100–
103]. Like humans, fish cannot synthesize PUFAs, but they obtain
them from feed (krill or algae that they consume) [91]. Lipids,
including PUFAs, are mostly extracted from fish and marketed as
fish oil. Fish oil is conventionally extracted with solvents or by wet
pressing, while recently supercritical fluids and fish silage have been
used [104]. Fish oil can also be extracted from fish byproducts and
discarded species [105–109]; byproduct fish oil can be produced
and refined to maintain high-quality PUFAs [110]. Microalgae
have a lower lipid content than fish, but they have a high content of
PUFAs and could therefore be a potential alternative source for
their extraction [111, 112].
The main natural pigments isolated from marine sources are
chlorophylls, carotenoids, and phycobiliproteins, which have health
benefits due to their antioxidant, anti-inflammatory, anti-obesity,
antiangiogenic, anticancer, and wound healing properties
[101, 113]. Chlorophylls are synthesized by cyanobacteria and
algae and are mainly responsible for photosynthesis. Apart from
their potential use as substitutes for synthetic pigments in the food
industry, chlorophylls exert antioxidant, antibacterial, anti-
inflammatory, and antimutagenic activities, which make them
potential nutraceuticals [86, 101]. Promising marine sources for
the extraction of chlorophylls are microalgae [114]. The most
abundant carotenoids found in marine sources are fucoxanthin,
astaxanthin, lutein, zeaxanthin, neoxanthin, violaxanthin, and can-
thaxanthin [101]. Astaxanthin and fucoxanthin are the most abun-
dant carotenoids in seaweeds and microalgae [115]. Some fish
species, such as families of Salmonidae and Mullidae, are a good
source of astaxanthin, β-carotene, zeaxanthin, and canthaxanthin
[91], while crustaceans and their byproduct are a valuable source of
natural astaxanthin [78]. Carotenoids found in sponges, jellyfish,
mollusks, crustaceans, sea urchins, and tunicates come from their
diet, and in the case of sponges are also associated with their
symbionts [116]. Carotenoids exhibit various biological activities,
such as antioxidant, anti-inflammatory, anticancer, anti-obesity,
anti-diabetic, wound healing, and photoprotective activities,
which are the foundation for their beneficial effects on health
through the reduction of cardiovascular diseases and cancer risks,
atherosclerosis, non-communicable diseases, and macular degener-
ation [86, 101]. Phycobiliproteins are found in red seaweeds and
cyanobacteria and exhibit antioxidant properties. Research on their
extraction and bioactivity is increasing [117, 118].
Phenolic compounds are a large group of phytochemicals that
have recently gained attention because of their bioactivities and
health-promoting benefits, including antioxidant, antimicrobial,
anti-inflammatory, anti-tumor, anti-allergic, anti-hypertensive,
anti-cholesterol, antithrombotic, anti-diabetic, immunomodula-
tory, wound healing, neuroprotector, photoprotector, and algicidal
properties [119, 120]. This group of compounds can be divided
into simple phenols, benzoic acid derivatives, flavonoids, tannins,
stilbenes, lignins, and lignans. Marine sources of phenolic com-
pounds are microalgae and seaweeds. These organisms produce
them for protection against oxidative stress, biofouling, predators,
pathogens, and other external factors [101, 119, 121]. Among the
three groups of seaweeds, brown algae have been known to yield
more phenolic compounds [119, 122, 123].
Microalgae, seaweeds, and fish are rich sources of all vitamins

(water and lipid soluble) and minerals. They are essential for
humans as they maintain health through the coordination of phys-
iological functions. Besides, vitamins have a strong antioxidant
effect. Their deficiency leads to numerous diseases [91, 100, 114,
124]. Commonly found minerals in marine sources are iron, man-
ganese, zinc, selenium, sodium, potassium, calcium, phosphorus,
sulfur, copper, magnesium, and iodine. Their contribution to
human health and well-being is exceptional, especially for immu-
nity, transmission of nerve impulses, oxygen transport, maintaining
electrolyte balance, thyroid health, and formation of bones and
teeth [91, 125].
4 Food Application of Bioactive and Nutraceuticals Derived from Marine
With an increasing world population, a large share of elderly peo-

ple, and increasing incidence of chronic diseases, the global market
of functional foods and nutraceuticals is constantly increasing and is
expected to grow in the next decade. In the food and nutraceutical
industry, marine organisms are well recognized as a source of
valuable nutritive components that can be consumed as whole
foods or be used to extract valuable components. For example,
fish and marine algae are a well-known source of valuable nutra-
ceuticals such as PUFAs, proteins, and minerals. On the other
hand, these compounds can be utilized for enrichment of other
foods either to increase their nutritional value, to enhance their
oxidative stability, or even give them functional properties.
Table 2 provides an overview of laboratory trials based on the
applications of bioactive compounds and nutraceuticals from
marine sources in food models. Pigments isolated from microalgae,
seaweeds, and shrimps have been used in various meat, dairy, and
bakery products to improve their oxidative stability. Protein hydro-
lysates and peptides from fish, shrimp, and seaweeds were success-
fully used in meat and bakery products, improving their nutritional
value and sensory properties while retarding lipid oxidation. Oils
from marine sources (fish and shrimp) improved the nutritional
value of meat, dairy, and bakery products, without affecting their
sensory properties, and in some cases reduced lipid oxidation.
Powdered seaweed and microalgae were used to increase the nutri-
tional value of products such as pasta, bread, snacks, cheese, tomato
puree, and tuna jerky.
The effect of the seaweed extracts application in food models
[126–128] or in edible films [129, 130] has also been investigated;
however, in most cases, the chemical identification of the com-
pounds from the extracts was not reported. Interestingly, the phe-
nolic compounds purified from marine algae have not yet been used
in food models, although they are the most studied secondary
64
Table 2
Food application of bioactive and nutraceuticals derived from marine sources
Bioactive molecules
or nutraceuticals Marine source Food application Main findings References
Astaxanthin Pink deep-water shrimp Marinated chicken Natural astaxanthin was effective as vitamin C in [133]
(Parapenaeuslongirostris) by-products steaks preventing lipid oxidation of marinated
(heads, cephalothorax and appendices) chicken steaks
Texture of samples was improved
Nikheel Rathod et al.
Microbiological stability was improved

Astaxanthin Microalga Haematococcus pluvialis Raw and cooked The oxysterol levels decreased [134]
lamb patties The amount of volatiles was reduced
Oxidative stability of raw and cooked lamb
patties was improved
Astaxanthin Microalga H. pluvialis Cookies The oxidative stability of cookies during storage [135]
was improved
The addition of astaxanthin did not affect the
cookies’ taste acceptability
Astaxanthin White shrimp (Litopeneusvannamei) shells Yogurt Astaxanthin was encapsulated with alginate and [136]
chitosan and added to yogurt
Yogurt maintained desirable sensory attributes
Extract rich in Microalga H. pluvialis Raw ground pork Application of extract delayed lipid oxidation [137]
astaxanthin meat and improved color stability of raw ground
pork meat during 7 days of refrigerated
storage
Fucoxanthin Microalga Phaeodactylumtricornutum Whole and skimmed In terms of stability and bioavailability, skimmed [138]
milk milk was an excellent food matrix for
application of fucoxanthin
Fucoxanthin Laminariales sp. Goat whole and skim Pasteurization did not affect fucoxanthin [139]
milk stability
Addition of fucoxanthin did not affect the
physicochemical properties of milk
Milk color was influenced
Goat milk can be a suitable matrix for
fucoxanthin supplementation
Extract containing Sargassum polycystum Snack bar When used in combination with cacao butter [140]
fucoxanthin and 1% stevia, the extract maintained its
stability and antioxidant properties; it was the
combination that panelists preferred in
organoleptic evaluation
Biomass and Spirulina (Arthrospiraplantensis) Biscuits Biscuits had a high oxidative stability during [141]
phycocyanin 30 days of storage
extracts Biscuits had good nutritional and sensory
profiles
Protein-procyanidin Spanish mackerel (Scomberomorus maculatus) Surimi Protein-procyanidin hybrid nanoparticles were [142]
hybrid used for stabilization of high internal phase
nanoparticles pickering emulsions that were applied in
surimi; surimi showed good cooking stability
Peptides and peptide- Tuna (Thunnusobesus) skin Pork patties Inhibition of lipid oxidation in pork patties was [143]
loaded observed during 14 days of refrigerated
nanoliposomes storage
Protein hydrolysate Pacific white shrimp (L.vannamei) Biscuits Biscuits fortified with shrimp protein [144]
cephalothorax hydrolysate powder had higher nutritional
value and increased sensory properties
Protein hydrolysate Palmariapalmata Bread The texture and sensory properties of wheat [145]
bread were not affected by the addition of
P. palmata protein hydrolysate
Extraction of Bioactive and Nutraceuticals from Marine Sources. . .
Renin inhibitory bioactivity of hydrolysate was

retained after the baking process
(continued)
65
66
Table 2
(continued)
Bioactive molecules
Protein hydrolysate Skipjack tuna (Katsuwonuspelamis) roe Emulsion sausage Lipid oxidation-reduction of sausages during [146]
from broadhead 12 days of storage was observed
catfish Skipjack roe protein hydrolysate did not affect
(Clarias sausages’ organoleptic properties
macrocephalus)
Fish concentrate Sea bass (Dicentrachuslabrax) filleting Fresh pasta Nutritional value of pasta was improved; n - 3 [147]
by-products (trimmings) fatty acids remained in satisfactory quantities
during the shelf-life
Enriched pasta had acceptable sensory
properties
n - 3-rich oil Sardine (Sardinapilchardus) gill and viscera Wheat flour chips Lipid oxidation of chips was prevented and [148]
antioxidant enzymes were activated
Chips’ lipid profile was improved
Fish oil Cod liver Cooked and Fish oil was microencapsulated [149]
dry-cured Nutritional value of sausages (n - 3 content)
sausages was improved without influence on oxidative
stability, physicochemical characteristics, and
acceptability
Fish oil Cod liver Chicken nuggets Bulk fish oil and microencapsulated fish oil were [150]
added to nuggets
Microencapsulated fish oil nuggets had lower
levels of lipid and protein oxidation, and
volatile compounds
No change in sensory quality was observed for
microencapsulated fish oil nuggets when
compared to control
Fish oil Not specified Yogurt Yogurt was fortified with fish oil and nano- [151]
encapsulated fish oil
Higher n - 3 contents and better sensory
characteristics were found for yogurt fortified
with nanoencapsulated fish oil
Shrimp oil Pacificwhite shrimp (L. vannamei) Skim milk Shrimp oil was encapsulated in nanoliposomes [152]
cephalothorax β-glucan was added to mask bitterness caused by
shrimp oil
In vitro digestion showed that n - 3 fatty acids
were bioaccessible for absorption in the gut
after digestion
Oxidative stability was enhanced throughout
the storage
Shrimp oil Pacific white shrimp (L.vannamei) Biscuits Shrimp oil was encapsulated with sodium [153]
hepatopancreas caseinate, fish gelatin, and glucose syrup
before addition to biscuits
Nutritive value of biscuits was improved
No adverse effect on biscuit quality and
sensorial properties was observed after
addition of micro-encapsulated shrimp oil
Powder and sulfated Ulva intestinalis Fish fingers Lipid oxidation was retarded over 6 months of [154]
polysaccharide storage when compared to control
Products were acceptable organoleptically
Sulphated polysaccharides had better impact on
products’ texture preservation
Powder Spirulina (A. plantensis) Pasta Nutritional value of pasta was significantly [155]
improved
Pasta with spirulina had high acceptability
scores
(continued)
Extraction of Bioactive and Nutraceuticals from Marine Sources. . .
67
68
Table 2
(continued)
Bioactive molecules
Powder Spirulina (A. plantensis) Cheese When cheese was fortified with A. platensis [156]
powder, the amount of protein and iron
increased
The positive effects on the survival of probiotic
bacteria were observed during the storage

Powder Ascophyllum nodosum Gluten-free bread Bread with seaweed powder showed favorable [157]
changes in the texture
Antioxidant activity of bread was increased
Powder Fucusvesiculosus Bread Bread density and bread crumb firmness were [158]
enhanced
Biomass Nannochloropsissp. Tomato puree Higher oxidative stability of tomato puree with [159]
Nannochloropsis sp. biomass was observed
when compared to control (fish oil)
Powder Spirulina sp. Snacks Nutritional value of snacks was improved [160]
without impacting physical parameters
significantly
Snacks had high sensory acceptance index
Powder Sargassum wightii Tuna (Thunnus Tuna jerky had improved antioxidant quality [161]
albacores) jerky Organoleptic quality was not affected
metabolites with many proven bioactive properties. On the other

hand, seaweed polysaccharides (agar, alginate, carrageenan) are
widely used in technological processes as emulsifiers, stabilizers,
and thickeners, but not as nutraceuticals. They are used in the
production of various foods, dairy products, wine, jam, jelly, and
bakery products [131]. In addition, seaweed polysaccharides are
used for the development of biodegradable packaging (e.g., edible
films) as natural compounds to replace synthetic polymers [132].
Overall, it is evident that the application of marine bioactive
components and nutraceuticals will be investigated in the future.
Their application is sometimes limited due to the marine or fishy
odor they produce; however, formulation and adaptation of the
recipes are needed as well as industrial trials that will fabricate new
functional products.
5 Conclusion
The extraction of bioactive and nutraceuticals from marine sources

is currently being pursued due to the potential resources of several
compounds with well-known functional and biological activities.
Ample research has proven the undeniable health benefits of sea
product extracts, thus attracting food industry and pharmaceutical
interest. The marine-extracted chemical compounds have a wide
range of functional characteristics, including neuroprotection,
wound healing, antioxidative properties, and many more. The
intensive demand for marine product-derived bioactive compounds
in the food industry has compelled researchers and industries to
employ various methods for extracting these compounds with high
efficiency. In the application of emerging extraction technology, the
extraction yield of functional compounds from marine products is
higher with the minimization of byproducts. The aforementioned
methods are environmentally friendly extraction techniques due to
the minimal use of synthetic and organic chemicals, reduced extrac-
tion time, better yield, and excellent quality of the extract. Thus,
these extraction methods have gained significant popularity and are
commercialized at an industrial scale. Many standards and proto-
cols have been used for the optimal extraction of the bioactive from
sea resources. However, the exploration of the optimization and
quantification of the bioactive from marine sources is still limited.
Moreover, the combined application of assisting technology and
green solvents in the extraction of those bioactive compounds
needs to be explored.
References
1. Eghtedari M, Jafari Porzani S, Nowruzi B 10. Chemat F, Rombaut N, Sicaire A-G,
(2021) Anticancer potential of natural pep- Meullemiestre A, Fabiano-Tixier A-S, Abert-
tides from terrestrial and marine environ- Vian M (2017) Ultrasound assisted extraction
ments: a review. Phytochem Lett 42:87–103. of food and natural products. Mechanisms,
https://doi.org/10.1016/j.phytol.2021. techniques, combinations, protocols and
02.008 applications. A review. Ultrason Sonochem
2. Vasconcelos A, Pomin V (2018) Marine 34:540–560
carbohydrate-based compounds with medici- 11. Tiwari BK (2015) Ultrasound: a clean, green
nal properties. Mar Drugs 16(7):233. extraction technology. TrAC Trends Anal
https://doi.org/10.3390/md16070233 Chem 71:100–109
3. Kumar S, Magnusson M, Ward L, Paul N, 12. Ghosh S, Sarkar T, Pati S, Kari ZA, Edinur
Brown L (2015) Seaweed supplements nor- HA, Chakraborty R (2022) Novel bioactive
malise metabolic, cardiovascular and liver compounds from marine sources as a tool for
responses in high-carbohydrate, high-fat fed functional food development. Front Mar Sci
rats. Mar Drugs 13(2):788–805. https://doi. 9(832957):10–3389
org/10.3390/md13020788 13. Huang G, Chen S, Dai C, Sun L, Sun W,
4. Rathod NB, Elabed N, Punia S, Ozogul F, Tang Y, Xiong F, He R, Ma H (2017) Effects
Kim S-K, Rocha JM (2023) Recent develop- of ultrasound on microbial growth and
ments in polyphenol applications on human enzyme activity. Ultrason Sonochem 37:
health: a review with current knowledge. Plan 144–149
Theory 12(6):1217. https://doi.org/10. 14. Mahindrakar KV, Rathod VK (2022)
3390/plants12061217 Ultrasound-assisted extraction of lipids, caro-
5. Wali AF, Majid S, Rasool S, Shehada SB, tenoids, and other compounds from marine
Abdulkareem SK, Firdous A, Beigh S, resources. In: Innovative and emerging tech-
Shakeel S, Mushtaq S, Akbar I, Madhkali H, nologies in the bio-marine food sector. Else-
Rehman MU (2019) Natural products against vier, San Diego, pp 81–128
cancer: review on phytochemicals from 15. Sharayei P, Azarpazhooh E, Zomorodi S,
marine sources in preventing cancer. Saudi Einafshar S, Ramaswamy HS (2021) Optimi-
Pharm J 27(6):767–777. https://doi.org/ zation of ultrasonic-assisted extraction of
10.1016/j.jsps.2019.04.013 astaxanthin from green tiger (Penaeus semi-
6. Rathod NB, Nirmal NP, Abdullah S, Surasani sulcatus) shrimp shell. Ultrason Sonochem
VKR, Ranveer RC, Kumar S, 76:105666
Chunhavacharatorn P, Benjakul S, Al-Asmari 16. Wang J, Shi S, Li F, Du X, Kong B, Wang H,
F (2023) Extraction of natural bioactive com- Xia X (2022) Physicochemical properties and
pounds using clean label technologies and antioxidant activity of polysaccharides
their application as muscle food preservatives. obtained from sea cucumber gonads via
Front Sustain Food Syst 7:1207704. https:// ultrasound-assisted enzymatic techniques.
doi.org/10.3389/fsufs.2023.1207704 LWT 160:113307
7. Cikoš A-M, Jokić S, Šubarić D, Jerković I 17. Bruno SF, Kudre TG, Bhaskar N (2019)
(2018) Overview on the application of mod- Impact of pretreatment-assisted enzymatic
ern methods for the extraction of bioactive extraction on recovery, physicochemical and
compounds from marine macroalgae. Mar rheological properties of oil from Labeo
Drugs 16(10):348. https://doi.org/10. rohita head. J Food Process Eng 42(3):
3390/md16100348 e12990
8. Vlčko T, Rathod NB, Kulawik P, Ozogul Y, 18. Kotnik T, Rems L, Tarek M, Miklavčič D
Ozogul F (2022) The impact of aromatic (2019) Membrane electroporation and elec-
plant-derived bioactive compounds on sea- tropermeabilization: mechanisms and models.
food quality and safety. In: Advances in food Annu Rev Biophys 48:63–91
and nutrition research. Elsevier, 19. Kranjc M, Miklavčič D (2017) Electric field
p S1043452622000341. https://doi.org/ distribution and electroporation
10.1016/bs.afnr.2022.05.002 threshold. In: Handbook of electroporation,
9. Wen C, Zhang J, Zhang H, Dzah CS, vol 80. Springer, Cham, pp 1043–1058
Zandile M, Duan Y, Ma H, Luo X (2018) 20. Wang L, Boussetta N, Lebovka N, Vorobiev E
Advances in ultrasound assisted extraction of (2020) Cell disintegration of apple peels
bioactive compounds from cash crops–a induced by pulsed electric field and efficiency
review. Ultrason Sonochem 48:538–549
of bio-compound extraction. Food Bioprod 31. Bordoloi A, Goosen N (2020) Green and
Process 122:13–21 integrated processing approaches for the
21. Tylewicz U (2020) How does pulsed electric recovery of high-value compounds from
field work? In: Pulsed electric fields to obtain brown seaweeds. In: Advances in botanical
healthier and sustainable food for tomorrow. research, vol 95. Elsevier, pp 369–413
Elsevier, San Diego, pp 3–21 32. Da Silva RP, Rocha-Santos TA, Duarte AC
22. González-Casado S, Martı́n-Belloso O, Elez- (2016) Supercritical fluid extraction of bioac-
Martı́nez P, Soliva-Fortuny R (2018) Enhanc- tive compounds. TrAC Trends Anal Chem
ing the carotenoid content of tomato fruit 76:40–51
with pulsed electric field treatments: effects 33. Ei ZZ, Benjakul S, Buamard N, Visuttijai K,
on respiratory activity and quality attributes. Chanvorachote P (2022) Shrimp lipid pre-
Postharvest Biol Technol 137:113–118 vents endoplasmic reticulum-mediated endo-
23. Franco D, Munekata PE, Agregán R, thelial cell damage. Foods 11(19):3076.
Bermúdez R, López-Pedrouso M, https://doi.org/10.3390/foods11193076
Pateiro M, Lorenzo JM (2020) Application 34. Raju N, Sae-leaw T, Osako K, Benjakul S
of pulsed electric fields for obtaining antioxi- (2022) Improved cholesterol depletion with
dant extracts from fish residues. Antioxidants enhanced astaxanthin and polyunsaturated
9(2):90 fatty acids of lipid from Pacific white shrimp
24. He G, Yin Y, Yan X, Wang Y (2017) Semi- cephalothorax using prior ethanolic separa-
bionic extraction of effective ingredient from tion of polar lipid and β-Cyclodextrin. J
fishbone by high intensity pulsed electric Food Sci Technol 59(6):2255–2262.
fields. J Food Process Eng 40(2):e12392 https://doi.org/10.1007/s13197-021-
25. Gulzar S, Benjakul S (2020) Impact of pulsed 05238-7
electric field pretreatment on yield and quality 35. Haq M, Chun B-S (2018) Characterization of
of lipid extracted from cephalothorax of phospholipids extracted from Atlantic Salmon
Pacific white shrimp (Litopenaeus vannamei) by-product using supercritical CO2 with eth-
by ultrasound-assisted process. Int J Food Sci anol as co-solvent. J Clean Prod 178:186–195
Technol 55(2):619–630 36. Mezzomo N, Tenfen L, Farias MS, Friedrich
26. Zhou Y, He Q, Zhou D (2017) Optimization MT, Pedrosa RC, Ferreira SRS (2015) Evi-
extraction of protein from mussel by high- dence of anti-obesity and mixed hypolipi-
intensity pulsed electric fields. J Food Process demic effects of extracts from pink shrimp
Preserv 41(3):e12962 (Penaeus brasiliensis and Penaeus paulensis)
27. Ozogul Y, Ucar Y, Takadaş F, Durmus M, processing residue. J Supercrit Fluids 96:
Köşker AR, Polat A (2018) Comparision of 252–261
green and conventional extraction methods 37. Zhang J, Tao N, Wang M, Shi W, Ye B,
on lipid yield and fatty acid profiles of fish Wang X, Zhu Q, Hua C (2018) Characteriza-
species. Eur J Lipid Sci Technol 120(12): tion of phospholipids from Pacific saury
1800107 (Cololabis saira) viscera and their neuropro-
28. Afolabi HK, Mudalip SKA, Alara OR (2018) tective activity. Food Biosci 24:120–126
Microwave-assisted extraction and characteri- 38. Sila A, Bougatef A (2016) Antioxidant pep-
zation of fatty acid from Eel Fish (Mono- tides from marine by-products: isolation,
pterus Albus). Beni-Suef Univ J Basic Appl identification and application in food systems.
Sci 7(4):465–470 A review. J Funct Foods 21:10–26
29. de la Fuente B, Pinela J, Mandim F, Heleno 39. Sinthusamran S, Benjakul S,
SA, Ferreira IC, Barba FJ, Berrada H, Kijroongrojana K, Prodpran T, Kishimura H
Caleja C, Barros L (2022) Nutritional and (2020) Protein hydrolysates from Pacific
bioactive oils from salmon (Salmo salar) side white shrimp cephalothorax manufactured
streams obtained by Soxhlet and optimized with different processes: compositions, char-
microwave-assisted extraction. Food Chem acteristics and antioxidative activity. Waste
386:132778 Biomass Valorization 11(5):1657–1670
30. Magnusson M, Yuen AK, Zhang R, Wright 40. Camargo TR, Mantoan P, Ramos P, Monser-
JT, Taylor RB, Maschmeyer T, de Nys R rat JM, Prentice C, Fernandes CC, Zambuzzi
(2017) A comparative assessment of micro- WF, Valenti WC (2021) Bioactivity of the
wave assisted (MAE) and conventional solid- protein hydrolysates obtained from the most
liquid (SLE) techniques for the extraction of abundant crustacean bycatch. Mar Biotechnol
phloroglucinol from brown seaweed. Algal 23(6):881–891
Res 23:28–36
41. Felix M, Romero A, Rustad T, Guerrero A 51. Lee SY, Hur SJ (2017) Antihypertensive pep-
(2017) Rheological properties and antioxi- tides from animal products, marine organ-
dant activity of protein gels-like systems isms, and plants. Food Chem 228:506–517
made from crayfish concentrate and hydroly- 52. Zayed A, Hahn T, Rupp S, Kr€amer R, Ulber R
sates. Food Bioprod Process 102:167–176 (2018) Fucoidan as a natural anticoagulant,
42. Catarino MD, Silva AM, Mateus N, Cardoso antiviral and anti-cancer drug. Naunyn
SM (2019) Optimization of phlorotannins Schmiedeberg’s Arch Pharmacol 391:S7–S8.
extraction from Fucus vesiculosus and evalua- Springer, New York
tion of their potential to prevent metabolic 53. Choudhary B, Chauhan O, Mishra A (2021)
disorders. Mar Drugs 17(3):162 Edible seaweeds: a potential novel source of
43. Lobine D, Rengasamy KR, Mahomoodally bioactive metabolites and nutraceuticals with
MF (2022) Functional foods and bioactive human health benefits. Front Mar Sci 8:
ingredients harnessed from the ocean: current 740054
status and future perspectives. Crit Rev Food 54. Venkatesan M, Arumugam V, Pugalendi R,
Sci Nutr 62(21):5794–5823 Ramachandran K, Sengodan K, Vijayan SR,
44. Menaa F, Wijesinghe U, Thiripuranathar G, Sundaresan U, Ramachandran S, Pugaz-
Althobaiti NA, Albalawi AE, Khan BA, Menaa hendhi A (2019) Antioxidant, anticoagulant
B (2021) Marine algae-derived bioactive com- and mosquitocidal properties of water soluble
pounds: a new wave of nanodrugs? Mar Drugs polysaccharides (WSPs) from Indian sea-
19(9):484 weeds. Process Biochem 84:196–204
45. Tortorella E, Giugliano R, De Troch M, 55. Sellimi S, Maalej H, Rekik DM, Benslima A,
Vlaeminck B, de Viçose GC, de Pascale D Ksouda G, Hamdi M, Sahnoun Z, Li S,
(2021) The ethyl acetate extract of the marine Nasri M, Hajji M (2018) Antioxidant, anti-
edible gastropod Haliotis tuberculata cocci- bacterial and in vivo wound healing properties
nea: a potential source of bioactive com- of laminaran purified from Cystoseira barbata
pounds. Mar Biotechnol 23(6):892–903 seaweed. Int J Biol Macromol 119:633–644
46. Sedighi M, Jalili H, Darvish M, Sadeghi S, 56. Draget KI, Taylor C (2011) Chemical, physi-
Ranaei-Siadat S-O (2019) Enzymatic hydro- cal and biological properties of alginates and
lysis of microalgae proteins using serine pro- their biomedical implications. Food Hydro-
teases: a study to characterize kinetic coll 25(2):251–256
parameters. Food Chem 284:334–339 57. Alghazwi M, Smid S, Musgrave I, Zhang W
47. Ennaas N, Hammami R, Beaulieu L, Fliss I (2019) In vitro studies of the neuroprotective
(2015) Production of antibacterial fraction activities of astaxanthin and fucoxanthin
from Atlantic mackerel (Scomber scombrus) against amyloid beta (Aβ1-42) toxicity and
and its processing by-products using commer- aggregation. Neurochem Int 124:215–224
cial enzymes. Food Bioprod Process 96:145– 58. Zhou L, Ouyang L, Lin S, Chen S, Liu Y,
153 Zhou W, Wang X (2018) Protective role of
48. Alsenani F, Tupally KR, Chua ET, Eltanahy E, β-carotene against oxidative stress and neu-
Alsufyani H, Parekh HS, Schenk PM (2020) roinflammation in a rat model of spinal cord
Evaluation of microalgae and cyanobacteria as injury. Int Immunopharmacol 61:92–99
potential sources of antimicrobial com- 59. Olatunde OO, Benjakul S (2022) Antioxi-
pounds. Saudi Pharm J 28(12):1834–1841 dants from crustaceans: a panacea for lipid
49. Lee S-H, Qian Z-J, Kim S-K (2010) A novel oxidation in marine-based foods. Food Rev
angiotensin I converting enzyme inhibitory Intl 38(1):1–31
peptide from tuna frame protein hydrolysate 60. Cherry P, O’Hara C, Magee PJ, McSorley
and its antihypertensive effect in spontane- EM, Allsopp PJ (2019) Risks and benefits of
ously hypertensive rats. Food Chem 118(1): consuming edible seaweeds. Nutr Rev 77(5):
96–102 307–329
50. Gildberg A, Arnesen JA, Sæther B-S, Rauø J, 61. Guzmán F, Wong G, Román T, Cárdenas C,
Stenberg E (2011) Angiotensin I-converting Alvárez C, Schmitt P, Albericio F, Rojas V
enzyme inhibitory activity in a hydrolysate of (2019) Identification of antimicrobial pep-
proteins from Northern shrimp (Pandalus tides from the microalgae Tetraselmis suecica
borealis) and identification of two novel (Kylin) butcher and bactericidal activity
inhibitory tri-peptides. Process Biochem improvement. Mar Drugs 17(8):453
46(11):2205–2209
62. Phadke GG, Rathod NB, Ozogul F, 73. Qureshi D, Nayak SK, Anis A, Ray SS, Kim D,
Elavarasan K, Karthikeyan M, Shin K-H, Hanh Nguyen TT, Pal K (2020) Introduction
Kim S-K (2021) Exploiting of secondary raw of biopolymers. In: Biopolymer-based formu-
materials from fish processing industry as a lations. Elsevier, pp 1–45. https://doi.org/
source of bioactive peptide-rich protein 10.1016/B978-0-12-816897-4.00001-1
hydrolysates. Mar Drugs 19(9):480. https:// 74. Iosageanu A, Oancea A, Ilie D, Anton E, Cra-
doi.org/10.3390/md19090480 ciunescu O (2021) The effect of fish bone
63. Wu J, Gu X, Yang D, Xu S, Wang S, Chen X, bioactive peptides on the wound healing pro-
Wang Z (2021) Bioactive substances and cess: an in vitro study on keratinocytes. Rom
potentiality of marine microalgae. Food Sci Biotechnol Lett 26:2692–2699
Nutr 9(9):5279–5292 75. Ramesh C, Vinithkumar NV, Kirubagaran R
64. Rauf A, Khalil AA, Khan M, Anwar S, (2019) Marine pigmented bacteria: a prospec-
Alamri A, Alqarni AM, Alghamdi A, tive source of antibacterial compounds. J Nat
Alshammari F, Rengasamy KR, Wan C Sci Biol Med 10:104–113
(2022) Can be marine bioactive peptides 76. Sathasivam R, Ki J-S (2018) A review of the
(MBAs) lead the future of foodomics for biological activities of microalgal carotenoids
human health? Crit Rev Food Sci Nutr 62: and their potential use in healthcare and cos-
7072–7116 metic industries. Mar Drugs 16(1):26
65. Manzoor M, Singh J, Gani A (2022) Explora- 77. Han JH, Lee YS, Im JH, Ham YW, Lee HP,
tion of bioactive peptides from various origin Han SB, Hong JT (2019) Astaxanthin ame-
as promising nutraceutical treasures: in vitro, liorates lipopolysaccharide-induced neuroin-
in silico and in vivo studies. Food Chem 373: flammation, oxidative stress and memory
131395 dysfunction through inactivation of the signal
66. Nurdiani R, Vasiljevic T, Singh T, Donkor O transducer and activator of transcription
(2017) Bioactive peptides from fish 3 pathway. Mar Drugs 17(2):123
by-products with anticarcinogenic potential. 78. Šimat V, Rathod NB, Čagalj M, Hamed I,
Int Food Res J 24(5):1840–1849 Generalić Mekinić I (2022) Astaxanthin
67. Mao W, Zang X, Li Y, Zhang H (2006) Sul- from crustaceans and their byproducts: a bio-
fated polysaccharides from marine green algae active metabolite candidate for therapeutic
Ulva conglobata and their anticoagulant activ- application. Mar Drugs 20(3):206. https://
ity. J Appl Phycol 18(1):9–14 doi.org/10.3390/md20030206
68. El-Baky H, Baz F, Baroty G (2009) Potential 79. Rahman SO, Panda BP, Parvez S, Kaundal M,
biological properties of sulphated polysac- Hussain S, Akhtar M, Najmi AK (2019) Neu-
charides extracted from the macroalgae Ulva roprotective role of astaxanthin in hippocam-
lactuca L. Acad J Cancer Res 2(1):1–11 pal insulin resistance induced by Aβ peptides
69. Marques J, Vilanova E, Mourão PA, Fernàn- in animal model of Alzheimer’s disease.
dez-Busquets X (2016) Marine organism sul- Biomed Pharmacother 110:47–58
fated polysaccharides exhibiting significant 80. Lu Y, Wang X, Feng J, Xie T, Si P, Wang W
antimalarial activity and inhibition of red (2019) Neuroprotective effect of astaxanthin
blood cell invasion by plasmodium. Sci Rep on newborn rats exposed to prenatal maternal
6(1):1–14 seizures. Brain Res Bull 148:63–69
70. Mousavian Z, Safavi M, Azizmohseni F, 81. Huang C, Wen C, Yang M, Gan D, Fan C,
Hadizadeh M, Mirdamadi S (2022) Charac- Li A, Li Q, Zhao J, Zhu L, Lu D (2019)
terization, antioxidant and anticoagulant Lycopene protects against T-BHP-induced
properties of exopolysaccharide from marine neuronal oxidative damage and apoptosis via
microalgae. AMB Express 12(1):1–16 activation of the PI3K/Akt pathway. Mol Biol
71. Cheng S, Tu M, Chen H, Xu Z, Wang Z, Rep 46(3):3387–3397
Liu H, Zhao G, Zhu B, Du M (2018) Identi- 82. Cao Z, Wang P, Gao X, Shao B, Zhao S, Li Y
fication and inhibitory activity against (2019) Lycopene attenuates aluminum-
α-thrombin of a novel anticoagulant peptide induced hippocampal lesions by inhibiting
derived from oyster (Crassostrea gigas) pro- oxidative stress-mediated inflammation and
tein. Food Funct 9(12):6391–6400 apoptosis in the rat. J Inorg Biochem 193:
72. Yegappan R, Selvaprithiviraj V, 143–151
Amirthalingam S, Jayakumar R (2018) Carra- 83. Man H, Bi W (2018) Protective effect of lyco-
geenan based hydrogels for drug delivery, tis- pene in a mouse model of Parkinson’s disease
sue engineering and wound healing. via reducing oxidative stress and apoptosis.
Carbohydr Polym 198:385–400
Anal Quant Cytopathol Histopathol 40:253– (2022) Microalgae polysaccharides: an alter-

258 native source for food production and sustain-
84. Hua Y, Xu N, Ma T, Liu Y, Xu H, Lu Y (2019) able agriculture. Polysaccharides 3(2):
Anti-inflammatory effect of lycopene on 441–457. https://doi.org/10.3390/
experimental spinal cord ischemia injury via polysaccharides3020027
cyclooxygenase-2 suppression. Neuroimmu- 95. Choi B-D, Choi YJ (2012) Nutraceutical
nomodulation 26(2):84–92 functionalities of polysaccharides from marine
85. Wen S-X (2019) Protective effects of lycopene invertebrates. In: Advances in food and nutri-
on cerebral vessels and neurons of hyperlipi- tion research, vol 65. Elsevier Inc., pp 11–30.
demic model rats. Chin J Pharmacol Toxicol https://doi.org/10.1016/B978-0-12-
33:93–101 416003-3.00002-0
86. Šimat V, Elabed N, Kulawik P, Ceylan Z, 96. Krichen F, Karoud W, Sila A, Abdelmalek BE,
Jamroz E, Yazgan H, Čagalj M, Regenstein Ghorbel R, Ellouz-Chaabouni S, Bougatef A
JM, Özogul F (2020) Recent advances in (2015) Extraction, characterization and anti-
marine-based nutraceuticals and their health microbial activity of sulfated polysaccharides
benefits. Mar Drugs 18(12):1–40. https:// from fish skins. Int J Biol Macromol 75:283–
doi.org/10.3390/md18120627 289. https://doi.org/10.1016/j.ijbiomac.
87. Nalini S, Sandy Richard D, Mohammed Riyaz 2015.01.044
SU, Kavitha G, Inbakandan D (2018) Anti- 97. Cermeño M, Kleekayai T, Amigo-Benavent-
bacterial macro molecules from marine organ- M, Harnedy-Rothwell P, FitzGerald RJ
isms. Int J Biol Macromol 115:696–710. (2020) Current knowledge on the extraction,
https://doi.org/10.1016/j.ijbiomac.2018. purification, identification, and validation of
04.110 bioactive peptides from seaweed. Electropho-
88. Sharanagat VS, Singla V, Singh L (2020) Bio- resis 41(20):1694–1717. https://doi.org/
active compounds from marine sources. In: 10.1002/elps.202000153
Goyal MR, Rasul Suleria HA, Kirubanandan 98. Kiran BR, Venkata Mohan S (2021) Microal-
S (eds) Technological processes for marine gal cell biofactory—therapeutic, nutraceutical
foods-from water to fork: bioactive com- and functional food applications. Plan Theory
pounds, industrial applications and genomics. 10(5):836. https://doi.org/10.3390/
Apple Academic Press, Inc., Oakville plants10050836
89. Ganesan AR, Tiwari U, Rajauria G (2019) 99. Ganesan AR, Mohanram MSG,
Seaweed nutraceuticals and their therapeutic Balasubramanian B, Ho Kim I, Seedevi P,
role in disease prevention. Food Sci Human Mohan K, Kanagasabai S, Valan Arasu M,
Wellness 8(3):252–263. https://doi.org/10. Abdullah Al-Dhabi N, Ignacimuthu S
1016/j.fshw.2019.08.001 (2020) Marine invertebrates’ proteins: a
90. Nicoletti M (2016) Microalgae nutraceuti- recent update on functional property. J King
cals. Foods 5(4):54. https://doi.org/10. Saud Univ – Sci 32(2):1496–1502. https://
3390/foods5030054 doi.org/10.1016/j.jksus.2019.12.003
91. Ashraf SA, Adnan M, Patel M, Siddiqui AJ, 100. Suleria H, Osborne S, Masci P, Gobe G
Sachidanandan M, Snoussi M, Hadi S (2020) (2015) Marine-based nutraceuticals: an inno-
Fish-based bioactives as potent nutraceuticals: vative trend in the food and supplement
exploring the therapeutic perspective of sus- industries. Mar Drugs 13(10):6336–6351.
tainable food from the sea. Mar Drugs 18(5): https://doi.org/10.3390/md13106336
2 6 5 . h t t p s : // d o i . o r g / 1 0 . 3 3 9 0 / 101. Shahidi F, Santhiravel S (2022) Novel marine
md18050265 bioactives: application in functional foods,
92. Šimat V (2021) Nutraceuticals and pharma- nutraceuticals, and pharmaceuticals. J Food
ceuticals from marine fish and invertebrates. Bioact 19. https://doi.org/10.31665/JFB.
Mar Drugs 19(7):401. https://doi.org/10. 2022.18316
3390/md19070401 102. Lange KW (2020) Omega-3 fatty acids and
93. Tanna B, Mishra A (2019) Nutraceutical mental health. Glob Health J 4(1):18–30.
potential of seaweed polysaccharides: struc- https://doi.org/10.1016/j.glohj.2020.
ture, bioactivity, safety, and toxicity. Compr 01.004
Rev Food Sci Food Saf 18(3):817–831. 103. Lange KW (2020) Lipids in the treatment of
https://doi.org/10.1111/1541-4337. neurodegenerative diseases. In: Bailey’s
12441 industrial oil and fat products. Wiley,
94. Moreira JB, Vaz B d S, Cardias BB, Cruz CG, New York, pp 1–17. https://doi.org/10.
Almeida ACA d, Costa JAV, Morais MG d 1002/047167849x.bio118
104. Bonilla-Méndez JR, Hoyos Concha JL, de 113. Pangestuti R, Kim SK (2011) Biological activ-
Extracción M (2018) Refinación y Concen- ities and health benefit effects of natural pig-
tración de Aceite de Pescado Como Fuente de ments derived from marine algae. J Funct
Ácidos Grasos Omega-3. Cienc Tecnol Agro- Foods 3(4):255–266. https://doi.org/10.
pecuaria 19(3). https://doi.org/10.21930/ 1016/j.jff.2011.07.001
rcta.vol19_num2_art:684 114. Galasso C, Gentile A, Orefice I, Ianora A,
105. Ozyurt G, Ozk AS, Durmus M (2017) Fatty Bruno A, Noonan DM, Sansone C, Albini A,
acid composition and oxidative stability of oils Brunet C (2019) Microalgal derivatives as
recovered from acid silage and bacterial fer- potential nutraceutical and food supplements
mentation of fish (sea bass – Dicentrarchus for human health: a focus on cancer preven-
labrax) by-products. Int J Food Sci Technol tion and interception. Nutrients 11(6):1226.
53(5):1255–1261. https://doi.org/10. https://doi.org/10.3390/nu11061226
1111/ijfs.13705 115. Rengasamy KR, Mahomoodally MF,
106. Özyurt G, Özkütük AS, Uçar Y, Durmuş M, Aumeeruddy MZ, Zengin G, Xiao J, Kim
Ozogul Y (2019) Evaluation of the potential DH (2020) Bioactive compounds in sea-
use of discard species for fish silage and assess- weeds: an overview of their biological proper-
ment of its oils for human consumption. Int J ties and safety. Food Chem Toxicol 135
Food Sci Technol 54(4):1081–1088. https:// (September):111013. https://doi.org/10.
doi.org/10.1111/ijfs.13954 1016/j.fct.2019.111013
107. Haq M, Ahmed R, Cho YJ, Chun BS (2017) 116. Maoka T (2011) Carotenoids in marine ani-
Quality properties and bio-potentiality of edi- mals. Mar Drugs 9(2):278–293. https://doi.
ble oils from Atlantic salmon by-products org/10.3390/md9020278
extracted by supercritial carbon dioxide and 117. Tan HT, Yusoff FM, Khaw YS, Ahmad SA,
conventional methods. Waste Biomass Valori- Shaharuddin NA (2021) Uncovering research
zation 8(6):1953–1967. https://doi.org/10. trends of phycobiliproteins using bibliometric
1007/s12649-016-9710-2 approach. Plan Theory 10(11):2358. https://
108. Soldo B, Šimat V, Vlahović J, Skroza D, doi.org/10.3390/plants10112358
Ljubenkov I, Generalić Mekinić I (2019) 118. Manivasagan P, Bharathiraja S, Santha
High quality oil extracted from sardine Moorthy M, Mondal S, Seo H, Dae Lee K,
by-products as an alternative to whole Oh J (2018) Marine natural pigments as
sardines: production and refining. Eur J potential sources for therapeutic applications.
Lipid Sci Technol 121(7):1–10. https://doi. Crit Rev Biotechnol 38(5):745–761. https://
org/10.1002/ejlt.201800513 doi.org/10.1080/07388551.2017.
109. Šimat V, Vlahović J, Soldo B, Generalić 1398713
Mekinić I, Čagalj M, Hamed I, Skroza D 119. Generalić Mekinić I, Skroza D, Šimat V,
(2020) Production and characterization of Hamed I, Čagalj M, Perković ZP (2019) Phe-
crude oils from seafood processing nolic content of brown algae (Pheophyceae)
by-products. Food Biosci 33:100484. species: extraction, identification, and quanti-
https://doi.org/10.1016/j.fbio.2019. fication. Biomol Ther 9(6). https://doi.org/
100484 10.3390/biom9060244
110. Šimat V, Soldo B, Skroza D, Ljubenkov I, 120. Cotas J, Leandro A, Monteiro P, Pacheco D,
Generalić Mekinić I (2019) Production and Figueirinha A, Gonca˛lves AMM, Da Silva GJ,
refinement of omega-3 rich oils from proces- Pereira L (2020) Seaweed phenolics: from
sing by-products of farmed fish species. Foods extraction to applications. Mar Drugs 18(8).
3:1–14 https://doi.org/10.3390/MD18080384
111. Zhou J, Wang M, Saraiva JA, Martins AP, 121. Shahidi F, Naczk M (2014) Antioxidant
Pinto CA, Prieto MA, Simal-Gandara J, properties of food phenolics. CRC Press,
Cao H, Xiao J, Barba FJ (2022) Extraction Boca Raton. https://doi.org/10.1201/
of lipids from microalgae using classical and 9780203508732.ch8
innovative approaches. Food Chem 384: 122. Čagalj M, Skroza D, Tabanelli G, Özogul F,
132236. https://doi.org/10.1016/j. Šimat V (2021) Maximizing the antioxidant
foodchem.2022.132236 capacity of Padina pavonica by choosing the
112. Frleta R, Popović M, Smital T, Šimat V right drying and extraction methods. Pro-
(2022) Comparison of growth and chemical cesses 9(4):587. https://doi.org/10.3390/
profile of diatom Skeletonema grevillei in bio- pr9040587
reactor and incubation-shaking cabinet in two 123. Čagalj M, Skroza D, Del Carmen M, Razola-
growth phases. Mar Drugs 20(11):697. Dı́az VV, Bassi D, Frleta R, Mekinić IG,
https://doi.org/10.3390/md20110697
Tabanelli G, Šimat V (2022) Variations in the 11(17):2654. https://doi.org/10.3390/

composition, antioxidant and antimicrobial foods11172654
activities of Cystoseira compressa during sea- 132. Muthukumar J, Chidambaram R, Sukumaran
sonal growth. Mar Drugs 20(1):64. https:// S (2021) Sulfated polysaccharides and its
doi.org/10.3390/md20010064 commercial applications in food industries—
124. Godswill AG, Somtochukwu IV, Ikechukwu a review. J Food Sci Technol 58(7):
AO, Kate EC (2020) Health benefits of 2453–2466. https://doi.org/10.1007/
micronutrients (vitamins and minerals) and s13197-020-04837-0
their associated deficiency diseases: a system- 133. Abdelmalek BE, Sila A, Ghlissi Z, Taktak MA,
atic review. Int J Food Sci 3(1):1–32. https:// Ayadi MA, Bougatef A (2016) The influence
doi.org/10.47604/ijf.1024 of natural astaxanthin on the formulation and
125. Lozano Muñoz I, Dı́az NF (2022) Minerals storage of marinated chicken steaks. J Food
in edible seaweed: health benefits and food Biochem 40(4):393–403. https://doi.org/
safety issues. Crit Rev Food Sci Nutr 62(6): 10.1111/jfbc.12224
1592–1607. https://doi.org/10.1080/ 134. Carballo DE, Caro I, Andrés S, Giráldez FJ,
10408398.2020.1844637 Mateo J (2018) Assessment of the antioxidant
126. Agregán R, Barba FJ, Gavahian M, Franco D, effect of astaxanthin in fresh, frozen and
Khaneghah AM, Carballo J, Ferreira ICFR, da cooked lamb patties. Food Res Int 111:342–
Silva Barretto AC, Lorenzo JM (2019) Fucus 350. https://doi.org/10.1016/j.foodres.
vesiculosus extracts as natural antioxidants for 2018.05.054
improvement of physicochemical properties 135. Al-Tarifi BY, Mahmood A, Assaw S, Sheikh
and shelf life of pork patties formulated with HI (2020) Application of astaxanthin and its
oleogels. J Sci Food Agric 99(10): lipid stability in bakery product. Curr Res
4561–4570. https://doi.org/10.1002/jsfa. Nutr Food Sci J 8(3):962–974. https://doi.
9694 org/10.12944/CRNFSJ.8.3.24
127. Agregán R, Franco D, Carballo J, 136. Taksima T, Limpawattana M, Klaypradit W
Tomasevic I, Barba FJ, Gómez B, (2015) Astaxanthin encapsulated in beads
Muchenje V, Lorenzo JM (2018) Shelf life using ultrasonic atomizer and application in
study of healthy pork liver pâté with added yogurt as evaluated by consumer sensory pro-
seaweed extracts from Ascophyllum nodo- file. LWT Food Sci Technol 62(1):431–437.
sum, Fucus vesiculosus and Bifurcaria bifur- https://doi.org/10.1016/j.lwt.2015.
cata. Food Res Int 112:400–411. https:// 01.011
doi.org/10.1016/j.foodres.2018.06.063 137. Pogorzelska E, Godziszewska J,
128. Barbosa RG, Trigo M, Campos CA, Aubourg Brodowska M, Wierzbicka A (2018) Antioxi-
SP (2019) Preservative effect of algae extracts dant potential of Haematococcus pluvialis
on lipid composition and rancidity develop- extract rich in astaxanthin on colour and oxi-
ment in brine-canned Atlantic Chub mackerel dative stability of raw ground pork meat dur-
(Scomber colias). Eur J Lipid Sci Technol ing refrigerated storage. Meat Sci 135:54–61.
121(8):1–8. https://doi.org/10.1002/ejlt. https://doi.org/10.1016/j.meatsci.2017.
201900129 09.002
129. Vala M, Augusto A, Horta A, Mendes S, Gil 138. Mok I-K, Lee JK, Kim JH, Pan C-H, Kim SM
MM (2017) Effect of tuna skin gelatin-based (2018) Fucoxanthin bioavailability from
coating enriched with seaweed extracts on the fucoxanthin-fortified milk: in vivo and
quality of tuna fillets during storage at 4 °C. in vitro study. Food Chem 258:79–86.
Int J Food Stud 6(2):201–221. https://doi. https://doi.org/10.1016/j.foodchem.2018.
org/10.7455/ijfs/6.2.2017.a7 03.047
130. Albertos I, Martin-Diana AB, Burón M, Rico 139. Nuñez de González MT, Attaie R, Mora-
D (2019) Development of functional Gutierrez A, Woldesenbet S, Jung Y (2021)
bio-based seaweed (Himanthalia elongata Stability of fucoxanthin in pasteurized skim
and Palmaria palmata) edible films for extend- and whole goat milk. Foods 10(7):1647.
ing the shelflife of fresh fish burgers. Food https://doi.org/10.3390/foods10071647
Packag Shelf Life 22(August):100382. 140. Nurcahyanti ADR, Puika VI, Pramitasari R,
https://doi.org/10.1016/j.fpsl.2019. Notario D (2021) Bioprospecting brown
100382 algae (Sargassum polycystum C. Agardh) as a
131. Lomartire S, Gonçalves AMM (2022) Novel potential antioxidant additive in snack bar and
technologies for seaweed polysaccharides its sensory evaluation. Food Res 5(6):58–65.
extraction and their use in food with thera- https://doi.org/1 0.2665 6/fr.20 17.5
peutically applications—a review. Foods (6).266
141. Abd El Baky HH, El Baroty GS, Ibrahem EA 149. Solomando JC, Antequera T, Perez-Palacios
(2015) Functional characters evaluation of T (2020) Evaluating the use of fish oil micro-
biscuits sublimated with pure phycocyanin capsules as omega-3 vehicle in cooked and
isolated from Spirulina and Spirulina biomass. dry-cured sausages as affected by their proces-
Nutr Hosp 32(1):231–241. https://doi.org/ sing, storage and cooking. Meat Sci 162:
10.3305/nh.2015.32.1.8804 108031. https://doi.org/10.1016/j.mea
142. Zhou C, Zhang L, Zaky AA, Tie S, Cui G, tsci.2019.108031
Liu R, Abd El-Aty AM, Tan M (2022) High 150. Pérez-Palacios T, Ruiz-Carrascal J, Jiménez-
internal phase Pickering emulsion by Spanish Martı́n E, Solomando JC, Antequera T
mackerel proteins-procyanidins: application (2018) Improving the lipid profile of ready-
for stabilizing astaxanthin and surimi. Food to-cook meat products by addition of omega-
Hydrocoll 133:107999. https://doi.org/10. 3 microcapsules: effect on oxidation and sen-
1016/j.foodhyd.2022.107999 sory analysis. J Sci Food Agric 98(14):
143. Bai J-J, Lee J-G, Lee S-Y, Kim S, Choi M-J, 5302–5312. https://doi.org/10.1002/jsfa.
Cho Y (2017) Changes in quality characteris- 9069
tics of pork patties containing antioxidative 151. Ghorbanzade T, Jafari SM, Akhavan S,
fish skin peptide or fish skin peptide-loaded Hadavi R (2017) Nano-encapsulation of fish
nanoliposomes during refrigerated storage. oil in nano-liposomes and its application in
Korean J Food Sci Anim Resour 37(5): fortification of yogurt. Food Chem 216:
752–763. https://doi.org/10.5851/kosfa. 146–152. https://doi.org/10.1016/j.
2017.37.5.752 foodchem.2016.08.022
144. Sinthusamran S, Benjakul S, 152. Gulzar S, Benjakul S, Hozzein WN (2020)
Kijroongrojana K, Prodpran T (2019) Chem- Impact of B-glucan on debittering, bioacces-
ical, physical, rheological and sensory proper- sibility and storage stability of skim milk for-
ties of biscuit fortified with protein tified with shrimp oil nanoliposomes. Int J
hydrolysate from cephalothorax of Pacific Food Sci Technol 55(5):2092–2103.
white shrimp. J Food Sci Technol 56(3): https://doi.org/10.1111/ijfs.14452
1145–1154. https://doi.org/10.1007/ 153. Takeungwongtrakul S, Benjakul S (2017)
s13197-019-03575-2 Biscuits fortified with micro-encapsulated
145. Fitzgerald C, Gallagher E, Doran L, Auty M, shrimp oil: characteristics and storage stabil-
Prieto J, Hayes M (2014) Increasing the ity. J Food Sci Technol 54(5):1126–1136.
health benefits of bread: assessment of the https://doi.org/10.1007/s13197-017-
physical and sensory qualities of bread formu- 2545-4
lated using a renin inhibitory Palmaria pal- 154. Jannat-Alipour H, Rezaei M, Shabanpour B,
mata protein hydrolysate. LWT Food Sci Tabarsa M, Rafipour F (2019) Addition of
Technol 56(2):398–405. https://doi.org/ seaweed powder and sulphated polysaccha-
10.1016/j.lwt.2013.11.031 ride on shelf_life extension of functional fish
146. Intarasirisawat R, Benjakul S, surimi restructured product. J Food Sci Tech-
Visessanguan W, Wu J (2014) Effects of skip- nol 56(8):3777–3789. https://doi.org/10.
jack roe protein hydrolysate on properties and 1007/s13197-019-03846-y
oxidative stability of fish emulsion sausage. 155. Muresan C, Pop A, Man S, Socaci S, Fărcas A,
LWT Food Sci Technol 58(1):280–286. Nagy M, Rus B (2016) The influence of dif-
https://doi.org/10.1016/j.lwt.2014. ferent proportions of Spirulina (Arthrospira
02.036 platensis) on the quality of pasta. J Agroali-
147. Ainsa A, Marquina PL, Roncalés P, Beltrán ment Process Technol 22(1):24–27
JA, Calanche MJB (2021) Enriched fresh 156. Mazinani S, Fadaei V, Khosravi-Darani K
pasta with a sea bass by-product, a (2016) Impact of Spirulina platensis on phys-
novel food: fatty acid stability and sensory icochemical properties and viability of Lacto-
properties throughout shelf life. Foods bacillus acidophilus of probiotic UF feta
10(2):255. https://doi.org/10.3390/ cheese. J Food Process Preserv 40(6):
foods10020255 1318–1324. https://doi.org/10.1111/jfpp.
148. Benkhoud H, Baâti T, Njim L, Selmi S, Hosni 12717
K (2021) Antioxidant, antidiabetic, and anti- 157. Różyło R, Hameed Hassoon W, Gawlik-
hyperlipidemic activities of wheat flour-based Dziki U, Siastała M, Dziki D (2017) Study
chips incorporated with omega-3-rich fish oil on the physical and antioxidant properties of
and artichoke powder. J Food Biochem gluten-free bread with brown algae. CyTA – J
45(3). https://doi.org/10.1111/jfbc.13297 Food 15(2):196–203. https://doi.org/10.
1080/19476337.2016.1236839
158. Arufe S, Della Valle G, Chiron H, Chenlo F, https://doi.org/10.1016/j.foodchem.2018.

Sineiro J, Moreira R (2018) Effect of brown 12.026
seaweed powder on physical and textural 160. Lucas BF, de Morais MG, Santos TD, Costa
properties of wheat bread. Eur Food Res JAV (2018) Spirulina for snack enrichment:
Technol 244(1):1–10. https://doi.org/10. nutritional, physical and sensory evaluations.
1007/s00217-017-2929-8 LWT 90:270–276. https://doi.org/10.
159. Gheysen L, Lagae N, Devaere J, Goiris K, 1016/j.lwt.2017.12.032
Goos P, Bernaerts T, Van Loey A, De 161. Hanjabam MD, Zynudheen AA, Ninan G,
Cooman L, Foubert I (2019) Impact of Nan- Panda S (2017) Seaweed as an ingredient for
nochloropsis Sp. dosage form on the oxida- nutritional improvement of fish jerky. J Food
tive stability of n-3 LC-PUFA enriched Process Preserv 41(2):e12845. https://doi.
tomato purees. Food Chem 279:389–400. org/10.1111/jfpp.12845
Chapter 4
Microwave-Assisted Extraction of Bioactive

and Nutraceuticals
Moufida Chaari, Sarra Akermi, Khaoula Elhadef, Hussein A. H. Said-Al Ahl,
Wafaa M. Hikal, Lotfi Mellouli, and Slim Smaoui
Abstract
The increasing consumer awareness about the link between nutrition and health has led the food industry to
produce fortified food with bioactive compounds. Considering that not all bioactive compounds are freely
available and in the light of increasing attention to preserve environmental resources, the new trend
consisted of waste recovery of industrial food processing residues with active potential. Currently, clean
label and eco-friendly extraction methods have realized reputation accounts for the removal of solvent
usage and reduction in energy consumption. In this context, microwave-assisted extraction (MAE) evolved
as a novel procedure for the extraction of bioactives and nutraceuticals. With higher extraction efficiency,
this process was noted to consume less time and energy, and interestingly, the bioactive compound’s
functionality has not degraded. In this chapter, MAE’s potential as an eco-friendly technique was explored.
To improve its efficiency, microwave-assisted extraction has been coupled with conventional techniques.
Accessible data stress the significance of various hybrid techniques: microwave/conventional ones for the
extraction of bioactive compounds. Information about this topic could help students and scientific
researchers who are engaged in chemical engineering, chemistry, and meat technology communities to
approach the complex theme of microwave-assisted extraction.
Key words Microwave-assisted extraction, Bioactive compounds, Nutraceuticals, Eco-friendly,

Hybrid technique extraction
1 Introduction
Nowadays, extraction and production of various bioactive com-

pounds have gained momentum as there is increased demand for
herbal products globally. This is because they are safe, possess
various biological activities as compared to synthetic formulations,
and are cost-effective. To provide higher recoveries and greater
reproducibility, the chief tendencies in analyte extraction was to
reduce solvent and energy consumption and to provide higher
79
80 Moufida Chaari et al.
recoveries and greater reproducibility. The implementation of tra-

ditional extraction methods may be more time-consuming, requir-
ing large volumes of solvents, and are principally related with the
degradation of heat-sensitive compounds [1]. In this sense, to
overcome the drawbacks of these extraction methods, it is decisive
to explore contemporary techniques. Green solvent extraction
methods have been developed, including microwave-assisted
extraction (MAE), which has gained a wide attention due to its
various advantages, namely, a reduced solvent consumption, a
shorter operation time, and an enhanced recovery yield [2]. Numer-
ous comparative studies have shown that MAE allowed better
performance in terms of compound recoveries [3–7]. By applying
MAE, plentiful kinds of compounds, comprising essential oils,
antioxidants, pigments, and other organic compounds have been
successfully isolated from various natural plant resources [5, 8–
10]. These previous studies displayed that MAE could be a
promising alternative to conventional extraction of plant pigments
(carotenoids and anthocyanins) [9, 11], polyphenols, polysacchar-
ides [12–14], essential oils [5, 15, 16], and proteins and lipids
[17]. Compared to maceration and Soxhlet extraction, it was estab-
lished that MAE approach was more effective [9]. It was found that
the obtained extracts using MAE had a greater concentration of
volatile terpenoids (α- and β-pinene) [18]. Microwaves have electric
and magnetic fields since they are electromagnetic devices
[19]. MAE employs microwave radiation to heat solvents and
facilitates the transfer of target compounds from the sample matrix
to the extractant by inducing polar molecules, ions, and dipoles
movement and rotation [20]. Microwaves can penetrate the sample
and incite cell molecules to absorb their energy, resulting in an
increase in temperature and pressure. Then, it facilitates the cell’s
rupture and the reachability of the components into the solvent
solution [21]. In fact, there are several categories of MAE such as
solvent-free MAE (SFM), focused-MAE (FMAE), ionic liquid-
based MAE (ILMAE), ultrasonic MAE (UMAE), microwave
hydro-distillation (MHD), microwave hydro-diffusion and gravity
(MHG), and microwave-assisted subcritical extraction (MASE)
[12, 22–27]. The extraction techniques employed for this purpose
are highly dependent on the following factors: microwave power,
time, solvent, sample-to-solvent ratio, temperature, and matrix
characteristics [28]. The aim of this chapter is to spotlight the
versatility of MAE in the recovery of bioactive compounds and
nutraceuticals from various types of vegetal materials using differ-
ent techniques of MAE with a special focus on the factors that could
influence its processing and efficiency.
Microwave-Assisted Extraction of Bioactive and Nutraceuticals 81
2 Mechanism of MAE Process
Microwaves have electric and magnetic fields since they are electro-
magnetic devices. These fields lead to a heating effect via two
mechanisms, dipolar rotation and ionic conduction [19].
(i) Dipolar rotation refers to the phenomenon that occurs when
molecules with uneven distribution of charge, known as a dipole
moment, attempt to align themselves with the alternating electric
field produced by microwaves. The oscillation of these dipolar
molecules results in collisions with other molecules in the sur-
rounding medium, which then generates heat. This process hap-
pens quickly and repeatedly, making it an efficient way to convert
electromagnetic energy to thermal energy [29].
On the other hand, (ii) ionic conduction is defined as a process
that occurs when charged particles, such as ions and electrons,
move through a medium in response to an electric field produced
by microwaves. This movement or migration generates friction
between the ions and the medium, which results in the generation
of heat. The degree of heat generated by this process depends on
factors such as the strength of the electric field and the conductivity
of the medium [20].
The relative contribution of these two mechanisms to the
overall heating of the sample is largely dictated by temperature.
Specifically, as the contribution of dipole rotation decreases, the
temperature of the sample increases, while the contribution of ionic
conduction increases. It means that if a sample contains both polar
molecules and ions, then as it is heated by microwave energy, the
heating will initially be dominated by dipole rotation. The relative
contribution of these two mechanisms also depends on the mobility
and concentration of the ions within the sample [20]. Conse-
quently, these mechanisms induced the destruction of hydrogen
bonds in organic molecules, which increased solvent penetration
into the plant matrix [30] and thereby dissolution of extractable
molecules. In fact, microwave-assisted extraction (MAE) may be
summarized in two main steps as Chemat et al. [21] and Vinatoru
et al. [20] mentioned:
1. Penetration of the solvent into the plant cell by diffusion:
Initially, in the equilibrium phase, solubilization and partition-
ing phenomena come into play, which leads to the detachment
of the substrate from the particle’s outer surface at a relatively
consistent rate. This step is then followed by an intermediate
phase of transition to diffusion, where resistance to mass trans-
fer begins to appear at the interface between the solid and
liquid phases. During this period, a mass transfer occurs
through convection and diffusion.
2. Cell rupture and leaching out of cell components into the

solvent solution: When the dielectric loss tangent of the plant
cell is higher than that of the solvent, the vegetal material could
absorb more electrical energy, which can lead to an increase in
the temperature of the plant material and subsequently an
increase in cell pressure. As the extract is removed mainly
through diffusion, it is typically regarded as the limiting step
of the process.
Throughout the extraction process, a variety of forces and
relationships can be observed, including dispersion forces, intersti-
tial diffusion, driving forces, and chemical interactions, with the
persistence and strength of these phenomena often closely linked to
the solvent’s properties, such as solubilization power, solubility in
water, purity, and polarity [21].
3 Factors Affecting MAE
Numerous types of compounds, including essential oils, antioxi-

dants, pigments, and other organic compounds, have been effec-
tively isolated from various natural plant resources using MAE
[5, 8–10]. The extraction techniques employed for this purpose
are highly dependent on the following factors (Fig. 1).
3.1 Microwave In a reaction medium, the amount of electromagnetic energy that is

Power transformed into heat depends, in a practical sense, on the permit-
tivity and permeability of the chemical compounds or mixture, as
well as the intensity of the electromagnetic field [21].
Microwaves belong to the electromagnetic spectrum, and their
frequency range spans from 300 MHz (classified as radio radiation)
up to 300 GHz. In scientific research, two specific frequencies are
usually utilized: 2.45 GHz, which is commonly used in laboratory
equipment, and 915 MHz, which is mostly used in industrial
equipment [20, 31]. It has been shown that the range of power
delivered was between 60 and 960 W (Table 1). Increasing
Fig. 1 Factors affecting MAE

Table 1
MAE extraction of different analytes from various vegetal materials
Materials Solvent Ratio Temperature Time Power Analyte References

Mango peel Diluted acidic solution 2 g/600 mL – 3 min 700 W Pectin: 1485.78 mg/mol [58]
(distilled H2O, with 2 M
HCl)
Sapindus mukorossi 40% Ethanol 1 g/19 mL – 13 min 425 W Saponins yield: 280.55 mg/g [59]
Red cabbage 50% Ethanol 1 g/20 mL – 10 min 600 W Total monomeric anthocyanin [10]
content: 220 mg cyanidin-3-
glycoside/L
Melissa officinalis L 25.9% Ethanol 300 mg/ – 29 min 400 W Rosmarinic acid: 49.5 mg RA/g [60]
10 mL of DW
Melastoma 31.33% Ethanol 0.5 g/30 mL 52.24 °C 45 min 500 W TPC: 39.02 mg GAE/g of DW [55]
sanguineum
fruit
Chaya (Cnidoscolus 99.8% Ethanol 1 g/20 mL 140 °C 10 min 850 W TPC: 57 mg GAE/g [61]
aconitifolius
Mill.) leaves
Tomato pericarps 100% Ethanol 45 g/L 180 °C 20 min 200 W TPC: 66.8 mg GAE1/g [62]
TFC: 3.89 mg CE/g
Hibiscus sabdariffa 60% Ethanol 3 g/30 mL 164 °C 22 min 850 W Flavonoids yield: 55% [63]
Banana peel Water 2 g/ – 6 min 960 W TPC: 50.55 mg GAE/g DM [64]
100 mL
Apple dust 40% Ethanol – 15 min 600 W TPC: 36.99 mg GAE/g DW [65]
by-product
Microwave-Assisted Extraction of Bioactive and Nutraceuticals
Grape juice waste Water 1 g/ – 2.23 min 428 W Total monomeric anthocyanin [66]
18.43 mL yield: 1.32 mg/g
Onion peels Choline chloride: urea: 1 g/ – 15.03 min 100 W TPC: 80.45 mg GAE/g DW [67]
83
water (1:2:4) 54.97 mL
(continued)
84
Table 1
(continued)
Materials Solvent Ratio Temperature Time Power Analyte References

Pomelo peels 10 mmol/L [HO3S 1 g/26 mL – 15 min 331 W Yield of pectin: 291.60 mg/g [68]
(CH2)4 mim] HSO4 Yield of naringin: 8.38 mg/g
aqueous solution
Spruce (Picea 50% Ethanol – – 4 min 300 W Catechin: 1.91 mg/g of VM [18]
abies) bark Epicatechin: 6.72 mg/g of VM
Moufida Chaari et al.
α-pinene: 2198.33 μg/g of VM

β-pinene: 2997.66 μg/g of VM
Camphene: 71.6 μg/g of VM
Myrcene: 105.46 μg/g of VM
Limonene: 101.07 μg/g of VM
Quercus cerris bark Water 10 g/ – 30 min 850 W TPC: 382.26 mg GAE/g of DW [4]
extracts 200 mL Total tannin content: 49.14%
Quercus cerris bark 70% Ethanol 10 g/ – 18 min 650 W TPC: 403.73 mg GAE/g of DW [4]
extracts 200 mL Total tannin content: 45.68%
Avocado 58% Ethanol 1 g/20 mL – 5 min 400 W TPC: 82.36 mg GAE/g [69]
(Persea americana TFC: 19.93 mg QE/g
Mill.) seeds
Olive tree leaves Solvent-free 5g – 2 min 250 W TPC: 2.480 ppm [70]
Oleuropein yield: 0.060 ppm
Coleus aromaticus Solvent-free 500 g – 30 min 500 W Essential oil yield: 0.54% [16]
leaves
Lagenaria Solvent-free 20 g – 60 s 480 W TPC: 288.9 mg GAE/g DW [22]
siceraria fruit TFC: 214.1 mg rutin
equivalent/g DW
Pegagan (Centella Solvent-free 20 g – 60 min 450 W Yield: 4.5474% [1]
Asiatica L.)
leaves
Cinnamomum Solvent-free 200 g – 23 min 580 W Essential oil yield: 3.51% [5]
camphora leaves
Black carrot 19.8% Ethanol 1 g/ 9.3 mL – 9.8 min 348.07 W Total anthocyanin content: [47]
pomace 753.4 mg/L
TPC: 264.9 mg GAE/100 mL
Black currant 60% Ethanol 1 g/28.3 mL – 3 min 551 W Flavonols: 2323.3 μg/g [71]
Anthocyanins: 473.7 μg/g
Patchouli Water 0.15 g/mL – 51.61 min 634.024 W Patchouli oil yield: 2.8% [72]
(Pogostemon
cablin)
Ananas comosus H2SO4 (0.5 N, pH 1.83) 1 g/10 mL 80 °C 2.5 min 600 W Yield of pectin: 2.43% [73]
peel Anhydrouronic acid content:
54.61%
Brown seaweeds 0.1 M HCl containing 2 M 1 g/25 mL – 1 min 560 W Sugar content: 0.47 mg glucose [74]
CaCl2 equivalent/mg extract
Eucalyptus globulus Water 1 g/48.5 mL 141 °C 15 s 500 W TPC: 350 mg of GAE/g of [75]
bark extract
Curcuma longa Choline chloride-citric 1 g/20ML – 6 min 60 W Curcuminoids yield: 89.87 mg/ [53]
acid (1:1) g
Pineapple peel 50% 1 g/20 mL – 40 min 600 W TPC: 14.188 mg GAE/g DW [76]
waste Ethanol TFC: 12.925 mg QE/g DW
Total Tannin Content:
371.25 mg TAE/g DW
Protein content: 59.49 mg
BSAE/g DW
Db dry basis, DM dry matter, DW dry weight, VM vegetal material, TPC total phenolic content, TFC total flavonoids content
Microwave-Assisted Extraction of Bioactive and Nutraceuticals
85
microwave power from 180 to 300 W gave rise to high trans-

lycopene and β-carotene contents [32]. Additionally, Vu et al.
[33] reported that high phenolic compounds were acquired when
the power was raised from 240 to 960 W. These radiations led to
the disruption of cell wall and then cell membrane followed by the
release of bioactive compounds [2]. Hence, it permitted the grad-
ual efflux of plant exudates. Consequently, it affected the yield of
bioactive compounds [33]. However, increasing microwave power
beyond 300 W decreased the contents of trans-lycopene and
β-carotene [32]. In fact, there is a risk of losing/degrading plant
bioactive components caused by the usage of higher power with
extended exposure [34]. Thus, microwave power and irradiation
time are completely opposed [2].
Nisca et al. [4] found that the TPC of Quercus cerris bark
extracts was improved when the microwave power was increased.
The variation of microwave power from 200 to 850 W had a
significant influence on the content of the total phenolics and
tannins.
Microwaves could be influenced by different types of materials,
which can be categorized as follows [35]:
Opaque materials: Conductive materials that possess free electrons,
like metals, have a tendency to reflect electromagnetic waves,
preventing them from passing through. These materials are
utilized in constructing microwave applicants [36].
Transparent materials: Materials that have a low dielectric loss or
insulating properties, such as ceramics and glass, only absorb
and reflect electromagnetic waves to a minimal extent, thus
enabling microwaves to pass through with minimal attenuation
[37]. These materials are typically used in reactors that are
placed within microwave applicants.
3.2 Extraction Time Heating time is an essential factor that affects the extraction mech-
anism [19]. Furthermore, increasing time augmented extraction
efficiency and quantity of analytes [2]. However, it also increases
the possibility of the degradation of thermolabile compounds. For
the extraction of different kinds of plant matrices, various time
scales are required [38]. Sometimes, 60 s-60 min are required
for the maximum production of analytes in MAE (Table 1). Several
findings agreed that extended exposure to microwave irradiation
resulted in a greater release of phenolic compounds from Hibiscus
sabdariffa, Aegle marmelos, and Myrtus communis leaves [39–
41]. The amount of polyphenols extracted increased by over 30%
when the extraction time was changed from 5 to 29.5 min
[42]. Whereas, Belwal et al. [8] reported that 2 min was suitable
for MAE of alkaloids, berberine and palmatine with concentrations
of 46.38 mg/g DW and 20.54 mg/g DW, respectively. In the
investigation of Kadi et al. [9], it was noticed that the total
carotenoid content from Citrus clementine peel reached its maxi-

mum (186.55 μg/g DM) at 7.64 min; then the compounds of
interest easily decomposed as a result of the long exposure. Accord-
ing to Samanta et al. [3], MAE technique has exhibited an increase
in yield of 70% compared to other traditional methods, resulting in
higher TFC and TPC levels, which was accomplished in a shorter
time frame.
3.3 Extraction The proper selection of extraction solvent is one of the key elements
Solvent and Sample- that have a significant impact on MAE’s total output. The proper-
to-Solvent Ratio ties of the solvent (nature, polarity, solubilization, purity, etc.) are
other variables that affect the process of extraction [2]. Several
forces, such as the physicochemical interactions, may be strongly
associated with the properties of the solvent [21]. Typically, when a
solvent has a high dielectric constant and dielectric loss, it tends to
have a greater ability to absorb microwave energy. Essentially, the
capacity of the solvent to absorb this energy increases as the dielec-
tric constant and dielectric loss increase, resulting in a faster heating
rate for the solvent relative to the plant material [43]. In order to
measure relative solubility, the Hildebrand solubility parameter
scale is commonly used. This parameter is the measure of the
cohesive energy between the solvent and the matrix in a solution
[20]. In fact, δ is linked to the hydrogen-bonding capacity, the
polarity, and the dispersion coefficient. As a result, there is a signifi-
cant correlation between the polarity and the Hildebrand solubility
parameter (δ) [20]. The solvent volume is also a crucial component
to take into account since it needs to be sufficient to ensure that the
entire sample is submerged in the solvent during the whole irradia-
tion process [21]. In addition, the selected solvent must be more
selective toward the target analyte than the other matrix constitu-
ents [44]. MAE can use water as a solvent for both polar and
nonpolar compounds, making it an attractive option for more
environmentally friendly extraction processes [43]. By blending
different solvents, it is possible to alter the properties of the solvent,
resulting in differing selectivity for various compounds [43]. In
fact, the use of an ethanol-water mixture as an extraction solvent
facilitated the recovery of TPC due to its high dielectric constant
and dissipation factor, which enables the effective absorption of
microwave energy. Furthermore, this solvent mixture increased
the penetration of the solvent into the sample matrix, thereby
enhancing heating efficiency. These results are in line with the
findings of Nisca et al. [4], who carried out an optimization of
extraction parameters for aqueous and hydroalcoholic extractions,
and the total polyphenolic and tannin contents were determined.
The results indicated that the optimal extraction conditions for
aqueous (30 min at 850 W) and hydroalcoholic (18 min at
650 W) extracts were different. The hydroalcoholic bark extract
exhibited a higher yield of total polyphenols (403.73 mg GAE/g
dried weight) compared to the aqueous extract that had a lower

level of tannins. Hence, MAE may yield higher levels of polyphe-
nols when mixtures of solvents are used due to the increased
solubility of target compounds and better penetration into the
plant material [4].
In addition, optimizing the solvent-to-solid ratio (S/S) is a
crucial parameter. It is necessary to ensure that the solvent volume
is adequate to fully immerse the sample during the entire irradiation
process, particularly when dealing with a matrix that may expand
during the extraction process [45]. As the ratio of sample to solvent
increased from 2:100 g/mL to 8:100 g/mL, the TPC decreased by
nearly 50% [33]. The reason coming behind these results is that
when a smaller sample ratio is utilized, the plant material swells,
which leads to an increase in the contact area between the plant
matrix and the solvent [46]. While Kumar et al. [47] stated that the
retrieval of phenolic content showed a notable upsurge when the
ratio of solvent to solid (S/S) increased, achieving the maximum at
20:1 and declining afterward at higher levels. Hence, 20:1 was
considered the best ratio for subsequent process parameters. The
range of 10–30 (v/w) was then used to refine the process para-
meters through response surface methodology (RSM) optimiza-
tion. Choosing the appropriate ratios can have significant difficulty
in MAE. This decision is typically influenced by various factors,
including the solvent’s selectivity toward the target analyte,
its ability to absorb microwaves, its interaction with the sample
matrix, and its compatibility with the analytical methods used
downstream [48].
3.4 Matrix MAE depends on the type of plant utilized as a raw material, which
Characteristics can produce a variety of valuable compounds as well as the compo-
sition of the chosen plant tissue/cell or part of the plant that
incorporates different kinds of components. Moreover, bioactive
and nutraceutical compounds are typically bound to other com-
pounds within plant structures, such as polyphenols, which are
uncommonly found in their unbound form. Instead, they are
often covalently linked to the plant cell wall, may exist in waxes or
on the exterior surfaces of plant organs, and are linked via glyco-
sides [49]. For example, plants’ leaves contain high content of
phenols [50]. Moreover, Rahmawatii et al. [1] reported that the
yield of extraction from Pegagan (Centella Asiatica L.) leaves is
significantly impacted by the quantity of material present. Also, the
particle size of the plant matrix is an important factor [51]. Several
studies reported that the extraction yield improved when matrix
particle size decreased [51–53]. According to Poureini et al. [52],
the apigenin extraction yield was enhanced by decreasing the parti-
cle size from 0.75 to 0.10 mm. A similar trend was depicted by Patil
et al. [53]. These authors detected an optimal range of particle size
between 0.150 and 0.212 μm for curcuminoids extraction. Thus,

fine matrix particles promote the deeper penetration of the micro-
wave [38]. This improvement can be explained by the raise of the
contact area between the solvent and the plant matrix. In fact,
reducing the size of the particles decreased the distance that the
solvent needs to diffuse, which in turn accelerated the rate of mass
transfer between the solute and the solvent [54].
The level of moisture in the sample matrix affects the extraction
efficiency. The presence of water can increase the microwave-
absorbing ability of the sample and facilitate heating by making
the extractant more polar [40]. By utilizing, the RSM combined
with a Box–Behnken design to extract essential oil from Cinnamo-
mum camphora leaves by MAE, Liu et al. [5] showed that the
optimal moisture content was found to be 60%.
3.5 Temperature Increasing temperature until a certain level increases the extraction
yield of some bioactive compounds. In fact, Zhao et al. [55] men-
tioned that the impact of extraction temperatures was examined
while holding other variables constant (30% ethanol, 30 mL/g,
30 min, 500 W). As the temperature increased (20–50 °C), there
was a significant increase in TPC value from 23.88 to 34.46 mg
GAE/g DW. Elevated temperatures have the potential to accelerate
intermolecular interactions and molecular movement, which may
lead to increased solubility of solutes in the solvent [43]. Therefore,
the TPC value depicted a marked improvement as the extraction
temperature increased from 40 to 50 °C, but subsequently
decreased as the temperature continued to rise. Kapoore et al.
[48] noticed that an increase in temperature resulted in decreased
yields of phycoerythrin, which confirmed that thermal damage can
occur over 40 °C, while carotenoids degrade at temperatures over
60 °C. The extraction of phenolic acids from green tea was found to
be more effective at a temperature of 100 °C, whereas the flavanols
and flavonols, which are sensitive to high temperatures, displayed
better extraction yield at a lower temperature of 80 °C [56]. In
addition, according to these authors, the extraction of quercetin
glycosides is more efficient at 80 °C compared to 100 °C. This
finding could be explained by the fact that quercetin glycosides
have an oxidizable catechol ring (B-ring), making them more sus-
ceptible to thermal degradation than kaempferol glycosides, which
have a mono-phenolic B-ring. Moreover, when extracted using
MAE at a temperature of 90 °C, the sulfated polysaccharides
obtained from Ulva prolifera using an acidic solvent (0.05 M
HCl) exhibited superior water- and oil-holding capacities. Con-
versely, the polysaccharides extracted at a higher temperature of
150 °C demonstrated the best foaming properties as well as the
highest antioxidant and pancreatic lipase inhibition activities [57].
4 Some Techniques of MAE
Coupling MAE with other extraction methods was proved to have

potential applications due to the popular effectiveness of MAE
(Fig. 2).
4.1 Solvent-Free This method involves using microwaves to perform a dry distilla-
MAE (SFM) tion on a fresh matrix, without adding any water or organic solvent.
The process involves heating of the raw material with water to
release the essential oil from glands, which is then carried away by
steam produced from the matrix water. The distillate, made up of
water and essential oil, is continuously condensed using a cooling
system placed outside the microwave oven. Any excess water is
returned inside the balloon to maintain the appropriate humidity
level of the matrix [21]. This straightforward approach allows the
efficient extraction of essential oils without the use of additional
solvents. The findings of Iftikhar et al. [22] revealed that the SFM
technique, which does not require solvents and utilizes a power
setting of 480 W and a duration of 60 s, is an efficient approach for
extracting antioxidant compounds from gourd fruit. Likewise, Wei
et al. [15] mentioned that the combination of SFM and moisture
regulation was a potent approach to extract essential oil from
deciduous leaves of C. longepaniculatum. In addition, compared
to conventional hydro-distillation, Liu et al. [5] depicted that SFM
exhibited better performance in terms of various parameters such
as extraction efficiency (3.51% in 23 min vs. 3.35% in 240 min),
initial extraction rate (3.3772 vs. 0.1868), extraction rate constant
(0.3002 vs. 0.0152), extraction capacity (3.67% vs. 3.51%),
oxygenated compound content (83.93% vs. 74.81%), energy
Fig. 2 Recent methods of MAE

consumption (0.22 kW h vs. 4 kW h), and environmental impact

(177.87 g CO2 vs. 3200 g CO2). These findings demonstrated that
SFM is a time-efficient, energy-saving, and eco-friendly method
that has great potential as a preferable alternative to traditional
methods for the extraction of essential oil from C. camphora leaves.
Hence, SFM was suggested as it showed higher yield and volumet-
ric mass transfer coefficient, greater proportions of oxygen com-
pounds, lower electricity consumption, and less CO2 emission and
water waste compared to conventional hydro-distillation [77].
4.2 Focused-MAE In FMAE, the sample is placed in an opened vessel and a specific
(FMAE) area is exposed to microwave radiation. This system functions at
atmospheric pressure [78], while the maximum temperature is
provided by the boiling point of the extraction solvent utilized
[79]. Hence, it can be used for the extraction of thermolabile
components. In addition, this system is composed of a condenser
that is set on the top of the vessel to avoid the loss of volatile
compounds [80]. Therefore, the microwave reactor’s configuration
influences heat production in the reaction medium [21]. In fact,
using a central composite experimental design, the extraction of
betulinic acid from Zizyphus joazeiro was optimized by employing
FMAE technology. This analysis confirms the applicability of
FMAE extraction as a speedy, environmentally friendly, and effec-
tive extraction method. As per the study, the optimal temperature
and duration of extraction are 70 °C and 15 min, respectively
[81]. By directing microwave energy to a small region of the sample
[82], FMAE attained a more efficient extraction with less energy
consumption [81].
4.3 Ionic Liquid- The merging of microwave irradiation with ionic liquids (ILs)
Based MAE (ILMAE) presents an influential approach toward achieving high effectiveness
and less harmful procedures. ILs are liquefied salts that retain their
liquid form at low temperatures, frequently under 100 °C, and they
are comprised of organic cations and organic or inorganic anions
[83]. In comparison to conventional organic solvents, ILs exhibit
numerous distinguishing characteristics, such as trivial vapor pres-
sure, elevated temperature stability, low volatility, chemical stability,
wide electrochemical stability window, and ionic conductivity
[84]. Thus, ILs are considered as outstanding microwave absor-
bers. As stated by Li et al. [85], ILMAE can enhance the extraction
efficiency of total biflavonoids in a shorter time and with a reduced
amount of solvents, compared to conventional soxhlet extraction.
In fact, according to Motlagh et al. [6], when compared to the
commonly used conventional Soxhlet method, the protein yield
obtained under optimized conditions using choline acetate ([Ch]
[Ac])-mediated water-based MAE technique (26.35%) is much
higher, indicating the superiority of this approach over the Soxhlet
extraction method (0.63%). The results indicated that [Ch]
[Ac]-based MAE of proteins from Nannochloropsis oceanica is

superior to the conventional method of Soxhlet methods, making
it a highly recommended innovative approach for protein separa-
tion. The findings of this investigation had the potential to aid in
identifying and utilizing important biochemical compounds from
microalgae through IL-based MAE, leading to the development of
new and enhanced bioproduct technologies. Furthermore, the
major obstacle in astaxanthin extraction is the effective disruption
of the thick and resistant cell walls of Haematococcus pluvialis.
However, the utilization of biocompatible protic ionic liquids-
based microwave-assisted liquid-solid extraction (PILs-MALSE)
has resolved this issue in the study of Fan et al. [7]. One of the
protic ionic liquids, ethanolammonium caproate (EAC), has the
ability to dissolve mannan, which is one of the key components of
the cell walls of Haematococcus pluvialis. Fan et al. [7] elucidated
that compared to traditional extraction techniques, the PILs-
MALSE method is more efficient for extracting astaxanthin. In
addition, the effectiveness of MAE combined with protic ionic
liquids (PILs) in obtaining phycobiliproteins was assessed by
Rodrigues et al. [24]. The most efficient solvent was a combination
of 2-hydroxyethylammonium acetate (2-HEAA) and
2-hydroxyethylammonium formate (2-HEAF), using a process
conducted at 62 W power and a ratio of 10 mL/g. These authors
found that MAE using PILs could be effective for the extraction of
phycobiliproteins with concentrations of 33 g L-1, 0.84 g L-1, and
0.41 g L-1 of allophycocyanin, phycocyanin, and phycoerythrin,
respectively. Guo et al. [86] successfully utilized ILMAE to extract
6-, 8-, and 10-gingerols and 6-, 8-, and 10-shogaols from
ginger. The highest extraction yields of gingerols and shogaols
were obtained using 1-decyl-3-methylimidazolium bromide
[C10MIM]Br. The efficiency of extraction of these compounds
was greatly influenced by the alkyl chain length and anions of
cations. Compared to methanol-based MAE (MMAE), ILMAE
not only produced higher extraction yields but also had a shorter
extraction time. The same tendency was observed when 1-octyl-3-
methylimidazolium acetate [Omim][OAc] was added to the water
as the extracting solvent. This IL has been demonstrated to
enhance lipid extraction ability when exposed to microwave irradi-
ation. [Omim][OAc] at 2.5% allowed the extraction of 19.2% of
lipids [87]. Despite many accomplishments, there is still insufficient
knowledge about the precise mechanism linking microwaves, ILs,
and nanostructures or polymers [83].
4.4 Ultrasonic MAE The concurrent utilization of ultrasonic and microwave extraction
(UMAE) methods led to a notably greater quantity of bioactive compounds
in comparison to the traditional decoction extraction techniques,
highlighting the synergistic effects of these novel approaches
(Kwansang et al. 2022). As reported by Sun et al. [12], the
UMAE approach, developed for the extraction of polysaccharides

from Camptotheca acuminata fruit (CAFP), yielded higher
amounts in a shorter duration than traditional hot water extraction
(HWE) techniques. The CAFP yield obtained via UMAE was
6.81%, which is 1.04 times greater than the yield from HWE.
Furthermore, the UMAE approach required a shorter extraction
time of 20 min compared to HWE, which necessitated 120 min of
extraction. In this line, Zheng et al. [88] indicated that the extrac-
tion yield of polysaccharides from Trametes orientalis was 7.52%. In
addition, the investigation of Zhang et al. [13] revealed that the
research results showed that UMAE had a greater degree of damage
to the cell wall of Dictyophora indusiata polysaccharides (DPs) and
better antioxidant capacity. The UMAE method had the highest
polysaccharides yield, which was related to the conformational
stretching and degradation avoidance of DPs in the higher molec-
ular weight components under the simultaneous action of micro-
wave and ultrasonic. In the same line, according to Shen et al. [89],
Panax notoginseng polysaccharides (PNPS) were extracted using
UMAE, and RSM was utilized to optimize the extraction para-
meters. The ideal extraction conditions were identified as 10 min
ultrasonic duration, 50 W ultrasonic power, 4 min microwave
duration, and 540 W microwave power, which led to a PNPS
extraction rate of 11.03%. Characterization of PNPS using SEM,
FTIR, and UV-Vis indicated that the UMAE method did not cause
any degradation to the polysaccharides.
The findings of Xu et al. [90] suggested that UMAE resulted in
a greater yield of pectin compared to conventional heating. The
ideal parameters for UMAE were identified as an extraction tem-
perature of 86 °C, an extraction time of 29 min, and a solid-liquid
ratio of 1:48 (w/v), resulting in a maximum pectin yield of 21.5%.
The study of Lu et al. [91] aimed to optimize the extraction of
various degrees of polymerized oligosaccharides from lotus seeds
using UMAE through RSM. The results demonstrated that the
optimal UMAE conditions for lotus seed oligosaccharides were
determined to be an extraction time of 325 s, a liquid-solid ratio
of 10.00 mL/g, ultrasonic power of 300.46 W, and microwave
power of 250 W. These conditions resulted in a 76.59% increase
in the yield of total oligosaccharides, with a 17.47% increase in
trisaccharides and a 27.21% increase in tetrasaccharides. Addition-
ally, the extraction time was significantly reduced compared to
traditional hot water, ultrasonic-assisted, and MAE methods.
Regarding oil extraction, Wang et al. [92] found that the utilization
of UMAE resulted in higher oil yield and greater superoxide radical
scavenging activity of white pepper compared to MAE and UAE,
indicating its superior efficiency as an extraction method. In terms
of the specific components extracted, UMAE generally yielded
more monoterpenes and sesquiterpenes than MAE and UAE.
Therefore, UMAE has the potential to become a prominent
eco-friendly method for extracting essential oil from P. nigrum due

to its maximum extraction yields and short extraction time. Fur-
thermore, Yu et al. [93] employed the UMAE method to extract
polyphenols, flavonoids, triterpenoids, and vitamin C from Clina-
canthus nutans. The optimized conditions for the extraction pro-
cess included the use of distilled water, a solid-liquid ratio of 1:
55 g/mL, an irradiation power of 90 W, and an extraction cycle
lasting 75 s. With the previously described conditions, the extrac-
tion yields of polyphenols, flavonoids, triterpenoids, and vitamin C
were found to be 8.893, 25.936, 16.789, and 0.166 mg/g, respec-
tively. These findings suggest that UMAE is a highly efficient
method for the extraction of bioactive substances from Clina-
canthus nutans. Overall, these findings suggest that UMAE has
the potential to be a more efficient and effective technique for
bioactive compounds extraction as compared to traditional
methods.
4.5 Microwave The mechanism of MHD produces heat by absorbing microwave

Hydro-distillation radiation from the plant material, resulting in the evaporation of
(MHD) essential oil components [21]. The condensed vapor is then col-
lected as a liquid that consists of essential oil. Newer research has
examined the possibility of MHD for extracting essential oils from
diverse aromatic plants [94–97]. Elyemni et al. [97] compared the
efficiency of two extraction methods, microwave-assisted hydro-
distillation (MHD) and Clevenger hydro-distillation (CH), for
obtaining essential oils from Rosmarinus officinalis L. MAH only
requires 20 min to obtain the same yield of essential oils that takes
CH 180 min. Furthermore, the quality of the essential oil was
enhanced by an increase of 1.14% in oxygenates. Additionally, as
reported by Megawati et al. [95], the utilization of microwave-
assisted hydro-distillation (MHD) for the extraction of mace essen-
tial oil was proven to be more effective than hydro-distillation
(HD). MHD resulted in 8.62% essential oils in just 42 min, whereas
HD only produced 7.03% in 73 min. In addition, MHD consumed
less energy (756 kJ) compared to HD (1095 kJ). As the power
input is increased, a higher yield of essential oil is obtained. At
300, 600, and 800 W within 10 min, yields obtained were 2.68,
4.56, and 5.41%, respectively, while at 20 min, yields obtained were
5.13, 7.39, and 6.83%. The findings of Mollaei et al. [25] devoted
that MHD may be a useful technique for extracting essential oil
from F. angulata due to its ability to decrease the distillation
duration, minimize energy usage, and enhance biological proper-
ties when compared to the HD method.
4.6 Microwave The MHG apparatus is essentially a microwave unit that operates
Hydro-diffusion and similarly to a standard commercial model. It utilizes a combination
Gravity (MHG) of microwave radiation and the force of gravity at ambient pressure
to perform extraction from fresh plant material [98]. As described
by De Castro and Peinado et al. [54], this method involved the

introducing of a matrix into a reactor inside a microwave oven.
Microwaves trigger the heating of the water present in the matrix,
leading to the elimination of cells that contain essential oil. The
hydro-diffusion process took place, wherein both the essential oil
and internal water of the matrix were released from the plant’s
interior to its exterior. To condense the distillate, a cooling system
situated outside the microwave oven is employed and then col-
lected under gravity. In addition, MHG extraction has demon-
strated optimal outcomes in diverse applications aimed at
extracting active compounds, including antioxidant molecules
from aromatic plants such as Cuminum cyminum, Cytisus scoparius,
Brassica rapa, Quercus robur, and Pleurotus ostreatus [17, 26,
99]. As detailed by Benmoussa et al. [99], the use of MHG resulted
in a higher yield of essential oil (1.579%) in a shorter time
(16 min vs. 150 min required for HD) as compared to conventional
hydro-distillation (HD) (1.550%). Additionally, the MHG tech-
nique requires less electricity, emits less carbon dioxide, and gen-
erates less wastewater. Examination via GC-MS affirmed that the
quality of cumin essential oils procured by MHG and HD were
comparable. According to this investigation, the effectiveness of
MHG and ethanolic solid-liquid extraction methods were com-
pared using various plant sources, that is, Cytisus scoparius, Brassica
rapa, Quercus robur, and Pleurotus ostreatus [17] (Table 2). The
results illustrated that MHG technology is suitable for generating
extracts with interesting antioxidant characteristics.
4.7 Microwave- By combining microwave-assisted and subcritical water extraction

Assisted Subcritical (MASE), Moirangthem et al. [11] found that anthocyanins could
Extraction (MASE) be extracted from straw with an 85.8% efficiency when exposed to a
temperature of 90 °C for 5 min. This combination was also
observed to have superior antioxidant activity as compared to a
conventional methanol extract. Both the straw and bran’s micro-
wave extracts did not exhibit any noticeable cytotoxicity on Jurkat
cells in vitro.
In addition, Yang et al. [27] used MASE to extract steviol
glycosides from Stevia rebaudiana (Bertoni). The results depicted
that the yields of major steviol glycoside, including rebaudioside A
and stevioside, and rebaudioside C were comparable to those
obtained by the conventional extraction method that used 70%
ethanol under sonication for 45 min, within just 1 min of reaching
subcritical water conditions at 140 °C. This method can be a cost-
effective alternative for producing high-purity steviol glycoside
sweeteners. Moreover, Cai et al. [28] attempted to enhance oil
extraction efficiency by utilizing seed pretreatments, such as micro-
wave assistance, to increase subcritical extraction fluid penetration
without affecting the physicochemical properties of the phyto-
chemicals in oilseeds. These authors established that using
Table 2
MAE methods for bioactive compounds extraction
Methods Compounds Plants References

Solvent-free MAE TPC/TFC Lagenaria siceraria fruit [22]
Essential oil Coleus aromaticus [16]
TPC Centella asiatica L. [1]
Essential oil Cinnamomum longepaniculatum [15]
leaves
FMAE (Focused MAE) Betulinic acid Zizyphus joazeiro bark [81]
Sesamol Sesame seed [23]
ILMAE (Ionic liquid- Lipids and Nannochloropsis oceanica [101]
based MAE) eicosapentaenoic acid Passion fruit and mango leaves [102]
Flavonoids Nothopanax scutellarium leaves [103]
Quercetin Coriander foliage [104]
Heneicos-1-ene Arthrospira platensis [24]
Phycobiliproteins Selaginella sinensis [105]
Amentoflavone/ Haematococcus pluvialis [7]
hinokiflavone
Astaxanthin
Ultrasonic MAE Polysaccharides Trametes orientalis [88]
Caffeic and ferulic acids Clinacanthus nutans [106]
Polysaccharides Camptotheca acuminata fruits [12]
α-mangostin Garcinia mangostana [107]
Polysaccharides Pericarp [13]
Dictyophora indusiata
Microwave hydro- Essential oils Rosmarinus officinalis L. [97]
distillation (MHD) Clove (Syzgium aromaticum) stem [96]
Myristicae arillus [95]
Ferulago angulate [25]
Pelargonium graveolens [94]
Microwave hydro- Essential oil Cuminum cyminum L. [99]
diffusion and gravity Phenolic compounds Blackberries (Rubus spp.) [26]
(MHG) Carotenoid/phenolic/ Cytisus scoparius;Brassica rapa; [17]
lipid/protein contents Quercus robur; Pleurotus ostreatus
Microwave-assisted Anthocyanins Manipur black rice [11]
subcritical extraction Steviol glycosides Stevia rebaudiana leaves [27]
Oil Tigernut [28]
Berberine hydrochloride Berberis aristata roots [100]
microwaving as a pretreatment prior to subcritical extraction is an

uncomplicated technique to improve oil output while producing
high-quality oil. In fact, Cai et al. [28] employed subcritical
n-butane extraction with the aid of microwave pretreatment to
extract tigernut oil from tigernut meal. Microwaving (560 W,
6 min) substantially increased subcritical extraction efficiency. The
most oil was obtained by subcritical extraction of tigernut oil at a
temperature of 52 °C for 32 min after three extraction cycles, with a
liquid-solid ratio of 3.62 kg/(kg of tigernut meal), resulting in a

maximum yield (24.736%) of tigernut oil. The oil’s high-quality
attributes, including a ratio of unsaturated to saturated fatty acids of
4.68 UFA/SFA, low acid value (3.30 mg KOH/g oil), low perox-
ide value (0.28 meq.kg–1), and a predominance of oleic acid, were
identified. Additionally, an optimization of the conditions for
extracting berberine from Berberis aristata roots by microwave-
assisted subcritical water extraction (MASCW) was conducted by
Manikyam et al. [100]. This method was employed to extract
berberine at temperatures ranging from 110 to 170 °C using
various combinations of five subcritical parameters. The experimen-
tal data concentration of berberine (223.82 μg/mL) was found to
be significantly correlated under specific subcritical parameters.
This new hybrid extraction technique can be an eco-friendly option
for producing high-purity compounds [27].
5 Conclusion
The MAE has minimized energy, time, and solvent consumption,

which makes it a sustainable technology. Furthermore, it has been
coupled with other extraction techniques such as ultrasonic, hydro-
distillation, and subcritical extraction. These combinations had
considerably reduced energy and time, and improved bioactive
compound yields. The versatility of MAE in the recovery of bioac-
tive and nutraceuticals from various kinds of vegetal materials could
be applied in advanced practical applications in food and pharma-
ceutical fields.
References
1. Rahmawati A, Fachri BA, Oktavia S et al microbial activity in Quercus cerris bark
(2021) Extraction bioactive compound of extracts. Plants (Basel, Switzerland) 11:240
pegagan (Centella Asiatica L.) using solvent- 5. Liu Z, Deng B, Li S et al (2018) Optimization
free microwave-assisted extraction. IOP of solvent-free microwave assisted extraction
Conf Ser: Mater Sci Eng 1053:012125 of essential oil from Cinnamomum camphora
2. Akhtar I, Javad S, Yousaf Z et al (2019) leaves. Ind Crop Prod 124:353–362
Microwave assisted extraction of phytochem- 6. Motlagh SR, Elgharbawy AA, Khezri R et al
icals an efficient and modern approach for (2021) Ionic liquid-based microwave-assisted
botanicals and pharmaceuticals. Pak J Pharm extraction of protein from Nannochloropsis
Sci 32:223 sp. biomass. Biomass Conv Bioref 13:8327
3. Samanta R, Ghosh M (2023) Optimization of 7. Fan Y, Niu Z, Xu C et al (2019) Biocompati-
microwave-assisted extraction technique for ble protic ionic liquids-based microwave-
flavonoids and phenolics from the leaves of assisted liquid-solid extraction of astaxanthin
Oroxylum indicum (L.) Kurtz using Taguchi from Haematococcus pluvialis. Ind Crop
L9 orthogonal design. Pharmacogn Mag 19: Prod 141:111809
97–104 8. Belwal T, Pandey A, Bhatt ID et al (2020)
4. Nisca A, Ștefănescu R, Moldovan C et al Optimized microwave assisted extraction
(2022) Optimization of microwave assisted (MAE) of alkaloids and polyphenols from
extraction conditions to improve phenolic Berberis roots using multiple-component
content and in vitro antioxidant and anti- analysis. Sci Rep 10:917
9. Kadi A, Boudries H, Bachir-bey M et al natural products isolation. In: Sarker SD,

(2022) Optimization of microwave-assisted Nahar L (eds) Natural products isolation.
extraction of carotenoids from Citrus clemen- Humana Press, Totowa, pp 89–115
tina peels. Curr Bioact Compd 18: 20. Vinatoru M, Mason TJ, Calinescu I (2017)
e301221199690 Ultrasonically assisted extraction (UAE) and
€ Turabi Yolaçaner E, Hamzalıoğlu A
10. Yiğit U, microwave assisted extraction (MAE) of func-
et al (2022) Optimization of microwave- tional compounds from plant materials. TrAC
assisted extraction of anthocyanins in red cab- Trends Anal Chem 97:159–178
bage by response surface methodology. J 21. Chemat F, Cravotto G (eds) (2013)
Food Process Preserv 46:e16120 Microwave-assisted extraction for bioactive
11. Moirangthem K, Ramakrishna P, Amer MH compounds. Springer US, Boston
et al (2021) Bioactivity and anthocyanin con- 22. Iftikhar S, Tariq Qamar M, Aydar AY et al
tent of microwave-assisted subcritical water (2022) Optimization of solvent-free micro-
extracts of Manipur black rice (Chakhao) wave-assisted extraction of antioxidant com-
bran and straw. Future Foods 3:100030 pounds from Lagenaria siceraria fruit by
12. Sun H, Li C, Ni Y et al (2019) Ultrasonic/ response surface methodology. Folia Hortic
microwave-assisted extraction of polysacchar- 34:163–171
ides from Camptotheca acuminata fruits and 23. Sharma M, Patel D, Meghwal K et al (2022)
its antitumor activity. Carbohydr Polym 206: Focused microwave assisted extraction setup
557–564 and extraction of sesamol from sesame seed. J
13. Zhang Y, Lei Y, Qi S et al (2023) Ultrasonic- Postharvest Technol 10:134–141
microwave-assisted extraction for enhancing 24. Rodrigues RDP, Silva ASE, Carlos TAV et al
antioxidant activity of Dictyophora indusiata (2020) Application of protic ionic liquids in
polysaccharides: the difference mechanisms the microwave-assisted extraction of phycobi-
between single and combined assisted extrac- liproteins from Arthrospira platensis with
tion. Ultrason Sonochem 95:106356 antioxidant activity. Sep Purif Technol 252:
14. Al-Ajalein A-HAS, Shafie MH, Yap P-G et al 117448
(2023) Microwave-assisted extraction of poly- 25. Mollaei S, Sedighi F, Habibi B et al (2019)
saccharide from Cinnamomum cassia with Extraction of essential oils of Ferulago angu-
anti-hyperpigmentation properties: optimiza- lata with microwave-assisted hydrodistillation.
tion and characterization studies. Int J Biol Ind Crop Prod 137:43–51
Macromol 226:321–335 26. Moraes DP, Machado ML, Farias CAA et al
15. Wei C, Wan C, Huang F et al (2023) Extrac- (2020) Effect of microwave hydrodiffusion
tion of Cinnamomum longepaniculatum and gravity on the extraction of phenolic com-
deciduous leaves essential oil using solvent- pounds and antioxidant properties of black-
free microwave extraction: process optimiza- berries (Rubus spp.): scale-up extraction.
tion and quality evaluation. Oil Crop Sci 8:7– Food Bioprocess Technol 13:2200–2216
15 27. Yang Z, Uhler B, Lipkie T (2019) Microwave-
16. Gunny AAN, Fang LP, Misnan NM et al assisted subcritical water extraction of steviol
(2021) Microwave-assisted solvent-free glycosides from Stevia rebaudiana leaves. Nat
extraction of essential oil from Coleus aroma- Prod Commun 14:1934578X19860003
ticus: anti-phytopathogenic potential for fruit 28. Cai X, Zhao X, Miao W et al (2022) Optimi-
post-harvesting. 3 Biotech 11:166 zation and kinetics modeling of microwave-
17. López-Hortas L, Falqué E, Domı́nguez H assisted subcritical n-butane extraction of
et al (2019) Microwave hydrodiffusion and tigernut oil. J Oleo Sci 71:1799–1811
gravity (MHG) extraction from different raw 29. Zhang H-F, Yang X-H, Wang Y (2011)
materials with cosmetic applications. Mole- Microwave assisted extraction of secondary
cules 25:92 metabolites from plants: current status and
18. Nisca A, Ștefănescu R, Stegăru DI et al (2021) future directions. Trends Food Sci Technol
Phytochemical profile and biological effects of 22:672–688
spruce (Picea abies) bark subjected to ultra- 30. Hudaib M, Gotti R, Pomponio R et al (2003)
sound assisted and microwave-assisted extrac- Recovery evaluation of lipophilic markers
tions. Plants (Basel, Switzerland) 10:870 from Echinacea purpurea roots applying
19. Delazar A, Nahar L, Hamedeyazdan S et al microwave-assisted solvent extraction versus
(2012) Microwave-assisted extraction in conventional methods. J Sep Sci 26:97–104
31. Moret S, Conchione C, Srbinovska A et al 42. Quiles-Carrillo L, Mellinas C, Garrigos MC

(2019) Microwave-based technique for fast et al (2019) Optimization of microwave-
and reliable extraction of organic contami- assisted extraction of phenolic compounds
nants from food, with a special focus on with antioxidant activity from carob pods.
hydrocarbon contaminants. Foods 8:503 Food Anal Methods 12:2480–2490
32. Lasunon P, Phonkerd N, Tettawong P et al 43. Routray W, Orsat V (2012) Microwave-
(2021) Effect of microwave-assisted extrac- assisted extraction of flavonoids: a review.
tion on bioactive compounds from industrial Food Bioprocess Technol 5:409–424
tomato waste and its antioxidant activity. 44. Tzanova M, Atanasov V, Yaneva Z et al
Food Res 5:468–474 (2020) Selectivity of current extraction tech-
33. Vu HT, Scarlett CJ, Vuong QV (2019) Max- niques for flavonoids from plant materials.
imising recovery of phenolic compounds and Processes 8:1222
antioxidant properties from banana peel using 45. Alara OR, Abdurahman NH, Obanijesu EO
microwave assisted extraction and water. J et al (2020) Extract-rich in flavonoids from
Food Sci Technol 56:1360–1370 Hibiscus sabdariffa calyces: optimizing
34. Vladić J, Janković T, Živković J et al (2020) microwave-assisted extraction method and
Comparative study of subcritical water and characterization through LC-Q-TOF-MS
microwave-assisted extraction techniques analysis. J Food Process Eng 43:e13339
impact on the phenolic compounds and 46. Hayat K, Hussain S, Abbas S et al (2009)
5-hydroxymethylfurfural content in pome- Optimized microwave-assisted extraction of
granate peel. Plant Foods Hum Nutr 75: phenolic acids from citrus mandarin peels
553–560 and evaluation of antioxidant activity
35. Singh C, Khanna V, Singh S (2023) Sustain- in vitro. Sep Purif Technol 70:63–70
ability of microwave heating in materials pro- 47. Kumar M, Dahuja A, Sachdev A et al (2019)
cessing technologies. Mater Today: Proc 73: Valorisation of black carrot pomace: micro-
241–248 wave assisted extraction of bioactive phyto-
36. Kumar S, Walia R, Kumar A et al (2021) ceuticals and antioxidant activity using Box–
Hybrid structure of MWCNT/ferrite and Behnken design. J Food Sci Technol 56:995–
GO incorporated composites for microwave 1007
shielding properties and their practical appli- 48. Kapoore RV, Butler TO, Pandhal J et al
cations. RSC Adv 11:9775–9787 (2018) Microwave-assisted extraction for
37. Elmahaishi MF, Azis RS, Ismail I et al (2022) microalgae: from biofuels to biorefinery. Biol-
A review on electromagnetic microwave ogy 7(18):25
absorption properties: their materials and per- 49. Krakowska-Sieprawska A, Kiełbasa A, Rafińska
formance. J Mater Res Technol 20:2188– K et al (2022) Modern methods of
2220 pre-treatment of plant material for the extrac-
38. Bagade SB, Patil M (2019) Recent advances in tion of bioactive compounds. Molecules 27:
microwave assisted extraction of bioactive 730
compounds from complex herbal samples: a 50. Feduraev P, Chupakhina G, Maslennikov P
review. Crit Rev Anal Chem 51:138 et al (2019) Variation in phenolic compounds
39. Purbowati ISM, Maksum A (2019) The anti- content and antioxidant activity of different
oxidant activity of Roselle (Hibiscus sabdariffa plant organs from Rumex crispus L. and
Linii) phenolic compounds in different varia- Rumex obtusifolius L. at different growth
tions microwave-assisted extraction time and stages. Antioxidants (Basel) 8:237
power. IOP Conf Ser: Earth Environ Sci 406: 51. Elhag HEA, Naila A, Ajit A et al (2019) Opti-
012005 mization of protein yields in water extracts of
40. Sonar MP, Rathod VK (2020) Microwave Jackiopsis ornata roots by response surface
assisted extraction (MAE) used as a tool for methodology using microwave assisted
rapid extraction of Marmelosin from Aegle extraction (MAE), Presented at the materials
marmelos and evaluations of total phenolic characterization using X-rays and related tech-
and flavonoids content, antioxidant and anti- niques, Kelantan, Malaysia
inflammatory activity. Chem Data Collect 30: 52. Poureini F, Mohammadi M, Najafpour GD
100545 et al (2020) Comparative study on the extrac-
41. Dahmoune F, Nayak B, Moussi K et al (2015) tion of apigenin from parsley leaves (Petrose-
Optimization of microwave-assisted extrac- linum crispum L.) by ultrasonic and
tion of polyphenols from Myrtus communis microwave methods. Chem Pap 74:3857–
L. leaves. Food Chem 166:585–595 3871
53. Patil SS, Rathod VK (2023) Extraction and 64. Vu HT, Scarlett CJ, Vuong QV (2018) Phe-
purification of curcuminoids from Curcuma nolic compounds within banana peel and their
longa using microwave assisted deep eutectic potential uses: a review. J Funct Foods 40:
solvent based system and cost estimation. Pro- 238–248
cess Biochem 126:61–71 65. Pavlić B, Naffati A, Hojan T et al (2017)
54. Luque De Castro MD, Castillo-Peinado LS Microwave-assisted extraction of wild apple
(2016) Microwave-assisted extraction of fruit dust-production of polyphenol-rich
food components. In: Innovative food pro- extracts from filter tea factory by-products:
cessing technologies. Elsevier, Amsterdam, PAVLIÇ et al. J Food Process Eng 40:e12508
pp 57–110 66. Varadharajan V, Shanmugam S, Ramaswamy
55. Zhao C-N, Zhang J-J, Li Y et al (2018) A (2017) Model generation and process opti-
Microwave-assisted extraction of phenolic mization of microwave-assisted aqueous
compounds from Melastoma sanguineum extraction of anthocyanins from grape juice
fruit: optimization and identification. Mole- waste: VARADHARAJAN et al. J Food Pro-
cules 23:2498 cess Eng 40:e12486
56. Nkhili E, Tomao V, El Hajji H et al (2009) 67. Pal CBT, Jadeja GC (2019) Microwave-
Microwave-assisted water extraction of green assisted deep eutectic solvent extraction of
tea polyphenols. Phytochem Anal 20:408– phenolic antioxidants from onion (Allium
415 cepa L.) peel: a Box–Behnken design
57. Yuan Y, Xu X, Jing C et al (2018) Microwave approach for optimization. J Food Sci Tech-
assisted hydrothermal extraction of polysac- nol 56:4211–4223
charides from Ulva prolifera: functional prop- 68. Liu Z, Qiao L, Gu H et al (2017) Develop-
erties and bioactivities. Carbohydr Polym ment of Brönsted acidic ionic liquid based
181:902–910 microwave assisted method for simultaneous
58. Wongkaew M, Sommano SR, Tangpao T et al extraction of pectin and naringin from pom-
(2020) Mango peel pectin by microwave- elo peels. Sep Purif Technol 172:326–337
assisted extraction and its use as fat replace- 69. Weremfo A, Adulley F, Adarkwah-Yiadom M
ment in dried Chinese sausage. Foods (Basel, (2020) Simultaneous optimization of
Switzerland) 9:450 microwave-assisted extraction of phenolic
59. Deng B, Liu Z, Zou Z (2019) Optimization compounds and antioxidant activity of Avo-
of microwave-assisted extraction saponins cado (Persea americana Mill.) seeds using
from Sapindus mukorossi pericarps and an response surface methodology. J Anal Meth-
evaluation of their inhibitory activity on xan- ods Chem 2020:1–11
thine oxidase. J Chem 2019:1–11 70. Şahin S, Samli R, Tan ASB et al (2017)
60. Caleja C, Barros L, Prieto MA et al (2017) Solvent-free microwave-assisted extraction of
Extraction of rosmarinic acid from Melissa polyphenols from olive tree leaves: antioxi-
officinalis L. by heat-, microwave- and dant and antimicrobial properties. Molecules
ultrasound-assisted extraction techniques: a 22:1056
comparative study through response surface 71. Li X, Chen F, Li S et al (2016) An efficient
analysis. Sep Purif Technol 186:297–308 homogenate-microwave-assisted extraction of
61. Rodrigues LGG, Mazzutti S, Siddique I et al flavonols and anthocyanins from
(2020) Subcritical water extraction and blackcurrant marc: optimization using combi-
microwave-assisted extraction applied for the nation of Plackett-Burman design and
recovery of bioactive components from Chaya Box-Behnken design. Ind Crop Prod 94:
(Cnidoscolus aconitifolius Mill.). J Supercrit 834–847
Fluids 165:104976 72. Kusuma HS, Mahfud M (2015) Box-Behnken
62. Pinela J, Prieto MA, Barreiro MF et al (2017) design for investigation of microwave-assisted
Valorisation of tomato wastes for develop- extraction of patchouli oil, Presented at the
ment of nutrient-rich antioxidant ingredients: international conference of chemical and
a sustainable approach towards the needs of material engineering (ICCME) 2015: Green
the today’s society. Innovative Food Sci technology for sustainable chemical products
Emerg Technol 41:160–171 and processes, Semarang, Indonesia
63. Pimentel-Moral S, Borrás-Linares I, Lozano- 73. Zakaria NA, Abd Rahman NH, Rahman RA
Sánchez J et al (2018) Microwave-assisted et al (2023) Extraction optimization and
extraction for Hibiscus sabdariffa bioactive physicochemical properties of high methoxyl
compounds. J Pharm Biomed Anal 156: pectin from Ananas comosus peel using
313–322 microwave-assisted approach. J Food Meas
Charact 17:3354
74. Zayed A, Finkelmeier D, Hahn T et al (2023) 85. Li D, Qian Y, Tian Y-J et al (2017) Optimiza-
Characterization and cytotoxic activity of tion of ionic liquid-assisted extraction of bifla-
microwave-assisted extracted crude fucoidans vonoids from Selaginella doederleinii and
from different brown seaweeds. Mar Drugs evaluation of its antioxidant and antitumor
21:48 activity. Molecules (Basel, Switzerland) 22:
75. Tomasi IT, Santos SCR, Boaventura RAR et al 586
(2023) Microwave-assisted extraction of poly- 86. Guo J-B, Fan Y, Zhang W-J et al (2017)
phenols from Eucalyptus Bark—a first step for Extraction of gingerols and shogaols from
a green production of tannin-based coagu- ginger (Zingiber officinale Roscoe) through
lants. Water 15:317 microwave technique using ionic liquids. J
76. Bansod SP, Parikh JK, Sarangi PK (2023) Food Compos Anal 62:35–42
Pineapple peel waste valorization for extrac- 87. Krishnan S, Ghani NA, Aminuddin NF et al
tion of bio-active compounds and protein: (2020) Microwave-assisted lipid extraction
microwave assisted method and Box Behnken from Chlorella vulgaris in water with 0.5%–
design optimization. Environ Res 221: 2.5% of imidazolium based ionic liquid as
115237 additive. Renew Energy 149:244–252
77. Liu Z, Li H, Zhu Z et al (2022) Cinnamo- 88. Zheng Y, Cui J, Chen A-H et al (2019) Opti-
mum camphora fruit peel as a source of essen- mization of ultrasonic-microwave assisted
tial oil extracted using the solvent-free extraction and hepatoprotective activities of
microwave-assisted method compared with polysaccharides from Trametes orientalis.
conventional hydrodistillation. LWT 153: Molecules 24:147
112549 89. Shen S, Zhou C, Zeng Y et al (2022) Struc-
78. Vernès L, Vian M, Chemat F (2020) Ultra- tures, physicochemical and bioactive proper-
sound and microwave as green tools for solid- ties of polysaccharides extracted from Panax
liquid extraction. In: Liquid-phase extraction. notoginseng using ultrasonic/microwave-
Elsevier, Amsterdam, pp 355–374 assisted extraction. LWT 154:112446
79. Kumar A, Kuang Y, Liang Z et al (2020) 90. Xu S-Y, Liu J-P, Huang X et al (2018)
Microwave chemistry, recent advancements, Ultrasonic-microwave assisted extraction,
and eco-friendly microwave-assisted synthesis characterization and biological activity of pec-
of nanoarchitectures and their applications: a tin from jackfruit peel. LWT 90:577–582
review. Mater Today Nano 11:100076 91. Lu X, Zheng Z, Li H et al (2017) Optimiza-
80. Yadav A, Kumar N, Upadhyay A et al (2020) tion of ultrasonic-microwave assisted extrac-
Effect of mango kernel seed starch-based tion of oligosaccharides from lotus (Nelumbo
active edible coating functionalized with lem- nucifera Gaertn.) seeds. Ind Crop Prod 107:
ongrass. J Indian Chem Soc 97:1751–1756 546–557
81. Fonseca FS, Reis LB, dos Santos JG et al 92. Wang Y, Li R, Jiang Z-T et al (2018) Green
(2017) Betulinic acid from Zizyphus Joazeiro and solvent-free simultaneous ultrasonic-
bark using focused microwave-assisted extrac- microwave assisted extraction of essential oil
tion and response surface methodology. Phar- from white and black peppers. Ind Crop Prod
macogn Mag 13:226 114:164–172
82. Reis LCB, Carneiro LM, Branco CRC et al 93. Yu Q, Li C, Duan Z et al (2017) Ultrasonic
(2015) Comparison of conventional micro- microwave-assisted extraction of polyphenols,
wave and focused microwave-assisted extrac- flavonoids, triterpenoids, and vitamin C from
tion to enhance the efficiency of the extraction Clinacanthus nutans. Czech J Food Sci 35:
of antioxidant flavonols from jocote pomace 89–94
(Spondias purpurea L.). Plant Foods Hum 94. Wei L, Yang H, Li H et al (2022) Comparison
Nutr 70:160–169 of chemical composition and activities of
83. Wang Y, Hou Q, Ju M et al (2019) New essential oils from fresh leaves of Pelargonium
developments in material preparation using a graveolens L′Herit. extracted by hydrodistil-
combination of ionic liquids and microwave lation and enzymatic pretreatment combined
irradiation. Nanomaterials 9:647 with a solvent-free microwave extraction
84. Delińska K, Yavir K, Kloskowski A (2021) method. Ind Crop Prod 186:115204
Ionic liquids in extraction techniques: deter- 95. Megawati, Fardhyanti DS, Sediawan WB et al
mination of pesticides in food and environ- (2019) Kinetics of mace (Myristicae arillus)
mental samples. TrAC Trends Anal Chem essential oil extraction using microwave
143:116396
assisted hydrodistillation: effect of microwave 102. Moučková K, Pacheco-Fernández I, Ayala JH

power. Ind Crop Prod 131:315–322 et al (2020) Evaluation of structurally differ-
96. Haqqyana VFWT, Altway MM (2020) Kinetic ent ionic liquid-based surfactants in a green
study in the extraction of essential oil from microwave-assisted extraction for the flavo-
clove (Syzgium aromaticum) stem using noids profile determination of Mangifera
microwave hydrodistillation. Morrocan J sp. and Passiflora sp. leaves from Canary
Chem 8:8–071 Islands. Molecules (Basel, Switzerland) 25:
97. Elyemni M, Louaste B, Nechad I et al (2019) 4734
Extraction of essential oils of Rosmarinus offi- 103. Rahmi I, Jufri M, Mun’im A et al (2020)
cinalis L. by two different methods: hydrodis- Extraction of Quercetin from Nothopanax
tillation and microwave assisted scutellarium leaves via ionic liquid-based
hydrodistillation. Sci World J 2019:1–6 microwave-assisted extraction. Pharm J 12:
98. López-Hortas L, Conde E, Falqué E et al 1512–1517
(2016) Flowers of Ulex europaeus L. – com- 104. Priyadarshi S, Balaraman M, Naidu MM
paring two extraction techniques (MHG and (2020) Ionic liquid-based microwave-assisted
distillation). C R Chim 19:718–725 extraction of Heneicos-1-ene from coriander
99. Benmoussa H, Elfalleh W, He S et al (2018) foliage and optimizing yield parameters by
Microwave hydrodiffusion and gravity for response surface methodology. Prep Biochem
rapid extraction of essential oil from Tunisian Biotechnol 50:246–251
cumin (Cuminum cyminum L.) seeds: opti- 105. Li D, Sun C, Yang J et al (2019) Ionic liquid-
mization by response surface methodology. microwave-based extraction of biflavonoids
Ind Crop Prod 124:633–642 from Selaginella sinensis. Molecules 24:2507
100. Manikyam HK, Ramesh C, Poluri KM et al 106. Chamutpong S, Chen C-J, Chaiprateep E
(2017) Microwave assisted Subcritical water (2021) Optimization ultrasonic–microwave-
extraction of Berberine hydrochloride from assisted extraction of phenolic compounds
the roots of Berberis Aristata using Harmony from Clinacanthus nutans using response sur-
search algorithm. J Herb Med Res 2:1–9 face methodology. J Adv Pharm Technol Res
101. Rezaei Motlagh S, Harun R, Awang Biak DR 12:190
et al (2021) Ionic liquid-based microwave- 107. Kwansang J, Chen C-J, Chaiprateep E (2022)
assisted extraction of lipid and eicosapentae- Optimization of water-based ultrasonic-
noic acid from Nannochloropsis oceanica bio- microwave assisted extraction (UMAE) of
mass: experimental optimization approach. J bioactive compounds from Garcinia mangos-
Appl Phycol 33:2015–2029 tana pericarp. J Complement Integr Med 19:
225–231
Chapter 5
Ultrasound-Assisted Extraction for Food, Pharmacy,

and Biotech Industries
Manab Jyoti Goswami, Utpal Dutta, and Dwipen Kakati
Abstract
Extraction is an important step in the route of phytochemical processing for the discovery of bioactive
constituents from plant materials. The extraction technique plays a significant role in the yield, chemical
structure, and bioactivity of the extracts. Ultrasound-assisted extraction (UAE) has been widely applied as a
novel, green, and rapidly developing extraction method suitable for upscaling and improving the extraction
efficiency of bioactive compounds. UAE has substantial advantages such as low consumption of solvent and
energy, simplification of manipulation and work-up, high extraction yield and purity of the final product,
and fewer damages of active compounds, over the conventional Soxhlet extraction and cold maceration.
UAE can also provide the opportunity for enhanced extraction of heat-sensitive bioactive and food
components at lower processing temperatures. Ultrasound-assisted herbal extracts exhibit higher antican-
cer, antimicrobial, and antidiabetic activities than extracts prepared through conventional methods. Nowa-
days most of the industry-based extractions are carried out using UAE as full extraction can be completed in
minutes with high reproducibility. UAE of herbal, oil, protein, polysaccharide, bioactive compounds, such
as phenolics, flavonoids, and natural colors, which have importance in food, pharmaceutical, and allied
industries, is discussed here.
Key words Ultrasound-assisted extraction, Green extraction, Cavitation effect, Bioactive compounds,
Industrial application
1 Introduction
Extraction is the first step toward isolating and identifying the

desired natural products from the raw materials. Plants are the
richest source of bioactive compounds that serve many purposes.
Extracts of plant origin play an important role as natural additives
or industrial inputs to food, pharmaceutical, cosmetic, perfumery,
and other allied industries. Plant extracts have been widely used in
traditional medicine, perfumes, food flavor, and preservatives from
ancient times. The medicinal properties of the plant extracts result
from the synergistic effect of the bioactive compounds present in
them. These bioactive compounds, commonly known as secondary
103
104 Manab Jyoti Goswami et al.
metabolites, exhibit potent antioxidant, antidiabetic, anti-

inflammatory, and anticancer activities [1]. Great interest has been
paid to extract these valuable compounds from different parts of
the plants using various extraction techniques. According to the
extraction principle, extraction methods include the distillation
method, solvent extraction, pressing, and sublimation. Solvent
extraction is the most extensively used method for the preparation
of plant extracts [2]. Preparation of the plant extract using solvent
extraction involves: (a) the solvent penetrates into the plant matrix;
(b) the solute dissolves in the solvent; (c) the solute diffused out of
the solid plant matrix; (d) the extracted solutes are collected and
concentrated. Conventional solvent extraction techniques, such as
cold maceration, Soxhlet extraction, and percolation, have been
used for a long time. The use of only organic solvents, consumption
of large quantities of solvents, and longer extraction time are some
disadvantages associated with these conventional methods. Some
modern extraction techniques, such as ultrasound-assisted extrac-
tion, microwave-assisted extraction, pressurized liquid extraction,
and supercritical fluid extraction, have been developed and exten-
sively used for the natural products extraction. These modern
extraction methods offer many advantages such as lower consump-
tion of solvent, shorter extraction time, and improved yields of
natural extracts over the conventional methods [3].
The use of ultrasound technology has been considered as an
innovative and one of the most promising technologies of the
twenty-first century. Ultrasonic waves have been extensively used
in the fields of pharmaceuticals, chemistry, cosmetics, and nourish-
ment since after the Second World War. Nowadays it is hard to find
a production line for food, pharmaceuticals, and biotech-related
industries that does not use ultrasonic waves. In general, use of
conventional extraction techniques is less efficient for industrial
applications. For example, conventional extraction techniques
have certain scientific and technological drawbacks to overcome:
often demanding up to 50% of investments in a new plant and more
than 70% of total process energy used in food industries [4]. The
use of efficient, enhanced, and greener extraction techniques such
as ultrasonic-assisted extraction (UAE) proved to be more promi-
nent in such industrial extraction processes. Indeed, such modern
techniques are ideal to compensate for the increasing energy costs
and to reduce greenhouse gas emissions. UAE has substantial
advantages such as low consumption of solvent and energy, simpli-
fication of manipulation and work-up, high extraction yield and
purity of the final product, and fewer damages of active compounds
over the conventional methods.
For achieving the objective of green extraction of natural pro-
ducts, ultrasound is recognized as a key technology. “Green extrac-
tion” includes the extraction process which reduces energy
consumption, allows use of alternative solvents and renewable
Ultrasound-Assisted Extraction for Food, Pharmacy, and Biotech Industries 105
natural products. It also refers to use of a minimal quantity of

solvent, reducing risk of environmental pollution and waste, while
ensuring a safe and high-quality extract [5]. Driven by the green
extraction goals, food and pharmaceutical industries have paid
special interest in using ultrasound technology for extraction pur-
poses. Higher product yield, low maintenance cost, and shorter
processing time are the green impacts of UAE on extraction. The
application of UAE as a green technology for the extraction of
different classes of food components such as pigments, aromas,
antioxidants, and other organic compounds are also found in
food and pharma industries. Technologically, integration of ultra-
sound with already available extraction techniques is quite simple.
Due to this fact, the implementation of UAE for improving the
extraction efficiency of flavonoids, flavonols, polyphenols, sugars,
minerals, and carotenoids in juices is commonly seen for industrial
purposes [6, 7].
Using ultrasound technology, the extraction of organic com-
pounds from natural matrices such as plants and seeds can be
improved significantly. The intensification of ultrasonic solid-liquid
extraction is attributed to the physical and chemical effects caused
by high-power ultrasound; more specifically the cavitation phe-
nomenon. The cavitation effects of ultrasound provide better pen-
etration of solvent into cellular materials and thereby results in
higher mass transfer than other extraction methods. Cavitation
phenomena also lead to high shear forces in the media. When
high-power ultrasound is applied to the surface of the materials,
the asymmetric collapse of the cavitation bubbles occurs. This
results in rapid micro-jetting toward the surface of the solid matrix.
This effect causes surface peeling, erosion, breakdown of cell walls,
and the exudation of cellular contents. It facilitates the extraction of
various compounds from natural products [8]. Ultrasound-
induced cavitation bubbles provide hydrophobic surfaces in the
extraction liquid and thereby net hydrophobic character of the
extraction medium increases. Hence extraction of polar compo-
nents into a hydrophilic aqueous media becomes possible through
UAE. It also reduces the need for normally undesirable hydropho-
bic and strongly polar solvents [9].
Phytochemicals have less toxicity risk compared to their syn-
thetic counterparts. Plant-based foods, including fruits, vegetables,
nuts, grains, seeds, and legumes, may contain hundreds of different
phytochemicals. Phytochemicals-rich functional food can provide
necessary health benefits when consumed regularly through diets.
Therefore, the extraction of bioactive phytochemicals from natural
resources is still a hot topic of research [10]. Using UAE, biologi-
cally active compounds from Salvia officinalis were extracted effi-
ciently with some 60% of the target compounds within 2 h at
ambient temperature [11]. UAE of tea solids from dried tea leaves
using water as a solvent increased the yield up to 20% at 60 °C in
comparison to the thermal extraction at 100 °C [12]. Due to such

benefits, it has been suggested that UAE could be used to prepare a
variety of herbal extracts for the phytopharmaceutical industry.
Applications of UAE in biotech industries are very common at
present time. For example, Ana et al. reported UAE to be a reliable
technology for genipin extraction from Genipa americana. Genipin
is an excellent natural cross-linker for proteins, gelatin, collagen,
and chitosan cross-linking. Due to its low acute toxicity, genipin is
used as a regulating agent for drug delivery. Ana et al. reported that
ultrasound improves the liberation of genipin and proteins from the
plant matrix as well as enhances the formation of polyelectrolyte
complexes. They found eight times improved yield of non-cross-
linked genipin while using UAE at 10 °C for 15 min [13]. This also
explains the effectiveness of UAE at low temperatures. Lutein is the
major carotenoid found in the human eye. Lutein improves
age-related macular disease that causes blindness and vision
impairment. Egg yolk is one of the major sources of lutein in our
foods. UAE improved the yield of lutein when saponified solvent
was used [14]. Adam et al. reported a solvent-free UAE method for
lipid recovery from fresh Nannochloropsis oculata microalgae bio-
mass. With a simple and scalable pre-industrial device, they proved
the better efficiency of UAE for lipid recovery than conventional
(e.g., Bligh and Dyer) methods [15]. Reports of UAE of various
bioactive compounds, such as pigments, polysaccharides, lipids,
acids, and antioxidants, from different microorganisms, are found
in the literature [4].
2 Ultrasonic System for Extraction
Ultrasonic cleaning baths (Fig. 1a) and the more powerful probe
systems (Fig. 1b) are the two most common ultrasound equipment
used for extraction. For large volumes of fluids, ultrasound baths or
continuous or recycled-flow sonoreactors are commonly used.
While an ultrasound horn with the tip submerged in the fluid is
sufficient for small extraction volumes. Ultrasonic devices for
extraction have also been manufactured by several companies on
an industrial scale. These devices usually have a volume capacity of
30–1000 L with a power range of 500–16,000 W [14]. Usually,
laboratory ultrasonic systems are used in batch mode, whereas flow
mode is often used in operating industrial systems.
2.1 Bath Systems 1. The basic component of an ultrasonic bath consists of a tank, an
electronic generator, and a transducer. The generator supplies
electrical power to the transducer. Generally, multiple transdu-
cers are attached to the tank on sides by epoxy resin in today’s
practice. A thermostatically controlled heater can also be
provided for the bath.
Fig. 1 Commonly used ultrasonic extraction systems (a) ultrasound bath (b) ultrasound probe. (Reproduced
from Chemat et al. [4] with permission from Elsevier)
2. For industrial use, units for stuffing and de-stuffing, rinsing,

washing, drying, solvent recovery, transport, etc., can be
incorporated with these systems.
3. The vessel with raw matrix and extractant is placed inside the
bath for extraction to be carried out. Suitable ultrasonic power
must be provided for achieving cavitation within the vessel. The
frequency is the key factor for determining the cavitation and
the energy released during the extraction process. For commer-
cial purposes, most of the ultrasonic baths operate at a single
frequency; such as 40–45 kHz during extraction [16].
2.2 Probe Systems 1. Probe systems comprise a generator, transducer, and horn. The
generator supplies alternating electrical frequencies, usually
20 kHz. It allows the regulation of the amplitude and also
controls the cooling of the medium. The horn may consist of
an upper horn made of titanium and a detachable horn.
Although the detachable horn can be made of different materi-
als, titanium alloy is often used.
2. The extraction operation is carried out by immersing the probe
into the vessel with raw material and extractant. The total
volume of the mixture to be sonicated governs the choice of
the detachable horn to be used. Sonochemical effects were
found to be stronger near the tip, as most of the energy
transmitted to the medium occurs near the tip. Stepped probes
are commonly used, whereas the use of spiral probes is also
seen [17].
3. Probes for industrial use can be developed with different boos-
ters to adjust the ultrasound energy transmitted to the
medium. For continuous sonication, a closed flow cell is used
and thereby a homogeneous sonication can be achieved.
2.3 Probes Versus 1. The extraction process in the probe is a type of direct sonica-
Baths tion, while the process involving baths is regarded as indirect
sonication.
2. The system arrangement in the probe enables amplification and
concentration of ultrasonic energy. This in turn enhances the
sonication effectiveness compared to bath systems, sometimes
up to 100 times greater.
3. In probe systems, higher energy is transmitted to the medium
giving rise to better performance. This helps to minimize the
extraction time than in bath systems. This shortening of extrac-
tion time increases the reproducibility of probe systems.
4. Sonication in a probe leads to the generation of heat and hence
this system may not be suitable for the recovery of volatile
compounds. So, bath systems are generally preferable for the
extraction of materials containing volatile compounds.
5. Cross-contamination is easier with ultrasonic probes than with
ultrasonic baths [16].
2.4 Online UAE Online UAE is a considerably faster approach for carrying out
System extraction. It comprises an open system where fresh solvent contin-
uously flows through the sample. It leads to the displacement of
mass transfer equilibria toward the solubilization of analyzing sub-
stances into the liquid media. The extract is then passed to the
continuous manifold for online analysis. The analytical procedure
involves preconcentration, derivatization, filtration, and finally
detection of the active compounds by various available techniques
(e.g., gas chromatography mass spectrum) [18].
Advantages of online UAE
1. Sample contamination, as well as losses of analytes, is minimum
in online UAE.
2. Less consumption of reagents is observed in online UAE as
compared to offline UAE.
3. Centrifugation or the filtration step is not required in online
UAE to separate the liquid phase from the solid particles.
Thereby sample preparation can be completed in a short dura-
tion of time [19].
3 Extraction Mechanism
3.1 Basic Principle Ultrasound is sound waves having frequencies greater than 20 kHz,
which is higher than the upper audible limit of human hearing.
Usually, the output source of ultrasound is a vibrating body that
causes vibration of the surrounding medium and then the transfer
of energy from the ultrasonic wave to the neighboring particles
occurs. Physical parameters including power, frequency, and ampli-

tude play vital roles in the ultrasonic process. The energy level at
which ultrasonic waves propagate through the medium is expressed
in terms of ultrasound power (W), ultrasound intensity (W/cm2),
or acoustic energy density (W/cm3) [20].
3.2 UAE Mechanism 1. In UAE, ultrasound frequency in the range of 20–50 kHz is
normally used. In extracting media, ultrasound can produce
vibration, cavitation, crushing, mixing, and some comprehen-
sive effect. These effects can break the cell wall and results in
successful extraction of natural product components from plant
matrices [21, 22].
2. Normally it is believed that cavitation effects, thermal effects,
and mechanical effects have a substantial influence on UAE.
The combined results of these effects lead to the destruction of
the cell wall, reduction in particle size, and enhance the reac-
tion rate through the mass transfer of the cell wall. In general,
these effects do not cause any changes in the structure and
functions of the extracts [23, 24].
3.3 Cavitation Effect 1. Cavitation effect in ultrasound is a unique physical phenome-

in UAE non and was discovered first by Thornycroft [25].
2. This effect arises due to the propagation of strong ultrasonic
waves in liquid media.
3. The ultrasound cavitation arises from the negative pressure,
which is a distinct critical value when the liquid can be dragged
out to form a vapor or gas cavity in the local domain. This
critical negative pressure that drags the liquid out is termed as
the cavitation threshold.
4. There are two types of cavitation effect—(a) stable cavitation
and (b) transient cavitation.
(a) Ultrasonic wave propagates longitudinally in the liquid
phase and its alternating pressure is stretched and com-
pressed periodically in the liquid. Due to the constant
compression and decompression cycle, the cavitation bub-
bles produced have different frequencies of the sound
wave pulse. This phenomenon is called “stable
cavitation.”
(b) The cavitation bubbles so produced keep on growing and
after reaching their critical value (high temperature,
5000 K and high pressure, 100 MPa), a cavitation zone
will be generated. This cavitation phenomenon is known
as “transient cavitation” [26, 27].
5. Cavitation phenomenon leads to high shear forces in the
media. The collapse of cavitation bubbles on the surface of a
solid matrix results in micro-jetting and creates several effects
such as surface peeling, erosion, and particle breakdown. Addi-

tionally, the crumbling of cavitation bubbles in a liquid media
lead to macro-turbulences and micro-mixing.
6. Cavitation effect can alter the chemical processes in the system.
In some cases, the cavitation effect causes the formation of
various free radicals. For example, hydroxy free radicals are
primarily generated when water is used as a solvent. The gen-
erated free radicals can modify other molecules such as proteins
present in the extract. Thus, it is important to control para-
meters such as power and external temperature that affect the
generation of radicals.
7. Depending on the extraction parameters and nature of the
natural products matrix, some physical effects of ultrasound
have been identified. These physical effects are fragmentation,
erosion, sonocapillary effect, sonoporation, local shear stress,
and detexturation as described by Chemat et al. [4]. These
influences of ultrasound can be attributed to the cavitation
effect (Fig. 2).
Fig. 2 Scheme of ultrasound cavitation physical process. (Reproduced from Wen et al. [26] with permission
from Elsevier)
3.4 Factors Effecting Several parameters effect the process of UAE. To obtain a high
UAE extraction efficacy, the study of these parameters is of great impor-
tance. However, it is not always necessary to consider that yield is
the only objective of the extraction process. Moreover, minimal use
of non-renewable resources along with low energy consumption
should be taken into account. The influencing factors in UAE
include the shape and size of the ultrasonic reactor device, extrac-
tion process parameters, solvent type, temperature, and matrix
particle size [26].
3.4.1 Shape and Size of When contacts a solid surface, ultrasonic waves are reflected. In the
the Ultrasonic Reactor UAE extraction using an ultrasonic bath, the shape of the reaction
Device vessel is crucial. In order to attain a minimum reflection of waves
the use of a flat bottom vessel such as a conical flask would be the
best choice. A vessel with minimal thickness should be used to
reduce attenuation. It is necessary to compute the optimal reactor
dimension and the location of the transducer-related elements to
obtain maximum energy transfer to the fluid. In the case of ultra-
sonic probes, a rapid decrease in intensity is detected both axially
and radially. Thus, it is preferred to keep a minimal space between
the ultrasonic probe and the wall of the container. The probe
should not touch the container to avoid damage to the
material [28].
3.4.2 Extraction Process Several studies reveal that high ultrasonic power induces greater
Parameters: Power and shear forces that cause major alterations in materials. However, in
Frequency the food industry optimization of this parameter is usually per-
formed. Optimization is done to achieve the best results using
minimum ultrasonic power [29]. Generally, improvement in yield
and composition of the extract in UAE can be achieved by increas-
ing the ultrasound power.
Ultrasound frequency may also influence the extraction process
and have to be optimized. The optimal choice of ultrasound fre-
quency enables to obtain the desired cavitation effect. An increase
in the ultrasound frequency or intensity in the extraction process
leads to the gradual decrease of the liquid cavitation bubbles. The
high frequency suppresses the compression-rarefaction cycles,
which is more difficult to induce acoustic cavitation bubbles due
to the short period. While the low frequency may reduce the
formation of transient cavitation bubbles [30]. The effect of fre-
quency not only influences the cavitation bubble size but is also
related to the mass transfer in the extraction process. Ultrasound
frequencies in the range of 20–100 kHz are commonly used in
UAE. Thus, optimization of the frequency is important for ultra-
sound extraction [4].
3.4.3 Solvent Selection of the most appropriate solvent for extracting the analytes
from the sample matrix is an important step of UAE. Ultrasound
extraction is largely influenced by the amount and type of solvent
used, concentration, and the ratio of solvent and solute. These
factors contribute to the transmission of ultrasonic energy. The
initiation of cavitation is affected by the physical properties of the
solvent such as viscosity, surface tension, and vapor pressure
[31]. The collapse of the cavitation bubble is more intense in
solvents with low vapor pressure compared to that of the solvents
with high vapor pressure. So, a solvent with low vapor pressure is
generally preferred in UAE [4].
3.4.4 Temperature and Sonochemical effects are favored by low temperatures as the cavita-
Time tion effect is prominent at lower extraction temperatures. Although
high temperature enhances solvent diffusion rates, it could lead to
the degradation of thermolabile compounds. At higher tempera-
tures, the collapse of cavitation bubbles may be reduced and conse-
quently, sonochemical effects are less effective. Therefore, the
temperature of the solvent should be controlled within a suitable
range to obtain the highest yield of the target compounds. Simi-
larly, the extraction time should be optimized. Short time may
result in incomplete extraction and very long extraction time may
induce undesirable reactions and less selective extractions [32].
3.4.5 Particle Size of the UAE yield may also be effected by the size of the matrix particles.
Matrix Usually, the reduction of particle size increases the surface contact
area. When particles of the matrix are small enough, most cell walls
are disrupted by ultrasound, thus facilitating better extraction [32].
4 Application of UAE in the Perspectives of Food, Pharmacy, and Biotech Industries
4.1 UAE of Fruits, Fruits and vegetables are the sources of a wide range of secondary
Vegetables, and Their metabolites present in their pulp, seed, peel, and bark. Various
By-Products phytochemicals, antioxidants, lipids, pigments, aromas, and other
molecules of industrial importance have been extracted from fruits
and vegetables. Laboratory-based extraction of these molecules can
be integrated further for their applications in the food, pharmaceu-
ticals, biotech, and cosmetics industries. Extensive use of UAE for
the extraction of such bioactive components has been observed.
UAE has substantial advantages for extracting such compounds
from fruit and vegetable matrices over conventional extraction
techniques. Using UAE, bioactive component extraction can be
performed in very short time, at a relatively low temperature, with
optimal energy and solvent requirement. As a non-thermal extrac-
tion technique, UAE facilitates the retention of the functionality of
bioactive compounds [33].
In the food industry antioxidants are broadly used for the

preservation and extension of the shelf life of foodstuffs [34]. Anti-
oxidants are substances that may prevent or delay the oxidation
processes taking place in our bodies. Antioxidants can prevent cell
damage by reacting with the oxidizing species formed due to
different biochemical reactions inside the body. Synthetic antiox-
idants such as butylated hydroxytoluene (BHT) and butylated
hydroxyanisole (BHA) have some negative side effects on human
health. As such, there is a visible interest in the food, pharmaceuti-
cal, and allied industries for the isolation and use of natural food
grade antioxidants as alternatives for synthetic antioxidants [35]. In
this context, UAE serves as an influential tool for the extraction of
such antioxidants mainly from plant matrices. The concentration of
the extracted antioxidant compounds depends on the studied food
matrix and the applied extraction method [36]. Many researchers
have reported that the application of UAE significantly increases
the antioxidant capacity of the prepared extract in comparison to
conventional extraction techniques. Table 1 represents the reports
of using UAE, in which improvement of the residual antioxidant
capacities of food and by-product extracts have been found.
In the same way, Mayra et al. reported the improved antioxi-
dant capacity of hybrid mandarin peels using ultrasound extraction
[44]. Recently, Selahvarzi et al. claimed significant enhancement in
the antioxidant activities of ultrasound extracted phenolic com-
pounds from pomegranate and orange peels [45]. The antioxidant
capacity of the common bean (Phaseolus vulgaris L.) was found to
be increased when UAE was used [46].
The extraction of valuable bioactive compounds from fruit and
by-products of vegetable processing has been carried out over the
last decade. Fruits such as apple, orange, grapefruit, pineapple, and
chokeberry, and vegetables such as carrots, potatoes, onions, and
asparagus are processed to yield different value-added products.
By-products of such fruits and vegetables, namely, skin, seed,
rind, and pomace contain valuable phytochemicals and secondary
metabolites. In some cases, it was found that these non-consumable
by-products contain a higher concentration of bioactive com-
pounds than their edible parts [47, 48]. For example, 50% of the
total phenolics in potato are present only on the potato peel
[49]. Ultrasound extraction using high-intensity sound waves is
found to be effective for the extraction of bioactive components
from fruit and vegetable waste and by-products. UAE of different
classes of valuable compounds from the viewpoint of their impor-
tance in food, pharmaceutical, and biotech industries are
discussed here.
4.1.1 Extraction of Pectin Pectin is an important heteropolysaccharide with multiple applica-

tions in food, pharmaceutical, and other industries. Pectin is used in
jellies, jams, frozen foods, and edible films. It is also used as a fat and
sugar replacement in low-calorie foods. In pharmaceutical
114
Table 1
Enhancing effect of UAE on antioxidant capacity compared to conventional extraction methods
Treatment conditions of UAE
Frequency Temperature Increase in antioxidant

Plant matrix Power (W) (kHz) (°C) Time (min) Solvent ratio capacity Reference
Manab Jyoti Goswami et al.
Apple pomace 150 25 10–40 5–55 Water 30% [37]

Orange peel 50–150 25 10–40 60 Ethanol: water 30% (DPPH) [38]
(20–80:80–20) 40% (ORAC)
Black chokeberry 100 30.8 20–80 250 Ethanol: water 85% [39]
(50:50)
Pomegranate peel 2.4–59.2 20 25 2–90 Ethanol: water 22–24% (per [40]
(30–70:70–30) antioxidant
yield)
160% (FRAP)
Rice bran 140 35 40–60 15–45 Ethanol: water 80% (FRAP) [41]
(50–90:50–10) 229% (DPPH)
Grape by-products 35 70 60 Ethanol: water 100% [42]
(50:50)
Origanum 1500 20 15–35 5–15 Methanol: water 106% (FRAP) [43]
majorana L. (80:20)
industries, pectin is mainly used to reduce cholesterol levels in the

blood and gastrointestinal disorders [50]. Pectin is present in many
fruits, vegetables, and their by-products. In present times, pectin is
mostly extracted through UAE. Reports suggest that above 25%
yield of pectin was obtained from grape pomace, pomegranate peel,
orange peel, grapefruit peel, eggplant peel, and tomato waste using
ultrasonic extraction. The quality of ultrasound extracted pectin
was found to be better than pectin extracted through heat-based
conventional process [51]. UAE of pectin from various fruit and
vegetable by-products reported by different research workers is
presented in Table 2.
4.1.2 Extraction of Polysaccharides are composed of several smaller monosaccharides

Polysaccharides and Other and are the most abundant carbohydrates found in foods. They are
Functional Compounds commonly used in food industries as functional ingredients. Poly-
saccharides possess various bioactivities including immunomodula-
tory, antioxidant, antitumor, and hypoglycemic activities
[33]. Hence, they are equally important materials for the pharma-
ceutical and biotech industries. In earlier days, conventional heat-
based extraction methods were used to extract hemicellulose, cel-
lulose, and xyloglucan components from plant and food
by-products. UAE of these components is more convenient than
the conventional techniques. Ultrasound extraction of these com-
pounds accelerates the extraction process and preserves their struc-
tural and molecular properties. UAE enhanced the extractability of
hemicellulose from sugarcane bagasse. Destruction of cell walls and
cleavage of links between hemicellulose and lignin by ultrasound
improved the extraction [66]. In the ultrasonic aqueous extraction
of polysaccharides from edible fungus, glycan-chitin complexes
with high average molecular weight are extracted. Whereas, no
such compounds were obtained in simple hot water extraction.
These compounds are reported to have antitumor activities
[67]. UAE of water-soluble polysaccharides from litchi seed and
their bioactivity studies were reported by Chen et al. [68]. Raja
et al. extracted antioxidant polysaccharides from the stem of Trapa
quadrispinosa stem ultrasonically. The extracted polysaccharides
exhibited higher total antioxidant capacity compared to the hot
water extracted polysaccharides in terms of ABTS and DPPH radi-
cal scavenging activity [69]. High-pressure UAE of polysaccharides
from Hovenia dulcis having antioxidant and hypoglycemic proper-
ties was reported by Yang et al. [70].
4.1.3 Extraction of UAE is a promising tool for the extraction of phenolic compounds
Polyphenols for laboratory and industrial purposes. Phenolic compounds are
widely used in wine industries for providing characteristic color
and flavor in wine samples. Most of the plant-derived polyphenolic
compounds exhibit antioxidant properties and can serve as natural
Table 2
UAE of pectin from different fruit and vegetable by-products
The optimum condition for UAE
By-product/ Power Frequency Temperature Time Yield

waste (W) (kHz) (°C) (min) pH Solvent (%) References
Grapefruit 200 24 70 25 1.5 Acidified water 17.92 [52]
peel (0.1 N HCl)
Grapefruit 12.56 20 67 28 1.5 0.5 M 27.34 [53]
peel hydrochloric
acid
Grapefruit 0.4 20 60 60 1.5 0.5 M 18.11 [53]
peel hydrochloric
acid
Grape 140 37 75 60 2 Citric acid 32.3 [54]
pomace solution
Pomegranate 130 20 61.9 28 1.2 Citric acid 23.87 [55]
peel solution
Banana peel 323 20 27 3.2 Citric acid 8.99 [56]
solution
Mango peel 497.4 20 85 10 1 M nitric acid 8.6 [57]
solution
Passion fruit 664 20 85 10 2 1 M nitric acid 12.67 [58]
peel solution
Orange peel 150 20 10 1.5 Citric acid 28.07 [59]
solution
Jackfruit peel 130 20 60 24 1.6 Citric acid 14.5 [60]
solution
Eggplant peel 50 W 30 1.5 Acidified water 33.64 [61]
Tomato 37 60 15 Ammonium 35.7 [62]
waste oxalate
(16 g/L)
Sunflower 375 20 32 3.2 Citric acid 8.89 [63]
head solution
Durian rind 85 240 2.3 1 N HCl 8.8 [64]
solution
Lemon peela 60–75 15–45 0.5 M nitric 10.11 [65]
acid solution
Mandarin 60–75 15–45 0.5 M nitric 11.29 [65]
peela acid solution
Kiwi peela 60–75 15–45 0.5 M nitric 17.3 [65]
acid solution
a
Optimum condition is not reported
antioxidants. Phenolic compounds are able to inhibit in vitro oxi-

dation of low-density lipoproteins and hence associated with
reduced risk of cardiovascular diseases. Polyphenolics also exhibit
anti-inflammatory, anti-ulcer, antimutagenic, and anticarcinogenic
properties. Due to the significant benefits of phenolic compounds
on human health, pharmaceutical industries have paid particular
interest in their use [71]. As UAE is a rapid process, consumes a
minimal quantity of solvent, and has a comparable recovery rate, it
is useful for industrial- or large-scale extraction of polyphenolic
compounds.
Selection of the appropriate solvent systems is a crucial step in
UAE to obtain a high yield of phenolic compounds. Methanol was
found to be the most appropriate solvent to extract phenolic com-
pounds from the leaves of Centaurea species [72]. While in most
cases, ethanol was proved to be the best solvent for ultrasonic
extraction of phenolic compounds; such as from mango peel
[73], lime peel [74], grape seed [75], and olive leaf [76].
4.1.4 Extraction of Flavonoids are a class of polyphenolic plant secondary metabolites.

Flavonoids These are found in a variety of fruits and vegetables. Flavonoids
exhibit a broad spectrum of health-promoting effects and are con-
sidered an indispensable component in a variety of pharmaceutical,
nutraceutical, and medicinal applications. Flavonoids are important
for human health because of their antioxidative, antibacterial, anti-
inflammatory, anticarcinogenic, and antimutagenic properties.
They can modulate key cellular enzyme functions. Applications of
flavonoids in the food industry include the preservation of foods
and the making of dietary supplements. Flavonoids are also used to
provide color and flavor of food products [77].
UAE of valuable flavonoids has been carried out to enhance the
extraction yield and their recovery. Some of the UAE-extracted
flavonoids having pharmaceutical importance are listed in Table 3.
All the cited flavonoid compounds have different biological
activities as reported by the respective authors. It can be concluded
that flavonoids are associated with significant medicinal properties
and may be used as natural antioxidants in the pharmaceutical and
food industries. UAE provides a high yield of flavonoid compounds
than the conventional extraction methods.
4.1.5 UAE of Anthocyanins and carotenoids are the natural color pigments pres-
Anthocyanins and ent mostly in fruits and vegetables. In present times, natural colors
Carotenoids are very demanding in the food industry. Increasing use of antho-
cyanins as natural colorants mostly in food products and beverages
has been observed. Anthocyanins are also used as bioactive com-
pounds in pharmaceutical industries which further boosted their
market requirements. Similarly, carotenoids have been used as bio-
active compounds in pharmaceutical and allied industries.
Table 3
UAE of bioactive flavonoids
Extraction parameters
Power Temperature Time

Plant (W) (°C) (min) Reported flavonoids References
Olive leaves 180–270 50 30 Luteolin-4′-O- [78]
(Olea europaea) glucoside
Apigenin-7-O-
glucoside
Rutin
Ocimum tenuiflorum 50 W 40 30 Apigenin [79]
leaves Luteolin
Rutin
Curry leaves 80–150 40–80 20 Catechin [80]
(Murraya koenigii L.) Myricetin
Quercetin
Moringa oleifera leaves 88 30 20 Catechin [81]
Hyperoside
Kaempferol-3-O-
rutinoside
Euonymus alatus 90 15 Catechin [82]
Dihydromyricetin
Lycium barbarum 50 90 Myricetin [83]
L. fruits Morin
Rutin
Chestnut peels 50 35–55 120 Luteolin [84]
(Eleocharis dulcis) Eriodictyol
Fisetin
Carotenoids are the colorants found in special food items

[85, 86]. Pinela et al. reported the ultrasonic-assisted extraction
of anthocyanins from Hibiscus sabdariffa calyces. They obtained
higher levels of anthocyanin than previously reported literature.
They also suggested that extracted anthocyanins may be used as a
natural food colorant in industrial applications [87]. Ochoa et al.
carried out the extraction of anthocyanin from purple yam (Dios-
corea alata) by both UAE and conventional methods. According to
their findings, UAE proved to be the feasible technique for obtain-
ing anthocyanin rich extracts from purple yam [88]. UAE enhanced
the concentration of corn carotenoids as reported by Jun et al. They
got a 3.6 times higher concentration of corn carotenoids with UAE
than extraction carried out without ultrasound assistance. Their
report also determined that UAE did not affect the structure of
the corn carotenoids [89].
4.1.6 Extraction of Edible UAE has been recognized as a valuable extraction method in the
Oils edible oil industries to improve yield and reduce the duration of
extraction time. Rapid extraction of oil from soybeans using high
frequency ultrasound waves was reported in the literature. The
yield was significantly improved in UAE [90]. Garcia and Castro
reported that ultrasound-assisted Soxhlet extraction is an effective
method for the extraction of a higher amount of oil from raw
matrices. They used this combined methodology and extracted a
higher amount of total fat content from soybean, sunflower, and
rape seeds than the Soxhlet extraction performed alone [91]. The
benefit of ultrasonic pre-treatment of oleaginous seeds before oil
extraction was also reported. Ultrasonic pre-treatment of the
almond and apricot seeds prior to oil extraction provided better
yield with a reduction in extraction time [92]. Application of UAE
on the extraction of oils from different food by-products, such as
rice bran, soybean germ, and papaya seed, has also been reported.
4.1.7 Extraction of Protein extraction is very much associated with pharmaceutical

Proteins industries. All the big pharmaceutical industries now develop and
sell recombinant protein as a drug. UAE has been becoming the
most well-known technology for the extraction of proteins for the
last four decades from laboratory-based research to industrial appli-
cations. In 1982, Moulton and Wang studied both batch and
continuous ultrasonic extraction of soybean protein. They reported
that both ultrasonic processes gave a higher yield of protein than
the conventional methods. The continuous ultrasonic extraction
provided 54% and 23% higher yields of protein than the batch
process for aqueous and alkaline extraction, respectively. These
findings are very helpful for the industrial use of the UAE at that
time. Later on, many researchers reported the ultrasound extrac-
tion of protein from various natural sources. Some of them also
optimized the extraction process for industrial applications. UAE
also influences the functional properties of the protein. In a recent
study, Wang et al. carried out the ultrasound-assisted alkaline
extraction of pea protein and found significant improvements in
the functional properties of extracted protein. They reported that
the extracted protein was associated with increased solubility, high
water retention, gel formation, and emulsifying capacity and stabil-
ity. The biological activities of the protein were also found to be
enhanced. The hydroxyl radical scavenging capacity of the extracted
protein was doubled as reported by them [93]. Improvement in the
techno-functional characteristics of bitter melon seed protein was
reported by Naik et al. They employed pulsed ultrasound-assisted
extraction for this purpose. Their findings are also similar to those
described by Wang et al. Pulsed UAE is an innovative green tech-
nology used to extract and recover specific active compounds from
biological materials [94].
4.2 UAE for In the phytopharmaceutical extraction industry, UAE is considered

Phytopharmaceutical a key extraction technique for the preparation of a wide range of
Extraction herbal extracts. Such extracts contain various compounds of inter-
est such as antioxidants, aromas, capsaicinoids, and volatile com-
pounds. Herbal extracts have extensive pharmaceutical
applications [4].
4.2.1 Extracts with Anticancer activities of herbal extracts are mainly associated with
Anticancer Properties the polyphenolic compounds they possess. Polyphenols have the
ability to prevent cancer by diminishing or hindering the harmful
effects of free radicals on cells through their scavenging properties.
Their diverse chemical structures allow them in neutralizing free
radicals produced in the body. Polyphenolic compounds can pre-
vent oxidative stress to a level that does not harm cellular DNA and
regulatory protein synthesis metabolism. The importance of UAE
in cancer studies is mainly due to its capability in preserving the
anticarcinogenic properties of polyphenols in plant extracts. Activ-
ities of ultrasound extracted polyphenols have been studied exten-
sively in both in vitro and in vivo systems [95]. Polyphenols
extracted with ultrasound assistance from Thelephora ganbajun
exhibited superior antiproliferative activities toward human breast
(MCF-7), liver (HepG2), lung (A549), and colon (HT-29) cancer
cells compared to Soxhlet and maceration extraction methods
[96]. Polyphenolic compounds extracted from Trapa quadrispi-
nosa Roxb. showed effective antitumor action against Hela,
HepG2, and U251 tumor cells [97]. Berkani et al. used UAE to
prepare the extract of the herbal plant Zizyphus lotus. The extract
was found to contain a high amount of total phenolic and flavonoid
contents. The herbal extract significantly inhibited cell proliferation
on the MCF-7 and HepG2 tumor cell lines with IC50 values of
<0.05 and 3 ± 0.55 mg/mL, respectively [98]. Extracts of Oci-
mum basilicum and Ocimum canum were found to reduce the
proliferation of human breast cancer cells MCF-7, when extracts
were prepared ultrasonically as reported by Koolamchal et al.
[99]. Thus, UAE can preserve and enhance the anticarcinogenic
and antitumor activities of polyphenol extracts.
4.2.2 Extracts with Many ultrasound-assisted herbal extracts containing polyphenolic

Antimicrobial Properties compounds reported to have antimicrobial properties. As the use of
toxic organic solvents is less common in UAE, many extraction
protocols have accepted the use of UAE as a treatment method in
this regard. It has been observed that UAE not only improve the
extraction efficiency of the extracts but also improves their
biological activities [100]. UAE was used by Hu et al. to extract
flavonoids from Cyclocarya paliurus after initial enzymolysis.
Extracted flavonoids exhibited higher antimicrobial properties
against Salmonella typhi, Staphylococcus aureus, and Escherichia coli
compared to that of the extracts prepared by conventional extrac-
tion techniques [101]. Hydroethanolic extract of Erodium
glaucophyllum obtained through UAE and Soxhlet extraction was

analyzed for its antibacterial effects. The effect was studied on
Salmonella aureus, Salmonella enterica, Lactobacillus casei, Listeria
innocua, and Bifidobacterium lactis. The ultrasound extracted
hydroethanolic extract possessed higher amount of phenolic con-
tents and showed the highest antimicrobial activities. The antiviral
effect of the extract was also studied on hepatitis A virus and murine
norovirus. Antiviral activity of the UAE extract was found to be
higher than that of the Soxhlet extract [102].
Hashemi et al. carried out both continuous and pulsed
ultrasound-assisted extraction to extract essential oils from Aloysia
citriodora Palau leaves. They reported that the antibacterial and
antioxidant properties of the extracts increased in both the ultra-
sonic extraction processes compared to non-sonicated extraction
[103]. Four different extraction techniques, namely, UAE, macer-
ation, assisted solvent extraction (ASE), and supercritical fluid
extraction (SFE) were used to prepare different extracts from Lepi-
dium sativum. When the antimicrobial activities of the extracts
were analyzed, it was found that SFE extract exhibited showed
the highest antimicrobial activity for freeze-dried and air-dried
sprouts, followed by UAE, maceration, and ASE [104]. Ricardo
et al. evaluated the antibacterial activities of Annona cherimola Mill
leave extracts prepared with ultrasound assistance, Soxhlet, and
maceration extraction methods. They obtained the best results
against gram-positive bacteria using UAE water extract [105].
4.2.3 Extracts with Reports suggest that UAE improves the antidiabetic properties of
Antidiabetic Properties herbal extracts. Sunita et al. investigated the in vivo antidiabetic
properties of Gymnema sylvestre leave extracts prepared through
UAE and Soxhlet extraction. It was found that insulin released
from rat pancreatic RINm-5 F β cells was affected by the extracts
prepared by both the methods and the amount of extract used. The
ultrasound-assisted extract at a concentration of 100 μg/mL
showed up to about four times more insulin production from
RINm-5 F β cells than extracts obtained from Soxhlet extraction
[106]. Hypoglycaemic properties of ultrasonically extracted aque-
ous crude extracts of Azadirachta indica, Bryophyllum pinnatum,
Carica papaya, and Mikania cordata were measured by Sadat et al.
They have carried out in vivo studies on artificially developed
diabetic mice. According to their report, A. indica, B. pinnatum,
and C. papaya significantly reduced the plasma glucose level below
126 mg/dL. The effect was observed almost similar to the standard
antidiabetic drug glibenclamide, which reduced the plasma glucose
level to 100.35 ± 12.32 mg/dL [107].
These observations accomplished that UAE significantly
enhances the anticancer, anti-inflammatory, and antidiabetic prop-
erties of herbal extracts. This explains the importance of the phar-
maceutical application perspectives of the UAE.
5 Hybridization of UAE for Industrial Application
UAE has been combined with several other technologies to

improve extraction efficiency. In the food and beverage industries,
ultrasonic technology has already been expanded into various pro-
cessing technologies [4].
5.1 Combination of For fast and efficient extraction, the Combination of UAE with
UAE with Microwave Assisted Extraction utilizing simultaneous irradiation is
Microwave Assisted one of the most promising hybrid techniques. It is commonly used
Extraction for the extraction of oils from vegetable sources (Fig. 3a).
Fig. 3 Hybrid extraction techniques (a) ultrasound-microwave extraction, (b) ultrasound-supercritical fluid
extraction, and (c) ultrasound-DIC extraction. (Reproduced from Chemat et al. [4] with permission from
Elsevier)
5.2 Combination of Supercritical fluid extraction is based on the improved solvent

UAE with Supercritical power of fluids above their critical point. When UAE is combined
Fluid Extraction with supercritical fluid extraction, ultrasound enhances the mass
transfer of the targeting species from the solid phase to the solvent
for extraction (Fig. 3b).
5.3 Combination of Applications of this hybrid technique have been seen in vegetable
Ultrasound and oil industries and the production of sugar, wine, and fruit juices.
Extrusion Extraction
5.4 Combination of Application of this combined technology was seen in the sequential
UAE and Instantaneous extraction of oil and antioxidants. Using this hybrid technology, it
Controlled Pressure was possible to improve the kinetics and yields of antioxidant
Drop Process (DIC) extraction [108] (Fig. 3c).
6 Conclusion
Ultrasound-assisted extraction is the most valuable and promising

extraction technique widely used in laboratory-based research and
industrial applications. It has substantial advantages over conven-
tional extraction techniques in terms of yield, time of extraction,
selectivity, extract quality, and safety. Using UAE, bioactive com-
ponent extraction can be performed in very less time, at a relatively
low temperature, with optimal energy and solvent requirement. As
a non-thermal extraction technique, UAE facilitates the retention
of the functionality of bioactive compounds. UAE can be consid-
ered as a green extraction technology that may produce green
extract in concentrate form. UAE can preserve the anticarcinogenic
properties of polyphenolic extracts. Ultrasound-assisted herbal
extracts exhibit higher anticancer, antimicrobial, and antidiabetic
activities. The use of toxic organic solvents was less common in
UAE. Volatile compounds can be retained in ultrasound-assisted
extract as UAE operates at a lower temperature. The importance of
UAE in the extraction of various components for food industries
and bioactive compounds having significant pharmacological activ-
ities is discussed in this chapter. It may be concluded that UAE is
one of the most powerful tools that can be used as a versatile
extraction technique in the food, pharmaceutical, and biotech
industries.
References
1. Rasul MG (2018) Extraction, isolation and products: a comprehensive review. Chin Med
characterization of natural products from 13(1):1–26
medicinal plants. Int J Basic Sci Appl Comput 3. Chemat F, Rombaut N, Fabiano-Tixier AS,
2(6):1–6 Pierson JT, Bily A (2015) Green extraction:
2. Zhang Q-W, Lin L-G, Ye W-C (2018) Tech- from concepts to research, education, and
niques for extraction and isolation of natural economical opportunities. In: Chemat F,
Strube J (eds) Green extraction of natural 15. Adam F, Abert-Vian M, Peltier G, Chemat F
products. Wiley-VCH, Weinheim, pp 1–36 (2012) “Solvent-free” ultrasound-assisted
4. Chemat F, Rombaut N, Sicaire A-G, extraction of lipids from fresh microalgae
Meullemiestre A, Fabiano-Tixier A-S, Abert- cells: a green, clean and scalable process. Bior-
Vian M (2017) Ultrasound assisted extraction esour Technol 114:457–465
of food and natural products. Mechanisms, 16. Lavilla I, Bendicho C (2017) Fundamentals of
techniques, combinations, protocols and ultrasound-assisted extraction. In: Water
applications. A review. Ultrason Sonochem extraction of bioactive compounds. Elsevier,
34:540–560 Amsterdam, pp 291–316
5. Chemat F, Vian MA, Cravotto G (2012) 17. Crum LA, Mason TJ, Reisse JL, Suslick KS
Green extraction of natural products: concept (1998) Sonochemistry and sonolumines-
and principles. Int J Mol Sci 13(7): cence, vol 524. Springer Science & Business
8615–8627 Media, Dordrecht
6. Patist A, Bates D (2008) Ultrasonic innova- 18. Priego-Capote F, de Castro L (2007)
tions in the food industry: from the labora- Ultrasound-assisted digestion: a useful alter-
tory to commercial production. Innov Food native in sample preparation. J Biochem Bio-
Sci Emerg Technol 9(2):147–154 phys Methods 70(2):299–310
7. Barba FJ, Zhu Z, Koubaa M, Sant’Ana AS, 19. Chemat F, Tomao V, Virot M (2008)
Orlien V (2016) Green alternative methods Ultrasound-assisted extraction in food
for the extraction of antioxidant bioactive analysis. In: Handbook of food analysis
compounds from winery wastes and instruments. CRC Press, Boca Raton, pp
by-products: a review. Trends Food Sci Tech- 85–103
nol 49:96–109 20. Sanderson BJ (2004) Applied sonochemistry–
8. Mason TJ, Paniwnyk L, Lorimer J (1996) The the uses of power ultrasound in chemistry and
uses of ultrasound in food technology. Ultra- processing. By Timothy J. Mason and John
son Sonochem 3(3):S253–S260 P. Lorimer, Wiley-VCH Verlag, Weinheim,
9. Vilkhu K, Mawson R, Simons L, Bates D 2002, 303 pp, ISBN 3-527-30205-0. J
(2008) Applications and opportunities for Chem Technol Biotechnol 79(2):207–208
ultrasound assisted extraction in the food 21. Ruiz-Jiménez J, Priego-Capote F, de Castro
industry—a review. Innov Food Sci Emerg ML (2004) Identification and quantification
Technol 9(2):161–169 of trans fatty acids in bakery products by gas
10. Lourenço SC, Moldão-Martins M, Alves VD chromatography–mass spectrometry after
(2019) Antioxidants of natural plant origins: dynamic ultrasound-assisted extraction. J
from sources to food industry applications. Chromatogr A 1045(1–2):203–210
Molecules 24(22):4132 22. Cravotto G, Boffa L, Mantegna S, Perego P,
11. Sališová M, Toma Š, Mason T (1997) Com- Avogadro M, Cintas P (2008) Improved
parison of conventional and ultrasonically extraction of vegetable oils under high-
assisted extractions of pharmaceutically active intensity ultrasound and/or microwaves.
compounds from Salvia officinalis. Ultrason Ultrason Sonochem 15(5):898–902
Sonochem 4(2):131–134 23. Toma M, Vinatoru M, Paniwnyk L, Mason TJ
12. Mason TJ, Zhao Y (1994) Enhanced extrac- (2001) Investigation of the effects of ultra-
tion of tea solids using ultrasound. Ultrason- sound on vegetal tissues during solvent
ics 32(5):375–377 extraction. Ultrason Sonochem 8(2):
13. Ramos-De-La-Pena AM, Renard CM, 137–142
Wicker L, Montanez JC, Garcia-Cerda LA, 24. Ashokkumar M (2015) Applications of ultra-
Contreras-Esquivel JC (2014) Environmental sound in food and bioprocessing. Ultrason
friendly cold-mechanical/sonic enzymatic Sonochem 25:17–23
assisted extraction of genipin from genipap 25. Mason TJ (2012) Trends in sonochemistry
(Genipa americana). Ultrason Sonochem and ultrasonic processing. AIP Conf Proc
21(1):43–49 1433:21–26. American Institute of Physics
14. Pingret D, Fabiano-Tixier A-S, Chemat F 26. Wen C, Zhang J, Zhang H, Dzah CS,
(2013) Ultrasound-assisted extraction. In: Zandile M, Duan Y, Ma H, Luo X (2018)
Natural product extraction: principles and Advances in ultrasound assisted extraction of
applications, RSC green chemistry, no. 21. bioactive compounds from cash crops–a
Royal Society of Chemistry, Cambridge, pp review. Ultrason Sonochem 48:538–549
89–112
27. Priego-Capote F, De Castro ML (2004) Ana- 38. Khan MK, Abert-Vian M, Fabiano-Tixier
lytical uses of ultrasound I. Sample prepara- A-S, Dangles O, Chemat F (2010)
tion. Trends Anal Chem 23(9):644–653 Ultrasound-assisted extraction of polyphenols
28. Lorimer JP, Mason TJ (1987) Sonochemistry. (flavanone glycosides) from orange (Citrus
Part 1—the physical aspects. Chem Soc Rev sinensis L.) peel. Food Chem 119(2):
16:239–274 851–858
29. Bermúdez-Aguirre D, Mobbs T, Barbosa- 39. d’Alessandro LG, Kriaa K, Nikov I, Dimitrov
Cánovas GV (2011) Ultrasound applications K (2012) Ultrasound assisted extraction of
in food processing. In: Ultrasound technolo- polyphenols from black chokeberry. Sep
gies for food and bioprocessing. Springer, Purif Technol 93:42–47
New York, pp 65–105 40. Pan Z, Qu W, Ma H, Atungulu GG, McHugh
30. Mason TJ, Cobley A, Graves J, Morgan D TH (2012) Continuous and pulsed
(2011) New evidence for the inverse depen- ultrasound-assisted extractions of antioxi-
dence of mechanical and chemical effects on dants from pomegranate peel. Ultrason Sono-
the frequency of ultrasound. Ultrason Sono- chem 19(2):365–372
chem 18(1):226–230 41. Tabaraki R, Nateghi A (2011) Optimization
31. Esclapez M, Garcı́a-Pérez JV, Mulet A, Cárcel of ultrasonic-assisted extraction of natural
J (2011) Ultrasound-assisted extraction of antioxidants from rice bran using response
natural products. Food Eng Rev 3(2): surface methodology. Ultrason Sonochem
108–120 18(6):1279–1286
32. Sun Y, Liu D, Chen J, Ye X, Yu D (2011) 42. Corrales M, Toepfl S, Butz P, Knorr D,
Effects of different factors of ultrasound treat- Tauscher B (2008) Extraction of anthocya-
ment on the extraction yield of the all-- nins from grape by-products assisted by ultra-
trans-β-carotene from citrus peels. Ultrason sonics, high hydrostatic pressure or pulsed
Sonochem 18(1):243–249 electric fields: a comparison. Innov Food Sci
33. Kumar K, Srivastav S, Sharanagat VS (2021) Emerg Technol 9(1):85–91
Ultrasound assisted extraction (UAE) of bio- 43. Hossain MB, Brunton NP, Patras A, Tiwari B,
active compounds from fruit and vegetable O’donnell C, Martin-Diana AB, Barry-Ryan
processing by-products: a review. Ultrason C (2012) Optimization of ultrasound assisted
Sonochem 70:105325 extraction of antioxidant compounds from
34. Weng C-J, Yen G-C (2015) Natural plant marjoram (Origanum majorana L.) using
extracts as antioxidants for food response surface methodology. Ultrason
preservation. In: Handbook of antioxidants Sonochem 19(3):582–590
for food preservation. Elsevier, Amsterdam, 44. Anticona M, Blesa J, Lopez-Malo D,
pp 235–249 Frigola A, Esteve MJ (2021) Effects of
35. Sarikurkcu C, Tepe B, Daferera D, ultrasound-assisted extraction on physico-
Polissiou M, Harmandar M (2008) Studies chemical properties, bioactive compounds,
on the antioxidant activity of the essential oil and antioxidant capacity for the valorization
and methanol extract of Marrubium globo- of hybrid Mandarin peels. Food Biosci 42:
sum subsp. globosum (lamiaceae) by three 101185
different chemical assays. Bioresour Technol 45. Selahvarzi A, Ramezan Y, Sanjabi MR,
99(10):4239–4246 Namdar B, Akbarmivehie M,
36. Roselló-Soto E, Galanakis CM, Brnčić M, Mirsaeedghazi H, Azarikia F (2022) Optimi-
Orlien V, Trujillo FJ, Mawson R, zation of ultrasonic-assisted extraction of phe-
Knoerzer K, Tiwari BK, Barba FJ (2015) nolic compounds from pomegranate and
Clean recovery of antioxidant compounds orange peels and their antioxidant activity in
from plant foods, by-products and algae a functional drink. Food Biosci 49:101918
assisted by ultrasounds processing. Modeling 46. Rodrı́guez L, Plaza A, Méndez D, Carrasco B,
approaches to optimize processing condi- Tellerı́a F, Palomo I, Fuentes E (2022) Anti-
tions. Trends Food Sci Technol 42(2): oxidant capacity and antiplatelet activity of
134–149 aqueous extracts of common bean (Phaseolus
37. Pingret D, Fabiano-Tixier A-S, Le vulgaris L.) obtained with microwave and
Bourvellec C, Renard CM, Chemat FJ ultrasound assisted extraction. Plants 11(9):
(2012) Lab and pilot-scale ultrasound- 1179
assisted water extraction of polyphenols from 47. Banerjee J, Singh R, Vijayaraghavan R,
apple pomace. J Food Eng 111(1):73–81 MacFarlane D, Patti AF, Arora A (2017)
Bioactives from fruit processing wastes:
green approaches to valuable chemicals. Food ultrasound assisted method. Int J Biol Macro-
Chem 225:10–22 mol 125:621–629
48. Galanakis CM (2012) Recovery of high 60. Moorthy IG, Maran JP, Ilakya S, Anitha S,
added-value components from food wastes: Sabarima SP, Priya B (2017) Ultrasound
conventional, emerging technologies and assisted extraction of pectin from waste Arto-
commercialized applications. Trends Food carpus heterophyllus fruit peel. Ultrason
Sci Technol 26(2):68–87 Sonochem 34:525–530
49. Friedman MJ (1997) Chemistry, biochemis- 61. Kazemi M, Khodaiyan F, Hosseini SS (2019)
try, and dietary role of potato polyphenols. A Eggplant peel as a high potential source of
review. J Agric Food Chem 45(5):1523–1540 high methylated pectin: ultrasonic extraction
50. Thakur BR, Singh RK, Handa AK, Rao M optimization and characterization. LWT 105:
(1997) Chemistry and uses of pectin—a 182–189
review. Crit Rev Food Sci Nutr 37(1):47–73 62. Grassino AN, Brnčić M, Vikić-Topić D,
51. Bagherian H, Ashtiani FZ, Fouladitajar A, Roca S, Dent M, Brnčić SR (2016) Ultra-
Mohtashamy M (2011) Comparisons sound assisted extraction and characterization
between conventional, microwave-and ultra- of pectin from tomato waste. Food Chem
sound-assisted methods for extraction of pec- 198:93–100
tin from grapefruit. Chem Eng Process 63. Ponmurugan K, Al-Dhabi NA, Maran JP,
Process Intensif 50(11–12):1237–1243 Karthikeyan K, Moothy IG, Sivarajasekar N,
52. Wang W, Ma X, Xu Y, Cao Y, Jiang Z, Ding T, Manoj JJB (2017) Ultrasound assisted pectic
Ye X, Liu D (2015) Ultrasound-assisted heat- polysaccharide extraction and its characteriza-
ing extraction of pectin from grapefruit peel: tion from waste heads of Helianthus annus.
optimization and comparison with the con- Carbohydr Polym 173:707–713
ventional method. Food Chem 178:106–114 64. Wai WW, Alkarkhi AF, Easa AM (2009) Opti-
53. Xu Y, Zhang L, Bailina Y, Ge Z, Ding T, Ye X, mization of pectin extraction from durian rind
Liu DJ (2014) Effects of ultrasound and/or (Durio zibethinus) using response surface
heating on the extraction of pectin from methodology. J Food Sci 74(8):C637–C641
grapefruit peel. J Food Eng 126:72–81 65. Karbuz P, Tugrul N (2021) Microwave and
54. Minjares-Fuentes R, Femenia A, Garau M, ultrasound assisted extraction of pectin from
Meza-Velázquez J, Simal S, Rosselló C various fruits peel. J Food Sci Technol 58(2):
(2014) Ultrasound-assisted extraction of pec- 641–650
tins from grape pomace using citric acid: a 66. Sun J-X, Sun R, Sun X-F, Su Y (2004) Frac-
response surface methodology approach. Car- tional and physico-chemical characterization
bohydr Polym 106:179–189 of hemicelluloses from ultrasonic irradiated
55. Moorthy IG, Maran JP, Muneeswari S, sugarcane bagasse. Carbohydr Res 339(2):
Naganyashree S, Shivamathi CS (2015) 291–300
Response surface optimization of ultrasound 67. Zhang M, Zhang L, Cheung PCK, Ooi VEC
assisted extraction of pectin from pomegran- (2004) Molecular weight and anti-tumor
ate peel. Int J Biol Macromol 72:1323–1328 activity of the water-soluble polysaccharides
56. Maran JP, Priya B, Al-Dhabi NA, isolated by hot water and ultrasonic treatment
Ponmurugan K, Moorthy IG, Sivarajasekar from the sclerotia and mycelia of Pleurotus
N (2017) Ultrasound assisted citric acid tuber-regium. Carbohydr Polym 56(2):
mediated pectin extraction from industrial 123–128
waste of Musa balbisiana. Ultrason Sonochem 68. Chen Y, Luo H, Gao A, Zhu M (2011)
35:204–209 Ultrasound-assisted extraction of polysacchar-
57. Guandalini BBV, Rodrigues NP, Marczak ides from litchi (Litchi chinensis Sonn.) seed
LDF (2019) Sequential extraction of pheno- by response surface methodology and their
lics and pectin from mango peel assisted by structural characteristics. Innov Food Sci
ultrasound. Food Res Int 119:455–461 Emerg Technol 12(3):305–309
58. de Oliveira CF, Giordani D, Lutckemier R, 69. Raza A, Li F, Xu X, Tang J (2017) Optimiza-
Gurak PD, Cladera-Olivera F, Marczak LDF tion of ultrasonic-assisted extraction of anti-
(2016) Extraction of pectin from passion fruit oxidant polysaccharides from the stem of
peel assisted by ultrasound. LWT Food Sci Trapa quadrispinosa using response surface
Technol 71:110–115 methodology. Int J Biol Macromol 94:335–
59. Hosseini SS, Khodaiyan F, Kazemi M, Najari 344
Z (2019) Optimization and characterization 70. Yang B, Wu Q, Luo Y, Yang Q, Wei X, Kan J
of pectin extracted from sour orange peel by (2019) High-pressure ultrasonic-assisted
extraction of polysaccharides from Hovenia surface methodology. BMC Complement

dulcis: extraction, structure, antioxidant activ- Altern Med 14(1):1–10
ity and hypoglycemic. Int J Biol Macromol 81. Lin X, Wu L, Wang X, Yao L, Wang L (2021)
137:676–687 Ultrasonic-assisted extraction for flavonoid
71. Albuquerque BR, Heleno SA, Oliveira MBP, compounds content and antioxidant activities
Barros L, Ferreira ICFR (2021) Phenolic of India Moringa oleifera L. leaves: simulta-
compounds: current industrial applications, neous optimization, HPLC characterization
limitations and future challenges. Food and comparison with other methods. J Appl
Funct 12(1):14–29 Res Med Aromat Plants 20:100284
72. Bouafia M, Colak N, Ayaz FA, Benarfa A, 82. Pi J, Jin P, Tang X, Liu Y, Mai X, Xie X, Li P,
Harrat M, Gourine N, Yousfi M (2021) The Fan H, Luo L (2022) Microwave-assisted
optimization of ultrasonic-assisted extraction polyethylene glycol-based aqueous
of Centaurea sp. antioxidative phenolic com- two-phase extraction of gallic acid and ellagic
pounds using response surface methodology. acid from Euonymus alatus: process optimiza-
J Appl Res Med Aromat Plants 25:100330 tion, quantification analysis and antioxidant
73. Martı́nez-Ramos T, Benedito-Fort J, Watson activity. Chem Eng Process Process Intensif
NJ, Ruiz-López II, Che-Galicia G, Corona- 172:108772
Jiménez E (2020) Effect of solvent composi- 83. Ali MC, Chen J, Zhang H, Li Z, Zhao L, Qiu
tion and its interaction with ultrasonic energy H (2019) Effective extraction of flavonoids
on the ultrasound-assisted extraction of phe- from Lycium barbarum L. fruits by deep
nolic compounds from Mango peels (Mangi- eutectic solvents-based ultrasound-assisted
fera indica L.). Food Bioprod Process 122: extraction. Talanta 203:16–22
41–54 84. Xu L, He W, Lu M, Yuan B, Zeng M, Tao G,
74. Rodsamran P, Sothornvit R (2019) Extrac- Qin F, Chen J, Guan Y, He Z (2018)
tion of phenolic compounds from lime peel Enzyme-assisted ultrasonic-microwave syner-
waste using ultrasonic-assisted and gistic extraction and UPLC-QTOF-MS anal-
microwave-assisted extractions. Food Biosci ysis of flavonoids from Chinese water chestnut
28:66–73 peels. Ind Crop Prod 117:179–186
75. Ghafoor K, Choi YH, Jeon JY, Jo IH (2009) 85. Zakynthinos G, Varzakas T (2016) Carote-
Optimization of ultrasound-assisted extrac- noids: from plants to food industry. Curr Res
tion of phenolic compounds, antioxidants, Nutr Food Sci 4(1):38–51
and anthocyanins from grape (Vitis vinifera) 86. Belwal T, Singh G, Jeandet P, Pandey A,
seeds. J Agric Food Chem 57(11): Giri L, Ramola S, Bhatt ID, Venskutonis PR,
4988–4994 Georgiev MI, Clément C (2020) Anthocya-
76. Şahin S, Şamlı R (2013) Optimization of olive nins, multi-functional natural products of
leaf extract obtained by ultrasound-assisted industrial relevance: recent biotechnological
extraction with response surface methodol- advances. Biotechnol Adv 43:107600
ogy. Ultrason Sonochem 20(1):595–602 87. Pinela J, Prieto M, Pereira E, Jabeur I, Bar-
77. Panche AN, Diwan AD, Chandra SR (2016) reiro MF, Barros L, Ferreira IC (2019) Opti-
Flavonoids: an overview. J Nutr Sci 5:e47 mization of heat-and ultrasound-assisted
78. Wang B, Qu J, Luo S, Feng S, Li T, Yuan M, extraction of anthocyanins from Hibiscus sab-
Huang Y, Liao J, Yang R, Ding C (2018) dariffa calyces for natural food colorants.
Optimization of ultrasound-assisted extrac- Food Chem 275:309–321
tion of flavonoids from olive (Olea europaea) 88. Ochoa S, Durango-Zuleta MM, Osorio-
leaves, and evaluation of their antioxidant and Tobón JF (2020) Techno-economic evalua-
anticancer activities. Molecules 23(10):2513 tion of the extraction of anthocyanins from
79. Upadhyay R, Nachiappan G, Mishra HN purple yam (Dioscorea alata) using
(2015) Ultrasound-assisted extraction of fla- ultrasound-assisted extraction and conven-
vonoids and phenolic compounds from Oci- tional extraction processes. Food Bioprod
mum tenuiflorum leaves. Food Sci Biotechnol Process 122:111–123
24(6):1951–1958 89. Ye J, Feng L, Xiong J, Xiong Y (2011)
80. Ghasemzadeh A, Jaafar HZ, Karimi E, Rah- Ultrasound-assisted extraction of corn carote-
mat A (2014) Optimization of ultrasound- noids in ethanol. Int J Food Sci Technol
assisted extraction of flavonoid compounds 46(10):2131–2136
and their pharmaceutical activity from curry 90. Li H, Pordesimo L, Weiss J (2004) High
leaf (Murraya koenigii L.) using response intensity ultrasound-assisted extraction of oil
from soybeans. Food Res Int 37(7):731–738
91. Luque-Garcıa J, De Castro ML (2004) activity of polyphenol extracts: a review.

Ultrasound-assisted soxhlet extraction: an Food Biosci 35:100547
expeditive approach for solid sample treat- 101. Hu W-B, Yang Z-W, Wang W-J (2019)
ment: application to the extraction of total Enzymolysis-ultrasonic assisted extraction of
fat from oleaginous seeds. J Chromatogr A flavanoid from Cyclocarya paliurus (Batal)
1034(1–2):237–242 Iljinskaja: HPLC profile, antimicrobial and
92. Sharma A, Gupta M (2006) Ultrasonic antioxidant activity. Ind Crop Prod 130:
pre-irradiation effect upon aqueous enzymatic 615–626
oil extraction from almond and apricot seeds. 102. Abdelkebir R, Alcántara C, Falcó I,
Ultrason Sonochem 13(6):529–534 Sánchez G, Garcia-Perez JV, Neffati M, Lor-
93. Wang F, Zhang Y, Xu L, Ma H (2020) An enzo JM, Barba FJ, Collado MC (2019)
efficient ultrasound-assisted extraction Effect of ultrasound technology combined
method of pea protein and its effect on pro- with binary mixtures of ethanol and water
tein functional properties and biological activ- on antibacterial and antiviral activities of Ero-
ities. LWT 127:109348 dium glaucophyllum extracts. Innov Food Sci
94. Naik M, Natarajan V, Modupalli N, Emerg Technol 52:189–196
Thangaraj S, Rawson AJL (2022) Pulsed 103. Hashemi SMB, Khaneghah AM, Koubaa M,
ultrasound assisted extraction of protein Barba FJ, Abedi E, Niakousari M, Tavakoli J
from defatted Bitter melon seeds (Momardica (2018) Extraction of essential oil from Aloysia
charantia L.) meal: kinetics and quality mea- citriodora Palau leaves using continuous and
surements. LWT 155:112997 pulsed ultrasound: kinetics, antioxidant activ-
95. Dei Cas M, Ghidoni R (2018) Cancer preven- ity and antimicrobial properties. Process Bio-
tion and therapy with polyphenols: chem 65:197–204
sphingolipid-mediated mechanisms. Nutri- 104. Rafińska K, Pomastowski P, Rudnicka J,
ents 10(7):940 Krakowska A, Maruśka A, Narkute M, Bus-
96. Xu D-P, Zheng J, Zhou Y, Li Y, Li S, Li H-B zewski B (2019) Effect of solvent and extrac-
(2016) Extraction of natural antioxidants tion technique on composition and biological
from the Thelephora ganbajun mushroom activity of Lepidium sativum extracts. Food
by an ultrasound-assisted extraction tech- Chem 289:16–25
nique and evaluation of antiproliferative activ- 105. Aguilar-Villalva R, Molina GA, España-Sán-
ity of the extract against human cancer cells. chez BL, Dı́az-Peña LF, Elizalde-Mata A,
Int J Mol Sci 17(10):1664 Valerio E, Azanza-Ricardo C, Estevez M
97. Li F, Mao Y-D, Wang Y-F, Raza A, Qiu L-P, (2021) Antioxidant capacity and antibacterial
Xu X-Q (2017) Optimization of ultrasonic- activity from Annona cherimola phytochem-
assisted enzymatic extraction conditions for icals by ultrasound-assisted extraction and its
improving total phenolic content, antioxidant comparison to conventional methods. Arab J
and antitumor activities in vitro from Trapa Chem 14(7):103239
quadrispinosa Roxb. residues. Molecules 106. Sheoran S, Panda BP, Admane PS, Panda AK,
22(3):396 Wajid S (2015) Ultrasound-assisted extrac-
98. Berkani F, Serralheiro ML, Dahmoune F, tion of gymnemic acids from Gymnema syl-
Ressaissi A, Kadri N, Remini H (2020) Ultra- vestre leaves and its effect on insulin-
sound assisted extraction of phenolic com- producing RINm-5 F β cell lines. Phytochem
pounds from a jujube by-product with Anal 26(2):97–104
valuable bioactivities. Processes 8(11):1441 107. Sadat A, Sultana A, Rahman MA, Sharma D,
99. Anusmitha KM, Aruna M, Job JT, Khan MA, Khan MMR, Azad MAK (2021)
Narayanankutty A, Benil P, Rajagopal R, Study of hypoglycemic properties of aqueous
Alfarhan A, Barcelo D (2022) Phytochemical fresh leaves extracts of Azadirachta indica,
analysis, antioxidant, anti-inflammatory, anti- Bryophyllum pinnatum, Carica papaya &
genotoxic, and anticancer activities of differ- Mikania cordata prepared by ultrasound
ent Ocimum plant extracts prepared by assisted extraction method. Int J Innov Sci
ultrasound-assisted method. Physiol Mol Res Technol 6(10):942–946
Plant Pathol 117:101746 108. Allaf T, Tomao V, Ruiz K, Chemat F (2013)
100. Dzah CS, Duan Y, Zhang H, Wen C, Instant controlled pressure drop technology
Zhang J, Chen G, Ma H (2020) The effects and ultrasound assisted extraction for sequen-
of ultrasound assisted extraction on yield, tial extraction of essential oil and antioxidants.
antioxidant, anticancer and antimicrobial Ultrason Sonochem 20(1):239–246
Chapter 6
Super- and Subcritical Fluid Extraction of Nutraceuticals

and Novel Phytocompound
Pankaj Koirala, Saphal Ghimire, Sampurna Rai,
Abstract
Alternative methods are currently being investigated to reduce the overuse of organic solvents, which have
serious environmental and health consequences. Certain promising green technology-based alternative
extractions are employed in a wide range of bioactive and nutraceutical extractions, including ultrasonica-
tion, microwave-assisted extraction, pressurized liquid extraction, and enzyme-assisted extraction. Super-
and subcritical fluid extractions, on the other hand, are sufficiently sophisticated green technologies that
have superior efficacy and selectivity for the extraction of nonpolar and low-polar constituents. In this
regard, this chapter gives an overview of the process, the theory of super- and subcritical extraction, and the
role of pivotal variables for optimal extraction. Additionally, recent findings of the principal phytochemical
and bioactive compounds extracted by this process with their nature, biological activities, and stability
during and after processing are discussed.
Key words Supercritical fluid extraction, Subcritical fluid extraction, Natural products, Pharmaceuti-
cal, By-products
1 Introduction
Naturally available bioactive compounds, mainly from plants, algae,

microorganisms, and other by-products, have potent health-
promoting compounds. These compounds include essential oils,
peptides, phytosterols, phenols, flavonoids, alkaloids, and terpe-
noids, which have excellent antioxidant, antimicrobial, anticancer-
ous, and anti-inflammatory properties. The extraction of these
bioactive constituents from plants is still a topic of interest due to
its potential in the research and production of functional ingredi-
ents and nutraceuticals. Yet challenges to maintaining structural
stability and functionality after extraction exist because of its sensi-
tivity to environmental condition.
129
130 Pankaj Koirala et al.
The optimal extraction of these bioactive with preserved

functional characteristics is crucial. The recovery of functional
compounds is dependent on parameters such as thermal exposure,
type of solvent, time, matrix properties, pressure, and ratio of
solvent to matrix [1]. Traditionally employed extraction techniques
(maceration, distillation, and Soxhlet extraction with organic or
inorganic solvents) has been reported to have several drawbacks,
such as low efficiency, higher organic solvent utilization, prolonged
extraction time, and targeted compounds degradation. To over-
come these drawbacks, methods such as sub- and supercritical fluid
extraction are being explored. Supercritical fluids are extensively
explored because their low or absent surface tension results in no
boundary between the liquid and gas phases. In this state, the
compound is dispersed like a gas and dissolves bioactive com-
pounds as in liquid. Supercritical fluid-based extraction improves
the extraction speed, density-dependent selectivity, and eventually
providing high-quality extracts with higher extraction yield when
compared to traditional extraction. Several fluids under supercriti-
cal conditions, including carbon dioxide, ethane, ethanol, propane,
water, ammonia, and methanol, are used for the recovery of bio-
active compounds. For instance, supercritical carbon dioxide
(SC-CO2) based extraction of essential oils allows for a higher
quality oil collection with preserved functional properties such as
antioxidants, antimicrobials, and other properties of essential
oils [2].
Besides supercritical fluid extraction, subcritical fluid extraction
of bioactive molecules has also gained interest. Certain solvents,
such as propane, ethanol, water, and carbon dioxide, are commonly
utilized in subcritical form for nutraceutical recovery from plants
because they are promptly recycled, minimally or nontoxic,
eco-friendly, and mostly nonflammable. Subcritical waters are
most abundantly used for extraction because they improve the
extraction yield and reduce the extraction time compared to tradi-
tional solvents [1, 3]. Under subcritical conditions, the fluid has
low dielectric constant, surface tension, and viscosity, which affect
polarity, whereas it has increased diffusivity, mass transfer, and
dissociation constant, which have high affinity for nonpolar solutes.
Subcritical water, especially, shows the required properties under
the specified conditions as mentioned above and is most abun-
dantly utilized for the optimal extraction of phenols and other
phytochemicals [1]. At specific conditions, the polarity of subcriti-
cal fluids varies, which can dissolve a wide range of compounds with
medium- to low-polarity. Both methods can be applied for the
extraction of compounds such as proteins, polysaccharides, antiox-
idants, essential oils, and other phytochemicals, depending on the
polarity of the intended compound. The polarity of the both sub-
and supercritical fluids can be adjusted by the addition of different
co-modifiers, so optimal extraction can be achieved without mod-
ifying functionality [4].
Super- and Subcritical Fluid Extraction of Nutraceuticals. . . 131
2 Principle of Super- and Subcritical Fluid Extraction
A pressure-volume-temperature (PVT) diagram provides an over-

view of the different states of a pure substance. The known phases
(solid, liquid, and gas) are separated by boundaries called “phase
boundaries,” where the two phases co-exist under specific
temperature-pressure combinations. Herein, for pure substances,
it has only a degree of freedom as in the two phases (liquid-vapor,
solid-liquid, or solid-vapor), thus the equilibrium pressure is a
function of the temperature in each case. Under the phase diagram,
the three states co-exist at a point called the “triple point,” whereby
the liquid-vapor boundary ceases to exist at a point called the
“critical point,” and the temperature and pressure at this point are
called the “critical temperature” and “critical pressure,” respec-
tively. At this point (the critical point), the liquid and gas phases
have the same density, and further increasing temperature or pres-
sure causes the liquid-gas phase boundary to disappear. Beyond the
critical point, separation of the fluid and gas phases is no longer
possible, and the state is called the “supercritical fluid state,” where
the characteristics of the fluids are drastically changed. In a super-
critical fluid, solubilities vary to a great extent with a minor change
in density, just as different solvents have different solubilities under
the same circumstances. Solubility of any fluid is directly propor-
tional to the fluid density at constant pressure. Based on the tar-
geted phytochemicals, the solvent and its parameter selection
should be made wisely, thereby allowing selective bioactive to be
extracted via supercritical fluid extraction. In general, these fluids
are broadly classified into two groups: (1) low critical
temperature—CO2, ethane, and propane and (2) high-critical
temperature—alkanes, methanol, and water. Solvent power of
high critical temperature is much higher than low critical
temperature.
Subcritical fluid, on the other hand, is a pressurized hot fluid
whose temperature falls between the critical point temperature and
the atmospheric boiling point. Under such conditions, the fluid has
a low dielectric constant and an increased dissolution property due
to the weakening of the hydrogen bonds by high temperature and
pressure, and this makes subcritical fluid more like less-polar
organic solvents. Subcritical water (SWE), for example, uses liquid
water as extractant at temperatures ranged in between 100 °C/
273 K, 0.1 MPa and 374 °C/647 K, 22.1 MPa with pH 6 and
extraction time 30 min has excellent yield in comparison to the
ethanol extraction [5]. Any fluid can be kept in a liquid state by
applying enough pressure at high temperatures. Changes in tem-
perature and pressure have a large effect on the dielectric constant.
Additionally, through the processes of diffusion and convection,
subcritical fluids are noted to promote mass transfer. Disruption in
the solute-solute or solute-solvent interaction is usually seen in the

application of subcritical fluid as it lowers the activation energy for
the desorption process. Also, elevated pressure in subcritical fluid
can help with extraction by forcing water into the matrix via pores,
which is impossible at normal pressure [1]. Extraction principle of
the subcritical fluid can be summarized as it is governed by solute-
solvent interaction in sample matrix and mass transfer principle
(diffusion, convection, and partitioning equilibrium) [3]. Although
numerous fluids are experimented with for nutraceutical extraction,
supercritical CO2 and subcritical water are the most common fluids
used for the extraction of the bioactive. This is due to the fact that
supercritical CO2 has fast diffusivity and nearly zero surface tension,
leading to extremely efficient extraction, while water is nontoxic
and eco-friendly by nature.
3 Properties of Sub- and Supercritical Fluids
The properties of both sub- and supercritical fluids depend on the

types of solvent, such as ethanol, methanol, water, carbon dioxide,
ammonia, or n-hexane, used. Thus, to determine the solute speci-
ficity and high solvation power of sub- and supercritical fluids,
understanding of the solvent-dependent properties of subcritical
and supercritical fluids under the aforementioned conditions is
required.
Viscosity For both sub- and supercritical fluids, with increasing

temperature, viscosity decreases by diffusion and partitioning equi-
librium. The viscosity of the supercritical fluid is similar to that of
gas, which means it is approximately 1/10 that of liquid.
Dielectric Constant (ε) In general, the dielectric constant of sub-

critical and supercritical fluids varied widely with temperature and
pressure. For example, water has a high dielectric constant (80) at
room temperature because of its extensive hydrogen bonding
structure, while subcritical water at the same time has a lower
dielectric constant, which is 25 at 250 °C and 25 bar pressure,
similar to methanol (ε = 33) and ethanol (ε = 24), that can dissolve
bioactive compounds with low to medium polarity [1]. This indi-
cates that the dielectric constant of the fluids is reduced to that of
organic solvents.
Fluid Surface Tension and Diffusivity Since the vapor-liquid

boundary is terminated in supercritical fluids, there is no surface
tension. Such fluids have high diffusivity that allows easy diffusion
as gas through the solid matrix. In regard to subcritical fluid, with
the increase of temperature, surface tension of fluid will decrease
steadily. Moreover, the diffusivity of the solvent increases as a result

of lower surface tension with temperature, and the solvating capac-
ity thus rises proportionally with increasing diffusivity.
Density The density of a supercritical fluid is usually between that

of a liquid and that of a gas and is closer to that of a liquid. Factors
such as pressure and temperature fluctuate the fluid’s density of
both (sub- and supercritical fluids). Herein, density of fluid
decreases with increasing temperature under constant pressure.
While, in the case of temperature, increasing pressure increases
fluid density when the odd factor remains constant, and vice versa.
Solvating Power The solubility of any fluid is directly propor-

tional to the fluid density at constant pressure. In supercritical
fluid, solvation power of fluid increases with density and reaches
maximum in the critical pressure. Solvent power of high critical
temperature solvents is much higher than low critical temperature
solvent. Likewise, investigation in subcritical fluid showed that the
solvating power of subcritical fluid is similar to that of organic
solvents. Solubility and mass transfer of subcritical fluids, especially
water, are dependent on temperature; at higher temperatures, the
dielectric constant is lower, allowing better solubility of moderately
polar compounds [6].
Polarity Polar solutes are most soluble in polar solvents; however,

nonpolar fluids can be effective solvents for many moderately polar
molecules. Especially for supercritical CO2, several cosolvents
(nonpolar organic solvents) are typically added to increase polarity
since polar compounds have low selectivity [7]. In terms of subcrit-
ical fluids, their properties are comparable to those of organic
solvents, resulting in an increased solvating power to dissolve com-
pounds with varying polarity, that is, medium- and low polarity.
The polarity of the fluid decreases with increasing pressure, while it
increases with increasing temperature, and low- or nonpolar com-
pounds are easily dissolved.
4 Factors Affecting Extraction Yields
4.1 Sample and In both subcritical and supercritical fluid extraction, a sample with a
Its Preparation smaller particle size has an increased interfacial area and minimal
diffusion paths of raw material, which improves the solute extrac-
4.1.1 Sample Matrix
tion rate. An investigation by Peng et al. [8] on the extraction of
and Size
tocopherol from roselle seed oil by SC-CO2 extraction under the
given conditions (temperature: 40 °C and pressure: 30 MPa)
demonstrated that the yield was lowered with increasing particle
size. The size of the solid matrix governs the mass transfer kinetics,
as small molecules diffuse easily and access the supercritical fluid
throughout the sample matrix. However, extraction from the small
sample matrix has the drawbacks of compound re-absorption and
clumping, which can decrease fluidized bed velocity, clog the filters,
and eventually decrease extraction efficiency. Thus, the optimal
particle size of the sample is recommended to maximize extraction
yield while minimizing the possibility of particle agglomeration
[3, 9].
4.1.2 Moisture and The extraction rate and yield are significantly influenced by the
Equilibrium Time moisture content of the sample. The extraction yield is generally
unaffected by a small amount of moisture, but yield steadily
decreases as the moisture percentage increases in the sample.
Kostrzewa et al. [10] investigated the effect of moisture content
on extraction yield and found that moisture had no effect on
carotenoid extraction and extraction efficiency until the moisture
content was less than 7.5 g/100 g. However, the higher moisture
in the sample showed lower carotenoid recovery and also necessi-
tated carotenoid concentration after separation.
4.2 Cosolvent/ Cosolvents, when dissolved in the fluid in different proportions,

Modifier aids the solvation power of sub- and supercritical fluids toward
targeted compounds by modifying the fluid density and polarity.
The cosolvent, either polar or nonpolar, helps to change the polar-
ity of the supercritical fluid. Also, the cosolvent percentage in the
extractant has an impact on the yield of selected compounds
because it affects the solubility of the targeted compounds
[11]. Herein, supercritical CO2 has low polarity and limits the
extraction of nonpolar and lipophilic compounds. Therefore, a
cosolvent (ethanol) can be added to enhance the affinity and solu-
bility of polyphenolic compounds, which eventually increases
extraction yield [12]. In the case of subcritical fluid extraction,
the addition of solvent modifiers, such as natural deep eutectic
solvents, increased the phenolic compound recovery as compared
to the exclusive use of subcritical water [13]. The cosolvent is most
important for maximum extraction, but it also reduces the specific-
ity of the intended molecules, so careful consideration should be
given to cosolvent selection.
4.3 Extraction The extraction yield of the bioactive compounds is proportionate

Procedure to the extraction process employed. Supercritical and subcritical
fluid extraction methods overcome the cons of several traditional
extraction methods. However, these methods still have limitations,
which could be counteracted by the parallel assistance of several
green technologies. An integration of either of the novel
technologies—enzyme, ultrasound, microwave, pulse electrified,
etc.—into supercritical fluid extraction has proven to be efficacious
in nutraceutical extraction specificity with the structural modifica-

tion of targeted compounds [14, 15]. Enzyme-assisted supercritical
fluid extraction, for instance, produces high-quality bioactive com-
pounds, though the choice of enzyme and operational parameters
must still be considered for maximum yield [16].
Similarly, novel technology assistance promotes excessive reten-
tion of functional quality and also increases extraction efficiency in
sub- and supercritical fluid extraction. For example, microwave-
assisted subcritical extraction of anthocyanin increased efficiency
to 85.8% with higher antioxidant activity [17].
4.4 Extraction The crossover behavior of the supercritical fluid in the solubility
Parameters isotherms is observed by the pressure-temperature relationship and
its effect on density and volatility. On increasing the temperature,
4.4.1 Temperature
the density decreases under the isobaric process, while the volatility
of the solute increases with temperature, thus reducing the extrac-
tion rate. Inversion pressure (crossover pressure) has the critical
role in defining temperature selection for optimum extraction. To
clarify, low temperature permits density to dominate under the
crossover pressure that leads higher extraction yield, while over
crossover pressure, higher temperature allows solute vapor pressure
to dominate, resulting higher extraction [3].
At the subcritical phase, increasing temperature improves the
dissociation constant by decreasing surface tension, where mass
transfer is supported by the disruption of intermolecular forces in
the sample matrix and increased solubility. The diffusivity of sub-
critical water, for example, increases with increasing temperature,
whereas the dielectric constant, surface tension, and viscosity are
significantly reduced. Low dielectric constants facilitate the extrac-
tion of low-polar organic compounds due to high solubility in
low-polarity fluids [18]. On the adjustment of pressure-
temperature, one should be conscious of the maximum tempera-
ture for the extraction of selected compounds because high tem-
peratures change the structural composition by causing thermal
degradation reactions and the neoformation of Maillard com-
pounds [13, 19].
4.4.2 Pressure Under the constant temperature, the solubility of the bioactive
compounds increases with increasing pressure due to an increment
in density and solvation power, which successively increases the
extraction kinetics. However, extremely high pressure hinders
fluid diffusion [20], which could reduce yield. Thus, optimal pres-
sure based on extract specificity is preferred. The effect of the
pressure, which is also dependent on temperature variation,
depends on the types of selected compounds. If the pressure is
near the critical pressure, solubility increases with decreasing tem-
perature, whereas at higher pressures, solubility increases with
increasing temperature.
Regarding subcritical extraction, pressure has a negligible influ-

ence as compared to the temperature on the phase and character-
istics of some subcritical solvents. Water, in particular, is relatively
incompressible below 300 °C, which thereby does not affect the
physical properties in the liquid phase [1]. However, pressure is still
important for subcritical solvents such as sub-CO2 and sub-ethanol
because the pressure in such fluids has a significant impact on
extraction yield [4].
4.4.3 Flow Rate The flow rate of the sub- and supercritical solvents has a direct
impact on the solvent-to-feed ratio, contact time, and mass transfer
resistance. Increasing the flow rate improves extraction efficiency by
minimizing the mass transfer resistance. With the low flow rate, the
solute saturates in the solvent as the result of axial dispersion, while
for the very high flow rate, yield is minimal due to insufficient
contact time [21], indicating the appropriate flow rate as a crucial
factor for maximum yield. However, too high solvent flow rate
cause substantial dilution of the extracts, necessitating an additional
concentration step following extraction [1].
4.4.4 Time Initially, the extraction yield in super- and subcritical fluid extrac-
tion increases with time, but with prolonged extraction time, this
rate drops. An investigation of the tocopherol yield from roselle
seed oil by supercritical fluid extraction demonstrated an increment
in extraction yield with increasing temperature for a certain time,
which was limited afterward [8]. This can be explained by the fact
that the process reached the saturation point and/or by thermal
degradation of bioactive compounds [22]. Consequentially, the
yield tends to decline with increasing extraction time. In contrast,
too short extraction time results in minimal solid-solvent interac-
tion, leading to a low yield.
5 Comparison with Conventional Method
The conventional methods of extraction are maceration, hydrodis-

tillation, and Soxhlet extraction [3]. These methods are outdated
due to low efficiency, high targeted compound degradation,
requires considerable period of time and impact on the environ-
ment triggered by wastage of unrecovered solvents is massive
[23]. In general, conventional solid-liquid extraction (SLE) and
liquid-liquid extraction (LLE) methods utilize organic solvents,
such as ethyl acetate, hexane, ethanol, acetone, and methanol, of
which the latter reigns as the most toxic. Also, the majority of the
solvents used in conventional SLE and LLE methods are nonbio-
degradable, volatile, and highly flammable [24]. The solvent recov-
ery rate in conventional extraction is rarely satisfactory, which
prompts solvents to be dispensed into the environment [25, 26].
The current advancement of green technology-based extrac-

tion has allowed for the selective extraction of phytochemicals with
low labor intense, less solvent requirement, and ease of automation.
These have overcome the challenges of the nutraceuticals and
pharmaceutical industries, where the results of conventional extrac-
tion technology adoption were not satisfying [23, 26, 27]. Pharma-
ceutical industries are now more concerned about the stability and
functionality of bioactive compounds during and after extraction as
they are susceptible to structural modification due to minor envi-
ronmental changes. The consideration of the process of deploying
solvent in green technology dictates not only the efficiency but also
the economic aspect [28]. The neoteric/green solvents, such as,
supercritical CO2, deep eutectic solvent (DES), gas-expanded sol-
vents (GXLs), switchable solvents, liquid polymer solvents (Poly-
ethylene glycol), and bio-based/renewable solvent
(γ-valerolactone) are also referred as green solvent, as they have
minimum contribution on the organic pollution due to solvent loss
[26, 28]. Efficiency, selectivity, and diligency are very important
factors in an extraction process for bioactives and nutraceuticals
[29]; hence, the industrial inclination toward sub- and supercritical
fluids is increasing.
At the supercritical state, CO2 acts as both a gas and a liquid,
rendering diffusion like a gas and solubility like a liquid. The
recovery process of the solvent after extraction is convenient as it
transforms from its supercritical state into its gaseous state at room
temperature, which enables the omission of an energy-intensive
recovery process. The interphase change due to the shift in the
thermodynamic state of the solvent suitably supports spontaneous
separation, resulting in a high degree of extract purity. The crucial
reasons for selecting CO2 for supercritical extraction are its moder-
ate critical temperature and pressure (31 °C and 7.38 MPa) and the
preservation of its potential oxidizing component. The selectivity
and solubility of the supercritical CO2 can be modulated by adjust-
ing its temperature, pressure, and cosolvent [29, 30], which is not
achievable in a conventional extraction technique. Pereira et al.
[31] studied the extraction yield and antioxidant capacity of Portu-
guese myrtle (Myrtus communis L.) leaves and fruits using SFE and
liquid phase extract (LPE) with diisopropyl ether. It was found that
the average extract yields of leaves and fruits using SFE were around
36 and 70 folds higher, as compared to LPE with diisopropyl ether.
The mean extraction yield using SFE was found to be 10.8% and
14.1% for leaves and fruits respectively, whereas only 0.3% and 0.2%
for LPE. Also, the extract obtained by SFE contains a significantly
higher content of polyphenols as well as possesses a higher antioxi-
dant capacity in contrast to LPE. Naturally, the bioactive com-
pound occurs in a multi-component state, so the prudential
selectivity of the extraction process is a decisive attribute
[32]. Because CO2 has a low polarity, it cannot be used to extract
polar compounds [33]; therefore, a supplementary cosolvent is

required to boost the extraction, especially when the target com-
pound is polar and has a long molecular structure.
Similarly, water, CO2, propane, etc., at subcritical state is also
eligible for its application as a solvent to extract nutraceutical
relevant bioactive compounds. In case of water, it is a polar solvent
and has high dielectric constant at an ambient temperature and
pressure, hence it is not applicable for the extraction of nonpolar
compounds. However, subcritical phase of water has low dielectric
constant, which is in par with that of organic solvents. This allows
water to behave like conventional organic solvents applicable for
low-polar target compound [1]. Additionally, it has perks of being
high selective without compromising economic and environmental
aspects. The cohesive (solute-solute) and adhesive (solute-matrix)
interaction in matrix is interrupted by subcritical water, due to
which desorption process is activated on the energy level far less
than that of conventional extraction. Ko et al. [34] extracted bio-
active compounds from Crassulaceae (Orostachys japonicus
A. Berger) and came into the conclusion that SWE was efficient
on extracting total phenols by 3.7–11.5 folds, flavonoids by
1.8–3.2 folds against methanol, and ethanol extraction done at
25 and 60 °C for 2 h. Thus, apparent superiority was reflected
against conventional (methanol, Soxhlet, hot-water) extraction
techniques [35]. Moreover, the quality characteristics, bioactive
phytochemicals, volatile compounds, and antioxidant capacities of
virgin avocado oil extracted using a couple of green methods,
namely, subcritical CO2 extraction and ultrasound-assisted aqueous
extraction (UAAE), are compared with the oil extracted using the
conventional solvent extraction. Results indicate the quality prop-
erties of avocado oil are unaffected by extraction methods. The
total phenolic content of avocado oil is in the range of
111.27–130.17 mg GAE/100 g and the major phytosterol is
β-sitosterol (1.91–2.47 g/kg). Avocado oil extracted using subcrit-
ical CO2 exhibits two to four times greater levels of α- and
γ-tocopherols than solvent extraction and UAAE. The volatile
components associated with nutty and grassy flavors are only
detected in avocado oil extracted under low-temperature extraction
conditions such as subcritical CO2 and UAAE. Based on the anti-
oxidant capacity tests, avocado oil obtained by subcritical CO2
exhibits the strongest antioxidant capacity compared with solvent
extraction and UAAE [36].
6 Novel Technology Integrated Sub- and Supercritical Fluid Extraction
Supercritical or subcritical fluid solely could not extract the polar

solvent, so it is necessary to combine these extraction process with
novel technologies such as ultrasonication, pulse electrified,
enzyme-assisted extraction, microwave-assisted extraction (MAE)
along with the other cosolvent [37]. In a biological system, the

intended bioactive compound exists in complex form. It might
be bound to a plethora of undesired compounds that hinder
the efficacy of the extraction process [3]. For instance, caffeine in
the ground coffee is bound with chlorogenic acid, while during
the decaffeination process, only caffeine is a target compound. The
challenge of freeing caffeine from chlorogenic acid is overcome by
coextracting with water. The moisture moisturizes the ground
coffee and facilitates the caffeine extraction process by loosening
up the chlorogenic acid-releasing caffeine [29].
Ultrasound assists the extraction process by enhancing mass
transfer in the system due to cavitation formation and collapse
[3]. Da Porto et al. [38] used a combined method of ultrasound-
assisted extraction (UAE) followed by re-extraction by SC-CO2 to
extract polyphenols from grape marc-a solid waste of the winery. A
maximum of 2336 mg GAE/100 g (dry matter) of total phenolic
compound (TPC) was extracted under optimized parameters of
UAE (80 °C and 4 min), and the combination with SC-CO2
increased the yield to 3493 mg GAE/100 g. Similarly, the total
flavonoid yield from Iberis amara was 18% higher with ultrasound-
assisted supercritical CO2 extraction than SC-CO2 extraction
solely. In addition to that, the extraction time was also reduced
from 100 min for SC-CO2 to 60 min [39].
Enzyme-assisted (EA) extraction refers to the use of enzymes to
compromise the structure of the pecto-cellulosic matrix and release
the target bioactive compound through hydrolysis. Phenols and
flavonoids are bound by hydrogen and hydrophobic bonds with the
polysaccharide of the cell wall. The degradation of polysaccharides
frees the phytochemicals, easing the extraction process [3].
Krakowska et al. [40] employed enzymes to assist the SC-CO2
extraction of polyphenols from Medicago sativa and determined
that SC-CO2 extraction yielded 94.17 μg/g, whereas enzyme-
assisted SC-CO2 yielded 142.69 μg/g of phenolic compounds.
Enzymatic pretreatment also evidently assisted on the extraction
of bioactive compounds from ginger. Nourbakhsh Amiri et al. [41]
pretreated ginger powder with α-amylase and extracted bioactive
compound via SWE. The enzyme-assisted SWE (EA-SWE) yielded
2.8-fold polyphenols and 2.22-fold more gingerols and shogaol
than that of extraction without the assistance of enzyme.
The SC-CO2 extraction has been paired with a microwave-
assisted extraction method to recover bioactive compounds. Sán-
chez-Camargo et al. [42] assessed the subsequent application of
MAE1 for the extraction of phenolic compounds from the
SC-CO2-exhausted mango peel. Prior to the MAE, mango peel
was subjected to the SC-CO2 extraction of phenolic compound,
and the subsequent re-extraction was carried out, which yielded
52.08 mg gallic acid equivalent/g (dry weight) of phenolic
compound.
7 Current Application of Super- and Subcritical Extraction
There is substantial use of sub- and supercritical fluid extraction for

bioactive compound extraction from diverse sources, including
microbial cells, macroalgae, and complex plant and animal tissues.
Macroalgae (seaweed) are abundant in bioactive compounds com-
prising of carotenoids, flavonoids, tocopherols, and phytosterols
[43]. More than 20% of the plants have been subjected to under-
stand their possible utilization as pharmaceuticals. The prominent
application of the sub- and supercritical extractions are found in
field of phytochemical extraction.
Till now, approximately 8000 of different polyphenolic com-
pounds from variety of sources has been on the radar of the phar-
maceutical study [44]. Green extraction technology is constantly
revolving around the process optimization as the different source
possess different set of condition which requires to be countered
with customized parameters. Statistical tool such as, response sur-
face methodology (RSM) has been employed to optimize the para-
meters such as type of solvent, type and ratio of cosolvent, duration
of extraction, temperature, and pressure. For example, Bobinaitė
et al. [45] extracted polyphenols from rowanberry (Sorbus aucu-
paria L.) pomace with the combination of SC-CO2 and pressurized
solvent (ethanol, acetone, and water) extraction techniques.
SC-CO2 extraction was optimized by modulating the parameters,
such as pressure (45 MPa), temperature (60 °C), and extraction
time (180 min) and achieved highest yield extract of 4.8 g/100 g
(dry weight). Furthermore, pressurized solvent (ethanol) extrac-
tion was carried out to recover residual polyphenol from exhausted
pomace yielding 16.04 g/100 g (dry weight). It evidently showed
that the best yield of the target extract was achievable with the
integrated approach. Similarly, Saravana et al. [46] carried out
SC-CO2 extraction of carotenoid from the seaweed using sun-
flower oil as a cosolvent. With RSM-guided optimized parameters,
such as temperature, pressure, and cosolvent flow rate, the recovery
of the carotenoids was comparatively higher.
Extraction of bioactive lipids has been done using conventional
methods and the recent advancement in supercritical fluid extrac-
tion technique has fuelled up the exploration of new horizon. The
study of dandelion seed’s extract for its bioactive compound ren-
dered the result, which has abundant polyunsaturated fatty acid
(PUFA), primarily linoleic acid [47]. The lipophilic extract of
SC-CO2 comprises of fatty acid, and the profiling identified linoleic
and oleic acid as the major one, followed by palmitic acid and stearic
acid. Whereas, other acids, such as myristic, α-Linolenic, behenic,
and palmitoleic were each below 1% [45]. Patil et al. [48] extracted
bio-oils from algae (Nannochloropsis salina) biomass via SC-CO2
extraction technique. Azeotropic mixture (hexane: ethanol, 1:1)
was added as cosolvent to enhance affinity for neutral and polar

lipids. Maximum yield was obtained when the parameters opti-
mized as temperature 80 °C, cosolvent to algal biomass ratio 12:
1, time 60 min and pressure 340 bar. It resulted in highest eicosa-
pentaenoic acid yield. Melloul et al. [49] extracted bioactive oil
from Peganum harmala by SCF extraction and analyzed the bioac-
tivity retention along with the yield. SC-CO2 extraction process
was operated at the condition of 300 bars of pressure, at the
temperature of 55 °C with the sample particle size of 0.3 mm,
and the extraction yield was maximum. However, the maximum
availability of the total phenols (79.04 mg GAE/g equivalent) with
IC50:172.199 μ was achieved at pressure (100 bar), temperature
(35 °C), and sample particle size (0.9 mm). Maximum flavonoid
(7.10 mg QE/g equivalent) was achieved at the similar condition
except pressure being raised to 170.7 bar. This gives an overview of
how crucial it is to be prudent about the optimization condition,
mainly pressure, to modulate the extraction efficiency.
Pharmaceuticals have been exploring bioactive compounds
from novel sources by using SCF extraction technique as it is
comparatively posing less severity to the viability of compound’s
bioactivity, hence, it does not overlook the presence of even trace
amount of high value compound.
Phytochemical such as polyphenols are the most studied class of
plant derived compound. Phenols and flavonoids are the major
secondary metabolites that has high bioactivity, hence has high
value. Polyphenol are composed of multiple hydroxy phenols
which are ubiquitously available in most of the whole grains, fruits,
and vegetables. They are highly bioactive and susceptible to the
extremities that they are subjected to. Flavonoids have been
extracted from the agricultural produce and its by-product, onion
skin [50], jujube leaves: 29.052 mg/g [51], grape seed: 7132 mg
GAE/100 g DM [52], tea leaves: 194.6 mg QE/100 mL
[53]. Majority of the investigation inclined toward the diversified
flavonoids on SCF extraction than that of conventional Soxhlet or
even outperformed some of the modern extraction techniques
(UAE). The major flavonoids, anthocyanin was extracted from
roselle (Hibiscus sabdariffa L.) using SC-CO2 extraction. Experi-
mentally, TPC and TFC extracted were 128.16 mg GAE/100 g
and 731 mg QUE/100 g, respectively [54]. Similarly, Rahmana
Putra et al. [18] conducted optimization study to maximize the
yield of phenolic and flavonoid compound from roselle using sub-
critical ethanol extraction (SEE). The optimization of the parame-
ter leads to the maximum yield of anthocyanin (921.43 mg/
100 g), TPC (40.57 mg/100 g), and TFC (559.14 g/100 g). In
terms of extraction efficiency, the major takeaway of phytochem-
icals extraction is the optimization of pressure, as the most of
investigation displayed that excess rise in pressure of supercritical
fluid increases mass transfer resistance and reduces the diffusion of
solvent into the feed matrix affecting the yield. Investigation for
cost cutting on the extraction technique has unfolded possibilities
on other method than SC-CO2 extraction. Comparative study
against SC-CO2 extraction has presented evidence on subcritical
alcohol extraction being less time intensive, lower cost to operate,
and more convenience on separating solvent from solute.
It has shown promising application on the valorization process
too. Agro-based by-products have massive potential as a bioactive
compound source, so they are going under an extensive up cycle
assay. Apart from that, industrial waste, such as herbal medicine
wastes (HMWs) are also eligible for valorization into high value
bioactive compounds [55]. By-products, such as spent coffee
grounds, were investigated for the extraction of phenolic com-
pounds by using SWE technique [56]. The extraction time was
crucial during the extraction process because the longer extraction
time yielded less phenols from the spent coffee grounds. Similar
drop in phenolic compound (ƞ-caffeoyl-quinic acid) was observed
when extraction time was extended. The extended time favors the
oxidation of phenolic compound and extensive extraction temper-
ature also aids oxidation process [57]. The innate requirement of
extended time and high temperature for SWE shown detrimental
effect on the bioactivity of the extracts, hence to minimize the loss-
SWE could be combined with the pretreatment (microwave, ultra-
sonication) of the raw material [58].
The combined application of SC-CO2 and sequential subcriti-
cal hydrothermal liquefaction (SC-HTL), Mathur et al. [59]
extracted high value products such as, PUFAs (eicosapentaenoic
acid, docosahexaenoic acid, etc.), aromatics, aldehydes, and alkynes
from the microalgal biomass. During the valorization of biomass
(Chlorella sp. and Phormidium sp.), the utilization of all the frac-
tion was achieved, and additionally, compared to conventional
(Soxhlet) extraction method—the quality was higher. Green extrac-
tion technologies are also enabling the exploration of other plant
biomass as a source of bioactive compound. Foliage—otherwise
waste of different plants are being assayed as a potential source of
bioactive compound [60]. Some of the example are as follows:
Goyeneche et al. [61] recovered total phenols from leaves of beet-
root (Beta vulgaris L.) by using SC-CO2 extraction technique.
Similarly, Essien et al. [62] used subcritical water extraction tech-
nique to recover phenolic compounds from Kānuka (Kunzea eri-
coides) leaves. The extraction efficiency was directly proportional to
the adjustment of the extraction parameters, especially, extraction
time, temperature, and solid-solvent ratio. Assessment displayed
that there is untapped possibilities for the cheap source of bioactive
compound which could be exploited via optimized green extrac-
tion technologies.
8 Characterization of Extracted Bioactive Compounds
Extracted solution includes a variety of bioactive compound mix-

tures, such as polyphenols, alkaloids, essential oils, vitamins,
enzymes containing carotenoids, tocopherols, sterols, fatty acids,
including flavonoids, antioxidants, bioactive peptides, a small
amount of digestive enzymes, and other compounds in minor
amounts [63, 64]. Characterization of these compounds is neces-
sary for obtaining specific compounds in pure form, quantification,
and bioactivity determination, which enables the application of
those actives in pharmaceutical and therapeutic products develop-
ment. The general overview of extracts along with in vitro/in vivo
bioactivity, toxicity, and stability of bioactive molecules from sub/
supercritical fluid extraction is articulated (Fig. 1).
In order to extract phlorotannins from Cystoseira abies-marina
seaweeds, subcritical (water, ethanol, and ethyl lactate) and super-
critical (SC-CO2 and SC-CO2 with varying proportions of
ethanol) conditions were used. Theoretically, the best solvent was
100% ethanol at a low temperature (25 °C). However, experi-
mentally, pure ethanol at 100 °C in a subcritical state (10.3 MPa)
showed the highest selectivity to extract phlorotannins among the
four solvents investigated using a thorough two-dimensional liquid
chromatography approach [66].
Typically, research in sub/supercritical fluid extraction includes
a thorough characterization of the extracts utilizing cutting-edge
methods such as high-performance liquid chromatography
(HPLC) or gas chromatography (GC) coupled to mass
Fig. 1 A general overview of the characterization of extracted bioactive compounds. (Source: Azmir et al. [65])
spectrometry (MS), for identifying and measuring bioactive com-

pounds. But in recent years, novel and more sophisticated tools
have developed that enable the simultaneous extraction and char-
acterization of molecules. For instance, [67] designed a SFE-UV/
Vis-ELSD apparatus that could quantify total lipids, carotenoids,
chlorophyll A, and ergosterol from a microalgae extract prepared
by SFE. This strategy not only simplifies the entire extraction-
identification-quantification process but also protects extracts
from possible damage throughout these processes.
Besides these, the other attributes (appearance, taste, viscosity,
and so on) of the supernatant and the residues during sub- and
supercritical extraction are desirable in comparison to other extrac-
tion methods. The high-pressure supercritical fluid extraction
extract exhibited the most desired sensory qualities (spicy, aro-
matic/herbaceous, pungent, and phenol-like). For example, the
optimized parameter for obtaining thymol-rich extracts with high
sensory qualities was the SFE at 40 °C and 16.7 MPa [68].
The optimum defatting performance was found for supercritical
CO2 defatting after comparing the effects of two defatting meth-
ods, namely, centrifugal defatting and supercritical carbon dioxide
defatting, on the color, size distribution, and flow properties of
the resulting insect powders. The supercritical CO2 powders had
lighter colors and larger particle sizes. With regard to flow char-
acteristics, supercritical CO2 powder had considerably higher
flowability and floodability indices than other processes [69].
Additionally, compared to native, hexane-extracted, and isopropyl
alcohol-extracted flours, supercritical fluid extracted flours dis-
played a higher peak viscosity. Defatting resulted in an improve-
ment in functional properties, with supercritical fluid extracted
flours exhibiting the most significant enhancement. Defatting
had an impact on both empirical and fundamental rheological
measurements, with supercritical fluid extracted flours exhibiting
the greatest change in viscoelasticity [70].
8.1 Extracts The most common assessment techniques for in vitro characteriza-
Bioactivity tion are antioxidant activity using DPPH, FRAP, and ABTS [71],
antidiabetic activity using an α-amylase inhibitory assay, anti-
8.1.1 In Vitro
inflammatory activity using the protein denaturation inhibition
Characterization
technique [72] and anticancer activity using cell viability determin-
ing 50% inhibition concentration (IC50) [73]. The most efficient
SC-CO2 extract had maximum cannabidiol (CBD) and was rich in
α-pinene, β-pinene, β-myrcene, and limonene. Under concentra-
tions from 10.42 to 66.03 μg/mL, extract doses exhibited inhibi-
tory effects against E. coli, P. aeruginosa, B. subtilis, and S. aureus
[74]. In addition, the observations of antioxidant activity show that
the percentage of antioxidant activity decreased in the order of
α-tocopherol > SCO2-extracted oil > solvent-extracted oil >
UAAE-extracted oil. In comparison to solvent- and UAAE-

extracted oils, SCO2-extracted oil had the highest antioxidant
activity, indicating a stronger capacity to scavenge free radicals [36].
The bioactivity of SWE extracted phenolics and flavonoids
exhibited the significant differences in comparison to Soxhlet
methods. However, methanol extracts from S. variolaris sea urchin
gonads demonstrated comparable antioxidant ability to SWE of
gonads [75]. Under specific temperature, α-Amylase inhibition
and inhibition of protein denaturation was higher than that of the
extracts from Soxhlet extraction, indicating superior antioxidant,
antidiabetic, and anti-inflammatory properties. Moreover, the
superior bioactivity can be attributed to the higher bioactive com-
pounds (chlorogenic acid, gallic acid, 5-HMF, 5-MF, and ferulic
acid) identified on HPLC analysis of phenolic composition. These
bioactive compounds were still preserved under high temperature
of 190 °C [72]. Beside these, the mean antioxidants activities,
DPPH-radical-scavenging ability, ABTS-radical-scavenging ability,
and FRAP antioxidant activity are higher in the extracts as com-
pared to liquid phase extraction with methanol and ethanol at both
25 °C and 60 °C, respectively for 2 h. This bioactivity is partly
correlated with the flavonoids and phenolics contents, and there
was higher content of flavonoid and phenolics in SW-extracted
extracts. Being thermal sensitive, several bioactive compounds are
unstable and destroyed at a high temperature of 240 °C, resulting
low aforementioned activity Ko et al. [34].
8.1.2 In Vivo Analysis For the newly identified bioactive molecules, to support this typical
(Clinical Trial/Animal Study) statement, clinical trials are required to show a bioactive com-
pound’s efficacy. Clinical trials designed to ascertain the pharmaco-
kinetics, bioavailability, efficacy, safety, and drug interactions of
newly created bioactive compounds and their formulations must
be considered (extracts). Before the treatment is widely adminis-
tered to patients, clinical trials are meticulously planned to safe-
guard the participants’ health as well as provide findings to
particular research questions by evaluating their results and check-
ing for both short- and long-term negative effects [76]. Raspberry
oil extracted by supercritical CO2 has potent anticarcinogenic prop-
erties via suppression potential against several carcinoma cell lines
(colon adenocarcinoma, doxorubicin-resistant colon adenocarci-
noma, breast cancer, doxorubicin-resistant breast cancer, and lung
cancer cell lines). Raspberry oil extraction using supercritical CO2
significantly increased free radical production and DNA strand
damage in cancer cells, particularly doxorubicin-resistant lines,
implying effective targets on cancer cell vulnerabilities [77]. Extrac-
tion of the bioactive under optimized SC-CO2 extraction has
potentially higher in vivo bioactivity. Animal studies have revealed
that compounds such as curcuminoids, oleoresins, and total vola-
tiles have potent bioactive functions [78].
8.1.3 Toxicity Assay Several toxicity assays are conducted to determine its safety before
clinical trials. Cytotoxicity test of the bioactive compounds are
generally carried out on VERO and MDCK cells using a MTT
assay. A decrease in the cells viability indicates the toxic effect of
that test compound. Acute oral toxicity is determined from LD50
technique. Other toxicity tests, such as body weight change (drastic
gain/loose), hematological and biochemical analysis, and in silico
prediction of toxicity, are carried out to evaluate short- and long-
term toxicological profiles [79].
It has been discovered that SC-CO2 is selective in the extrac-
tion of desirable chemicals, leaving no harmful residues in the
extracts and posing no threat to the processed product’s thermal
stability. Actually, the use of SC-CO2 extracts in food products is
frequently acknowledged as safe. With potential applications for the
extraction of important chemicals from solid plant matrices and
seed oil, SC-CO2 extraction has grown quite mature. While other
fluids, such as propane, can be used to extract plant material, CO2
offers more advantages due to its nontoxicity and favorable ther-
modynamic properties, which make it easier to employ under
supercritical conditions (over 31.1 °C and 7.4 MPa) [80]. Peterson
et al. [81] looked at how high temperatures affected the oil during
subcritical oil extraction. Peroxide readings were discovered to be
less than 5 ppm, which indicates no oxidation when compared to
Soxhlet. The FFA results also indicated that using the subcritical
approach did not result in any appreciable oxidation deterioration.
Only little amounts of ethanol (30 mg/day authorized daily expo-
sure) can be utilized for food extractions because it is a class
3 solvent and Petroleum ether is considered as class 4 because of
its less toxicity as compared to class 1 and 2 solvents [82]. Con-
trarily, water has no such health-based exposure restriction.
8.2 Stability of Bioactive compounds are mostly heat-liable. Heat treatment has a
Bioactive Compounds significantly destructive effect on polyphenols, similarly, other fac-
tors such as oxygen, light, and pH tend to accelerate the degrada-
tion of bioactive compounds causing a loss in their therapeutic
functions. Also, due to the acidic environment in our gastrointesti-
nal tract, that is, in the stomach, most of the bioactive compounds
lose their bioactivity [83]. So, for increasing the stability and bio-
availability different new techniques are used. Some of them cur-
rently used in food and pharmaceutical applications are protecting
bioactive compounds from severe exposure to external environ-
ments by storing them in moisture, air and light barrier containers,
storage at low temperatures, etc. [84]. Furthermore, encapsulation
(microencapsulation, nanoencapsulation) and emulsification are
the most widely used methods. The most modern techniques
involve microencapsulating bioactive and using nonthermal
manufacturing methods for increasing the stability of bioactive

compounds [84]. The process of microencapsulation involves
encasing an active ingredient, which could be tiny solid particles,
liquid drops, or gaseous substances, in an encasing substance that
will serve as a protective enclosure [85]. In the research carried out
by Šeregelj et al. [86], for the encapsulation of bioactive com-
pounds from sweet potato peel using a whey protein powder as a
coating material showed that the encapsulation process enables the
carotenoid and phenolic compounds retention and increases the
shelf life under different experimental conditions, on under light
and dark conditions.
9 Advances and Future Outlook
For the sustainable approach, rather than following one expensive

method of extraction, combined methods which can deal with
many bioactive compounds should be in more with maximum
flexibility. Several emerging extraction techniques can be combined
to further shorten the extraction period, boost the extraction yield,
and get around the drawbacks of the individual techniques
[87]. One of the often-used combination extraction methods is
to pretreat sample with a microwave, ultrasonic, or enzyme before
undergoing super- and subcritical fluid extraction process to hasten
the breakdown of cell walls. Similar to this, a combination of two or
more developing extraction methods can also be applied, such as
supercritical fluid extraction (SFE) and ultrasonic-assisted extrac-
tion (UAE), subcritical fluid extraction, microwave-assisted extrac-
tion (MAE) and solvent extraction, enzyme application prior to
pulsed electric field (PEF) treatment, and supercritical fluid treat-
ment prior to MAE followed by further solvent extraction
[88]. Additionally, these methods can be further developed in
terms of efficient extraction of specific bioactive compounds that
could be closely linked to the usage of novel food-grade solvents
such as ethyl lactate. Another intriguing strategy could emphasize
increasing selectivity by choosing the best solvents and cosolvents
based on how they interact with the bioactive metabolites. The
stability of bioactive compounds is found be shorter in ambient
condition, so for increasing the stability of bioactive compounds,
more research should be conducted. Research should be focused on
increasing the stability, bioavailability, and bio-accessibility of bio-
active compounds from the storage to the absorption in the
bloodstream.
References
1. Zhang J et al (2020) Recent advances in the 13. Loarce L et al (2020) Implementation of sub-
extraction of bioactive compounds with subcritical water extraction with natural deep
critical water: a review. Trends Food Sci Tech- eutectic solvents for sustainable extraction of
nol 95:183–195 phenolic compounds from winemaking
2. Yousefi M et al (2019) Supercritical fluid by-products. Food Res Int 137:109728
extraction of essential oils. TrAC Trends Anal 14. Rodrigues LGG et al (2020) Subcritical water
Chem 118:182–193 extraction and microwave-assisted extraction
3. Essien SO, Young B, Baroutian S (2020) applied for the recovery of bioactive compo-
Recent advances in subcritical water and super- nents from Chaya (Cnidoscolus aconitifolius
critical carbon dioxide extraction of bioactive Mill.). J Supercrit Fluids 165:104976
compounds from plant materials. Trends Food 15. Dassoff ES, Li YO (2019) Mechanisms and
Sci Technol 97:156–169 effects of ultrasound-assisted supercritical
4. Ghafoor K et al (2022) Extraction and evalua- CO2 extraction. Trends Food Sci Technol 86:
tion of bioactive compounds from date (Phoe- 492–501
nix dactylifera) seed using supercritical and 16. Patil PD et al (2021) Enzyme-assisted super-
subcritical CO2 techniques. Foods 11(12): critical fluid extraction: an integral approach to
1806 extract bioactive compounds. Trends Food Sci
5. Munir MT et al (2018) Subcritical water Technol 116:357–369
extraction of bioactive compounds from waste 17. Moirangthem K et al (2021) Bioactivity and
onion skin. J Clean Prod 183:487–494 anthocyanin content of microwave-assisted
6. Gan A, Baroutian S (2022) Current status and subcritical water extracts of Manipur black rice
trends in extraction of bioactives from brown (Chakhao) bran and straw. Future Foods 3:
macroalgae using supercritical CO2 and sub- 100030
critical water. J Chem Technol Biotechnol 18. Rahmana Putra N et al (2022) Optimization
97(8):1929–1940 and solubilization of interest compounds from
7. Putra NR et al (2018) Diffusivity optimization roselle in subcritical ethanol extraction (SEE).
of supercritical carbon dioxide extraction with Alex Eng J 65:59–74
co-solvent-ethanol from peanut skin. Malays J 19. Sarkar S, Gayen K, Bhowmick TK (2022)
Fundam Appl Sci 14(1):9–14 Green extraction of biomolecules from algae
8. Peng WL et al (2020) Optimization of process using subcritical and supercritical fluids. Bio-
variables using response surface methodology mass Conv Bioref. https://doi.org/10.1007/
for tocopherol extraction from Roselle seed oil s13399-022-02309-3
by supercritical carbon dioxide. Ind Crop Prod 20. Wang W et al (2019) Antioxidant activity and
143:111886 total polyphenols content of camellia oil
9. Putra NR et al (2018) Effect of particle size on extracted by optimized supercritical carbon
yield extract and antioxidant activity of peanut dioxide. J Am Oil Chem Soc 96(11):
skin using modified supercritical carbon diox- 1275–1289
ide and soxhlet extraction. J Food Process Pre- 21. Santos ÊRM et al (2017) Supercritical fluid
serv 42(8):e13689 extraction of Rumex Acetosa L. roots: yield,
10. Kostrzewa D, Dobrzyńska-Inger A, composition, kinetics, bioactive evaluation
Reszczyński R (2021) Pilot scale supercritical and comparison with conventional techniques.
CO2 extraction of carotenoids from sweet J Supercrit Fluids 122:1–9
paprika (Capsicum annuum L.): influence of 22. Esmaeelian M et al (2020) Optimization of
particle size and moisture content of plant experimental parameters in subcritical water
material. LWT 136:110345 extraction of bioactive constituents from the
11. Uwineza PA, Waśkiewicz A (2020) Recent saffron (Crocus sativus L.) corm based on
advances in supercritical fluid extraction of nat- response surface methodology. J Food Meas
ural bioactive compounds from natural plant Charact 14(4):1822–1832
materials. Molecules 25(17):3847 23. Garcia-Vaquero M, Rajauria G, Tiwari B
12. Quispe-Fuentes I et al (2022) A study of dried (2020) Conventional extraction techniques:
mandarin (Clementina orogrande) peel apply- solvent extraction. In: Torres MD, Kraan S,
ing supercritical carbon dioxide using Dominguez H (eds) Sustainable seaweed tech-
co-solvent: influence on oil extraction, pheno- nologies. Elsevier, Amsterdam, pp 171–189
lic compounds, and antioxidant activity. J Food 24. Vovers J, Smith KH, Stevens GW (2017)
Process Preserv 46(1):e16116 Bio-based molecular solvents. In: Pena-
Pereira F, Tobiszewski M (eds) The application 36. Tan CX et al (2018) Characterization of virgin
of green solvents in separation processes. Else- avocado oil obtained via advanced green tech-
vier, Amsterdam, pp 91–110 niques. Eur J Lipid Sci Technol 120(10):
25. Vauchel P et al (2018) Comparative LCA of 1800170
ultrasound-assisted extraction of polyphenols 37. Al-Maqtari QA et al (2021) Supercritical fluid
from chicory grounds under different opera- extraction of four aromatic herbs and assess-
tional conditions. J Clean Prod 196:1116– ment of the volatile compositions, bioactive
1123 compounds, antibacterial, and anti-biofilm
26. Cañadas R et al (2020) Overview of neoteric activity. Environ Sci Pollut Res 28(20):
solvents as extractants in food industry: a focus 25479–25492
on phenolic compounds separation from liquid 38. Da Porto C, Natolino A, Decorti D (2015)
streams. Food Res Int 136:109558 The combined extraction of polyphenols from
27. Murugan M, Tee L, Oh K (2021) Evaluation grape marc: ultrasound assisted extraction fol-
of the environment impact of extraction of lowed by supercritical CO2 extraction of
bioactive compounds from Darcyodes rostrata ultrasound-raffinate. LWT Food Sci Technol
using deep eutectic solvent (DES) using life 61(1):98–104
cycle assessment (LCA). J Phys Conf Ser 39. Liu X-Y et al (2022) Supercritical CO2 extrac-
2120:012005 tion of total flavonoids from Iberis amara
28. Clarke CJ et al (2018) Green and sustainable assisted by ultrasound. J Supercrit Fluids 184:
solvents in chemical processes. Chem Rev 105581
118(2):747–800 40. Krakowska A et al (2018) Enzyme-assisted
29. Perez-Vega S et al (2022) Influence of the optimized supercritical fluid extraction to
supercritical fluid extraction (SFE) on food improve Medicago sativa polyphenolics isola-
bioactives. In: Jafari SM, Capanoglu E (eds) tion. Ind Crop Prod 124:931–940
Retention of bioactives in food processing. 41. Nourbakhsh Amiri Z et al (2018) Subcritical
Springer International Publishing, Cham, pp water extraction of bioactive compounds from
309–340 ginger (Zingiber officinale Roscoe). Int J Eng
30. Villanueva-Bermejo D et al (2019) Production 31(12):1991–2000
of omega 3-rich oils from underutilized chia 42. Sánchez-Camargo AdP et al (2021)
seeds. Comparison between supercritical fluid Microwave-assisted extraction of phenolic
and pressurized liquid extraction methods. compounds with antioxidant and anti-
Food Res Int 115:400–407 proliferative activities from supercritical CO2
31. Pereira P et al (2016) Supercritical fluid extrac- pre-extracted mango peel as valorization strat-
tion vs conventional extraction of myrtle leaves egy. LWT 137:110414
and berries: comparison of antioxidant activity 43. Gomez-Zavaglia A et al (2019) The potential
and identification of bioactive compounds. J of seaweeds as a source of functional ingredi-
Supercrit Fluids 113:1–9 ents of prebiotic and antioxidant value. Anti-
32. Mahato N et al (2019) Modern extraction and oxidants 8(9):406
purification techniques for obtaining high 44. Altemimi A et al (2017) Phytochemicals:
purity food-grade bioactive compounds and extraction, isolation, and identification of bio-
value-added co-products from citrus wastes. active compounds from plant extracts. Plants
Foods 8(11):523 (Basel) 6(4):42
33. Pai S, Hebbar A, Selvaraj S (2022) A critical 45. Bobinaitė R et al (2020) Recovery of bioactive
look at challenges and future scopes of bioac- substances from rowanberry pomace by con-
tive compounds and their incorporations in the secutive extraction with supercritical carbon
food, energy, and pharmaceutical sector. Envi- dioxide and pressurized solvents. J Ind Eng
ron Sci Pollut Res 29(24):35518–35541 Chem 85:152–160
34. Ko M-J, Nam H-H, Chung M-S (2020) Sub- 46. Saravana PS et al (2017) Influence of
critical water extraction of bioactive com- co-solvents on fucoxanthin and phlorotannin
pounds from Orostachys japonicus A. Berger recovery from brown seaweed using supercriti-
(Crassulaceae). Sci Rep 10(1):10890 cal CO2. J Supercrit Fluids 120:295–303
35. Ho TC, Chun B-S (2019) Extraction of bioac- 47. Milovanovic S et al (2022) Dandelion seeds as a
tive compounds from Pseuderanthemum pala- new and valuable source of bioactive extracts
tiferum (Nees) Radlk. Using subcritical water obtained using the supercritical fluid extraction
and conventional solvents: a comparison study. technique. Sustain Chem Pharm 29:100796
J Food Sci 84(5):1201–1207
48. Patil PD et al (2018) Extraction of bio-oils compounds: a comprehensive review. Crit Rev
from algae with supercritical carbon dioxide Food Sci Nutr 63:5155–5193
and co-solvents. J Supercrit Fluids 135:60–68 61. Goyeneche R et al (2020) Recovery of bioac-
49. Melloul S, Zehioua R, Meniai A-H (2022) tive compounds from beetroot leaves by super-
Supercritical CO2 extraction of bioactive com- critical CO2 extraction as a promising
pounds from local Peganum Harmala plant bioresource. J Supercrit Fluids 155:104658
seeds and optimization of the extraction yield 62. Essien S, Young B, Baroutian S (2020) Subcrit-
and the antioxidant activities. Sustain Chem ical water extraction for selective recovery of
Pharm 28:100729 phenolic bioactives from kānuka leaves. J
50. Campone L et al (2018) Response surface Supercrit Fluids 158:104721
methodology to optimize supercritical carbon 63. Mena-Garcı́a A et al (2019) Green techniques
dioxide/co-solvent extraction of brown onion for extraction of bioactive carbohydrates. TrAC
skin by-product as source of nutraceutical com- Trends Anal Chem 119:115612
pounds. Food Chem 269:495–502 64. Marić M et al (2018) An overview of the tradi-
51. Song L et al (2019) Supercritical CO2 fluid tional and innovative approaches for pectin
extraction of flavonoid compounds from Xin- extraction from plant food wastes and
jiang jujube (Ziziphus jujuba Mill.) leaves and by-products: ultrasound-, microwaves-, and
associated biological activities and flavonoid enzyme-assisted extraction. Trends Food Sci
compositions. Ind Crop Prod 139:111508 Technol 76:28–37
52. Da Porto C, Natolino A (2017) Supercritical 65. Azmir J et al (2013) Techniques for extraction
fluid extraction of polyphenols from grape seed of bioactive compounds from plant materials: a
(Vitis vinifera): study on process variables and review. J Food Eng 117(4):426–436
kinetics. J Supercrit Fluids 130:239–245 66. Sánchez-Camargo A et al (2016) Application
53. Maran JP et al (2015) Box-Behnken design of Hansen solubility approach for the subcriti-
based multi-response analysis and optimization cal and supercritical selective extraction of
of supercritical carbon dioxide extraction of phlorotannins from Cystoseira abies-marina.
bioactive flavonoid compounds from tea RSC Adv 6(97):94884–94895
(Camellia sinensis L.) leaves. J Food Sci Tech- 67. Abrahamsson V, Jumaah F, Turner C (2018)
nol 52(1):92–104 Continuous multicomponent quantification
54. Idham Z et al (2022) Improvement of extrac- during supercritical fluid extraction applied to
tion and stability of anthocyanins, the natural microalgae using in-line UV/Vis absorption
red pigment from roselle calyces using super- spectroscopy and on-line evaporative light scat-
critical carbon dioxide extraction. J CO2 Util tering detection. J Supercrit Fluids 131:157–
56:101839 165
55. Jouyandeh M et al (2022) Green products 68. Morsy NF (2020) Production of thymol rich
from herbal medicine wastes by subcritical extracts from ajwain (Carum copticum L.) and
water treatment. J Hazard Mater 424:127294 thyme (Thymus vulgaris L.) using supercritical
56. Pedras BM et al (2019) Semi-continuos extrac- CO2. Ind Crop Prod 145:112072
tion/hydrolysis of spent coffee grounds with 69. Laurent S et al (2022) Effect of two defatting
subcritical water. J Ind Eng Chem 72:453–456 processes on the physicochemical and flow
57. Morais ARC, da Costa Lopes AM, Bogel-Łuka- properties of Hermetia illucens and Tenebrio
sik R (2015) Carbon dioxide in biomass pro- molitor larvae powders. J Food Process Preserv
cessing: contributions to the green biorefinery 46(10):e16853
concept. Chem Rev 115(1):3–27 70. Awasthi T et al (2022) Effect of solvents and
58. Getachew AT, Chun BS (2017) Influence of supercritical-CO2 extraction of lipids on
pretreatment and modifiers on subcritical physico-chemical, functional, pasting and rhe-
water liquefaction of spent coffee grounds: a ological properties of hard, medium hard and
green waste valorization approach. J Clean soft wheat varieties. Int J Food Sci Technol
Prod 142:3719–3727 57(8):5057–5067
59. Mathur M et al (2022) Valorization of micro- 71. Roy VC et al (2020) Recovery and
algal biomass to value-added products using bio-potentialities of astaxanthin-rich oil from
integrated supercritical CO2 extraction and shrimp (Penaeus monodon) waste and mack-
sub-critical hydrothermal liquefaction. J Clean erel (Scomberomous niphonius) skin using
Prod 373:133925 concurrent supercritical CO2 extraction. J
60. Yeasmen N, Orsat V (2023) Green extraction Supercrit Fluids 159:104773
and characterization of leaves phenolic
72. Chamika WAS et al (2021) In vitro characteri- 80. Lefebvre T, Destandau E, Lesellier E (2021)
zation of bioactive compounds extracted from Selective extraction of bioactive compounds
sea urchin (Stomopneustes variolaris) using from plants using recent extraction techniques:
green and conventional techniques. Food a review. J Chromatogr A 1635:461770
Chem 361:129866 81. Peterson J et al (2009) Extraction of oil from
73. Niyonizigiye I et al (2020) Characterization oilseeds using accelerated solvent extraction
and in vitro cytotoxicity of phytochemicals (ASE). LCGC N Am 27:36
from Aspilia africana obtained using green 82. Witschi C, Doelker E (1997) Residual solvents
extraction techniques. S Afr J Bot 128:231– in pharmaceutical products: acceptable limits,
238 influences on physicochemical properties, ana-
74. Jokić S et al (2022) Terpenes and cannabinoids lytical methods and documented values. Eur J
in supercritical CO2 extracts of industrial hemp Pharm Biopharm 43(3):215–242
inflorescences: optimization of extraction, anti- 83. Rivas JC, Cabral LMC, Rocha-Leão MH
radical and antibacterial activity. Pharmaceuti- (2020) Stability of bioactive compounds of
cals 15(9):1117 microencapsulated mango and passion fruit
75. Archana A, Babu KR (2016) Nutrient compo- mixed pulp. Int J Fruit Sci 20(sup2):S94–S110
sition and antioxidant activity of gonads of sea 84. Shinwari KJ, Rao PS (2018) Stability of bioac-
urchin Stomopneustes variolaris. Food Chem tive compounds in fruit jam and jelly during
197:597–602 processing and storage: a review. Trends Food
76. Venturella G et al (2021) Medicinal mush- Sci Technol 75:181–193
rooms: bioactive compounds, use, and clinical 85. Gharsallaoui A et al (2007) Applications of
trials. Int J Mol Sci 22(2):634 spray-drying in microencapsulation of food
77. Grajzer M et al (2021) Bioactive compounds of ingredients: an overview. Food Res Int 40:
raspberry oil emulsions induced oxidative stress 1107–1121
via stimulating the accumulation of reactive 86. Šeregelj V et al (2020) Encapsulation and deg-
oxygen species and NO in cancer cells. Oxida- radation kinetics of bioactive compounds from
tive Med Cell Longev 2021:5561672 sweet potato peel during storage. Food Tech-
78. Nagavekar N, Singhal RS (2019) Supercritical nol Biotechnol 58(3):314–324
fluid extraction of Curcuma longa and Cur- 87. Zuin VG, Ramin LZ (2018) Green and sus-
cuma amada oleoresin: optimization of extrac- tainable separation of natural products from
tion conditions, extract profiling, and agro-industrial waste: challenges, potential-
comparison of bioactivities. Ind Crop Prod ities, and perspectives on emerging approaches.
134:134–145 Top Curr Chem (Cham) 376(1):3
79. Ortiz-Andrade R et al (2020) Toxicological 88. Dias JL et al (2019) Extraction of umbu (Spon-
screening of four bioactive citroflavonoids: dias tuberosa) seed oil using CO2, ultrasound
in vitro, in vivo, and in silico approaches. Mole- and conventional methods: evaluations of com-
cules 25(24):5959 position profiles and antioxidant activities. J
Supercrit Fluids 145:10–18
Chapter 7
Novel Solvent Based Extraction

Ratnnadeep C. Sawant, Shun-Yuan Luo, and Rahul B. Kamble
Abstract
Solvent extraction techniques have a wide range of uses in analytical chemistry, including large-scale
industrial separations, waste management, the pharmaceutical and biochemical industries, and both inor-
ganic and organic chemistry. For the extraction of nutraceuticals from plants, a number of novel techniques
have been developed, including accelerated solvent extraction, supercritical fluid extraction, ultrasound-
assisted extraction, and microwave-assisted extraction. These techniques aim to reduce extraction time,
increase extraction yield, and improve the quality of extracts while also consuming less solvent. Solvent
extraction methods are currently frequently used in separation technologies. Recovery, concentration, and
separation of organic acids and acid mixtures are of great interest to researchers. Both the conventional and
traditional precipitation techniques and various more recent efforts to develop extraction-based process
technologies. The main problem with current solvent extraction separation is that the majority of methods
are empirical, distinctive, and exclusive to particular application domains, requiring a lot of testing. This
review offers a succinct look at the most recent developments in new solvent extraction separation
techniques for the application of bioactive extraction in the food and nutraceutical industries.
Key words Solvent, Extraction, Bio-active extraction, Organic solvents
1 Introduction
Solvent extraction is an effective separation technique widely used

in a variety of applications, ranging from separations in analytical
chemistry to industrial processes in hydrometallurgy pharmaceuti-
cal, food engineering, and waste treatment [1]. Separate processes
of solvent extraction in Fig. 1 have been extensively described using
a variety of theoretical chemistry tools. There is still no comprehen-
sive theory and no effective method for combining the extraction
phases due to the complexity of solvent extraction systems. Under-
standing the fundamental solution chemistry of food items and
their behaviors in both aqueous and organic phases is necessary
for the development of new reagents for bioactive solvent extrac-
tion methods. Recent developments have altered the solvent extrac-
tion landscape in the food and nutraceutical industries, opening up
153
154 Ratnnadeep C. Sawant et al.
Fig. 1 Basic processes in solvent extraction
new avenues and extending our understanding of how the use of

solvents has developed into one of the most effective methods in
the study of bioactive extraction. For the separation of solutes from
relatively concentrated feeds, such as those used in the industrial
synthesis of chemicals and metals by hydrometallurgy, conventional
solvent extraction is a tried-and-true process. On the other hand,
diluted streams provide a problem. In order to effectively treat
these streams using traditional liquid-liquid extraction, the distri-
bution ratio must be quite high; otherwise, the amount of the
organic phase would be too great for reasons of safety and the
environment. These restrictions are attempted to be overcome by
the revolutionary solvent extraction technologies developed in
recent years. Hazardous organic solvents can be swapped out with
environmentally friendly solvents to make processes greener and
more environmentally sustainable. In a recent review, sampling
techniques and thorough, modern methodologies for chemical
characterization of antibiotic residues in various sample matrices
are presented. With applications in antibiotic residue analysis,
solvent-based sample preparation methods using green solvents
Novel Solvent Based Extraction 155
are explored in particular [2]. Rajapaksha et al. investigated the

applicability of semi-continuous subcritical solvent extraction
(SSE) for the extraction of polyphenols from wasted black tea at
pilot size [3]. SSE improved the phenolics’ diffusivity and solubility.
When compared to hot water extraction (HWE), the extraction of
phenolic content from discarded black tea using a 1:1 ethanol-
water solvent at 125 °C and 0.3 MPa yielded considerably more
phenol. This chapter analyses their potential to enhance conven-
tional solvent extraction’s performance in light of current develop-
ments in solvent development and theory.
2 Water as a Solvent
The universality of water as the solvent for living systems is usually

justified by arguing that water supports the rich organic chemistry
that seeds life. Nevertheless, it has been recently pointed out that
alternative chemistries are possible in other organic solvents
[4]. Water is undoubtedly the ideal substitute for organic solvents
from an environmental standpoint because it is affordable, secure,
nontoxic, noninflammable, and recyclable. While polar molecules
can be dissolved in water, the majority of the often-examined
organic compounds are hydrophobic and have poor to very poor
water solubility. This is the fundamental drawback of using water as
a solvent. Between the normal boiling point (100 °C) and the
critical temperature (374 K), subcritical water, also known as pres-
surized hot water, delivers liquid water under pressure. Water has a
minimal environmental impact because it is harmless to human
health and the environment, safe to handle, and can be transported,
thanks to existing infrastructure. Water is, therefore, regarded as
one of the most environmentally friendly solvents. Additionally, it
does not require drying the starting material prior to an extraction
process that will be conducted in aqueous conditions [5]. The
variation of the dielectric constant with temperature is the most
crucial aspect to take into account in this kind of extraction
method. With a dielectric constant close to 80, water is a strongly
polar solvent when it is at normal temperature. With the right
pressure, water can be heated to a temperature of 250 °C while
remaining in its liquid condition, significantly lowering this value to
numbers near to 27. According to Miller et al. this dielectric
constant value is comparable to that of ethanol [6]. Steam needs
to be pressurized at high temperatures (above boiling point:
100–374 °C) in order to move through the material effectively.
The instrumentation basically consists of an oven where the extrac-
tion cell is installed and extraction takes place, a water reservoir
connected to a high-pressure pump to inject the solvent into the
system, and a restrictor or valve to maintain the pressure. The vial at
the conclusion of the extraction mechanism is where the extracts
are collected. The system can also be fitted with a coolant mecha-
nism to allow for the quick cooling of the extracted product.
Despite the fact that this method has often been applied as a
batch process, research of continuous methods and the online
coupling of a subcritical water extraction (SWE) system to an
HPLC apparatus via a solid phase trapping have been reported [7].
3 Organic Solvents
In separation techniques such as solvent extraction, sonication,

microwave-assisted extraction, and pressurized fluid extraction,
organic solvents are frequently utilized. The selection of the solvent
is mostly determined by the advised standard procedures. Accord-
ing to the Environmental, Health, and Safety assessment results for
26 regularly used pure organic solvents, formaldehyde, dioxane,
formic acid, acetonitrile, and acetic acid all received high marks
overall. Dioxane had a high persistency, acetic and formic acids
had high ratings for irritation, and formaldehyde had high values
for acute and chronic toxicity. Methyl acetate, ethanol, and metha-
nol, which in particular pose modest environmental dangers and
relatively low health hazards, on the other hand, received poor total
scores.
From a life-cycle perspective, the use of tetrahydrofuran, butyl
acetate, cyclohexanone, and 1-propanol is not advised because
these solvents have significant negative environmental effects
throughout petrochemical manufacture. In addition, solvents
with a very high environmental impact include formic acid, ethyl
acetate, acetonitrile, dioxane, 1-butanol, and dimethylformamide.
On the other hand, solvents that are good for the environment
include hexane, heptane, and diethyl ether. These outcomes in
relation to alkanes are brought about by energy recovery through
incineration along with the comparatively negligible environmental
effects of their manufacturing. Following the use of supercritical
fluids as substitute solvents, the use of renewable organic or ILs, the
use of aqueous solutions of amphiphilic compounds or supramole-
cules, and the use of water under particular temperature and pres-
sure conditions must all be taken into consideration, as shown in
Fig. 2. For their transparency in this spectrum range and for extrac-
tion, carbon tetrachloride, chloroform, and dichloromethane were
the solvents that had been commonly employed in spectroscopy.
However, they are no longer used due to their toxicity, carcino-
genic effects, and ozone-depleting properties. Since then, signifi-
cant research has been put into developing chlorinated solvent
substitutes. With volatile organic compounds (VOCs), the issue is
the same. Common solvents used in solvent extraction are fre-
quently volatile organic compounds (VOCs), and during the past
few decades, they have been linked to a number of direct and
indirect environmental and health harms (Table 1).
Fig. 2 Greener alternatives to the organic toxic solvents in solvent extraction
The following should be highlighted as direct impacts

of VOCs: their toxicity and carcinogenic effects, which depend on
the compound, the exposure, and the length of exposure; their
flammability and associated fire hazards; and the potential for per-
oxide generation, particularly in the case of ethers. The following
are the three main indirect environmental issues caused by VOCs:
(i) their ozone-depletion properties, particularly in the case of
chlorofluorocarbons, which are currently being phased out;
(ii) the potential for global warming because of the role that
VOCs play in the creation of photochemical smog; and (iii) their
environmental persistence. The usage of environmentally friendly
solvents must be strictly enforced in substitution of regularly used
ones for the aforementioned causes. Table 2 demonstrates that the
US EPA’s list of harmful air pollutants from 2002 includes popular
solvents used in solvent extraction.
3.1 Subcritical Hot The physicochemical transformations of water from ambient to

Water as a Solvent for near-critical conditions, under which subcritical water extraction
Extraction (SWE, Fig. 3) can be carried out, are one of the key factors that
make water an intriguing solvent [5]. Subcritical water extraction
or extraction with hot water under pressure at the critical point of
water (22.4 MPa and 374 °C) has become a practical instrument to
replace the conventional extraction techniques. SWE is a technol-
ogy that doesn’t harm the environment and can increase extraction
yields from solid samples [8]. To keep water in the liquid condition,
SWE uses hot water (between 100 and 374 °C) under high pressure
(often between 10 and 60 bar). At temperatures between 80 and
250 °C and high pressure, the polarity of water reduces signifi-
cantly, replacing organic modifiers and offering environmentally
Table 1
Common organic solvents present in the list of hazardous air pollutants published in 2002 by the
US EPA
Chemical name Effects on humans and environment

Acetonitrile Acid rain
Benzene Recognized human carcinogen (Group 1)
Ethyl benzene Photochemical smog formation
Toluene Skin irritation
Xylene Skin irritation
Phenol Moderately toxic
Cresol Moderately toxic
Carbon disulfide Coronary heart disease
Carbon tetrachloride, Chloroform, Ozone-depleting agents/toxic to the liver, heart and
Dichloromethane, Trichloroethylene, kidneys/reasonably anticipated to be a human
Tetrachloroethylene, Chloroethane carcinogen (Group 2B)
Bromoform, Dibromoethane, Bromoethane Recognized human carcinogen (Group1)
Methanol Produces formaldehyde, which causes headache,
insomnia, gastrointestinal problems and blindness
Methyl ethyl ketone Potentiates toxicity of haloalkanes and n-hexane
Methyl isobutyl ketone Potentiates toxicity of haloalkanes and n-hexane
n-Butyl ketone Nerve-cell degeneration
Methyl tert-butyl ether Possible human carcinogen (Group 2B)
Diethanolamine Negative effects on liver, kidney and blood
Formaldehyde Allergic contact dermatitis/reasonably anticipated to
be a human carcinogen
Triethylamine Reversible corneal edema
1,3- Butadiene Recognized human carcinogen (Group1)
beneficial green solutions [9]. It is not a novel idea to employ liquid

water at temperatures higher than its boiling point to increase the
solubility of organic molecules.
It has long been used as an environmentally friendly substitute
for organic solvents in the cleaning process to improve the extrac-
tion of oil shale [10], sulfur from ore bodies (Williams et al. 1999),
and essential oils from plant materials [11]. Steam needs to be
pressurized at high temperatures (above boiling point: 100–374 °
C) in order to move through the sample effectively. A potent
substitute for the extraction of solid materials is the SWE. It has
been used to remove pesticides and polycyclic aromatic hydrocar-
bons from soil samples as well as contaminants with a variety of
polarities from environmental samples.
Table 2
Critical properties of several solvents used in supercritical fluid extraction (SFE)
Critical Property
Solubility parameter
Solvent Temperature (°C) Pressure (atm) Density (g/mL) δSFC (cal-1/2 cm-3/2)
Ethene 10.1 50.5 0.200 5.8
Water 101.1 217.6 0.322 13.5
Methanol -34.4 79.9 0.272 8.9
Carbon dioxide 31.2 72.9 0.470 7.5
Ethane 32.4 48.2 0.200 5.8
Nitrous oxide 36.7 71.7 0.460 7.5
Sulphur hexafluoride 45.8 37.7 0.730 5.5
n-Butane -139.9 36.0 0.221 5.2
n-Pentane -76.5 33.3 0.237 5.1
Fig. 3 Diagram of a subcritical water extraction (SWE) system. SR solvent

reservoir, PV purge valve, RV pressure relief valve, EC extraction cell, SV static
valve, CV collector vial, WV waste vial
Basile et al. suggested using SWE as a very promising substitute

for traditional and supercritical CO2 extraction procedures for the
isolation of essential oils [12]. Since that time, the method has
demonstrated its applicability in the field of essences in comparison
to more traditional methods like solvent extraction and steam
distillation, which have a number of well-known drawbacks, includ-
ing low extraction efficiency, extended extraction times, and signifi-
cant quantities of toxic solvent waste. Subcritical water, compressed
hot water, or pressurized hot water is defined as water that remains
liquid in the temperature range of 100–374 °C. Compared to
ambient water, this kind of water has special characteristics. A low
relative dielectric constant and a high ion product are the two types.
These characteristics allow for the extraction of useful substances
from natural resources using this water. This chapter reviews the use
of subcritical water for extracting chemicals from agricultural waste
or products [13].
A large variety of substances can be employed as supercritical
fluids (see Table 2 for a list of some of the critical features of various
solvents utilized in SFE). Supercritical carbon dioxide solvent is the
subject of great interest since it is nontoxic, acceptable to the
environment, affordable, and has low and moderate critical pres-
sures (72.9 atm and 31.3 °C, respectively). Carbon dioxide is a
relatively nonpolar solvent that mostly dissolves nonpolar solutes.
Advantages and Disadvantages of Supercritical Extraction

SFE’s biggest drawback is its high pressure, which necessitates
more expensive production equipment. The critical pressures, how-
ever, are lower than many of the high-pressure processes now
employed in the petrochemical sector. It demonstrates the advan-
tageous mass transport qualities that, when compared to the liquid
phase, can be attained in the supercritical region due to low viscos-
ity and high diffusivity. Both the solutes and the solvent of choice
affect the separation properties in SFE [14].
4 Renewable Water-Based Solvents
Selective extractants, which can be created, for example, by creating

organic compounds specifically with preorganized metal-binding
sites, are still in high demand. Such extractants may be developed
using computer-based models. The extractants and diluents
employed must be either nontoxic, nonvolatile, or recovered dur-
ing the process due to environmental reasons. There are many new
demands placed on the diluents utilized by the increasing combi-
nation of extraction and distillation used in biotechnology. It is
important to take precautions while extracting biologically active
chemicals to prevent the activity loss that frequently results from

contact with organic diluents. As a result, a number of systems have
been created with these chemicals in mind.
4.1 Aqueous Two- The first of these employs two-phase separation systems, which
Phase Systems as combine aqueous solutions with polymers and inorganic salts to
Extractants form two phases that are primarily composed of water. Using
supercritical circumstances, a second system converts the initial
two-phase system into a single phase under unique temperature-
pressure conditions. Additionally, by encapsulating the active
organic compound inside the aqueous center of a micelle of
surface-active chemicals, the active organic compound can be pro-
tected from the organic diluent. As is explained below, all of these
systems are currently the subject of active research.
4.2 Supercritical A fluid becomes a supercritical fluid when it is pushed to pressure

Fluid Extractants and temperature over its critical point. A substance at a temperature
and pressure over its critical point is referred to as a supercritical
fluid. In these circumstances, the fluid’s characteristics are posi-
tioned somewhere between those of a gas and a liquid. Although
a supercritical fluid’s density is comparable to that of a liquid and its
viscosity is comparable to that of a gas, its diffusivity is in between
those two states. The most effective and efficient method for
extracting important component botanicals is supercritical fluid
extraction. The king of botanical extraction solvents is CO2. The
critical temperature and critical pressure for supercritical CO2
extraction are higher than 31 °C and 74 bar, respectively. Super-
critical fluids are extremely compressed gases that have intriguingly
mixed properties of both gases and liquids. Reactions that are
challenging or perhaps impossible to achieve in normal solvents
can occur in supercritical fluids. It is a quick process that takes
10–60 min to finish. Simply releasing pressure can separate a super-
critical fluid from an analyte, leaving nearly no residue and produc-
ing a pure residue [15].
The extraction of polar solutes from supercritical CO2 is less
successful than it is with lipophilic molecules. In order to signifi-
cantly increase the solubility of amphiphilic molecules in supercriti-
cal CO2, CO2 must be coupled with a polar cosolvent for the
purpose of isolating them. The two cosolvents most frequently
utilized are ethanol and methanol. At room temperature, carbon
dioxide is a gas; therefore, after the extraction is finished and the
system is decompressed, a significant amount of CO2 is eliminated
without leaving any residues, producing a solvent-free extract.
When carbon dioxide consumption is high on an industrial scale,
the process can be managed to recycle it. Supercritical CO2, on the
other hand, performs less well in the extraction of highly polar
chemicals from natural matrices due to its low polarity. Modifiers
(also known as cosolvents) are frequently employed to remedy this
issue. Modifiers are highly polar chemicals that can significantly

alter the solvent characteristics of pristine supercritical CO2 when
introduced in small amounts. The high investment costs of SFE as
compared to conventional atmospheric pressure extraction meth-
ods are another disadvantage. The hydroperoxide method used at
PAO to extract valuable components (methyl phenyl carbinol,
acetophenone, ethylbenzene, phenol, and propylene glycol) from
industrial wastewater created during the coproduction of styrene
and propylene oxide was used as an example to develop and discuss
strategies for improving the efficiency of the extraction process
carried out under supercritical fluid conditions beyond the
binodal [16].
5 Ionic Liquids as Solvents for Extraction
Organic salts with a melting point less than 100 °C are ionic liquids
(ILs). If the salt is liquid at room temperature, they are referred to
as room-temperature ILs. They typically consist of an organic or
inorganic anion and a bulky, poorly coordinating organic cation.
When air- and water-stable ILs were first synthesized in the early
1990s, chemists were interested in ILs [17]. ILs have a wide range
of uses in chemistry and are sometimes referred to as a “green
alternative.” They are frequently combustible and explosive and
have low vapor pressures, which suggests they have less of an impact
on the environment than VOCs. ILs are helpful in liquid–liquid
extraction (LLE), liquid phase microextraction (LPME), and solid
phase microextraction (SPME) due to their low vapor pressure and
strong solubility for both inorganic and organic chemicals
[18]. Studies of the deep eutectic solvent (DES) extraction mecha-
nism, particularly extraction by the creation of a deep eutectic
system (DESys), have found similarities between the DES- and
ionic liquids (IL)-based extraction systems. Xiao et al. presented
new uses for ILs and DES in the extraction of nutritious natural
compounds [19]. In the mechanochemical extraction of target
chemicals from Moringa oleifera leaves, the extraction behavior of
choline chloride (ChCl) and 1-(2-hydroxyethyl)-3-methylimidazo-
lium chloride ([HMIm][Cl]) in DES and IL, respectively, was
thoroughly investigated.
5.1 Properties of ILs ILs have a variety of fascinating and distinctive characteristics, one
of which is extremely low vapor pressure. ILs fundamentally do not
evaporate under typical process operating conditions. The use of
ILs as industrial solvents to replace VOCs and so remove a source of
air pollution as well as risks from inhalation and explosion has thus
attracted a lot of interest [20]. Additionally, ILs maintain their
liquid state throughout a huge temperature range (e.g., 70–400 °
C). Both of these characteristics, a negligible vapor pressure and a
wide liquidus range, will make it easier to recover and repurpose ILs
in the context of LLE and hence bring financial advantages (such as
a make-up required and solvent loss that are incredibly low). Some
ILs can remove hydrophobic substances due to their hydrophobic
nature [21]. However, using the proper chelators or ligands that
could form complexes to improve the hydrophobicity of the metal
species, cationic compounds can be successfully recovered from
hydrophobic ILs [22]. The following factors are those that deter-
mine how well metal ions are extracted: (1) The side chain length
and IL structure, which alter the hydrophobicity and increase
partition coefficients [23]. (2) Selectivity should be maximized by
the ligand utilized. (3) The system’s pH [24].
5.2 Toxicology The possibilities provided by ILs, with their several million possible
Considerations structures created by combining various cations and anions, also
pose an uncommon issue in terms of defining or identifying the
toxicity and adverse impacts of those compounds on the environ-
ment. ILs are novel compounds that have not yet gained wide-
spread acceptance. It is challenging for traditional scientists to
abandon theories that have sprung out from the fertile ground of
molecular solvents over millennia. The number of laboratories that
work with ILs has increased significantly in recent years, particularly
in China. Of course, it’s crucial to have a complete awareness of the
potential health risks and environmental effects of ILs.
Unfortunately, research has shown that the initial generation of
ILs, which were based on imidazolium or pyridinium cations, were
oftentimes much more harmful than conventional solvents. The
goal for the elimination of the most harmful IL structures is the
development of simple toxicity tests to enable quick and affordable
identification of the best IL structures. Selecting only the most
suitable candidates for application, the remaining candidate struc-
tures can be examined using minimum inhibitory concentrations
(MICs) and minimum biocidal concentrations (MBCs) tests,
growth rate measurements, and EC50 (medium effective concen-
tration) calculations.
Imidazolium, pyridinium, phosphonium, and ammonium
cations were not found to have low freshwater toxicity with EC50
values below 100 mg/L, according to certain researches [25]. No
matter the kind of cation, the length of the substituted alkyl chain
on the cation had a substantial impact on the toxicity; for example,
ILs with eight carbon atoms (C8) were shown to be more toxic
than those with six and four. Due to their extensive structural
variety, ILs appear to have a particularly promising future in sepa-
ration approaches. Technologists need thorough research to hasten
the introduction of new nontoxic ILs in the created new separation
processes because certain ILs are harmful and cannot be considered
as general green replacement solvents.
6 Regeneration of Organic Phase
Unwanted components are not always stripped. Then it accumu-

lates in the organic phase to the point where it substantially impedes
extraction. In this instance, a more forceful strip is applied to the
solvent to remove the impurity and renew its performance. The
contaminant may be allowed to accumulate in the extract for some
time before the solvent is regenerated in batches, or a tiny side
stream may be constantly bled from the recycled organic phase and
regenerated. Regeneration of the organic phase can be expensive
due to its robust nature, but the contamination can occasionally be
highly valuable. Complexes of platinum, gold, and cobalt have
occasionally behaved as pollutants, and their recovery has covered
the cost of regeneration.
7 Bio-Derived Solvents in Water
Among all bio-solvents, bio-based ethanol is now the most widely

manufactured. It is made by biologically transforming sugars. Both
edible (sugarcane and maize) and inedible (cellulose) feedstocks are
used in these procedures [26]. However, there has been a move
towards improving the processes that produce cellulosic ethanol
due to worries that edible feedstock is driving up food prices [27],
and in fact, Beta Renewable launched the first cellulosic ethanol
commercial-scale production plant in the world in 2013. This
facility (Biochemtex, Crescentino Biorefinery) in Milan, Italy, uses
ProesaTM technology to pre-treat agricultural waste (such as rice
straw, giant cane (Arundo donax), and wheat straw) for the gener-
ation of ethanol at a rate of 60,000 tons per year.
The most widely used applications for ethanol are as a biofuel
and solvent in consumer goods such as perfumes, food coloring and
flavoring, alcoholic beverages, and some types of mediation. Both
natural and manufactured drugs may contain the latter. A highly
effective method of extracting the active antimalarial medication
artemisinin from Artemisia annua has been shown [28]. Methanol
is currently produced on a large scale from fossil fuels by hydro-
genating carbon monoxide in the presence of a catalyst like ZnO/-
Cr2O3 or Cu/ZnO/Al2O3 [29]. It can be found in trace
concentrations in fermentation broths and when biomass is gasified
to create bio-based syngas, which is then converted to methanol.
The latter’s economic potential is still being researched
[30]. Energy density for n-butanol is 29.2 MJ/L, which is compa-
rable to gasoline’s (32.5 MJ/L) and has a low level of toxicity. It is
not surprising that this alcohol is occasionally blended with petrol
to enhance its qualities because it is likewise miscible with petrol. In
addition to being used as a solvent for paints, varnishes, resins, and
Table 3
Solubilities of bio-derived solvents in water
Solvents Examples Solubility in Water

Alcohols Ethanol Soluble
Glycerol Soluble
Glycerol derivatives Over 60 species Mostly soluble
Esters Biodiesel Insoluble
Ethyl Lactate Soluble
Acids Gluconic acid Soluble
Terpenes D-limonene Insoluble
α-pinene Insoluble
p-cymene p-cymene Insoluble
Furfural family Furfural Soluble
Furfural alcohol Soluble
Levulinic acid Soluble
Ethyl levulinate Soluble
Butyl levulinate Soluble
γ-valerolactone γ-valerolactone Soluble
Furan derivatives 2-Methyltetrahydrofuran Soluble
2,5-Dimethylfuram (DMF) Insoluble
Dihydrolevoglucosenone Dihydrolevoglucosenone (Cyrene) Soluble
waxes, n-butanol is also used to make plasticizers like butyl phtha-

lates, solvents like butyl propanoate, dibutyl ether, and butyl ace-
tate, as well as coatings (varnishes, resins, and waxes). Table 3 lists a
variety of bio-derived solvents and their water solubilities; it
demonstrates that biodiesels, terpenes, and 2,5-dimethylfuran
(DMF) are water insoluble and may, therefore, be employed in
solvent extraction procedures [1]. Energy crops like corn, forest
goods like wood, aquatic biomass like microalgae, and waste pro-
ducts like urban wastes are all examples of biomass. These
bio-derived solvents can be destroyed after usage (Fig. 4).
8 Application of Solvent Extraction in Biotechnological Separations
Traditional biotechnological product recovery strategies have cen-

tered on separation techniques like electrophoresis or column liq-
uid chromatography. These techniques are costly for medium- and
low-value products and challenging to scale up to production
levels. The primary recovery of fermentation cell culture products,
such as carboxylic acids, proteins, and amino acids, has thus been
recognized as a possible application for liquid extraction. However,
Fig. 4 Life cycle of bio-derived solvents
the separation issue is challenging because the product combina-

tions, which frequently contain cell waste and enzymes, are compli-
cated. Proteins can be handled in aqueous two-phase systems or by
extraction in reverse micellar systems but are not appropriate for
conventional solvent extraction due to incompatibility with organic
solvents. Although it is frequently claimed that biotechnological
processes are energy efficient since the reaction temperature is low,
it is crucial to understand that the product concentrations are low
and that the product recovery step is frequently the one that
requires the most energy.
8.1 Carboxylic Acids For the purpose of separating organic and amino acids from fer-
Separation mentation broth, solvent extraction has been suggested as an alter-
native [31]. High molecular weight amines and organophosphate
solvating agents can both be used to extract citric acid. Important
requirements have been defined for the solvent extraction process
used to produce citric acid: A distribution coefficient of 10 or below
enables simple water stripping. Although the citric acid may be
recovered using a base, the production of a citrate salt would
require additional processing as in the typical flow sheet, eliminat-
ing the benefits of solvent extraction. This is why stripping with
water is crucial.
8.2 Amino Acids Because amino acids include both carboxyl (-COOH) and amino
(-NH2) groups, they exhibit cation-like behavior at low pH,
anionic behavior at high pH, and zwitterion behavior at intermedi-
ate pH levels. Their solubility in nonpolar diluents is limited despite
the fact that they have no net charge in this intermediate pH range
due to their hydrophilicity. Additionally, its extraction using oxygen
donor extractants with carbon bonds is subpar. In order to extract
an amino acid, it is typically necessary to change it into one of its
ionic forms and then utilize an appropriate ion-pair extractant.
Because the amino acid has a net negative charge in high pH
conditions, an anionic extractant, often an alkylammonium salt,

such as tri-n-octylamine hydrochloride (R3NHCl), can be used to
remove it. Here, (R3NH) (RNH2COO) is the extracted species
[32]. Both times, regeneration of the extracting species is simple by
the use of diluted alkaline or acidic solutions, respectively.
8.3 Citric Acid For the purpose of separating organic and amino acids from fer-
mentation broth, solvent extraction has been suggested as an alter-
native [33]. High molecular weight amines and organophosphate
solvating agents can both be used to extract citric acid. Important
requirements have been specified for the solvent extraction proces-
sing of citric acid: A distribution coefficient of 10 allows for simple
water stripping. Although the citric acid may be recovered using a
base, the production of a citrate salt would require additional
processing as in the typical flow sheet, eliminating the benefits of
solvent extraction. This is why stripping with water is crucial.
Temperature had an impact on a diluent’s extraction, which
decreased as temperature rose. So, an effective technique was devel-
oped by extraction from the broth at room temperature and water
stripping at a higher temperature (60–70 °C). The creation of
emulsions and the resulting poor separation made it difficult to
extract using long-chain amines.
8.4 Extraction of Oil A difficult task in calculating the total economics of fuel production
from Algae Biomass is the extraction of fuels from microalgae biomass. It has been
discovered that the commonly used extraction techniques call for
either more sophisticated machinery or challenging processing
conditions. The goal of the innovative extraction technique is to
remove the oil from the biomass using a combination of solvent
extraction and magnetic stirred agitation. When compared to
extraction techniques such as supercritical extraction and nano-
assisted extraction, which are actively being researched, this tech-
nology has been shown to be more cost-effective. If magnetic
stirred or electromagnetic-assisted agitation is used on a commer-
cial scale, the use of magnetic stirrer-based extraction for sustain-
able biofuel production may lead to new dimensions [34]. This
method uses natural algae biomass for oil extraction.
8.5 Bioactive from In a variety of culinary products, compounds from marine algae
Marine Algae have been employed as gelling, thickening, and emulsifying agents.
Apart from being a rich source of iodine historically, sea algae were
not recognized as a source of health-promoting chemicals in the
West [35]. Marine algae are a rich source of nutraceuticals with a
variety of biological activities, according to a recent study on func-
tional food ingredients [36]. Dietary fiber, sulfated polysaccharides,
omega-3 fatty acids, amino acids, bioactive peptides, vitamins,
minerals, and carotenoids are all abundant in marine algae. These
algae are also widely distributed in nature. New and improved

innovative extraction technologies must be developed in order to
fully realize this promise. Traditional extraction methods need a lot
of time and employ organic solvents, which are not environmentally
friendly [37].
9 Pharmaceutical Separations
For the creation of pharmaceuticals and the isolation of natural

compounds, liquid-liquid extraction is widely utilized [38]. Since
these compounds are frequently heat-sensitive, techniques like air
distillation or evaporation cannot be used to recover them. Due to
competition, there is a lack of specific information about ongoing
business operations. The purification and concentration of penicil-
lin is a well-known and best-documented example of a process that
ran into issues common to medicinal substances [39].
9.1 Production of Before being given to the first extraction step, where it is in touch
Penicillin with substances like butyl acetate, the fermentation broth contain-
ing penicillin is first filtered to remove mycelium and pH-adjusted
to 2–2.5 to convert it to the mostly undissociated penicillanic acid
[40]. The partition coefficient and penicillin stability have been
compromised to determine the exact pH that is employed. How-
ever, if it becomes necessary to create pure penicillin for pharma-
ceutical use, it can be refined by reextraction at pH 2–2.5 and
further stripping with a phosphate solution at pH 6. The majority
of penicillin is utilized as intermediates in the synthesis of, for
example, cephalosporin. This penicillin extraction is an illustration
of the direct partitioning of a solute using a polar organic molecule.
Utilizing organic chemicals that form ion pairs with penicillin is a
different method that has been taken into consideration. Here, the
authors discovered that in the pH range 5–7, where the product is
most stable, the penicillin anion could be extracted effectively with
a secondary amine (Amberlite LA-2). This method, which is widely
employed in hydrometallurgy, can be utilized to extract either
cationic species using organic acid anions or anionic species utiliz-
ing cations, as was previously demonstrated. While a hydrocarbon
diluent is typically used in hydrometallurgy, more polar diluents are
typically needed for medicinal applications. Ion pair creation is used
in a number of other biotechnology systems, even though the
adoption of such chemically assisted extraction procedures is
unlikely to replace the current extraction processes for the commer-
cial extraction of penicillin.
10 Future Trends in the Development of New Solvents
Concern over the effects of chemical activities on the environment

will only grow in the future. The liquid effluents must be made
environmentally safe. The main efforts focus on enhancing the
water’s solvent properties at the proper temperature and pressure
or using cosolvents or additions that are water-miscible, including
surfactants. However, other options, like the creation of new sol-
vents with improved separation capabilities, switching to renewable
organic solvents like alcohols from petroleum-derived ones, or the
use of supercritical fluids and ILs, can provide less expensive,
extremely inventive, and untapped environmentally friendly
options.
11 Concluding Remarks
So long as the processes are properly built, solvent extraction is, in

theory, an environmentally pleasant process that doesn’t pollute the
air or the water. Therefore, it might replace a lot of the current
polluting operations. The solvent extraction effluents, however, are
a specific issue since they may contain biochemically active chemi-
cals that present “new” environmental dangers. These can be pro-
cessed using a variety of solid sorbents, which can subsequently be
burned, although the benefit of solvent extraction may be lost.
Therefore, there is a need for environmentally friendly and biode-
gradable solvent phases. Future research in this area will need more
focus. Numerous fields of chemistry have used the principle of
solvent extraction, which divides chemical species between two
immiscible liquid phases. One such example is liquid partition
chromatography, whose widespread application has given rise to a
whole field (and industry!) as a result of the principle of solvent
extraction providing the most effective separation procedure now
available to organic chemistry. Fundamental studies on solvent
extraction are anticipated to continue to influence the creation of
new, selective analytical methods. Chemical kinetics, final equilib-
rium distribution of the contents between the two phases, and
transit of chemical compounds from one phase into another are
all topics covered by solvent extraction. The mechanisms that
enable such transport and distribution are what sustain life in
biological systems. Fundamental investigations of these “solvent
extraction” processes advance our knowledge of all natural pro-
cesses. Here, only a lack of creativity prevents the development of
significant new scientific findings. The focus of future research
efforts in this field should be on overcoming the barriers to imple-
menting these cutting-edge technologies on a large-scale basis so
that the enormous advantages of better bioactive extraction from
food and their use in the nutraceutical sectors may be realized.
Continued research on extraction technologies that are both

economically and environmentally viable is driven by the need to
extract nutraceuticals from plant-based materials. Traditional solid-
liquid extraction techniques take a long time and a lot of solvent.
The extensive use of solvent raises operating costs and contributes
to more environmental issues. As an alternative to traditional
extraction procedures, a number of unique extraction techniques
have been developed, giving benefits in terms of extraction time,
solvent usage, extraction yields, and reproducibility. However, only
a small number of applications have made use of innovative extrac-
tion approaches. More study is required to better comprehend the
mechanisms of extraction, overcome technological obstacles,
enhance the design, and scale up novel extraction systems for use
in industrial settings.
References
1. Li Z, Smith KH, Stevens GW (2016) The use 10. Ogunsola OM, Berkowitz N (1995) Extrac-
of environmentally sustainable bio-derived sol- tion of oil shales with sub-critical and near-
vents in solvent extraction applications—a critical water. Fuel Proc Technol 45:95–107
review. Chin J Chem Eng 24(2):215–220 11. Cassel E, Vargas RMF, Martinez N,
2. Khataei MM, Epi SBH, Lood R, Spégel P, Lorenzo D, Dellacassa E (2009) Steam distilla-
Yamini Y, Turner C (2022) A review of green tion modeling for essential oil extraction pro-
solvent extraction techniques and their use in cess. E Ind Crop Prod 29:171–176
antibiotic residue analysis. J Pharm Biomed 12. Basile A, Jiménez CMM, Clifford AA (1998)
Anal 209:114487 Extraction of rosemary by superheated water. J
3. Rajapaksha S, Shimizu N (2022) Pilot-scale Agric Food Chem 46:5205–5209
extraction of polyphenols from spent black tea 13. Wiboonsirikul J, Adachi S (2008) Extraction of
by semi-continuous subcritical solvent extrac- functional substances from agricultural pro-
tion. Food Chem X 13:100200 ducts or by-products by subcritical water treat-
4. Bains W (2004) Many chemistries could be ment. Food Sci Technol Res 14(4):319–319
used to build living systems. Astrobiology 14. Kislik VS (2011) Solvent extraction: classical
4(137):167 and novel approaches. Elsevier
5. Castro-Puyana M, Marina ML, Plaza M (2017) 15. Sapkale GN, Patil SM, Surwase US, Bhatbhage
Water as green extraction solvent: principles PK (2010) Supercritical fluid extraction. Int J
and reasons for its use. Curr Opin Green Sus- Chem Sci 8(2):729–743
tain Chem 5:31–36 16. Gumerov FM, Khairutdinov VF, Zaripov ZI
6. Miller DJ, Hawthorne SB (2000) Solubility of (2021) An additional condition of efficiency
liquid organic flavor and fragrance compounds of the supercritical fluid extraction process.
in subcritical (hot/liquid) water from 298 to Theor Found Chem Eng 55:348–358
473 K. J Chem Eng Data 45:315–318 17. Wilkes JS, Zaworotko MJ (1992) Air and water
7. Li B, Yang Y, Gan Y, Eaton CD, He P, Jones stable 1-ethyl-3-methylimidazolium based
AD (2000) On-line coupling of subcritical ionic liquids. J Chem Soc Chem Commun 13:
water extraction with high-performance liquid 965–967
chromatography via solid-phase trapping. J 18. Chapeaux A, Simoni LD, Ronan T, Stadtherr
Chromatogr A 873:175–184 MA, Brennecke JF (2008) Extraction of alco-
8. Luque de Castro MD, Jime ´nez-Carmona hols from water with 1-hexyl-3-methylimida-
MM, Ferna ´ndez-Pe ´rez V (1999) Towards zolium bis(trifluoromethyl sulfonyl)imide.
more rational techniques for the isolation of Green Chem 10:1301–1306
valuable essential oils from plants. Trends. 19. Xiao Z, Liu M, Bi W, Chen DDY (2023) Ionic
Anal Chem 19:708–716 liquid as hydrogen bond acceptor in the extrac-
9. Smith RM (2002) Extractions with super- tion of nutritional natural products. Food
heated water. J Chromatogr A 975:31–46 Chem 412:135589
20. Fadeev AG, Meagher MM (2001) Opportu- 30. Morone P, Cottoni L (2016) Biofuels: technol-
nities for ionic liquids in recovery of biofuels. ogy, economics, and policy issues. In: Hand-
Chem Commun 3:295–296 book of biofuels production, 2nd edn.
21. Fredlake CP, Crosthwaite JM, Hert DG, Aki Woodhead Publishing, Duxford
SNVK, Brennecke JF (2004) Thermophysical 31. Wennersten R (1983) The extraction of malic
properties of imidazolium-based ionic liquids. acid from fermentation broth using a solution
J Chem Eng Data 49:954 of tertiary amine. J Chem Tech Biotechnol 33:
22. Visser AE, Swatloski RP, Griffin ST, Hartman 85
DH, Rogers RD (2001) Liquid/liquid extrac- 32. Hano T, Matsumoto M, Ohtake T, Sasaki K,
tion of metal ions in room temperature ionic Hori F, Kawano Y (1990) Separation of lactic
liquids. Sep Sci Technol 36:785–804 acid from aqueous solution. J Chem Eng Jpn
23. Chun S, Dzyuba SV, Bartsch RA (2001) Influ- 23(6):734
ence of structural variation in room- 33. Schugerl K (1994) Solvent extraction in bio-
temperature ionic liquids on the selectivity technology. Springer-Verlag, Berlin, pp
and efficiency of competitive alkali metal salt 113–125
extraction by a crown ether. Anal Chem 73: 34. Govindarajan L, Raut N, Alsaeed A (2009)
3737–3741 Novel solvent extraction for extraction of oil
24. Wei GT, Yang Z, Chen CJ (2003) Room tem- from algae biomass grown in desalination reject
perature ionic liquid as a novel medium for stream. J Algal Biomass Util 1(1):18–28
liquid/liquid extraction of metal ions. Anal 35. Yvonne Y (2007) Marine algal constituents. In:
Chim Acta 488:183–192 Marine nutraceuticals and functional foods.
25. Zhao D, Liao Y, Zhang Z (2007) Toxicity of CRC Press, Boca Raton, pp 259–296
ionic liquids. Clean 35:42–48 36. Smit AJ (2004) Medicinal and pharmaceutical
26. Muñoz I, Flury K, Jungbluth N, Rigarlsford uses of seaweed natural products: a review. J
GI, Canals LM, King H (2014) Life cycle Appl Phycol 16:245–262
assessment of bio-based ethanol produced 37. Kadam SU, Tiwari BK, O’Donnell CP (2013)
from different agricultural feedstocks. Int J Application of novel extraction technologies
Life Cycle Assess 19:109–119 for bioactives from marine algae. J Agric Food
27. Lynd LR, Liang X, Biddy MJ, Allee A, Cai H, Chem 61(20):4667–4675
Foust T, Himmel ME, Laser MS, Wang M, 38. Verrall MS (1992) In: Thornton JD
Wyman CE (2017) Cellulosic ethanol: status (ed) Science and practice in solvent extraction.
and innovation. Curr Opin Biotechnol 45: Oxford Science Publications, Oxford. Chap. 3
202–211 39. Kirk RE, Othmer DF, Kroschwitz JI, Howe-
28. Babacan U, € Cengiz MF, Bouali M, Tongur T, Grant M (1993) Kirk-Othmer encyclopedia of
Mutlu SS, Gülmez E (2022) Determination, chemical technology, vol 10, 4th edn. Wiley,
solvent extraction, and purification of artemisi- New York, p 125
nin from Artemisia annua L. J Appl Res Med 40. Bajpai RK, Reuss M (1980) A mechanistic
Aromatic Plants 28:100363 model for penicillin production. J Chem Tech-
29. Hansen JB, Nielsen PEH (2008) Methanol nol Biotechnol 30(1):332–344
synthesis. In: Handbook of heterogeneous
catalysis, vol 1, 2nd edn. Wiley-VCH Verlag
GmbH & Co. KgaA, Weinheim
Chapter 8
Enzyme-Assisted Extraction
Sadhana B. Maled, Ajay R. Bhat, Subrahmanya Hegde, Yuvaraj Sivamani,
Arunachalam Muthuraman, and Sumitha Elayaperumal
Abstract
The extraction of compounds from plant and animal sources that have medicinal properties are known as
bioactive compounds, which can either improve health or can be hazardous. As novel natural compounds
from various plants are uncovered, we are discovering more biologically active compounds. Enzymes can be
employed to extract such bioactive compounds by rupturing plant cells. The extraction of a bioactive
compound such as stevioside from a stevia plant with the assistance of enzymes is a better example of
processing with potential utility for the food sector. The extraction of bioactive compounds from plant
sources with the help of environment-friendly enzymes, especially for food, nutraceutical, or pharmaceuti-
cal uses, is a cutting-edge technology used in these industries. This study explains the overall idea about
bioactive compounds, their extraction processes, importance, and uses.
Key words Bioactive, Stevioside, Stevia, Enzymes, Enzyme-assisted extraction, Nutraceutical, Bio-
technological applications
1 Introduction
The chemical compounds that are produced in the plant, which are
not involved in the metabolic activity of the plant, are mostly
secondary metabolites. These are biologically active compounds
[1]. These are produced by the subsidiary pathways. These bioac-
tive compounds have antioxidant, anti-inflammatory, and antimi-
crobial properties and greater immunomodulatory potential
properties. Detailed information about secondary metabolites and
their application is mentioned in Table 1. These compounds pos-
sess additional nutritional value to the food that is generally found
in less amount, which provides health-related benefits over the
product’s basic nutritional value [2]. These compounds include
pharmaceuticals, flavors, fragrances, cosmetics, food additives,
feedstocks, and antimicrobials. These compounds are produced
173
174 Sadhana B. Maled et al.
Table 1
Type of chemical class, plant source, and enzyme used for the extraction of specific bioactive
compounds
Type of bioactive Class of bioactive Enzyme used for

compound compound Plant source extraction
Glycosides Sugar Grapefruit peel Cellulose and pectinase
Oligosaccharides waste Cellulose
Inulin Rice bran Inulinase
Starch Jerusalem artichoke Pectinase
Pectin Cassava Xylanase
Pumpkin Pectinesterase
Endopolygalacturonase
Beta glucosidase
Cellulose
Oil and carotenoids Oil Grape seed Cellulose
Carotenoids Marigold flower Xylase
Lycopene Tomato Pectinase
Anthocyanin Grape’s skin Protease
Capsaicin Chilly Viscozyme
Carotene Carrot pomace Pectin
Nutrase
Corolase
Ht-Proteolytic
Pancreatin
Cellulose
Pectinase
Pectin ax BE3-l
Cellulose
Hemicellulose
Pectinase
Pectin ultra SP-L
Others Flavonoids Kinnow peel Recombinant
Phenolics Citrus peel rhamnosidase
Soluble fiber Carrot pomace CelluzymeMX
Protein Lentils and white Cellulose-rich crude
Catechins beans preparation
D beverage Glucoamylase
Pepsin
by the plant cell through metabolic pathways such as the malonic

acid pathway, shikimic acid pathway, methylerythritol phosphate
pathway, and mevalonic acid pathway.
The extraction of bioactive compounds is becoming much
easier nowadays, and these extracted compounds are utilized in
the drug industries for the production of novel drugs against life-
threatening diseases and also utilized in food industries to enhance
the quality of the food. Other potential properties of bioactive
compounds, for example, antimicrobial, anti-inflammatory, anti-
cancer, antimicrobial, and antidiabetic activities are utilized for
Enzyme-Assisted Extraction 175
various applications. For the extraction of these compounds, the

traditional method of extraction gives less yield, and also there is no
proper method for extracting the bioactive compounds.
Biotechnologists and other chemists started to find a way to
extract bioactive compounds with a greater yield on an industrial
scale, which can also be helpful commercially. After so many
attempts of research, enzyme-assisted extraction came out to be
an efficient way to extract bioactive compounds [3]. The enzymes
will disrupt the cell wall and make the cell wall permeable that can
be easier to get more yield of bioactive compounds such as flavo-
noids, terpenoids, and lectins, which are having applications in
various types of industries. This method of using specific enzymes
for the extraction of compounds seems to be very efficient as
compared to the other extraction techniques.
2 Examples of Plant-Based Bioactives
The major classes of bioactive compounds are alkaloids, terpenoids,

and phenolic compounds. Other basic examples include bioactive
compounds that are flavonoids, terpenes, polyphenols, lycopene,
resveratrol, lignan, tannins, and indoles [4]. These types of com-
pounds are mainly used in food industries, for example, marigold
flowers are the most significant source of carotenoids. Nevertheless,
the efficacy of the extraction depends on the circumstances for
collection, drying, and the solvent extraction procedure, which
results in 50% losses of the carotenoids. Macerating enzymes have
been used before with good results to increase the extraction yield
of effective chemicals from natural materials. In this study, a differ-
ent extraction technique for carotenoids was discovered that
involved both a simultaneous enzymatic procedure and solvent
extraction [5]. By skipping the ineffective matter and drying pro-
cedures, the suggested method uses freshly milled flowers as raw
material. A carotenoid recovery yield of 98% was achieved using the
described technique at the 80 L scale, under typical testing condi-
tions [6]. Lycopene, a carotenoid found in high concentration in
tomatoes and tomato-derived products, is receiving a lot of atten-
tion these days because epidemiological evidence suggests that it
may protect against cancer and some degenerative diseases that are
impacted by free radical reactions. Recent epidemiological studies
have shown that consumption of tomatoes and blood lycopene
levels are inversely related to the risk of getting cancer at several
anatomical sites, including the prostate gland, stomach, and lungs.
By successfully delocalizing trapped free radical species, conjugated
carbon-carbon double bonds endow lycopene with their antioxi-
dant characteristics. Hence, food industries, pharmaceutical indus-
tries, and cosmetic industries all have a significant demand for
lycopene [7].
Salvia officinalis, which is communally called sage, is the most

commonly known herb used in the kitchen, and it belongs to the
family Labiatae. Sage has a long history of helping people stay
healthy and alleviate illnesses. Sage essential oil has shown efficiency
in the treatment of Alzheimer’s disease and efficiency to enhance
memory, according to current research and scientific studies. Alz-
heimer’s is a neurological disease. Sage is one of the kitchen plants
with an aromatic nature and contains an oil-producing capacity.
Sage has been the subject of much research for its phenolic antioxi-
dant components since its early days, roughly 20 years ago. Sage has
several powerful antioxidants, according to numerous types of
research [8]. Sage is a plant of commercial relevance for scientific
research because of its essential oil and antioxidant components. All
food, cosmetic, and pharmaceutical products start with sage essen-
tial oil and flavorings as their primary ingredient [9]. Sage antiox-
idants can be utilized as a different approach to the popular
rosemary antioxidants to protect and preserve specific foods and
nutraceutical items to lengthen their shelf life [8]. Other different
types of bioactive compounds and their respective plant source and
their uses are mentioned in Table 2.
Bioactive compounds can also be extracted from other sources
such as microbes, fungi, and animals. Fishes are one of the sources
for bioactive compounds. Different types of bioactives extracted
from different fish species and their application are listed in Table 3.
2.1 Extraction The process of extraction involves the separation of our desired
Process natural constituents from the available raw materials. There are
different types of extraction methods commonly used in industries
for the extraction of compounds from plant and animal sources, for
example, distillation extraction, solvent-based extraction, pressing
method, and sublimation process, which are categorized based on
their working principle. The reason behind the process of extrac-
tion of bioactive material is to get the therapeutically valuable
molecule and the inert part is eliminated with the help of a selective
solvent treatment known as the menstruum. Menstruum is the
solvent used in the extraction. The materials that remain after the
process of extraction are called “marc.” For the extraction process,
we need to prepare concentrations of raw materials using various
solvents. The solutions such as alcohol and hydro alcohol sols are
prepared from raw materials or chemical substances such as
belladonna [11].
Using various solvents with different polarities is the key feature
of this process. However, to date, there is no single extraction
method for extracting all the metabolites at a given time. Some of
the normal methods are listed as follows:
1. Maceration: In this method of extraction, the plant material is
crushed or chopped into small pieces. Sometimes the dried
Table 2
List of bioactive compounds and their plant sources with uses
Product Plant species Uses

Digoxin Digitalis lanata Cardiovascular disorders
Vanillin Vanilla sp. Vanilla
Jasmine Jasminum sp. Perfume
Taxol Taxus brevifolia Anticancer
Baccharine Baccharis megapotanica Anticancer
Cesaline Caesalpinia gillisesii Anticancer
Pyrithrins Tagetus erecta Insecticide
Saffron Crocus sativus Food color and flavoring agent
Stevioside Stevia rebaudiana Sweetener
Berberine Coptis japonica Antibacterial
Quinine Cinchona officinalis Antimalarial
Atropine Atropa belladonna Muscle relaxant
Reserpine Rauwolfia serpentina Hypotensive
Diosgenin Dioscorea deltoidea Antifertility
Vinblastine Catharanthus Anticancer
Maytansine Maytenus bucchananii Anticancer
Thaumatin Thaumatococcus danielli Sweetener
Capsaicin Capsicum frutescens Chilli
powder of plants is used to increase the efficiency of extraction.

This method is performed in the closed system of the men-
struum, which is the solvent used in the extraction. The plant
material and this solvent are placed together; then it is incu-
bated for some days with shaking at certain time intervals. After
5–7 days, the fluidics strained off and we can see the two phases
in the system, namely, one supernatant and the residue part.
Then the residue part is pressed to get the more fluidic content
out and further followed by the filtration. Once the filtration is
done, the solution is taken and solidified by evaporation
method for ease of analysis of the compound followed by
concentrating it and sent for further analysis and preparation
of drugs in the pharmaceutical industries.
2. Percolation: This method is preferable for the production of
vegetable drugs in the food industry. In this method, the
uniform moistening of drugs with solvent for a period of
3–4 h is done in a separable vessel, which is performed in the
Table 3
List of the bioactive compounds derived from different fish species [10]
Source Origin Bioactives

Acipenser schrenckii Skin Antioxidant, Cry protective
Navodon Skin Antioxidant
Gadus macrocephalus Gelatin Antioxidant, ACE inhibitory
Johnius belengerii Skin Antioxidant
Theragra chalcogramma Skin ACE inhibitory
Oncorhynchus keta Skin Neurobehavioral
Priacanthus macracanthus Skin Antioxidant
Lutjanus vitta Skin Antioxidant
Parastromateus niger Viscera Antioxidant
Johnius belengerii Bone Calcium binding
Exocoetus volitans Bone Antioxidant, antiproliferative
Raja porosa Cartilage Antioxidant
Oreochromis niloticus Skin Antioxidant
Oncorhynchus keta Skin Long bone development
Chanos chanos Collagen Iron binding
Oncorhynchus keta Collagen Learning and memory
Oreochromis sp. Collagen Facial skin quality
Gadus morhua Bone Antioxidant
Clupea harengus Whole, body, head gonads Antioxidant
closed system. A piece of filter paper is placed on the surface,

then picked up so that the top layers of drugs are not disrupted.
The solvent is poured on the sample slowly at some specific
time interval, allowing the bottom area to percolate easily and
which is collected in the bottom of containers. This process
followed for one complete day and fluid percolated through the
plant sample is collected and followed by the evaporation of the
sample to get the powder form to quantify the sample then is
concentrated and sent for analysis which is further sent for
production industries for the production of compounds at an
industrial scale.
3. Soxhlet apparatus: This apparatus is used in the small-scale
extraction of bioactive compounds in laboratory conditions.
These steps are involved in bioactive extraction. The plant
sample of holanerrhea pubescus, terminalia elliptica, or any
other plant species such as mulberry which have high medicinal
properties is first dried in the microwave and then the pow-

dered, dry powder of the plant sample is then placed in the
pocket made up of a blotting paper like a pouch. This pouch is
loaded in the main chamber of Soxhlet apparatus, the chamber
that is attached to the condenser on the top, for the cooling
effect, and in the bottom heating mantle for heating the sam-
ple. The solvent used here is ethanol or methanol. The phyto-
chemicals in the sample are drawn into the solution and
collected at the bottom, and after boiling the sample is cooled
by the condenser. The process is continued until the drug or
the phytochemicals are extracted and the extracted compounds
in the flask are processed by evaporating and followed by the
concentration method and sent for analysis [3].
2.2 Solvents Used in In the process of extraction of bioactive compounds, different

Extraction solvents are used. There are two types of solvents, namely, nonpolar
solvents and polar solvents. The nonpolar solvents used in the
extraction are cyclohexane, toluene, hexane, benzene, ether, chlo-
roform, and ethyl acetate; hence, the products extracted from the
nonpolar solvents are alkaloids, terpenoids, coumarins, fatty acids,
flavonoids, and terpenoids [12]. The polar solvents used in the
extraction are acetone, acetonitrile, butanol, propanol, ethanol,
and methane. The products that are extracted by using polar sol-
vents are flavonols, lectins, alkaloids, quassinoids, flavones, poly-
phenols, tannins, and saponins [12].
3 Process Development
The production of bioactive compounds has a commercial demand

in the market; hence, there demand toward the plant tissue culture
is at higher rates. The bioactive compounds in plants are obtained
in the lower quantity with high-value pharmaceutical properties
and food-beneficial properties. Hence, to get the bioactive com-
pounds in the greater quantity for commercial use, biotechnolo-
gists go for the method called extraction of bioactive compounds
facilitated by enzymes [13]. In an enzymatic method, there will be
efficient extraction and a greater yield of bioactive compounds. In
this method, specific lytic enzymes are used to damage the outer
membrane of the plant cells to improve the productivity of the
extraction [6].
The use of enzymes for the extraction of various bioactive
compounds, for example, extracting vanillin from vanilla grape
seed, extracting carotenoids either from tomato peel or marigold
flower, extracting polysaccharides from Sterculia foetida, extracting
oil from grape seeds, etc. has shown significantly good results. This
method of extraction has greater potential and is commercially
attractive [13]. Prior to the adoption of traditional extraction tech-
niques, enzymes were utilized specifically to treat plant material
[7]. It is frequently necessary to use a variety of enzymes, such as

pectinases, cellulases, also hemicelluloses, to break the structure
[14]. Improving the extraction of bioactives from plants while
maintaining the rigidity of the plant cell wall is challenging. These
enzymes enhance cell wall permeability by hydrolyzing cell wall
constituents, leading to increased bioactive extraction yields
[13]. Extracts of fungi, bacteria, vegetables, or animal organs,
also fruits can all be used to make enzymes. Understanding an
enzyme’s catalytic features and mode of action, ideal performing
circumstances, and the enzyme or combination of the enzyme to
utilize in an extraction application will help you use enzymes more
effectively [11].
3.1 The It is the crucial step in the process of getting the bioactive molecules
Disintegration of the out of cells. Cells are the basic unit of life; cells store valuable
Cell Wall by the Action bioactive compounds such as flavonoids, anthocyanin, carotene,
of Enzymes and many more which has significant value in both food and
nutraceutical industries; there is a variety of other classes of bioac-
tive compounds from medicinal plants, which have pharmacologi-
cal importance. Hence, it is important to extract them without
denaturation and also to increase the extraction yield and produc-
tivity. Therefore, as the science is developing, there are various
techniques that have been introduced for the easy extraction of
phytoconstituents from various sources. The enzyme-assisted
extraction is one such method that is commonly used nowadays
to better intracellular compound extraction methods.
During the process of extraction of intracellular compounds
from the cell, it is important to break open the cell so that it allows
the intracellular compounds to come out of the cell. The capacity of
the enzymes to degrade the cell wall component and also to disin-
tegrate the systematic integrity of the plant cell wall forms the basis
for enzyme-assisted extraction. When enzymes and substrates bind
to one another, the shape of the enzyme molecule adapts as better
as possible with the substrate to improve the interaction. The
change in form could put the substrate under strain and stress,
which could lead to the bonds breaking and speed up the reaction.
When the substrate concentration is high, the enzyme can acceler-
ate the process until the substrate concentration becomes limiting.
The operational characteristics of each investigation of enzyme-
assisted extraction are taken into consideration, including system
pH, enzyme extraction time, substrate particle size, concentration,
and reaction temperature. All study findings demonstrate that
enzyme-assisted extraction leads to a decrease in extraction time
and solvent volume in addition to an increase in yield and product
quality (Fig. 1).
Bioactive compounds in plants are secondary metabolites of
plants that have therapeutic or harmful effects on living things.
Secondary metabolites are created in plants in addition to the
basic biosynthetic and metabolic pathways for the substances
Plant sample Aquous extraction of Breaking down of cell Centrifugation

bioactive through enzyme wall due to the action
assisted method of enzymes
Downstreat processes
Filtration
Fig. 1 Picture depicting enzyme-assisted extraction
related to plant growth and development. These by-products of the

plant cell are not necessary for the regular operation of the plant,
but some of them have been identified to play crucial roles in the
survival of living plants, such as signaling and protection. It appears
that most plant species can synthesize these compounds. But some
chemical groups that make up a plant’s bioactive compounds show
toxicological and pharmacological effects on both animals and
humans.
Bioactive substances can be used for a variety of purposes,
including enhancing the nutritional, sensory, and technological
qualities of conventional food, creating functional foods that have
proven physiological benefits, creating nutraceuticals from food or
agro-industrial waste, key nutritional components, and creating
films for use as active, smart, and/or bioactive food packaging.
3.2 Steps Involved in • Raw material selection

Step 1
the Extraction of
Bioactive Compounds Step 2
• Sample preservation
Step 3
• Bioactive compound extraction
Step 4
• Detection and separation compounds
Step 5
• Purification of compunds by Chromatography
Step 6
• Purified bioactive compound
The purified bioactive compounds can be sent for structural

elucidation by NMR, FTIR, and LC-MS/GC-MS and
biochemical characterization such as in vitro toxicity,
in vitro evaluation, and clinical research.
3.3 Other Different Nowadays, the importance and commercial demands for the
Techniques Combined enzyme-assisted extracted bioactive compounds from plants are at
with EAE to Enhance a high rate. To fulfill the demands of the market, researchers are
the Extraction Process finding novel techniques, which are going to solve the problem
with high commercial value and usage. Some of the novel techni-
ques are microwave-assisted extraction, supercritical fluid extrac-
tion, and ultrasound-assisted enzymatic extraction. These
techniques are combined with enzyme-assisted extraction to
increase the efficiency of extraction to get more yield. These tech-
niques are eco-friendly and efficient with a high yield for the
commercial purpose to fulfill the demands of the market.
1. UAEE (Ultrasound-assisted enzymatic extraction):
Ultrasonic irradiation was utilized to expedite the treat-
ment of enzymes due to the increased effectiveness of
enzyme-used reactions and the convenience of practical work.
As innovative approaches for extracting bioactive compounds,
enzyme-assisted extraction methods linked with ultrasonogra-
phy have been devised [15]. Acoustic cavitation occurs when
ultrasound waves flow through a solvent solution, resulting in
increased extraction yield [16]. When ultrasonic waves are
exposed to a solvent media, tiny vapor-filled bubbles form. It
is named cavitation. Whenever the bubbles reach a particular
structure, they explode forming in localized high temperatures
and pressures. Whenever these bubbles break at the plant cells’
surface, the high temperature and pressure released create liq-
uid jets with shear forces that also are directed toward the plant
cells’ surface. The liquid jets and shear pressures that occur
during this procedure lead to physical harm to the cell’s wall
or cell membrane’s integrity. Ultrasound enhances cell wall
penetration, allowing more solvent to penetrate the cell wall
and leach compounds into the liquid phase. Furthermore,
under ultrasonic treatment optimal conditions (proper fre-
quencies and intensities) can result in increased enzyme work.
This is owing to favorable arrangement shifts and structural
rigidity, to facilitate bimolecular production [17].
Ultrasound-assisted enzymatic extraction is found to be
one of the easiest and quick methods of isolation. An indirect
sonication (ultrasonic wash) and also direct sonication, or
ultrasound horn can be used. The extraction yield in UAEE
of bioactives is related to factors such as ultrasound power
enzyme concentration. This technique will minimize the time
taken for extraction and can be inculcated in commercial use.
The researchers discuss the source of bimolecular extraction, as

well as the different aspects that influence ultrasonic extraction,
including ultrasound strength, frequency, irradiation period,
and extraction yield [15]. Researchers used an enzymolysis-
ultrasound-aided extraction (EUAE) technique to extract poly-
saccharides from corn silk. The ultrasonic treatment will
change the chemical makeup and morphological aspects of
maize-derived silk polysaccharide. Moreover, it modifies the
molecular weight distribution. This is proved based on the
previous studies. The polysaccharide output improved from
4.56% to 7.10% under optimal extraction conditions. In addi-
tion, polysaccharides isolated using EUAE showed modifica-
tions in morphological aspects in the cell wall as well as an
increased anticancer and antioxidant activity when observed
side by side with polysaccharides isolated through boiling
water extraction. Carotenoids were isolated using carrot sap
utilizing ultrasound in conjunction with therapy using enzymes
by intermittent radiation. This technique increases productivity
without causing heat denaturation of carotenoids.
2. Enzyme-assisted high-pressure extraction:
The high-pressure extract (HPE) technique comprises pro-
cessing the plant matter with solvent, then treating the combi-
nation using isostatic ultra-high hydrostatic pressure and
purifying the mixture to remove the solids [18]. To acquire
the biomolecule of interest, the extracted extract is concen-
trated, dried, or filtered further. Plants undergo structural
alterations as a result of high-pressure treatment due to physical
damage to cell membranes, which increases cell wall permeabil-
ity and phytochemical diffusion through solvents [19]. This
results in a higher extraction rate as well as efficiency. In con-
trast to traditional extraction, HPE can easily separate heat-
sensitive chemicals [20]. With this approach, volatile chemicals
can be removed quickly and easily without degradation. Most
biological biomolecules become more soluble under high pres-
sure [21]. HPE operates at pressures ranging from 100 to
1000 MPa. When compared to other removal technologies,
HPE operates at the maximum pressure, allowing it to extract
greater phytoconstituent [22]. To investigate the extraction of
chemicals from plants using natural sources, researchers com-
bined the benefits of biochemical extraction at high
pressures [23].
3. Enzyme-assisted ionic liquid extraction:
Ionic liquids (ILs) are liquid at room temperature and are
composed of bulky organic ligands and inorganic anions
[24]. Because of their outstanding qualities of low vapor pres-
sure, the flexibility of recycling, miscibility using common
organic solvents, thermal and chemical stabilities, and high
solubility and extraction rate in organic compounds, they have

recently gained prominence in a variety of sectors. Because of
the pressing need for environmental preservation, ionic liquids
are seen as safer alternatives to standard solvents in the extrac-
tion of biomolecules from plants. ILs have previously been used
to extract bioactive substances including alkaloids, lignans, and
polyphenols [25]. The viscosity of ILs is also important in their
application for separating bioactive chemicals from plants. At
high temperatures, the fluidity of ILs decreases. ILs can interact
with both polar and nonpolar compounds and are helpful when
paired with a variety of extraction methods [25]. Chemical
composition, concentrations, moisture level, loading rate,
pH, column temperature, and enzymes to target ratio are all
crucial parameters to monitor during IL extraction. IL-assisted
enzymatic extracting has been utilized successfully to extract a
wide range of chemicals, including curcumins and phenolic
compounds [26]. It has been demonstrated that ILs have a
higher viscosity than standard organic solvents. The fluidity of
ILs affects the stability and activity of enzymes. The researchers
and the scientific community recently confirmed how enzyme
conformation changes are delayed in very viscous IL solvents.
This maintains enzyme stabilities for long periods. In general,
high solvent viscosities lead to effective enzyme stability. The
remarkable thermal stability of enzymes in viscoelastic ILs has
opened the door to the extraction of pharmaceuticals from
plants [26]. The durability of a broad range of enzymes, includ-
ing thermolyzing, lysozyme, and chymotrypsin, was shown to
be much higher in this solvent than in typical organic solvents.
From a critical standpoint, enzymes in ILs exhibit good func-
tional and temperature stability in many circumstances [27].
3.4 Types of 1. Polyphenols:

Bioactives That Can Be Plants with a phenolic composition one and phenolic ring
Extracted Using (e.g., polyphenolic compounds or phenolic alcohols) include a
Enzyme-Assisted diverse range of phenolics. The extraction of phenolic chemi-
Extraction Method cals was done either traditionally using mixed solvents (ace-
tone/water, dioxane/ethanol, biofuel, and methanol/water)
or alkaline/acidic techniques. Antioxidants are a large class of
phenolics that are among the most coveted bioactive chemicals
[28]. They are quite interesting and also serve a variety of
biological purposes. They disrupt oxidative cycles to prevent
or delay oxidative damage to macromolecules. Many studies
have shown that fruit peel, seeds, pomace, and leaves are high
in polyphenolic chemicals [29]. Because of the possible toxicity
of some chemical preservatives, researchers have made greater
attempts to uncover and use naturally produced antioxidant
properties from agricultural residues. The early extraction pro-
cesses are critical during the separation of bioactive compounds
from vegetables and fruits. Among these procedures, dehydra-

tion and grinding have the greatest impact on removal capacity
and mass transfer to obtain polyphenols. Drying, in general,
entails the elimination of all bound water from the peel by
enhancing the permeability of the cellular matrix, enabling
diffusion rate and promoting interaction with enzymes,
thereby improving phenol extraction. The extraction efficiency
of polyphenols is affected by phenolic component properties,
operational drying circumstances, and drying [30].
The citrus industry generates huge amounts of peel and
seed wastes (up to 50% of total fruit weight), which could be
the main sources of phenolic chemicals in certain peels. Taking
this into account, total phenolic extract using food-safe enzyme
cellulase, the bioactives from five distinct fruit peels (mandarin,
Yen Ben lemons, grapefruit, orange, and Meyer lemon) were
extracted. Similarly, Gomez-Garcia et al. performed cellulase-
assisted extraction of antioxidative polyphenolic using grape
(Vitis vinifera L.) leftovers in an attempt to valorize fruit
waste. Not only did protease extraction produce more pheno-
lics (O-coumaric acid as assessed by high-performance liquid
chromatography-electrospray mass spectrometry), but it also
demonstrated free radical-scavenging ability. These experi-
ments show that enzymatic extraction of phenolics performs
far superior to traditional approaches. Various methods are
being employed to use agricultural industry trash as a renew-
able source for high value-added (primarily polyphenolic che-
micals) products. The use of enzymes offers an alternate
method for producing these beneficial chemicals from agro-
industrial waste. Enzyme-assisted extraction has been used to
extract antioxidant components from a variety of sources such
as lemon balm (Melissa officinalis), red algal leftover (Palmaria
palmata), Agaricus blazei Murill (rice bran), and pumpkin peel
waste (Cucurbita moschata). Plant cell walls are largely made up
of interconnected polysaccharides such as starch, cellulose,
hemicellulose (xyloglucans), and pectin, which act as a barrier
to the escape of intracellular chemicals [31].
Flavonoids are entrapped in polysaccharide complexes via
hydrogen bonding and hydrophobic interactions. A combina-
tion of carbohydrate-hydrolyzing enzymes, including cellulose,
hemicellulose, and protease, may be a more effective opportu-
nity to sufficiently damage the cell walls, break down compli-
cated interior storage materials, and therefore facilitate the
release of free polyphenols. Some polyphenols are always cova-
lently coupled with glucose in the form of glucosides with
glycosidic connections. Glucosidase is capable of breaking the
-1,4 glucoside bonds in glucosides. Cellulose, hemicellulose,
and pectin can be hydrolyzed using the enzymes, cellulase,
-glucosidase, and pectinase. To improve the extraction yield
of intracellular contents, enzyme-aided treatment was com-

bined with solvent extraction. The authors pretreated Prunus
species with various enzymes (cellulase, pectinase, protease,
and alpha-amylase) before extracting anthocyanins. Flavonoids
are entrapped within polysaccharide complexes via hydrogen
bonding and hydrophobic interaction [32]. A combination of
carbohydrate-hydrolyzing enzymes including cellulose, hemi-
cellulose, and pectinase may be more effective. By solvent
extraction, there is a possibility to adequately damage the wall
of the cell and break down complicated inner storage. Cellulase
produced the highest anthocyanin content when compared to
other enzymes. Furthermore, the quantity of the targeted
enzyme has a considerable impact on the extraction. Lower
enzyme concentrations result in less enzyme interaction with
the peels/pulp, resulting in less extraction of the desirable
polyphenols. A larger concentration of enzyme, on the other
hand, can successfully extract the necessary polyphenols. How-
ever, in some circumstances, using more enzymes for extract
may result in final product inhibition. To evaluate the effects of
varying cellulase concentrations (ranging from 1% to 10%) on
the recovery of lycopene from pumpkin tissues, one researcher
observed that as the enzyme concentration increased, lycopene
yield increased up to 7% within the enzymatic reaction. In
contrast, no notable decrease in anthocyanin extraction yield
from saffron tepals was observed as the Pectinex content
increased from 1% to 10%. However, the proper enzyme and
its quantity are determined by the nature of fruit yield cell
walls [33].
The combined impacts of numerous enzymes can result in
greater cell wall breakdown, leading to higher removal yields.
Several enzyme mixes are commercially available. The synergis-
tic effect of the enzyme combinations Pectinex Ultra Clear and
Lallzyme Beta for grapes extraction of juice of Vitis labrusca
L. variety Concord was investigated. They found that when
two enzymatic preparations were combined, it increased juice
yield and the release of bioactive components compared to
using individual enzyme preparations. To increase extraction
yield, available commercial enzymes are typically mixed with
two or more enzymes. Phenolic components and antioxidants
were recovered from rice bran in one study by employing
available commercial carbohydrates: AMG, Cell clast, Pento-
pan, Viscozyme, Termamyl, and Ultralow. The ferric-reducing
capacity of phenolics released by all carbohydrates increased
significantly (1.5–3.3 times). Among the enzyme mixtures
listed above, Pentopan (a mixture of Endo-1-4-xylanase as
well as feruloyl esterase, caffeoyl esterase, as well as pectinase)
emerged to be the most efficient in increasing antioxidant
activity, so although Cell clast, Ultralow, as well as Viscozyme
appeared to be more effective in increasing phenolic content as

well as radical-scavenging activity. Commercial carboxylases
successfully boosted phenolic acid extraction yield through
enzymatic hydrolysis of cell-wall components as well as the
release of free phenolic acids. Bioactive chemicals, particularly
anthocyanins as well as other polyphenols, are affected by
processing and subsequent storage. As a result, it must be
considered while evaluating the possible health advantages of
foods and beverages. Using different commercial enzymes, the
researchers extracted phytonutrients from bilberry skin and
examined the stability in the form of half-life values. Due to
enzyme-assisted extraction, the half-life value increased (from
12% to 64%). Because of the electrostatic attraction between
anthocyanin and hydrolyzed pectin molecules, this suggested
good pigment stability. Other polyphenols (especially flavanols
and polyphenols) were also produced after the significant enzy-
matic degradation of polysaccharide cell walls in bilberry skin.
These polyphenols served as co-pigments to enhance anthocy-
anin retention, complexes (carbohydrates) can transform
water-insoluble cell wall and cell membrane substances into
water-soluble components (short polysaccharide fragments),
resulting in a variety of bioactive characteristics observed. The
chemical structure, structural morphology, and physiochemical
properties of insoluble and soluble residues are critical in iden-
tifying optimal conditions to enhance extraction yield [33].
2. Polysaccharides:
Aside from polyphenols, polysaccharides are indeed the
group of metabolites that have been investigated the most.
Water-soluble, high-molecular-weight polysaccharides can
modify the rheological properties of food and are frequently
employed in a variety of food applications as stabilizers, emul-
sifiers, thickeners, and texture modifiers. Additionally, dietary
fibers contain a wide range of bio-functional properties, includ-
ing immunomodulatory, hematopoiesis-promoting, antioxi-
dant, antibacterial, and anticancer activities, according to
several recent studies [34]. The most typical technique for
removing polysaccharides is traditional heated reflux extrac-
tion. In general, the recovery time and temperature have a
significant impact on the yield of this approach. However,
prolonged use at high temperatures can weaken polysacchar-
ides, which in turn reduces their biological activity. The enzy-
matic extraction for bioactive biodegradable polymers has been
extensively studied [21]. There are two ways to perform
enzyme-assisted carbohydrate extraction. The first is to use
enzymes capable of destroying biological membranes such as
cell walls and membranes to aid in the isolation of desired
polysaccharides. To facilitate extraction, enzymes that partially
break down desired polysaccharides to minute fragments are

used. Alkaline was used to extract carrageenan from Mastocar-
pus stellatus (a type of protease). The gelling characteristics of
the isolated polysaccharides were excellent [35]. The extraction
procedure resulted in a combined extraction of important phy-
tochemicals such as polyphenols adding value to the extract. As
a result, enzymes can be used to selectively extract bioactive
polysaccharides, potentially allowing for the targeted creation
of certain gelation characteristics and desired physical
features [22].
Another work used a response surface approach to do
cellulase-assisted extraction of water-soluble Malva Sylvester’s
carbohydrates (MSP). The maximum MSP yield (10.40%) was
obtained at 5.64% cellulase, 55.65 °C temps, 3.4 h, and
5.22 ph. These homogenous polysaccharide fractions were
then purified using chromatography. In a dose-dependent pat-
tern, the fractions dramatically improved antioxidant, antican-
cer (tested on HepG2 and A549), and antibacterial activity.
Using enzymes thereby improves not only the removal effi-
ciency but also the medicinal characteristics of polysaccharides.
Because of their unique physical and biological qualities, sea-
weed (algal) disaccharides and polysaccharides are gaining pop-
ularity in the functional food and pharmaceutical industries.
The researchers conducted enzyme-assisted carbohydrate
extraction from the brown cyanobacteria Ecklonia radiata
using a combination of six commercial enzyme mixtures: Vis-
cozyme L, Cellclast 1.5 L, Ultraflo L, Alacalase 2.4 L, Neutrase
0.8 L, and Flavourzyme 1000 L. They discovered that total
sugar output was unaffected by enzyme type or ph. The above-
mentioned parameters, however, govern the molecular mass of
the isolated polysaccharides. High buffer salt concentrations
were discovered to impede polysaccharide extraction. As a
result, buffers should not be used in enzyme-aided algal carbo-
hydrate extraction for increased extraction efficiency [22].
It is commonly accepted that the chemical components,
structural, molecular mass, and conformation of polysacchar-
ides can influence their bioactivity and other therapeutic qua-
lities. To separate polysaccharides from Epimedium
acuminatum, hot water extraction was optimized. Nonenzy-
matic extracting and heating extraction, on the other hand,
resulted in a lower polysaccharide yield (30.2%) as compared
to enzyme-aided water extraction (82.4%). Furthermore,
unlike previous methods, enzymatic water extraction enhanced
time efficiency, reduced solvent usage, and functioned at lower
extraction temperatures. SEM images demonstrated that after
enzyme-assisted extraction, the cell walls became thin and dis-
ordered, facilitating mass transfer among polysaccharides and
solvent and therefore increasing yield. The impact of various
extraction procedures on the makeup of both acidic and neutral

pectic polysaccharides was investigated during bulk pectic
extraction from agroindustry leftovers. The most common
method for freeing pectin from plant sources is acidic extrac-
tion. Previous research has shown that the extraction method
can cause significant pectin breakdown, resulting in a low yield
and loss of gelling characteristics. Furthermore, they are used
in crude form instead of purified pectin to cut production costs.
The influence of different extraction procedures (acids,
enzymes, and chelators) on the recovery and content of bulk
pectic polysaccharides was studied using four distinct wastes as
substrates: berry pomace, onions hulk, compressed pumpkin,
and sugar beet pulp. Enzymatic hydrolysis employing Cell
class, which contains cellulases (endoglucanase), was found to
be successful in removing pectin through plant cell walls. Sugar
beet pulp or onion hulk was discovered to be appropriate
substrates for purifying pectin and manufacturing pectin-
based products based on the mechanism of recovering pectic
sugars following enzymatic extraction. In addition, the incuba-
tion conditions of the different substrates with enzymes were
shown to be significantly milder and more time efficient than
extraction with acidic and chelators. The enzyme extraction
method has demonstrated an advantage over conventional
procedures in the sustained extraction of polysaccharides with
increased pharmacological of the extract. Complex enzymes
have also been shown to increase polysaccharide output and
extract quantity. Another benefit of enzyme-aided extraction is
that less alcohol is required again for the precipitation of target
polysaccharides from the reaction mixture, making the proce-
dure more environmentally friendly.
3. Oils:
Oil extraction via seeds employs traditional procedures
including mechanical press by hydrolytic method, solvent
extraction, and extruder pressing. In certain circumstances,
the oil can be obtained or produced directly from the fruits
using a normal mechanical pressing and is taken without fur-
ther procedures and experimental analysis, such as virgin olive
oil. Hexane is commonly used for oil extraction due to its ease
of recovery, low boiling point (63–69 °C), and high solubiliz-
ing ability. Unfortunately, due to health, safety, and environ-
mental considerations, hexane is not a preferred solvent in the
extraction process. Researchers are seeking alternatives to hex-
ane that will not reduce oil yield or quality. As a result, the use
of green solvents and fluids may be a viable alternative to oil
extraction. Aqueous enzymatic extract (AEE) is an effective
method for extracting oils from oil seeds. This approach is
straightforward to use, consumes less energy, and is
economically viable. In general, oil droplets are surrounded by

protein, which is a major component of the cell wall. Proteins
and pectin were the main components of cell walls in soybeans
and rapeseeds. As a result, degrading these components with
proteolytic, cellulase, pectinase, as well as other enzyme mixes
increased oil output. The chemical structure, anatomy of the
cell wall, and placement of the oil droplet within the seed must
all be considered while selecting enzymes [36]. Furthermore,
in oil extraction optimization trials, the oil-to-water ratio must
be considered in addition to general characteristics such as pH,
temperature, and particle size. Enzymes as well as their concen-
tration require moisture content to function, and the presence
of low humidity in oilseeds causes the formation of a thick
suspension, which inhibits enzyme activity [37].
In the literature survey scientifically, there are various
examples and illustrations available in which an enzyme mix-
ture has been proven to function synergistically to enhance and
improve the extraction efficiency of required oils including
phenolics from bay leaf (Laurus nobilis L.). By disintegrating
plant cell walls, the enzyme mixture comprising cellulose,
hemicellulose, and xylanase improved the removal efficiency
of biomolecules of plant matrix. The extraction rate of essential
oils was enhanced by 243%, 227%, 240.54%, and 248% when
the substrate was treated using hemicellulose, cellulase, and
xylanase individually and their ternary mixture. Further inves-
tigation found that essential oils increased the antioxidant
activity of enzyme-pretreated substrates. The addition of
enzymes altered the proportions of separate components and
increased the number of oxygenated monoterpenes. As a result,
the essential oils produced from enzyme-treated samples had
higher antioxidant activity. Similarly, oil was extracted using
bush mango kernel flour by treating it with commercially avail-
able enzyme mixtures such as Alkalize, Pectinex, and Visco-
zyme, yielding oil yields of 35%, 42.2%, and 68.0%, respectively.
In addition to boosting oil extraction output, the enzymatic
method increased oil sample quality by increasing the number
of bioactive components (such as carotenoids and other phe-
nolics) along with their antibacterial activity. The use of tannase
increased the total phenolics in the hydrophilic and lipophilic
fractions, resulting in the oil having a better antioxidant capac-
ity. Essential oils with enhanced antioxidant capabilities and
antibacterial activity have a high potential for usage in the
food and pharmaceutical industries. Enzymatic processing
was shown to drastically reduce the production of oil left in
Chilean hazelnut food in a few situations [38].
Clove essential oil is one of the most important essential
oils. Recently, research has concentrated on the pretreatment of
clove buds’ powder with enzymes including cellulase,
lignocellulose, pectinase, and amylase before extraction. For

the recovery of clove oil, the typical procedure of steam distil-
lation yields 10.1%. In recent investigations, enzymes specific
for cell wall activity were utilized in preprocessing before
extraction to enhance the purity and quantity of the phyto-
chemicals recovered. The extraction of oil from discarded
pomegranate seeds using protease (from Aspergillus oryzae)
treatment yielded a more than 50% good return than the
control samples in terms of quantity of extracted essential oil.
SEM images of discarded pomegranate seeds were obtained
before and after protease treatment to better understand the
extraction mechanism. The micrographs of SEM revealed that
even after protease therapy, the cell wall is damaged as a result
of the enzymatic treatment, which renders the surface of the
seeds very porous, facilitating the recovery of physiologically
entrapped oil. Furthermore, the protease-derived oil had 1.4
times the phenolic content and 4% more antioxidant activity
than hexane-extracted oil. The preceding example implies that
pretreatment with enzymes before resource extraction can
boost oil extraction yield. Enzymatic pretreatment before sol-
vent extraction and mechanical pressing softens the cell wall
and increases oil extraction [37]. This method reduces the
potential of oil production in water emulsion following
extraction.
The parameters for enzyme selection are determined by the
location of oil inside the cellular structure as well as the chemi-
cal composition of the substances surrounding it. The simulta-
neous assessment of each of these criteria is critical in
determining the appropriate enzyme combination for a partic-
ular oil-containing substrate. Despite its numerous benefits,
the application of AEE is still limited due to the lengthy pro-
cessing time and difficult drying process following enzymatic
treatment. A substantial amount of the enzyme is required
(usually more than 1% of the weight of an oilseed ingested),
which contributes to the expensive expense. Furthermore, the
lack of commercially available enzymes has hampered the devel-
opment of these methods. An additional issue with AEE is the
difficulty in avoiding emulsification of the oil extracted, which
necessitates post-extraction demulsification step toward
recovering and increasing oil yield. Tabtabaei and Diosady
employed aqueous enzymatic emulsion de-emulsion approach
to destabilize the oil-in-water emulsions to recover access to
the oil before the industrial application [39]. The researchers
used enzymatic demulsification therapy by several proteases
and phospholipases in their work to test its ability to release
free oil. The targeted emulsifiers are hydrolyzed. Protex 6 Land
phospholipase therapies proved successful in collecting over
91% of the oil inside the emulsion.
3.4.1 Flavors and Colors Colors and flavors improve the quality of a food product by influ-
encing its visual appearance and taste. In the food sector, there is an
ever-increasing need for natural flavors and colors. Synthetic dyes
were suspected of emitting hazardous compounds that are highly
polluting, allergenic, carcinogenic, and toxic to humans. Given the
health and environmental concerns associated with industrial
chemical dyes, researchers redirected their focus away from artificial
(synthetic) colorants and toward the excitation of natural colorants
derived from plant sources [40]. Colorants taken from roots, bark,
foliage, nuts, berries, and flowers include anthocyanins, betalains,
chalcones, chlorophyll, carotenoid, and flavones. Traditional natu-
ral dye extraction procedures include water and alkali extraction,
fermentation, and solvent extraction. If the right enzymes are
chosen and the operating conditions are tuned, enzyme-assisted
extraction method has a lot of promise for pigment isolation. In the
past, commercially accessible enzymes including cellulase, amylase,
and pectinase were investigated. This method may hasten the
extraction of pigments through tough and compact plant matter
such as barks and roots. Conventional means of natural dye extrac-
tion, in addition to being time-consuming and ineffective, result in
the co-extraction of unwanted compounds such as chlorophyll and
waxes [41].
3.5 Enzyme- 1. Extraction of bioactive from by-products.

Enhanced Processes By enhancing the permeability of plant cell walls, enzyme-
for Plant Materials assisted extraction (EAE) makes it possible to extract pectin
from waste and by-products [42]. Many phenol chemicals,
such as flavonoids and anthocyanidins, can be extracted using
enzymes [43]. For enzymatic treatment to be as effective as
possible, factors such as enzyme activity, treatment time, sub-
strate ratio, and particle size are crucial [44]. Scientists have
addressed that ideal condition for extracting pistachio green
hull. For the extraction of cellulose, tannases, pectinases, and
their mixtures were utilized. Results have revealed that the
three enzymes used simultaneously to extract phenolics gave
the best result [45].
2. Production of fermented drinks and plant-based drinks from
grains.
The production of high-protein food items using grains
and cereals, involves a process that requires the degradation of
cell walls with the assistance of enzymes to obtain the desired
food product. Yearly, dairy-related product consumption is
growing by 10%. In the year 2019, America reached to the
extent of 1.8 billion dollar spending on dairy products
[46]. This is due to the high release of sugars forming the
acceptant-sensory organoleptic features [47]. The enzymes
which are majorly used to start the earlier steps pectinase-

denatured rapeseed fibers and cellulose for the growth of the
cultures such as L. johnsonii L63, reuteri L45, Plantarum L47
are some examples for the starter culture and growth of bacte-
ria for high production of nutrients in fermenter tanks
[48]. Furthermore, plant-based fermented goods have antibac-
terial qualities, and their pH is lower than that of typical plant-
based drinks, impacting product stability [49].
3.6 A Review of Usually, bioactive compounds in natural products exist as either

Enzymes and Factors soluble or insoluble conjugates (glycosides). In food industries, for
Influencing Bioactive example, the majority of phenolics (24% of overall phenolic con-
Extraction tent) are found as bounded phenolics [50]. The majority of pheno-
lic compounds are imprisoned inside cell wall polysaccharides such
as cellulose, hemicellulose, and pectin, which are connected by
chemical bonding and hydrogen bonds. Other phenolic acids cre-
ate ether links with lignin via their aromatic ring hydroxyl groups
and ester links with structural polysaccharides and proteins via their
carboxylic groups [51]. Flavonoids are covalently bonded to sugar
moieties via glycosidic bonds or carbon-carbon bonds. Tannins
have a proclivity to create powerful complexes with proteins.
Here, enzymes such as cellulase, hemicellulose, pectinase, and pro-
tease are used to solubilize the plant cell wall, consequently speed-
ing the discharge of intracellular biomolecules [45]. Hydrolases,
such as lipases, work in the water that is present inside the reaction
system. They also act in the presence of additional substrates such as
alcohols, amines, and oximes. During the separation of flavonoids
in Ginkgo biloba, Penicillium decumbens cellulase outperformed
T. reesei cellulase in the condition of maltose as the glycosyl
donor. Cellulases and hemicelluloses can be used to separate oils
and proteins. These enzymes attack the interior locations of the
polysaccharide chains at random. This results in the formation of
tiny oligosaccharides of varying lengths, which allow for the easy
liberation of entrapped molecules [52].
The extraction efficiency is determined by the solvent system,
temperature, enzyme mode of action, substrate availability, extrac-
tion length, enzyme loading, and pH condition. Each enzyme has a
different optimal pH for enzymatic hydrolysis [53]. Many enzymes
have an optimal pH that is close to the neutral pH of proteins.
Because proteins are particularly insoluble in this pH range, bio-
molecule release may be hampered. As a result, pH must be selected
in such a manner that not only does it inhibit enzyme action but
also does not fall within the range of protein isoelectric point.
Temperature, in addition to pH, is an essential aspect to consider
during extraction [16].
3.7 Advantages and The enzyme-assisted extraction method is a highly efficient tech-
Disadvantages of nique compared to the other extraction techniques as it takes very
Enzyme-Assisted less time with the high productivity of extracted compounds. It
Extraction Method requires the knowledge of the basic and simple methodology for
the extraction of compounds. The extracted compounds will retain
3.7.1 Advantages of their structural and physiochemical and configure stability during
Enzyme-Assisted and after the process of extraction. Consequently, the concept of
Extraction Method “green chemistry” is being pursued. People are searching for an
effective, ecologically friendly way to increase bioactive recovery
rates. Due to its improved extraction capabilities and environmental
friendliness, enzymatic extraction has demonstrated many benefits.
Through this process, we can exactly figure out our required intra-
cellular compound and can be extracted with high accuracy so that
purity of the extracted compound is enhanced. The process
required for the extraction of intracellular bioactive chemicals
using this unique technology is regarded as lenient, meaning
there are no precise requirements to be maintained. The cellular
barrier that is the cell wall, which processes cellulose, hemicellulose,
and pectin, can efficiently be degraded with enzymes, namely,
cellulase, hemicellulose, and pectinase, respectively, without affect-
ing the bioactives [54, 55].
3.7.2 Disadvantages of • Costly equipment is needed; hence, this method is expensive

Enzyme-Assisted compared to other extraction methods.
Extraction Method • We establish this technique at a small-scale level and cannot be
used at the industrial level.
• This technique is not suitable for the extraction of by-products
such as fibers, phenolic compounds, carotenoids, and
anthocyanin.
• Some plants having different cell wall compositions rather than
usual cell wall compositions will bring a difficulty to break open
the cell wall so that it allows the intracellular compounds to
come out of the cell. There are no such novel enzymes that are
available to break open those cell walls with different biochemi-
cal compositions.
• The process of extraction and purification of enzymes for the
treatment process is a difficult and crucial step, and it is difficult
to store and maintain those enzymes in large quantity, which is
the drawback in this method [56].
The industrial importance of bioactives: Bioactive compounds
are having more importance in the food industries and pharmaceu-
tical industries.
3.8 Some of the (a) Antidiabetic activity: Diabetes is concerned with a group of
Pharmaceutical conditions characterized by a high level of blood glucose,
Activities of Bioactive commonly known as blood sugar [57]. Too much sugar in
Compounds the blood causes serious health problems, sometimes even it
may lead to death. In diabetics, there are two types: type 1 and
type 2. Type 1 diabetes destroys by the immune system by
mistake [58]. The reason is insulin binds to its receptor on
target cells; hence, less glucose is taken into the cells so that
more glucose stays in the blood. Therefore, this type of diabe-
tes is called insulin-dependent. Another type, known as type
2 diabetes, is characterized by insulin resistance. This condi-
tion is often associated with factors such as obesity, a sedentary
lifestyle, and an unhealthy disposition. Type 2 is related to
endocrine metabolism. In such circumstances, plants or natu-
ral products containing antidiabetic properties, such as insuli-
nogenic or secretagogue properties, hold significant promise
and potential for the development of novel pharmaceuticals.
There are so many plants with the antidiabetic properties.
Some of them are Acacia arabica, Aegle marmelos, agrimonia
eupatoria, allium cepa, Allium sativum, Aloe vera, Azadir-
achta indica, and Benincasa hispida [59]. The various parts
of medicinal plants can treat diabetes in various ways, includ-
ing insulin secretagogue activity, the insulin release from the
pancreas, insulin-like activity, an increase in plasma insulin
concentration, an increase in insulin binding to insulin recep-
tors, a decrease in plasma triglyceride levels, insulin-sensitizing
activity, and an antihyperglycemic mechanism to stimulate islet
insulin release [60]. Additionally, a sizable population world-
wide has switched to this complementary method of treating
illness because of its varied flora, affordability, and simplicity of
use with little negative effects. Studies show that medicinal
plants are multitargeting and least likely to fail during treat-
ment, which is supported by the evidence [61].
(b) Anticancer activity: Cancer is the result of uncontrolled, rapid
cell division. Numerous types of cancer can be found
[62]. Cancer is one the most deadly disease caused due to
metabolism. This disease is not completely curable. We can
just increase the life span of the patient with chemotherapy and
some antibiotics [63]. Presently, ten million people lose their
lives every year with this deadly disease, and this may exceed in
the future according to the WHO (World Health Organiza-
tion) [64]. Topoisomerase inhibitors such as irinotecan and
doxorubicin and alkylating drugs such as oxaliplatin, carbo-
platin, and cisplatin are used in chemotherapy. Irinotecan’s
adverse effects include neutropenia and sensory neuropathy
(side effects include nephron, gastrointestinal, cardiovascular,
pulmonary, and hematologic toxicity). Apart from their com-

plexity, expense, and non-eco-friendliness, the aforemen-
tioned medications’ main drawbacks are their side effects and
toxicity because they also target normal cells [65]. In order to
treat cancer naturally and herbally, a group of photochemical
known as “vinca alkaloids,” which were extracted from Cath-
aranthus roseus, are used. Vinorelbine, vindesine, vincristine,
and vinblastine are the four primary alkaloids found in vinca.
Vinblastine and vincristine are particularly effective at stop-
ping the cell cycle in metaphase and interfering with microtu-
bule function. Currently, vinorelbine, vindesine, and
vinfosiltine are semisynthetic derivatives of the vinca alkaloids.
Consequently, we can predict a bright future for photochemi-
cal research because, over the next 10 years, it is anticipated
that these compounds will completely change how cancer is
treated [66].
(c) Diuretic properties: Today, heart disease, kidney disease, and
excessive blood pressure are all fairly frequent. Acute renal
failure, edema or an increase in blood calcium or potassium,
acute left ventricular failure or heart failure, and acute pulmo-
nary edema are common medical conditions that patients
frequently experience. Different medications are used to
treat these issues because they assist the body to excrete
more electrolytes and urine, which helps to lessen fluid reten-
tion. In actuality, many medications have negative long-term
effects and are unable to treat the threat of high blood pres-
sure. In contrast, green plants with high flavonoid and poly-
phenol content have been discovered to have high salt and
potassium excretion properties, including Cynodon dactylon,
Emblica officinalis, Kalanchoe pinnata, and Bambusa nutans.
Hence, many of the plants are shrubs are to be identified and
used in the preparation of drugs for the curing of
diseases [67].
4 Conclusion
Considering all the extraction methods of the bioactive com-

pounds, enzyme-assisted method seems to be the most efficient
method for extracting the bioactive compounds. The enzymes
make the cell wall most permeable and which leads to the high
yield of metabolites that are having more applications in the food
and pharmaceutical industries [28]. This method consists of efforts
of food technologists, food chemists, nutritionists, and toxicolo-
gists. For improving the level of release of bioactive compounds,
synthesis of new enzymes and their purification is important.
Compared to earlier extraction methods, enzymes-assisted

extraction is more efficient and reliable. Extracting the terpenoids,
polyphenols, and lectins need more research work to uncover the
hidden potential, without the release of toxic substances. The
extraction methods should use metabolites, which are eco-friendly
nature. The approach of genetic engineering also plays an impor-
tant role in this process to produce on a larger scale. Still, there is a
need for finding the available enzymatic processes for further
enhancement of the yield of the bioactive compounds.
References
1. Bernhoft A (2010) Bioactive compounds in phenolic compound content in virgin olive oils
plants: benefits and risks for man and animals: by using enzymes during malaxation. J Food
proceedings from a symposium held in Norwe- Eng 48(3):189–194. https://doi.org/10.
gian Academy of Science and Letters, Oslo, 1016/S0260-8774(00)00157-6
13–14 November 2008. Novus Forlag, Oslo 10. Atef M, Mahdi Ojagh S (2017) Health benefits
2. Biesalski H-K et al (2009) Bioactive com- and food applications of bioactive compounds
pounds: definition and assessment of activity. from fish byproducts: a review. J Funct Foods
Nutrition 25(11–12):1202–1205. https://doi. 35:673–681. https://doi.org/10.1016/j.jff.
org/10.1016/j.nut.2009.04.023 2017.06.034
3. Sasidharan S, Chen Y, Saravanan D, 11. Zhi Fu G, Chan A, Minns D (2005) Prelimi-
Sundram K, Latha L (2010) Extraction, isola- nary assessment of the environmental benefits
tion and characterization of bioactive com- of enzyme bleaching for pulp and paper making
pounds from plants’ extracts. Afr J Tradit (7 pp). Int J Life Cycle Assess 10(2):136–142.
Complement Altern Med 8(1). https://doi. https://doi.org/10.1065/lca2004.06.162
org/10.4314/ajtcam.v8i1.60483 12. Sun H, Ge X, Lv Y, Wang A (2012) Application
4. Patil BS, Jayaprakasha GK, Chidambara of accelerated solvent extraction in the analysis
Murthy KN, Vikram A (2009) Bioactive com- of organic contaminants, bioactive and nutri-
pounds: historical perspectives, opportunities, tional compounds in food and feed. J Chroma-
and challenges. J Agric Food Chem 57(18): togr A 1237:1–23. https://doi.org/10.1016/
8142–8160. https://doi.org/10.1021/ j.chroma.2012.03.003
jf9000132 13. Sowbhagya HB, Chitra VN (2010) Enzyme-
5. Ramadan MF et al (2009) Chromatographic assisted extraction of flavorings and colorants
analysis for fatty acids and lipid-soluble bioac- from plant materials. Crit Rev Food Sci Nutr
tives of Derris indica crude seed oil. Chroma- 50(2):146–161. https://doi.org/10.1080/
tographia 70(1–2):103–108. https://doi.org/ 10408390802248775
10.1365/s10337-009-1141-9 14. Wu Y, Cui SW, Tang J, Gu X (2007) Optimiza-
6. Barzana E et al (2002) Enzyme-mediated sol- tion of extraction process of crude polysacchar-
vent extraction of carotenoids from Marigold ides from boat-fruited sterculia seeds by
flower (Tagetes erecta). J Agric Food Chem response surface methodology. Food Chem
50(16):4491–4496. https://doi.org/10. 105(4):1599–1605. https://doi.org/10.
1021/jf025550q 1016/j.foodchem.2007.03.066
7. Choudhari SM, Ananthanarayan L (2007) 15. Arvayo-Enrı́quez H, Mondaca-Fernández I,
Enzyme aided extraction of lycopene from Gortárez-Moroyoqui P, López-Cervantes J,
tomato tissues. Food Chem 102(1):77–81. Rodrı́guez-Ramı́rez R (2013) Carotenoids
https://doi.org/10.1016/j.foodchem.2006. extraction and quantification: a review. Anal
04.031 Methods 5(12):2916. https://doi.org/10.
8. Durling N et al (2007) Extraction of phenolics 1039/c3ay26295b
and essential oil from dried sage (Salvia offici- 16. Nadar SS, Rathod VK (2017) Facile synthesis
nalis) using ethanol–water mixtures. Food of glucoamylase embedded metal-organic fra-
Chem 101(4):1417–1424. https://doi.org/ meworks (glucoamylase-MOF) with enhanced
10.1016/j.foodchem.2006.03.050 stability. Int J Biol Macromol 95:511–519.
9. Garcı́a A, Brenes M, José Moyano M, Alba J, https://doi.org/10.1016/j.ijbiomac.2016.
Garcı́a P, Garrido A (2001) Improvement of 11.084
17. Bansode SR, Rathod VK (2017) An investiga- 26. Mann JP, McCluskey A, Atkin R (2009) Activ-
tion of lipase catalysed sonochemical synthesis: ity and thermal stability of lysozyme in alkylam-
a review. Ultrason Sonochem 38:503–529. monium formate ionic liquids—influence of
https://doi.org/10.1016/j.ultsonch.2017. cation modification. Green Chem 11(6):785.
02.028 https://doi.org/10.1039/b900021f
18. Corrales M, Toepfl S, Butz P, Knorr D, 27. Correia RT, Borges KC, Medeiros MF, Genov-
Tauscher B (2008) Extraction of anthocyanins ese MI (2012) Bioactive compounds and
from grape by-products assisted by ultrasonics, phenolic-linked functionality of powdered
high hydrostatic pressure or pulsed electric tropical fruit residues. Food Sci Technol Int
fields: a comparison. Innov Food Sci Emerg 18(6):539–547. https://doi.org/10.1177/
Technol 9(1):85–91. https://doi.org/10. 1082013211433077
1016/j.ifset.2007.06.002 28. Azmir J et al (2013) Techniques for extraction
19. Prasad KN, Yang E, Yi C, Zhao M, Jiang Y of bioactive compounds from plant materials: a
(2009) Effects of high pressure extraction on review. J Food Eng 117(4):426–436. https://
the extraction yield, total phenolic content and doi.org/10.1016/j.jfoodeng.2013.01.014
antioxidant activity of longan fruit pericarp. 29. Acosta-Estrada BA, Gutiérrez-Uribe JA, Serna-
Innov Food Sci Emerg Technol 10(2): Saldı́var SO (2014) Bound phenolics in foods,
155–159. https://doi.org/10.1016/j.ifset. a review. Food Chem 152:46–55. https://doi.
2008.11.007 org/10.1016/j.foodchem.2013.11.093
20. Kim JH, Park Y, Yu KW, Imm J-Y, Suh HJ 30. Londoño-Londoño J et al (2010) Clean recov-
(2014) Enzyme-assisted extraction of cactus ery of antioxidant flavonoids from citrus peel:
bioactive molecules under high hydrostatic optimizing an aqueous ultrasound-assisted
pressure: enzyme-assisted extraction of cactus extraction method. Food Chem 119(1):
under HHP. J Sci Food Agric 94(5):850–856. 8 1 – 8 7 . h t t p s : // d o i . o r g / 1 0 . 1 0 1 6 / j .
https://doi.org/10.1002/jsfa.6317 foodchem.2009.05.075
21. del Pilar Sánchez-Camargo A et al (2016) Con- 31. Li BB, Smith B, Hossain MM (2006) Extrac-
siderations on the use of enzyme-assisted tion of phenolics from citrus peels. Sep Purif
extraction in combination with pressurized Technol 48(2):189–196. https://doi.org/10.
liquids to recover bioactive compounds from 1016/j.seppur.2005.07.019
algae. Food Chem 192:67–74. https://doi. 32. Yadav K, Singh MR, Rai VK, Srivastava N, Pra-
org/10.1016/j.foodchem.2015.06.098 sad Yadav N (2020) Commercial aspects and
22. Ezeh O, Gordon MH, Niranjan K (2016) market potential of novel delivery systems for
Enhancing the recovery of tiger nut (Cyperus bioactives and biological agents. In: Advances
esculentus) oil by mechanical pressing: mois- and avenues in the development of novel car-
ture content, particle size, high pressure and riers for bioactives and biological agents. Else-
enzymatic pre-treatment effects. Food Chem vier, pp 595–620. https://doi.org/10.1016/
194:354–361. https://doi.org/10.1016/j. B978-0-12-819666-3.00020-1
foodchem.2015.07.151 33. Lotfi L, Kalbasi-Ashtari A, Hamedi M, Ghor-
23. Bai Y, Liu L, Zhang R, Huang F, Deng Y, bani F (2015) Effects of enzymatic extraction
Zhang M (2017) Ultrahigh pressure-assisted on anthocyanins yield of saffron tepals (Crocos
enzymatic extraction maximizes the yield of sativus) along with its color properties and
longan pulp polysaccharides and their acetyl- structural stability. J Food Drug Anal 23(2):
cholinesterase inhibitory activity in vitro. Int J 210–218. https://doi.org/10.1016/j.jfda.
Biol Macromol 96:214–222. https://doi.org/ 2014.10.011
10.1016/j.ijbiomac.2016.11.105 34. Giavasis I (2014) Bioactive fungal polysacchar-
24. Bogdanov MG (2014) Ionic liquids as alterna- ides as potential functional ingredients in food
tive solvents for extraction of natural and nutraceuticals. Curr Opin Biotechnol 26:
products. In: Chemat F, Vian MA (eds) Alter- 162–173. https://doi.org/10.1016/j.copbio.
native solvents for natural products extraction. 2014.01.010
Springer Berlin Heidelberg, Berlin, Heidel- 35. Rostami H, Gharibzahedi SMT (2017)
berg, pp 127–166. https://doi.org/10.1007/ Cellulase-assisted extraction of polysaccharides
978-3-662-43628-8_7 from Malva sylvestris: process optimization and
25. Dai Y, van Spronsen J, Witkamp G-J, potential functionalities. Int J Biol Macromol
Verpoorte R, Choi YH (2013) Ionic liquids 101:196–206. https://doi.org/10.1016/j.
and deep eutectic solvents in natural products ijbiomac.2017.03.078
research: mixtures of solids as extraction sol- 36. Chair of Food Plant Chemistry and Processing,
vents. J Nat Prod 76(11):2162–2173. https:// Faculty of Food Sciences, University of Warmia
doi.org/10.1021/np400051w
and Mazury in Olsztyn, Pl. Cieszyński 46. Tangyu M, Muller J, Bolten CJ, Wittmann C
1, 10-726 Olsztyn, Poland et al (2016) Opti- (2019) Fermentation of plant-based milk alter-
mization of pumpkin oil recovery by using natives for improved flavour and nutritional
aqueous enzymatic extraction and comparison value. Appl Microbiol Biotechnol
of the quality of the obtained oil with the qual- 103(23–24):9263–9275. https://doi.org/10.
ity of cold-pressed oil. Food Technol Biotech- 1007/s00253-019-10175-9
nol 54(4). https://doi.org/10.17113/ftb.54. 47. M€akinen OE, Wanhalinna V, Zannini E,
04.16.4623 Arendt EK (2016) Foods for special dietary
37. Mat Yusoff M, Gordon MH, Niranjan K needs: non-dairy plant-based milk substitutes
(2015) Aqueous enzyme assisted oil extraction and fermented dairy-type products. Crit Rev
from oilseeds and emulsion de-emulsifying Food Sci Nutr 56(3):339–349. https://doi.
methods: a review. Trends Food Sci Technol org/10.1080/10408398.2012.761950
41(1):60–82. https://doi.org/10.1016/j.tifs. 48. Zhu X, Wang L, Zhang Z, Ding L, Hang S
2014.09.003 (2021) Combination of fiber-degrading enzy-
38. Womeni HM, Ndjouenkeu R, Kapseu C, matic hydrolysis and lactobacilli fermentation
Mbiapo FT, Parmentier M, Fanni J (2008) enhances utilization of fiber and protein in
Aqueous enzymatic oil extraction from Irvin- rapeseed meal as revealed in simulated pig
gia gabonensis seed kernels. Eur J Lipid Sci digestion and fermentation in vitro. Anim
Technol 110(3):232–238. https://doi.org/ Feed Sci Technol 278:115001. https://doi.
10.1002/ejlt.200700172 org/10.1016/j.anifeedsci.2021.115001
39. Soto C, Concha J, Zuniga ME (2008) Antioxi- 49. Panghal A, Janghu S, Virkar K, Gat Y,
dant content of oil and defatted meal obtained Kumar V, Chhikara N (2018) Potential
from borage seeds by an enzymatic-aided cold non-dairy probiotic products – a healthy
pressing process. Process Biochem 43(6): approach. Food Biosci 21:80–89. https://doi.
696–699. https://doi.org/10.1016/j. org/10.1016/j.fbio.2017.12.003
procbio.2008.02.006 50. Hari Krishna S, Karanth NG (2002) Lipases
40. Tabtabaei S, Diosady LL (2013) Aqueous and and lipase-catalyzed esterification reactions in
enzymatic extraction processes for the produc- nonaqueous media. Catal Rev 44(4):499–591.
tion of food-grade proteins and industrial oil https://doi.org/10.1081/CR-120015481
from dehulled yellow mustard flour. Food Res 51. Sharma A, Tewari R, Rana SS, Soni R, Soni SK
Int 52(2):547–556. https://doi.org/10. (2016) Cellulases: classification, methods of
1016/j.foodres.2013.03.005 determination and industrial applications.
41. Li Y, Jiang L, Sui X, Wang S (2011) Optimiza- Appl Biochem Biotechnol 179(8):
tion of the aqueous enzymatic extraction of 1346–1380. https://doi.org/10.1007/
pine kernel oil by response surface methodol- s12010-016-2070-3
ogy. Procedia Eng 15:4641–4652. https://doi. 52. Talley K, Alexov E (2010) On the
org/10.1016/j.proeng.2011.08.872 pH-optimum of activity and stability of pro-
42. Wilkins MR, Widmer WW, Grohmann K, teins. Proteins Struct Funct Bioinforma:n/a–
Cameron RG (2007) Hydrolysis of grapefruit n/a. https://doi.org/10.1002/prot.22786
peel waste with cellulase and pectinase 53. Peterson ME, Daniel RM, Danson MJ,
enzymes. Bioresour Technol 98(8): Eisenthal R (2007) The dependence of enzyme
1596–1601. https://doi.org/10.1016/j.bio activity on temperature: determination and val-
rtech.2006.06.022 idation of parameters. Biochem J 402(2):
43. Bai J et al (2021) Source of gut microbiota 3 3 1 – 3 3 7 . h t t p s : // d o i . o r g / 1 0 . 1 0 4 2 /
determines oat β-glucan degradation and BJ20061143
short chain fatty acid-producing pathway. 54. Cheng X, Bi L, Zhao Z, Chen Y (2015)
Food Biosci 41:101010. https://doi.org/10. Advances in enzyme assisted extraction of nat-
1016/j.fbio.2021.101010 ural products. In: Proceedings of the 3rd inter-
44. Catalkaya G, Kahveci D (2019) Optimization national conference on material, mechanical
of enzyme assisted extraction of lycopene from and manufacturing engineering, Guangzhou,
industrial tomato waste. Sep Purif Technol China. https://doi.org/10.2991/ic3me-15.
219:55–63. https://doi.org/10.1016/j. 2015.72
seppur.2019.03.006 55. Wang L, Wu Y, Liu Y, Wu Z (2017) Complex
45. Urbonaviciene D, Viskelis P (2017) The enzyme-assisted extraction releases antioxida-
cis-lycopene isomers composition in supercriti- tive phenolic compositions from guava leaves.
cal CO2 extracted tomato by-products. LWT – Molecules 22(10):1648. https://doi.org/10.
Food Sci Technol 85:517–523. https://doi. 3390/molecules22101648
org/10.1016/j.lwt.2017.03.034
56. Zhang M et al (2021) Optimization of pathway. Diabetes 55(4):1043–1050. https://

enzyme-assisted extraction and purification of doi.org/10.2337/diabetes.55.04.06.
flavonoids from Pinus koraiensis nut-coated db05-1089
film and antioxidant activity evaluation. Mole- 62. Barzegar E, Fouladdel S, Movahhed TK,
cules 26(7):1950. https://doi.org/10.3390/ Ghahremani MH, Ostad SN, Azizi E (2015)
molecules26071950 Effects of berberine on proliferation, cell cycle
57. Patel D, Prasad S, Kumar R, Hemalatha S distribution and apoptosis of human breast
(2012) An overview on antidiabetic medicinal cancer T47D and MCF7 cell lines. Iran J
plants having insulin mimetic property. Asian Basic Med Sci 18(4):9
Pac J Trop Biomed 2(4):320–330. https://doi. 63. Weaver BA (2014) How Taxol/paclitaxel kills
org/10.1016/S2221-1691(12)60032-X cancer cells. Mol Biol Cell 25(18):2677–2681.
58. Patel D, Kumar R, Prasad S, Sairam K, Hema- https://doi.org/10.1091/mbc.e14-04-0916
latha S (2011) Antidiabetic and in vitro antiox- 64. Duthie GG, Duthie SJ, Kyle JAM (2000) Plant
idant potential of Hybanthus enneaspermus polyphenols in cancer and heart disease: impli-
(Linn) F. Muell in streptozotocin–induced dia- cations as nutritional antioxidants. Nutr Res
betic rats. Asian Pac J Trop Biomed 1(4): Rev 13(1):79–106. https://doi.org/10.
316–322. https://doi.org/10.1016/S2221- 1079/095442200108729016
1691(11)60051-8 65. Zhaung ZP, McCauley R (1989) Ubiquitin is
59. Yang B, Jiang Y, Shi J, Chen F, Ashraf M involved in the in vitro insertion of monoamine
(2011) Extraction and pharmacological prop- oxidase B into mitochondrial outer mem-
erties of bioactive compounds from longan branes. J Biol Chem 264(25):14594–14596.
(Dimocarpus longan Lour.) fruit – a review. https://doi.org/10.1016/S0021-9258(18)
Food Res Int 44(7):1837–1842. https://doi. 63734-2
org/10.1016/j.foodres.2010.10.019 66. Almagro L, Fernández-Pérez F, Pedreño M
60. Sy GY, Cissé A, Nongonierma RB, Sarr M, (2015) Indole alkaloids from Catharanthus
Mbodj NA, Faye B (2005) Hypoglycaemic roseus: bioproduction and their effect on
and antidiabetic activity of acetonic extract of human health. Molecules 20(2):2973–3000.
Vernonia colorata leaves in normoglycaemic h t t p s : // d o i . o r g / 1 0 . 3 3 9 0 /
and alloxan-induced diabetic rats. J Ethnophar- molecules20022973
macol 98(1–2):171–175. https://doi.org/10. 67. Sohgaura A, Bigoniya P, Shrivastava B. Diuretic
1016/j.jep.2005.01.024 potential of Cynodon dactylon, Emblica offici-
61. Liu D, Zhen W, Yang Z, Carter JD, Si H, nalis, Kalanchoe pinnata and Bambusa nutans,
Reynolds KA (2006) Genistein acutely stimu- p7
lates insulin secretion in pancreatic β-cells
through a cAMP-dependent protein kinase
Chapter 9
Pulsed Electric Fields as a Green Technology

for the Extraction of Bioactive Compounds
Radhika Theagarajan, Susindra Devi Balendran, and Priyanka Sethupathy
Abstract
In recent years, several innovative extraction procedures have been developed to carry out more effective
and sustainable extraction of bioactive compounds. Sustainability is a vital concept for social, technological,
and economic advancement, which strives to establish a circular economy. Thus, the significance and
demand of eco-friendly methods used for producing plant extracts is currently booming. However, the
food industries are strictly expected to implement manufacturing techniques that are highly effective and
energy-efficient because of the rising cost of energy and other utilities. Pulsed electric field (PEF) processing
is very efficient, more eco-friendly and sustainable compared to conventional extraction techniques such as
solvent extraction and steam distillation. Further, PEF is also a desirable and effective nonthermal method
with improved functioning, extractability, and retrieval of phytochemicals with nutritional benefits. Thus,
swapping the conventional techniques with PEF can minimize or eradicate the utilization of harmful
solvents and utilize less energy and water, and in return preserve the environment for the future genera-
tions. Therefore, PEF extraction technology has been considered as a sustainable strategy and green
technology to extract the bioactive compounds. Additionally, cutting-edge nonthermal extraction techni-
ques such as PEF make it intelligible and more effective to identify, characterize, and analyze bioactive
components. PEF also has various advantages when compared to conventional extraction techniques such
as cost efficiency, reduced extraction times, and greater yields with less solvent use. This chapter focuses on
the employability of pulsed electric fields as a suitable green technology for bioactive extraction.
Key words Pulsed electric field, Green technology, Nonthermal processing, Novel extraction tech-
nique, Bioactive compounds
1 Introduction
Pulsed electric field (PEF) technology, a nonthermal technique,

employs electric pulses to mostly conserve foods with higher elec-
trical conductivity such as liquid or semi-liquid foods. PEF treat-
ment also increases the membrane permeability of food material’s
cell wall by inducing permanent irreversible perforation in the cell
membrane using a pulsated electric field. PEF is purely utilized in
201
202 Radhika Theagarajan et al.
Fig. 1 Impact of PEF on the process of cell membrane permeabilization [2]
the food and nutraceutical industries to expedite extraction rates

and to reduce the extraction time [1].
Extraction of bioactive components using a pulsed electric field
(PEF) is viewed as a substitute for thermal methods, due to the
absence of thermal destruction of bioactive compounds. Subse-
quently, the enhancement of nutrient benefit is also one of the
main advantage of PEF extraction. Further, it also aids in the
reduction and/or prevention of quality deterioration of bioactive
food ingredients. PEF technology is depicted in Fig. 1. PEF results
in the permeation of cell membranes when they are treated with
short treatment times and with minimal energy consumption. The
fundamental idea behind PEF technology revolves around passing
the pulsed electric force through the cell membrane which gener-
ates a charge on the molecules, enabling them to separate based on
charge mass resulting in the electroporation of the cell membrane
and in return increasing the extraction yield significantly [3].
Moreover, PEF’s continuous extraction process will aid in the
proficient and rapid extraction of the components from biological
tissue. Further, this novel nonthermal food processing approach
has a great potential to be expanded to pilot plant and industrial
stages. Also, the PEF extraction process is very quick and uses less
organic solvent when put together as a continuous process. Also,
pollution is prevented when using PEF continuous extraction.
Additionally, the entire procedure can be completed at or slightly
higher than the ambient temperature. Thus, thermal deterioration
of bioactive compounds are minimized [4].
The nonthermal processing capability of PEF technology mini-
mizes the adverse chemical reactions that emerge from the deterio-
ration of bioactive components present in processed foods. Further,
it has been used in several food industries and food-related sectors
Pulsed Electric Fields as a Green Technology for the Extraction. . . 203
for various applications such as food pasteurization, fruit juices

clarification, and preservation. For instance, PEF is a great replace-
ment for thermal pasteurization of liquid foods, due to the negative
impacts associated with several other conventional thermal proces-
sing procedures.
This chapter discusses the major key technological advance-
ments in PEF in the field of bioactive compounds extraction, as
well as the significant advantages of the technology. Additionally,
the fundamentals of PEF are discussed, and special emphasis was
placed on technology evolution over a period with regard to appli-
cations in bioactive extraction [5].
2 Extraction Approaches in Nutraceuticals and Bioactive Extraction: Conventional

and Novel Techniques
In recent times, bioactive components can be retrieved utilizing

several novel extraction techniques such as solvent extraction,
supercritical fluid extraction, subcritical water extraction, and
PEF, enzymes assisted-, ultrasound-, and microwave- extraction
methods. Moreover, the easier accessibility to these methods offers
greater chance to employ any of them most effectively for the
recovery of distinctive bioactive compounds [6].
In particular, the conventional extraction methods employ cell-
damaging techniques which consume a large quantity of mechani-
cal or thermal energy. Also, these processes lead to the formation of
dangerous redox compounds over an extended period due to ther-
mal degradation and oxidation. Additionally, these traditional
methods are inadequate for selective extraction of particular bioac-
tive compounds from the plant-based food materials. So, the qual-
ity of recovered products such as terpenes and terpenoids, alkaloids,
carotenoids, phenolic compounds, polyphenols, proteins, and
polysaccharides may be damaged or reduced by these standard
methods. In recent years, numerous studies explore various sustain-
able nonthermal methods of extracting food components. These
eco-friendly extraction techniques result in higher yields and
supreme-quality of extracted products, since they utilize minimal
to no organic solvents depending on the selected novel nonthermal
extraction technique and also consume less time and energy [7].
The conventional extraction process of bioactive compounds
has various unit operations such as soaking, maceration, boiling,
grinding, magnetic stirring, water percolation, heat reflux, and
Soxhlet extraction. These approaches have a variety of disadvan-
tages and constraints, including excessive processing times, poor
extraction recoveries, increased solvent consumption, and low
extraction efficiency. Some of these also entail the possibility of
thermally degrading thermolabile bioactive compounds. To
address the above-mentioned drawbacks of conventional extraction

methods, many novel methods have been investigated. The effi-
ciency and performance of newly developed technique in compari-
son to existing extraction techniques such as the Soxhlet method
was demonstrated in a review work [8]. Therefore, it is essential to
employ novel nonthermal techniques especially PEF as a preferable
strategy to achieve improved extractability in order to overcome the
significant limitations in the extraction process of bioactive; further,
this study elucidates the efficacy of the PEF application for the
nutraceuticals and bioactive extraction [8].
3 PEF-Based Bioactive Extraction Technique: A Sustainable Greener Technology
Sustainability is a key idea for social, technological, and economic

advancement which aims to create a circular economy and maintain
the ability to meet society’s demands. Consumers in developed
nations are becoming more and more demanding for a diet that is
safe, wholesome, and appealing to the senses. PEF technology has
been considered as a sustainable choice for the extraction of bioac-
tives from foods due to the critical implications (higher energy and
water usage) of conventional thermal extraction methods. PEF
tends to support a strategic advantage for the food industry by
advancing sustainable food processing without sacrificing product
quality or safety. Technically, PEF technology’s sustainability in
each food industry is assessed through life cycle assessments
(LCAs) [9].
Numerous studies have identified PEF technology as a smart
substitute for products extraction and preservation (through pas-
teurization and sterilization) and also it can decrease or even pre-
vent the generation of harmful chemical compounds. Indeed,
PEF-assisted extraction methods adhere to all sustainability and
green chemistry concepts. PEF is a green extraction method that
makes it possible to extract natural colorants using nontoxic sol-
vents rather than contaminating food and beverages with hazard-
ous chemicals. PEF-assisted extraction technologies are much more
energy-efficient than traditional methods in terms of sustainability
concepts. One of the key features of this cutting-edge technology
that encourages energy savings is the low treatment time observed
in PEF-based operations. There are, however, assessment studies
examining the environmental effects of PEF technology on various
manufacturing sectors, such as bioactive component extraction
procedures [10]. Therefore, it is crucial to concentrate on the
sustainable attributes of the food processing industry, such as uti-
lizing the PEF technique for the extraction process, rather than
employing a conventional approach and delivering harmful residues
to the ecosystem.
4 Role of PEF Processing System in Bioactive Extraction
PEF as a technology can cause permeabilization of cell membranes

when applied for shorter times and it consumes low energy. The
fundamental idea behind PEF technology is the electroporation of
the cell membrane resulting in high extraction yield. The electro-
poration theory is the primary mechanism used in the exploration
of PEF exaction applications. Generally, plant, animal, or microbial
cells are briefly exposed to high-voltage electric field pulses that may
cause the lipid bilayer and proteins in cell membranes to become
unstable. Additionally, the plasma membranes of the cells become
permeable to tiny molecules after being exposed to strong electric
fields; this causes the cell to inflate and finally ensues the cell
membrane to rupture. The charging mechanism at the membrane
interfaces causes the potential of the transmembrane to rise when a
sphere-shaped biological cell is exposed to an external electric
field [11].
Electroporation produced by the dielectric breakdown of cell
membranes is the central principle of PEF-assisted extraction. Due
to the presence of free charges with opposing polarities throughout
the membrane, it is believed that cell membranes operate like a
capacitor with a lower dielectric constant and a natural transmem-
brane voltage. The accumulation of charges across the membrane
increases the transmembrane potential when the external electric
field is supplied. The potential is further raised by repeated expo-
sures to electric fields, which causes electrostatic attraction between
opposite charges to move across the membrane and narrow it. If the
external field intensity increases past the critical breakdown voltage,
which results in the creation of transmembrane pores, the mem-
brane will break down [12].
The continuous and short voltage pulses induces a permeability
in cell membranes that makes it easier for bioactive components to
be released from the interior of the cells and this occurs when plant
tissue is exposed to an electric field of moderate intensity
(0.5–10 kV/cm) and remarkably reduced energy (1–10 kJ/kg),
applied repeatedly in the form of very brief voltage pulses (generally
from few second to 1 ms). PEF treatment may impose a selective
permeability of the membranes (tonoplast and plasma membrane)
while the cell wall persists due to its nonthermal action on foods,
which enhances the purity and yield of the extracts. Therefore, PEF
treatment has been demonstrated to improve the quantity and
quality of the juice collected from fruits and vegetables when com-
bined with mechanical extraction [13].
The effects of high-intensity PEF processing on physicochemi-
cal and antioxidant properties were evaluated. High-intensity PEFs
(HIPEF) help in protecting the quality of juice throughout storage,
which is preferable compared to heat treatment [3]. Figure 2 illus-
trates the processing system of PEF.
Voltage Voltage
Sample transport
control device power supply
system
Treatment Treated
Samples
chamber samples
Temperature Temperature
inductors control system
Fig. 2 Schematic illustration of the PEF treatment system [11]
4.1 Mechanism of The application of rapid pulses (μs to ms) of moderate electric
PEF-Based Bioactive voltage (usually 0.5–20 kV/cm) to a suitable substrate situated
Extraction Unit between two electrodes is known as the pulsed electric field
(PEF)-assisted extraction. The method has been used for preserva-
tion, enzyme, and microbial inactivation using high electric voltage
(5–50 kV/cm). Particularly, in cell cultures and plant systems, low
to medium PEF treatment intensities are frequently regarded as an
efficient pretreatment technique for improving secondary metabo-
lite extraction yields.
In the batch system of the PEF technique, the electric field
intensity ranges from 100 to 300 V/cm, and in the continuous
mode of extraction, it ranges from 20 to 80 kV/cm. Several
hypotheses share two points of view regarding the putative PEF
mechanism. One involves accelerating chemical reactions compris-
ing numerous substances in the biological cell membrane to
increase the solvent’s solubility, and the other is the electroporation
process (Fig. 3). An external electrical force is used in electropora-
tion or electro-permeabilization to increase the permeability of cell
membranes. A high-voltage electric field is positioned between the
electrodes and food or any other targeted materials. By generating
hydrophilic holes, which activate protein channels, the cell mem-
brane is pierced. When high-voltage electrical pulses are applied to
the sample, a force per unit charge known as the electric field
occurs. When high-voltage electrical pulses are applied across the
electrodes, the sample feels a force per unit charge known as the
electric field. When the membrane no longer serves as a structural
component, the plant material is removed [14].
Fig. 3 Electroporation mechanism of extraction
4.2 Design and The main PEF variables were intensively evaluated to determine the
Fabrication of PEF- optimal PEF for the specific system to achieve the maximum poly-
Based Bioactive phenol content of the extracts. For specific extraction duration, it
Extraction Unit was shown that the key PEF parameters that affect permeability are
field intensity, pulse duration, and pulse period. For electropora-
tion, the uniform electric field chambers expose each cell in the
sample to the same electric field. High-yield intracellular chemical
extraction is feasible when the field strength is sufficient and close
to the ideal value. Due to the possibility that the best extraction
yields could differ noticeably above or below the ideal value, the
electric field strength set point must be determined by systematic
experimental design [15]. Figure 4 shows the schematic represen-
tation of an ordinary PEF-based processing system for the treat-
ments of food systems.
For instance, in a study conducted by Zhu et al. [16] PEF
pretreatment for protein extraction was performed with a PEF
generator system that was self-built, Cardamine violifolia was pre-
treated with PEF. A peristaltic pump was used to pump 4 g of
sample, dissolved in 120 mL of water, at a rate of 1.35 mL/s into
the treatment chamber. The operating parameters were as follows:
The frequency was 1.01 kHz, the half-peak width was 48 s, and the
pulsed electric field strength was 6.67 kV/cm. After six cycles, a
NaOH solution was used to bring the pH value to 9.0 ± 0.2, and
the treated solution was then extracted using conventional extrac-
tion (CE), ultrasound (US)-assisted extraction. After extraction,
the extracts were centrifuged at 4000 rpm for 20 min at room
temperature to get the supernatant.
Similarly, in a study performed by Athanasiadis et al. [17] 4.0 g
of freshly cleaned plant material (not dried) was crushed into
smaller pieces and combined with 80 mL of the solvent (at a ratio
Trigger signal
Control and
High voltage Energy
monitoring Power switch Pulse forming
DC supply storage
system
High voltage pulse generator
Current and voltage measurement
Flow/speed control for a continuous processing
Pre-processing measurement of sample food Treatment

Sample Processed
food chamber
food
Flow/speed
Post-processing measurement of sample food control unit
Fig. 4 Schematic representation of a typical PEF-based processing system for the treatments of food
application [7]
of 20:1 mL/g) for the extraction of total polyphenols. 100% water,

25%, 50%, and 75% ethanol in water, and 100% ethanol were the
different extraction solvents. The mixture was thoroughly mixed
before being inserted into the PEF chamber for a 20 min extrac-
tion. 10 and 100 μs were used as the pulse durations for the
extraction of total polyphenols. One (m) of the period (1000 Hz
frequency) and 100 pulse cycles were accomplished. At 1.0 kV/cm,
the electric field density was established. When the temperature was
measured before and after PEF, there was no discernible difference
(less than 1 °C). The mixture was put in a Falcon tube when PEF
was finished, and it was centrifuged at 4500 g for 10 min. The
supernatant was then immediately subjected to further studies.
4.3 Configuration The high-voltage pulses are delivered to the treatment chamber
and Requirements of containing the food sample using a pulse modulator. To transmit
PEF-Based Extraction the stored energy in an economically sound way, power switches are
Unit required. Most significantly, it has an impact on the electrical sys-
tems’ overall design. Initially, various food sample components
were extracted using batch devices with PEF pretreatment (for
solid-liquid extraction). However, Yin et al. [18] successfully
applied extraction in a continuous-flow treatment chamber. Since
then, there has been a considerable advancement in technology that
resulted in the invention of a continuous PEF extraction system,
making it simpler to extract continuous products. According to
reports, the PEF continuous extraction system has efficiently
extracted fishbones, eggshells, tomato juice, and other materials
[19]. However, it has not been widely implemented in food
Pulsed electric field

Treatment chamber
Cooling
coil
Pump
Untreated Treated
Generator of
product product
high voltage
pulse
Fig. 5 Fundamental components of PEF processing treatment [10]
processing, though. When the electric field applied voltage and

associated strength are higher than the necessary critical transmem-
brane potential, PEF-based extraction is possible. Figure 5 exhibits
the basic components required for the PEF-based bioactive
treatment.
The final application and the food that needs to be processed
determine this crucial transmembrane potential. The development
of pores takes place in the membrane of biological cells, including
those of plants, animals, microorganisms, and algae, when the
necessary critical transmembrane potential has been applied. Once
the pores have formed and have a radius of around 0.5 nm, they
may enlarge in response to the applied electric field, leading to
irreversible electroporation, which is the disruption of the cell.
After the applied voltage is removed in irreversible electroporation,
the cell cannot move back to its initial location.
4.4 PEF—Batch and The treatment chambers can be separated into batch treatment
Continuous Treatment chambers (Fig. 6) and continuous treatment chambers (Fig. 7)
Chamber based on the type of the treated product (solid, semisolid, liquid,
and semiliquid). The latter form is far more practical for
manufacturing applications since it enables the pumping through
the chamber of liquid and semiliquid products. A central computer
manages the process. It sets the parameters, manages the operation
of the pump, and collects data from the sensors inserted within the
chamber. The main issue with liquid products handled with PEF is
the nonuniformity of the electric field distribution inside the treat-
ment chamber, which is brought by the design of the chamber, the
occurrence of bubbles and other contaminants, and the thermo-
physical characteristics of the product itself. As a result, some areas
Control Panel/
PEF Generator Data Processing
Electrodes
Sample to be Placed/
PEF Chamber
Fig. 6 Schematic representation of batch extraction system of PEF
Oscilloscope
Pump Flowmeter
Thermometer
Raw Sample Treated Sample
Fig. 7 Schematic representation of continuous extraction system of PEF
of the liquid volume may either receive inadequate or excessive

treatment, typically in the center or in dead spots, often in bound-
ary regions [20].
Generally, batch chambers are limited in the amount of liquid
and solid food they can process. However, the productivity
observed with the traditional continuous processing of liquid sam-
ples needed in industrial applications can be achieved in a dynamic
chamber. Common electrode arrangements utilized in PEF include
parallel, coaxial, and colinear electrode configurations. The adja-
cent plate pairs each electrodes and biaxial electric field lines
[21]. However, compared to a setup with only two electrodes,
this later configuration does not appear to offer any noticeable
advantages. Multiple electrode rings on alternating potentials are
segregated by insulating rings in colinear electrode designs. Two
insulators and three conductors make up the colinear chamber. The
physical structure of the insulator positioned between the electro-

des regulates the heterogeneous distribution of temperature and
electric field intensity. This arrangement of electrodes offers a high
resistance that is beneficial for continuous bioactive extraction and a
significant treatment capacity with a smaller effective cross-sectional
area of the electrodes. This design is advantageous for PEF systems
since it requires limited current from the pulse modulator [7].
5 Factors Influencing the PEF-Based Bioactive Extraction
It is essential to recognize that the consequences during bioactive

extraction will depend on the strength of a given pulse as well as the
total damage sustained by a progressive succession of pulses that
results in cell malfunction when thinking about the shape and
spatial distribution of PEF therapeutic effects. Similar to charging
a capacitor, the electric field-induced change in the cell transmem-
brane potential depends on the strength of the electric field and the
length of the charge mobility. As a result, the strength of the
applied electric field and the length of time that a certain pulse is
exposed to the electric field can directly influence cellular effects at a
specific segment (Fig. 8). For corresponding waveform character-
istics (fundamental frequency, packet active period, and the total
number of packets) and a specific electrode arrangement, an effec-
tive electric field limit may be outlined to account for changes in
Insertion of proteins into the cell membrane
Insertion of small molecules into the

Pulsed electric field treatment intracellular environment
Insertion of large molecules into the

intracellular environment
Cell fusion
Cell destruction
Fig. 8 Effect on the cell structure subjected to PEF [10]

treatment size, with changes to a delivered voltage having an impact

on the electric field intensity. The length of the electric field pulse,
for a given voltage, can also modify how long the cell is exposed to a
different environment [10].
6 Overall Application of PEF Techniques in Bioactive Extraction
The sustainability of the world’s food supply depends on the utili-

zation of agro-industrial byproducts, which are abundant in natu-
rally occurring bioactive chemicals. Numerous unique strategies
have been developed and optimized to help with the effective and
sustainable extraction of the bioactive alongside more traditional
ways. Table 1 briefly elucidates the application of PEF in the extrac-
tion of various bioactive from different food systems.
7 Integrated Extraction Technologies in Combination to PEF
Integration of PEF and Ultrasonication (US) technology is

reported to be the most extensively utilized strategy for enhancing
bioactive extraction, since this method elucidates many notable
instances, particularly in the field of bioactive extraction from
food ingredients, as both strategies primarily focus on the cellular
levels discharging of the food material and hence accelerate the
extraction process.
A study conducted by Manzoor et al. [41] looks at the com-
bined effects of PEF and US to assess the physicochemical proper-
ties, bioactive components, and chemical composition of almond
extract. First, PEF was used to treat the almond extract, and then an
US. In comparison to all other treatments, combined treatment
(PEF-US) achieved the highest value of total phenolics, total flavo-
noids, concentrated tannins, anthocyanin contents, and antioxidant
activity in DPPH. All of those treatments had a marginal differences
in hue. Furthermore, according to FT-IR spectra, PEF-impact US
on almond extract did not result in the production of any new
carbonyl compounds, but rather in their concentration increases.
This study showed that the PEF-US could help with volatile com-
ponent stability improvement as well as bioactive chemical
extraction.
Another widely administered technique along with PEF is the
solvent treatment for improved bioactive extraction. A study by
Quagliariello et al., [42] intends to show that PEF-assisted brown
rice extraction results in higher antioxidant component yields,
including oryzanol, polyphenols, and phenolic acids, as well as
saturated and unsaturated fatty acid yields and cytotoxic effects on
cancer cells. The first PEF-assisted extraction conditions were
established by measuring the DPPH antioxidant activity and the
cell permeabilization index using impedance. The cytotoxicity and
Table 1
Application of PEF in the bioactive extraction from various food systems
Treatment intensity
P: Pulse width t: Treatment

EF: Electric field intensity F: N: Number of time T:
Category Bioactive compound Commodity E: Energy Frequency pulse S: Solvent Temperature References
Fruits and Phenolics, total Blueberry fruits (Vaccinium EF: 1–5 kV/cm 10 Hz P: 1–23 μs 50% ethanol; T: 20 ± 1 °C [13]
vegetables anthocyanins, myrtillus L.) E: 10 kJ/kg 0.5% HCl, v/v
and antioxidant
activity
Carotenoids, Date palm fruit extract EF: 1, 2, and 3 kV/cm 10 Hz N: 30 t: 100 s [3]
anthocyanins,
flavonoids, and
phenolics
Polyphenols Grape vine (Vitis vinifera); Greek EF: 1.2–2.0 kV/cm P: 10 μs t: 1 ms [22]
mountain tea (Sideritis scardica)
and Saffron crocus (Crocus
sativus)
Phenolic Cocoa bean shell and coffee silver EF: 1.5–3 kV/cm for N: 500–1000 S: water for CBS; t: 5–20 μs [23]
compounds skin CBS and ethanol/water
1.30–4.40 kV/cm solution for
for CS CS
Phenolic Brewers’ spent grain EF: 2.5 kV/cm 50 Hz P: 10 μs, N: Ethanol/water t: 14.5 s [24]
compounds 500–2250
Phenolic Onion 2.5 kV/cm 1 Hz 90 pulses Water 45 °C [25]
compounds and 100 μs
flavonoid
compounds
TPC, AA, and total Blackcurrant 1318 V/cm 315 pulses Cold pressing 10 and 22 °C [26]
monomeric
anthocyanins
(continued)
Pulsed Electric Fields as a Green Technology for the Extraction. . .
213
Table 1
214
(continued)
Treatment intensity
P: Pulse width t: Treatment

EF: Electric field intensity F: N: Number of time T:
Category Bioactive compound Commodity E: Energy Frequency pulse S: Solvent Temperature References
Pigments Betanin and Red beetroot tissue P: 4.38 kV/cm 10 μs Acidic buffer 9.2 °C. [27]
vulgaxanthin E: 4.10 kJ/kg 10, 20, 30 (pH: 6.5)
Turmeric crude Rhizomes of turmeric 0.5–7.5 kV/cm 100 ms 20 ms [28]
extracts
Pigments Spirulina 30 kV 300 Hz 4–32 μs [29]
Radhika Theagarajan et al.
Green leaves Polyphenols Olive leaves 1 kV/cm 1000 Hz 1000 μs Aqueous ethanol 30 min [30]
Phenolics Fresh rosemary and thyme 1.1 ± 0.2 kV/cm 10 Hz 67 bipolar Aqueous NaCl 40 °C [31]
by-products pulses of
30 μs
Bioactive Custard apple (Annona squamous) Electric field strengths 100–300 Ethanol (70%, 2.5–5 min [32]
compounds leaf extract (2–6 kV/cm) pulse pulses v/v)
energies (45–142 kJ/
kg)
Antioxidant Drumstick (Moringa oleifera) plant 7 kV/cm 37 kHz Aqueous 40 min [33]
activity extraction
By-products Nonpurified Peach pomace waste 0.8–10 kV/cm 0.1 Hz 4–30 Ethanol: water 20–22 °C [34]
bioactive monopolar 70:30
extracts pulses of
4 μs
Total phenolic Cocoa bean shell (CBS) and coffee 1.5–3 kV/cm 50 Hz 500–1000 0.1% formic acid 5–20 μs [23]
contents silver skin and 100%
methanol
Polyphenol and Grape stems 1 kV/cm 1 Hz 30 min [35]
volatile
compounds
Phenols, Fresh thinned peaches EF: 0–5 kV/cm 1 Hz P: 30–150 μs Water t: 3 μs [36]
flavonoids, and E: 0.61–9.98 kJ/kg N: 10–50 T: 15–35 °C
antioxidant
compounds
Algae Polyphenols and Brown macroalga (Alaria esculenta) 0, 720 pulses Ethanol [37]
carbohydrates concentration
(0%, 15%)
Starch Green macroalga (Ulva ohnoi) 1 kV/cm 3 Hz 200 pulses [38]
Phycocyanin and Spirulina platensis 15–25 kV/cm 2–6 Hz 1 μs [39]
protein
Pigments and total Tetraselmis chuii and Phaedoactylum 1–4 kV/cm 2 Hz 45–400 pulses Aqueous or 100 ms [40]
phenolic tricornutum 100 kJ/kg dimethyl
compounds sulfoxide
(DMSO)
215
anti-inflammatory capabilities of PEF have then been assessed in

the human colon cancer cell line HT29. The findings demonstrate
that PEF-assisted extraction increases the quantity of bioactive
chemicals in comparison to untreated extracts. Additionally,
brown rice PEF extracts significantly reduce interleukin production
and gene expression in colon cancer cells, suggesting that they
could be used as a natural antioxidant. Therefore, it appears that
incorporating PEF pretreatment into the solvent extraction process
of brown rice bioactive is a promising strategy to greatly increase
their biological activity.
Further, this review focuses on the idea that combining non-
thermal technologies is a beneficial alternative strategy in the bio-
active extraction sectors; however, it is economically less cost-
effective than using them individually. Apart from these techniques,
various researchers have discovered that this cutting-edge technol-
ogy works well in the extraction process when combined with other
methods such as osmotic shock and mechanical press [43]. More-
over, Table 2 depicts the applications of other nonthermal proces-
sing combined with PEF for the extraction of bioactive
components.
8 Benefits of PEF-Based Bioactive Extraction
The application of PEF-based extraction has become established

during the last ten years in both bioactive extraction and novel food
processing techniques. Compared to many of the present methods
used in the bioactive extraction, PEF-based solutions are more
efficient and sustainable for extraction. The numerous application
of pulsed electric fields is due to its advantages in terms of cost and
the environment; PEF processing is more acceptable because it uses
less specific energy per processed product. For instance,
PEF-treated juices are purer than untreated juices, which is proba-
bly due to the careful extraction of penetrated cells. Similarly,
PEF-based extraction frequently has higher selectivity for certain
bio-compounds, is quicker, more sustainable, causes less tempera-
ture rise, and uses less energy. Therefore, PEF technologies are
known to provide increased earnings and perhaps be able to effec-
tively produce bioactive components with added value from food
waste [43].
Moreover, the use of PEF as a nonthermal approach for food
processing is one of the key areas of research in the context of
biofuel methods. Although cooling is required to retain a low
temperature of the treated product during PEF treatment, it should
be noted that the energy of the electric pulses generates heat owing
to Joule heating. However, this phenomenon can be used in a
delicate preservation procedure. The inactivation efficiency is
increased by combining high temperature with PEF membrane
Table 2
Integration of PEF and other nonthermal techniques in extraction of bioactive compounds
Integrated
Commodity Bioactive compounds treatment Treatment conditions Significant findings Reference
Canola seed Total phenolic content, Microwave and Microwave: Liquid to soil ratio of 6.0 and 633.3 W Due to their reduced solvent usage, shorter [44]
cake total flavonoids PEF for 5 min extraction times for moderate microwave power,
PEF-assisted extraction: 30 V, 30 Hz, 10% ethanol lower electroporation voltage and frequency, and
concentration and 10 s greater efficiency in extracting polyphenols
Almond Condensed tannins, PEF and US PEF: Flow rate of 40 mL/min, 18 kV/cm electric Permeabilization, increases the yield, extraction [41]
extract anthocyanin, total field strength for 500 L/s, 1 kHz pulse frequency; efficiency, and the extraction of intracellular
phenolics, total US: 40 kHz ultrasonic frequency, 200 W radiation, metabolites. Due to the release of bound
flavonoids and 35 °C temperature for 20 min phenolics mediated by cavitation-induced cell
membrane rupture, ultrasound improves the
bioactive component
Grape stem Polyphenols PEF and US PEF: low electric field strength of 1 kV/cm Compared to solely ultrasound-assisted extraction- [35]
(30 min). derived extracts, this integrated technique helps
US: 35 kHz frequency and a 320 W high frequency to improve the yield of volatile compounds in the
peak extracts
Spirulina Phenolics, chlorophyll, and PEF and PEF: 44 pulses, 3 kV/cm, 99 kJ/kg PEF has the power to damage the structural and [45]
carotenoids pressurized PLE: preheating time of 1 min, heating time of functional integrity and obliterate the
liquid 5 min, flush volume of 60%, nitrogen purge time microalgae’s adherent filaments, which helps to
extraction of 60 s, extraction pressure of 103.4 bars, facilitate more effective PLE extraction. The
(PLE) extraction temperature of 40 °C, extraction time polyphenol content is increased by this integrated
of 15 min technique (PEF + PLE)
Ziziphus lotus Total phenolics, Supercritical fluid – Extraction of pharmaceutical drugs and nutritional [46]
fruits, chlorophyll, and extraction and supplements from natural sources, such as
leaves, and carotenoids PEF aromatic and medicinal plants, spices, and herbs
roots
217
electroporation. Enzymes and bacteria are mostly inactivated in

research on the usage of PEF [47].
High-voltage impulses induce the cell membrane to rupture,
allowing tiny molecules to get through and leading to the swelling
and breaking of the cells. For items that are liquid or semisolid,
such as soups, liquid eggs, or fruit juices, PEF can be employed. In
2005, fruit juices produced using this method was made available
on the US market. For solid items, the industry that processes
potatoes has seen the most success with PEF technology. PEF alters
the structural stability of tissues, causing a better-controlled release
of intracellular substances such as reducing sugars or amino acids
involved in Maillard reactions, which lowers the risk of acrylamide
content in fried or cooked potato products [48].
It is claimed that those certain electrical factors, such as pulse
frequency and the make up of the processed product (such as the
presence of halides), have an impact on the quantity of metal
discharged from the electrodes. Unfavorable electrode reactions
can be prevented or at least avoided by determining the ideal
conditions for PEF treatment on an industrial scale, as well as the
electrode material and geometry. The high-voltage pulse generator,
treatment chamber, fluid-handling system, control, and monitor-
ing devices make up the majority of a typical PEF machine. The
initial component provides the necessary shape, duration, and
intensity for the high-voltage pulses. A pair of electrodes in the
treatment chamber is used to apply the generated pulses, and the
product being treated is then positioned between them [20].
9 Future Perspective of PEF-Based Bioactive Extraction
Consumers today demand fresh, healthy products that can maintain

their nutritional profile during storage, which highlights the signif-
icance of creating novel and green processing methods [49].
The PEF-based extraction has been created as an alternative to
traditional extraction techniques, offering benefits such as faster
processing, greater extraction yield, fewer extract contaminants,
and reduced solvent and energy usage. However, there are still
certain defects in the PEF-based extraction method. As a result,
the PEF-based extraction still experiences a lot of obstacles that will
need to be solved in the future. Research regarding validation of the
PEF’s extraction mechanisms and creation and assessment of its
extraction kinetics models are highly required. Similarly, PEF
mechanism and kinetics are still up for debate and require more
research. For future industrial use, a deeper comprehension of the
mechanism and the development of a scientific model of
PEF-assisted extraction unit are necessary. Further, to scale up its
extraction technique for industrial applications and optimize the
treatment chamber shape of PEF, more research is required. The
extraction system’s industrial applicability will be facilitated by

further development of the technology. In the future, we anticipate
that PEF-based extraction will be a competitive method for bioac-
tive material extraction. More research is required on bioactive and
nutraceuticals extraction inferred from food processing industries.
These surpluses might constitute excellent alternatives for the crea-
tion of innovative functional foods. Consequently, it is essential to
research components extracted, with the PEF extraction technique
providing substantial support [4].
According to earlier studies, it has been demonstrated that PEF
treatment can increase the effectiveness of extraction in plant items;
however, the outcomes vary depending on the type of extraction
medium used. Therefore, in future research, the following aspects
should be considered when applying the PEF in the extraction of
bioactive compounds: When employing the PEF technology, mate-
rials properties of the diverse plants and extracted chemicals should
be taken into account. Even though the PEF technology has some
advantages in the extraction processes, it is still necessary to develop
optimal processing to integrate with certain other methods, and a
compatible extraction strategy should also be put into consider-
ation. This is due to the differences in the food materials adminis-
tered and processing parameters of the PEF equipment [11].
10 Conclusions
The demand for natural products extracted from plants and other
materials is booming, especially for those nutrients derived from
by-products, as a result of customers’ concerns about conceivable
hazards in the extracts and health risks associated with employing
organic solvents as an extraction medium. Therefore, researchers
are increasingly focusing their attention on investigating more
complex and efficient green-extraction processes as a result of the
rising demand for sustainably extracted bioactive compounds. Like-
wise, PEF technology is a recently developed nonthermal method
that has been mainly used in the food industry for various applica-
tions. The PEF procedure used to extract bioactive and nutraceu-
ticals has evolved recently. Moreover, the dielectric breakdown
theory is the chief principle for bioactive extraction on a theoretical
basis using PEF and is one of the widely recognized mechan-
isms. There has not been much research conducted on the mecha-
nism underlying PEF dependent bioactive extraction, however,
these techniques have been used as assisting technique for the
improved extraction.
References
1. Naliyadhara N, Kumar A, Girisa S, Daimary and colorings from plant matrices. Food
UD, Hegde M, Kunnumakkara AB (2022) Chem X 15:100398
Pulsed electric field (PEF): avant-garde extrac- 11. Zeng X, Zhang Z (2019) Pulsed electric field
tion escalation technology in food industry. assisted extraction of bioactive compounds. In:
Trends Food Sci Technol 122:238–255. Advances in food processing technology.
https://doi.org/10.1016/j.tifs.2022.02.019 Springer, pp 125–135
2. Arshad RN, Abdul-Malek Z, Roobab U, Qur- 12. Kumari B, Tiwari BK, Hossain MB, Brunton
eshi MI, Khan N, Ahmad MH, Liu Z-W, Aadil NP, Rai DK (2018) Recent advances on appli-
RM (2021) Effective valorization of food cation of ultrasound and pulsed electric field
wastes and by-products through pulsed electric technologies in the extraction of bioactives
field: a systematic review. J Food Process Eng from agro-industrial by-products. Food Bio-
44(3):e13629 process Technol 11(2):223–241
3. Siddeeg A, Faisal Manzoor M, Haseeb Ahmad 13. Bobinaitė R, Pataro G, Lamanauskas N, Šat-
M, Ahmad N, Ahmed Z, Kashif Iqbal Khan M, kauskas S, Viškelis P, Ferrari G (2015) Applica-
Aslam Maan A, Zeng X-A, Ammar A-F (2019) tion of pulsed electric field in the production of
Pulsed electric field-assisted ethanolic extrac- juice and extraction of bioactive compounds
tion of date palm fruits: bioactive compounds, from blueberry fruits and their by-products. J
antioxidant activity and physicochemical prop- Food Sci Technol 52(9):5898–5905
erties. Processes 7(9):585 14. Ranjha MMAN, Kanwal R, Shafique B, Arshad
4. Xi J, Li Z, Fan Y (2021) Recent advances in RN, Irfan S, Kieliszek M, Kowalczewski PŁ,
continuous extraction of bioactive ingredients Irfan M, Khalid MZ, Roobab U et al (2021)
from food-processing wastes by pulsed electric A critical review on pulsed electric field: a novel
fields. Crit Rev Food Sci Nutr 61(10):1738– technology for the extraction of phytoconsti-
1750 tuents. Molecules 26(16):4893
5. Aoude C, Lammerskitten A, Parniakov O, 15. Pappas VM, Palaiogiannis D, Athanasiadis V,
Zhang R, Grimi N, El Zakhem H, Vorobiev E Chatzimitakos T, Bozinou E, Makris DP, Lalas
(2022) Equipment and recent advances in SI (2022) Optimization of pulsed electric-
pulsed electric fields. In: Innovative and field-based total polyphenols extraction from
emerging technologies in the bio-marine food Elaeagnus pungens limelight leaves using
sector. Academic Press, London, pp 149–172 hydroethanolic mixtures. Oxygen 2(4):537–
6. Kumar K, Yadav AN, Kumar V, Vyas P, Dhali- 546
wal HS (2017) Food waste: a potential biore- 16. Zhu Z, Li S, Cai J, Cong X, Yu T, Yang W, He
source for extraction of nutraceuticals and J, Cheng S (2020) Green recovery of Se-rich
bioactive compounds. Bioresour Bioprocess 4 protein and antioxidant peptides from Carda-
(1):1–14 mine Violifolia: composition and bioactivity.
7. Arshad RN, Abdul-Malek Z, Munir A, Buntat Food Biosci 38:100743
Z, Ahmad MH, Jusoh YMM, Bekhit AE-D, 17. Athanasiadis V, Pappas VM, Palaiogiannis D,
Roobab U, Manzoor MF, Aadil RM (2020) Chatzimitakos T, Bozinou E, Makris DP, Lalas
Electrical systems for pulsed electric field appli- SI (2022) Pulsed electric field-based extraction
cations in the food industry: an engineering of total polyphenols from Sideritis raiseri using
perspective. Trends Food Sci Technol 104:1– hydroethanolic mixtures. Oxygen 2(2):91–98
13 18. Yin Y, He G (2008) A fast high-intensity pulsed
8. Yan L-G, He L, Xi J (2017) High intensity electric fields (PEF)-assisted extraction of dis-
pulsed electric field as an innovative technique soluble calcium from bone. Separ Purif Tech-
for extraction of bioactive compounds: a nol 61(2):148–152
review. Crit Rev Food Sci Nutr 57(13):2877– 19. Góngora-Nieto MM, Sepúlveda DR, Pedrow
2888 P, Barbosa-Cánovas GV, Swanson BG (2002)
9. Arshad RN, Abdul-Malek Z, Roobab U, Munir Food processing by pulsed electric fields: treat-
MA, Naderipour A, Qureshi MI, Bekhit AE-D, ment delivery, inactivation level, and regulatory
Liu Z-W, Aadil RM (2021) Pulsed electric aspects. LWT Food Sci Technol 35(5):375–
field: a potential alternative towards a sustain- 388. https://doi.org/10.1006/fstl.2001.
able food processing. Trends Food Sci Technol 0880
111:43–54 20. Nowosad K, Sujka M, Pankiewicz U, Kowalski
10. Bocker R, Silva EK (2022) Pulsed electric field R (2021) The application of PEF technology in
assisted extraction of natural food pigments food processing and human nutrition. J Food
Sci Technol 58(2):397–411. https://doi.org/ 31. Tzima K, Brunton NP, Lyng JG, Frontuto D,
10.1007/s13197-020-04512-4 Rai DK (2021) The effect of Pulsed Electric
21. Bermaki H, Ziane M, Semmak A, Bellebna Y, Field as a pre-treatment step in Ultrasound
Belhassaini H, Tilmatine A (2017) Experimen- Assisted Extraction of phenolic compounds
tal analysis of monoaxial and biaxial pulsed from fresh rosemary and thyme byproducts.
electric field treatment chambers for food pro- Innov Food Sci Emerg Technol 69:102644
cessing. Carpathian J Food Sci Technol 9 32. Ahmad Shiekh K, Odunayo Olatunde O,
(1):43–50 Zhang B, Huda N, Benjakul S (2021) Pulsed
22. Lakka A, Bozinou E, Makris DP, Lalas SI electric field assisted process for extraction of
(2021) Evaluation of pulsed electric field poly- bioactive compounds from custard apple
phenol extraction from Vitis vinifera, Sideritis (Annona squamosa) leaves. Food Chem
scardica and Crocus sativus. ChemEng 5(2):25 359:129976. https://doi.org/10.1016/j.
23. Barbosa-Pereira L, Guglielmetti A, Zeppa G foodchem.2021.129976
(2018) Pulsed electric field assisted extraction 33. Bozinou E, Karageorgou I, Batra G, Dourto-
of bioactive compounds from cocoa bean shell glou G, Lalas SI (2019) Pulsed electric field
and coffee silverskin. Food Bioprocess Technol extraction and antioxidant activity determina-
11(4):818–835. https://doi.org/10.1007/ tion of Moringa oleifera dry leaves: a compara-
s11947-017-2045-6 tive study with other extraction techniques.
24. Martı́n-Garcı́a B, Tylewicz U, Verardo V, Pasini Beverages 5(1):8. https://doi.org/10.3390/
F, Gómez-Caravaca AM, Caboni MF, Dalla beverages5010008
Rosa M (2020) Pulsed electric field (PEF) as 34. Plazzotta S, Ibarz R, Manzocco L, Martı́n-Bel-
pre-treatment to improve the phenolic com- loso O (2021) Modelling the recovery of bio-
pounds recovery from brewers’ spent grains. compounds from peach waste assisted by
Innov Food Sci Emerg Technol 64:102402 pulsed electric fields or thermal treatment. J
25. Liu ZW, Zeng XA, Ngadi M (2018) Enhanced Food Eng 290:110196
extraction of phenolic compounds from onion 35. Ntourtoglou G, Drosou F, Chatzimitakos T,
by pulsed electric field (PEF). J Food Proc Athanasiadis V, Bozinou E, Dourtoglou VG,
Preserv 42(9):e13755 Elhakem A, Sami R, Ashour AA, Shafie A, Lalas
26. Gagneten M, Leiva G, Salvatori D, Schebor C, SI (2022) Combination of pulsed electric field
Olaiz N (2019) Optimization of pulsed electric and ultrasound in the extraction of polyphenols
field treatment for the extraction of bioactive and volatile compounds from grape stems.
compounds from blackcurrant. Food Bioproc Appl Sci 12(12):6219. https://doi.org/10.
Technol 12:1102–1109 3390/app12126219
27. Nowacka M, Tappi S, Wiktor A, Rybak K, Mis- 36. Redondo D, Venturini ME, Luengo E, Raso J,
zczykowska A, Czyzewski J, Drozdzal K, Arias E (2018) Pulsed electric fields as a green
Witrowa-Rajchert D, Tylewicz U (2019) The technology for the extraction of bioactive com-
impact of pulsed electric field on the extraction pounds from thinned peach by-products.
of bioactive compounds from beetroot. Foods Innov Food Sci Emerg Technol 45:335–343
8 ( 7 ) : 2 4 4 . h t t p s : // d o i . o r g / 1 0 . 3 3 9 0 / 37. Einarsdóttir R, Þórarinsdóttir KA, Aðalbjörns-
foods8070244 son BV, Guðmundsson M, Marteinsdóttir G,
28. Poompavai S, Gowri Sree V (2022) Anti-pro- Kristbergsson K (2022) Extraction of bioactive
liferative efficiency of pulsed electric field trea- compounds from Alaria esculenta with pulsed
ted curcuma longa (Turmeric) extracts on electric field. J Appl Phycol 34(1):597–608.
breast cancer cell lines. IETE J Res 68 https://doi.org/10.1007/s10811-021-
(6):4555–4569 02624-8
29. K€aferböck A, Smetana S, de Vos R, Schwarz C, 38. Prabhu MS, Levkov K, Livney YD, Israel A,
Toepfl S, Parniakov O (2020) Sustainable Golberg A (2019) High-voltage pulsed electric
extraction of valuable components from Spiru- field preprocessing enhances extraction of
lina assisted by pulsed electric fields technology. starch, proteins, and ash from marine macro-
Algal Res 48:101914 algae Ulva ohnoi. ACS Sustain Chem Eng 7
(20):17453–17463
30. Pappas VM, Lakka A, Palaiogiannis D, Athana-
siadis V, Bozinou E, Ntourtoglou G et al 39. Jaeschke DP, Mercali GD, Marczak LDF,
(2021) Optimization of pulsed electric field as Müller G, Frey W, Gusbeth C (2019) Extrac-
standalone “green” extraction procedure for tion of valuable compounds from Arthrospira
the recovery of high value-added compounds platensis using pulsed electric field treatment.
from fresh olive leaves. Antioxidants 10 Bioresour Technol 283:207–212. https://doi.
(10):1554 org/10.1016/j.biortech.2019.03.035
40. Kokkali M, Martı́-Quijal FJ, Taroncher M, pressurized liquid extraction (PLE) and com-
Ruiz MJ, Kousoulaki K, Barba FJ (2020) bined PEF + PLE process evaluation: effects on
Improved extraction efficiency of antioxidant Spirulina microstructure, biomolecules recov-
bioactive compounds from Tetraselmis chuii ery and Triple TOF-LC-MS-MS polyphenol
and Phaedoactylum tricornutum using pulsed composition. Innov Food Sci Emerg Technol
electric fields. Molecules 25(17):3921 77:102989. https://doi.org/10.1016/j.ifset.
41. Manzoor MF, Zeng X-A, Rahaman A, Siddeeg 2022.102989
A, Aadil RM, Ahmed Z, Li J, Niu D (2019) 46. Ramos C (2022) Supercritical fluid extraction
Combined impact of pulsed electric field and and pulsed electric field assisted extraction of
ultrasound on bioactive compounds and FT-IR Ziziphus lotus fruits, leaves and roots {. Biol
analysis of almond extract. J Food Sci Technol Life Sci Forum 18:37. https://doi.org/10.
56(5):2355–2364. https://doi.org/10.1007/ 3390/Foods2022-12985
s13197-019-03627-7 47. Mohamed MEA, Eissa AHA (2012) Chapter
42. Quagliariello V, Iaffaioli RV, Falcone M, Ferrari 11: Pulsed electric fields for food processing
G, Pataro G, Donsı̀ F (2016) Effect of pulsed technology. In: Structure and function of
electric fields – assisted extraction on anti- food engineering. IntechOpen, Rijeka, pp
inflammatory and cytotoxic activity of brown 275–306
rice bioactive compounds. Food Res Int 48. Genovese J, Tappi S, Luo W, Tylewicz U, Mar-
87:115–124. https://doi.org/10.1016/j. zocchi S, Marziali S, Romani S, Ragni L, Roc-
foodres.2016.07.005 culi P (2019) Important factors to consider for
43. Vorobiev E, Jemai AB, Bouzrara H, Lebovka acrylamide mitigation in potato crisps using
NI, Bazhal MI (2005) Pulsed electric field pulsed electric fields. Innovative Food Sci
assisted extraction of juice from food plants. Emerg Technol 55:18–26. https://doi.org/
In: Novel food processing technologies. CRC 10.1016/j.ifset.2019.05.008
Press, Boca Raton, pp 105–130 49. Manzoor MF, Zeng X-A, Ahmad N, Ahmed Z,
44. Teh SS, Niven BE, Bekhit AEDA, Carne A, Rehman A, Aadil RM, Roobab U, Siddique R,
Birch EJ (2015) Microwave and pulsed electric Rahaman A (2020) Effect of pulsed electric
field assisted extractions of polyphenols from field and thermal treatments on the bioactive
defatted canola seed cake. Int J Food Sci Tech- compounds, enzymes, microbial, and physical
nol 50(5):1109–1115. https://doi.org/10. stability of almond milk during storage. J Food
1111/ijfs.12749 Process Preserv 44(7):e14541
45. Zhou J, Wang M, Berrada H, Zhu Z, Grimi N,
Barba FJ (2022) Pulsed electric fields (PEF),
Chapter 10
Pulsed Electric Field Extraction

Subrahmanya Hegde, Yuvaraj Sivamani, Arunachalam Muthuraman,
Abstract
An efficient nonthermal food processing method is called pulsed electric field processing. It involves
applying a short-duration, high-voltage pulse to a food product that is sandwiched between the two
electrodes. This method is used for microbial inactivation in food, making them last longer without the
need for preservatives or thermal treatments. This method results in many benefits for foods, such as
protection of their original nutritional value, taste, color, freshness, and flavor. Apart from these common
benefits, there are many other benefits of pulsed electric field (PEF) for specific foods. The PEF treatment
can increase the number of bioactive substances that can be extracted from plant tissues and their
by-products, including vitamins, minerals, polyphenols, anthocyanins, and plant oil, as well as the soluble
intracellular matter from microorganisms. In this chapter, we examine how the PEF method is used in the
extraction of bioactive compounds as well as their applications in the food and nutraceutical industries.
Key words Pulsed electric field, Nonthermal, High-voltage pulses, Extraction yield, Microbial inacti-
vation, Bioactive compounds, Intracellular matter
1 Introduction
One of the extending and enticing uses of high-voltage engineering

techniques is the pulsed electric field (PEF) technique. It is a
cutting-edge processing method that does not require any thermal
energy [1]. A pulsed electric field’s effect on a biological cell’s
electric potential gradient between its inside and exterior improves
the conductivity and permeability of the cell membrane [2] and
controls or prevents the devaluation of the quality of the food
compounds [1]. By heating food to a temperature between
60 and above 100 C for varying lengths of time, thermal processing
serves as a common method for biological stabilization. Although
the extra energy can be used to eliminate or prevent harmful germs,
numerous unexpected secondary reactions reduce the nutritional
and sensory quality of food [3]. Numerous studies have shown that
pulsed electric field (PEF) technology can be used to produce safe,
223
224 Subrahmanya Hegde et al.
high-quality foods that include considerable amounts of chemicals

that are good for health [2]. PEF technology can be used to process
both solid and liquid foods, as well as semisolid foods. Although
minimal information is offered about the impact of PEF on food
composition, quality, and acceptability, most researchers have
mainly concentrated on the element of food preservation with
particular reference to microbial control. Recent research has
been done to assess the potential of PEF for increasing the effi-
ciency of food processing, including enhancing juice extraction and
intensifying food dehydration or drying [4]. In addition, increased
emphasis has been given recently to the use of pulsed electric fields
to extract beneficial components from food waste and by-products
through osmosis, squeezing, and drying. It reduces the negative
effects of standard heating methods. Important chemicals have
been successfully separated, intensified, stabilized, and dehydrated
without losing their nutritional characteristics using PEF technol-
ogy, a possible alternative to traditional techniques. Pulsed electric
field (PEF) has just been introduced as a method to stress the plant
cells, which will boost the extraction process by enhancing the
production of active ingredients [5]. Traditional extraction techni-
ques typically take a long time, use a lot of solvents, and involve
heating. There has recently been an increase in the demand for
novel extraction techniques that are also energy- and environmen-
tally efficient to improve mass transfer processes, and the quality of
the extract, and to minimize extraction time and solvent consump-
tion while avoiding the use of organic solvents. High-voltage elec-
trical discharges (HVED), pulsed electric fields (PEF), ultrasound-
assisted extraction (UAE), microwave-assisted extraction (MAE),
high-pressure extraction (HPE), and microwave-assisted extraction
(MAE) are some of these new techniques that have shown promise
in improving the overall yield and selectivity of biomolecules from
various vegetal matrices [6]. In reaction to the administration of
pulsed electric fields of high intensity and duration between micro-
seconds and milliseconds, cell membrane permeabilization may
happen briefly or permanently. Since the usage of PEF has drawn
significant interest in several scientific fields, including cell biology,
biotechnology, medicine, and food technology, the effects of PEF
on biomembranes have been intensively investigated [7].
2 General Overview of PEF and Its Working Principle
The PEF has enormous advantages and benefits in different per-

spectives at different sectors. It can be exclusively used for the
preservation of different types of food. Through this process, it
follows its methodology and also it is utilized for the extraction of
various kinds of bioactive compounds that are produced by plants
and microorganisms. Therefore, this technique is being abundantly
Pulsed Electric Field Extraction 225
utilized in food, nutraceuticals, and laboratory extraction of bioac-

tive compounds [6]. We all know that every living thing in
the world is made up of various types of cells and that a cell is the
fundamental structural unit of life. As a result, cells serve as the
storage units for all the valuable materials needed for an organism’s
growth and development. If we want to obtain something valuable
from an organism, we must target its basic cell. From the cell, we
can get our desired product, for example, any bioactive compound
from the plant cell. So if we want to take out the desired product,
one has to disrupt the cell in order to allow the required com-
pounds to come out of the cell. To accomplish this, we require a
revolutionary technology that does not harm the organism, saves
time, costs less money, and is simple to use. Pulsed electric field
extraction is one such advanced technique; hence, the name itself
states that the PEF technology works based on the utilization of
short pulses of high electric fields for a very short interval of time
(milliseconds), the electric property associated with each point in
space when the charge is present in any form is known as electric
field. Here, the sample from which the bioactive compound has to
be extracted or any food product that has to be processed should be
kept between the two electrodes, which generate high electric field
pulses of about 100–300 V/cm in batch mode and 20–80 kV/cm
in continuous mode of extraction [5]. This process depends on the
number of pulses that we pass through the sample placed between
the electrodes [4]. The space between the two electrodes to place
the sample is known as the treatment gap of the chamber. Typically,
there are two ways to help the extraction of valuable bioactive
compound from the cell of an organism [7]: one is to achieve
the solubility of the solvent in a biological membrane one has to
speed up the chemical-based reaction from various compounds
and the another one is electroporation of cell membrane [8].
Electroporation is the method of permeabilization of the cell to
extract bioactive compound, by the external application of short-
term high-voltage electrical impulses, which will help to increase
the permeability of the cell membrane [5]. This happens through
the formation of hydrophilic pores. When a cell is suspended in an
electric field, an electric potential flows across the cell membrane
[9], and subsequently the high voltages will cause the pores in the
cell membrane of that cell and thus cellular structural functioning
will be lost and the desired product can be easily extracted [10]. We
can apply electric field in various ways like oscillatory square waves,
unipolar triangular, or bipolar pulses. The electroporation that
occurs is either temporary or permanent, but depending on the
application, this effect can be controlled [11]. Irreversible electro-
poration increases the extraction process; sometimes, this cell per-
meability depends on the size and geometry of the cell [12].
According to certain research, electrical fields with strengths
between 0.1 and 10 kV/cm are sufficient for fragile plant tissues
such as the pericarp or mesocarp of a few fruits, while seeds and
Cell before PEF treatment. Disintegrated cell by PEF Extraction of bioactive
Solvent
Bioactive compound
Fig. 1 Cellular disintegration and the extraction of bioactive compound from the ruptured cells upon the
application of pulsed electric field
other hard materials require higher intensities, between 10 and

20 kV/cm, to be extracted effectively [13]. It is still unclear how
electroporation caused by PEF works at the molecular level. The
most widely recognized theory, however, is based on Sale and
Hamilton’s “transmembrane potential (ΔΦ) breakdown model”
[14]. A high amount of transmembrane potential develops because
of the accumulation of electric charges on the membrane of the
cells as and when it is sandwiched between the electric field–pro-
ducing electrodes. But the cells have a critical endurance limit, that
is, without disruption plasma membrane of a cell can withstand
some level of electric field strength. When it crosses or exceeds, the
cell membrane gets disrupts [15]. Increased membrane permeabil-
ity causes cells to disintegrate, while an increase in mass transfer rate
facilitated the release of intracellular components [5]. High electric
field strength affects the electroporation rate, type of waveform,
duration of time, and the type of sample taken [16]. One can also
perform this method at various temperature ranges such as ambi-
ent, sub-ambient, and above ambient type of temperatures
(Fig. 1) [4].
3 Equipment Design of PEF-Assisted Extraction
To achieve this complex process, that is, the high voltage at a very
short interval of time, engineers have designed equipment that has
all the necessary components to meet our required purpose. The
entire unit of PEF consists of a generator that produces very high-
voltage pulses, a monitoring and control system, and a fluid man-
aging assembly with a treatment chamber. To convert alternate
Fig. 2 Design of pulsed-field extraction system
current into direct current, it has a converter/charger [5] and a

device which helps to store the energy in the generator. To produce
electrical pulses, scientists have designed the switch that helps in
the on and off mechanism of a high-voltage circuit. It consists of a
capacitor that is to be continuously monitored if there is any
interruption in the voltage flow [3]. The PEF unit consists of two
main electrodes in the treatment chamber where our desired sam-
ple to be treated is placed, among them, one electrode is connected
to a generator that produces very high voltage and another one is
attached to the ground. An electric field is generated because of the
difference in the electric potential on either side of the membrane.
In addition, this depends on the type of electrode and the distance
between those electrodes and the sample placed in the treatment
chamber (Fig. 2).
The effect of the electric field depends on other factors such as
the design or the layout of the treatment chamber, the nature of
electric pulses, and the conductivity of a product. The PEF extrac-
tion methods can be categorized into the following type of extrac-
tion methods based on the way of performed [7].
1. Batch method of PEF extraction
2. Continuous method of PEF extraction
3.1 Batch Method of In the batch method of extraction, the unit contains a pretreatment
PEF Extraction chamber for solid-liquid extraction. The cylindrical vessel of poly-
propylene is fixed in the pretreatment unit of PEF. It consists of two
electrodes made of stainless steel, which are parallelly arranged, and
the distance between them is 10 mm. Electrical field strength, shape
Fig. 3 Flowchart explaining the PEF batch extraction method
number, the width of the pulses, and frequency are the most
important parameters one should consider during processing [5]
(Fig. 3).
3.2 Continuous In order to carry out the extraction process in a well-organized

Method of PEF manner in an industrial level, the continuous method of PEF
Extraction extraction has been developed. The batch method of PEF extrac-
tion gave a better result only in terms of better extraction, but it
took considerably high operating time due to the low capacity of
the batch method. To overcome this problem, a new arrangement
and design is developed. The first extraction from this system was
done by Yong Guang et al. [3] to extract the polysaccharide from
the Rana temoraria chensinensis David in 2006 [17]. They got a
good result that PEF extraction gave 55.59% compared to usual
extraction methods. Nowadays, this method is extensively used in
laboratories to get various kinds of animal and plant by-products
such as fishbone, eggshell, and tomato juice [5].
Normally, the continuous PEF extraction system contains a
generator to produce high electric pulses; a treatment chamber to
place the desired sample to be treated; a product handling system; a
controlling system; output voltage reader (oscilloscope), which can
read about 40 kV pulse; and the frequency can be adjustable from
40 to 3000 Hz. In this method, at constant fluid speed, the mixture
of solvent is sucked into a treatment chamber through a pump
(peristaltic pump). During the extraction procedure, the thermo-

stat controls the cooling coil’s temperature (25 °C) in the water
bath. Nowadays coaxial and cofield continuous PEF units are
abundantly used as they are user-friendly [5, 7, 10].
4 Factors Affecting the PEF Extraction
Factors such as nature of the solvent, composition of the sample,

size, shape, conductivity, pH, extracted compound size, and their
location in the cell affect the PEF extracting system. The properties
of the cells and tissues also have a significant influence over the
effectiveness of pulsed electric field extraction [11, 18]. In case of
low ionic strength, the extraction procedure is improved. The
cytoplasmic system is impacted by ionic strength because of cell
shrinkage and pore formation. Even so, the conductivity of the
matrix has a substantial impact on how the electric field behaves
as it moves through the matrix [19]. As it influences the physical
characteristics of the target molecule, such as, surface tension,
viscosity, and solubility, EFS is a critical parameter in determining
the degree of extraction [20]. Ensuring that electric fields are
distributed evenly throughout the treatment chamber is crucial.
Electric field energy is transmitted as square, bipolar, exponentially
decaying, and oscillatory pulses. Due to their high energy and lethal
performance, exponential square wave pulses are among those that
are frequently used in the PEF extraction procedure. Square waves
are also the most typical waveform used in the extraction process.
Although the PEF intensity varies depending on the food’s char-
acteristics, typically 12–45 kV/cm is sufficient to remove the essen-
tial ingredients from food. With an increase in the EFS, the
extraction of target compounds also gets better because of the
considerable energy transfer in the food sample. Additionally, Lin
et al. [21] found a significant increase (5.042 0.04 to 6.996
0.03 mg/mL) in the calcium malate extraction from an eggshell
while using PEF at 0 to 10 kV/cm EFS. The PEF treatments,
according to the scientists, accelerated the interactions between
ionic groups and electrons with increased kinetic energy, speeding
up the extraction of calcium malate and malic acid. Another critical
element influencing the PEF extraction process is treatment tem-
perature [22]. As a nonthermal process, the PEF extraction tech-
nique works at or close to room temperature. Higher temperatures
typically cause liquid solvents’ viscosity to diminish, which is harm-
ful to the extraction process. Another criterion to gauge PEF
effectiveness is treatment time (pulse numbers and width)
[19]. The product’s temperature could rise with a longer treatment
period, though. In addition, choosing the right solvent is essential
for better PEF extraction. Numerous aspects, including solubility,
conductivity, and solvent polarity will affect the extraction process.
The extraction rate was ultimately boosted by the higher solvent

conductivity because it facilitated cell membrane electroporation.
The strong polarity of the solvent and the extract’s high solubility
in the solvent both accelerated mass transfer and extraction rates
[5, 23].
5 Factors Impacting the Effectiveness of Pulsed Electric Field Treatment
The physicochemical characteristics of the biomaterial (cell or tis-

sue), the treatment medium, and the pulse parameters all have an
impact on the potency of PEF treatment.
5.1 Tissue The effects of electroporation depend on the physical and chemical
Parameters characteristics of the cell and the tissues, such as the composition,
thickness, and size of the cell membrane. The value of the trans-
membrane potential depends on the type of tissue and the rigidity
of the membrane; it is inversely proportional to the size and rigidity
of the cell. Soft plant tissues typically require a moderate strength of
the electric field, whereas hard tissue parts require a comparatively
high amount of electric field strength to rupture the cell membrane
by exceeding transmembrane potential. The larger field strength
necessary is 20 kV/cm, and the moderate quantity of electric
strength required is (E 5 0.12 kV/cm) [15].
5.2 Media Parameter According to certain research, the medium’s conductivity and ionic
potential may have an impact on the membrane’s ability to separate.
According to existing lines of evidence, the conductivity of the
medium is inversely proportional to cell mortality in the case of
microbe inactivation and gene transfection. However, several inves-
tigations have shown the exact opposite phenomenon. In extrac-
tion studies, the medium’s conductivity is generally unaffected. The
plant tissues or dietary by-products do, however, contain large
amounts of extracellular ionic compounds (salts) when suspended
in the aqueous environment during PEF treatment, which may
change the cell size (owing to osmosis) and electrical conductivity.
The heat generated during the PEF treatment may have a favorable
or negative effect on extraction efficiency. Larger field amplitude
pulses can be employed in low-conducting mediums to achieve the
appropriate level of electroporation [14].
5.3 Pulse Parameter The degree of electroporation may be influenced by the pulse’s
electric field strength, polarity duration, and frequency. For paral-
lelly arranged electrodes, the electric field strength can be defined as
the ratio of voltage and the distance between the two parallelly
arranged electrodes.
Electric field strength ðE Þ

= Applied voltage ðV Þ=Distance between two electrodes ðd Þ ½4]:
By creating irreversible electroporation, the high electric field
strength for a long time results in better tissue damage. Typically,
the medium electric field strength beyond the critical field and
longer pulse durations are best for electroporation of plant cells.
As far as the pulse shape is concerned, it also has a significant
effect on the membrane disintegration and extraction of intracellu-
lar compounds from the cells. Due to its energy efficiency and
ability to produce homogenous electric fields, square wave pulse
has been used in the majority of laboratory-scale experiments.
Oscillatory pulses are not advised since cells are only marginally
injured under these circumstances because they are continuously
exposed for a long period [14, 24].
As far as pulse polarity is concerned, typically bipolar pulses are
found to be more efficient than those monopolar pulses. The
bipolar pulses are more efficient because in that mode the direction
of the provided electric field will be continuously reversed. In
addition, it processes half-negative and half-positive pulses. Overall
studies indicate that bipolar pulses with a square form may result in
a higher extraction yield [15, 25].
6 Extraction of Intracellular Bioactive Compound from Plant Sources

Through PEF-Assisted Method
Researchers’ interest in organism extraction has increased as a result

of the PEF treatment’s reputation as a viable method. In addition
to soluble intracellular matter from microorganisms, the PEF treat-
ment can boost the extraction yield of bioactive compounds from
plant tissues and their by-products, such as polyphenols, anthocya-
nins, and plant oil [1].
6.1 What Are Secondary metabolites of plants that have medicinal or toxic effects
Bioactive Compounds? on living organisms are known as bioactive substances in plants. In
addition to the primary biosynthetic and metabolic pathways for
compounds related to plant growth and development, secondary
metabolites are produced in plants. These by-products of plant cells
are not required for daily plant function, but some of them are
found to have important functions in living plants, including sig-
naling and protection. The majority of plant species appear to be
able to produce these substances. But some of the chemicals and
bioactive compounds from the plants have pharmacological and
toxicological effects on humans and animals [21].
Plants are a large reservoir of various kinds of bioactive com-
pounds; these are the extra constituents that are rich in nutritional
value. Therefore, it is important to utilize these compounds in
various fields of medicine as well as food and nutraceutical indus-

tries. Scientists have studied that these bioactive compounds show
antidiabetic, anticancerous, and antioxidant properties so that they
can be utilized to cure cancer [26], to increase bone strength, and
to build the strong immune system [21, 27]. As these bioactive
compounds have commercial importance, it is important to extract
them from the cell of the plant, which is a very big task. Hence,
scientists have done various types of research to find out the cost-
effective, and less harmful, methods to enhance the productivity of
these compounds from the plant sources. The novel technology
called pulsed electric field extraction is found to be more efficient
compared to the conventional method of extraction of bioactive
compounds [2].
When the cell is exposed to an electric field, the dipole nature of
the cell membrane will separate the molecules based on their charge
under the electric field. Initially, when the pulse of high voltage is
passed through the cell for a very short interval of time, it weakens
the lipid bilayer of the plasma membrane of the cell and also the
proteins present in the plasma membrane [28]. Eventually, the high
electrical pulses will create the temporary pore in the plasma mem-
brane, which leads to the transfer of large molecules along the cell
membrane. As the outer membrane of the cell encloses or shields
the intracellular membranes, the optimal level of pulses will not
affect the intracellular membranes [2, 25]. In order to achieve
increased extraction of bioactive compound from the desired sam-
ple, it should be treated with the optimal level of pulsed fields,
which leads to the reversible electroporation on the cell membranes
of the cell. The temporary pore formed by the pulses enables the
mass transfer of intracellular compounds into the surrounding
solution, primarily due to the high membrane permeability [29]
for the movement of an electric field it should need a media, that is,
ions as the foods contain several ions which help to provide con-
ductivity to the product to a certain extent. However, the PEF
method is most abundantly utilized nowadays for liquid foods as
the electric current can easily pass through the liquid media from
one point to the other point due to the presence of charged
molecules [4].
Free radicals are one of the most dangerous oxygen-containing
molecules with an uneven number of electrons. This property
allows free radicles to react easily with other molecules in the
body. Free radicles can cause a chain of chemical reactions in our
body, which may damage the cell, and it may be harmful to our
body. Moreover, this may later lead to tissue degradation and
followed by various kinds of diseases such as heart ailments, cancer,
and atherosclerosis-related diseases [30]. To overcome the harmful
effects of free radicles in the body, antioxidants are utilized, which
will play a major role in protecting the cells of our body from free
radicle damage by neutralizing the free radicles by donating the
electrons. The bioactive compounds present in the intracellular

matrix of food, animal, and plant cells possess these antioxidant
properties. By using the pulsed electric field extraction system, one
can easily extract valuable bioactive compounds from the cells
[2, 19, 25].
Table 1 shows the application of the optimal value of PEF for
the extraction of various bioactive compounds from plant sources
(Table 1).
Table 1
Utilization of the PEF method in the extraction of bioactive compounds from various sources
S. Bioactive
No compound Product The optimum value of the PEF application References
1. Polyphenol Red grape E-400 V/cm, n-2 tp -2000+_1 μs, delta [31]
pomace t-20 ms [32]
Orange juice E-35 kV/cm, f-800 Hz, tp-4 ms, tPEF- [33]
Orange peel 750 ms [34]
Tea leaves E-5 kV/cm, n-20, tp-3 μs, tPEF-60 μs [35]
Green grapes E-0.9 kV/cm, tPEF-0.5 s μs
E-10 kV/cm, tPEF-100 μs, n-15
2. Anthocyanin Merlot grapes U-7 kV, f-178 Hz, tPEF-150 s [36]
Raspberry E-3 kV/cm, tPEF-15 μs, n-420 [2]
Potato E-3.4 kV/cm, t-3 μs, n-35 tPEF-105 μs, [37]
Red cabbage 8.9 kJ/kg [38]
Blueberry E-2.5 kV/cm, tPEF-15 μs, n-50, [39]
W-15.63 J/g
E-3 kV/cm, W-10 kJ/kg, f-10 Hz, tp-20 μs
3. Carotenoids Carrot E-0.6 kV/cm, tPEF-3 ms, tp-20 μs, f-5 Hz [40]
Tomato E-25 kV/cm, tPEF-110 μs [41]
Orange juice E-35 kV/cm, f-800 Hz, tp-4 ms, tPEF- [42]
750 ms
4. Lycopene Tomato E-1.5 kV/cm, n- 40, tp- 500 μs [43]
Watermelon E-35 kV/cm, f- 200 Hz, tp- 50 μs [44]
5. Lutein Microalgae E-25 kV/cm, tPEF-150 μs [45]
6. Alkaloids Korean E-20 kV/cm, n-8, tPEF-60 s [46]
monkshood E-0.75 kV/cm, tPEF-600 μs [47]
Potato peel
7. Betulin Mushroom E- 40 kV/cm, tp- 2 μs [2]
8. Protein Microalgae E-38 kV/cm, f- 158 Hz, tp-232 μs [2]
9. Polysaccharides Corn silk E-30 kV/cm, tp- 6 μs [48]
10. Lipid Microalgae U-45 kV, E- 45 kV/cm, tp-10 s, [49]
tPEF-30 s, n-3
6.2 PEF-Assisted Fruits and vegetables are the reservoirs of valuable antioxidants,
Extraction of Bioactive minerals, vitamins, and fiber [50]. These contain beneficial antiox-
Compounds from idants such as phenolics, carotenoids, and anthocyanin [51].
Fruits and Vegetable Hence, these compounds have commercial and medicinal impor-
Sources tance, and it is necessary to extract them. To achieve this, PEF
method plays an important role in the extraction process. There
are various other types of traditional methods of extraction of
bioactive compounds such as the Soxhlet extraction method, mac-
eration techniques, and hydrodistillation [52]. However, all these
methods are time- and energy-consuming and are also less produc-
tive. Various studies have been undertaken to compare the extrac-
tion yield between normal extraction methods and PEF-assisted
extraction methods. In all the cases, the PEF-assisted extraction
gave the best result compared to normal traditional methods. One
of the study findings showed that PEF-assisted extraction has
improved the release of vitamin C, anthocyanin, and also increased
antioxidant property of the grape juice when compared to the
normal untreated sample [2, 5, 53]. Hence, the extracted sample
had a good composition of phytochemicals and helped to prevent
the oxidation stress of the cell [53].
Another study accounts for good quality extraction of phenols
and flavonoids from onion through PEF extraction. As the cells
have a plasma membrane, which affects the mobility of intracellular
substances among other cells, it is a difficult and time-consuming
process to extract the required compounds by traditional methods
of extraction. However, in contrast, in the PEF method, the high-
voltage pulses will cause a plasma membrane permeability by form-
ing a pore so that the substance inside the cells can be easily
removed out, leading to high yield in a short interval of time [54].
Similar studies show that the PEF extraction method provides a
greater yield of polysaccharides, proteins, and polyphenolic com-
pounds from white button mushrooms when compared to usual
thermal treatments [5].
6.2.1 From Plant Leaves One of the research studies has been done by Khursheed Ahmad
Shiek and his team about the examination of extraction yield of
bioactive compound from the leaf of custard apple. They prepared
the extraction by adding 70% of ethanol along with the help of a
pulsed electric field. Results showed the increased cell integration
index in the extraction of custard apple when they provided a pulse
electric field of 6 kV/cm of about 300 pulses with the energy of
142 kJ/kg for about 5 min. They also found that the productive
yield of extraction through the PEF method has shown an addi-
tional higher percentage of 5.2% than those of untreated extracts by
the PEF method, which is 13.28%. The concentration of chloro-
phyll A and B in the pulsed electric field–treated custard apple leaf
extraction was found to be very less or negligible [55].
Drying is an essential pretreatment before the lab and a time-

intensive process of extracting tea polyphenols from tea leaves. In
this study, pulsed electric field technology was employed to dehy-
drate phenolic compounds instead of the conventional thermal
technique. Evaluating the impact of various PEF settings on the
total polyphenol production from fresh tea leaves and a solid-liquid
extraction, Zhibin Liu and their team made this observation.
By applying PEF treatment with an electric field intensity of
1.00 kV/cm, 100 pulses of 100 s each, and 5 s of pulse repetition—
which supplied 22 kJ/kg and raised the temperature by 1.5 °C—it
was possible to further explore the kinetics of green tea catechin
extraction. Results showed that compared to oven drying, PEF
pretreatment nearly quadrupled the extraction rate [56].
Another case study was done by Segovia et al. for the extraction
of polyphenols in Borago officinalis L. leaves. They observed that
PEF treatment of about 300 Hz frequency with 30 kV voltage has
enhanced the extraction yield of polyphenols from 1.3% to 6.6%.
They have also found that it also enhanced the oxygen radical
absorption capacity from 2.0% to 13.7%. In addition, they have
observed that PEF treatment took less time for the extraction and
also it increased the antioxidant property of the extract [5].
6.2.2 Extraction of There is a variety of seeds from different plants that have numerous
Bioactive Compounds from beneficial applications in the day-to-day life as well, as they have
Plant Seeds commercial and medicinal importance. The seeds include ground-
nuts, cotton, safflower, and sunflower. These are the most impor-
tant commercially valuable seeds as they are the reservoir of oil and
fat [57]. These fats and oils contain triacylglycerol, which plays a
vital role in enhancing the immune system of the body [58];
moreover, these oils are the main source of calories and vitamins.
Usually, the oil from these seeds is extracted by squeezing the seeds.
Once the oil is extracted, the press cake is then subjected to solvent
extraction. This is usually done by normal Soxhlet extraction meth-
ods by using a large quantity of hexane [59]. However, nowadays,
the PEF extraction method is incorporated at an industrial level to
increase the extraction yield without any negative impact on the
nutritional value of the product [5, 60].
Studies show that from the PEF extraction method oil yield of
55.9% from sunflower seeds [5], an yield of 0.105% with a 50%
increase in essential oil from damask rose flower seeds [5], 48.24%
with hexane (40 mL) solvent from sunflower seeds, [55] 22.66 kg/
100 kg from Arroniz variety of olive fruit seeds, [61] and 85.5% oil
yield from Nocellara del Belice variety of olive fruit seeds [62].
6.2.3 Extraction of Soxhlet extraction is the most popular approach for obtaining
Bioactive Compounds from bioactive compounds from plant sources. Other classical extraction
Herbs and Spices methods include maceration, etching, and cohobating. However,
the commercial production of essential oils has historically relied
heavily on steam distillation [63]. The conventional methods are

intricate multistage procedures that use more organic solvent, take
longer time, and use more energy while losing analytes. These
elements contribute to the limited selectivity of traditional extrac-
tion techniques. By increasing osmotic dehydration with less
energy input and improving nutrient recovery, the current PEF
approach, on the other hand, improved the extraction capacity of
bioactive compounds from metabolically active tissues. Further-
more, solvent diffusion and freeze-drying made PEF extraction
easier [64].
6.2.4 Extraction of Microorganisms, including bacteria, yeast, and algae, are excellent
Bioactive Compounds from sources of extremely valuable substances such as enzymes, pig-
Microorganisms ments, and nutrients. Since the majority of these chemicals are
found inside the cell, it is crucial to isolate and refine them before
using. Microorganisms are capable of a wide range of reactions and
may adapt to a broad spectrum of environmental factors. They can
be introduced into the laboratory from nature to build advanta-
geous molecules using inexpensive materials such as carbon and
nitrogen. Bioactive compounds produced by bacteria are extremely
advantageous for human nutrition and health due to their
biological activity. The screening of naturally occurring microbial
products for the creation of novel medicinal medicines has
advanced in research [65].
Several studies have shown that PEF-assisted extraction of
bioactive compounds from various microorganisms, such as
Arthrospira platensis, fresh abalone (Haliotis discus hannai Ino)
viscera, fresh microalgae Auxenochlorella protothecoides studied,
which has resulted in increased extraction of
151.94 ± 14.22 mg/g, 42.35%, 175.20 mg/100 mL, and 97%,
respectively [66–68].
6.2.5 Extraction of In the recent times, due to the increase in the population and the
Bioactive Compounds from discovery of various food products, there is also increasing in the
Food Wastes rate of food waste. Food waste may be agricultural, bakery, dairy,
and industrial; these food wastes are nowadays leading to different
types of pollution around the world. Hence, the scientists pro-
pound ways to reduce food waste and utilize them for beneficial
aspects. Most of the food wastes from agricultural and dairy foods
are a large reservoir of valuable bioactive compounds such as car-
bohydrates, phenolic acids, flavonoids, anthocyanin, terpenoids,
limonoids, lipids, catechins, tannins, vitamins, alkaloids enzymes
such as amylase, cellulase, pectinase, and invertase. Hence, it is
important to extract those valuable bioactive compounds without
simply discarding them. Conservation of energy and production of
energy are the need of the hour. As science and technology have
developed, scientists have invented many extraction methods,
Table 2
Bioactive compounds from fruit wastes
Name
of the fruit Waste type Bioactive compound extracted References
Citrus fruits Peel, seeds Carbohydrates, limonoids [69, 70]
Apple Pomace Carbohydrates, phenolic acids, flavonoids, anthocyanins, [70–72]
dihydrochalcones, triterpenoid
Mango Kernel seed Flavonoids, phenolic acids, tannins, xanthanoids, catechins, [70]
Peel hydrolysable
Carotenoids
Banana Peel Flavonols, catechins, catecholamines, phenolic acids [70]
Elderberry Branch waste Phenolic acids, flavonols, anthocyanins [70]
Table 3
Bioactive compounds from vegetable wastes [70]
Name of the vegetable Waste type Bioactive compound extracted

Carrot Discarded carrots α-carotene, β-carotene, lycopene lutein,
lutein γ-tocopherol
Potato Pulp and peel Carbohydrate
Phenolic acids
Glycoalkaloid
Carotenoids
Beetroot Pomace Phenolic acids
Aerial parts including stem and leaves Flavonoids
Betalains, phenolic compounds
Cauliflower Leaves and stem Phenolic acids
Flavonoids
Isothiocyanate
Proteins
Broccoli Industrial residues: stalks and floret Phenolic acids
Waste from agriculture activity: leaves Flavonoids
Glucosinolates
which have been already discussed. Pulsed electric field extraction is

one such effective method that is commonly used at the industrial
level for the extraction of bioactive compounds from food wastes; it
is mentioned in Tables 2 and 4.
Bioactives extracted from different types of food waste are
listed in Table 2.
Bioactive compounds extracted from vegetable wastes are listed
in Table 3
Table 4
Bioactive elements found in dairy, marine, and animal waste products [70]
Industrial product Waste type Bioactive compounds

Meat products Blood: plasma hemoglobin cuttings Bioactive peptides from protein hydrolysate
and trimmings Bioactive peptides from protein hydrolysate
Horns Collagen hydrolysate
Bones
Skin
Marine products Salmon nasal cartilage Astaxanthin
Heads, tails, and shrimp shells Polyunsaturated fatty acids Ω3
Salmon skin and trimmings Proteoglycans
Bioactive peptides from protein hydrolysate
Dairy products Whey Bioactive milk
Colostrum Galactooligosaccharides
Lactoferrin
Oligosaccharides
Bioactive elements found in dairy, marine, and animal waste

products are listed in Table 4 [70].
7 Utilization of the PEF Extraction Method in Food and Nutraceutical Industries
The demand for safe and high-quality food items has led to the
development of numerous unique food processing processes in
recent years. Consumers today have high standards for the items’
olfactory quality, usability, and nutritional worth. They also place a
high value on the utilization of environmentally sustainable food-
producing methods. The demand for products that resemble fresh-
ness and for food produced using environmentally friendly prac-
tices, as well as the growing customer interest in food with high
nutritional value, all stimulated the incorporation of pulsed electric
field (PEF) technology into food production [6, 73].
Pulsed electric fields (PEFs) have been utilized successfully and
safely on a range of different products in the food and bioproces-
sing industries. In Germany, Ukraine, and Moldova, the first stud-
ies on the use of pulsed electric fields (PEFs) were published in the
1950s, but it took decades for the technology to be used in indus-
trial settings. Since the 1990s, more than 20 research organizations
have been investigating the basic mechanisms of action, influencing
variables, and potential applications [74].
Applications of the pulsed electric field (PEF) can be used to
disintegrate biological tissues or microorganisms. There are many
uses for this technology, including improving mass transfer during
extraction or drying processes and mild food preservation. The
method has acquired its initial commercial uses. The reliability
and cost-effectiveness of the equipment have improved thanks to

the development of equipment based on semiconductor technol-
ogy. The technology is moving toward more widespread
industrial use.
8 Benefits in Food Industries
Due to its exceptional effectiveness in microbial inactivation and

quality preservation, the food processing and preservation indus-
tries have paid close attention to PEF technology. Gene transfec-
tion, drying, pasteurization, and juice extraction from fruits and
vegetables have all been accomplished with this technology. Pre-
dominantly, this method is employed in industries during freezing,
drying, food preservation by microbial inactivation, spore, and
enzyme inactivation, starch modification, etc., over the past few
years [14].
8.1 Inactivation of The inactivation of microorganisms is crucial for extending the

Microorganisms product shelf life in the field of food processing. To do this, a
cutting-edge technique called pulsed electric field is used. High-
voltage electric impulses are utilized in this method to make the
membranes of the bacteria permeable. Here, the microorganisms’
membrane serves as a capacitor that is filled with a dielectric liquid.
There is a transmembrane potential difference when a high voltage
is applied. When the crucial threshold value, or 1 V, is reached,
electroporation occurs, which results in the membrane of the
microorganisms rupturing and killing it. These days, this technol-
ogy is often used to destroy bacteria in liquid food products,
although this method is only suitable for materials with poor con-
ductivity and no air bubbles, such as milk, soya milk, fruit juice, and
wine [75].
9 French Fry Manufacturing Saves Water and Energy by Utilizing the PEF Method
PEF has created new benchmarks for the manufacturing of French

fries and chips. The method replaces previously used pre-heating
and reduces water and energy consumption by up to 90%. PEF is
applied cold, with treatment periods of less than 1 s, as opposed to
gradually warming the substance up to 60 °C, loss of turgor pres-
sure and tissue softening during hydro jet or slicing cutting result-
ing in a clean cut are the main advantages of treatment with an
intensity of less than 1 kJ/kg. Less product breakage and feathering
are caused by the smooth cut, which improves product quality and
yield and enables the use of various product configurations or
building materials. Additionally, a smoother product surface
means that up to 10% less oil will penetrate the product. More than
90 PEF systems are currently in operation by the global fries
industry [76].
9.1 Perfect Vegetable chips are a fad, but it can be difficult to make them
Vegetable Chips consistently well. However, the use of PEF results in significant
quality improvements along the entire production chain: less starch
is discharged into the processing water, slicer blade wear is reduced,
and so on. Users of PEF also get from 1% to 2% yield boost. Quality
benefits range from better crunch to improved color to more of the
original vegetable’s natural flavor. PEF makes it possible to use raw
materials and shapes that were not before. Accelerated moisture
release following PEF permits lower frying temperatures in less
time, preserving the color of natural food while reducing the devel-
opment of acrylamide for products such as sweet potato, carrot,
parsnip, or beetroot chips [77].
10 PEF Enhanced Extraction in Different Food Items
Extracellular materials are extracted during numerous food proces-

sing processes. PEF-induced cell membrane permeabilization can
be used to improve extraction yield in the extraction of fruit juices,
sugar, colors, pigments, and oils. PEF can be used to replace
enzymatic maceration or mechanical disintegration in the genera-
tion of juice. A lower extraction temperature can be used in the
sugar sector, and in the processing of olive oil, yield increases of up
to 10% and shorter malaxation durations have been noted.
A higher extraction yield of anthocyanins and polyphenols from
processed grapes allows for a significant reduction in maceration
times and an improvement in production capacity. Two distinct
equipment configurations can be used depending on the type of
raw material. Pipe systems with diameters ranging from 50 to
200 mm can process materials such as vegetable mashes, semisolid
products, or precut products. Beets, tubers, and entire fruits are
examples of solid food items that are usually processed on PEF belt
systems [78].
10.1 Premium One of the earliest methods for preventing microbial development
Quality Drying and preserving a food product’s quality is drying. The process is
typically constrained by moisture diffusion. PEF removes this
restriction, enabling a decrease in temperature and a reduction in
drying protocol time as it opens the cell membrane. For the major-
ity of plant-based products, a drying time reduction of about 20% is
achieved because water may migrate from the product, preventing
the formation of a core crust and product shrinkage. Less adverse
effects on product color, shape, flavor, and energy savings result
from lowering the drying temperature. Additionally, PEF
treatment can be used with any drying process, including tradi-

tional drying by air, drying by vacuum, and drying by osmotic or
freezing method [77].
10.2 Peel Removal: PEF also has an impact on the structural qualities of plant matter.
The Easy Way Fruits, kernels, and seeds can vary in their elastic characteristics and
peel attachment after treatment. The tomato skin may be readily
removed without any connected tomato flesh following treatment
at 2 kJ/kg. Increased flexibility improves kernel integrity and
reduces breakage during shelling for nuts and seeds such as
cashews [79].
10.3 Superior Quality PEF not only affects microbial cells but also plant-based cells.
Juices Microbial inactivation results from the loss of membrane barrier
function in yeasts, molds, and bacteria. To ensure the juice’s micro-
biological safety and to provide a particular shelf life, the PEF
treatment reduces the microbial load of the juice. Reduced treat-
ment temperature and duration are the main advantages over tra-
ditional thermal processing. Thus, heat-sensitive flavors, colors,
and nutrients are unaffected. PEF can be run continuously and is
simple to integrate into existing processing lines, in contrast to
other nonthermal processing choices. The majority of industrial
PEF lines in the juice business use this technology since it works
well with mild temperatures. With inlet temperatures ranging from
30 to 40 °C and maximum product temperatures well below 60 °C,
the majority of industrial PEF lines in the juice industry employ
preheating and cooling systems that are already in place or that have
been specially designed for them. Compared to a high-
temperature, brief treatment, the temperature reduction—typically
more than one z-value—leads to a premium appearance and
flavor [80].
10.4 Use of PEF in Treating animal cells such as meat has positive impacts as well. PEF
Meat Processing modifies the structure of meat to make it more softer and delicious,
whereas tumbling mechanically can speed up the process. The
production process is more efficient and meat quality improves as
a result of the about 50% faster brine uptake following PEF treat-
ment of the meat [74].
11 Application of PEF in Nutraceutical Industries
Foods or food additives with medicinal qualities are known as

nutraceuticals. Nutraceuticals are categorized into three types,
namely, nutrients, herbals, and dietary supplements. The dietary
components with well-established roles, such as vitamins, minerals,
amino acids, and fatty acids, are collectively known as nutrients,
extracts, and concentrations made from herbs or other botanical
products have come under herbals and dietary nutraceuticals are
the oral products that contain dietary ingredients that are meant to
enhance the flavor of the foods we eat. Mostly all these nutraceu-
ticals come from organic materials, mostly plants and microbes
[81]. “Nutraceuticals” is a term that is occasionally used inter-
changeably or in conjunction with the terms “functional foods,”
“bioactive chemicals,” “natural food additives,” and “dietary sup-
plements.” To give a variety of health benefits and to ward against
diseases, certain proteins, fatty acids, fiber, plant extracts, and sec-
ondary metabolites have been employed as nutraceuticals. The
global nutraceutical industry is constantly growing as a result of
the rising demand for functional foods and nutraceuticals, particu-
larly in industrialized nations. To develop nutraceuticals, businesses
are concentrating on mild and effective (in terms of productivity
and purity) technologies. Some of the important applications of
plant bioactive compounds in nutraceutical compounds are men-
tioned in Table 5 [14].
11.1 Extraction of The most significant class of nutraceutical chemicals is thought to

Polyphenols be dietary polyphenols. They are secondary metabolites generated
by plants, and they are important for oxidant, pathogen, and UV
radiation defense mechanisms. In humans, polyphenols display a
wide range of biological effects, including anti-inflammatory, anti-
cancer, and antiaging effects. Foods such as vegetables, fruits,
seeds, oils, tea, wine, and chocolate often include dietary polyphe-
nols. More than 8000 phenolic compounds in vascular plants have
been discovered so far.
Applying an electric field of 450 V/cm for 10 ms with a specific
energy of under 3 kJ/kg improved the yields of polyphenols from
apple mash [86]. PEF with 5 pulses per second at 30 to 60 kV
(at 35 C) doubled the number of polyphenols recovered from grape
skin [86]. It was discovered that certain polyphenols needed to be
isolated from grape pomace and peeled using PEF because they
could not be extracted using alternative methods [87]. To maxi-
mize the recovery of polyphenols from fresh tea leaves, several
parameters, including pulse duration (0.05 s), several pulses [88],
pause between pulses (PBP, 0.5–3 s), and pulse intensity
(0.4–0.9 kV/cm), were evaluated [86].
11.2 Extraction of According to some studies, PEF is used to extract various impor-
Nutraceuticals from tant chemicals from microalgae (Chlorella vulgaris, Chlamydomo-
Microalgae nas reinhardtii, and Dunaliella salina). In dietary supplements,
nutraceuticals, and food additives, lipids derived from microalgae
can be a valuable source of essential fatty acids. Some research
studies and patents have revealed the possible use of PEF for the
microalgae’s pretreatment to boost lipid production. After PEF
treatment, lipid droplets may remain inside cells. However, algal
lipids can be extracted with suitable solvents following the separa-
tion of other extractives [89].
Table 5
Plant bioactive compounds and their function and applications in nutraceutical industries [82–87]
Food source Extracted bioactive compound Function and benefits in nutraceuticals

Red wine, grapes Isoflavonoids Polyphenols Immunomodulator, antiosteoporotic, anticancerous, antioxidant
property
Wheat Derived immunopeptides, wheat gluten Enhance the activity of natural killer cells
Oats Dietary fiber Helps in the achievement of lipid lowering
Flaxseed, soybean and soy-based Phytoestrogens Antiosteoporotic, anticancerous, antiproliferative, antiestrogen
products, tea legumes, cabbage
Cauliflower, broccoli, sprouts, Brussels, Diallyl sulfides, isothiocyanates, Anticancer, immunomodulator, antimicrobial, detoxification
onions, garlic glucosinolates
Corn, carrots, papaya Carotenoids It acts as an antioxidant immunomodulator
Seeds, nuts, vegetable oil Phytosterols, tocotrienols, and tocopherols Lipid lowering, immunomodulator, antioxidant
Tender coconut Phytosterols, triglycerides Anti-inflammatory, antihelminthic, antioxidant, antinociceptive,
antitumor, antimicrobial, antifungal, analgesic, antiarthritic,
antibacterial, antipyretic, antidiarrheal, hypoglycemic,
antiseizure, hepatoprotective, cytotoxicity, nephroprotective,
and antiosteoporosis effects, vasodilation
Chlorella vulgaris Uncharacterized peptides Hemopoiesis, activation of humoral immune functions, activation
of monocyte and macrophage system
Garlic Ajoene, allicin Anticancer, antimicrobial, helps to lower cholesterol; antistatic and
antibiotic properties
Rice Phenolic compounds, alkaloids, essential It acts as anti-inflammatory, hypocholesterolemic, helps to prevent
oils, aromatic carbons cancer, nematicide, antihistaminic, antiarthritic, anticoronary,
antieczemic, and antiandrogenic Activities
Pulsed Electric Field Extraction
Turmeric Curcuminoids Antioxidant, anti-inflammatory, antimutagenic, anticarcinogenic,

antibacterial, antifertility, antidiabetic, anticoagulant, antifungal,
antiviral, antivenom, antifibrotic, antiulcer, antiprotozoal,
243
hypocholesteremic, hypotensive
(continued)
Table 5
244
(continued)
Food source Extracted bioactive compound Function and benefits in nutraceuticals

Fenugreek Apigenin, isovitexin vitexin Antioxidant, lipid-lowering activities, hypoglycemic
Cinnamon Polyphenols, cinnamaldehyde Antibacterial, anti-inflammatory, antifungal
Catechins
Black Pepper Piperidines, piperine It helps to improve digestibility
Utilized to treat vertigo, indigestion, congestion, asthma, diarrhea,
fever. It has antimicrobial activity
Subrahmanya Hegde et al.
Ginger Shogaols, gingerols Anti-inflammatory, anticancer, antioxidant, neuroprotective,

antimicrobial, respiratory protective, cardiovascular protective,
antidiabetic, antiobesity, antinausea, and antiemetic activities
Citrate fruits Vitamin C Antifungal, antibacterial, anticancer, anti-inflammatory,
Flavonoids cardioprotective, hepatoregenerating
Natural honey Coumaric acid, chrysin, luteolin, abscisic Regenerative, anti-inflammatory, antifungal, antibacterial
acid, apigenin, caffeic acid
Allium sativum Allicin Antioxidant, antibacterial, and arteriosclerosis
Green leafy vegetables and germinated Betaine (trimethyl glycine) Homocysteine
grains
Ananas Bromelain Prevent heart disease, arthritis, and inflammation
Capsicum annuum Capsaicin Antioxidant
Strawberries and raspberries Ellagic acid Anticancer
Curcuma longa Curcumin Alzheimer’s disease, anticancer
11.3 Extraction of There are several bioactive compounds from plants that can be used
Various Bioactive as nutraceuticals and can be extracted through the various available
Compounds from extraction techniques including pulsed electric field extraction. The
Plants Having chemical compounds such as allicin from Allium sativum, betaine
Nutraceutical Value (trimethyl glycine) from green leafy vegetables and germinated
grains, bromelain from Ananas sp., capsaicin or trans-8-methyl-
N-vanillyl-5 noneamide from Capsicum annum, ellagic acid from
strawberries and raspberries, curcumin from Curcuma longa,
omega 3 fatty acids from Linum spp., and resveratrol especially
high in grape skin were extracted and are giving a huge contribu-
tion to the nutraceutical industries [82].
12 PEF Aided Extraction: Pros and Cons
As PEF-assisted extraction method is a novel method of bioactive

extraction that uses high-voltage pulses to disintegrate the cell wall
and cell membrane to allow the intracellular compounds to come
out of the cell. During the process of PEF extraction, the electrical
pulses will generate the heat because of joule heating, therefore, the
cooling down the processes product during PEF treatment to
balance a low temperature. Hence, this process can be implicated
in the process of gentle preservation. High temperature and PEF
membrane electroporation together enhance the inactivation rate.
Products, namely, fruit juices, soups, or liquid eggs can all be
processed using PEF, as it involves the inactivation of enzymes
and microorganisms by breaking the cell membrane and making it
permeable to small molecules thus leading the microorganism’s
death by utilizing high-voltage pulses. Because no harmful chemi-
cal reactions have been found, the pulsed electric field technology is
widely regarded as being safe for humans [6]. Other important
advantages of the PEF extraction method include benefits such as
selective extraction, speedy extraction, and clean extraction. The
PEF’s operational parameters are easy to manage. It is possible to
change operating parameters (such as electric field intensity, pulse
duration and number, and the size of membrane pores) so that only
the desired components can be released while keeping other com-
ponents inside the cell. The recovery factor is greatly increased
under ideal circumstances with the shortest extraction time. In
addition, it does not involve a drying or dehydration process,
which lowers the operational cost. It does not affect the quality of
the food, which has been processed. Highly scalability, and with this
novel technique, one can reduce the amount of solvent [90].
Some limited drawbacks have been considered with the indus-
trial level of PEF extraction, which is depicted. Air bubbles in the
treatment chamber can affect the uniformity of the PEF treatment
and hinder the dielectric breakdown. Depending on the strength of

the applied electric field, the cell membranes during the electropo-
ration mechanism can be irreversible or reversible. The gap
between the electrodes and applied electric strength has a signifi-
cant impact on the PEF’s effectiveness [90]. Additional constraints
encompass cost, limited availability in commercial units, and
reduced effectiveness in treating solid foods, as it relies on the
electrical conductivity of the food. Hence, it is better suited for
liquid food treatment. Another significant drawback involves the
release of heavy metals and toxic particles from outdated electrodes,
which subsequently impacts the extraction and processing techni-
ques [6].
13 Application of Bioactive Compounds in Food and Nutraceutical Industries
Bioactive substances have become important food constituents for

maintaining health and preventing disease. Noncommunicable dis-
eases are on the rise as the population ages and become less physi-
cally active. In some circumstances, bioactive chemicals are thought
to be an intriguing alternative to traditional illness-preventive treat-
ment methods [83]. This is being supported more and more by
customers’ growing demand for natural products as they look for
long-term solutions to improve the quality of life through indivi-
dualized nutrition. The creation of innovative solutions for this
sector depends on an understanding of the chemistry of natural
products and their mechanistic approach. According to research,
poor lifestyle choices and stress raise the risk of several ailments,
including cerebrovascular diseases, cardiovascular diseases, infec-
tions, and diseases such as cancer. The expanding understanding
of how nutrition impacts health has resulted in a significant increase
in the demand for functional foods and nutraceuticals. For their
antibacterial qualities as well as humoral- and cell-mediated immu-
nological functions, specific bioactive components have been added
as supplements to functional foods, nutraceuticals, and pharmaceu-
ticals, where their biological activities can aid in disease control and
prevention. As a result, the therapeutic potential of many food
ingredients has grown in significance. Secondary plant metabolites
with biological properties such as antimicrobial activity, enzyme
detoxification regulation, antioxidant activity reduced platelet
aggregation, immune system modulation, anticancer property,
and hormone metabolism activity are successfully used
(Fig. 4) [83].
Plant bioactive compounds
Primary metabolite Secondary metabolite
Phenolics Terpenes Nitrogen

Proteins
compounds
Lipids
Carbohydrates Lignin Monoterpenes

Tannins Diterpenes
Nucleic acid phenolics Sesquiterpenes Cyanogenic
Miscellaneous Tetraterpenes Alkaloids
Flavanoids Polyterpenes Glucosinolates
Glycosidic
Amino acids
Flavones
Isoflavones
Flavonols
anthocyanins
Fig. 4 Classification of plant bioactive compounds
14 Bioactive Compounds Extracted from Animal Sources and Their Application in

Nutraceutical Industries
Animals are a rich source of bioactive substances with different

types of biological functions to maintain healthy human life. The
production of these bioactive chemicals might either be necessary
for an animal’s survival or increase its value to other living things.
To stop, lessen, or treat many diseases and their associated symp-
toms, numerous natural chemicals have been identified, and classi-
fied, from animal sources and are used as dietary and medical
supplements.
Some of the bioactive compounds from animal sources and
their application are listed in Table 6.
Table 6
Bioactive compounds from animal sources and their application [83, 88, 91, 92]
Food
source Extracted bioactive compounds Function and benefits in nutraceuticals
Meat Fatty acids, minerals, vitamins, peptides Antioxidant activities and antihypertensive
Fish Proteins, fatty acids, polyether, peptides, Antithrombotic, immunomodulatory,
enzymes and lectins, polysaccharides antimicrobial, anticancer, and antioxidant
activities
Egg Avidin, ovalbumin lysozyme, ovomucin, Antimicrobial, immunomodulatory,
ovotransferrin, phospholipids antihypertensive, and anticancer activities
Milk Whey protein Helps to modulate both innate and adaptive
immune responses
15 Conclusion
This chapter summarizes the revolutionary technologies that have

been developed in recent decades for the recovery of our interesting
chemical compounds from various sources, which process signifi-
cant value in different sectors. The pulsed electric field extraction
method for various bioactive and functional compounds represents
an innovative, eco-friendly, user-friendly, energy-efficient, time-sav-
ing approach with high yield potential. Its role is crucial in produc-
ing significant quantities of essential compounds with nutritional,
pharmaceutical, and medicinal value, benefitting the global popu-
lation for sustainable, healthy living. Since plants, animals, and
microorganisms process valuable bioactive compounds, it is neces-
sary to extract and purify them with high accuracy without wasting
them. Hence, new technologies should be invented, which should
be more efficient in terms of yield, time, and energy. One such
technology in the present era is pulsed electric field extraction. In
summary, this method finds efficient industrial application for
improved extraction of intracellular functional compounds from
living organisms. These compounds are subsequently purified and
used in the food processing and nutraceutical industries to enhance
both nutritional and pharmaceutical aspects, thereby benefiting
food quality and the development of nutrient and pharmaceutical
products.
References
1. Zeng X, Zhang Z (2019) Pulsed electric field for the recovery of oil and high-value com-
assisted extraction of bioactive compounds. In: pounds from vegetable oil industry: energy
Jia J, Liu D, Ma H (eds) Advances in food and economic cost implications. Food Res Int
processing technology [Internet]. Springer 80:19–26
Singapore, Singapore; 2019 [cited 2022 Oct 12. Barba FJ, Boussetta N, Vorobiev E (2015)
3], pp 125–35. Available from: http://link. Emerging technologies for the recovery of iso-
springer.com/10.1007/978-981-13-6451-8_ thiocyanates, protein and phenolic compounds
5 from rapeseed and rapeseed press-cake: effect
2. Thulasidas JS, Varadarajan GS, Sundararajan R of high voltage electrical discharges. Innov
(2019) Pulsed electric field for enhanced Food Sci Emerg Technol 31:67–72
extraction of intracellular bioactive compounds 13. Shorstkii I, Khudyakov D (2020) Influence of
from plant products: an overview. Nov Appro pulsed electrical discharge, hydrostatic pressure
Drug Des Dev 5:6 and temperature on rheological properties of
3. Ricci A, Parpinello G, Versari A (2018) Recent sunflower cake during oil pressing. Heliyon
advances and applications of pulsed electric 6(1):e03046
fields (PEF) to improve polyphenol extraction 14. Poojary MM, Lund MN, Barba FJ (2020)
and color release during red winemaking. Bev- Pulsed electric field (PEF) as an efficient tech-
erages 4(1):18 nology for food additives and nutraceuticals
4. Abbas Syed Q (2017) Pulsed electric field tech- development. In: Pulsed electric fields to
nology in food preservation: a review. J Nutr obtain healthier and sustainable food for
Health Food Eng [Internet]. 2017 June tomorrow [Internet]. Elsevier; 2020 [cited
19 [cited 2022 Oct 3];6(6). Available from: 2022 Nov 13], pp 65–99. Available from:
https://medcraveonline.com/JNHFE/ https://linkinghub.elsevier.com/retrieve/pii/
pulsed-electric-field-technology-in-food-pres B9780128164020000045
ervation-a-reviewnbsp.html 15. Teh SS, Niven BE, Bekhit AEDA, Carne A,
5. Ranjha MMAN, Kanwal R, Shafique B, Arshad Birch EJ (2015) Microwave and pulsed electric
RN, Irfan S, Kieliszek M et al (2021) A critical field assisted extractions of polyphenols from
review on pulsed electric field: a novel technol- defatted canola seed cake. Int J Food Sci Tech-
ogy for the extraction of phytoconstituents. nol 50(5):1109–1115
Molecules 26(16):4893 16. Moreira SA, Alexandre EMC, Pintado M, Sar-
6. Nowosad K, Sujka M, Pankiewicz U, Kowalski aiva JA (2019) Effect of emergent non-thermal
R (2021) The application of PEF technology in extraction technologies on bioactive individual
food processing and human nutrition. J Food compounds profile from different plant materi-
Sci Technol 58(2):397–411 als. Food Res Int 115:177–190
7. Xi J, Li Z, Fan Y (2021) Recent advances in 17. Yongguang Y, Yuzhu H, Yong H (2006)
continuous extraction of bioactive ingredients Pulsed electric field extraction of polysaccha-
from food-processing wastes by pulsed electric ride from Rana temporaria chensinensis
fields. Crit Rev Food Sci Nutr 61(10): David. Int J Pharm 312(1–2):33–36
1738–1750 18. Arshad RN, Abdul-Malek Z, Roobab U, Qur-
8. Barbosa-Pereira L, Guglielmetti A, Zeppa G eshi MI, Khan N, Ahmad MH et al (2021)
(2018) Pulsed electric field assisted extraction Effective valorization of food wastes and
of bioactive compounds from cocoa bean shell by-products through pulsed electric field: a sys-
and coffee silverskin. Food Bioprocess Technol tematic review. J Food Process Eng [Internet].
11(4):818–835 2021 Mar [cited 2022 Nov 11];44(3).
9. Selvamuthukumaran M, Shi J (2017) Recent Available from: https://onlinelibrary.wiley.
advances in extraction of antioxidants from com/doi/10.1111/jfpe.13629
plant by-products processing industries. Food 19. Zia S, Khan MR, Shabbir MA, Aslam Maan A,
Qual Saf 1(1):61–81 Khan MKI, Nadeem M et al (2022) An inclu-
10. Redondo D, Venturini ME, Luengo E, Raso J, sive overview of advanced thermal and non-
Arias E (2018) Pulsed electric fields as a green thermal extraction techniques for bioactive
technology for the extraction of bioactive com- compounds in food and food-related matrices.
pounds from thinned peach by-products. Food Rev Int 38(6):1166–1196
Innov Food Sci Emerg Technol 45:335–343 20. Sánchez-Vega R, Elez-Martı́nez P, Martı́n-Bel-
11. Puértolas E, Koubaa M, Barba FJ (2016) An loso O (2015) Influence of high-intensity
overview of the impact of electrotechnologies pulsed electric field processing parameters on
antioxidant compounds of broccoli juice. antioxidant activity of orange juice in compari-

Innov Food Sci Emerg Technol 29:70–77 son with traditional thermal processing. J Agric
21. Kumar A, Ahmad F, Zaidi S (2019) Impor- Food Chem 53(11):4403–4409
tance of bioactive compounds present in plant 33. Luengo E, Álvarez I, Raso J (2013) Improving
products and their extraction – a review. Agric the pressing extraction of polyphenols of
Rev [Internet]. 2019 Dec 30 [cited 2022 Nov orange peel by pulsed electric fields. Innov
1 1 ] ; 4 0 ( 0 4 ) . A v a i l a b l e f r o m : h t t p : // Food Sci Emerg Technol 17:79–84
arccjournals.com/journal/agricultural- 34. (2013) ICheaP11- Special ISSUE 11th Inter-
reviews/R-1926 national Conference on Chemical and Process
22. Zhao Y, Zheng Y, He H, Sun Z, Li A (2021) Engineering: 2–5 June 2013, Milan, Italy.
Effective aluminum extraction using pressure Associazione italiana di ingegneria chimica,
leaching of bauxite reaction residue from coag- Florence
ulant industry and leaching kinetics study. J 35. Shree JT, Sree GV, Sundararajan R (2018)
Environ Chem Eng 9(2):104770 Enhancement of bioactive compounds from
23. Pintać D, Majkić T, Torović L, Orčić D, green grapes extract using pulsed electric field
Beara I, Simin N et al (2018) Solvent selection treatment. J Cancer Prev Curr Res [Internet].
for efficient extraction of bioactive compounds 2018 Sep 14 [cited 2022 Nov 11];9(5).
from grape pomace. Ind Crop Prod 111:379– Available from: https://medcraveonline.com/
390 JCPCR/a-systematic-outline-for-the-osseous-
24. Amiali M et al (2010) Physicochemical prop- industries-in-the-north-pontic-palaeometallic-
erty changes and safety issues of foods during age.html
pulsed electric field processing. In: Physico- 36. Delsart C, Ghidossi R, Poupot C, Cholet C,
chemical aspects of food engineering and pro- Grimi N, Vorobiev E et al (2012) Enhanced
cessing. CRC Press, Boca Raton, pp 117–218 extraction of phenolic compounds from merlot
25. Azmir J, Zaidul ISM, Rahman MM, Sharif KM, grapes by pulsed electric field treatment. Am J
Mohamed A, Sahena F et al (2013) Techniques Enol Vitic 63(2):205–211
for extraction of bioactive compounds from 37. Puértolas E, Cregenzán O, Luengo E,
plant materials: a review. J Food Eng 117(4): Álvarez I, Raso J (2013) Pulsed-electric-field-
426–436 assisted extraction of anthocyanins from
26. Nuccitelli R (2019) Application of pulsed elec- purple-fleshed potato. Food Chem 136(3–4):
tric fields to cancer therapy. Bioelectricity 1(1): 1330–1336
30–34 38. Gachovska T, Cassada D, Subbiah J, Hanna M,
27. Remali J, Aizat WM (2021) A review on plant Thippareddi H, Snow D (2010) Enhanced
bioactive compounds and their modes of action anthocyanin extraction from red cabbage
against coronavirus infection. Front Pharmacol using pulsed electric field processing. J Food
11:589044 Sci 75(6):E323–E329
28. Kotnik T, Kramar P, Pucihar G, Miklavcic D, 39. Bobinaitė R, Pataro G, Lamanauskas N,
Tarek M (2012) Cell membrane electropora- Šatkauskas S, Viškelis P, Ferrari G (2015)
tion- part 1: the phenomenon. IEEE Electr Application of pulsed electric field in the pro-
Insul Mag 28(5):14–23 duction of juice and extraction of bioactive
29. Sensoy I, Sastry SK (2004) Extraction using compounds from blueberry fruits and their
moderate electric fields. J Food Sci 69(1): by-products. J Food Sci Technol 52(9):
FEP7–FE13 5898–5905
30. Qin B-L, Zhang Q, Barbosa-Canovas GV, 40. Roohinejad S, Everett DW, Oey I (2014)
Swanson BG, Pedrow PD (1994) Inactivation Effect of pulsed electric field processing on
of microorganisms by pulsed electric fields of carotenoid extractability of carrot purée. Int J
different voltage waveforms. IEEE Trans Die- Food Sci Technol 49(9):2120–2127
lectr Electr Insul 1(6):1047–1057 41. Luengo E, Ãlvarez I, Raso J (2014) Improving
31. El Darra N, Grimi N, Vorobiev E, Louka N, carotenoid extraction from tomato waste by
Maroun R (2013) Extraction of polyphenols pulsed electric fields. Front Nutr [Internet].
from red grape pomace assisted by pulsed 2014 Aug 12 [cited 2022 Nov 11];1.
ohmic heating. Food Bioprocess Technol Available from: http://journal.frontiersin.
6(5):1281–1289 org/article/10.3389/fnut.2014.00012/
abstract
32. Sánchez-Moreno C, Plaza L, Elez-Martı́nez P,
De Ancos B, Martı́n-Belloso O, Cano MP 42. Torregrosa F, Cortés C, Esteve MJ, Frı́gola A
(2005) Impact of high pressure and pulsed (2005) Effect of high-intensity pulsed electric
electric fields on bioactive compounds and fields processing and conventional heat
treatment on orange-carrot juice carotenoids. juices after pulsed electric fields. Food Chem
J Agric Food Chem 53(24):9519–9525 196:833–841
43. Madia VN, De Vita D, Ialongo D, Tudino V, 54. Liu ZW, Zeng XA, Ngadi M (2018) Enhanced
De Leo A, Scipione L et al (2021) Recent extraction of phenolic compounds from onion
advances in recovery of lycopene from tomato by pulsed electric field (PEF). J Food Process
waste: a potent antioxidant with endless bene- Preserv 42(9):e13755
fits. Molecules 26(15):4495 55. Shorstkii I, Mirshekarloo MS, Koshevoi E
44. Oms-Oliu G, Odriozola-Serrano I, Soliva- (2017) Application of pulsed electric field for
Fortuny R, Martı́n-Belloso O (2009) Effects oil extraction from sunflower seeds: electrical
of high-intensity pulsed electric field proces- parameter effects on oil yield: PEF-assisted oil
sing conditions on lycopene, vitamin C and extraction from sunflower seeds. J Food Pro-
antioxidant capacity of watermelon juice. cess Eng 40(1):e12281
Food Chem 115(4):1312–1319 56. Liu Z, Esveld E, Vincken JP, Bruins ME
45. Luengo E, Martı́nez JM, Bordetas A, Álvarez I, (2019) Pulsed electric field as an alternative
Raso J (2015) Influence of the treatment pre-treatment for drying to enhance polyphe-
medium temperature on lutein extraction nol extraction from fresh tea leaves. Food Bio-
assisted by pulsed electric fields from Chlorella process Technol 12(1):183–192
vulgaris. Innov Food Sci Emerg Technol 29: 57. Mushtaq A, Roobab U, Denoya GI, Inam-Ur-
15–22 Raheem M, Gullón B, Lorenzo JM et al (2020)
46. Bai Y, Li C, Zhao J, Zheng P, Li Y, Pan Y et al Advances in green processing of seed oils using
(2013) A high yield method of extracting alka- ultrasound-assisted extraction: a review. J Food
loid from Aconitum coreanum by pulsed elec- Process Preserv [Internet]. 2020 Oct [cited
tric field. Chromatographia 76(11–12): 2022 Nov 12];44(10). Available from:
635–642 h t t p s : // o n l i n e l i b r a r y. w i l e y. c o m /
47. Hossain MB, Aguiló-Aguayo I, Lyng JG, doi/10.1111/jfpp.14740
Brunton NP, Rai DK (2015) Effect of pulsed 58. Yusuf AK (2016) A review of methods used for
electric field and pulsed light pre-treatment on seed oil extraction. Int J Sci Res. 7(12):6
the extraction of steroidal alkaloids from potato 59. Chemat F, Rombaut N, Sicaire AG,
peels. Innov Food Sci Emerg Technol 29:9–14 Meullemiestre A, Fabiano-Tixier AS, Abert-
48. Zhao W, Yu Z, Liu J, Yu Y, Yin Y, Lin S et al Vian M (2017) Ultrasound assisted extraction
(2011) Optimized extraction of polysacchar- of food and natural products. Mechanisms,
ides from corn silk by pulsed electric field and techniques, combinations, protocols and appli-
response surface quadratic design. J Sci Food cations. A review. Ultrason Sonochem 34:540–
Agric:n/a–n/a 560
49. Zbinden MDA, Sturm BSM, Nord RD, Carey 60. Sitzmann W, Vorobiev E, Lebovka N (2016)
WJ, Moore D, Shinogle H et al (2013) Pulsed Applications of electricity and specifically
electric field (PEF) as an intensification pre- pulsed electric fields in food processing: histor-
treatment for greener solvent lipid extraction ical backgrounds. Innov Food Sci Emerg Tech-
from microalgae. Biotechnol Bioeng 110(6): nol 37:302–311
1605–1615 61. Puértolas E, Martı́nez de Marañón I (2015)
50. Sharmin T, Ahmed N, Hossain A, Hosain M, Olive oil pilot-production assisted by pulsed
Mondal SC, Haque R et al (2016) Extraction electric field: impact on extraction yield, chem-
of bioactive compound from some fruits and ical parameters and sensory properties. Food
vegetables (pomegranate peel, carrot and Chem 167:497–502
tomato). Am J Food Nutr 4:12 62. Tamborrino A, Urbani S, Servili M,
51. Altemimi A, Lakhssassi N, Baharlouei A, Romaniello R, Perone C, Leone A (2019)
Watson D, Lightfoot D (2017) Phytochem- Pulsed electric fields for the treatment of olive
icals: extraction, isolation, and identification pastes in the oil extraction process. Appl Sci
of bioactive compounds from plant extracts. 10(1):114
Plan Theory 6(4):42 63. Calleja-Gómez M, Castagnini JM, Carbó E,
52. Tiwari BK (2015) Ultrasound: a clean, green Ferrer E, Berrada H, Barba FJ (2022) Evalua-
extraction technology. TrAC Trends Anal tion of pulsed electric field-assisted extraction
Chem 71:100–109 on the microstructure and recovery of nutri-
53. Leong SY, Burritt DJ, Oey I (2016) Evaluation ents and bioactive compounds from mushroom
of the anthocyanin release and health- (Agaricus bisporus). Separations 9(10):302
promoting properties of Pinot Noir grape
64. Giacometti J, Bursać Kovačević D, Putnik P, Impact of pulsed electric field (PEF) pretreat-
Gabrić D, Bilušić T, Krešić G et al (2018) ment on process performance of industrial
Extraction of bioactive compounds and essen- French fries production. J Food Eng 235:16–
tial oils from mediterranean herbs by conven- 22
tional and green innovative techniques: a 77. Ostermeier R, Giersemehl P, Siemer C, Töpfl S,
review. Food Res Int 113:245–262 J€ager H (2018) Influence of pulsed electric
65. Shukla R (2015) Studies on bioactive com- field (PEF) pre-treatment on the convective
pounds from different microorganisms. Int J drying kinetics of onions. J Food Eng 237:
Sci Eng Res 6(6):9 110–117
66. Martı́nez JM, Luengo E, Saldaña G, Álvarez I, 78. Abenoza M, Benito M, Saldaña G, Álvarez I,
Raso J (2017) C-phycocyanin extraction Raso J, Sánchez-Gimeno AC (2013) Effects of
assisted by pulsed electric field from Artro- pulsed electric field on yield extraction and
sphira platensis. Food Res Int 99:1042–1047 quality of olive oil. Food Bioprocess Technol
67. Li M, Lin J, Chen J, Fang T (2016) Pulsed 6(6):1367–1373
electric field-assisted enzymatic extraction of 79. Puértolas E, Saldaña G, Raso J (2017) Pulsed
protein from abalone (Haliotis Discus Hannai electric field treatment for fruit and vegetable
Ino) viscera: PEF-assisted extraction of abalone processing. In: Miklavčič D (ed) Handbook of
viscera protein. J Food Process Eng 39(6): electroporation [Internet]. Springer Interna-
702–710 tional Publishing, Cham; 2017 [cited 2022
68. Silve A, Kian CB, Papachristou I, Kubisch C, Nov 13], pp 2495–2515. Available from:
Nazarova N, Wüstner R et al (2018) Incuba- http://link.springer.com/10.1007/978-3-31
tion time after pulsed electric field treatment of 9-32886-7_181
microalgae enhances the efficiency of extrac- 80. Heinz V, Toepfl S, Knorr D (2003) Impact of
tion processes and enables the reduction of temperature on lethality and energy efficiency
specific treatment energy. Bioresour Technol of apple juice pasteurization by pulsed electric
269:179–187 fields treatment. Innov Food Sci Emerg Tech-
69. Anderson JW, Baird P, Davis RH Jr, Ferreri S, nol 4(2):167–175
Knudtson M, Koraym A et al (2009) Health 81. Granato D, Nunes DS, Barba FJ (2017) An
benefits of dietary fiber. Nutr Rev 67(4): integrated strategy between food chemistry,
188–205 biology, nutrition, pharmacology, and statistics
70. Ben-Othman S, Jõudu I, Bhat R (2020) Bioac- in the development of functional foods: a pro-
tives from agri-food wastes: present insights posal. Trends Food Sci Technol 62:13–22
and future challenges. Molecules 25(3):510 82. TY – BOOK AU – Patel, Minal AU – Desai,
71. Wang Z, Xu B, Luo H, Meng K, Wang Y, Liu Bimal AU – Jha, Suman AU – Tandel, M B
M et al (2020) Production pectin oligosacchar- PY – 2018/04/17 SP – T1 – Nutraceuticals
ides using Humicola insolens Y1-derived from plant sources: a review ER -
unusual pectate lyase. J Biosci Bioeng 129(1): 83. Mondal S, Soumya NPP, Mini S, Sivan SK
16–22 (2021) Bioactive compounds in functional
72. Lavelli V, Corti S (2011) Phloridzin and other food and their role as therapeutics. Bioact
phytochemicals in apple pomace: stability eval- Compd Health Dis 4(3):24
uation upon dehydration and storage of dried 84. Mie˛kus N, Marszałek K, Podlacha M, Iqbal A,
product. Food Chem 129(4):1578–1583 Puchalski C, Świergiel AH (2020) Health ben-
73. Soliva-Fortuny R, Balasa A, Knorr D, Martı́n- efits of plant-derived sulfur compounds, gluco-
Belloso O (2009) Effects of pulsed electric sinolates, and organosulfur compounds.
fields on bioactive compounds in foods: a Molecules 25(17):3804
review. Trends Food Sci Technol 20(11–12): 85. Anurag P, Rajamohan T (2003) Cardioprotec-
544–556 tive effect of tender coconut water in experi-
74. Kantono K, Hamid N, Oey I, Wang S, Xu Y, mental myocardial infarction. Plant Foods
Ma Q et al (2019) Physicochemical and sen- Hum Nutr 58(3):1–12
sory properties of beef muscles after Pulsed 86. Intahphuak S, Khonsung P, Panthong A
Electric Field processing. Food Res Int 121: (2010) Anti-inflammatory, analgesic, and anti-
1–11 pyretic activities of virgin coconut oil. Pharm
75. https://www.newfoodmagazine.com/arti Biol 48(2):151–157
cle/109295/the-benefits-of-pulse-electric- 87. Jose SP, Asha S, Im K, Ratheesh M, Santhosh S,
fields/ Sandya S et al (2017) Nephro-protective effect
76. Fauster T, Schlossnikl D, Rath F, of a novel formulation of unopened coconut
Ostermeier R, Teufel F, Toepfl S et al (2018) inflorescence sap powder on gentamicin
induced renal damage by modulating oxidative electric field (PEF) technology as the cell dis-
stress and inflammatory markers. Biomed Phar- ruption method to extract lipid from microal-
macother 85:128–135 gae for biodiesel production. Int J Renew Eneg
88. Gill HS, Doull F, Rutherfurd KJ, Cross ML Res 25:599–621
(2000) Immunoregulatory peptides in bovine 91. Achour A, Lachgar A, Astgen A, Chams V,
milk. Br J Nutr 84(S1):111–117 Bizzini B, Tapiero H et al (1997) Potentializa-
89. Barba FJ, Grimi N, Vorobiev E (2015) New tion of IL-2 effects on immune cells by oyster
approaches for the use of non-conventional cell extract (JCOE) in normal and HIV-infected
disruption technologies to extract potential individuals. Biomed Pharmacother 51(10):
food additives and nutraceuticals from micro- 427–429
algae. Food Eng Rev 7(1):45–62 92. Kim SK, Wijesekara I (2010) Development and
90. Joannes C, Sipaut CS, Dayou J, Yasir S, Mansa biological activities of marine-derived bioactive
RF (2015) The potential of using pulsed peptides: a review. J Funct Foods 2(1):1–9
Chapter 11
Case Studies and Application of Different Novel Extraction

Methods
Muskaan Sharma, Sakshi Vaishkiyar, and Sunidhi Kumari
Abstract
This chapter focuses on the case studies of the bioactive compounds that is defining the promising pathway
of compounds that can be used as functional units. This chapters allows to gain an insight of the steps taken
to make the optimization of the parameters and synthesis pathway. This chapter also focuses on the selection
of the material, sorting, and process of extraction. The highest percentage of bioactive compounds required
to achieve this extraction process and the point of consideration in implementation are also taken into
considereation A case study on polyphenols, alkaloids, and terpenoids will be discussed. This chapter
focuses on the novel extraction method by which an efficient amount of bioactive compounds are obtained.
The main focus is to explore the extraction of bioactive compounds from inexpensive resources or residues
that give cost-efficient products. Various novel methods of bioactive compound extraction and their
application in different domains will be discussed. This chapter provides a wholesome view of the steps in
the bioactive compounds by case studies and will carry forward to the novel extraction methods.
Key words Bioactive compounds, Polyphenols, Novel extraction method, Application, Case study,
Alkaloids, Terpenes
1 Introduction
Bioactive compounds are defined as the nutrients and non-nutrient

components that are present in the food matrix whether it is of
vegetable or animal that is leading to physiological effects [1]. The
compounds are secondary metabolites, mostly hydrophobic and
poorly soluble compounds. The properties of nutrients are seen in
terms beyond their classical nature [2]. These compounds are
emerging as the key components in the food-related and
medicine-related components that are leading to outstanding con-
tributions to health status and in the prevention of diseases and also
promote green technology. In recent times, it is seen that the
population is getting aged and involvement in physical activity is
little to moderate, which is increasing the potential chances of
noncommunicable diseases. The trend is leading to the increased
255
256 Muskaan Sharma et al.
demand for natural products that are boosting consumer needs.

Reduction in cost, efficient time, and amount of solvent used is an
effective method over traditional method [3, 4]. Along with these
variable options of bioactive compounds are present, presenting the
sustainable option for improving the better quality of life. The main
focus of the development of bioactive compounds is to increase
personalized nutrition. With the application of the knowledge of
chemistry in natural products, a fruitful outcome is derived that is
termed as the bioactive compounds. These compounds are consid-
ered as the extra nutritional constituents that are there in a very
small portion. Some examples of bioactive compounds are carote-
noids, flavonoids, carnitine, choline, and so on. The occurrence of
bioactive compounds is found in a variety of foods and plant
materials that can be extracted by microwave-assisted extraction
[5]. Most of the bioactive compounds are having antioxidant,
anticarcinogenic, anti-inflammatory, and antimicrobial properties.
Following are some of the crucial compounds listed that will
improve the quality of life and allows the prevention of noncom-
municable diseases.
• Phenolic compound: It is comprised of a sub-category of flavo-
noids that are present in almost all plants. Cereals, legumes,
nuts, and olives are a few materials that are having an abundance
of flavonoids. The category of compounds is having antioxidant
activities that impose a favorable impact on cardiovascular risk
factors [6].
• Phytoestrogens: It is found in flaxseed, whole grains, fruits, and
vegetables. The compounds are displaying the dual nature that
are the antioxidant properties and estrogen at the molecular
levels. Plant-based compounds that are having estrogen-like
properties and isoflavones, stilbene, coumestan, and lignan
[5, 7].
• Carotenoids: It is one of the efficient free radical scavengers
present that is showing potent antioxidant compounds. The
majority is found in fruits and vegetables such as apricot, carrot,
mangoes, and pumpkins.
• Glucosinolates are the natural form present in many pungent
plants such as mustard, cabbage, and horseradish. The role is
seen in the induction of phase 1 and 2 enzymes and inhibition of
enzyme activation, and currently ongoing research is going to
investigate the role in the mitigation of cancer.
• Vitamins are the influential nutrient present that any organism
requires in a limited amount. The compounds are not synthe-
sized by the human body but must be obtained by diet. It offers
a diverse role in various metabolic activities and health functions
[8]. Activities such as regulation and catalysis are offered by
vitamins, and they also act as an antioxidant (Malva sylvestris
leaves).
Case Studies and Application of Different Novel Extraction Methods 257
Area of application: Bioactive compounds are used for many

purposes as there are numerous functional and structural properties
that are displayed. One of the potential areas is Neutraceutical as
vegetable-based proteins [9]. It is a combination of nutrition and
pharmaceutics that comprise the properties of medicinal and other
(in terms of physiological benefits). The use of Neutraceuticals
leads to the improvement in health factors, delays the process of
aging, prevents chronic disease, increases the expectancy of life, and
supports the structure and function of the body [7]. Great advan-
tages are seen in complex curative disorder that relates to oxidative
stress. It includes the allergy, Alzheimer’s cardiovascular condi-
tions, diabetes, cancer, and obesity. The food industry is the second
largest existing domain of bioactive compounds where food bioac-
tive compounds are referred to as the extranational constituents
that are occurring in a very small percentage of food [10].
It encopasses different functional categories that act as flavonoids
need to be mention. Morphine, quinine, and nicotine are a few
examples of bioactive compounds.
Functional bioactive compounds are classified into the follow-
ing categories that are produced by plants and grouped among
polyphenols(one of abundant class), triterpenes, phytosterols, and
polysaccharides, lupine, oleanane, and ursane, which seem to pos-
sess anticancer agents. Terpenoids, also called isoprenoids, are con-
sidered the second largest group of secondary metabolites. They are
particularly abundant in living cells and organisms [11]. It is one of
the responsible components present for the fragrance of plants and
fruits. Beta-carotene or carotenoids are grouped as the constituents
that are depicting an important part of the human diet [12]. They
will be serving as the direct precursor of vitamins A and E. The
function played by carotenoids is in terms of providing resistance
against diseases and enhancing the immune system [13]. Sulforaph-
ane, a compound that is an important agent found in broccoli, leads
to the anticancerous effects [14]. Apart from this, a polysaccharide
that is a polymeric chain of the monosaccharide lies and is linked
with the glycosidic bonds. Taking an adequate amount of func-
tional bioactive food is very favorable and important for the growth
of cells [15]. At the same time, consideration needs to be taken that
the excess is not there. Otherwise, it will lead to mutation and
toxicity. Relation of a bioactive compound with the human system
and health: An imbalance in the production of the reactive oxygen
species in the system leads to the creation of oxidative stress. To
reduce the impact, the reactive oxygen species aim to make the
promotion of the antioxidant and co-factors interaction that allows
the maintenance of health and will prevent aging and age-related
disorders. Antioxidants are obtained by the bioactive compounds
resulting in the deactivation of the free radicals and serve as the
redox biomarker that controls the redox state of the functional
proteins. With the evidence, it is found that bioactive compounds
hold a great place in human welfare. This chapter focuses on “Case

studies and application of different novel extraction methods.” The
gain of insight into the case studies will help in enhancing the
overview that what leads to the discovery of new bioactive com-
pounds [16]. The section is followed by a description of novel
extraction processes that define the effectiveness and efficiency of
various processes and what research going on that makes increases
the output of the techniques [17]. Understanding the extraction
process allows the transformation on an industrial scale.
Case studies will be discussing the process of extraction of the
various classes of bioactive compounds, their parameters, obtained
products, and optimization of the process that is resulting in the
high yield. The case studies section will provide information about
polyphenols, terpenoids, carotenoids, and so on. One of the classi-
cal case studies is of polyphenols; it is one of the most abundant
classes of bioactive compounds that exist. It is a plant-based food
category lies that is possessing the ability of antioxidant, anti-
inflammatory, antimicrobial, and cardioprotective [18]. Polyphe-
nols are found in several components that are there in the normal
diet and will contribute to the metabolization, transport, and dis-
tribution to the target organs [11]. The case study will be giving
evidence that what are the sample preparation techniques is there
that increase pre-prepare the sample. Extraction techniques are
comprised of the recovery of analytes with the intake of the classical
and advanced emerging techniques that is supporting the high yield
of the polyphenol bioactive compounds. After this comes the
cleanup which will be making the elimination of the atoms and
entities that are interfering with the functioning of the bioactive
compounds. The last step is evaporation which is reducing the
extract volume. Similarly, the case study of catenoid, terpenoids,
and polysaccharides is going to be discussed that highlights the
similarity and difference in the process of recovery, gives facts about
the unique properties of the bioactive compounds, and what will
components used in the extraction process that is evolving in the
yield percentage of 80–90%. Furthermore, a novel extraction
method will be discussed and its application in various areas. The
case study will be forming a basis that how the extraction process
happens while the next section allows the understanding that what
are the factor lies that make contributes to the optimization of the
extraction process that makes it suitable for the pilot scale or
industrial purpose. Novel extraction techniques are designed in
such a manner that extraction of bioactive compounds will take
place with less solvent consumption and extraction time. It
improves the extraction quality and extraction yield. Extraction is
the foremost step that lies in the differentiation of the desired
natural products by the selected raw material. Out of all extraction
processes, solvent extraction is the most widely used method
[19]. It comprises the stages that are as follows penetration of
solvent into a solid matrix, dissolution of the solute into the sol-
vent, diffusion out of the process from the solid matrix, and extrac-
tion of the solute [20]. In this process, the factor that comes into
the role is the diffusivity and solubility of the material that is
facilitating the extraction of the bioactive compounds. The law of
similarity and inter miscibility is the basic principle of extraction
protocol. The laws will be used to scale up the production of
bioactive in food industries and pharmaceuticals [21]. Extraction
is an advantageous process that is adding value to industrial growth.
The ongoing trend of bioactive compounds due to emerging con-
sumer needs will lead to the motivation to perform the new experi-
ment and optimize the process of extraction via nanocarrier [17].
2 Case Studies on the Nutraceuticals and Bioactive Compounds
2.1 Case Study 1: It has been evidenced that bioactive compounds comprise many
Fruit Seed-Based applications in the culinary industries as well as pharmaceutical
Bioactive Compounds industries. The long interest is in the functional feature of the
for the Formulation of diverse fruit seeds such as the tomato, guava, dates, as well as
Nutraceuticals apple. Some of the bioactive compounds in this include bioactive
peptides, lycopene polysaccharides, phytochemicals, and vitamins
[22]. These are abundant in fruit and its by-products and have
many health benefits. The role of bioactive compounds that are
obtained from the tomato and the seeds is that they are processed
into cans, products, and sauces. The seeds and the peels comprise
the bioactive compound named beta-carotene as well as lycopene.
They have a diverse phytochemical composition and the seeds of
tomatoes are known to be a natural source of antioxidants and
phenolic compounds and carotenoids. Another one is peel and
pulp which has lycopene, flavonoids, and phenolic acids. It can be
done by the methods of crushing, heating, and stirring. The con-
tent of moisture was 8.5%, fat was 20%, ash was 3.1%, and dietary
fiber was 35.1% in the tomato. When in the case study the phyto-
chemical analysis was done, the presence of the 14 flavonoids was
there such as quercetin, kaempferol, and isorhamnetin. They have
the power to alter many numerous cellular signal transduction
pathways as well as stimulation of endogenous defensive activities
are there [23, 24]. They have anticancer, antibacterial, antimuta-
genic, and stimulating endogenous defensive activities. They are
also known to have cardioprotective and antiplatelet properties. In
this case, there were many approaches used, such as the valorization
of unused parts in tomato, and there were methods for extraction
of the carotenoid compounds. The seed extracts of tomatoes have
antibacterial properties, and pharmaceutical and food sectors can
use them for the preservation of food and microbial deterioration is
there. The waste from tomatoes can be significantly used by indus-
tries to develop various by-products or functional foods, as well as
the processing manufacture of tomatoes can benefit from it [25]. It

was discovered that the peels are high in lycopene, fiber, and
phenols, and the seeds are high in crude protein and fat. Investiga-
tion were carried out for their roles and applications as food sources
and bioactive phytochemical constituents are the nutritional, phar-
macological, therapeutic uses, functional properties, and bioactive
contents of the seeds of various fruits. Allaqaband et al. [1] show
that alkaloids, carotenoids, flavonoids, glycosides, saponins, terpe-
noids, tannins, steroids, and polyphenolic compounds, which have
anti-inflammatory, antioxidant, anticancer, antidiabetic, antihyper-
lipidemic, anti-obesity, neurological disorders, cardiovascular, skin
diseases, and chronic diseases properties, are essential bioactive
components found in seeds.
2.2 Case Study 2: Pretreatment of seed will lead to the production of the phytosterol
Pomegranate Seed Oil composition. In addition, it holds the ability of antioxidant. For the
extraction of the bioactive compounds, seed oil is extracted with
the mechanical procedure. It will not lead to the effective results,
apart from this other extraction procedure will help. The extraction
procedure is known as the important step and it puts a great impact
on analysis as well as characterization. There are majorly four steps
that are used in food analysis such as sample preparation, data
acquisition, data analysis, and statistical integration. The sample
preparation and/or treatment, the extraction process, the extract
cleanup, and in most cases, extract concentration by evaporation
are all done in the first phase [26]. Depending on the purpose of
the study and the targeted analytes, the second stage involves the
collecting of data utilizing high-resolution analytical techniques,
such as gas chromatography-mass spectrometry (GC-MS), liquid
chromatography/mass spectrometry (LC-MS), and nuclear mag-
netic resonance (NMR). The equipment software processes the
data acquisition in the third stage. Depending on the goal of the
study, the final phase may include multivariate statistical analysis,
which might include, among other things, analysis of variance
(ANOVA), principle components analysis (PCA), partial linear
square analysis mixed with discriminant analysis, and cross-
validation. As per the case study, there are four strategies for the
treatment and extraction of the bioactive compounds from the food
matrices. It includes the treatment, in which the different methods
are milling, filtration, drying, hydrolysis, centrifugation, as well as
adjustment of pH. In the extraction part, analytes will be recovered
from the sample, and the classical advanced emerging techniques
are used. In the cleanup process, the elimination of the compounds
is done with the analysis. The different methods that can be used
include liquid-liquid extraction, dispersive solid-phase extraction,
as well as solid phase extraction. There are then advanced extraction
techniques that comprise microextraction techniques and these
comprise a wide range of applications. The different features are
simplicity, versatility, as well as high extraction efficiency and envi-

ronmental friendliness [27]. It is used for the determination of
polyphenols. Some of the relevant improvements have been done
and it was done by the QuEChERS method, which comprises the
food matrices. Ultrasound-assisted extraction involves the use of
US radiation and it has devices such as sonoreactors, probes, and
water baths. Microwave-assisted extraction is such that, it is based
on the solvent heating that occurs due to the interaction of polar
molecules in the media. It includes dipole rotation and ionic con-
duction. Electrotechnologies are nonthermal methods of pasteuri-
zation and sterilization in the food industry. They use the current
that flows across samples to electroporate membranes and harm
living tissues. The electrical field induces electropermeabilization
and the release of the target analytes by causing charge accumula-
tion and transmembrane potential. Water is used as a solvent in
subcritical water extraction (SbFE), a pressurized liquid extraction
method, which speeds up the mass transfer and extraction rate from
the solid matrix [28]. High- and ultra-performance liquid chroma-
tography is one of the most widely used techniques for the separa-
tion, identification, and quantification of analytical processes. It is
difficult to analyze and characterize the bioactive compounds found
in nature due to the wide variety in their number and chemical
makeup. Câmara et al. [11] advanced and developed extraction
procedures that are faster, more sensitive, and reproducible,
employed to address several extractions and analysis/characteriza-
tion-related shortcomings. The direct analysis functionality of some
ambient approaches has proven to be very helpful for their direct
analysis, identification, and characterization. The next-green
extraction methods present a great extractive performance regard-
ing polyphenols.
2.3 Case Study 3: A The bioactive compounds are known to be found in fruit biowaste
Case Study of as well as they provide low-cost, integrated, and environment-
Raspberry Fruit friendly alternatives. The fruit named raspberry is known to be
Pomace—A Bioactive rich in antioxidant compounds which inculcate fiber, anthocyanin,
Compound and ellagitannins. The content of water is 80% and carbohydrates
are predominantly found. The chemical properties are dependent
on the edaphoclimatic conditions. The majority of antioxidants in
raspberry fruit pomace are squandered during juice manufacturing
(RFP). According to reports, RFP has 77.5% of the total nutritional
fiber found in fresh fruit [29]. The RFP also still contains a signifi-
cant amount of phenols. Tocopherols and -linoleic acid are both
abundant in the seeds. A sizable amount of biological potential is
also lost when RFP is wasted, including its antioxidant, antiproli-
ferative, and antihyperglycemic properties. Due to its capacity to
“spare” heat-sensitive chemicals, cheap cost, wide availability, and
efficiency, traditional maceration is the most widely used extraction
method for the exploration of berry fruits and their biopotential.

Investigating the total phenolic, flavonoid, and anthocyanin con-
tent is the basis for the case study. The case study is based on
research into the antioxidant activity and total phenolic, flavonoid,
and anthocyanin content of Rubus idaeus L. pomace obtained in the
Mediterranean region, specifically Montenegro. The effectiveness
of conventional maceration and ultrasound-assisted extraction as
extraction procedures were compared [30]. Moreover, a high-
performance liquid chromatography analysis was used to assess
the polyphenolic profile of the obtained extracts. The plant material
and the sample preparation include the raspberry which was har-
vested. The usage of fruits was in the extraction of juice by the
destoning machine. The two different extraction methods that
were used here were ultrasound-assisted extraction as well as con-
ventional maceration. Initially, the homogenization of pomace was
comprised of formic acid and methanol.
The methods that were used in the evaluation of the pomace
extracts were the colorimetric Folin-Ciocalteu method. The units
in which the results were expressed include Gallic acid per volume.
The flavonoid content was determined by the aluminum chloride
colorimetric method. The pH differential method is used for deter-
mining the anthocyanin content. The original procedure for the
ferric reducing antioxidant power test (FRAP) was followed. Using
an Agilent Technologies 1100 liquid chromatograph fitted with a
diode-array detector, the chemical characterization of the analyzed
extract and the quantification of the chosen components were
carried out both before and after hydrolyses. Even specimens of
the same plant species having different phenolic contents and envi-
ronmental factors such as climate, soil type, proximity to the shore
can affect the outcomes. Some plant species tend to create more
phenols than the same species growing in conditions that are dif-
ferent from those seen in colder climes, higher elevations, and more
dry environments [31]. Lysis was carried out of several significant
polyphenolic compounds, including gallic, p-coumaric, caffeic,
chlorogenic, ellagic acid, and quercetin. According to the results,
the raspberry pomace’s output of total phenolics, flavonoids, and
anthocyanins—particularly caffeic, chlorogenic, ellagic, and gallic
acids—was increased by ultrasound-assisted extraction (UAE). The
production of quercetin and p-coumaric acid is maximized during
traditional maceration. Raspberry fruit pomace has a great indus-
trial potential for biowaste valorization and may be helpful in the
creation of dietary supplements that are high in antioxidants [32].
2.4 Case Study 4: The in vivo bioavailability of phenolic compounds is increased

Bioactive Potential of when they are delivered via a carrier vehicle, and the quantity of
Phenolic Compounds: biotransformations that restrict the expression of bioactivity is
A Case Study decreased. The bioavailability of these substances is increased
when probiotic yeast biomass, such as Saccharomyces boulardii, is

used for assimilation [20]. In these situations, the biological action
may be accomplished by directly affecting the microbial pattern in
the colon. The biotransformation into an intermediary molecule
occurs during this process. Following the evaluation of the bioac-
tivity and chemical characterization of the natural substrate repre-
sented by medicinal herbs, new sources of biologically active
chemicals have been discovered [6]. The vegetal material, the prod-
uct conditioning mode, the meteorological circumstances, and the
makeup of the soil all influence the number of useful chemicals
(such as polyphenolcarboxylic acids) that are present. The utiliza-
tion of six dried samples of A. linearis leaves, P. cupana seeds,
A. chilensis berries, I. paraguariensis leaves, S. aromatic cloves,
and wild berries as alternatives for treating diseases brought on by
oxidative stress was examined and chosen. The studies were carried
out using a high-pressure liquid chromatograph called ELITE-
LaChrom and analytical scales for DAD (Diode-Array Detection)
detectors. By measuring the DPPH (2,2-diphenyl-1-picrylhydra-
zyl) scavenging activity and the chelating activity, the antioxidant
capacity of each hydroalcoholic extract was evaluated. The distribu-
tion of bioactive components was associated with some nutraceu-
ticals’ capacity to mitigate the physiological effects of oxidative
stress (like phenolic acids and flavonoids) [33]. The discovery of
the crucial point of contact prompted the development of novel
methods for the exploitation of bioactive substances, which may
ultimately increase the target compound’s bioaccessibility and bio-
availability. Additional research should be done to verify this aspect.
This in vivo indicator demonstrated the product’s potential.
Understanding the stability of the functional components and the
significance of the pattern and amount of these chemicals in vivo
came about as a result of the ability to digest yeast cells. Thus, one
viewpoint made clear by this study was the distribution of the full
range of nutraceuticals in the evaluated product—rather than
increasing the concentration of a compound. Bioassimilation and
bioavailability of phenolic compounds characterize the in vivo com-
plex effect that phenolic compounds exert on contact with eukary-
otic cells in addition to the increase in oxidative stress stability
[34, 35]. Because there was a species-specific link, a correlation
between bioactive substances and various activities in vitro/in vivo
was not found in the case of high levels. Dabulici et al. [17] provide
information about the capacity of probiotic biomasses to release
useful components will be determined by a precise understanding
of bioassimilation and bioavailability processes, which will also
improve the formulation of nutraceuticals.
3 Different Novel Extraction Methods
There is a versatile range of structures and functionalities of natural

bioactive compounds present. It will serve as the excellent pool of
the atoms and molecules that will be supporting the production of
nutraceuticals, functional food, and additive. In nature, some of the
compounds are found in abundance like polyphenols while the rest
are found in very low levels. To make the extraction of compounds,
the use of the chemical synthesis processes is leading to unprofitable
outcomes. As in setting up the chemical reaction, the process
comprises one or more than one reaction that is aiming to make
the conversion of the reactant from the starting material to multiple
products [36]. One of the setbacks that are found in chemical
synthesis is the use of material and maintaining conditions such as
pressure, temperature, and ions. Therefore, the outcomes are pre-
senting an insufficient option at the pilot scale. Taking the focus on
the massive harvesting of bioactive compounds in the desired
amount will occur by developing technologies. It overcomes the
challenges of the screening and production of the compounds.
Advanced technologies have been developed that will be commonly
used for the extraction of bioactive compounds [37]. A variety of
innovative and novel technologies is there that integrate the use of
natural resources and methods that is economically advantageous
to chemical synthesis. Conventional liquid-liquid or solid-liquid
extraction and the advanced include pressurized-liquid extraction,
subcritical and supercritical extractions, and microwave- and
ultrasound-assisted extractions are commonly used technologies
present [36]. In addition, enzyme-and instant controlled pressure
drop-assisted extractions are currently been used. The advantages
that the technology offers are in terms of the release of the com-
pounds from the matrix. In the future, the innovative approach will
be leading to an increase in the production of the specific condition
that is further used as a nutraceutical and as a bioactive compound
(like supercritical carbon dioxide extraction) [32]. This section will
be discussing the novel extraction method that is followed by a
comparative analysis of the Soxhlet extraction.
3.1 Solvent In this extraction technique (HPLC), different organic solvents are
Extraction Technique introduced to effectively size the raw material that will take up the
desired components such as anthocyanins. These components are
preferring the properties of anticancerous agents and anti-
inflammatory [38]. For this, samples will undergo centrifugation
and then be filtered for the removal of the solid residues [39]. The
extract that is obtained is used as an additive, supplemental food,
and as an ingredient in functional food. Low processing cost and
ease of operation will be serving as the advantage over other
techniques. Agro-waste (fruit peel, food, and grains) is found to be

the potential source for the extraction of nutraceutical and bioac-
tive compounds.
The procedure of using solvent extraction is as follows: selec-
tion of the plant material. Taking fresh fruit without any physical or
microbial damage is figured out. Extraction protocol in which the
peel of the fruit will be boiled will be in a ratio of 1:20 (water).
Further, it is homogenized and filtered out under a vacuum with
the use of the Whatman paper [38, 40]. The sample will be lyophi-
lized and kept at a freezing temperature. The next step is determin-
ing the total phenolic component, gallic acid is used as the standard
one that is mixed with the Folin-Ciocalteu reagent (1:1), 7.5%
(w/v) sodium carbonate before the UV-vis spectrometry
(760 nm) that will be repeated for five times. After this, FRAP
(Ferric Reducing Antioxidant Power Assay) is done that is leading
to the formation of the calibration curve. Sample extracts and
standards were analyzed with the DPPH Free Radical Scavenging
Capacity [41]. Further, analysis of the compounds will take place
with the HPLC technique. It will determine the detection in differ-
ent wavelengths that is 280, 320, and 370 nm. Once, the HPLC is
performed; statistical analysis will be done that is allowing the
presentation of the results in the form of standard deviation and
variance [42]. The correlational data will be calculated with Pear-
son’s coefficient.
3.2 Supercritical It is one of the environmental-friendly technologies present that is

Fluid Extraction used for the extraction of bioactive compounds. The reason that
lies behind the high efficiency is the use of natural matter which are
plants and algae. One of the qualities offered by the technique is
final product extraction, which is very easy due to solubilizing
lipophilic substances [42, 43]. In the process of extraction, the
material is placed in a container that is comprised of the pressure
and temperature control that is needed for maintaining certain
conditions. The protocol is comprised of the dissolution of the
fluid that is transported in the separator. The product further gets
collected in the tap that is there in the separator. The main focus of
the technique is a selection of the supercritical fluid that is allowing
a wide range of compounds to get extracted [44]. For the extrac-
tion of the compounds, the plant material has been used in the
ground state and will be placed in the vessels, CO2 gas is used that
undergoes high temperature and pressure [45]. After that, a pump
forces the supercritical phase CO2 to get into the extraction vessel
where it meets the plant and breaks the trichomes and allows the
plant material to get break down. Over the period, carbon dioxide
based techniques have gained a significant place in extraction that
whether it is the use of nontoxic compounds or giving a cost-
effective process the techniques also gained a significant value that
contributes in the growth of various industries [13]. Supercritical
carbon dioxide is serving as the novel extraction method as it will be

strengthening the antioxidant extraction process by making the rise
in superior abilities of the cells.
3.3 Subcritical Water This technique called the subcritical water extraction technique is
Extraction an alternative technology that is used for the extraction of the
phenolic compound from different food. Subcritical water also
refers to the temperature of the water which is between 100 and
374 °C, the pressure is also high and it is maintained in a liquid
state. The major advantage of the SCW over any other conventional
extraction technique is that it requires a short extraction time, the
solvent cost is low, the quality of extraction is high, and the major
advantage is that it is environment friendly. Subcritical water extrac-
tion and microwave-assisted technology is the most effective engi-
neering approach and also it offers some environment-friendly
techniques so several other plant components can be extracted
[46]. An author Tunchaiyaphum has extracted some phenolic com-
pound from the mango peels with the help of SCW technology.
The amount of phenolic compound, which is present in the mango
peel, was much higher than using the Soxhlet extraction technique.
Hence, SCW extraction is also known as an alternative green tech-
nology for phenolic compound extraction from agricultural waste.
It also substitutes the conventional method using the organic sol-
vent. There are mainly eight extracted phenolic compounds and
these are gallic acid, caffeic acid, chlorogenic acid, syringic acid,
protocatechuic acid, benzoic acid, p-hydroxyl benzoic acid, flavo-
noid, and coumaric acid [47].
3.4 Enzyme-Assisted There are a number of methods of enzymes that can be used for the
Extraction extraction of bioactive compounds from food waste. However, the
main source of extraction of the antioxidants is the plant tissue
[48]. The plant cells contain polysaccharides and these are hemicel-
lulose, cellulose, and pectins, all these act as barriers so the intracel-
lular substance can be released. There are several enzymes such as
cellulase, xylanase, beta-glucosidase, and beta-glucose held to
degrade the cell wall structure and then depolymerize the plant
cell wall polysaccharide [49]. As water is used as a solvent, in place
of chemicals, it is an eco-friendly approach that is used for the
extraction of some bioactive compounds along with oil. Several
bioactive compounds are released from the plant cell following
the cell disruption and extraction method. It also optimizes enzyme
preparation and it is done either alone or in the form of a mixture.
The enzyme-assisted extraction is a highly promising technique
when compared to conventional solvent-based extraction. It is
mainly the ability of the enzyme to catalyze the reaction, and mild
processing conditions should be used in an aqueous solution. It
leads to the production of high efficiency molecules that is cost-
efficient and organic rich [50].
3.5 Extraction with The extraction done with ultrasound is a relatively simple and
the Help of Ultrasound effective technique when it is compared to the traditional extraction
method. All the bioactive compounds are obtained from natural
products. The ultrasound method induces a much greater method
in which diffusion of the solvent in the cellular material is done.
Adopting this technique improves the mass transfer and along with
it disrupts the cell wall and facilitates the release of bioactive com-
pound. The amount of extraction is highly influenced by the ultra-
sound frequency, which mainly depends on the plant material
which needs to be extracted. Wang et al. [51] have demonstrated
the use of ultrasound-assisted extraction. In this method, the
extraction of three dibenzyl butyrolactone lignans, which are
trichloride, hemislienoside, and action, is done from Hemistepta
lyrate [52]. To determine the corresponding extract, high-
performance liquid chromatography is been done. In another
study by Fahmi et al. [53], the extraction efficiency of the four
isoflavone derivatives is studied; these are glycerin, daidzin, genis-
tin, and malonyl genistin from the soybean. To carry out this
extraction method mix-stirring method is used. As this technique
also used ultrasound, it has been known to improve the extraction
yield and it depends on the solvent. Azka et al. [54] have extracted
anthocyanins and other phenolic compounds from the grape peel.
This is also done using ultrasound technology as it takes care of
even the smallest details. Pineiro et al. [55] have optimized and
validated ultrasound-assisted extraction while extracting stilbenes
from grape canes.
Using this method, the stilbenes in the grape cane will be
extracted in 10 min. The temperature required will be 75 °C with
an ethanol concentration of 60% in the extraction solvent. From the
study, it was concluded that the grape cane by-product is a potential
source of the bioactive compound and it plays a vital role in the
food and pharmaceutical industry [56]. Aguilo Aguayo et al. [57]
also studied the effect of ultrasound technology while extracting
water from a soluble polysaccharide. These are obtained from the
milled and dried product which is generated from Agaricus bis-
porus. It has been observed that beta-Glucan has been obtained in a
specific amounts such as 1.01 and 0.98 g/100 g dry mass in the
particle size of about 355–250 and 150–125 μm, respectively; these
are the by-products of mushroom. The highest extraction was done
at 4.7% and this was achieved when there is an extraction time of
15 min, with an amplitude of 100 um and 1 h of precipitation in
about 80% ethanol [58].
3.6 The Microwave- This is a new extraction method that mainly combines microwave
Assisted and traditional solvent extraction. It is a highly advantageous tech-
Extraction (MAE) nique due to the short extraction time, high extraction rate, and
low cost of the traditional method. However, the major advantage
of ultrasonic-assisted extraction and solvent extraction is that the
plant metabolite requires a shorter interval of time. Padmapriya

et al. [59] stated that the extracted mangiferin presented in the
Curcuma amada uses ethanol as a solvent. The mangiferin which is
extracted has increased to 500 W; however, it has decreased when
the microwave power was increased. An adequate mangiferin yield
of about 41 μg/ml is obtained with an extraction time of 15 s with a
microwave power of 500 W [60]. Kerem et al. [61] extracted
saponins from the chickpeas by using MAE and found that this
method is far better and superior to the Soxhlet extraction. How-
ever, to complete this process adequate time and energy must be
required and utilized. The chickpea saponin has exhibited that
prominent inhibitory activity should be used against Penicillium
digitatum along with additional filamentous fungi. The Mangifera
indica leaves have also been extracted from the mangiferin by
Kulkarni and Rathod [62]. They used microwave-assisted extrac-
tion conditions and used water as a solvent. The maximum-assisted
extraction condition which is used has adopted water as a solvent.
However, the maximum extraction yield which is obtained is
55 mg/g and it is obtained with an extraction time of about
5 min [63]. The solid-to-solvent ratio which is used is 1:20 and
the microwave power required is 272. When sequential batch
extraction and Soxhlet extraction are compared, it has been noticed
that MAE has increased the yield of extraction in a much shorter
period. It has also reduced the solvent requirement time to a much
shorter level when compared to the conventional method. Smiderle
et al. [64] have also studied MAE along with pressurized liquid
extraction (PLE) as an advanced technique to obtain polysacchar-
ides mainly biologically active beta-glucan. This component is
obtained from the Pleurotus ostreatus and Ganoderma lucidum;
these are the fruiting bodies and in this beta and alpha glucan
have been detected in all the extracts. In another study by Kolář
et al. [65], MAE is optimized in response to the surface methodol-
ogy to enhance the extraction of polyphenols from the basil. It has
been then found that the extraction can be done with 50% ethanol,
at a microwave power of 442 W, and with an extraction time of only
15 min. Under this condition, basil liquid extraction is obtained
and it contains 4.299 g GAE/100 g polyphenol and 0.849 g
catechin which is equivalent to about 100 g of DDW of total
flavonoid [66].
Hence, from the above discussion, it can be concluded that the
microwave-assisted method offers several advantages when com-
pared to another extraction method. This is a highly efficient
method because it takes less extraction time, is less labored, and is
highly sensitive; hence, it makes it a favorable method of extraction
in the bioactive compound.
3.7 Pulsed-Electric This method is a highly novel extraction technology that is used for
Field-Assisted the extraction of bioactive compounds. This is a preferred method
Extraction (PEFAE) because less energy is required in it and also it is an environment-
friendly solvent. This method is a nonthermal extraction process, as
all the natural compounds can only be recovered at a minimum
temperature so quality and nutrition value can be obtained. Elec-
troporation is the primary mechanism that has been followed
behind the pulse electric field extraction method. In this process
of PEFAE, the electric energy is utilized to create nano- or micro-
poration of the cell membrane so all the bioactive compounds
which are present in the cell plasma can be extracted easily
[16]. An electric pulse is necessary for the transfer of the molecules
or the ions from inside the cell to the cell membrane as it acts like an
insulator [67].
In another study, it was observed that the apple peels were
treated by the pulsed electric field using several electric intensities
and time to extract the phenolic compound. The extraction is
analyzed using several electric conductivity and confocal laser
extraction. Results were obtained depending on the cell integration
index along with electric field intensity. This study has also con-
firmed that higher the intensity, the cell integration will be con-
stant, and the soluble matter recovery will be higher. Parniakov
et al. [68] has stated that the yield of the antioxidants, proteins, and
carbohydrates obtained from the mango peel was maximum when
PEFAE is at the intensity of 13.3 kV/cm. This process is carried out
with extraction at a 50 °C temperature for about 5 h at about
6 pH [69].
3.8 CE (Combination All the extraction technologies which are discussed above are men-
of Extraction) Process tioned and used in combination. When all the methods are used,
this reduces the extraction time, increases extraction yield, and
overcomes any limitation if present. The pre-treatment of any
fruit by-product by using the ultrasound or enzyme before any
CE process. Likewise, two or more emerging extraction processes
are used in combination such as UAE and SFE.
Techniques Solvents Advantage Disadvantage Application

Soxhlet Ethanol (highest) Low processing cost The disadvantage is Anticancerous
extraction Hexane (lowest) and easy seen in terms of agents and anti-
operations using toxic solvents inflammatory and
and extended time functional foods
to carry out the
process. In
addition, it also
requires the
evaporation and
concentration step
for recovery
(continued)
Techniques Solvents Advantage Disadvantage Application

Subcritical Acetonitrile, Short extraction When high water flow Extraction of
water methanol, time, the solvent is applied, it bioactive
extraction ethanol, liquid cost is low, and the increases extract compounds from
ammonia, and quality of volume and lowers Orostachys
dichloromethane extraction is high the concentration japonicus known
of the final extract as rock pine
Enzyme- Methanol or Eco-friendly Enzymes relatively Use specific
assisted ethanol and approach expensive, cannot enzymes to
extraction polyphenol break plant cells destroy the cell
completely, not wall of the source
feasible material and
hence improve
extraction yield
Extraction Acetone and Improves mass A low quantity of oil Effective in cell wall
with the dichloromethane transfer is produced disruption and
help of mass transfer
ultrasound
Microwave- Ethanol Short extraction High maintenance The active
assisted time, high cost in commercial- component of
extraction extraction rate, and scale settings medicinal plants
(MAE low cost of the uses microwave
traditional method energy
Pulsed- Ethanol-water High yield utilizes High maintenance Increase extraction
electric mixture and fewer solvents and yield
field- propylene glycol energy, and it saves
assisted a lot of time
extraction compared to
(PEFAE) traditional
extraction
methods
3.9 Interpretation Bioactive compounds are the type of chemical composition occur-
ring in plants and animals in small amounts. A required percentage
is needed for the efficient functioning of an individual. Bioactive
compounds are abundant in nature and considered superfoods.
The demand and growing interest of consumers lead to the inclina-
tion of the scientific community toward the discovery of versatile
compounds. To extract a single molecule from a plant source, a
series of steps is going to take place. This is a tedious process that
requires the sources and costs that make the availability of the
compounds to get restricted. At the same time, it is important to
overcome the restriction as the survival of an individual is depen-
dent on the consumption of bioactive compounds. Bioactive com-
pounds are comprising ample amounts of molecules that are
significantly contributing to the production of nutraceuticals. It
will act as the functional moiety in the variable industries whether
it is the food industry or pharmaceutical industry. Nutraceuticals
hold variable properties that are seen in terms of physiological

activities and will be helping in the prevention of cancer therapies.
The journey of bioactive compounds to the nutraceutical requires
many steps that allow the conversion into a functional form.
References
1. Allaqaband S, Dar AH, Patel U, Kumar N, 9. Bernardi S, Lupatini-Menegotto AL, Kalschne
Nayik GA, Khan SA et al (2022) Utilization DL, Moraes Flores ÉL, Bittencourt P, Colla E,
of fruit seed-based bioactive compounds for Canan C (2021) Ultrasound: a suitable tech-
formulating the nutraceuticals and nology to improve the extraction and techno-
functional food: a review. Front Nutr functional properties of vegetable food pro-
9 . h t t p s : // w w w. f r o n t i e r s i n . o r g / a r t i teins. Plant Foods Hum Nutr (Dordrecht,
cles/10.3389/fnut.2022.902554/full Netherlands) 76(1):1–11. https://doi.org/
2. Ahmadian-Kouchaksaraie Z, Niazmand R 10.1007/s11130-021-00884-w
(2017) Supercritical carbon dioxide extraction 10. Dilshad R, Bahrami A, Assadpour E,
of antioxidants from Crocus sativus petals of Williams L, Jafari SM (2021) Nano/microen-
saffron industry residues: optimization using capsulated natural antimicrobials to control the
response surface methodology. J Supercrit spoilage microorganisms and pathogens in dif-
Fluids 121:19–31. https://doi.org/10.1016/ ferent food products. Food Control 128:
j.supflu.2016.11.008 1 0 8 1 8 0 . h t t p s :// d o i . o r g / 1 0 . 1 0 1 6 / j .
3. Akhtar I, Javad S, Yousaf Z, Iqbal S, Jabeen K foodcont.2021.108180
(2019) Review: microwave assisted extraction 11. Câmara JS, Albuquerque BR, Aguiar J,
of phytochemicals an efficient and modern Corrêa R, Gonçalves JL, Granato D, Pereira J,
approach for botanicals and pharmaceuticals. Barros L, Ferreira I (2020) Food bioactive
Pak J Pharm Sci 32(1):223–230 compounds and emerging techniques for their
4. Cheng Y, Xue F, Yu S, Du S, Yang Y (2021) extraction: polyphenols as a case study. Foods
Subcritical water extraction of natural pro- (Basel, Switzerland) 10(1):37. https://doi.
ducts. Molecules (Basel, Switzerland) 26(13): org/10.3390/foods10010037
4 0 0 4 . h t t p s : // d o i . o r g / 1 0 . 3 3 9 0 / 12. Goleroudbary MG, Ghoreishi SM (2016)
molecules26134004 Response surface optimization of Safranal and
5. Bagade SB, Patil M (2021) Recent advances in Crocin extraction from Crocus sativus L. via
microwave assisted extraction of bioactive com- supercritical fluid technology. J Supercrit Fluids
pounds from complex herbal samples: a review. 108:136–144. https://doi.org/10.1016/j.
Crit Rev Anal Chem 51(2):138–149. https:// supflu.2015.10.024
doi.org/10.1080/10408347.2019.1686966 13. Quiroz JQ, Torres AC, Ramirez LM, Garcia
6. Chhouk K, Quitain AT, Pag-asa DG, Marid- MS, Gomez GC, Rojas J (2019) Optimization
able JB, Sasaki M, Shimoyama Y, Goto M of the microwave-assisted extraction process of
(2016) Supercritical carbon dioxide-mediated bioactive compounds from Annatto seeds
hydrothermal extraction of bioactive com- (Bixa orellana L.). Antioxidants (Basel, Swit-
pounds from Garcinia Mangostana pericarp. J zerland) 8(2):37. https://doi.org/10.3390/
Supercrit Fluids 110:167–175. https://doi. antiox8020037
org/10.1016/j.supflu.2015.11.016 14. Desmawati D, Sulastri D (2019) Phytoestro-
7. Barkat AK, Naveed A, Akhtar R, Haroon K, gens and their health effect. Maced J Med Sci
Ghulam M, Atif A et al (2012) Human skin, 7(3):495–499. https://doi.org/10.3889/
aging and antioxidants. J Med Plants Res 6(1): oamjms.2019.044
1–6. https://doi.org/10.5897/JMPR11. 15. Chugh B, Kamal-Eldin A (2020) Bioactive
1427 compounds produced by probiotics in food
8. Bimakr M, Ganjloo A, Zarringhalami S, Ansar- products. Curr Opin Food Sci 32:76–82.
ian E (2017) Ultrasound-assisted extraction of https://doi.org/10.1016/j.cofs.2020.02.003
bioactive compounds from Malva sylvestris 16. Fan R, Wang L, Fan J, Sun W, Dong H (2022)
leaves and its comparison with agitated bed The pulsed electric field assisted-extraction
extraction technique. Food Sci Biotechnol enhanced the yield and the physicochemical
26(6):1481–1490. https://doi.org/10.1007/ properties of soluble dietary fiber from orange
s10068-017-0229-5
peel. Front Nutr 9:925642. https://doi.org/ 26. Maqsood S, Adiamo O, Ahmad M, Mudgil P
10.3389/fnut.2022.925642 (2020) Bioactive compounds from date fruit
17. Dabulici CM, Sârbu I, Vamanu E (2020) The and seed as potential nutraceutical and func-
bioactive potential of functional products and tional food ingredients. Food Chem 308:
bioavailability of phenolic compounds. Foods 1 2 5 5 2 2 . h t t p s :// d o i . o r g / 1 0 . 1 0 1 6 / j .
(Basel, Switzerland) 9(7):953. https://doi. foodchem.2019.125522
org/10.3390/foods9070953 27. Chaouch MA, Benvenuti S (2020) The role of
18. Joana Gil-Chávez G, Villa JA, Fernando Ayala- fruit by-products as bioactive compounds for
Zavala J, Basilio Heredia J, Sepulveda D, Yahia intestinal health. Foods 9(11):1716. https://
EM, González-Aguilar GA (2013) Technolo- doi.org/10.3390/foods9111716
gies for extraction and production of bioactive 28. Dantas TL, Alonso Buriti FC, Florentino ER
compounds to be used as nutraceuticals and (2021) Okra (Abelmoschus esculentus L.) as a
food ingredients: an overview. Compr Rev potential functional food source of mucilage
Food Sci Food Saf 12(1):5–23. https://doi. and bioactive compounds with technological
org/10.1111/1541-4337.12005 applications and health benefits. Plan Theory
19. Carpentieri S, Režek Jambrak A, Ferrari G, 10(8):1683. https://doi.org/10.3390/
Pataro G (2022) Pulsed electric field-assisted plants10081683
extraction of aroma and bioactive compounds 29. Kaseke T, Opara UL, Fawole OA (2021)
from aromatic plants and food by-products. Effects of enzymatic pretreatment of seeds on
Front Nutr 8:792203. https://doi.org/10. the physicochemical properties, bioactive com-
3389/fnut.2021.792203 pounds, and antioxidant activity of pomegran-
20. Cui H, Abdel-Samie MAS, Lin L, Jafari SM ate seed oil. Molecules 26(15):4575. https://
(2021) Application of antimicrobial-loaded doi.org/10.3390/molecules26154575
nano/microcarriers in different food 30. Drinić Z, Mudrić J, Zdunić G, Bigović D,
products. In: Application of nano/microen- Menković N, Šavikin K (2020) Effect of pome-
capsulated ingredients in food products. Aca- granate peel extract on the oxidative stability of
demic, pp 469–517. https://doi.org/10. pomegranate seed oil. Food Chem 333:
1016/B978-0-12-815726-8.00012-X 1 2 7 5 0 1 . h t t p s :// d o i . o r g / 1 0 . 1 0 1 6 / j .
21. Chemat F, Vian MA, Fabiano-Tixier AS, foodchem.2020.127501
Nutrizio M, Jambrak AR, Munekata PE et al 31. Rojo-Gutiérrez E, Carrasco-Molinar O,
(2020) A review of sustainable and intensified Tirado-Gallegos JM, Levario-Gómez A, Chá-
techniques for extraction of food and natural vez-González ML, Baeza-Jiménez R,
products. Green Chem 22(8):2325–2353. Buenrostro-Figueroa JJ (2021) Evaluation of
https://doi.org/10.1039/C9GC03878G green extraction processes, lipid composition
22. Zhang QW, Lin LG, Ye WC (2018) Techni- and antioxidant activity of pomegranate seed
ques for extraction and isolation of natural oil. J Food Measur Character 15(2):
products: a comprehensive review. Chin Med 2098–2107. https://doi.org/10.1007/
13:20. https://doi.org/10.1186/s13020- s11694-020-00804-7
018-0177-x 32. Krivokapić S, Vlaović M, Damjanović
23. Zia S, Khan MR, Shabbir MA, Aslam Maan A, Vratnica B, Perović A, Perović S (2021) Bio-
Khan MKI, Nadeem M et al (2022) An inclu- waste as a potential source of bioactive
sive overview of advanced thermal and non- compounds – a case study of raspberry fruit
thermal extraction techniques for bioactive pomace. Foods 10(4):706. https://www.
compounds in food and food-related matrices. mdpi.com/2304-8158/10/4/706
Food Rev Intl 38(6):1166–1196. https://doi. 33. Szymanowska U, Baraniak B (2019) Antioxi-
org/10.1080/87559129.2020.1772283 dant and potentially anti-inflammatory activity
24. Rangel-Huerta OD, Pastor-Villaescusa B, of anthocyanin fractions from pomace obtained
Aguilera CM, Gil A (2015) A systematic review from enzymatically treated raspberries. Antiox-
of the efficacy of bioactive compounds in car- idants 8(8):299. https://doi.org/10.3390/
diovascular disease: phenolic compounds. foods10040706
Nutrients 7(7):5177–5216. https://doi.org/ 34. Mildner-Szkudlarz S, Bajerska J, Górnaś P,
10.3390/nu7075177 Segliņa D, Pilarska A, Jesionowski T (2016)
25. Zulkifli KS, Abdullah N, Abdullah A, Physical and bioactive properties of muffins
Aziman N, Kamarudin WSSW (2012) Bioac- enriched with raspberry and cranberry pomace
tive phenolic compounds and antioxidant powder: a promising application of fruit
activity of selected fruit peels. Int Conf Environ by-products rich in biocompounds. Plant
Chem Biol 49(14):66–70. https://doi.org/ Foods Hum Nutr 71(2):165–173. https://
10.7763/IPCBEE doi.org/10.3390/antiox8080299
35. Leontopoulos S, Skenderidis P, Kalorizou H, 330. https://doi.org/10.1016/j.foodres.

Petrotos K (2017) Bioactivity potential of poly- 2018.03.006
phenolic compounds in human health and their 44. Nasri H, Baradaran A, Shirzad H, Rafieian-
effectiveness against various food borne and Kopaei M (2014) New concepts in nutraceuti-
plant pathogens. A review. J Food Biosyst Eng cals as alternative for pharmaceuticals. Int J
7:1–19. https://www.researchgate.net/pro Prev Med 5(12):1487–1499. https://www.
fi l e / S t e f a n o s - L e o n t o p o u l o s / p u b l i c a ncbi.nlm.nih.gov/pmc/articles/PMC433
tion/321425983_Bioactivity_Potential_of_ 6979/
Polyphenolic_Compounds_in_Human_ 45. Peterson CT, Rodionov DA, Osterman AL,
Health_and_their_Effectiveness_Against_Vari Peterson SN (2020) B vitamins and their role
ous_Food_Borne_and_Plant_Pathogens_A_ in immune regulation and cancer. Nutrients
Review/links/5a215d244585150259b950 12(11):3380. https://doi.org/10.3390/
67/Bioactivity-Potential-of-Polyphenolic- nu12113380
Compounds-in-Human-Health-and-their-
Effectiveness-Against-Various-Food-Borne- 46. Rahaman A, Kumari A, Farooq MA, Zeng XA,
and-Plant-Pathogens-A-Review.pdf Hassan S, Khalifa I et al (2021) Novel extrac-
tion techniques: an effective way to retrieve the
36. Ko MJ, Nam HH, Chung MS (2020) Subcriti- bioactive compounds from saffron (Crocus
cal water extraction of bioactive compounds sativus). Food Rev Intl 1–29. https://doi.
from Orostachys japonicus A. Berger (Crassu- org/10.1080/87559129.2021.1967377
laceae). Sci Rep 10(1):10890. https://doi.
org/10.1038/s41598-020-67508-2 47. Sachdeva V, Roy A, Bharadvaja N (2020) Cur-
rent prospects of nutraceuticals: a review. Curr
37. Kostrzewa D, Dobrzyńska-Inger A, Turczyn A Pharm Biotechnol 21(10):884–896. https://
(2020) Optimization of supercritical carbon d o i . o r g / 1 0 . 2 1 7 4 /
dioxide extraction of sweet paprika (Capsicum 1389201021666200130113441
annuum L.) using response surface methodol-
ogy. Chem Eng Res Des 160:39–51. https:// 48. Zhang M, Ma W, Wang C, Yang X, Lou Y,
doi.org/10.1016/j.cherd.2020.05.005 Xia X, Xu H (2021) Optimization of enzyme-
assisted extraction and purification of flavo-
38. Kumar K, Srivastav S, Sharanagat VS (2021) noids from Pinus koraiensis nut-coated film
Ultrasound assisted extraction (UAE) of bioac- and antioxidant activity evaluation. Molecules
tive compounds from fruit and vegetable pro- (Basel, Switzerland) 26(7):1950. https://doi.
cessing by-products: a review. Ultrason org/10.3390/molecules26071950
Sonochem 70:105325. https://doi.org/10.
1016/j.ultsonch.2020.105325 49. Sadef Y, Javed T, Javed R, Mahmood A, Alwa-
hibi MS, Elshikh MS et al (2022) Nutritional
39. Kumar K, Yadav AN, Kumar V, Vyas P, Dhali- status, antioxidant activity and total phenolic
wal HS (2017) Food waste: a potential biore- content of different fruits and vegetables’
source for extraction of nutraceuticals and peels. PLoS One 17(5):e0265566. https://
bioactive compounds. Bioresour Bioprocess doi.org/10.1371/journal.pone.0265566
4(1):1–14. https://doi.org/10.1186/
s40643-017-0148-6 50. Saha S, Barua B, Sikdar D (2017) Phytochemi-
cal screening, phenolic content and antioxidant
40. Mao QQ, Xu XY, Cao SY, Gan RY, Corke H, activity of wild date palm (Phoenix sylvestris
Beta T, Li HB (2019) Bioactive compounds Roxb.) fruit extracted with different solvents.
and bioactivities of ginger (Zingiber officinale Int Food Res J 24(6) https://www.
Roscoe). Foods 8(6):185. https://doi.org/10. researchgate.net/profile/Dwaipayan-Sikdar/
3390/foods8060185 publication/322254798_Phytochemical_
41. Michalak M, Pierzak M, Kre˛cisz B, Suliga E screening_phenolic_content_and_antioxidant_
(2021) Bioactive compounds for skin health: a activity_of_wild_date_palm_Phoenix_
review. Nutrients 13(1):203. https://doi.org/ sylvestris_Roxb_fruit_extracted_with_differ
10.3390/nu13010203 ent_solvents/links/5a5654bfaca272bb6963
42. Molino A, Larocca V, Di Sanzo G, Martino M, e925/Phytochemical-screening-phenolic-con
Casella P, Marino T et al (2019) Extraction of tent-and-antioxidant-activity-of-wild-date-
bioactive compounds using supercritical car- palm-Phoenix-sylvestris-Roxb-fruit-extracted-
bon dioxide. Molecules 24(4):782. https:// with-different-solvents.pdf
doi.org/10.3390/molecules24040782 51. Wang, X., Wu, Y., Chen, G., Yue, W., Liang,
43. Nadar SS, Rao P, Rathod VK (2018) Enzyme Q., & Wu, Q. (2013). Optimisation of ultra-
assisted extraction of biomolecules as an sound assisted extraction of phenolic com-
approach to novel extraction technology: a pounds from Sparganii rhizoma with response
review. Food Res Int (Ottawa, ON) 108:309– surface methodology. Ultrasonics sonochemis-
try, 20(3), 846–854
52. Shang A, Cao SY, Xu XY, Gan RY, Tang GY, in food processing: a review. Ultrason Sono-
Corke H et al (2019) Bioactive compounds chem 73:105506. https://doi.org/10.1016/
and biological functions of garlic (Allium sati- j.ultsonch.2021.105506
vum L.). Foods 8(7):246. https://doi.org/10. 61. Kerem Z, German-Shashoua H, Yarden O
3390/foods8070246 (2005) Microwave-assisted extraction of bioac-
53. Fahmi R, Khodaiyan F, Pourahmad R, Emam- tive saponins from chickpea (Cicer arietinum
Djomeh Z (2014) Effect of ultrasound assisted L). Journal of the Science of Food and Agricul-
extraction upon the Genistin and Daidzin con- ture 85(3):406–412. https://doi.org/10.
tents of resultant soymilk. Journal of food sci- 1002/jsfa.1989
ence and technology 51(10):2857–2861. 62. Kulkarni VM, Rathod VK (2018) Exploring
https://doi.org/10.1007/s13197-012-0744-6 the potential of Mangifera indica leaves extract
54. Azka G, Tahir MQ, MAK, Syed TH (2011) versus mangiferin for therapeutic application.
Transformational leadership, employee Agriculture and Natural Resources. https://
engagement and performance: Mediating doi.org/10.1016/j.anres.2018.07.00
effect of psychological ownership. African 63. Streimikyte P, Viskelis P, Viskelis J (2022)
Journal of Business Management 5(17):7391– Enzymes-assisted extraction of plants for sus-
7403. https://doi.org/10.5897/ajbm11.126 tainable and functional applications. Int J Mol
55. Piñeiro, Z., Marrufo-Curtido, A., Serrano, M. Sci 23(4):2359. https://doi.org/10.3390/
J., & Palma, M. (2016). Ultrasound-assisted ijms23042359
extraction of stilbenes from grape canes. Mole- 64. Smiderle FR, Morales D, Gil-Ramı́rez A, de
cules, 21(6), 784 Jesus LI, Gilbert-López B, Iacomini M, Soler-
56. Sharifi-Rad J, Rodrigues CF, Sharopov F, Rivas C. Evaluation of microwave-assisted and
Docea AO, Can Karaca A, Sharifi-Rad M et al pressurized liquid extractions to obtain β-d-
(2020) Diet, lifestyle and cardiovascular dis- glucans from mushrooms. Carbohydr Polym.
eases: linking pathophysiology to cardioprotec- 2017 Jan 20;156:165–174. https://doi.org/
tive effects of natural bioactive compounds. Int 10.1016/j.carbpol.2016.09.029. Epub 2016
J Environ Res Public Health 17(7):2326. Sep 11. PMID: 27842810.
https://doi.org/10.3390/ijerph17072326 65. Kolář F, Čertner M, Suda J, Schönswetter P,
57. Aguiló-Aguayo, I., Walton, J., Viñas, I., & Husband BC (2017) Mixed-Ploidy Species:
Tiwari, B. K. (2017). Ultrasound assisted Progress and Opportunities in Polyploid
extraction of polysaccharides from mushroom Research. Trends in plant science 22
by-products. Lwt, 77, 92–99 (12):1041–1055. https://doi.org/10.1016/j.
58. Sharma K, Mishra K, Senapati KK, Danciu C tplants.2017.09.011
(eds) (2021) Bioactive compounds in nutra- 66. Suleria HA, Barrow CJ, Dunshea FR (2020)
ceutical and functional food for good human Screening and characterization of phenolic
health. BoD–Books on Demand. https:// compounds and their antioxidant capacity in
books.google.co.in/books?hl=en&lr=&id= different fruit peels. Foods 9(9):1206.
CG0tEAAAQBAJ&oi=fnd&pg=PR12&dq= https://doi.org/10.3390/foods9091206
Bioactive+Compounds+in+Nutraceutical+and 67. Wen C, Zhang J, Zhang H, Dzah CS,
+Functional+Food+for+Good+Human Zandile M, Duan Y et al (2018) Advances in
+Health++View+Chapters++Share++Cite+Bio ultrasound assisted extraction of bioactive
active+Compounds+in+Nutraceutical+and compounds from cash crops – a review. Ultra-
+Functional+Food+for+Good+Human son Sonochem 48:538–549
+Health+Bioactive+Compounds+in+Nutraceu 68. Parniakov O, Bals O, Lebovka N, Vorobiev E
tical+and+Functio nal+Food+for+Good (2016) Pulsed electric field assisted vacuum
+Huma&ots=09FYMlY-bu&sig= freeze-drying of apple tissue. Innovative Food
nhMvbgLpb9OGa-w8dwGperyVZsE&redir_ Science & Emerging Technologies, 35, 52–57.
esc=y#v=onepage&q&f=false https://doi.org/10.1016/j.ifset.2016.04.00
59. Padmapriya K, Dutta A, Chaudhuri S, Dutta D 69. Wen L, Zhang Z, Sun DW, Sivagnanam SP,
(2011). Microwave assisted extraction of man- Tiwari BK (2020) Combination of emerging
giferin from Curcuma amada. 3 Biotech 2 technologies for the extraction of bioactive
(1):27–30. https://doi.org/10.1007/ compounds. Crit Rev Food Sci Nutr 60(11):
s13205-011-0023-7 1826–1841. https://doi.org/10.1080/
60. Singla M, Sit N (2021) Application of ultra- 10408398.2019.1602823
sound in combination with other technologies
Chapter 12
Pressurized Liquid Extraction for the Isolation of Bioactive

Compounds
Rakesh Barik, Sinoy Sugunan, and Mohd Affendi Bin Mohd Shafri
Abstract
Pressurized liquid extraction (PLE), an advanced extraction technique, employs solvent extraction at
elevated temperatures and pressures, consistently under their individual critical points, so the solvent is
sustained in the liquid state during the entire extraction process. As a result of utilizing these exact
conditions of pressure and temperature, an alteration in the physicochemical properties of the solvent
arises. It is an alternative and advanced preparation technique compared to conventional extraction
methods in many areas, such as environmental, food, and pharmaceutical analysis. Medicinal plants are
the sources of numerous compounds that can tackle numerous diseases when they are used in a reasonable
combination. Every single plant contains one or more major bioactive compounds that are responsible for
various biomedical functionalities. This chapter summarizes the application of the PLE technique in
extraction and phytochemical analysis. The various advantages offered by this technique, such as low
solvent usage, less preparation time, high extraction efficiency and better reproducibility, have made it a
better alternative for the extraction and analysis of phytoconstituents.
Key words Extraction, Phytoconstituents, Efficiency, Selectivity, Solvent, Analysis
1 Introduction
Pressurized fluid extraction (PFE) or pressurized liquid extraction

(PLE) is an innovative extraction technique consisting of liquid
solvents at higher temperatures and pressures to formulate samples
for analysis by either gas chromatography or liquid chromatogra-
phy. This process is also called accelerated solvent extraction (ASE),
pressurized hot solvent extraction (PHSE), subcritical solvent
extraction (SSE), pressurized fluid extraction (PFE), high-pressure
solvent extraction (HPSE), and high-pressure high-temperature
solvent extraction (HPHTSE) [1].
The PLE was developed from Soxhlet extraction, established by
the German inventor Franz Ritter von Soxhlet in 1879. This
method was immensely successful in obtaining solutes even from
solid samples, which were previously impervious. Hence, the
275
276 Rakesh Barik et al.
Soxhlet method could become a reference standard in analytical

extraction for more than a century. Novel extraction techniques,
such as SFE (supercritical fluid extraction), PLE, and other pro-
cesses, were developed and demonstrated cleaner and more effi-
cient than Soxhlet extraction [2].
Pressurized liquid extraction is analogous to Soxhlet extrac-
tion, but the solvent condition inside the PLE cell extends to the
supercritical region during extraction, ensuring efficient extrac-
tions. The raised temperature alters the sample more soluble and
accomplishes a higher diffusion rate, while the high pressure keeps
the solvent below its boiling point [3]. Solvents could penetrate
solid models in an enhanced way at elevated pressures and tempera-
tures, lowering solvent usage. A pressurized liquid extraction, while
compared to a traditional Soxhlet extraction, exhibits a lessening of
extraction time to approximately to 20 min from 18 h and a decline
in total organic solvent consumption to 80 mL or less of organic
solvent from 300 mL [4].
The effectiveness of the extraction procedure generally varies
with the three correlated facets of the matrix, mass transfer, and
solubility. Concerning the matrix, its nature and the constituent of
interest to be extracted and its position within the matrix have an
influence [5]. The elevated temperature enhances solubility proper-
ties and mass transfer between the plant matrix and the extraction
solvent, leading to improved extraction kinetics. Lowering of sol-
vent viscosity facilitates the plant matrix’s hydration and increases
the bioactive compounds’ solubility. Higher temperature also leads
to the breakdown of bonds or interactive forces in the matrix
(dipoles, van der Waals, and hydrogen bridges), accelerates the
release of compounds, and produces high extraction yields [6].
1.1 Basic Principles The PLE method is a quick extraction technology due to the direct
of Pressurized Liquid interaction between the liquid solvent and the particles of the plant
Extraction (PLE) matrix under high pressure and subcritical temperature conditions
to extract the constituents of interest effectively. The efficiency of
the extraction technique mostly depends on the three correlated
facets of the matrix, mass transfer, and solubility. Related to the
matrix, its nature and the molecule of interest to be extracted and
its position within the matrix have an effect [7]. Elevated tempera-
ture significantly enhances solubility properties and mass transfer
between the plant matrix and the extraction solvent, resulting in
better extraction kinetics. Solvent viscosity decreases, facilitating
the plant matrix’s hydration and increasing the bioactive com-
pounds’ solubility. High temperature also causes the breakdown
of bonds or bonding forces in the matrix (dipoles, van der Waals,
and hydrogen bridges), facilitates the release of compounds, and
produces high extraction yields [8].
Pressurized Liquid Extraction for the Isolation of Bioactive Compounds 277
The PLE system is an extraction method designed to perform

the extraction of constituents from multiple samples simulta-
neously. The PLE system provides high recoveries and exceptional
precision for all analytes in a small amount of time. Inexpensive
stainless-steel extraction cells with end cap filtration keep working
costs at a minimum. Optional disposable end cap filtration
enhances productivity and prevents valuable time. This advanced
method (PLE) is preferred over other conventional methods of
extraction due to the following reasons [9]:
1. Decreased Solvent Costs
PLE uses as small as 15 mL of solvent compared to more
than 500 mL of solvent required to perform Soxhlet
extractions.
2. Lowered Running Costs
Speedy extraction and cleanup and decreased solvent use
and waste lower the running costs by around 70%.
3. Enhanced Productivity
The entire extraction and cleanup could be carried out in
less than 30 min. Conventional methods could consume
10–16 h.
4. Decreased Solvent Leftover
The PLE system reduces solvent waste by utilizing solvents
in an efficient way.
5. Purge Cross-Contamination
Optional low-cost disposable extraction cells and Teflon
filtration end caps guarantee trouble-free extraction and
exclude the risk of cross-contamination.
6. One-Step Sample Extraction and Cleanup
The optional in-line cleanup segment performs the com-
prehensive sample extraction and cleanup in one step with
augmented speed and reduced cost.
7. Automated Operation and Documentation
Real-time software lets plotting six pressure channels and
six temperature data channels simultaneously. This powerful
feature permits automatic documentation of all extraction
data. Temperature and pressure data can be overlaid, printed
in graphic or tabular format, and stored for future reference.
8. Modular Assembly Provides for Easy Maintenance
The modular design of the PLE system mixed with its
exposed plumbing facilitates effective system maintenance.
The PLE system’s channels are designed to operate indepen-
dently. This adaptability ensures easy replacement with no
down time.
9. Leakage and Clog-Free Operation

Simple design combined with large bore plumbing enables
the PLE system for n operation free from clogging and leakage.
10. Numerous Extractions
The programming of varying pressure and temperature lets
the extraction of a variety of compounds.
Pressurized fluid extraction is comparable to Soxhlet extrac-
tion, except the solvents are used near their supercritical region
with increased extraction properties. In that physical region, the
elevated temperature enables higher solubility and increased diffu-
sion rate of lipid solutes in the solvent. By keeping the solvent
below its boiling point, the high pressure allows an enhanced
solvent penetration in the sample [10]. Thus, PFE permits a high
extraction efficiency with a decreased solvent volume and a quick
extraction time. That technique is called accelerated solvent extrac-
tion (ASE). Dionex first developed this method and validated it on
a commercially available automated extraction system
(Dionex ASE).
With the similar solvent blend employed in the Folch proce-
dure, the elevated pressure solvent extraction of total lipids in
poultry meat decreased the consumption of solvents and the time
extraction. Concurrently, it has given comparable lipid recoveries
and fatty acid compositions.
The PFE device contains an extraction cell (1 up to 100 mL)
kept at a temperature between 80 and 200 °C into which a solvent
is injected and held at 10–20 MPa for some minutes. Then, the
extract is pushed into a collection vial by a second volume of
solvent, and finally, the complete solvent is driven with an inert
gas flow [11].
In the beginning, PFE was used for environmental contami-
nants in soils, sediments, and animal tissues, but it is currently used
for food (meat, seeds, feeds), pharmaceutical products, and several
other biological samples. This technique successfully replaced the
Folch extraction for oxysterols in food. The polar and nonpolar
lipids’ efficiency of extractions with pressurized solvents (hexane,
methylene chloride, isopropanol, ethanol) was assessed in corn and
oats kernels. The solvent polarity and temperature properties were
tested on the recovery of total lipids, triglycerides, glycolipids, and
phytosterols [12].
1.2 Mechanism and The PLE involves circulating the solvent through the extraction cell
Components of or column with a high-performance liquid chromatography
Pressurized Liquid (HPLC) pump where the plant matrix is placed to remove the
Extraction (PLE) bioactive molecules of interest [13]. The pretreated and
conditioned sample within the extraction column is exposed to
the designated temperature using an electrothermal liner while
being compressed to the specified pressure. In order to stabilize
the system and facilitate solvent diffusion through the plant matrix,
the pressure and temperature are kept constant, marking the begin-
ning of static extraction [14]. Afterward, the required pressure and
solvent flow rate are maintained, commencing dynamic extraction.
The extraction procedure is conducted through multiple cycles. At
the conclusion of the process, the extraction column containing the
sample is substituted with a new column consisting solely of an
inert element. The system is then cleaned by pumping out the
solvent and passing nitrogen or carbon dioxide through it [15].
As a result of the constraints posed by commercial equipment,
the only feasible method to handle liquid samples is by converting
them into solids, typically achieved through the addition of an
absorbent or adsorbent substance. The extraction process for ana-
lytes from semisolid and solid samples can be outlined using the
following five steps [16]:
1. Dampening of the sample (analytes to be extracted and matrix)
with menstruum
2. Dislodging of compounds from the matrix (including or not
the breakdown of chemical bonds)
3. Dissolution of the compounds in the menstruum
4. Dissemination of the compounds from the matrix
5. Propagation across the immediate solvent layer enveloping the
matrix, culminating in the bulk solvent
The extraction efficiency is contingent upon both kinetic and
thermodynamic factors. Consequently, the efficacy of extraction is
influenced by three interconnected facets [17]:
A. Matrix effect
B. Mass transfer
C. Solubility
The characteristics of PLE are subject to several factors that
impose limitations, including the careful selection of temperature,
pressure, flow rate, and extraction duration required for achieving
comprehensive extraction.
2 Instrumentation
The measurement process is simple to perform the liquid extraction

process. Anti-corrosion products should be used, as high pressure
(35–200 bar) and temperature (room temperature to 200 °C) are
frequently used [18].
The most common representation of the instrument is shown
in Fig. 1. It comprises of a pump, solvent container a furnace with
the extraction cell, valves and chokes, and a collection vessel.
Fig. 1 Representative diagram of a pressurized liquid extraction (PLE) system showing configurations for the
development of static system (a) and a dynamic system (b)
The solvent tank is first connected to the high-pressure pump.

The pump introduces the solvent into the system and helps remove
the extract when the process is complete. The extraction process
takes place inside the extraction cell. If needed, a filter paper is
inserted into the stainless-steel extraction cell, followed by the
sample, which is sometimes mixed with a dispersant [19].
The cell is manually or automatically placed in the oven sup-
ported by valves and restrictors to control the entire pressure
throughout the extraction process. Finally, there is a collection
vial placed at the end of the extraction system. Instrumentation
can be more or less sophisticated depending on the process require-
ments. For example, a solvent controller is needed if there are
multiple solvent reservoirs (to obtain solvent mixtures), or an
inert gas loop (usually nitrogen) can help purge the menstruum
from the lines after extraction. In addition, a cooling bath can be
used for the collection vessel, which lowers the temperature of the
extractant to minimize thermal degradation. A dynamic pressurized
liquid extractor requires a somewhat more complicated high-
pressure pump to control solvent flow, solvent preheat coils, and a
pressure limiter (backpressure regulator) or micrometer valve
instead of a static open or close valve as in static pressurized liquid
extractor [20].
3 Factors Affecting Pressurized Liquid Extraction (PLE)
3.1 Effect of Temperature is the most important factor because it changes the
Temperature solvents’ physical and chemical properties and affects the extraction
efficiency. Temperature lowers the dielectric constant of the solvent
and changes its polarizability. It also reduces the viscosity and
density of the menstruum, enhances diffusion and penetration
into the matrix, and causes a higher mass transfer rate. Breakdown
of the structure of the vegetable matrix is brought about by high
temperatures, which reduces the surface tension between the men-
struum, sample, and the compounds during the extraction process.
This change is desirable to form cavities in the menstruum into
which the desired compounds are transported [21].
On the other hand, very high temperatures bring about the
degradation of thermolabile compounds. Too high temperatures
(>150 °C) may lead to the formation of toxic compounds such as
hydroxymethylfurfural (HMF) [22]. Hence, proper optimization
of temperature is essential after knowing the nature of compound
to be extracted, to avoid undesirable compounds, and get high
extraction yields [23].
3.2 Effect of Chemical affinity of the compound to be extracted for the extrac-
Menstruum tion solvent is the essential factor for proper selection of the men-
struum. This leads to high diffusion and mass transfer leading
further to high extraction yields. Additionally, the menstruum
should be nontoxic or less toxic in nature, inexpensive, accessible,
and easily disposable. The most environmentally acceptable solvent
system is the water–ethanol mixture [24].
3.3 Effect of High-pressure technology is a significant merit in the entire process

Pressure of pressurized liquid extraction as it maintains the menstruum in
the liquid state even after subjecting to temperatures above their
respective boiling points [25]. The highest pressures result in
higher extraction yields because they help hydrate the plant matrix
by keeping the solvent in a liquid state. However, their effect is less
than that of temperature, since air bubbles can form at higher
pressure, which reduces the solubility of the compounds in
question [26].
3.4 Nature of the Plant matrices are usually subjected to unique treatments such as air
Plant Matrix drying, freeze drying, grinding, and screening before extraction.
Drying type and conditions directly affect the extraction yield. The
small size of the model makes it easy to change sizes. The larger
contact area of the material with the extraction solvent improves the
adsorption and separation of the target compounds. However, in
some studies, it is recommended not to reduce the particle size too
much in order not to prevent the diffusion of the solvent from the
compressed structure [27].It is also essential to know the nature
(nature and moisture) of the target compound in the plant matrix.
Many studies have shown that samples with high water content can
improve performance compared to dry models. This may be due to
cell disruption during drying, inhibiting the release of the target
compound. However, other studies have shown that water com-
petes with solvent extraction by reducing the recovery of bioactive
compounds in plant matrices [28].
3.5 Effect of The time the solvent is in contact with the matrix at a specific
Extraction Time temperature, pressure, and flow rate is the extraction time. Matrix
structure, type of target composition, temperature, pressure,
weight, etc. are some of the factors affecting its efficiency. Many
factors affect it. The static or dynamic extraction method is the
most specific parameter matrix to understand the extraction time
required to separate bioactive compounds from plants [29].
3.6 Impact of Energy The extraction process uses high pressure and temperature, which
and Environment means more energy. But it is lower than other extraction methods
such as supercritical fluid extraction or traditional Soxhlet extrac-
tion. According to some reported studies, the chemical effects
related to the type of solvent applied in the extraction of Rosemary
plants and Soxhlet extraction shows that PLE is less energy-
intensive than Soxhlet extraction. However, the temperature of
PLE is higher (183 °C vs. 78 °C for Soxhlet) [30].
3.7 Chemical and Pressurized liquid extraction using specific menstruum such as
Sensory Factors ethanol and others can produce some undesirable compounds
such as hydroxymethylfurfural (HMF), a nonenzymatic brown
indicator [31]. Similarly, HMF was associated with the induction
of colon cancer precursors in mice but was not toxic as found in
some laboratory tests. Ethanol concentration had a significant
effect on the overall performance of the PLE process. The lower
the ethanol content of the PLE extract, the higher the recovery of
phenolic acids and flavanols. Therefore, it is recommended to
recover at ethanol concentrations of 15%, 32.5%, and 50% for
phenolic acids, stilbenes, and flavanols, respectively. The extract
showed highest total polyphenol content and antioxidant activity
at 150 °C and 32.5% ethanol [32].
4 Advantages and Disadvantages of Pressurized Liquid Extraction Technique
The benefits of this approach are demonstrated by applying a

solvent at the right pressure and temperature to interact with
plant molecules and extract them via mass transfer and solubility
[33]. Pressurized liquid extraction uses solvents that are GRAS-
type and environmentally benign, such as water and alcohol. In
comparison to current procedures, which employ harmful solvents,
it also utilizes less extraction solvent [34]. This extraction tech-
nique is carried out using equipment that is basic, practical, and
easy to operate. Many of them feature semiautomatic designs that
can be connected to instruments for analytical measurement and
separation, such as liquid phase chromatography (HPLC) and gas
chromatography (GC) [35]. Additionally, to increase extraction
efficiency, this novel technique can be used in conjunction with
other techniques including supercritical fluid extraction (SFE) and
ultrasonic technology.
5 Applications
5.1 Isolation of Tocopherols were obtained from apple seeds and kiwi fruit using
Tocopherols this technique. A more pure extract was obtained, and the recovery
rate was similar to or higher than that of the existing method.
Extraction parameters optimized to maximize the effects of grain
tocopherols and tocotrienols or carotenoids from microalgae have
been described [36–38].
5.2 Determination of Pressurized liquid extraction technique has gained popularity as a

Organic Pollutants green extraction method for analyzing a variety of organic pollu-
tants, including personal care products and pharmaceuticals, nano-
particles, flame retardants, and endocrine-disrupting chemicals,
which are frequently found in environmental samples since it was
first introduced as an official US Environmental Protection Agency
(EPA) method for determining persistent organic pollutants in
solid environmental samples [39].
The majority of applications concentrate on removing organic
pollutants from sediment and sewage sludge [40]. For the extrac-
tion of persistent organic pollutants from nonbiological materials
such as soils, sediments, silt, and dust, analytical methods based on
the pressurized liquid extraction method have been developed.
Furthermore, comparable types of samples have revealed the pres-
ence of numerous novel contaminants, including nitrosamines
[41], alkylphenols, bisphenol A, and UV filters [42]. Using pres-
surized liquid extraction to create sludge samples, researchers were
able to find additional contaminants that have been classified as
endocrine disruptors, such as homologs of bisphenol and bisphenol
A [43], hormonal steroids, and flame retardants such as brominated
and chlorinated flame retardants [44].
Similar to other extraction methods, the identification of the
target pollutants can be hampered by the coextraction of nontarget
analytes from the pressurized liquid extraction matrix. Due to this,
a post-extraction cleanup step is often necessary before the deter-
mination stage [45]. This can be accomplished using various solid
phase extraction cartridges, gel permeation chromatography, or
packed chromatographic columns. Sulfur is a characteristic elemen-
tal interference in soil and sediment matrices, and in some applica-
tions, pressurized liquid extraction processes employ the right
adsorbents in the extraction cell to retain it [46]. The pressured
liquid extraction throughput is greatly increased by the option of
including in-cell cleanup. In order to get clean extracts for the
examination of nonpolar compounds such as polycyclic aromatic
hydrocarbons and flame retardants such as polybrominated diphe-
nyl ethers, silica gel was thus demonstrated to be a successful
sorbent. In order to determine UV filters, activated carbon was
effective in removing sulfur under reducing circumstances [47].
Emerging pollutants in cosmetics, personal care items, and

environmental pressured liquid extractions include fragrance aller-
gies, UV filters, and cosmetic preservatives. Pressurized liquid
extraction was shown to be a successful method in this instance
for removing these compounds from pressurized liquid extraction
cosmetic matrices [48]. Pressurized liquid extraction-based tech-
nologies enable simultaneous in-cell derivatization and extraction
of multiclass cosmetic preservatives (such as parabens, triclosan,
bronidox, and bronopol) before GC-MS analysis [41]. Hence, in
a study of dust analysis, parabens and triclosan were found after
eliminating nonpolar interferences with hexane at low temperatures
and pressure and extracting the target analytes with polar solvents
[49].
5.3 Estimation of Although the advantages of this technology have been reviewed for
Pesticides the study of biological and food samples, pressurized liquid extrac-
tion applications were initially focused on the extraction of envi-
ronmental pollutants [50]. Since this technique enables the
simultaneous extraction of many residue types with a wide range
of polarity, numerous applications for identifying pesticide residues
have been documented [51]. Thus, samples of animal and vegeta-
ble tissues are analyzed using pressured liquid extraction. Pesticides
are typically found in nonfat foods with a medium to high water
content, such as fruits, vegetables, and cereal-based diets. As a
result, it is frequently necessary to add a drying agent (such as
sodium sulfate or diatomaceous earth) [52, 53].
Pressurized liquid extraction has been used to identify a wide
range of pesticide residues in various agricultural and food matrices,
such as honey [54], organophosphorus pesticide residues in corn
[35], pyrethroid residues in feed samples [55], multiclass pesticide
residues in food commodities and grain, or herbicide residues in
soybeans [56].
By combining this method with gas chromatography/high
resolution isotope dilution mass spectrometry, common organo-
chlorine, organophosphorus, and pyrethroid pesticide residues in
herbal liquid extractions such as tea may be accurately
determined [57].
The analytical complexity of tea sample matrices can be reduced
by using pressurized liquid extraction followed by gel permeation
chromatography due to the high concentration of caffeine, pig-
ments, polyphenols, etc., in tea samples. Applying a selective pres-
sured liquid extraction technique, many pesticides as well as other
lyophilic pollutants can be recovered and identified from lipid-rich
matrices. The homogenized lipid sample is then placed in the
extraction cell on top of basic alumina, silica gel, and florisil. The
target chemicals are then extracted utilizing a single automated
process employing a 1:1 (v/v) dichloromethane/hexane ratio [58].
Comparative assessment of pressurized liquid extraction per-

formance and other pressurized sample preparation procedures
such as supercritical fluid extraction or the traditional Soxhlet
extraction revealed higher recoveries of pesticides by pressurized
liquid extraction, compared with conventional analytic techniques,
such as QuEChERS and buffered ethyl acetate extraction.
Pressurized liquid extraction methods also provided superior
performance for extracting pesticide residues [59]. In these cases,
the removal of lipids and other co-extractable materials was
achieved by adding fat-retaining sorbents to the pressurized liquid
extraction cell, such as florisil, alumina, or sulfuric acid–impreg-
nated silica gel [60].
5.4 Determination of Numerous fungi create a group of toxicologically significant poi-

Toxins sons known as mycotoxins. Through the use of pressurized fluid
extraction, mycotoxins, such as zearalenone, ochratoxin A, and
aflatoxin, have been discovered in a variety of foods [61]. Before
analyzing mycotoxins using chromatography, a cleaning procedure
is typically advised. Compared to traditional sorbents, molecular
imprinted polymer solid phase extraction, which is based on custo-
mized polymers with particular binding sites corresponding to the
target chemicals, is more selective. This idea led to the successful
detection of two Alternaria mycotoxins, such as alternarisol and
alternariol monomethyl ether, in tomato samples using a modest
amount of methanol (8 mL per sample) and a short period (13 min
each sample) [62].
5.5 Determination of Metal and organometallic compounds have been determined using
Metals liquid extraction under pressure using Envi-Carb-based dispersing
agents and speciation of polar arsenic species in Seafood. The
recovery of four arsenic species was evaluated using silica, C-18,
sea sand, diatomaceous earth, and alumina as a cleanup agent. On
the other hand, Envi-Carb was used as a cleanup sorbent and
dispersing agent to extract Mg, Al, Ti, Cu, Ag, Sn, and Pb in
lubricating oils without any additional cleanup step [47].
5.6 Estimation of The release of pharmaceutical products into the environment has
Antibiotics raised issues concerning their occurrence, fate, and effects on the
biota. Antibiotics are an essential group of pharmaceutical products
widely used in human and animal health care, which are reportedly
ubiquitous compounds in the aquatic environment. Pressurized
liquid extraction is a reliable technique for extracting antibiotics
and other drug residues associated with suspended solid matter
[63]. Multiresidue analysis of sulfonamide antibiotics and their
acetylated metabolites in soils and sewage sludge can be performed
using fully automated pressurized liquid extraction methods [64].
In these methods, a subsequent step for preconcentration and
purification is required. The extraction of quinolone and
sulfonamide residues, such as lomefloxacin, enoxacin, sarafloxacin,

enrofloxacin, sulfadiazine, sulfamethoxydiazine, and sulfa
dimethylpyrimidine, in fish and shrimp was carried out by pressur-
ized liquid extraction using diatomaceous earth as a dispersing
agent and acetonitrile as the extraction solvent [65].
5.7 Standardization Pressurized fluid extraction is one of the most widely used techni-
of Polyphenols ques for extracting polyphenolic compounds from various sources
such as food, vegetables, seafood, and agro-industrial by-products.
Whether acidified or not, the hydrogen ethanol mixture (EtOH
> 50%) is the preferred solvent for the extraction of polyphenols by
liquid extraction. In addition, the temperatures of heat- and cold-
resistant phenolics are generally 40–60 and 75–220 °C,
respectively.
One study [62] used an optimized pressurized liquid extraction
to extract antioxidant phenoliccompounds from defatted peanut
shells using ethanol aqueous solution (60.5% v/v) as the solvent at
a temperature of 220 °C for a time period of 12.2 min. Under these
conditions, extracts with high phenolic yield like phenolic acids and
glycoside flavonoids were obtained.
Another study used pressurized liquid extraction method to
extract polyphenolic compounds with antioxidant activity from
Rubus fruticosus L. residues [66].
Anthocyanins were the main components recovered by using
water: ethanol (50 : 50) as extraction solvent in a dynamic extrac-
tion mode at 100 °C for a time period of 30 min.
Another example is the extraction of monomeric anthocyanins
and other phenolic compounds from grape (Vitis vinifera) pulp by
continuous liquid extraction [67]. The extraction was divided into
two consecutive parts to recover the different groups.
The first step was performed at 40 °C using water/ethanol
(50% w/w) pH 2.0 as solvent, and the second step was performed
at 100 °C using water/ethanol (50% w/w). The process yielded
two different sources: one rich in anthocyanins (first step) and one
rich in other phenolic compounds (second step). The low temper-
ature of the first step prevents thermal degradation of the antho-
cyanins before the second step, while the low pH aids the extraction
yield.
In the second step, efficient extraction of phenolic compounds
was found as high temperature increased the extraction of heat-
stable phenolic compounds.
Another interesting example is the recovery of biflavonoids and
anthocyanins from the dried fruit of Brazilian pepper (Schinus
terebinthifolius Raddi) after a defatting step in a continuous pres-
sure liquid extraction process [68]. The first step was to use petro-
leum ether at 60 °C for 6 min. In the second step, phenolic
compounds were extracted from the stone fruit and dried fruit
exocarp using acidified ethanol (5% v/v acetic acid) utilizing a static
extraction cycle (10 min each) at 75 °C and 100 °C. This continu-
ous supply of fluid facilitates the selective extraction of phenolic
compounds such as naringenin, biapigenin, and methylated
anthocyanins.
A combined application of pressurized liquid extraction and
ultrasonic-assisted extraction was used for the extraction of antho-
cyanins from Rubus fruticosus, Vaccinium myrtillus, and Eugenia
brasiliensis [69]. In this process, the sample is subjected to prelimi-
nary sonication before being extracted by pressurized liquid extrac-
tion. These samples were mixed by using hydroethanol solution
(50% or 70% ethanol) as menstruum and processed in an ultrasonic
bath at 80 °C for 8 min.
Pressurized liquid extraction is also used in biorefinery pro-
cesses to extract polyphenolic compounds. One study [70] carried
out the biorefining of hemp (Cannabis sativa L.) residues by
sequential supercritical carbon dioxide and pressurized liquid
extraction along with enzyme-assisted extraction. Lipophilic frac-
tion rich in cannabidiol and cannabidiolic acid was obtained in the
supercritical process, the pressurized liquid extraction method gave
a flavonoid-rich fraction, and the enzyme-assisted process gave a
sugar-rich fraction. The liquid extraction biotreatment process was
divided into two successive stages. In the first step, acetone was
used as the solvent and hydroethanol solution (4:1 v/v) was used in
the second step. Each step was performed at 100 °C for 45 min
(3 cycles × 15 min).
In another study [71], a continuous method was developed for
the isolation of bioactive compounds (human aromatase inhibitors)
from Cicer arietinum seeds using liquid extraction, countercurrent
chromatography, and preparative liquid chromatography.
The pressurized liquid extraction was performed using aqueous
ethanol (60% w/v) at 80 °C for 5 min. Thereafter, the extract was
transferred into the two chromatography sample loops. Both the
chromatography separations were optimized based on the polarity
of the active compounds already characterized in the pressurized
liquid extract.
The complementarity between countercurrent chromatogra-
phy and preparative liquid chromatography allowed the isolation
of 11 bioactive flavonoid-type compounds. This novel continuous
extraction method is effective and can be applied to other bioactive
compounds in various food or plants [72].
Recovery of phenolic compounds from various parts of medic-
inal plants has been made possible by pressurized liquid extraction.
The high temperatures and pressure and the right menstruum
bring about rapid and effective extraction of compounds of differ-
ent polarities [73].
Various fruits, vegetables, oils, such as Hibiscus sabdariffa caly-
ces, Sclerocarya birrea stem, pomegranate peel, sweet cherry stem,
and olive oil, have been subjected to pressurized fluid extraction to
isolate their phytochemicals and their by-products generated in the

process of production [74].
In these studies, surface response technique was used to opti-
mize phytochemicals combined with advanced extraction techni-
ques to improve the process of bioactive extracts.
Cassia grandis (Fabaceae), also called as Carao or Red flower, is
a legume native to Central and South America. The pods are edible
and the seeds are used to make chocolates [75]. Research reports
point to the antioxidant properties of the seeds, which explains
their use in traditional medicine [76]. Some of these functions
may be related to their use in bioactive substances such as phenols,
flavonoids, and tannins [77].
Many authors have reported the biological activities of phyto-
chemicals belonging to this chemical group [78]. Since scientific
information shows the extending and medicinal properties of the
content of phenolic compounds, they can be used to create food
antioxidants or as ingredients in nutraceuticals.
A research aimed at identifying and optimizing the extraction
of phenolic compounds from C. grandis seeds was performed by
combining advanced extraction techniques and analysis platforms.
Response surface technique of liquid chromatography was com-
bined with electrospray to extract oil from oil time-of-flight Mass
spectrometry [79].
For the extraction, the solvent was degassed for 15 min to
remove oxygen to prevent oxidation. For each extraction, the
sample was mixed with sand and loaded into a stainless-steel
extraction cell.
The selection options are sandwich type (5 g sand + mixed
sample - sand +5 g sand). Cellulose filters are installed at both
ends of the pool to prevent clogging with metal frits. The above
extraction procedure was used and the resulting product was col-
lected in glass bottles. These extracts were rapidly cooled to room
temperature, filtered, and evaporated in vacuo.
Using Statgraphics Centurion XV software version 15.1.02,
the response surface method was used to evaluate the effect of
wastewater on the recovery and yield of phenolic compounds.
The design pattern used is a basic mixed design model with two
pivot points and two levels (maximum and minimum) for each
independent variable. Temperature, percentage of ethanol, and
extraction time were chosen as independent variables, and the
experimental design consisted of a total of 14 experiments [80].
5.8 Isolation of Pressurized liquid extraction technology has been used recently for
Terpenoids the extraction of terpenoid compounds from various sources
including plants and microorganisms [81]. Owing to the chemical
diversity and polarity, different solvents and temperature ranges are
required.
However, different solvents and temperature ranges are

required due to their chemical diversity and polarity. The com-
monly used solvents include ethanol, water, hydroethanolic mix-
tures, and ethyl acetate. Certain renewable solvents such as
2-methyltetrahydrofuran at temperature ranging from 40 to 160 °
C are also used.
In a study [82], various pressurized hot water extraction para-
meters such as time, temperature, and the frequency of cycles were
optimized for the recovery of terpenoids such as steviol glycosides,
carotenoids, and other bioactive compounds from Stevia rebaudi-
ana Bertoni leaves.
Optimal conditions for the extraction of terpenoids were 160 °
C and 30 min (10 min per cycle), demonstrating that the technique
was found to be efficiently used to recover thermally labile and
nonpolar to polar components in Stevia leaves.
Pressurized liquid extraction has been used to extract other
terpenoids such as carotenoids from microbes. One such instance
is the extraction of additional carotenoids, including hydroxylated
and nonhydroxylated salinixanthin forms, from the marine bacteria
Rhodothermus marinus, under pressurized liquid extraction condi-
tions using ethanol as the solvent for 6 min (3 cycles of 2 min each).
A different investigation extracted carotenoids and chloro-
phylls from the microalgae Chlamydomonas sp. using pressured
liquid extraction [83]. In this instance, the pressured liquid extrac-
tion extract’s primary carotenoid was identified as lutein under the
most stringent circumstances (100% ethanol, 40 °C for 20 min).
However, the chlorophyll/pheophytin content of this extract was
likewise high. The synthesis of terpenoids was studied chemically
using pressurized liquid extraction.
Neochloris oleoabundans microalgae were used in a research
study [84] that used pressurized liquid extraction as a reference
extraction method to examine the effects of various culture condi-
tions such as effects of nitrogen, light intensity, and carbon supply,
on the total carotenoid and carotenoid composition. Also assessed
was the pressure liquid extraction extracts’ capacity to inhibit the
proliferation of human colon cancer cells. At 100 °C and a static
extraction time of 20 min, ethanol was used for the extractions.
Pressurized liquid extraction helped to create the ideal circum-
stances for the cultivation of large quantities of carotenoids with
antiproliferative activity, such as lutein, carotenoids monoesters,
and violaxanthin.
Since different solvents can be used depending on the terpe-
noids’ chemical characteristics, pressurized liquid extraction is a
flexible method for terpenoids. In this regard,
2-methyltetrahydrofuran was first assessed for the pressured liquid
extraction of a number of carotenoids from Chlorella vulgaris
[85]. For the extraction of xanthophylls (violaxanthin, astaxanthin,
lutein, and canthaxanthin) and carotenoids (Carotene and
lycopene), a mixture of 2-methyltetrahydrofuran and ethanol (50:

50 V/V) was heated to 110 °C for 30 min. To identify and charac-
terize high-value chemicals from natural sources, a multianalytic
platform with pressurized liquid extraction was also included.
To generate extracts from Physalis peruviana L. calyces that are
rich in withanolide, in vitro antioxidant assays and Hansen solubil-
ity criteria were suggested [86]. In this investigation, pressured
liquid extraction solvents were chosen based on the Hansen solu-
bility parameters technique and target molecules
4-hydroxywithanolide E and withanolide E. The extraction tem-
perature, ethanol, ethyl acetate, and their combinations were
assessed in relation to the amount of withanolide present in the
pressured liquid extraction extracts. The best results were obtained
using a 75:25 v/v mixture of ethanol and ethyl acetate heated to
125 °C. The development of integrated solutions to increase pro-
cess selectivity toward the recovery of target compounds has been
accelerated by the quest for terpenoids with biological activity.
To obtain carnosic acid and carnosol-enriched rosemary (Ros-
marinus officinalis L.) extracts with antiproliferative activity on
colon cancer cell lines, another study developed an integrated
pressurized liquid extraction followed by supercritical antisolvent
fractionation at pilot plant scale and compared the process with
other sub- and supercritical methods. The pressurized liquid
extraction and supercritical process began with the production of
a hydroethanolic extract under pressurized liquid extraction condi-
tions (80:20 v/v, 150 °C, 20 min). Based on the antisolvent
properties of SC-CO2 in aqueous systems, the pressurized liquid
extraction extract was then diluted with water and fractionated.
High levels of phenolic terpenes were detected in the fractions
produced by pressurized liquid extraction and supercritical antisol-
vent fractionation, and they also demonstrated antiproliferative
solid activity [87].
5.9 Extraction of The extraction of lipids is one of the principal uses of pressured
Lipids liquid extraction. This technique has been used to extract lipids
from a variety of sources and chemical structures utilizing low- or
medium-polarity solvents such as hexane, (+)-limonene, ethyl ace-
tate, methyl acetate, ethanol, and hydroethanolic combinations.
The temperature used for lipid extraction typically ranged from
90 to 220 °C. For the purpose of resolving issues with traditional
extraction techniques utilizing hazardous organic solvents, pressur-
ized liquid extraction was assessed as an environmentally friendly
method for isolating edible oils. For the effective extraction of
3-rich oil from Echium plantagineum seeds utilizing hexane-free
processing methods, pressured liquid extraction, microwave-
assisted extraction, and ultrasound-assisted extraction have recently
been examined [88].
A range of solvents, including water, ethanol, ethyl acetate,

hexane, and mixtures of ethanol and water, were utilized at tem-
peratures between 60 and 200 °C. In a recent application, the effect
of (+)-limonene on lipid recovery in various microalgae (Arthros-
pira platensis, Phormidium sp., Anabaena planctonica, and Stigeo-
clonium sp.) was studied using this method. A mixture of
limonene/ethanol (1:1 by volume) under pressure liquid extrac-
tion conditions (200 °C for 15 min) was the selective solvent for
obtaining lipid extracts rich in valuable fatty acids from the sources
evaluated [89]. A sequential pressurized liquid extraction approach
was also used for lipid fractionation [90].
Another recent study developed a four-step sequential method
using pressurized liquid extraction to extract and fractionate lipid
compounds from Nannochloropsis gaditana [91]. This method was
based on increasing the temperature progressively and decreasing
solvent polarity through sequential steps. In the first and second
steps, the polar compounds (i.e., carbohydrates and peptides) were
eliminated using water and hydroethanolic mixture (5% v/v) at 90 °
C. In the third and fourth steps, lipid compounds were fractionated
using hexane/ethanol mixture (3:1 v/v) at 120 and 150 °C,
respectively. This method allowed to obtain fractions enriched in
neutral and polar lipids such as triacylglycerols, diacylglycerols,
monoacylglycerols, free fatty acids, and glycolipids.
In another study, pressurized liquid extraction with methyl
acetate was used for oil extraction from Crambe abyssinica
H. seeds. The technique at 140 °C in a dynamic process (solvent
flow 3.0 mL min-1 × 30 min) provided a high extraction efficiency
with a fatty acid composition similar to commercial C. abyssinica oil
obtained by mechanical pressing. Thus, the oil obtained by pressur-
ized liquid extraction had good quality and was found suitable for
biodiesel production [92].
5.10 Isolation of Pressurized liquid extraction has also been used to extract essential
Volatile Oils oils from plants. The most common method used for this applica-
tion is pressurized hot water extraction due to its high efficiency
and “green and clean” status for essential oil extraction. Due to the
large chemical composition of essential oils (terpenes, alcohols,
ethers, oxides, aldehydes, ketones, esters, amines, phenols, hetero-
cycles, etc.) in this technology, the temperature and cooling are
typically within 50–200 °C. The ability of this method to extract
essential oils has been analyzed and compared to hydrodistillation
and Soxhlet extraction methods [93].
In a study, pressurized hot water extraction method was used
with optimized conditions of temperature and flow rate for the
isolation of volatile oils from Matricaria chamomilla leaves. The
optimal conditions of temperature and flow rate (150 °C and
4 min mL-1 for 120 min) gave the best quality yield (14%) com-
prising of α-bisabolene oxides, β-trans-farnesene, and α-bisabolol
oxides A–B [94].
The same approach was used to obtain volatile oils from Cor-
iandrum sativum L. seeds [95]. The optimized conditions were
125 °C, 0.5-mm particle size, and 2.0 mL min-1 of water flow. The
extraction process showed an important volatile oils yield (14.1%);
however, hydrodistillation (21.7%) and Soxhlet (19.4%, using hex-
ane as solvent) methods presented the best performance. Neverthe-
less, it is worth mentioning that this technique obtains higher
quality volatile oils, since small amounts of hydrocarbons are
extracted. Another study used different extraction techniques
(hydrodistillation, Soxhlet, supercritical fluid extraction, and pres-
surized hot water extraction) to extract volatile oils from C. sativum
seeds [95]. In this case, supercritical fluid extraction (sc-CO2 at 40 °
C and 300 bar for 4 h) presented the best quality and yield;
however, under pressurized hot water extraction conditions
(200 °C for 20 min), it was possible to obtain an extract of volatile
oils rich in polyphenolic compounds with a higher added value.
Solvents other than water have also been explored for essential oil
extraction under pressurized liquid extraction conditions.
In a recent study, ethanol, ethyl acetate, and hexane were
evaluated for efficient extraction of α-bisabolol using pressurized
liquid extraction and ultrasonic extraction methods from the wood
of Eremanthus erythropappus.
α-Bisabolol is an important essential oil in many plants and is
used in skin preparations, cosmetics, fine perfumes, and shampoo
coatings. The highest purity content of α-bisabolol (64.23%) was
obtained under pressurized liquid extraction conditions (55 °C,
20 min extraction) [96].
6 Recent Advancements in Pressurized Fluid Extraction
6.1 Sequential Recently, a combination of alternating supercritical fluid extraction

Biorefining and pressurized fluid extraction methods has been successfully used
to isolate and purify bioactive components from waste. Combining
the two methods allows for the use and complete separation of all
bioactive compounds from waste.
In another study of cherry stems, the resulting extract was
found to contain 42 compounds out of which 20 compounds
were unknown and found suitable for incorporation into foods
and nutraceuticals [97].
6.2 Micro- The use of supercritical fluid extraction as a microencapsulation

encapsulation and tool for rapid expansion of supercritical solutions is a useful pro-
Nanoencapsulation by gram in which active ingredients and coatings are dissolved in
Combining supercritical fluid as solvents. The supercritical fluid containing
Pressurized Liquid the solvent is held at high temperature before expanding through
Extraction and a capillary device or orifice nozzle. At this point, supersaturation
Supercritical Fluid occurs and causes the layer of material placed on the active ingredi-
Extraction ent to dissolve and form microcapsules. In addition to obtaining
bioactive substances, lipid extracts, which are useful when used in

any food, are stored in microcapsules. There are other techniques,
such as supercritical melt micronization and microencapsulation,
which use supercritical antisolvents or combine the coating material
(coating, fluid bed coating) with supercritical CO2 [98].
7 Conclusion
Hence, pressurized fluid extraction is a technique performed to

extract solid or semisolid samples using organic solvents. Elevated
temperatures are used to increase the kinetics of the extraction
process while applying high pressures to maintain the organic sol-
vents in the liquid state. Compared with traditional extraction
techniques, it is unique because extractions are performed rapidly
with reduced solvent use. It can reduce the extraction time down to
20 min per sample versus hours using Soxhlet and reduce solvent
consumption to 30 mL per sample.
It is a recent technique of extraction of analytes from solid
samples. The extraction efficiency is widely influenced by factors
such as temperature, pressure, type and volume of menstruum, and
addition of other reagents. Further derivatization reactions can be
coupled for improving the analytical applications of this method.
The rate of reaction and throughput are highly enhanced. Faster
time of reactions reduces the exposure of labile samples to air and
light.
Recent technological developments in the design of equipment
at the industrial level contribute largely to the broadening of utili-
zation of this technique in various fields.
Hence, an exhaustive understanding of the mechanisms of
recently developed extraction techniques becomes necessary for
promoting their use as cost-effective and environment-friendly
measures to isolate bioactive-rich compounds.
References
1. Carabias-Martı́nez R, Rodrı́guez-Gonzalo E, 4. Plaza M, Turner C (2015) Pressurized hot

Revilla-Ruiz P, Hernández-Méndez J (2005) water extraction of bioactives. Trends Anal
Pressurized liquid extraction in the analysis of Chem 71:39–54
food and biological samples. J Chromatogr A 5. Pattnaik M, Pandey P, Martin GJO, Mishra
1089:1–17 HN, Ashok Kumar M (2021) Innovative tech-
2. Herrero M, Castro-Puyana M, Mendiola JA, nologies for extraction and microencapsulation
Ibañez E (2013) Compressed fluids for the of bioactives from plant-based food waste and
extraction of bioactive compounds. Trends their applications in functional food develop-
Anal Chem 43:67–83 ment. Foods 10:2–8
3. Mustafa A, Turner C (2011) Pressurized liquid 6. Gilbert-López B, Barranco A, Herrero M,
extraction as a green approach in food and Cifuentes A, Ibáñez E (2017) Development
herbal plants extraction: a review. Anal Chim of new green processes for the recovery of
Acta 703:8–18
bioactives from Phaeodactylum tricornutum. 19. Otero P, Quintana SE, Reglero G, Fornari T,
Food Res Int 99:1056–1065 Garcı́a-Risco MR (2018) Pressurized Liquid
7. Sun H, Ge X, Lv Y, Wang A (2012) Application Extraction (PLE) as an innovative green tech-
of accelerated solvent extraction in the analysis nology for the effective enrichment of galician
of organic contaminants, bioactive and nutri- algae extracts with high quality fatty acids and
tional compounds in food and feed. J Chroma- antimicrobial and antioxidant properties. Mar
togr A 1237:1–23 Drugs 16:156–162
8. Hoff RB, Pizzolato TM (2018) Combining 20. Mustafa A, Trevino LM, Turner C (2012) Pres-
extraction and purification steps in sample surized hot ethanol extraction of carotenoids
preparation for environmental matrices: a from carrot by-products. Molecules 17:1809–
review of matrix solid phase dispersion 1818
(MSPD) and pressurized liquid extraction 21. Spigno G, Tramelli L, Faveri DMD (2007)
(PLE) applications. Trends Anal Chem 109: Effects of extraction time, temperature and sol-
83–96 vent on concentration and antioxidant activity
9. Herrero M, Sánchez-Camargo AP, of grape marc phenolics. J Food Eng 81:200–
Cifuentes A, Ibáñez E (2015) Plants, seaweeds, 208
microalgae and food by-products as natural 22. Al-Farsi MA, Lee CY (2008) Optimization of
sources of functional ingredients obtained phenolics and dietary fibre extraction from date
using pressurized liquid extraction and super- seeds. Food Chem 108:977–985
critical fluid extraction. Trends Anal Chem 71: 23. Barkia I, Al-Haj L, Abdul Hamid A, Zakaria M,
26–38 Saari N, Zadjali F (2019) Indigenous marine
10. Carr AG, Mammucari R, Foster NR (2011) A diatoms as novel sources of bioactive peptides
review of subcritical water as a solvent and its with antihypertensive and antioxidant proper-
utilisation for the processing of hydrophobic ties. Int J Food Sci Technol 54:1514–1522
organic compounds. Chem Eng J 172:1–17 24. Zhao S, Zhang D (2013) A parametric study of
11. Hawthorne SB, Yang Y, Miller DJ (1994) supercritical carbon dioxide extraction of oil
Extraction of organic pollutants from environ- from Moringa oleifera seeds using response
mental solids with sub- and supercritical water. surface methodology. Sep Purif Technol 113:
Anal Chem 66:2912–2920 9–17
12. Teo CC, Tan SN, Yong JWH, Hew CS, Ong 25. Tan MC, Tan CP, Ho CW (2013) Effects of
ES (2010) Pressurized hot water extraction extraction solvent system, time and tempera-
(PHWE). J Chromatogr A 1217:2484–2894 ture n total phenolic content of henna (Law-
13. Kronholm J, Hartonen K, Riekkola ML (2007) sonia inermis) stems. Int Food Res J 20:3117–
Analytical extractions with water at elevated 3123
temperatures and pressures. TrAC Trends 26. Ahmad R, Ahmad N, Aljamea A, Abuthayn S,
Anal Chem 26:396–412 Aqeel M (2021) Evaluation of solvent and tem-
14. Hyotylainen T (2009) Critical evaluation of perature effect on green accelerated solvent
sample pretreatment techniques. Anal Bioanal extraction (ASE) and UHPLC quantification
Chem 394:743–758 of phenolics in fresh olive fruit (Olea europaea).
15. Hawthorne SB, Galy AB, Schmitt VO, Miller Food Chem 342:12824–12838
DJ (1995) Effect of SFE flow rate on extraction 27. Souza MC, Silva LC, Chaves JO, Salvador MP,
rates: classifying sample extraction behavior. Sanches VL, da Cunha DT, Foster Carneiro T,
Anal Chem 67:2723–2732 Rostagno MA (2021) Simultaneous extraction
16. Hashim YZH, Kerr PG, Abbas P (2016) Aqui- and separation of compounds from mate (Ilex
laria spp. (agarwood) as source of health ben- paraguariensis) leaves by pressurized liquid
eficial compound: a review of traditional use, extraction coupled with solid-phase extraction
phytochemistry and pharmacology. J Ethno- and in-line UV detection. Food Chem Mol Sci
pharmacol 189:331–360 2:1–11
17. Azah N, Chan YS, Mailina J, Abu SA, Majid JA, 28. Chaves JO, Sanches VL, Viganó J, de Souza
Saidatul HS, Nor HH, Nik YY (2008) Com- Mesquita LM, de Souza MC, da Silva LC,
parison of chemical profiles of selected gaharu Acunha T, Faccioli LH, Rostagno MA (2022)
oils from Peninsular Malaysia. Malays J Anal Sci Integration of pressurized liquid extraction and
12:338–340 in-line solid-phase extraction to simultaneously
extract and concentrate phenolic compounds
18. Subasinghe SMCUP, Hettiarachchi DS (2013) from lemon peel (Citrus limon L.). Food Res
Agarwood resin production and resin quality of Int 157:111–252
Gyrinops walla Gaertn. Int J Agric Sci 3:356–
362
29. Klejdus B, Plaza M, Šnóblová M, Lojková L pluvialis microalga. J Pharm Biomed Anal 51:
(2017) Development of new efficient method 456–463
for isolation of phenolics from sea algae prior 39. Andrich G, Zinnai A, Nesti U, Venturi F
to their rapid resolution liquid (2006) Supercritical fluid extraction of oil
chromatographic–tandem mass spectrometric from microalga Spirulina (arthrospira) platen-
determination. J Pharm Biomed Anal 135:87– sis. Acta Aliment 35:195–203
96 40. Canosa P, Perez-Palacios D, Garrido-Lopez A,
30. Viganó J, de Paula Assis BF, Náthia-Neves G, Tena MT, Rodriguez I, Rubi E (2007) Pressur-
dos Santos P, Meireles MAA, Veggi PC, Martı́- ized liquid extraction with in-cell clean-up fol-
nez J (2020) Extraction of bioactive com- lowed by gas chromatography-tandem mass
pounds from defatted passion fruit bagasse spectrometry for the selective determination
(Passiflora edulis sp.) applying pressurized of parabens and triclosan in indoor dust. J
liquids assisted by ultrasound. Ultrason Sono- Chromatogr A 1161:105–112
chem 64:104–119 41. Chiaia-Hernandez AC, Keller A, W€achter D,
31. Dias ALB, de Aguiar AC, Rostagno MA (2021) Steinlin C, Camenzuli L, Hollender J (2017)
Extraction of natural products using supercriti- Long-term persistence of pesticides and TPs in
cal fluids and pressurized liquids assisted by archived agricultural soil samples and compari-
ultrasound: current status and trends. Ultrason son with pesticide application. Environ Sci
Sonochem 74:105–114 Technol 51:10642–10651
32. Kraujalis P, Kraujaliene V, Kazernavičiūtė R, 42. Martı́n-Pozo L, de Alarcón-Gómez B, Rodrı́-
Venskutonis PR (2017) Supercritical carbon guez-Gómez R, Garcı́a-Córcoles MT, Çipa M,
dioxide and pressurized liquid extraction ˙ of Zafra-Gómez A (2019) Analytical methods for
valuable ingredients from Viburnum opulus the determination of emerging contaminants
pomace and berries and evaluation of product in sewage sludge samples. A review. Talanta
characteristics. J Supercrit Fluids 122:99–108 192:508–533
33. Bobinaite R, Kraujalis P, Tamkut L, 43. Ramirez N, Ozel MZ, Lewis AC, Marcé RM,
Urbonaviˇcien D, Viškelis P, Venskutonis PR Borrull F, Hamilton JF (2012) Determination
(2020) Recovery of bioactive substances from of nicotine and N-nitrosamines in house dust
rowanberry pomace by consecutive extraction by pressurized liquid extraction and compre-
with supercritical carbon dioxide and pressur- hensive gas chromatography–nitrogen chemi-
ized solvents. J Ind Eng Chem 85:152–160 luminiscence detection. J Chromatogr A 1219:
34. Viganó J, Zabot GL, Martı́nez J (2017) Super- 180–187
critical fluid and pressurized liquid extractions 44. Martinez-Moral MP, Tena MT (2011)
of phytonutrients from passion fruit Focused ultrasound solid-liquid extraction
by-products: economic evaluation of sequential and selective pressurised liquid extraction to
multi-stage and single-stage processes. J Super- determine bisphenol a and alkylphenols in sew-
crit Fluids 122:88–98 age sludge by gas chromatography-mass spec-
35. Das S, Nadar SS, Rathod VK (2021) Integrated trometry. J Sep Sci 34:2513–2522
strategies for enzyme assisted extraction of bio- 45. Rodil R, Moeder M (2008) Development of a
active molecules: a review. Int J Biol Macromol simultaneous pressurised-liquid extraction and
191:899–917 clean-up procedure for the determination of
36. Sánchez-Camargo ADP, Montero L, Stiger- UV filters in sediments. Anal Chim Acta 612:
Pouvreau V, Tanniou A, Cifuentes A, 152–159
Herrero M, Ibáñez E (2016) Considerations 46. Subedi B, Aguilar L, Robinson EM, Hageman
on the use of enzyme-assisted extraction in KJ, Björklund E, Sheesley RJ (2015) Selective
combination with pressurized liquids to pressurized liquid extraction as a sample-
recover bioactive compounds from algae. preparation technique for persistent organic
Food Chem 192:67–74 pollutants and contaminants of emerging con-
37. Satapathy AK, Gunasekaran G, Sahoo SC, cern. Trends Anal Chem 68:119–132
Amit K, Rodriques PV (2009) Corrosion inhi- 47. Wang N, Su M, Liang S, Sun H (2016) Sensi-
bition by Justicia gendarussa plant extract in tive residue analysis of quinolones and sulfona-
hydrochloric acid solution. Corros Sci 51: mides in aquatic product by capillary zone
2848–2856 electrophoresis using large-volume sample
38. Rodrı́guez-Meizoso I, Jaime L, Santoyo S, stacking with polarity switching combined
Señoráns F, Cifuentes A, Ibáñez E (2010) Sub- with accelerated solvent extraction. Food Anal
critical water extraction and characterization of Methods 9:1020–1028
bioactive compounds from Haematococcus
48. Fontanals N, Pocurul E, Borrull F, Marcé RM chromatography with fluorescence detection.

(2021) Clean-up techniques in the pressurized Food Addit Contam Part A Chem Anal Con-
liquid extraction of abiotic environmental solid trol Expo Risk Assess 35:2175–2182
samples. Trends Environ Anal Chem 29:54–59 58. Moreda-Piñeiro J, Alonso-Rodrı́guez E, Mor-
49. Khan Z, Kamble N, Bhongale A, Girme M, eda-Piñeiro A, Moscoso-Pérez C, Muniategui-
Bahadur Chauhan V, Banerjee K (2018) Anal- Lorenzo S, López-Mahı́a P, Prada-
ysis of pesticide residues in tuber crops using Rodrı́guez D, Bermejo-Barrera P (2010)
pressurised liquid extraction and gas Simultaneous pressurized enzymatic hydrolysis
chromatography-tandem mass spectrometry. extraction and clean up for arsenic speciation in
Food Chem 241:250–257 seafood samples before high performance liq-
50. Chiesa LM, Labella GF, Giorgi A, Panseri S, uid chromatography inductively coupled
Pavlovic R, Bonacci S (2016) The occurrence plasma-mass spectrometry determination.
of pesticides and persistent organic pollutants Anal Chim Acta 679:63–73
in Italian organic honeys from different pro- 59. Carballo-Paradelo S, Soto-Ferreiro RM,
ductive areas in relation to potential environ- Carlosena-Zubieta A, Terán-Baamonde J,
mental pollution. Chemosphere 154:482–490 Andrade-Gardaa JM, Prada-Rodrı́guezab D
51. Kettle A (2014) Recent advancement of pres- (2012) Pressurized liquid extraction to deter-
surized liquid extraction. Chromatography mine Mg, Al, Ti, Cu, Sn and Pb in lubricating
online com 31:10–18 oils by inductively coupled plasma mass spec-
52. Kostik V (2014) Development and validation trometry. J Anal At Spectrom 27:1694–1700
of a method for the simultaneous determina- 60. Ballesteros-Vivas D, Álvarez-Rivera G, Sán-
tion of 20 organophosphorus pesticide resi- chez-Camargo ADP, Ibáñez E, Parada-
dues in corn by accelerated solvent extraction Alfonso F, Cifuentes A (2019) A multi-
and gas chromatography with nitrogen phos- analytical platform based on pressurized-liquid
phorus detection. Am J Appl Chem 2:46–54 extraction, in vitro assays and liquid chroma-
53. Feng J, Tang H, Chen D, Dong H, Li L (2013) tography/gas chromatography coupled to
Accurate determination of pesticide residues q-TOF mass spectrometry for food
incurred in tea by gas chromatography-high by-products revalorization. Part 1:
resolution isotope dilution mass spectrometry. Withanolide-rich extracts from goldenberry
Anal Methods 5:4196–4204 (Ph). J Chromatogr A 1584:155–164
54. Robinson EM, Trumble SJ, Subedi B, 61. Jelić A, Petrović M, Barceló D (2009) Multi-
Sanders R, Usenko S (2013) Selective pressur- residue method for trace level determination of
ized liquid extraction of pesticides, polychlori- pharmaceuticals in solid samples using pressur-
nated biphenyls and polybrominated diphenyl ized liquid extraction followed by liquid
ethers in a whale earplug (earwax): a novel chromatography/quadrupole-linear ion trap
method for analyzing organic contaminants in mass spectrometry. Talanta 80:363–371
lipid-rich matrices. J Chromatogr A 1319:14– 62. Garcia-Galan MJ, Diaz-Cruz S, Barcelo D
20 (2013) Multiresidue trace analysis of sulfon-
55. Campone L, Piccinelli AL, Celano R, Russo M, amide antibiotics and their metabolites in soils
Valdes A, Ibanez C (2015) A fully automated and sewage sludge by pressurized liquid extrac-
method for simultaneous determination of tion followed by liquid chromatography-
aflatoxins and ochratoxin a in dried fruits by electrospray-quadrupole linear ion trap mass
pressurized liquid extraction and online solid- spectrometry. J Chromatogr A 1275:32–40
phase extraction cleanup coupled to ultra- 63. Bodoira R, Rossi Y, Montenegro M, Maestri D,
highpressure liquid chromatography-tandem Velez A (2017) Extraction of antioxidant poly-
mass spectrometry. Anal Bioanal Chem 407: phenolic compounds from peanut skin using
2899–2911 water-ethanol at high pressure and temperature
56. Urraca JL, Marazuela MD, Moreno-Bondi MC conditions. J Supercrit Fluids 128:57–65
(2004) Analysis for zearalenone and 64. Tlili I, Caria G, Ouddane B, Ghorbel-Abid I,
α-zearalenol in cereals and swine feed using Ternane R, Trabelsi-Ayadi M (2016) Simulta-
accelerated solvent extraction and liquid chro- neous detection of antibiotics and other drug
matography with fluorescence detection. Anal residues in the dissolved and particulate phases
Chim Acta 524:175–183 of water by an offline SPE combined with
57. Rico-Yuste A, Gomez-Arribas LN, Perez- on-line SPE-LC-MS/MS: method develop-
Conde MC, Urraca JL, Moreno-Bondi MC ment and application. Sci Total Environ 563:
(2018) Rapid determination of Alternaria 424–433
mycotoxins in tomato samples by pressurised 65. Jelić A, Petrović M, Barceló D (2009) Multi-
liquid extraction coupled to liquid residue method for trace level of
pharmaceuticals in solid samples using pressur- 75. Joshi H, Kapoor VP (2003) Cassia grandis
ized liquid extraction followed by liquid Linn. f. seed galactomannan: structural and
chromatography/quadrupole-linear ion trap crystallographical studies. Carbohydr Res 338:
mass spectrometry. Talanta 80:36–48 1907–1912
66. Pereira DTV, Tarone AG, Cazarin CBB, Bar- 76. Macı́a Fuentes JA, Fernández IM, Fernández
bero GF, Martı́nez J (2019) Pressurized liquid HZ, Sánchez JL, Alemán RS, Navarro-
extraction of bioactive compounds from grape Alarcon M, Borrás-Linares I, Saravia Maldo-
marc. J Food Eng 240:105–113 nado SA (2020) Quantification of bioactive
67. Agregán R, Munekata PE, Domı́nguez R, molecules, minerals and bromatological analy-
Carballo J, Franco D, Lorenzo JM (2017) sis in carao (Cassia grandis). J Agric Sci 12:88–
Effect of addition of the extracts on the oxida- 94
tive stability of canola oil under accelerated 77. Nastić N, Borrás-Linares I, Lozano-Sánchez J,
storage conditions. Food Res Int 99:986–994 Švarc-Gajić J, Segura-Carretero A (2020)
68. Feuereisen MM, Gamero Barraza M, Zimmer- Comparative assessment of phytochemical pro-
mann BF, Schieber A, Schulze-Kaysers N files of comfrey (Symphytum officinale L.) root
(2017) Pressurized liquid extraction of antho- extracts obtained by different extraction tech-
cyanins and biflavonoids from Schinus tere- niques. Molecules 25:837–842
binthifolius Raddi: a multivariate 78. Leyva-Jiménez FJ, Lozano-Sánchez J, Borrás-
optimization. Food Chem 214:564–571 Linares I, Arráez-Román D, Segura-Carretero
69. Machado APDF, Pereira ALD, Barbero GF, A (2018) Comparative study of conventional
Martı́nez J (2017) Recovery of anthocyanins and pressurized liquid extraction for recovering
from residues of Rubus fruticosus, Vaccinium bioactive compounds from Lippia citriodora
myrtillus and Eugenia brasiliensis by ultra- leaves. Food Res Int 109:213–222
sound assisted extraction, pressurized liquid 79. Leyva-Jiménez FJ, Lozano-Sánchez J, Cádiz-
extraction and their combination. Food Chem Gurrea MDLL, Arráez-Román D, Segura-
231:1–10 Carretero A (2019) Functional ingredients
70. Kitrytė V, Bagdonaitė D, Venskutonis PR based on nutritional phenolics. A case study
(2018) Biorefining of industrial hemp (Canna- against inflammation: Lippia genus. Nutrients
bis sativa L.) threshing residues into cannabi- 11:1646–1662
noid and antioxidant fractions by supercritical 80. Cádiz-Gurrea MDLL, Borrás-Linares I,
carbon dioxide, pres Cicer arietinum surized Lozano-Sánchez J, Joven J, Fernández-
liquid and enzyme-assisted extractions. Food Arroyo S, Segura- Carretero A (2017) Cocoa
Chem 267:420–429 and grape seed by products as a source of anti-
71. Zhang Y, He Y, Liu C, Liu C, Li S (2018) In oxidant and anti-inflammatory proanthocyani-
vitro screening and isolation of human aroma- dins. Int J Mol Sci 18:376–384
tase inhibitors from Cicer arietinum by a novel 81. Jiménez-Sánchez C, Lozano-Sánchez J, Gabal-
continuous online method combining chro- dón-Hernández JA, Segura-Carretero A, Fer-
matographic techniques. J Sep Sci 41:483–492 nández- Gutiérrez A (2015) RP-HPLCESI-
72. Garcı́a P, Fredes C, Cea I, Lozano-Sánchez J, QTOF/MS2 based strategy for the compre-
Leyva-Jiménez FJ, Robert P, Vergara C, Jime- hensive metabolite profiling of Sclerocarya bir-
nez P (2021) Recovery of bioactive com- rea (marula) bark. Ind Crop Prod 71:214–234
pounds from pomegranate (Punica granatum 82. Kovačević DB, Barba FJ, Granato D, Galanakis
L.) peel using pressurized liquid extraction. CM, Herceg Z, Dragović-Uzelac V, Putnik P
Foods 10:203–214 (2018) Pressurized hot water extraction
73. Cea Paze I, Lozano-Sánchez J, Borrás- (PHWE) for the green recovery of bioactive
Linares I, Nuñez H, Robert P, Segura- compounds and steviol glycosides from Stevia
Carretero A (2019) Obtaining an extract rich rebaudiana Bertoni leaves. Food Chem 254:
phenolic compounds from olive pomace by 15–26
pressurized liquid extraction. Molecules 24: 83. Montero L, Sedghi M, Garcı́a Y, Almeida C,
3108–3118 Safi C, Engelen-Smit N, Cifuentes A, Mendiola
74. Pimentel-Moral S, Borrás-Linares I, Lozano- JA, Ibáñez E (2018) Pressurized liquid extrac-
Sánchez J, Alañón ME, Arráez-Román D, tion of pigments from Chlamydomonas sp. and
Segura-Carretero A (2020) Pressurized GRAS chemical characterization by HPLC–MS/MS.
solvents for the green extraction of phenolic J Anal Test 2:149–157
compounds from Hibiscus sabdariffa calyces. 84. Castro-Puyana M, Herrero M, Urreta I, Men-
Food Res Int 137:109–116 diola JA, Cifuentes A, Ibanez E et al (2013)
Optimization of clean extraction methods to 91. Molino A, Martino M, Larocca V, Di Sanzo G,

isolate carotenoids from the microalga Neo- Spagnoletta A, Marino T, Karatza D, Iovine A,
chloris oleoabundans and subsequent chemical Mehariya S, Musmarra D (2019) Eicosapentae-
characterization using liquid chromatography noic acid extraction from Nannochloropsis gadi-
tandem mass spectrometry. Anal Bioanal Chem tana using carbon dioxide at supercritical
405:4607–4616 conditions. Mar Drugs 17:132–143
85. Awaluddin SA, Thiruvenkadam S, Izhar S, 92. de Mello BTF, Iwassa IJ, Cuco RP, dos Santos
Hiroyuki Y, Danquah MK, Harun R (2016) Garcia VA, da Silva C (2019) Methyl acetate as
Subcritical water technology for enhanced solvent in pressurized liquid extraction of
extraction of biochemical compounds from Crambe seed oil. J Supercrit Fluids 145:66–73
Chlorella vulgaris. Biomed Res Int 23:67–78 93. Xu L, Zhan X, Zeng Z, Chen R, Li H, Xie T,
86. Wang L, Lou G, Ma Z, Liu X (2011) Chemical Wang S (2011) Recent advances on supercriti-
constituents with antioxidant activities from cal fluid extraction of essential oils. Afr J Pharm
litchi (Litchi chinensis Sonn.) seeds. Food Pharmacol 5:1196–1211
Chem 126:1081–1087 94. Khajenoori M, Asl AH, Bidgoli HN (2013)
87. Sánchez-Camargo ADP, Garcı́a-Cañas V, Subcritical water extraction of essential oils
Herrero M, Cifuentes A, Ibáñez E (2016) from Matricaria chamomilla L. Int J Eng
Comparative study of green sub- and supercrit- Trans B Appl 26:489–494
ical processes to obtain carnosic acid and 95. Eikani MH, Golmohammad F, Rowshanzamir
carnosol-enriched rosemary extracts with S (2007) Subcritical water extraction of essen-
in vitro anti-proliferative activity on colon can- tial oils from coriander seeds (Coriandrum
cer cells. Int J Mol Sci 17:1–18 sativum L.). J Food Eng 80:735–740
88. Castejón N, Luna P, Señoráns FJ (2018) Alter- 96. Santos KA, Gonçalves JE, Cardozo-Filho L, da
native oil extraction methods from Echium Silva EA (2019) Pressurized liquid and
plantagineum L. seeds using advanced techni- ultrasound-assisted extraction of α -bisabolol
ques and green solvents. Food Chem 244:75– from candeia (Eremanthus erythropappus)
82 wood. Ind Crop Prod 130:428–435
89. Otero P, López-Martı́nez MI, Garcı́a-Risco 97. Abuzara SM, Hyun SM, Kim JH, Park HJ, Kim
MR (2019) Application of pressurized liquid MS, Park JS, Hwang SJ (2018) Enhancing the
extraction (PLE) to obtain bioactive fatty solubility and bioavailability of poorly water-
acids and phenols from Laminaria ochroleuca soluble drugs using supercritical antisolvent
collected in Galicia (NW Spain). J Pharm (SAS) process. Int J Pharm 538:1–13
Biomed Anal 164:86–92 98. Soh SH, Lee LY (2019) Microencapsulation
90. de Medeiros VPB, da Costa WKA, da Silva RT, and nanoencapsulation using supercritical
Pimentel TC, Magnani M (2021) Microalgae fluid (SCF) techniques. Pharmaceutics 11:21–
as source of functional ingredients in 32
new-generation foods: challenges, technologi-
cal effects, biological activity, and regulatory
issues. Crit Rev Food Sci Nutr 62:4929–4950
Chapter 13
Fruit Waste: Potential Bio-Resource for Extraction

of Nutraceuticals and Bioactive Compounds
Milan Dhakal, Saphal Ghimire, Geeta Karki, Gitanjali Sambhajirao Deokar,
Fahad Al-Asmari, and Nilesh Prakash Nirmal
Abstract
Globally, fruits and vegetables generate almost half of total food waste, which has become a major
environmental concern. Though the ample amounts of fruit by-products are considered as industrial
waste and usually disposed of or used as animal feed and biofuel, they can be a great source of nutraceuticals
and bioactive compounds. Studies have suggested that bioactive compounds from fruit by-streams can be
extracted using conventional methods such as solvent extraction, maceration, and enzyme-assisted extrac-
tion and emerging technologies such as supercritical-fluid extraction, pressurized-liquid extraction,
microwave-assisted extraction, ultrasound-assisted extraction, and electric pulse field. This chapter discusses
the potential of extraction of nutraceuticals and bioactive compounds from fruit waste and their possibilities
for further application in the food, feed, cosmetic, and pharmaceutical industries, with their future
perspectives.
Key words Fruit waste, Valorization, Bioactive compounds, Nutraceuticals, Extraction methods
1 Introduction
Each year, more than 1.3 billion tons of edible food are wasted
globally, which is equivalent to nearly one-third of the entire
amount of food produced and more than enough to feed one
billion people. Less developed nations account for 44% of global
food waste during the post-harvest and processing stages of the
food supply chain, while developed nations in Europe, North
America, Oceania, Japan, South Korea, and China account for the
remaining 56% of these losses, of which 40% occur at the pre- and
postconsumer stages [1]. According to a United Nations report in
2017, the world population is estimated to reach 9.8 billion by
2050, and studies show that the world will need 70–100% more
food by that time. The only way to attain food security is by
299
300 Milan Dhakal et al.
minimizing food waste and food loss. Apart from food security,
food waste leads to monetary losses. Around the globe, the loss
from food waste accounts for $750 billion, which would directly
affect farmers’ and consumers’ incomes. The final disposal of food
waste in landfills (uncontrolled methane release) as well as the
production, processing, manufacture, transportation, storage, and
distribution of food all contribute to the emission of greenhouse
gases. Additional unfavorable externalities brought on by food loss
and waste include nutrient depletion, soil erosion, salinization, and
air and water pollution [2].
Fruit wastes, including citrus fruit skins, pineapple leftovers,
sugarcane bagasse, and other fruit residues (mostly peels and
seeds), are produced in enormous amounts in metropolitan areas
because of high consumption and industrial processing. One of the
main causes of municipal solid waste (MSW), which has become a
more challenging environmental issue, is fruit waste. Such wastes
are disposed of by landfilling or incineration. However, both tech-
niques impose various risks to the environment as well as human
health by releasing methane or secondary pollutants such as furans,
dioxins, and acid gases [3, 4]. In order to minimize those hazards,
recovering the bioactive components from fruit wastes, notably the
phenolic compounds, and fully utilizing them in food, pharmaceu-
ticals, and cosmetics seems to be crucial. Furthermore, value addi-
tion to agri-food waste is quite cost-effective and has minimal
impact on the environment [5]. Epidemiologically, consumption
of enough fruit and vegetables (10 servings or more per day) is
confirmed to prevent the inflammation and oxidative stress that are
linked to heart disease and diabetes, both of which have significant
mortality rates worldwide. The presence of secondary metabolites
such as fibers, carotenoids, anthocyanins, and phenols in fruits and
vegetables exhibit various antioxidant, anti-inflammatory, and
anticarcinogenic as well as biological activities [6–9]. These second-
ary metabolites are intended to help the plants grow and increase
their capacity for survival (resistance to environmental stress, ill-
nesses, and UV radiation). More than 15% of phenolic concentra-
tions are found in the skin of oranges, grapes, and lemons, as well as
the seeds of mangoes, avocados, and jackfruit than in fruit pulp.
However, a total of 55 million metric tons of biowaste are antici-
pated to be produced during each processing stage, including
5.5 million metric tons from the processing of fruits and vegetables,
6 million metric tons from canning and freezing, 5–9 million metric
tons from the processing of wine, and other sources. These losses
occur from different compositions such as under-ripe, over-ripe
fruits and vegetables or inedible parts including peels, rind, seeds,
core, rag, stones, pods, vine, shell, skin, pomace [10]. Along with
the fruit pomace, secondary metabolites such as fibers, carotenoids,
anthocyanins, and phenols are lost during manufacturing [11].
Fruit Waste: Potential Bio-Resource for Extraction of Nutraceuticals and. . . 301
Food waste can be valorized into value-added goods using a

sustainable process that lowers the amount of food waste dumped
in landfills and reduces greenhouse gas emissions. Food waste
valorization is a growing sector as people become more conscious
of the negative environmental effects of food waste disposal.
Numerous decision-makers have embraced the concept of valoriza-
tion, and the results of their actions have been sustainable projects
in the social, business, and industrial spheres of society. Since
valorization is still in its early stages, these judgments must be
carefully considered; in addition, it is necessary to do extensive,
difficult assessments of the product’s life cycle, life-cycle costs, and
social life cycle [12]. Successful bioactive chemical extraction from
fruit or vegetable wastes is feasible, and their enormous potential in
pertinent industries can be realized through the manufacture of
various dietary supplements and functional foods as well as food
additives and food preservatives [13, 14]. To make use of this
bioactive-rich waste, extraction techniques should be novel, sim-
pler, sustainable, and cost-effective, and there have been continu-
ous efforts to identify such techniques. Some safer and more
environmentally friendly extraction methods are supercritical fluid
extraction, pulsed electric field, hydrodynamic cavitation, etc. Such
extracted bioactive have been currently utilized in food as coloring
and flavoring ingredients and have been identified as safe (GRAS)
[15]. By adding bioactive from fruit and vegetable waste, several
examples of functional food products, including extruded snacks,
bakery goods, beverages, breakfast cereals, and dairy products, have
been effectively produced [16]. Incorporating these bioactive com-
pounds has also prevented meat from spoiling by improving the
appearance and softness of the meat and providing an antibacterial
impact [17]. Dried fruit pomace can be added to bread, cakes,
muffins, cookies, biscuits, and other baked goods to increase their
dietary fiber content. Although the byproducts of the processing of
fruits are all included in the same category in this study, each
by-stream has difficulties for valorization. Utilizing fruit processing
byproducts raises the issue of microbial deterioration caused by
excessive moisture content [18, 19]. Drying of byproducts for
processing can lead to a concentration of pesticides. Additionally,
even at modest levels of water activity, molds can still produce
mycotoxins. The hazards that need to be considered before being
valued can also vary greatly across different product sections includ-
ing stems, leaves, and skins. The use of these by-streams as animal
feed is the focus of most current research on the subject. Data on
safety must be compiled if they are to be utilized in food applica-
tions, and existing food safety legislation must be expanded to
cover these by-products.
2 Fruit Byproducts
Portions of fruits, vegetables, and other food products are wasted

or lost because of the morphological qualities of the product,
improper handling techniques, or discarding for a variety of rea-
sons. Depending on the commodities and morphological compo-
nents, such as leaves, roots, tubers, skin, pulp, seeds, stones,
pomace, etc., there are different amounts and types of wastes and
losses. When it comes to waste, apples produce 89.09% of the
finished goods during slicing and 10.91% of seed and pulp as
byproducts [11]. The main processing waste left over from making
apple juice is called apple pomace. Apple pomace mostly consists of
peels/flesh (95%) and stem (1%), with 2–4% of seeds also present.
Pectins, cellulose, hemicelluloses, and lignins make up 35–60% of
the dietary fiber in apple pomace. Banana peels, which make up
about 35% of the weight of the fruit, have drawn attention in recent
years because of their high concentrations of phenolics (hydroxy-
cinnamic acids), flavonoids, phytosterols, carotenoids, anthocya-
nins, biogenic amines, vitamins (B3, B6, B12, C, and E) and
dietary fibers. Currently, industries use avocado to make guaca-
mole, spreadable puree, fresh and frozen chunks, and oil. Addition-
ally, processing produces a lot of by-products (peel and seed),
which consist of 33% of the fruit. Given their high concentration
of phenolic acids (hydroxybenzoic and hydroxycinnamic acids),
condensed tannins (procyanidins), and flavonoids (flavonols) with
recognized antioxidant and anti-inflammatory actions, avocado
peels and seeds have a high bioactive potential. Papaya biomass,
which includes the seeds and skin, is wasted to a greater than 20%
extent. Around 30% of the oil in papaya seeds is made up of mostly
palmitic, stearic, oleic, and linoleic acids, as well as tocopherols and
carotenoids, with beneficial nutritional and functional qualities.
Papaya seeds are an excellent source of beneficial compounds that
can be used to create food additions or supplements [20]. Mandar-
ins produce roughly 16% peels and 84% of the finished product after
being peeled [11]. There are a lot of byproducts produced, primar-
ily peel and seeds, which can make up as much as 50% of the original
fruit weight after processing citrus fruit to produce juice. Peel
(60–65%), internal tissues (30–35%), and seeds (0–10%) are
among the by-products. Citrus peel contains dietary fiber, poly-
phenols, essential oils, and vitamins, all of which have the potential
to be bioactive. The dry weight of peels can be up to 70% dietary
fiber. Citrus by-products are a rich source of natural flavonoids,
such as hesperidin, naringin, and narirutin, flavonols, such as rutin
and quercetin, flavones, such as diosmin and tangeretin, as well as
several other compounds, such as flavanones, flavanone glycosides,
and polymethoxylated flavones, which are specific to citrus. The
peel is rich in phenolic acids, particularly hydroxycinnamic (ferulic,
p-coumaric, sinapic, caffeic, and chlorogenic) and hydroxybenzoic

(vanillic, p-hydroxybenzoic, and gallic). In the process of proces-
sing pineapples, 14% of the peels, 9% of the core, 15% of the pulp,
15% of the top, and 48% of the overall product are produced
[11]. Pineapple peel (PP) wastes include a significant amount of
commercial bioproducts, including pectin, dietary fiber, and
enzymes, which might be used for value-added products
[21]. Mango processing yields roughly 11% peels, 13.5% seeds,
18% inedible pulp, and 58% finished product. Even more than the
fruit pulp, mango peels and seeds contain significant amounts of
bioactive substances such as dietary fibers, phenolic compounds,
carotenoids, and vitamins [11]. Around 5–9 MMT of solid waste is
produced annually by the processing of grapes and wine, which
accounts for 20–30% of all processed materials. The amount and
soluble/insoluble ratio of dietary fiber in grape pomace varies by
variety and includes hemicelluloses, cellulose, and pectin. The main
polyphenols are phenolic acids, flavanols, flavonols, stilbenes, and
anthocyanins. About38–52% (dry weight) of grape pomace is made
up of grape seeds, which make up 2–5% of the weight of the fruit.
Grape seeds also include polyphenols, primarily gallic and caftaric
acid, catechin, epicatechin, epicatechin gallate, and procyanidins B1
and B2, in addition to lipids (such as linoleic acid), proteins, carbs,
and vitamin E. By cold-pressing seeds, seed oil can be produced
while keeping more nutrients that are healthy. Approximately
6 MMT of solid waste from the canning and freezing of fruits and
vegetables is produced each year, with 20–30% of that waste being
made up of leaves, stalks, and stems. The waste of jackfruit is mainly
from rind and seeds, which accounts for 50–70%. Jackfruit peel is
reported to be rich in cellulose, pectin, protein, and starch com-
prising about 27.75%, 7.52%, 6.27%, and 4%, respectively [22]. The
weight of the dragon fruit is comprised of 22–44% skins. Betalains,
phenolics, and dietary fibers like pectin and oligosaccharides have
been identified as the key bioactive constituents in dragon fruit
peels. The identification of and possible health advantages of beta-
lains have drawn the most interest of these [23]. Guava is wasted
mainly from core, seeds, and skin. Seeds of guava are an important
byproduct while processing juice. According to reports, seeds have
a higher high-fat content than core and pulp. Studies have shown
that guava seeds’ mono- and polyunsaturated fatty acids have sev-
eral positive effects [24]. Nearly one-third of the watermelon is
made up of the watermelon rind (WMR) [25]. Alkaloids, saponin,
cardiac glycosides, flavonoids, phenols, lipids, proteins, citrulline,
fiber, carbs, vitamins, and mineral salts are all present in the WMR.
WMR has a wide range of advantages for metabolic health, includ-
ing antioxidative, antihypertensive, antidiabetic, hypolipidemic,
and vasodilating properties, according to epidemiological research.
Therefore, there should be a possibility for this solid waste to be
transformed into usable products that can be added to the human
diet [26]. To conclude, foods enriched with health-improving

ingredients (phenols, carotenoids and other colors, vitamins, die-
tary fibers, among other things) may be created through the right
use of waste materials obtained from horticultural products in order
to mitigate environmental issues and promote human health [11].
3 Bioactive Compounds in Fruits with Potential Health Benefits
Fruit processing produces two different types of waste: solid waste

made up of pulp remnants, rind, peel, pomace, skin, seeds, and
stones; and liquid waste made up of juice and wash waters, which
has a high proportion of bioactive compounds and a higher poten-
tial to be used as functional ingredients in other formulations. Due
to ignorance and lack of understanding of the need of turning
“waste into wealth,” these wastes are typically dumped or are
merely used as a feed source. The fruit’s varied parts, such as the
peel/skin or the kernels, are concentrated with distinct bioactive
substances in varying amounts. Various studies have found that
fruit kernels and skins are rich sources of phytochemicals and
essential minerals. For instance, the phenolic content of the skin
and seeds of many fruits and vegetables, such as grapes, lemons,
oranges, and avocados, as well as the seeds of jackfruit, mangoes,
and peaches, is significantly higher than that of the pulp [27–
30]. Bioactive compounds like phenolic compounds (phenolic
acid, carotenoids, flavonoids), bioactive proteins (peptide isolate,
amino acids), biogenic amines, fatty acids, fibers, and others can all
be found in abundance in fruit waste byproducts (Table 1). Phyto-
chemicals, phytosterols, and essential oils, for instance, can all be
found in large quantities in fruit seeds. Pectin, priceless fibers, and
minerals are also present in the peels [31]. The bioactive substances
reduce the risk of developing heart-related illnesses, cancer, catar-
acts, Alzheimer’s, Parkinson’s, and aging-related disorders. These
substances work defensively against chronic diseases, limiting the
development of carcinogenic molecules and balancing the immune
system because of their high antioxidant and antibacterial activity.
These substances are advantageous whether taken as dietary sup-
plements or as an ingredient in functional foods. In addition to
their benefits for nutraceuticals, natural antioxidants and colorants
may be preferable to synthetic antioxidants in the processing and
pharmaceutical industries [32–34]. The most abundant bioactive
molecules present in fruit wastes are enzymes, oils, carotenoids,
vitamins, polyphenols with minor portions of biologically active
proteins, and biogenic amines. The presence of these bioactive
molecules in different portions of fruit and their potential health
benefits with examples is discussed in a later session.
Table 1
Summary of bioactive compounds in major fruit waste
Major bioactive compounds

Fruits Parts and antioxidant activity Concentration References
Apple Pomace Malic acid 1.08 g/100 g [63–66]
Total polyphenol 4620 mg/ kg
Quercetin, phloridzin, phloretin 3.31 mg/g
Chlorogenic acid 6.89 mg/g
Flavonoids 2153–3734 mg/kg dw
Anthocyanins 50–130 mg/kg dw
Dihydrochalcones 688–2535 mg/kg dw
Seed Linoleic acid 63.76 g/100 g [64, 67]
Phloridzin 2.96 μg/g
Oleic acid 34.84 g/100 g
Antioxidant activity (DPPH) 0.71 μg TROLOX/g
Total phenolic acid 1.61 μg GAE/g
Hydroxybenzoic acid 2.99 mg/g
Banana Peel Total phenolic content 872.7 mg of [68–74]
Gallocatechin GAE/100 g DM
Epigallocatechin 91.9 mg/100 g DM
Gallic acid 65.9 mg/100 g DM
Rutin 47 mg of (GAE)/g
Myricetin DM
Kaempferol 973.08 mg/100 g DE
Isoquercitrin 11.52 mg/100 g DE
Naringenin 9.30–28.80 μg/mL
Ferulic acid 10.47–14.54 μg/mL
Cinnamic acid 8.47 mg/100 g DE
Alpha-hydroxycinnamic acid 1.63–60 mg/100 g
Sinapic acid DM
p-Coumaric acid 1.93 ng/g
Dopamine 40.66 ng/g
L-dopa 10.29 ng/g
8.05 ng/g
1.17–1.72 mg/g DM
0.31 mg/g DM
Stem Phenols 5.5743 mg/kg [75]
Phytate 1.2967 mg/kg
Hemaglutinin 1.8814 mg/kg
Ascorbic acid 0.44 mg/mL
β-Carotene 0.066 mg/100 mL
Lycopene 0.006 mg/100 mL
Total flavonoid 0.032 mg EQ/g
Saponin 14.494%
Alkaloid 0.347%
Flavonoid 0.253%
Tannin 67.594%
Hydroxyl scavenging activity 0.543%
(IC 50%) 1.318%
Chelating effect of ferrous iron 1.296%
(IC 50%)
Hydrogen peroxide scavenging
activity (IC50%)
(continued)
Table 1
(continued)

Citrus Peel Phenolic acids 103 mg/100 g [76]
(orange) Gallic acid 3 mg/100 g
Hydroxybenzoic acid 2 mg/100 g
Chlorogenic acid 12 mg/100 g
Syringic acid 2 mg/100 g
Vanillic acid 2 mg/100 g
Rosmarinic acid 8 mg/100 g
Trans-2-hydroxycinnamic acid 4 mg/100 g
Trans-cinnamic acid 2 mg/100 g
p-Coumaric acid 34 mg/100 g
Ferulic acid 33 mg/100 g
Flavonoids 33 mg/100 g
Epicatechin 4 mg/100 g
Catechin 4 mg/100 g
Rutin 14 mg/100 g
Naringin 7 mg/100 g
Flavone 3 mg/100 g
Seed Total phenolic compounds 5.60 mg/kg [77]
Total carotenoids 7.85 μg β-carotene/g
Phytosterol 158.21 mg/100 g
Tocopherols 153.67 mg/kg
Dates Seed Lignin 24.34% [67, 78]
Cellulose 20.63%
Hemicellulose 13.49%
Protocatechuic 7.9 mg/100 g
Caffeoylshikimic 28.3 mg/100 g
Date press Total phenolic content 11.79 mg of GAE/g [79]
cake Total flavonoid content 1.89 mg of quercetin/
IC50 g
Cupric reducing antioxidant capacity 1.42 mg/mL
Ferric reducing antioxidant potential 1.57 mg Vit C/g
Chelating effect 9.32 mg Vit C/g
7.87%
Guava Peel Total phenolic compounds 589.49 mg [67]
Anthocyanin GAE/100 g
Total flavonoids 121.85 Eq. mg CyGE/
Carotenoids 100 g
374.05 mg de
catechin/100 g
4.47 mg of
β-carotene/mL
Seed Vitamin C 87.44 mg/100 g [67, 80]
Carotenoids totals 1.25 mg/100 g
Soluble dietary fiber 0.39 g/100 g
Insoluble dietary fiber 63.55 g/100 g
Linoleic acid 75.25%
Oleic acid 11.40%
Palmitic acid 6.6%
(continued)
Table 1
(continued)

2-Chloroethyl linoleate 44.53%
2-Pentanone 4-hydroxy-4-methyl 23.56%
n-Hexadecanoic acid 9.85%
Tocopherol (α-tocopherol) 654 ppm
Carotenoids (β-carotene) 19.24 ppm
Phytosterol (stigmasterol, 420 mg/100 g
campesterol) 9.6 mg/100 g
Vanillin 3.9 mg/100 g
Vanillic acid 2.4 mg/100 g
Cinnamic acid 9.4 mg/100 g
Cinnamaladehyde 1048.9 mg/100 g
β-sitisterol 82.6 mg/100 g
γ-tocopherol
Jackfruit Peel Phenolic content 10.0 mg CE/g [81, 82]
Flavonoids 158 mg GAE/g
Gallic acid 12.08 μg/g
Ferulic acid 13.41 μg/g
Tannic acid 5.73 μg/g
Seed Polyphenols 243 mg GAE/100 g [81, 82]
Flavonoids 2.03 mg EC/100 g
Tannins 0.06–0.229 mg/100 g
Gallic acid 1.105 mg/100 g
Ferulic acid 0.216 mg/100 g
Flesh Gallic acid 19.31 μg/g [81, 82]
Ferulic acid 2.66 μg/g
Papaya Peel Gallic acid 18.06 μg/g DW [83, 84]
Caffeic acid 29.28 μg/g DW
P-coumaric acid 38.16 μg/g DW
Ferulic acid 95.46 μg/g DW
Quercetin 3.17 μg/g DW
Seed Total phenolic content 1080 mg GAE/100 g [83, 84]
Total flavonoid content DW
117.7 mg GAE/100 g
DW
Pineapple Peel Phenolic content 5803.221 mg GAE/g [85–87]
Gallic acid 31.76 mg/100 g DW
Catechin 58.51 mg/100 g DW
Epicatechin 50.00 mg/100 g DW
Ferulic acid 19.5 mg/100 g DW
Bromelain 13.158 μm/mL
Carotenoids 1.82 μg/g DM
Core Phenolic content 1543.51 mg GAE/g [86, 87]
Ferulic acid 19.5 mg/100 g DW
Carotenoids 0.35 μg/g DM
Crown Total phenolic content 41.34 mg GAE/g DM [86, 87]
Total flavonoid content 33.69 mg QE/g DM
(continued)
Table 1
(continued)

Pomegranate Peel Total phenolic content 18–510 mg/g DW [88, 89]
Tannin 193–420 mg/g DW
Flavonoids 84–134 mg/g DW
Gallic acid 123.79 mg/100 g DW
Ellagic acid 35.89 mg/100 g DW
Caffeic acid 20.56 mg/100 g DW
Seed Total polyphenol 11.84 mg GAE/g [90]
Total flavonoid content 6.79 mg RE/g
Total anthocyanin 40.84 mg CGE/g
Tannins 29.57 mg TAE/g
Tomato Peel Chlorogenic der 33–141.10 mg/kg [91–93]
p-Coumaric 07.38–26.58 mg/kg
p-Coumaric der 16.70–101.99 mg/kg
Quercetin 5.04–13.68 mg/kg
Rutin 107.06–410.13 mg/
Rutin der kg
Naringenin 36–109.75 mg/kg
Lycopene 73.52–287.62 mg/kg
B-carotene 167.43 mg/kg DW
Lutein 55.20 μg/g DW
Tocopherols 0.65–1.54 mg/100 g
Caffeic acid-glucoside isomer 1.62 g/100 g dw
Caffeic acid 0.74 mg/100 g
Syringic acid 0.55 mg/100 g
Di-Caffeoylquinic acid 0.547–1.122 mg/
Tri-Caffeoylquinic acid 100 g
0.812–1.113 mg/
100 g
0.591–0.662 mg/
100 g
Seed Gallic acid 0.11–6.94 mg/100 g [91, 94–
Ferulic acid 1.67–9.08 mg/100 g 99]
Kaempferol <0.001–2.01 mg/
Quercetin 100 g
Rutin <0.001–0.90 mg/
Coumaric acid 100 g
Phloridzin 0.065–3.53 mg/100 g
Phloretin 2.58 mg/100 g
Procyanidin B2 1.35 mg/100 g
Naringenin 26.72 mg/100 g
Myricetin 76.62 mg/100 g
Caffeic acid 0.16–0.35 mg/100 g
Vanillic acid 0.34–0.88 mg/100 g
Sinapic acid 0.95–2.19 mg/100 g
Chlorogenic acid 2.01 mg/100 g
Lycopene 1.82–3.56 mg/100 g
ß-Carotene 0.05–1.41 mg/100 g
1.6–16.70 mg/100 g
0.093–5.500 mg/
100 g
(continued)
Table 1
(continued)

Watermelon Rind Total phenolic content 385–507 mg CAE/kg [100]
Polyphenols 63.33 mg TAE/g
Citrulline 24.7 mg/g
Flavonoids 1.105 mg RE/g
Seed Total phenolic content 0.087 mg GAE/g [101–105]
Sinapic acid 152.330 μg/mL
Gallic acid 2.559 μg/mL
Caffeic acid 1.33 μg/g
Lignans pinoresinol 1.02 μg/g
Alkaloids 28.33 mg/g
Flavonoids 0.67 mg/g
Tannins 50.6–64.5 mg/g
3.1 Enzymes Enzymes are used in a biochemical reaction as a biocatalyst, with

several health benefits that aid in the digestion process by a partial
breakdown of complex biomolecules, that is, fat, carbohydrate, and
protein, into its smaller building blocks which ultimately makes our
stomach easier to breakdown and absorb necessary nutrients. The
most abundant enzymes in the fruits are amylase, protease, and
lipase. Amylases are enzymes responsible for the breakdown of
carbohydrates into their simpler sugar, whereas proteases are
responsible for the breakdown of protein molecules into their
small peptides as well as in the simpler form of amino acids, and
lipases are responsible for the breakdown of fats into three fatty
acids along with a glycerol molecule [35]. Some examples of fruits
that contain enzymes are:
• Pineapple consists of protease enzymes, especially bromelain.
• Papaya also consists of protease enzymes called papain.
• Mango consists of amylase enzymes.
• Banana consists of amylase and glucosidases.
• Avocado consists of lipase enzymes and polyphenol oxidase in
small quantities.
• Kiwi fruit consists of a protease called actinidain.
Lack of these enzymes in the stomach causes difficulty in the
breakdown process and can lead to digestive issues, or problems like
food intolerances in some cases. Thus, consuming fruit enzymes
that are natural digestive enzymes can help to get rid of this prob-
lem. There are several additional health benefits of fruit enzymes as
these are found to act as antioxidants having anti-carcinogenic
effect and also have been proven to maintain cardiovascular health,
increase the buffering capacity of blood by maintaining the pH level

of the bloodstream, and increase the body’s immune system to
prevent common illness and several forms of infections [36–39].
3.2 Oils Fruit waste, especially seed/kernel parts, has a higher amount of
bioactive oil concentrated in it as compared to other parts. In the
research conducted by da Silva and Jorge [40] for different fruit
seeds, the lipid content ranged from 7.0% to 40.4%. Kernels/seeds
from different citrus fruits, apples, guava, tomato, grape, mango,
pumpkin, passion fruit, orange, melon, kumquat, etc. contain a
different proportion of fatty acids in their oil. These oils are poten-
tial sources of essential fatty acids. Major fatty acids in fruit oil are
palmitic acid (C16:0), palmitoleic acid (C16:1), stearic acid (C18:
0), oleic acid (C18:1), linoleic acid (C18:2), linolenic acid (C18:3),
and arachidic acid (C20:0). For example, in mango seed oil, steric
acid (C18:0) and oleic acid (C18:1) are the major fatty acids. While
in papaya, mango, and soursop seed oil, oleic acid (C18:1) and
linoleic acid (C18:2) are the major fatty acids. These oils are utilized
more frequently because of their greater health advantages in the
chemical, pharmaceutical, cosmetic, and food industries, as well as
for the direct development of functional foods. Fruit seeds and
their oils have added nutritional and health benefits due to phyto-
nutrients and phytochemicals, which are natural chemicals that take
part in a variety of biological processes, improve health status, or
prevent or treat sickness situations. Similar advantageous effects
have also been connected to other substances, including phenolics,
tocopherols, and carotenoids as well as lipids like FA, sterols, or
polar lipids. Fruit seed oils have been shown to have antioxidant,
antiproliferative, anti-inflammatory, and anti-diabetic properties.
Tocopherols act as primary antioxidants, as they donate one hydro-
gen atom to the peroxide radical, thus, interrupting the chain
oxidative process. There are 4 types of tocopherols mostly present
in fruit oil, namely: α-Tocopherol, γ -Tocopherol, β- Tocopherol,
and Δ-Tocopherol. α-Tocopherol is the most active homologous in
humans, and it performs the biological role of vitamin
E. Phytosterols and phytostanols present in fruit oil perform hypo-
cholesterolemic activity after ingestion by reducing cholesterol
absorption by the intestine.
3.3 Carotenoids Carotenoids are present in abundant amounts in both edible and
inedible portions of fruits and vegetables, but significantly higher in
inedible portions like peels and pomace. Different types of conven-
tional and novel green techniques are used to extract carotenoids
from fruit waste. Carotenoids in higher concentration have been
successfully extracted from many fruits wastes, that is, carrot bio-
waste (8.27 mg/100 g), mango pulp (2.18 mg/100 g), passion
fruit peels (1.18 mg/100 g), seabuckthorn pomace (28.3 mg/

100 g), red jalapeno pepper (230.54 mg/100 g), tomato bypro-
ducts (631.1 mg/100 g), sweet peppers (41.37 mg/100 g), etc.
[41]. The amount of total carotenoids ranged from 0, in mango
seed oil, to 49.40 mg β-carotene/g, in papaya seed oil [40]. These
are lipid soluble components, mainly exerts pro-vitamin A function,
along with this they support the immune system, protect skin from
harmful UV radiations, fasten wound healing process, and show
antioxidant capacity, as they protect the cell against lipid oxidation,
preventing the risk of degenerative diseases, such as cancer, heart
diseases, and cell degeneration [41]. So, these properties made
carotenoids to be used in the food, pharmaceutical, cosmetic, and
feed industries.
3.4 Vitamins Fruit waste is a rich source of several water-soluble vitamins, includ-
ing thiamine, niacin, riboflavin, vitamin C, and niacin, as well as
lipid-soluble vitamins, including vitamins A, D, E, K, and carote-
noids [42–44]. All three antioxidant vitamins A, C, and E can be
found in abundance in berries. Ascorbic acid can be found in a wide
variety of fresh fruits [45]. Vitamin C plays a crucial role in the
prevention of cancer because of its potent antioxidant action, which
shields our cells from oxidative damage. It is an effective electron
donor in biological systems in addition to possessing redox poten-
tial. Vitamin C lowers oxidative stress on the stomach’s mucosa,
DNA damage, and inflammation via scavenging reactive oxygen
species (ROS). By converting nitrous acid in the stomach to nitric
oxide and generating dehydroascorbic acid, it also prevents gastric
nitrosation and the creation of N-nitroso compounds. And finally,
it strengthens the host’s immune system. It inhibits stomach cell
growth and induces apoptosis, which directly affects the growth
and virulence of Helicobacter pylori [46]. The B-complex vitamins
(thiamine, riboflavin, and niacin) are crucial cofactors in biochemi-
cal processes and are necessary for healthy skin, normal body
growth and development, appropriate heart and neuron function,
and the production of red blood cells. Since individuals who suffer
from heart failure have a vitamin B deficit, vitamin Bs are directly
engaged in energy metabolism, and there is growing interest in
their potential to prevent heart failure [47]. Antioxidant-rich vita-
mins, such as lipid-soluble vitamins A, D, E, and K, can reduce the
risk of cardiovascular disease, cancer, and neurological disorders
[48]. Red and purple fruits have significant quantities of anthocya-
nins, which have bacteriostatic and bactericidal activity against
many pathogens (including Staphylococcus sp., Klebsiella sp., and
Helicobacter and Bacillus). Anthocyanins possess a variety of
biological effects, including anti-tumor, anti-inflammatory, antiox-
idant, anti-diabetic, and neuroprotective [49].
3.5 Alkaloids and Alkaloids and polyphenols are secondary metabolites of plants
Polyphenols which act directly by reducing activity, scavenging the free radical
and indirectly by chelating the prooxidant metal ions. Alkaloids are
water soluble compounds containing one or more nitrogen atom in
their molecule and possess significant biological activity. They
directly interact with neurotransmitters and results in various psy-
chological and physiological responses in human body [50]. Caf-
feine, theobromine, pipernine, quinine, capsaicin, solasodine,
solamargine, and solasonine are some examples of alkaloids com-
monly present in fruit waste [51]. Among all phenolics, dietary
phenolics, that is, polyphenols, flavonoids, and phenolic acids, are
considered to have major health benefits and preservative action in
food formulations. These compounds are rich in peel, rind, and
seeds of fruit waste. The major phenolic compounds present in fruit
seed oils are p-Coumaric acid, salicylic acid, and quercetin, with a
significant amount of gallic acid, catechin, caffeic acid, and epica-
techin in some fruits. Peels of citrus fruits contain bioactive com-
ponents and have been used traditionally in various places to treat
cough, muscle pain, digestive issues, and skin inflammation. Peels
of pomegranate contain punicagranine, an anti-inflammatory pyr-
rolizine alkaloid. Annona crassiflora fruit peels’ polyphenol-rich
fraction exhibits antioxidant properties that may find use in the
treatment of diabetes in clinical settings [52].
3.6 Bioactive These part of the fruit waste does not have a nutritional function as
Polysaccharides and they are resistant to enzymatic digestion in our body but has a vital
Dietary Fibers role in digestive function commonly known as cellulose and non-
cellulosic polysaccharides, that is, pectic substances, hemicellulose,
gums, mucilages, and other noncarbohydrate portions, such as
lignin, categorized as dietary fiber [53]. These compounds possess
several health benefits, such as regulating the feces output; reducing
the risk of obesity by providing a satiety effect, diabetics, and
hypertension; and lowering the occurrence of colorectal cancers
by trapping mutagenic substances and not allowing them to reach
the bloodstream [54].
3.7 Bioactive Protein The nonedible portion of fruits is a good source of bioactive
and Peptides proteins. For example, peels of citrus fruits ranged from 2.5% to
9.0% protein content [55, 56], whereas peels of papaya, kiwi, and
avocado fruit were found to be 1.55%, 1.79%, and 1.57%, respec-
tively [57]. Some enzymes have been discovered, which exert bio-
activity like actinidin from the seeds of kiwi fruit [58], vicilin-like
protein watermelons seeds [59], and leptin from seeds of jackfruit
[60]. Citrus natural peptides have been investigated as a potential
source of novel atheroprotective medicines due to their compara-
tive benefits over small molecules in the creation of anti-
inflammatory and cardioprotective drugs.
3.8 Biogenic Amines The biogenic amines represent the nitrogenous compounds result-
ing from several enzymatic reactions, such as reductive amination,
decarboxylation, transamination, and degradation of the
corresponding precursor amino acids. Vasoactive amines, that is,
tryptamine, histamine, and tyramine have a positive role in blood
pressure control [61]. Five amines have been found in the study
conducted by [62] for pineapple, papaya, guava, mango, and pas-
sion fruit, that is, spermidine, putrescine, agmatine, serotonin, and
spermine. The total amine levels varied from 0.77 to 7.53 mg/
100 g in mango and passion fruits, respectively. Among these
amines, spermidine was detected in every test fruit sample, whereas
spermine and putrescine were detected in most of the samples.
Agmatine and serotonin were found in abundant amounts in pas-
sion fruits, papaya, and pineapple. Passion fruit seemed to be a
good source of polyamines spermine and spermidine, with 2.43
and 3.05 mg/100 g, which has an important role in maintaining
health, growth, and antioxidant activity and regulating membrane
permeability. Serotonin governs your mood and is responsible for
happiness. It also controls when you sleep and wake up, helps you
think, keeps your mood stable, and regulates your sexual drive. It is
also linked to the stomach to mediate reflex action and to lower the
risk of thrombosis.
4 Extraction Technologies for Bioactive Compounds from Fruit Waste
In order to utilize the bioactive compounds from fruit waste,

extraction is the most important step. Generally, extraction is car-
ried out using different solvents, acids, alkalis, steam diffusion, and
hydro-distillation. Selection of method of extraction depends on
the bioactive compound to be extracted, source (raw materials) and
desired purity. Extraction of bioactive compounds could be affected
by several factors, such as extraction conditions (temperature, pres-
sure), part of the fruit (peel, pomace, seed), particle size, extraction
time, and solvent used for extraction. The methods and condition
of extraction play a significant role in terms of purity, yield, time of
extraction, cost-effectiveness, and effect on the structure of bioac-
tive compounds [106]. Therefore, the extraction method and para-
meters should be selected based on the target bioactive compound
and source (raw material). Bioactive compounds from fruit waste
can be extracted mainly by conventional extraction techniques and
emerging/novel extraction techniques. Extraction is an effective
way to obtain bioactive compounds. However, high temperature
and long extraction duration cause instability and loss of function-
ality of extracted bioactive compounds. Figure 1 shows the sche-
matic diagram of different extraction technologies for the
extraction of bioactive compounds from fruit waste. Table 2
Fig. 1 Schematic diagram of different extraction technologies for the extraction of bioactive compounds from
fruit waste
shows the extraction of bioactive compounds from different fruit

waste using conventional as well as emerging technologies. Both
technologies are discussed below.
4.1 Conventional Conventional extraction techniques are the traditional extraction

Extraction methods that are in use since ancient times and are the most
Technologies frequently used techniques for extracting bioactive compounds
from plants. The main principle of this extraction technique is
thermal treatment and solvent extraction. Most of the conventional
method depends upon the extraction potential of solvent and
application of heat or agitation. Moreover, the process is strongly
dependent on the polarity of the extracting solvent. However, this
method has low efficiency, longer extraction time, and high tem-
perature and requires a large volume of organic solvent
[13, 128]. Most common conventional extraction techniques
include (a) Soxhlet extraction, (b) hydro-distillation, and
(c) maceration.
4.1.1 Soxhlet Extraction Soxhlet extraction has been used as a classical method of extracting
bioactive compounds from plant parts since ancient times. Though
the Soxhlet extraction method was designed for lipid extraction,
this method has been popularly used to extract bioactive com-
pounds from plants part including fruits. The advantages of Soxhlet
are it is simple and inexpensive. Nevertheless, this is time-
consuming and requires a large amount of solvents; thus, it is not
environmentally friendly. Alcohols, mostly ethanol and methanol,
Table 2
Extraction of bioactive compounds from different fruit waste using conventional and emerging technologies
Extraction
technique Fruit waste Bioactive compounds Extraction conditions Results References
Soxhlet Grape peel Phenolic compounds 80 mg GAE/g [107]

extraction
Maceration Grape and orange residue Phenolic compounds 67.1 and 146.6 mg [108]
GAE/g (dry
weight)
Tangerine peels Total phenolic content 40 °C, 80% methanol [109]
Peels of apple, apricot, avocado, banana, custard Phenolic compounds 4 °C, 70% ethanol, 0.45–25.2 mg [110]
apple, dragon fruit, peach, pear, pineapple, 120 rpm agitation GA/g
plum, pomegranate
Mango seed kernel Total phenolic content 50% ethanol 107.7 mg GAE/g [111]
Microwave- Pomegranate peel Phenolic compounds 600 W 199.4 mg GAE/g [112]

assisted dry weight
extraction Pomegranate peel, carob fruit peel, banana peel Polyphenols [113–
(MAE) 115]
Avocado peels Phenolic compounds 500 W, 95.1 s 281.4 mg GAE/g [116]
dry weight
Ultrasound- Grape seeds Total phenolic contents 53.14% ethanol, 46.03 °C Higher yield [117]
assisted and 24.03 min
extraction Seeds and peels of avocado Trans-5-O-caffeoyl-D-quinicacid, Ethanol-water solvent, [118]
(UAE) procyanidin B1, catechin, and 15 min
epicatechin
Fermented grape pomace Malvidin-3-O-glucoside, peonidin-3- 24 kHz [119]
glucoside, petunidin-3-gucoside, and
delpinidin-3-glucoside
Pomegranate peel Phenolic content and total flavonoids 140 W, 30–40 kHz, 69.87 and 7 mg/g [113]
30 min
Tomato paste Lycopene 98 W (microwave), Extraction yield [120]
40 kHz, 50 W, 86.4 ° 97.4%
Fruit Waste: Potential Bio-Resource for Extraction of Nutraceuticals and. . .
C, 365 s
Grape cane byproducts Stilbenes 75 °C, ethanol (60%), [121]
10 min
315
(continued)
Table 2
316
(continued)
Extraction
technique Fruit waste Bioactive compounds Extraction conditions Results References
Supercitical Mango peel Flavonoids and carotenoids 30 MPa, 40 °C, carbon [122]
fluid dioxide flow rate
extraction 1.1 L/min, 7.7 h
(SFE)
Milan Dhakal et al.
Pulse electric Grape byproducts Anthocyanins 3 kV/cm 2 folds higher than [123]
field (PEF) control
Mango peel Mangiferin, quercetin, ellagic acid 13.3 kW/cm, 60 °C, Extracted with high [124]
1000 kJ/kg clarity and
colloidal stability
Enzyme- Guava leaves Phenolics Cellulase or beta- Yield increased by [125]
assisted glucosidase-assisted 103.2%
extraction extraction
(EAE) Pistachio green hull Phenolic compounds Cellulase, pectinase and Yield increased by [126]
tannase, pH 4.0, 37 °C 112%
Pressurized Pomegranate peel Phenolic compounds 3 MPa, 126.1 °C, solvent- [127]
liquid solid ratio 54.8 mL/g,
extraction 18.5 min
(PLE)
are the most common solvents used for extraction of bioactive

compounds. Other solvents used include chloroform, carbon tetra-
chloride, chlorobenzene, acetone, and acetonitrile [129]. The
selection of solvent depends on several factors, such as target com-
pounds, toxicity, cost, and polarity. This extraction technique is
widely used to extract alkaloids, terpenes, terpenoids, and phenolic
compounds. However, this process produces undesirable residue,
and the extract obtained might undergo oxidative transformation
during solvent removal. Moreover, this method requires longer
extraction time and a large amount of solvent [130]. The process
is mainly affected by solubility, mass transfer, and solid material
characteristics. A small quantity of dried sample is kept in thimble,
and the distillation beaker is filled with appropriate solvent. As the
solution attains overflow point, it is aspirated from thimble holder
and transferred to the distillation flask. The extract is held in this
combination, which transfers it into the liquid in bulk. The extract
solute stays in the distillation flask, whereas the solvent stays with
the solid sample. The process is repeated continuously until the
extraction is complete [11]. Caldas, Mazza [107] performed a
comparative study of conventional (Soxhlet) and nonconventional
extraction methods to obtain phenolic compounds from grape
peel. The authors reported that the Soxhlet method (80 mg
GAE/g) resulted in higher extraction yield than nonconventional
methods (around 65 mg GAE/g). However, the phenolics recov-
ered nonconventionally possessed the highest purity.
4.1.2 Hydro-distillation Several bioactive compounds including oils are extracted using
hydro-distillation technique. Water and steam distillation, water
distillation, and direct steam distillation are three different types
of hydro-distillation methods. In this extraction technique, the
sample is completely immersed in boiling water. This process
undergoes different physicochemical processes such as hydro-
diffusion, hydrolysis, and thermal decomposition. However, this
technique is not appropriate for heat-sensitive compounds as they
might be lost or degraded during the extraction process. The
advantages of this extraction technique are: no use of organic
solvents and shorter extraction time.
4.1.3 Maceration Maceration is a cost-effective extraction technique popularly used

at the household level to obtain bioactive compounds from plant
parts. In this method, ground samples with small particle size (for
increased surface area) and appropriate amount of menstruum
(solvent) are mixed in a closed container. Then the mixture is
pressed and an extract-rich liquid is strained. Occasional stirring is
performed to intensify the solvent diffusion and separate concen-
trated bioactives from sample surface. Maceration is one of the best
methods to extract thermolabile (heat-sensitive) bioactive com-
pounds [131]. Different types of maceration techniques are: simple
maceration, double maceration, and triple maceration. Soto,

Quezada-Cervantes [108] extracted 67.1 and 146.6 mg GAE/g
(dry basis) of total phenolic content from grape and orange resi-
dues, respectively. A higher amount of total phenolic content was
extracted from tangerine peels with maceration at a temperature of
40 °C using 80% methanol as solvent [109]. Suleria, Barrow [110]
extracted phenolic compounds using maceration in the range of
0.45–25.5 mg GAE/g from peels of apple, apricot, avocado,
banana, custard apple, dragon fruit, grapefruit, kiwifruit, mango,
lime, melon, nectarine, orange, papaya, passion fruit, peach, pear,
pineapple, plum, and pomegranate with 70% ethanol, agitation at
120 rpm and 4 °C. Furthermore, Lim, Cabajar [111] compared
maceration at different concentrations of ethanol to extract total
phenolic contents from mango seed kernel and found that maxi-
mum extraction (107.7 mg GAE/g) with 50% ethanol. Overall, the
factors affecting the yield and efficiency of conventional extraction
techniques are the type of solvent used, polarity of bioactive com-
pounds, and mass transfer rate.
4.2 Emerging Conventional extraction technologies possess some pitfalls, such as

Extraction the use of large quantities of costly solvents, longer extraction time,
Technologies thermal degradation of heat-sensitive bioactive compounds, low
extraction yield, and low purity and selectivity. Therefore, to over-
come those limitations of conventional extraction methods, novel/
emerging extraction technologies have been developed. Emerging
extraction technologies reduce the use of harmful organic solvents,
increase extraction rate, reduce energy use, increase heat and mass
transfer, improve extraction yield, and reduce processing steps
[132, 133]. Recently, emerging technologies such as ultrasound-
assisted extraction (UAE), microwave-assisted extraction (MAE),
enzyme-assisted extraction (EAE), pressurized liquid extraction
(PLE), supercritical fluid extraction (SFE), and pulse electric field
(PEF) are considered effective for the extraction of bioactive com-
pounds from fruits.
4.2.1 Microwave- Microwave-assisted extraction (MAE) reduces the amount of sol-

Assisted Extraction vent used and extraction time as compared with conventional
extraction methods. Microwave-assisted extraction involves direct
application of nonionizing electromagnetic waves in the range of
0.3–300 GHz to food samples. The common frequency used is
2450 MHz, which is equivalent to 600–700 W energy. MAE gen-
erally possesses several steps because of heat and mass gradients
generated into the matrix, which includes (a) penetration of the
solvent into the matrix, (b) solubilization and/or breakdown of the
components, (c) transport of the solubilized compounds from the
insoluble matrix to the bulk solution, and (d) separation of the
liquid and residual solid phase [134, 135]. This method can reduce
extraction time and volume of solvent required, and improve
recovery of bioactive compounds as compared with the conven-

tional Soxhlet extraction method because the localized pressure and
temperature can enhance the penetration of solvents into the plant
matrix and disrupt the cell wall to release bioactive compounds
[136–138]. MAE is considered an efficient and fast method to
extract bioactive compounds from plant matrices. Extraction of
compounds results because of dipole rotation and ionic conduction
by heating the sample in the meantime. Factors to be considered for
the extraction of bioactive compounds using microwave are the
amount of solvent, solid-solvent ratio, microwave power, dielectric
properties of samples, sample moisture content, stirring effect, the
temperature of extraction, and time. The stability of the extracted
compound depends on the microwave power used, extraction time,
and temperature. Most importantly, water, instead of organic sol-
vents, is sufficient to use as solvent in this extraction process. The
most common solvent used in microwave extraction is ethanol,
which is used either alone or with water.
The sample usually should be dried and powdered prior to
MAE as milling improves the extraction of phenolics. The decrease
in particle size increases the surface area of the sample in contact
with the solvent and reduces diffusion distances, resulting in greater
mass transfer and yield [139]. While if the particle size is too small
(<250 μm), a cleaning step might be required because this will
make it difficult to separate the extract from the residue
[135]. MAE has been widely used and studied for the extraction
of bioactive compounds such as carotenoids, polyphenol, etc. from
fruit byproducts. Studies have reported that pectin is the most
extracted compound from citrus fruit waste and byproducts (citrus
residues, peel, and bagasse) using MAE technology [140]. Kader-
ides, Papaoikonomou [112] used microwave-assisted extraction to
obtain phenolics (199.4 mg GAE/g dry basis) from
pomegranate peel.
MAE has been proved to be an effective process to extract
highly intact bioactive compounds with better recovery than con-
ventional methods. Recently, MAE has been gaining attention and
is considered an excellent green extraction technique. The MAE is
more effectively applied for the extraction of short-chain polyphe-
nols, such as phenolic acids and flavonoids, which remain stable at
microwave heating of 100 °C [141]. However, polyphenols that are
polymeric with hydroxyl conjugates, such as tannins, or are heat
sensitive, such as anthocyanins, could undergo structural damage
during microwave treatment [142].
Previous literature have suggested that polyphenolic com-
pounds from wastes such as pomegranate peel [113], carbo fruit
peel [114], and banana peel [115] were extracted efficiently using
MAE. Furthermore, coupling MAE with other extraction technol-
ogies could increase extraction yield and reduce cost when the
combination is designed with statistical and modeling resources

such as RSM [143]. Trujillo-Mayol, Céspedes-Acuña [116] com-
bined MAE with UAE for the extraction of bioactives from avocado
peels; the combination included 15 min of sonication at 60 C,
followed by 95.1 s of microwave irradiation (500 W). The combi-
nation resulted in maximum yield of total phenolic content
(281.4 ± 0.2 mg GAE/g dry extract) and had higher efficiency
compared with sonication and microwave application.
4.2.2 Ultrasound- Ultrasound-assisted extraction (UAE), also known as sonication,

Assisted Extraction uses a sound intensity of 5–1000 W/cm2 sound wave with fre-
quency from 20 kHz to 100 MHz [144]. Ultrasound creates
compression and expression in the medium. This induces acoustic
cavitation by forming collapsing bubbles in the solvent, generating
localized pressure, which can permeabilize the cell wall, release
intercellular content into the solvent, and improve mass transfer.
Ultrasound-assisted extraction reduces the extraction time and
volume of solvent required to extract bioactive compounds.
Many process parameters such as the composition of the solvent,
solvent-solid ratio, particle size, pressure, moisture, ultrasonication
time and temperature, influence the extraction process and yield.
Studies have suggested that use of frequency higher than 20 kHz
affects the physicochemical properties of the extracted compounds
because of formation of free radicals [145, 146]. Organic solvents,
namely, ethanol, methanol, acetone, and isopropanol, are fre-
quently used. To protect bioactive compounds from thermal deg-
radation, a low temperature of below 50 °C is recommended.
Ghafoor and Choi [117] used UAE to extract bioactive com-
pounds from grape seeds and found that UAE significantly
increased the yield of total phenolic contents, antioxidant activities,
and anthocyanin content. Bioactive compounds, namely, trans-5-
O-caffeoyl-D-quinicacid, procyanidin B1, catechin, and epicate-
chin, have been extracted from the seeds and peel of avocado with
ethanol-water as solvent and extraction period of 15 min
[118]. Moreover, phenolic compounds and anthocyanins such as
malvidin-3-O-glucoside, peonidin-3-glucoside, petunidin-3-guco-
side, and delpinidin-3-glucoside were extracted by Barba, Brian-
ceau [119] from fermented grape pomace using UAE. Pectins,
carotenoids, and lipids have been successfully extracted using
UAE [147–149]. Furthermore, previous studies have suggested
that UAE requires less energy, reduces extraction time, and
improves extraction yield compared to conventional extraction
technologies. More and Arya [150] extracted bioactive from pome-
granate peel and reported higher yield with noticeable antioxidant
and bioactive content. Similarly, phenolic content and total flavo-
noid content of 69.87 and 7 mg/g, respectively, with 67% antioxi-
dant activity were obtained from pomegranate peel using UAE at
140 W power, 30–40 kHz frequency for 30 min [113]. The

ultrasound-assisted extraction technology could be combined
with other extraction technologies to increase extraction yield.
Previous studies have reported that ultrasound-assisted extraction
when combined with supercritical fluid extraction for the extraction
of phenolic compounds from blackberry bagasse doubles the
extraction yield than only using supercritical fluid extraction
[151]. The extraction yield of lycopene from tomato paste of
97.4% was obtained when microwaves (98 W) were combined
with ultrasonication bath at 40 kHz and 50 W, 86.4 °C, and
extraction time of 365 s. However, 89.4% lycopene yield was
obtained with UAE [120]. Piñeiro, Marrufo-Curtido [121] opti-
mized and validated the ultrasound-assisted extraction process for
rapid extraction of stilbenes from grape canes. The optimal condi-
tions were extraction time of 10 min, extraction temperature of 75 °
C, and ethanol as extraction solvent (60%). The study suggested
that grape cane byproducts were potential sources of bioactive
compounds. Different from conventional methods, UAE possesses
the advantage of low operational temperature, which results in less
thermal degradation and reduced extraction time.
4.2.3 Supercritical Fluid Supercritical fluid is a hybrid media that possesses combined prop-
Extraction erties of liquid and gases in a single phase. Supercritical fluid extrac-
tion (SFE) does not require toxic organic solvent; hence, it is
considered a green technology. The advantage of using supercritical
fluids for recovery of bioactive compounds from fruit waste is that
supercritical fluid has low viscosity and high coefficient of diffusion
as compared to conventionally used liquid solvents. These proper-
ties promote efficient extraction of bioactive compounds by allow-
ing the fluid to penetrate into the matrix to a greater extent. Fluids
such as carbon dioxide, water, methanol, ethylene, ethanol,
n-butane, and n-pentane at pressure and temperature above their
critical points are used in SFE. The most used fluid in supercritical
fluid extraction is carbon dioxide as it is relatively inexpensive,
nonpolar in nature, nontoxic, and nonflammable. The low polarity
of carbon dioxide enhances the extraction of nonpolar or small
polar biomolecules such as lipid and volatile compounds
[152]. However, supercritical fluid extraction with carbon dioxide
could efficiently extract nonpolar or mid-polar compounds (essen-
tial oils and carotenoids). The yield and purity of extracted bioactive
compounds from fruit waste depend on several factors, such as
temperature, pressure duration, solvent flow rate, amount of
co-solvent used, co-solvent flow rate, the nature of fruit waste,
and prior processing technique (drying, lyophilization, etc.). Avail-
able literature suggests that supercritical fluid extraction has advan-
tage over conventional extraction in terms of selectivity, compound
stability, easy recovery, and time and energy saving. Pham [122]
extracted nonpolar flavonoids and carotenoids from mango peel

using SFE at pressure 30 MPa, extraction temperature 40 °C,
carbon dioxide flow rate 1.1 L/min, and extraction time of 7.7 h.
Extraction with supercritical fluid extraction technique is harmless
to both food components and human consumption. Moreover,
toxic solvents used in other extraction are completely avoided,
and the high energy required is reduced; hence, SEF has no envi-
ronmental impact. In recent years, supercritical carbon dioxide has
been considered the standard, powerful, and green extraction tech-
nology to valorize fruit waste because of safety and purity of
extracts [11].
4.2.4 Pulse Electric Field Pulse electric field extraction technology is an energy-efficient and
Extraction environmentally friendly process that uses high voltage pulses of
electricity, which is used in food processing to prolong shelf-life.
However, it has been equally used for the extraction of bioactive
compounds. In PEF treatment, the food sample is subjected to
electrical resistance of 20–80 kV/cm for very short time (<1 s) to
produce high energy discharges. When the electric pulse is applied
to plant tissue, it increases tissue softness through electroporation
of cell membranes. Electroporation depends on energy, time, and
number of pulses. The pore size and wall/membrane disintegration
increase with the intensity of electric pulses; the main factor in play
to govern optimization of PEF extraction is electric field/mass ratio
[144, 153–155]. Studies have suggested that use of PEF for bioac-
tive compound extraction improves the extraction yield. Corrales,
Toepfl [123] reported that the use of PEF to extract anthocyanins
from grape byproducts enhanced the yield of anthocyanins, and
PEF could also be used to selectively extract targeted bioactive
compounds. Moreover, the extraction depends on pH, extraction
time, electric field strength, pulse shape, pulse width, pulse fre-
quency, food matrix density, and the chemical properties of the
compound to be extracted. Parniakov, Barba [124] reported that
application of pulse electric field–assisted extraction significantly
increased the extraction yield of polyphenol (mangiferin, quercetin,
ellagic acid) from mango peel. Moreover, Delsart, Ghidossi [156]
extracted anthocyanins and polyphenols from grape skin using PEF
and observed that the extraction yield of these compounds was
higher than other extraction techniques. Plum and grape peels
[157], orange [158] and lemon [159] peel residues, or olive pom-
ace [160] are additional examples of byproduct valorization by
PEF-assisted polyphenol extraction. PEF has several advantages
over conventional extraction technology, which include: (a) no
water removal or dehydration of samples, (b) additional chemicals
not needed, (c) no heating needed, (d) less time-consuming,
(e) scalability. However, more research is still needed on the effects
of pulsed electric fields on pulsed matrices.
4.2.5 Enzyme-Assisted Enzymes are also used to extract bioactive compounds from food
Extraction waste including fruit waste. Enzymes such as cellulase, pectinase,
hemicellulase, xylanase, β-gluconsae, β-glucosidase, and alcalase can
decompose the cell wall materials (pectin, hemicellulose, and cellu-
lose) of fruit waste and can assist in the extraction of bioactive
compounds [161]. Since EAE utilizes water as solvent rather than
organic solvents, it is considered as the most ecologically friendly
extraction technique. Factors that should be considered during
enzyme-assisted extraction are type and concentration of enzyme,
extraction temperature, particle size, water-to-solid ratio, time, and
pH. More specifically, pH and temperature are critical to activate
catalytic potential. Further, the efficiency of enzyme-assisted extrac-
tion could be maximized when assisted with other processes such as
ultrasound, microwave, and so on. Wang, Wu [125] treated guava
leaves with cellulase or beta-glucosidase-assisted extraction and
found that the extraction of phenolics increased by 103.2%.
Enzyme-assisted extraction is gaining popularity in the valorization
of polyphenol-rich matrices from food-agroindustry since it is an
environmentally friendly approach [162]. Similarly, Ghandahari
Yazdi, Barzegar [126] used the combination of different enzymes,
cellulase, pectinase, and tannase, at optimal conditions (pH 4.0,
37 °C) to extract phenolic compounds from pistachio green hull
and compared to the conventional control extract. They observed
that the yield of phenolic compounds increased by 112%, and the
antioxidant capacity was found to be 71% higher than extract
without enzyme treatment [126, 163]. Treatment with cellulase
and tannase to recover polyphenols from Syrah grape pomace
increased the recovery of phenolics (up to 66%) as well as the
antioxidant activity (up to 80%), compared to classic hydroalcoholic
(50:50) extraction at 50 °C for 6 h [163, 164].
4.2.6 Pressurized Liquid Pressurized liquid extraction (PLE), also known as accelerated
Extraction solvent extraction (ASE), was introduced as an extraction technol-
ogy by Dionex Corporation for the extraction of anthocyanin from
seaweed [139]. PLE is also a green extraction technology that uses
water instead of organic solvent and reduces the amount of solvent
used to extract bioactive compounds from fruit waste. The pressur-
ized liquid extraction technique employs pressure (between 5 and
15 MPa) to keep the solvent at a temperature higher than its boiling
point. PLE, solid-liquid extraction technique, uses pressurized sol-
vents at high temperature (>100 °C). The high temperature and
pressure applied increases the solubility and mass transfer rate of
bioactive compounds. The high pressure used aids the penetration
of the solvent into the pores of the solid material and the high
temperature facilitates diffusion of the solvent into the solid mate-
rial. PLE has been frequently applied for the extraction of phenolic
compounds, carotenoids, and tocopherols from fruit waste. Xi, He
[127] have extracted phenolic compounds from pomegranate peel
with PLE at 3 MPa, 126.1 °C, solvent-solid ratio 54.8 mL/g, and
extraction time of 18.5 min. The extraction of bioactive com-
pounds using pressurized liquid extraction technique is the func-
tion of factors such as solvent polarity, toxicity of solvent, particle
size, mass transfer rate, sample moisture content, temperature,
pressure, and extraction time.
5 Current Application and Challenges
Fruit waste contains a variety of dietary, nutraceutical, and medici-

nal bioactive compounds. In the current scenario, these bioactive
compounds are used as novel functional ingredients for enrichment
and fortification in different food products for formulation of
functional and nutritional products, in the chemical industry, bio-
remediation, energy, cosmetics industries, and pharmaceutical and
nutraceutical applications. These bioactive compounds have sub-
stantial value to the food sector as being used as food and nutrient
supplements, as colorants, and as an additive in meat and meat
products for their preservative and enhancing action [165–
167]. Polyphenols are used as antioxidants and colorants in differ-
ent industrial applications. Fruit oils consist abundant quantity of
antioxidants and fatty acids, due to which they are used in salad
dressings, frying oil, food formulations, and skin care products as
they possess anti-ageing, anti-inflammatory, and skin reconstructive
properties. The bioactive chemicals derived from these oils may also
be useful to the pharmaceutical and cosmetic sectors in addition to
the food business. Several natural essences are being produced
utilizing bioactive components of fruit waste, which are used as
flavoring agents in pharmaceutical, food formulations, culinary,
cosmetics, and detergent industries, that is, vanillin (4-hydroxy-3-
methoxy benzaldehyde), furaneol (2,5-dimethyl-4-hydroxy-3
(2H)-furanone), ethyl acetate, decanal, citral, limonene, glucose
and fructose, different esters, aldehydes, alcohols, acid, and lac-
tones, etc. [11, 168, 169].
The present research trend is most inclined toward the isolation
and identification of bioactive compounds but, there are limited
studies conducted for the study of their toxicological effects,
digestibility, bioaccessibility, bioavailability, and metabolism. So,
these facts limit the potential use of bioactive compounds in indus-
trial applications. For exploiting the value of isolated bioactive, that
is, pigments, vitamins, polyphenols, oils, enzymes, etc., in vitro and
in invo studies are essential. Studies are being conducted on the
extraction, isolation, identification, purification, and characteriza-
tion of bioactive from a single source only; still, there is a lot to go
to understand the effect of the mixture of raw materials on the
bioactivity of extract, whether the extract will show the antagonistic
or synergistic effect, as fruit processing industries creates waste

combined of multiple sources. So, a sustainable approach will be
utilizing the natural bioactive compounds in industrial applications
from fruit wastes after a proper investigation of their bioactivity.
6 Conclusion and Future Perspectives
Based on the available literature and sophisticated state-of-the-art

technologies bioactive compounds in fruit waste can be identified
and quantified precisely. Fruit waste with higher content of specific
bioactive molecule could be utilized for extraction. The advance-
ment of food processing technologies opens the opportunity to
utilize the increasing amount of fruit waste as a source of bioactive
compounds, which eventually will contribute to economy and
waste management. Moreover, the processing waste of fruit from
industries and other different stages of food supply chain is very
high; therefore, the disposal of such waste is a serious issue. If not
disposed of properly, they might be hazardous to the environment.
On the other hand, such waste could be a potential bioresource to
extract bioactive compounds. This chapter discussed several con-
ventional as well as emerging extraction technologies employed to
obtain bioactive compounds that have high potential to be used as
nutraceuticals and dietary supplements. In conclusion, the bioac-
tive compounds present in the side stream of fruit processing have
great potential for application in food and nutraceutical industries
as bio-preservatives, stabilizers, and nutraceuticals agents. The uti-
lization of fruit byproducts will help fruit producers gain extra
revenue from fruit residue and help to mitigate the issue of proper
disposal of fruit waste.
References
1. Shurson GC (2020) “What a waste” – can we dioxins/furans in municipal waste incinera-
improve sustainability of food animal production. J Hazard Mater 62(1):1–33
tion systems by recycling food waste streams 5. Makris DP, Boskou G, Andrikopoulos NK
into animal feed in an era of health, climate, (2007) Recovery of antioxidant phenolics
and economic crises? Sustainability 12(17): from white vinification solid by-products
7071 employing water/ethanol mixtures. Biore-
2. Elimelech E, Ayalon O, Ert E (2018) What sour Technol 98(15):2963–2967
gets measured gets managed: a new method 6. Hosseini B et al (2018) Effects of fruit and
of measuring household food waste. Waste vegetable consumption on inflammatory bio-
Manag 76:68–81 markers and immune cell populations: a sys-
3. Qdais HA, Abdulla F, Qrenawi L (2010) tematic literature review and meta-analysis.
Solid waste landfills as a source of green Am J Clin Nutr 108(1):136–155
energy: case study of Al Akeeder landfill. 7. Bagattoli P et al (2016) Phytochemical, anti-
JJMIE 4(1):69–74 oxidant and anticancer activities of extracts of
4. Buekens A, Huang H (1998) Comparative seven fruits found in the southern Brazilian
evaluation of techniques for controlling the flora. Indian J Pharm Sci 78(1):34
formation and emission of chlorinated
8. Zhu F, Du B, Xu B (2018) Anti-inflammatory 21. Shivamathi CS et al (2022) Process optimiza-

effects of phytochemicals from fruits, vegeta- tion and characterization of pectin derived
bles, and food legumes: a review. Crit Rev from underexploited pineapple peel biowaste
Food Sci Nutr 58(8):1260–1270 as a value-added product. Food Hydrocoll
9. Jha AK, Sit N (2021) Extraction of bioactive 123:107141
compounds from plant materials using com- 22. Akter F, Haque MA (2020) Jackfruit waste: a
bination of various novel methods: a review. promising source of food and feed. Ann
Trends Food Sci Technol 119:579–591 Bangladesh Agric 23(1):91–102
10. Bisht B et al (2022) A review on holistic 23. Le NL (2022) Functional compounds in
approaches for fruits and vegetables biowastes dragon fruit peels and their potential health
valorization. Mater Today Proc 73:54–63 benefits: a review. Int J Food Sci Technol
11. Sagar NA et al (2018) Fruit and vegetable 57(5):2571–2580
waste: bioactive compounds, their extraction, 24. Khalifa I, Nawaz A (2022) Valorization of
and possible utilization. Compr Rev Food Sci guava fruit byproducts: chemical composi-
Food Saf 17(3):512–531 tion, bioactive components, and technical
12. Mak TMW et al (2020) Sustainable food concerns to the food industry. In: Ramadan
waste management towards circular bioecon- MF, Farag MA (eds) Mediterranean fruits
omy: policy review, limitations and opportu- bio-wastes: chemistry, functionality and tech-
nities. Bioresour Technol 297:122497 nological applications. Springer, Cham, pp
13. Jiménez-Moreno N et al (2020) Valorization 819–839
of selected fruit and vegetable wastes as bioac- 25. Chakrabarty N et al (2020) Assessment of the
tive compounds: opportunities and chal- potential of watermelon rind powder for the
lenges. Crit Rev Environ Sci Technol value addition of noodles. J Biosyst Eng
50(20):2061–2108 45(4):223–231
14. More PR, Jambrak AR, Arya SS (2022) 26. Zia S et al (2021) An update on functional,
Green, environment-friendly and sustainable nutraceutical and industrial applications of
techniques for extraction of food bioactive watermelon by-products: a comprehensive
compounds and waste valorization. Trends review. Trends Food Sci Technol 114:275–
Food Sci Technol 128:296–315 291
15. Alexandre EM et al (2018) Emerging tech- 27. Yilmaz Y et al (2014) Antioxidant activity and
nologies to extract high added value com- phenolic content of seed, skin and pulp parts
pounds from fruit residues: of 22 grape (Vitis vinifera L.) cultivars (4 com-
sub/supercritical, ultrasound-, and enzyme- mon and 18 registered or candidate for regis-
assisted extractions. Food Rev Int 34(6): tration). J Food Process Preserv 39:1682–
581–612 1691
16. Arya SS et al (2022) The wastes of coffee bean 28. Cosmulescu S et al (2015) Total phenolic,
processing for utilization in food: a review. J flavonoid distribution and antioxidant capac-
Food Sci Technol 59(2):429–444 ity in skin, pulp and fruit extracts of plum
17. Pateiro M et al (2018) Guarana seed extracts cultivars. J Food Biochem 39:64–69.
as a useful strategy to extend the shelf life of https://doi.org/10.1111/jfbc.12112
pork patties: UHPLC-ESI/QTOF phenolic 29. Inglett GE, Chen D (2011) Contents of phe-
profile and impact on microbial inactivation, nolics and flavonoids and antioxidant activ-
lipid and protein oxidation and antioxidant ities in skin, pulp, and seeds of miracle fruit.
capacity. Food Res Int 114:55–63 J Food Sci 76(3):C479–C482
18. Ala’a H et al (2010) Drying characteristics 30. Soong Y, Barlow P (2004) Antioxidant activ-
and quality change of unutilized-protein ity and phenolic content of selected fruit
rich-tomato pomace with and without seeds. Food Chem 88:411–417
osmotic pre-treatment. Ind Crop Prod 31. Mena-Garcı́a A et al (2019) Green techniques
31(1):171–177 for extraction of bioactive carbohydrates.
19. Garcı́a-Lomillo J, González-SanJosé M TrAC Trends Anal Chem 119:115612
(2017) Applications of wine pomace in the 32. Altemimi A et al (2017) Phytochemicals:
food industry: approaches and functions. extraction, isolation, and identification of bio-
Compr Rev Food Sci Food Saf 16:3–22 active compounds from plant extracts. Plants
20. Pathak PD, Mandavgane SA, Kulkarni BD (Basel) 6(4):42
(2019) Waste to wealth: a case study of papaya 33. Azmir J et al (2013) Techniques for extraction
peel. Waste Biomass Valorization 10(6): of bioactive compounds from plant materials:
1755–1766 a review. J Food Eng 117(4):426–436
34. Sasidharan S et al (2011) Extraction, isolation 48. Verhaar MC, Stroes E, Rabelink TJ (2002)
and characterization of bioactive compounds Folates and cardiovascular disease. Arterios-
from plants’ extracts. Afr J Tradit Comple- cler Thromb Vasc Biol 22(1):6–13
ment Altern Med 8(1):1–10 49. Suriyaprom S et al (2022) Antioxidants of
35. Chatterjee B, Sharma A (2018) Fruit enzymes fruit extracts as antimicrobial agents against
and their application: a review. Int J Clin pathogenic bacteria. Antioxidants (Basel)
Biomed Res 4:84 11(3):602
36. Brien S et al (2004) Bromelain as a treatment 50. Aniszewski T (2015) Chapter 2 – alkaloid
for osteoarthritis: a review of clinical studies. chemistry. In: Aniszewski T (ed) Alkaloids,
Evid Based Complement Alternat Med 1(3): 2nd edn. Elsevier, Boston, pp 99–193
251–257 51. Dillard C, German B (2000) Phytochemicals:
37. Pavan R et al (2012) Properties and therapeu- nutraceuticals and human health. J Sci Food
tic application of bromelain: a review. Bio- Agric 80:1744–1756
technol Res Int 2012:976203 52. Sarkar T et al (2022) Minor tropical fruits as a
38. Stremnitzer C et al (2015) Papain degrades potential source of bioactive and functional
tight junction proteins of human keratino- foods. Crit Rev Food Sci Nutr 63:1–45
cytes in vitro and sensitizes C57BL/6 mice 53. Dhingra D et al (2012) Dietary fibre in foods:
via the skin independent of its enzymatic a review. J Food Sci Technol 49(3):255–266
activity or TLR4 activation. J Invest Dermatol 54. DeVries JW (2019) Dietary fiber: the influ-
135(7):1790–1800 ence of definition on analysis and regulation. J
39. Bassinello PZ, Cordenunsi BR, Lajolo FM AOAC Int 87(3):682–706
(2002) Amylolytic activity in fruits: compari- 55. Pfaltzgraff LA et al (2013) Food waste bio-
son of different substrates and methods using mass: a resource for high-value chemicals.
banana as model. J Agric Food Chem 50(21): Green Chem 15(2):307–314
5781–5786
56. Mamma D, Christakopoulos P (2014) Bio-
40. da Silva AC, Jorge N (2017) Bioactive com- transformation of citrus by-products into
pounds of oils extracted from fruits seeds value added products. Waste Biomass Valori-
obtained from agroindustrial waste. Eur J zation 5(4):529–549
Lipid Sci Technol 119(4):1600024
57. Chitturi S, Talatam VG, Vuppu DS (2013)
41. Kultys E, Kurek MA (2022) Green extraction Studies on protein content, protease activity,
of carotenoids from fruit and vegetable bypro- antioxidants potential, melanin composition,
ducts: a review. Molecules 27(2):518 glucosinolate and pectin constitution with
42. Tedesco DEA et al (2021) Fruit and vegetable brief statistical analysis in some medicinally
wholesale market waste: safety and nutritional significant fruit peels. Pharm Lett 5:13–23
characterisation for their potential re-use in 58. Boland M (2013) Kiwifruit proteins and
livestock nutrition. Sustainability 13(16): enzymes: actinidin and other significant pro-
9478 teins. Adv Food Nutr Res 68:59–80
43. Khattak KF, Rehman T (2017) Analysis of 59. Wani AA et al (2008) Extraction optimization
vegetable’s peels as a natural source of vita- of watermelon seed protein using response
mins and minerals. Int Food Res J 24:292– surface methodology. LWT Food Sci Technol
297 41(8):1514–1520
44. Banasaz S et al (2020) Encapsulation of lipid- 60. Devalaraja S, Jain S, Yadav H (2011) Exotic
soluble bioactives by nanoemulsions. Mole- fruits as therapeutic complements for diabe-
cules 25(17):3966 tes, obesity and metabolic syndrome. Food
45. Skrovankova S et al (2015) Bioactive com- Res Int 44(7):1856–1865
pounds and antioxidant activity in different 61. de la Torre CAL, Conte-Junior CA (2018)
types of berries. Int J Mol Sci 16(10): Chapter 6 – detection of biogenic amines:
24673–24706 quality and toxicity indicators in food of ani-
46. Aburawi S (2021) Vitamin C and human dis- mal origin. In: Holban AM, Grumezescu AM
eases: an overview. Mediterr J Pharm Pharm (eds) Food control and biosecurity. Academic,
Sci 1:25–36 pp 225–257
47. Hrubša M et al (2022) Biological properties 62. Santiago-Silva P, Labanca R, Gloria MBA
of vitamins of the B-complex, part 1: vitamins (2011) Functional potential of tropical fruits
B(1), B(2), B(3), and B(5). Nutrients 14(3): with respect to free bioactive amines. Food
484 Res Int 44:1264–1268
63. Barreira JCM, Arraibi AA, Ferreira ICFR stem. Int J Biosci Biochem Bioinform 4:110–
(2019) Bioactive and functional compounds 115
in apple pomace from juice and cider 76. Jabri K, Marzouk B (2013) Characterization
manufacturing: potential use in dermal for- of bioactive compounds in Tunisian bitter
mulations. Trends Food Sci Technol 90:76– orange (Citrus aurantium L.) peel and juice
87 and determination of their antioxidant activ-
64. da Silva LC et al (2021) Recent advances and ities. Biomed Res Int 2013:345415
trends in extraction techniques to recover 77. Silva A, Jorge N (2019) Bioactive properties
polyphenols compounds from apple and antioxidant capacity of oils extracted from
by-products. Food Chem X 12:100133 citrus fruit seeds. Acta Aliment 48(2):
65. Sato M et al (2010) Apple pomace from 196–203
eleven cultivars: an approach to identify 78. Yasin BR, El-Fawal HAN, Mousa SA (2015)
sources of bioactive compounds. Acta Sci Date (Phoenix dactylifera) polyphenolics and
Agron 32:29–35 other bioactive compounds: a traditional
66. Ben-Othman S, Jõudu I, Bhat R (2020) Islamic remedy’s potential in prevention of
Bioactives from agri-food wastes: present cell damage, cancer therapeutics and beyond.
insights and future challenges. Molecules Int J Mol Sci 16(12):30075–30090
25(3):510 79. Majzoobi M et al (2019) Chemical composi-
67. Patra A, Abdullah S, Pradhan RC (2022) tion and functional properties of date press
Review on the extraction of bioactive com- cake, an agro-industrial waste. mdrsjrns
pounds and characterization of fruit industry 21(7):1807–1817
by-products. Bioresour Bioprocess 9(1):14 80. Silva J et al (2014) Chemical composition,
68. Mohd Zaini H et al (2022) Banana peels as a fatty acid profile and bioactive compounds of
bioactive ingredient and its potential applica- guava seeds (Psidium guajava L.). Cienc Tec-
tion in the food industry. J Funct Foods 92: nol Aliment 3:1–8
105054 81. Singh A et al (2015) Studies on the phenolic
69. Chueh CC et al (2019) Antioxidant capacity acid contents in different parts of raw and ripe
of banana peel and its modulation of Nrf2- jackfruit and their importance in human
ARE associated gene expression in broiler health. Int J Appl Sci Res Rev 2(2):69–73
chickens. Ital J Anim Sci 18(1):1394–1403 82. Zhang L et al (2017) Jackfruit (Artocarpus
70. Avram I, Gatea F, Vamanu E (2022) Func- heterophyllus Lam.) peel: a better source of
tional compounds from banana peel used to antioxidants and a-glucosidase inhibitors
decrease oxidative stress effects. PRO 10(2): than pulp, flake and seed, and phytochemical
248 profile by HPLC-QTOF-MS/MS. Food
71. Behiry SI et al (2019) Antifungal and antibac- Chem 234:303–313
terial activities of Musa paradisiaca L. peel 83. Castro-Vargas HI et al (2019) Valorization of
extract: HPLC analysis of phenolic and flavo- papaya (Carica papaya L.) agroindustrial waste
noid contents. PRO 7(4):215 through the recovery of phenolic antioxidants
72. González-Montelongo R, Gloria Lobo M, by supercritical fluid extraction. J Food Sci
González M (2010) Antioxidant activity in Technol 56(6):3055–3066
banana peel extracts: testing extraction condi- 84. Rodrigo U, Perera G (2018) Important
tions and related bioactive compounds. Food biological activities of papaya peel extracts
Chem 119(3):1030–1039 and their importance in formulation of a low
73. Saeed S et al (2021) Valorization of banana cost fish feed to enhance the skin colour and
peels waste into biovanillin and optimization the healthiness of guppies. Int J Sci Res Publ
of process parameters using submerged fer- 8:8490
mentation. Biocatal Agric Biotechnol 36: 85. Banerjee S et al (2018) Valorisation of pine-
102154 apple wastes for food and therapeutic applica-
74. Tallapally M et al (2020) GC-MS based tar- tions. Trends Food Sci Technol 82:60–70
geted metabolomics approach for studying 86. Sánchez-Pardo M et al (2014) Chemical char-
the variations of phenolic metabolites in arti- acterisation of the industrial residues of the
ficially ripened banana fruits. LWT 130: pineapple (Ananas comosus). J Agric Chem
109622 Environ 03:53–56
75. Apriasari ML, Iskandar, Suhartono E (2014) 87. Polanı́a AM et al (2023) Valorization of pine-
Bioactive compound and antioxidant activity apple waste as novel source of nutraceuticals
of methanol extract Mauli Bananas (Musa sp) and biofunctional compounds. Biomass
Convers Biorefin 13(5):3593–3618. https:// lanatus, Thunb). J Pharmacogn Phytochem

doi.org/10.1007/s13399-022-02811-8 6(5):801–803
88. Brar S, Dhillon G, Soccol C (2013) Biotrans- 101. Rezig L et al (2019) Chemical composition
formation of waste biomass into high value and bioactive compounds of Cucurbitaceae
biochemicals. Springer seeds: potential sources for new trends of
89. Garcı́a P et al (2021) Recovery of bioactive plant oils. Process Saf Environ Prot 127:73–
compounds from pomegranate (Punica gran- 81
atum L.) Peel using pressurized liquid extrac- 102. Fadimu GJ et al (2020) Ultrasound-assisted
tion. Foods 10(2):203 process for optimal recovery of phenolic com-
90. Elfalleh W et al (2011) Total phenolic con- pounds from watermelon (Citrullus lanatus)
tents and antioxidant activities of pomegran- seed and peel. J Food Meas Charact 14(3):
ate peel, seed, leaf and flower. J Med Plant Res 1784–1793
6(xx):4724–4730 103. Irabor E, Ebhoaye J (2020) Qualitative and
91. Gharbi E et al (2016) Salicylic acid differently quantitative screening of some phytochemical
impacts ethylene and polyamine synthesis in compounds in watermelon (Citrullus lana-
the glycophyte Solanum lycopersicum and the tus) seeds cultivated in ESAN west local gov-
wild-related halophyte Solanum chilense ernment area of Edo state. Int J Eng Sci
exposed to mild salt stress. Physiol Plant Technol 5:268–273
158(2):152–167 104. Juliet O (2018) Phytochemical screening and
92. Navarro-González I et al (2011) Chemical nutritional profile of Citrullus lanatus. Seeds
profile, functional and antioxidant properties 3:295–298
of tomato peel fiber. Food Res Int 44(5): 105. Addo PW, Agbenorhevi J, Adu-Poku D
1528–1535 (2018) Antinutrient contents of watermelon
93. Szabo K et al (2019) Screening of ten tomato seeds. MOJ Food Process Technol 6:237–
varieties processing waste for bioactive com- 239
ponents and their related antioxidant and 106. Putnik P et al (2017) Innovative “green” and
antimicrobial activities. Antioxidants (Basel) novel strategies for the extraction of bioactive
8(8):292 added value compounds from citrus wastes – a
94. Silva PA et al (2019) Characterization of review. Molecules 22(5):680
tomato processing by-product for use as a 107. Caldas TW et al (2018) Phenolic compounds
potential functional food ingredient: nutri- recovery from grape skin using conventional
tional composition, antioxidant activity and and non-conventional extraction methods.
bioactive compounds. Int J Food Sci Nutr Ind Crop Prod 111:86–91
70(2):150–160 108. Soto KM et al (2019) Fruit peels waste for the
95. Toor RK, Savage GP (2005) Antioxidant green synthesis of silver nanoparticles with
activity in different fractions of tomatoes. antimicrobial activity against foodborne
Food Res Int 38(5):487–494 pathogens. LWT 103:293–300
96. Eller FJ et al (2010) Extraction and analysis of 109. Safdar MN et al (2017) Extraction and quan-
tomato seed oil. J Am Oil Chem Soc 87(7): tification of polyphenols from kinnow (Citrus
755–762 reticulate L.) peel using ultrasound and mac-
97. Ferreres F et al (2010) Tomato (Lycopersicon eration techniques. J Food Drug Anal 25(3):
esculentum) seeds: new flavonols and cyto- 488–500
toxic effect. J Agric Food Chem 58(5): 110. Suleria HA, Barrow CJ, Dunshea FR (2020)
2854–2861 Screening and characterization of phenolic
98. Valdez-Morales M et al (2014) Phenolic con- compounds and their antioxidant capacity in
tent and antioxidant and antimutagenic activ- different fruit peels. Foods 9(9):1206
ities in tomato peel, seeds, and byproducts. J 111. Lim KJA et al (2019) Extraction of bioactive
Agric Food Chem 62(23):5281–5289 compounds from mango (Mangifera indica
99. Concha-Meyer A et al (2020) Platelet anti- L. var. Carabao) seed kernel with ethanol–
aggregant activity and bioactive compounds water binary solvent systems. J Food Sci Tech-
of ultrasound-assisted extracts from whole nol 56(5):2536–2544
and seedless tomato pomace. Foods 9(11): 112. Kaderides K et al (2019) Microwave-assisted
1564 extraction of phenolics from pomegranate
100. Dieng SIM et al (2017) Total polyphenols peels: optimization, kinetics, and comparison
and flavonoids contents of aqueous extracts with ultrasounds extraction. Chem Eng Pro-
of watermelon red flesh and peels (Citrullus cess 137:1–11
113. Motikar PD, More PR, Arya SS (2021) A green and sustainable recovery of nutrition-
novel, green environment-friendly cloud ally valuable compounds from mango peels.
point extraction of polyphenols from pome- Food Chem 192:842–848
granate peels: a comparative assessment with 125. Wang L et al (2017) Complex enzyme-
ultrasound and microwave-assisted extrac- assisted extraction releases antioxidative phe-
tion. Sep Sci Technol 56(6):1014–1025 nolic compositions from guava leaves. Mole-
114. Quiles-Carrillo L et al (2019) Optimization cules 22(10):1648
of microwave-assisted extraction of phenolic 126. Ghandahari Yazdi AP et al (2019) Optimiza-
compounds with antioxidant activity from tion of the enzyme-assisted aqueous extrac-
carob pods. Food Anal Methods 12(11): tion of phenolic compounds from pistachio
2480–2490 green hull. Food Sci Nutr 7(1):356–366
115. Vu HT, Scarlett CJ, Vuong QV (2019) Max- 127. Xi J, He L, Yan L-G (2017) Continuous
imising recovery of phenolic compounds and extraction of phenolic compounds from
antioxidant properties from banana peel using pomegranate peel using high voltage electri-
microwave assisted extraction and water. J cal discharge. Food Chem 230:354–361
Food Sci Technol 56(3):1360–1370 128. Yang Y-C et al (2013) Comparison of mod-
116. Trujillo-Mayol I et al (2019) Improvement of ified ultrasound-assisted and traditional
the polyphenol extraction from avocado peel extraction methods for the extraction of bai-
by assisted ultrasound and microwaves. J calin and baicalein from radix Scutellariae. Ind
Food Process Eng 42(6):e13197 Crop Prod 45:182–190
117. Ghafoor K, Choi YH (2009) Optimization of 129. Reshad AS, Tiwari P, Goud VV (2015)
ultrasound assisted extraction of phenolic Extraction of oil from rubber seeds for bio-
compounds and antioxidants from grape diesel application: optimization of para-
peel through response surface methodology. meters. Fuel 150:636–644
J Korean Soc Appl Biol Chem 52(3):295–300 130. Heleno SA et al (2016) Optimization of
118. Tremocoldi MA et al (2018) Exploration of ultrasound-assisted extraction to obtain
avocado by-products as natural sources of mycosterols from Agaricus bisporus L. by
bioactive compounds. PLoS One 13(2): response surface methodology and compari-
e0192577 son with conventional Soxhlet extraction.
119. Barba FJ et al (2015) Effect of alternative Food Chem 197:1054–1063
physical treatments (ultrasounds, pulsed elec- 131. Zhang Q-W, Lin L-G, Ye W-C (2018) Tech-
tric fields, and high-voltage electrical dis- niques for extraction and isolation of natural
charges) on selective recovery of products: a comprehensive review. Chin Med
bio-compounds from fermented grape pom- 13(1):1–26
ace. Food Bioprocess Technol 8(5): 132. Ganesh KS, Sridhar A, Vishali S (2022) Utili-
1139–1148 zation of fruit and vegetable waste to produce
120. Lianfu Z, Zelong L (2008) Optimization and value-added products: conventional utiliza-
comparison of ultrasound/microwave tion and emerging opportunities – a review.
assisted extraction (UMAE) and ultrasonic Chemosphere 287:132221
assisted extraction (UAE) of lycopene from 133. Chemat F et al (2019) Review of alternative
tomatoes. Ultrason Sonochem 15(5): solvents for green extraction of food and nat-
731–737 ural products: panorama, principles, applica-
121. Piñeiro Z et al (2016) Ultrasound-assisted tions and prospects. Molecules 24(16):3007
extraction of stilbenes from grape canes. 134. Veggi PC, Martinez J, Meireles MAA (2013)
Molecules 21(6):784 Microwave-assisted extraction for bioactive
122. Pham HNT (2017) Recovering bioactive compounds theory and practice. Springer, p 4
compounds from fruit and vegetable 135. Talmaciu AI, Volf I, Popa VI (2015) A com-
wastes. In: Recovering bioactive compounds parative analysis of the ‘green’ techniques
from agricultural wastes, pp 81–100. https:// applied for polyphenols extraction from bior-
doi.org/10.1002/9781119168850.ch4 esources. Chem Biodivers 12(11):
123. Corrales M et al (2008) Extraction of antho- 1635–1651
cyanins from grape by-products assisted by 136. Li H et al (2012) Microwave-assisted extrac-
ultrasonics, high hydrostatic pressure or tion of phenolics with maximal antioxidant
pulsed electric fields: a comparison. Innova- activities in tomatoes. Food Chem 130(4):
tive Food Sci Emerg Technol 9(1):85–91 928–936
124. Parniakov O et al (2016) Extraction assisted
by pulsed electric energy as a potential tool for
137. Routray W, Orsat V (2012) Microwave- antioxidant activity, and oxidation stability.
assisted extraction of flavonoids: a review. Food Chem 172:7–17
Food Bioprocess Technol 5(2):409–424 150. More PR, Arya SS (2021) Intensification of
138. Pérez-Serradilla J, De Castro ML (2011) bio-actives extraction from pomegranate peel
Microwave-assisted extraction of phenolic using pulsed ultrasound: effect of factors, cor-
compounds from wine lees and spray-drying relation, optimization and antioxidant bioac-
of the extract. Food Chem 124(4): tivities. Ultrason Sonochem 72:105423
1652–1659 151. Reátegui JLP et al (2014) Extraction of anti-
139. Mustafa A, Turner C (2011) Pressurized liq- oxidant compounds from blackberry (Rubus
uid extraction as a green approach in food and sp.) bagasse using supercritical CO2 assisted
herbal plants extraction: a review. Anal Chim by ultrasound. J Supercrit Fluids 94:223–233
Acta 703(1):8–18 152. Liu J et al (2018) Determination of garlic
140. Marić M et al (2018) An overview of the phenolic compounds using supercritical fluid
traditional and innovative approaches for pec- extraction coupled to supercritical fluid chro-
tin extraction from plant food wastes and matography/tandem mass spectrometry. J
by-products: ultrasound-, microwaves-, and Pharm Biomed Anal 159:513–523
enzyme-assisted extraction. Trends Food Sci 153. Soliva-Fortuny R et al (2009) Effects of
Technol 76:28–37 pulsed electric fields on bioactive compounds
141. Du F-Y et al (2009) Application of ionic in foods: a review. Trends Food Sci Technol
liquids in the microwave-assisted extraction 20(11–12):544–556
of polyphenolic compounds from medicinal 154. Vorobiev E, Lebovka N (2006) Extraction of
plants. Talanta 78(3):1177–1184 intercellular components by pulsed electric
142. Alara OR et al (2020) Extract-rich in flavo- fields. In: Pulsed electric fields technology
noids from Hibiscus sabdariffa calyces: opti- for the food industry. Springer, pp 153–193
mizing microwave-assisted extraction method 155. Boussetta N et al (2014) Valorization of oil-
and characterization through LC-Q-TOF- seed residues: extraction of polyphenols from
MS analysis. J Food Process Eng 43(2): flaxseed hulls by pulsed electric fields. Ind
e13339 Crop Prod 52:347–353
143. Chen C et al (2015) Optimization of dynamic 156. Delsart C et al (2012) Enhanced extraction of
microwave-assisted extraction of Armillaria phenolic compounds from Merlot grapes by
polysaccharides using RSM, and their pulsed electric field treatment. Am J Enol
biological activity. LWT-Food Sci Technol Vitic 63(2):205–211
64(2):1263–1269 157. Medina-Meza IG, Barbosa-Cánovas GV
144. Rodrı́guez De Luna SL, Ramı́rez-Garza R, (2015) Assisted extraction of bioactive com-
Serna Saldı́var SO (2020) Environmentally pounds from plum and grape peels by ultra-
friendly methods for flavonoid extraction sonics and pulsed electric fields. J Food Eng
from plant material: impact of their operating 166:268–275
conditions on yield and antioxidant proper- 158. Luengo E, Álvarez I, Raso J (2013) Improv-
ties. Sci World J 2020:6792069 ing the pressing extraction of polyphenols of
145. Shen X, Shao S, Guo M (2017) Ultrasound- orange peel by pulsed electric fields. Innova-
induced changes in physical and functional tive Food Sci Emerg Technol 17:79–84
properties of whey proteins. Int J Food Sci 159. Peiró S et al (2019) Improving polyphenol
Technol 52(2):381–388 extraction from lemon residues by pulsed
146. Kentish S, Feng H (2014) Applications of electric fields. Waste Biomass Valorization
power ultrasound in food processing. Annu 10(4):889–897
Rev Food Sci Technol 5(1):263–284 160. Andreou V et al (2020) Effect of pulsed elec-
147. Grassino AN et al (2016) Ultrasound assisted tric fields and high pressure on improved
extraction and characterization of pectin from recovery of high-added-value compounds
tomato waste. Food Chem 198:93–100 from olive pomace. J Food Sci 85(5):
148. Goula AM et al (2017) Green ultrasound- 1500–1512
assisted extraction of carotenoids from pome- 161. Singh JP et al (2016) Composition, bioactive
granate wastes using vegetable oils. Ultrason compounds and antioxidant activity of com-
Sonochem 34:821–830 mon Indian fruits and vegetables. J Food Sci
149. Samaram S et al (2015) Optimisation of Technol 53(11):4056–4066
ultrasound-assisted extraction of oil from 162. Gligor O et al (2019) Enzyme-assisted extrac-
papaya seed by response surface methodol- tions of polyphenols – a comprehensive
ogy: oil recovery, radical scavenging review. Trends Food Sci Technol 88:302–315
163. Gil-Martı́n E et al (2022) Influence of the 167. Chhikara N et al (2019) Bioactive com-
extraction method on the recovery of bioac- pounds of beetroot and utilization in food
tive phenolic compounds from food industry processing industry: a critical review. Food
by-products. Food Chem 378:131918 Chem 272:192–200
164. Meini M-R et al (2019) Recovery of phenolic 168. Pattnaik M et al (2021) Innovative technolo-
antioxidants from Syrah grape pomace gies for extraction and microencapsulation of
through the optimization of an enzymatic bioactives from plant-based food waste and
extraction process. Food Chem 283:257–264 their applications in functional food develop-
165. Pogorzelska-Nowicka E et al (2018) Bioac- ment. Foods 10(2):279
tive compounds in functional meat products. 169. Hikal W et al (2021) Sustainable and environ-
Molecules 23(2):307 mentally friendly essential oils extracted from
166. Talekar S et al (2018) From waste to wealth: pineapple waste. Biointerface Res Appl Chem
high recovery of nutraceuticals from pome- 12:6833–6844
granate seed waste using a green extraction
process. Ind Crop Prod 112:790–802
Chapter 14
Plant Seeds: A Potential Bioresource for Isolation

of Nutraceutical and Bioactive Compounds
Gitanjali Sambhajirao Deokar, Nilesh Prakash Nirmal ,
and Sanjay Jayprakash Kshirsagar
Abstract
The greatest difficulty facing the cosmos now is the ability to survive in excellent health. The idea that
nature preserves plant seeds when they are dormant and have been sitting in the soil for a long time is the
foundation for the current notion. Bacteria, fungus, and virus attack are deterred from attacking seeds with
the assistance of antimicrobial phytochemicals, plant defensins, other associated antimicrobial biopeptide
components, plant prebiotics, probiotics, postbiotics, etc., prepared by the seeds during its journey towards
the matured plant. Nature uses this technique to safeguard the offspring. We merely need to understand
what nature is trying to tell us. The aim of the chapter is to take us through this message of nature followed
for the well-being and growth of plant life cycle. Readers shall be acquainted with the current state of
knowledge on plant seeds as a potential bioresource for the extraction of nutraceutical and bioactive
chemicals. Moreover, the chapter will take the reader through Mother Nature’s ways of green extraction
techniques like sprouting, germination, and fermentation for the health benefits of human beings.
Key words Plant seeds, Antimicrobial biopeptide, Prebiotics, Probiotics, Postbiotics, Green extrac-
tion, Sprouting, Germination, Fermentation
1 Introduction
Healthy survival is the current biggest challenge for the universe

[1]. When it comes to people, animals, and plants, good survival is
relevant in this context. In the end, it applies to the entire cosmos.
Ecological equilibrium is the name we can give it [2]. The largest
difficulty the universe is currently facing is keeping this balance.
Humans are involved in the hunt for answers to address this imbal-
ance that is causing the turmoil in regard to everyone’s health since
they are morally responsible beings with more active minds than
animals and plants [3]. Researchers are deeply involved in figuring
out the answers for leading a healthy lifestyle, whether it is by
333
334 Gitanjali Sambhajirao Deokar et al.
medication, preventative measures, physical activity, yoga, eating

habits, etc. It is indeed time to reflect, comprehend, and pinpoint
the natural processes that have been in place to preserve ecological
balance. Let us imagine the seeds of a plant in relation to this. The
survival and lifespan of plant seeds will be key topics of the present
chapter. A plant’s whole defense system [4], which consists of a
number of defense mechanisms, keeps the seeds and, ultimately, the
progeny free from illness. In this context, we’ll take a closer look at
a number of seed defense systems that are involved in homeostatic
regulation and defense mechanisms throughout the plant’s life
cycle [5, 6]. In this sense, both edible [7] and non-edible plant
seeds [8] could be considered.
In order to comprehend seed defense mechanisms and the
significance of plant seeds as a bioresource for the extraction of
nutraceutical and bioactive substances [9], it is important to pros-
pect a wide range of bioactive components, including antimicrobial
biopeptides like defensins, thionins, lipid transfer proteins, cyclo-
tides, snakins, and hevein-like proteins [10]. When thinking about
health and the innate immune system, it’s also critical to look into
microflora-based defense mechanisms like prebiotics, probiotics,
and postbiotics connected to seeds [11]. The reader should pay
particular attention to how nature uses green extraction and isola-
tion techniques. The current chapter focuses on the in situ green
extraction methods that seeds use. The crucial ones are germination
and sprouting [12]. These are the bioprocesses for enhancing the
digestibility and bioactivity of the components [13]. Most of the
inactive or dormant components get activated during germina-
tion/sprouting process. The most notable one is conversion of
difficult to digest crude proteins into easily digestible amino acids
by various enzymes which get activated during the process. The
most crucial activity, fermentation, which forms the basis of innate
immune system of universe [14], also requires special attention
because it is Mother Nature’s green extraction strategy for preserv-
ing the ecological balance. The concept of fermentation should
help readers comprehend how prebiotics and probiotics fit into
the rules governing plant health and how it could be correlated in
terms of making maximum beneficial use of plant seeds for health
benefits of human beings. The present chapter will definitely acti-
vate thought process for the complete cost effective and appropri-
ate utilization of the components from seeds with zero waste
aspects [15] and health benefits. The present chapter discusses
seed to plant life cycle helping us realize the nature’s unique
arrangements to explore the homeostatic regulations and to think
if we can make beneficial use of it for the well-being of the universe
alongside with appropriate management of ecological homeostasis.
Figure 1 represent the schematic for understanding in situ green
extraction systems of nature and activation of nutraceutical &
Plant Seeds: A Potential Bioresource for Isolation of Nutraceutical and. . . 335
Fig. 1 Roadmap for understanding in situ green extraction systems of nature
bioactive resources from the plants seeds. Further, how these bio-
active compounds involved in defense and healthy growth of plant,
which could be beneficially utilized for commercial exploitation of
the products for human health benefits.
1.1 The Established In ecology, homeostasis refers to an ecosystem’s capacity to pre-

Mechanisms Used by serve its overall stability despite any disturbances. It is the result of
Nature to Preserve interactions between species on an ecological level as well as biodi-
Ecological versity. The attributes of a system consider the entire system. It is
Homeostasis independent of the species that make up the system. Complex
systems frequently exhibit homeostasis. Ecosystems are kept in a
state of balance, but since they are such a very diversified system,
they cannot be totally stable. In an ecological system, negative
feedback controls the maintenance of equilibrium. The adverse
feedback negates the environmental change. One of the main
causes of negative feedback is resource limitations. For instance,
when a resource is scarce or nearly exhausted, the death rate of
those who use it rises or the birth rate falls [16, 17]. Our main
concern in this case is ecological balance, which includes health
issues.
Homeostasis is described in regard to this as a self-balancing

mechanism through which biological systems retain stability while
adjusting altering external environment. Any organism can keep
internal conditions that are more or less constant in order to adapt
to and survive in challenging exterior environments. The principle
of homeostasis has evolved into the main organizing tenet of physi-
ology as a result of the mounting challenges of existence and
ongoing medical research conducted worldwide. Understanding
this self-regulating process is necessary in order to properly appre-
ciate how ecological homeostasis controls both health and sickness
in this intricate environment. Disruption of homeostatic processes
is the first sign of sickness. Therefore, human health-related
research must be focused on restoring these homeostatic circum-
stances by cooperating rather than competing with nature
[18]. Resource constraint in terms of health is lack of understand-
ing of how nature contributes to the human wellbeing indirectly
through its well-planned happenings and resources. Ecosystems are
the planet’s life-support systems—for the human species and all
other forms of life. Ecosystem services are indispensable to the well-
being and health of people everywhere. Fresh water, food, fiber,
fuel, biological products, nutrient and waste management, proces-
sing, and detoxification, control of infectious diseases, cultural,
spiritual, and recreational services, and climate regulation are a
few of the ecosystem services that should be mentioned.
The most important ecosystem function nature offers to pre-
serve the health equilibrium of the universe is the management of
infectious diseases [19]. The existence of plant seeds is the most
significant illustration of the ecosystem service that nature has given
to humans. The various chemical components that are present in
seeds and variety of roles of seed components during the plant’s life
cycle make up the entire package of defense mechanisms in plants.
The treasure contained in the form of diverse biologically useful
components enables the seeds to last longer and contributes to the
continuation of the generations. We must comprehend the idea
underlying plant seed survival and longevity in order to compre-
hend the significance of seeds.
1.2 Concept Behind Seeds are a crucial component of the ecosystem process because
Plant Seed Survival they enable plants to adapt to changing environments, maintain
and Longevity existing populations, and establish new populations in suitable areas
through a scatter network. Thus, seeds play a variety of significant
roles in the physiology, ecology, and geography of plants [20].
Two key factors, seed lifetime and seed dormancy, are used to
control seed quality. The most important quality of the seed is its
resistance to deterioration. Seed dormancy, which limits seed ger-
mination under unfavorable conditions and prevents the plant from
concluding its life cycle without losing its ability to grow in the
appropriate conditions, serves as the plant’s protective mechanism.
According to Dalling et al. (2011), seeds make use of four strategies

for resisting decay: (i) physical barriers that make seeds impervious
to pathogens; (ii) endogenous chemical defenses of seeds; (iii)
chemical defenses of beneficial seed-microbial interactions; and
(iv) rapid seed germination [21, 22]. A crucial mechanism for
seed survival and longevity in the soil is the array of inducible
enzyme-based biochemical defenses that are present on the exterior
and inside of seeds. Since they may be applicable to other defense
enzymes as well as to a variety of plant species and habitats, enzyme-
based biochemical defenses may have wider ramifications [22]. In
the framework of this chapter, the themes that demand consider-
ation are chemical and microbiological defense systems.
2 Chemical Defense System of Seeds
Structural and chemical traits influence predator rate prior to incor-

poration in the soil. Seeds contain diverse secondary chemicals,
mostly at concentrations much higher than elsewhere in the plant.
Chemical constituents found to be effective against microbial infec-
tion in the soil include phenolic compounds, alkaloids, antimicro-
bial peptides, etc. Consistent with an antimicrobial role, chemical
defenses often are allocated primarily to seed coats rather than
embryo or endosperm tissue protecting seeds from pathogen infec-
tion at the pre-dispersal stage [23]. Excellent insights into the idea
of seed defense are provided by research done in 2018 by team
Zalamea et al. In the seeds of pioneer species (pioneer tree species
from lowland tropical forests in Panama), they studied dormancy-
defense syndromes and trade-offs between physical and chemical
defenses. The physical and chemical properties of seeds determine
their ability to survive in the soil seed bank in the presence of
pathogens and predators. Seeds can survive in soil and even decades
after being dispersed, seeds can still germinate and recruit
[24]. Research data from Zalamea et al. (2018) mentions two
methods that can be used to create both short- and long-term
persistence of seeds. Impermeable seeds exhibit physically dormant
defensive syndrome while permeable seeds exhibit chemical defense
implying physiological dormancy. The quiescent defensive syn-
drome is mirrored by the decreased level of chemical and physical
defenses in transient seeds. Overall, they discovered a connection
between seed defense and seed dormancy, indicating that environ-
mental forces on seed persistence and delayed germination may be
able to select for trait combinations defining various dormancy-
defense syndromes [24].
When it comes to soil borne infections, the phenolic chemicals
that plants release from their roots and seeds frequently
exhibit strong antifungal, antibacterial, and antiviral properties.
The phenolic group of metabolites consists of terpenoids,
phenylpropanoids, cinnamic acids, lignin precursors, hydroxy-

benzoic acids, catechols, coumarins, flavonoids, isoflavonoids, and
tannins, among others [25]. Trigonella foenum-graecum is a
Leguminosae plant that is widely farmed in India and Egypt.
Trigonelline alkaloid was initially discovered in the seeds of this
species. Salmonella enteric and Escherichia coli are both susceptible
to the antibacterial effects of the coffee alkaloid trigonelline. Alka-
loids are crucial for plant protection in both sweet and wild types
of Lupinus. Lupin seeds contains about 5% quinolizidine alkaloids,
which are poisonous to insects [25]. Quinolizidine alkaloids (QAs)
are poisonous secondary metabolites that are present in the genus
Lupinus, some of which are significant grain legume crops.
QAs provide the plants with protection from insect pests. Based
on measurements of QA translocation and total QA levels in repro-
ductive tissues, it has been hypothesized that of the QAs that
accumulate in seeds, half are generated within the seed and half
are translocated [26]. In relation to this chemical defense associa-
tion of seeds let us have a look at bioactive compounds of seeds
which are actively involved in defense systems associated with seeds
and plants throughout the life cycle of plant.
2.1 Antimicrobial The barrier defense mechanism of plants includes antimicrobial

Peptides in Seeds peptides (AMPs). They have been isolated from the roots, seeds,
flowers, stems, and leaves of numerous different species, and they
have actions toward both phytopathogens and organisms that are
harmful to humans, such as viruses, bacteria, fungi, protozoa,
parasites, etc. The antibacterial action of AMPs has drawn a lot of
attention due to the rise of drug-resistant infections. Plant AMPs
are thus seen as promising antimicrobial substances with significant
biotechnological uses. Plant AMPs are thought to have a consider-
able impact on both plant growth and development as well as
pathogen defense mechanisms. Plant AMPs are divided into various
groups and share characteristics including a positive charge, disul-
fide links (which stabilize the structure), and a method of action
that targets multiple sites on the plasma membranes, intracellular
components of pathogen, and outer membrane structures [27]. In
the case of a mechanism involving membrane breakdown, it
advances by forming membrane pores, which causes ion and
metabolite leakage, depolarization, stoppage of the respiratory
processes, and cell death. Plant AMPs can be classified as anionic
(AAMPs) or cationic peptides (CAMPs) depending on their elec-
trical charge. The cationic residues electrostatically draw negatively
charged molecules, such as anionic phospholipids, lipopolysacchar-
ides, or teichoic acids, causing the peptide to assemble on the
surface of the membrane. When concentration reaches a certain
point, the collapse starts [28, 29]. These peptides are categorized
into distinct families mainly on the basis of their amino acid
sequence, identity, number of cysteine residues, and their spacing.
They exhibit a wide range of functions ranging from direct anti-

microbial properties to immunomodulatory effects [28, 29]. The
main families of AMPs comprise defensins, thionins, lipid transfer
proteins, cyclotides, snakins, and hevein-like proteins, according to
amino acid sequence homolog [28].
Note Representative examples of plant seed antimicrobial peptides

(AMPs) isolated using germination process with their classification
and bioactivities are given in Table 1.
2.1.1 Defensins Plant defensins are the vast antimicrobial peptide superfamily found
across the plant world [27]. Plant defensins are small, highly stable,
rich in arginine, lysine, and cysteine residues, that constitute a part
of the innate immune system. These are large family of cationic
host defense peptides (HDP) [30]. The first plant defensins were
isolated from wheat Triticum aestivum L and barley Hordeum
vulgare and initially classified as gamma thionins. Plant defensins
are small basic, cysteine-rich peptides ranging from 45 to 54 amino
acids with low molecular weight about 5 kDa. Biological activities
reported for plant defensins include antifungal, antibacterial, pro-
teinase, and insect amylase inhibitor activities [28]. Additionally, it
has been reported that these compounds mediate abiotic stress and
zinc tolerance, inhibit protein synthesis, impair ion channel func-
tion, and hinder microbial, root hair, and parasitic plant growth,
change the redox state of ascorbic acid, stimulate the perception of
sweetness, act as epigenetic factors, influence self-incompatibility,
and support male reproductive development [31]. Plant defensin
genes produce precursor proteins that target a signal at the amino
terminal and a pro-peptide at the c-terminal (CTPP) and have a
mature defensin domain.
This CTPP is optional; some defensins may contain it while
others do not. Class II plant defensins are defined as peptides with a
c-terminal pro-peptide signal (CTPP) of 27–33 amino acid resi-
dues. Glutamic and aspartic acids, which have a negative charge and
balance the positive charge of the defensin domain, are prevalent in
these amino acid residues. Class I is assigned to the other class that
is deficient in these signals. Class I plant defensins are only detected
in the seeds, but class II plant defensins are reported to be abun-
dantly produced in both the reproductive and vegetative sections of
the plant [32–34].
Activation Process of The study projects carried out by the co-researcher Terras F et al. in
Defensins in Seeds 1995 provided illuminating insight into the activation mechanism
of defensins for the execution of their functional tasks. Raphanus
sativus-antifungal protein-1 (RsAFPl) and Protein-2 (RsAFP2),
two homologous, 5-kD cysteine-rich proteins found in radish
seeds, both demonstrate strong antifungal action in vitro. In their
Table 1
Representative examples of plant seed antimicrobial peptides (AMPs) isolated using germination
process and their classification and bioactivities
Sr.
no. Plant seed AMP Peptide Function Reference
1. Radish seeds Defensins 5-kD cysteine-rich Antifungal activity [35]
[Raphanus proteins, Raphanus against foliar
sativus] sativus-antifungal pathogen Alternaria
protein-1 (RsAFPl), longipes
and Protein-
2 (RsAFP2)
2. Lentil seeds [Lens Defensins Lentil seed defensin Activity against [96]
culinaris] termed as Lc-def has Aspergillus niger
8 cysteines forming (Aspergillus niger
four disulfide bonds. causes sooty mold on
A 74-residue onions and
predefensin contains ornamental plants)
a putative signal
peptide (27 amino
acid) and a mature
protein
3. Pearl millets, Thionins Thionins (PR protein- Sporangia. graminicola [39]
[Pennisetum 13) are a class of zoospores lysis
glaucum (L.) Cys-rich polypeptides
R. Br.] of about 5 kDa
cultivars
IP18292
4. Black cumin Thionins Nigellothionins- Growth inhibitory [97]
seeds [Nigella dominant peptide, activity against
sativa (L.)] NsW2 with 8-Cys filamentous fungi
motifs and yeasts
5. Rice seeds [Oryza Lipid transfer OsLTPL36, a homolog Seed quality seed [45]
sativa] proteins of putative lipid development and
transport protein seed germination
6. Castor bean Lipid transfer Nonspecific lipid Regulation of fatty acid [98]
[(Ricinus proteins transfer protein, β oxidation through
communis (nsLTP) enhancement of
(L.)] acetyl CoA oxidase
activity in
glycosomes (nsLTP
as acetyl CoA carrier-
energy source and
helps in cellular
respiration
(continued)
Table 1
(continued)
Sr.
no. Plant seed AMP Peptide Function Reference
7. Lentil seeds [Lens Lipid transfer Lc-LTP 1–8, (92–93) Antibacterial: Inhibit [99]
culinaris] proteins amino acid residues, growth of
with four disulfide Agrobacterium
bonds tumefaciens.
(causative agent of
crown gall disease)
8. Sweet violet Cyclotides Peptides with cyclic, Act as defense and [47]
seeds [Viola cystine knot storage proteins
odorata] structural motif
9. Alfalfa seed Snakins MsSN1 has a putative Antibacterial and [51]
[(Medicago signal peptide of antifungal activity,
sativa (L.)] 25 amino acids and helps in plant innate
possesses a snakin/ immunity
GASA domain
(Pfam02704)
containing
12 cysteine residues
in conserved
positions within a
conserved C-terminal
region
10. Quinoa seeds Hevein-like Type III lectin Inhibition of [55]
[Chenopodium proteins precursor, CB-HLPs phytopathogenic
quinoa] (chitin-binding fungi
hevein-like peptides –
cysteine-rich
peptides) chenotides,
tandem repeats of the
mature peptide
domains, with a
cleavable Gly/Ala-
rich linker consisting
of 18 amino acids
research, it was shown that these proteins are found in the cell wall
and are more common in the lining of the outer cell layers of several
seed organs. Additionally, following breakdown of the seed coat,
RsAFPs are selectively released during seed germination. The num-
ber of proteins produced is sufficient to provide a microenviron-
ment that inhibits fungal development surrounding the seed
[35]. There is also documented additional material that supports
the idea that external stimuli activate defense mechanisms. It was
discovered that when a vegetative plant tissue is injured or detects
pathogens, a number of dynamic defense mechanisms can be acti-

vated. In response to attempts by fungus hyphae to penetrate the
cell wall, newly produced carbohydrate material may be deposited
at the location [35, 36]. Furthermore, injury and elicitor therapy
can cause preexisting cell wall proteins to become oxidatively cross-
linked [37]. At early germination, when the seed coat, an efficient
physical barrier against microorganisms, is destroyed and the tiny
seedling is exposed to the earth, is a particularly vulnerable time.
The importance of this event in relation to seedling defense against
fungal infections was evaluated using radish seed as a model system
for studying the release of Rs-AFPs during germination. A bioassay
was carried out in which seeds were permitted to sprout on a
medium promoting the formation of a fungal colony. A halo of
growth inhibition surrounded the seed as the borders of colony
grew closer to it. After their seed coat is damaged, either by germi-
nation or by mechanical incision, radish seeds produce a proteina-
ceous antifungal chemical [35].
2.1.2 Thionins Thionins are a family of antimicrobial peptides that are found in the
seeds, stems, roots, and leaves of cruciferous plants, mistletoe, and
cereals. They have a low molecular weight (approximately 5 kDa)
and are highly concentrated in arginine, lysine, and cysteine resi-
dues. Thionins are poisonous to yeast, fungus, and bacteria. It was
proposed that their activity resulted from the lysis of the mem-
branes of adhering cells. Antifungal activity shown by thionins, is
a result of electrostatic interactions between positively charged
thionins and negatively charged phospholipids in fungal mem-
branes, which lead to the creation of pores or a particular contact
with a particular lipid domain [28]. The typical 5 kDa, basic thionin
peptide with three or four disulfide bridges is obtained by first
producing them as preproproteins and then processing them. Tran-
scriptomes of more than 1000 plant species have been sequenced as
part of the “one thousand plant transcriptomes initiative” (1KP
project). New thionin sequences were sought using the data. The
four types of thionins that were previously identified, only two
classes and their variants are presently recognized using the data
from the 1KP study. Due to the fact that every variant was linked to
either class 1 (eight cysteines) or class 2 (six cysteines). Eighteen
versions in total were found by the 1KP project with all the distinct
variants [38].
Activation Process of Mobilization of thionins is very clearly elucidated by Chandrashe-

Thionins in Seeds khara S. et al. in 2010. Purified thionin from pearl millet was tested
on the fungus Sclerospora graminicola that causes downy mildew
disease and was more successful in rupturing the membrane, point-
ing towards a potential toxicity mechanism. It was shown that
resistant cultivars expressed thionin transcripts at a higher level
than susceptible cultivars. Immunofluorescence studies have
revealed strong fluorescence in vascular locations, confirming its

systemic translocation during contact, as well as in epidermal
regions, confirming its involvement in halting pathogen entrance.
Additionally, localization studies using the coleoptile epidermal
peeling of resistant and susceptible cultivars of pearl millet revealed
that thionin protein was more abundant in seedlings of the resistant
cultivar that had been inoculated with S. graminicola than in unin-
oculated seedlings at the papillar region of the cell wall. In suscep-
tible inoculation samples, no discernible localization was found
[39]. Thionins are created from bigger precursors, including a
C-terminal peptide and a signal peptide that have undergone
post-translational processing. These characteristics demonstrate
that thionins are released following pathogen infection, which
results in antimicrobial activity at the site of pathogen entry and
stops the pathogen from spreading the infection region [39, 40].
In conclusion, this study showed that thionin is crucial for pearl
millet systems’ ability to fight the downy mildew infection
[39]. Similar studies were done on wheat and barley thionins
against bacterial strains including Clavibacter michiganensis subsp.
Sepedonicus and fungal pathogens such Rosellinia nectatrix, Colle-
totrichum lagenarium, Phytophthora infestans, Fusarium solani,
Thievaliopsis paradoxa, and Drechslera teres. Thionins have been
shown to have an impact on several cell types, and there is evidence
that these proteins can alter the permeability of yeast membranes
[41, 42].
2.1.3 Lipid Transfer Nonspecific lipid transfer proteins(LTPs) are small, cysteine-rich
Proteins proteins that have a variety of functional roles in the growth and
development of plants, including the production of cutin wax,
adhesion of pollen tubes, cell expansion, seed development, germi-
nation, and adaptation to changing environmental conditions.
LTPs have a hydrophobic cavity with eight conserved cysteine
residues that allows for a wide range of lipid-binding specificities.
Many LTPs serve as positive regulators of plant disease resistance
because they are members of the pathogenesis-related protein
14 family (PR14) [43]. Researchers are interested in LTPs for
three basic reasons. Plant LTPs have two distinct properties. First,
they can bind and transfer lipids, which is how they received their
names and were grouped into one class. The second characteristic is
that LTPs are protective proteins that are part of innate plant
immunity. The third characteristic is that one of the most therapeu-
tically significant classes of plant allergens is represented by
LTPs [44].
Activation Process of Lipid In the studies reported in 2015 by Wang et al., a homolog of a
Transfer Proteins in Seeds putative lipid transport protein was revealed to exhibit unique
expression in the developing rice seeds. Homolog was predomi-
nantly expressed in developing seed coat and endosperm aleurone
cells, according to transcriptional profiling and in situ hybridization

studies [45]. By forming complexes with diverse lipid molecules,
LTPs are thought to be involved in the activation and control of
many signaling pathways in plants. One of the classes of signal
mediators in plants are oxylipins. Oxylipins also control the proce-
dures for neutralizing the harmful byproducts created during stress.
The concurrent activity of lipoxygenase and allene oxide synthase
results in the formation of covalent complexes between the barley
LTP1 and oxylipin of 9(S),10-epoxy-10,12(Z)-octadecadienoic
acid during seed germination, according to studies on the grain.
This connection may point to a cooperative role for LTPs and
oxylipins in the control of the signaling pathways that activate the
mechanism that protects plant cells from damage under stress
[44, 45].
2.1.4 Cyclotides A cystine knot made up of three disulfide bonds stabilizes the
globular microproteins known as cyclotides, which have a distinc-
tive head-to-tail cyclized backbone. Compared to other peptides of
comparable size, they exhibit excellent stability to chemical, ther-
mal, and biological degradation because of their distinctive circular
backbone topology and knotted arrangement of three disulfide
links. Multiple cyclotides (between 10 and 160) are typically pres-
ent in every tissue of a single plant, including the flowers, leaves,
stems, roots, and perhaps even the seeds [46].
Activation Process of Slazak, B., et al. (2020) described experiments involving variations
Cyclotides in Seeds in the quantity of cyclotides in developing seeds of Viola odorata.
To soften the seed coating, sterilized seeds were placed in a beaker
with wet paper and kept at 4 °C for 7 days. The seed coating was
then taken off, and the seeds were placed on half-strength MS
media that had been thickened with 7 g/l agar. Three distinct
stages of seedling development—the seed, a germinated seedling
with endosperm, and seedlings that utilized the entire
endosperm—were collected and freeze-dried after seeds were
grown in a culture chamber. The growing seedling appeared to
ingest cyclotides found in the seed endosperm. It was found that
the cyclotide pattern found in various tissues and surroundings is
shaped by degrading processes. The findings show that various
cyclotides have distinct functions, some of which are related to
defense and others which are related to storage proteins [47].
2.1.5 Snakins Snakins are typically tiny, positively charged, cysteine-rich proteins
with a molecular weight of about 7 kDa that play a number of roles
in plant defense responses, including antimicrobial action against a
wide variety of phytopathogens and animal diseases. Snakin-1
(StSN1), the first recognized Snakin peptide, was isolated from
potato tubers and given the name Snakin because it had sequence
features with snake venoms. The 12 cysteine residues that are
always present in the conserved GASA (Gibberellic Acid Stimulated

in Arabidopsis) domain in the C-terminal portion of the Snakin
family peptides give them their distinctive properties [48, 49].
Genes from the Snakin/Gibberellic Acid stimulated in Arabidopsis
(GASA) family are important for plant growth and development
as well as protection against pathogens. In one study by Deng
M. et al. in 2021, the effect of Snakin-2 (SN2) on tuber dormancy
and sprouting was examined. This work used a transgenic tech-
nique to regulate the level of SN2 expression in tubers, and it
showed that StSN2 strongly impacted tuber sprouting, silencing
StSN2 caused dormancy to be released, and overexpressing tubers
had a longer dormant period than the control [50].
Activation Process of Understanding the activation and use of Snakin peptides for anti-
Snakins in Seeds fungal action in alfalfa seeds was made possible by Garcia et al.
(2014). Sterilized seeds of both transgenic and wild types were
put in petri dishes with 1% agar water under 16 h of light
(100 moles/m2s) and 25 °C.
Plants were moved to 1:1 vermiculite: perlite and kept
in magenta vessels to preserve humidity after being incubated at
25 °C with a 16 h photoperiod for a month. Phoma medicaginis was
sprayed onto all aerial tissues of two-month-old plants to inoculate
them. The percentage of sick leaflets was examined 30 days after
vaccination, and 60 days later, the number of regrowing plants and
the percentage of heavily defoliated plants were counted. Snakin-1
produced by alfalfa (MsSN1)-overexpressing alfalfa transgenic
plants exhibits increased antimicrobial activity against virulent fun-
gal strains without changing the nitrogen-fixing symbiosis, paving
the way for the development of efficient alfalfa transgenic cultivars
for biotic stress resistance [51].
2.1.6 Hevein-Like A family of antifungal plant AMPs known as hevein-like antimicro-

Proteins bial peptides (AMPs) contain a chitin-binding site that interacts
with the chitin of fungal cell walls. Hevein-like peptides are mem-
bers of the AMP family that are structurally related to the antimi-
crobial peptide hevein, which is found in the latex of the Hevea
brasiliensis (Willd. ex A. Juss.). Müll. Arg. Chitin and similar oligo-
saccharides can bind to hevein-like AMPs’ chitin-binding site. It is
widely accepted that antifungal action is mediated by binding to
chitin and similar oligomers found in fungal cell walls [52, 53]. Anti-
microbial peptide (AMP) derived from cycad (Cycas revoluta)
seeds, Cy-AMP1, has been isolated and characterized. Antimicro-
bial peptide Cy-AMP1 was shown to have chitin-binding ability in
one of the researches conducted by Seiya Yokoyama et al. in 2008,
and the chitin-binding domain was found to be conserved in
knottin-type and hevein-type antimicrobial peptides. To investigate
the function of the chitin-binding domain, Escherichia coli was used
to generate and purify the recombinant Cy-AMP1. Cy-AMP1

mutations drastically reduced their antifungal activity compared
to native Cy-AMP1 and completely lost their capacity to bind
chitin [54].
Activation Process of Another work by Shining Loo 2021, examined the hyperstable
Hevein-Like Proteins in antifungal Chitin-binding hevein-like peptides (CB-HLPs) cheno-
Seeds tides found in quinoa seeds (Chenopodium quinoa). The biosynthe-
sis of chenotides was shown to be novel and to belong to a new
family of cleavable hololectins, which were designated as type III
lectin precursors. These precursors were tandem repeats of the
mature peptide domains with an 18-amino-acid cleavable linker
between them. Chenotides can also stop the growth of phyto-
pathogenic fungi because they link to chitin. It has been noted
that the presence of chenotides, which are naturally occurring
anti-microbial agents, in quinoa may be the underlying cause of
the grain’s prolonged shelf life and unintentional selection as a
staple food throughout human history [55].
3 Microbial Defense System of Seeds
The life cycle stage of seed germination and seedling development

is where all prior encounters with microbiota may ultimately have
an impact. The interactions of seed-associated bacteria with the soil
microbiota during seed germination may have the greatest effects
on overall plant fitness. The soil microbiota, together with the
endophytic and epiphytic seed microbiome, are all activated during
seed development. During the crucial seed-to-seedling stage of the
life cycle, imbibition and germination can produce a spermosphere
environment (microbiome zone in the soil surrounding a germi-
nating seed) that is either advantageous or deleterious for further
plant life cycle [56–58].
3.1 Endophytes of The genetic spread of endophytes across generations of hosts can
Seeds occur via seeds. Seed endophytes are founders of the juvenile plant
microbiome and can aid host defense during seed germination and
later phases. Endophytes are symbionts that live inside plant tissues,
including seeds. The findings were reported by Khalal and Raizada
2018, by using dual culture assay methodology wherein they exam-
ined the in-vitro antagonistic effects of endophytes from seeds of
various cultivated cucurbits against significant soil-borne pathogens
like Pythium aphani dermatum, Phytophthora capsici, Fusarium
graminarium, and Rhizoctonia solani. For the purpose of inducing
plant defense, the endophytes were also examined in-vitro for their
ability to secrete volatile organic compounds. It was discovered that
fungal infections were suppressed when extracellular ribonuclease
activity was also examined. These findings demonstrate that the
microorganisms that are packaged inside the seeds of grown cucur-

bits have a great ability to inhibit illness [56]. The term endophyte”
refers to a significant collection of many and diversified plant sym-
bionts that exist asymptomatically and occasionally repeatedly
within plant tissues without doing any damage or transmitting
illnesses to their host plants [57]. The complexity and dynamism
of seed microbiomes are the result of intricate interactions with
microbes throughout the plant life cycle. Exciting new potential for
research into plant-microbe interactions are being revealed by the
diversity and dynamics of seed microbiomes. The interactions that
seeds have with microbes, notably fungus, bacteria, and viruses,
have an effect on their tenacity, resistance to diseases, emergence
from dormancy, and subsequent seedling vigor [58, 59].
The endophytic microbiota (i.e., those microbial species that
live in internal seed tissues and are vertically transmitted to progeny
seedlings). The primary symbionts known as seed endophytes have
been shown to have the potential to impact the development of
secondary symbioses in maturing hosts, according to one of the
important findings by Ridoutel et al. in 2019. On Phialocephala
fortinii early root colonization of winter wheat seedlings, the most
prevalent primary symbionts had priority effects. The primary sym-
biont controls not only the effectiveness of early P. fortinii coloni-
zation but also, indirectly, the fitness of the seedling because
P. fortinii is very beneficial for wheat seedling growth [60]. The
germination and survival of seeds are affected by soil-borne fungus
that have a host affinity and host-specific effects. Plant species have
a greater impact on the organization of seed associated fungus
communities than do soil types, forest features, or time in the soil.
These fungi have effects on seed viability and germination that
are specific to their hosts [61]. A distinct pattern has evolved from
culture-based studies demonstrating the diversity of bacteria and
fungus recovered from seeds, leaves, and roots, while the micro-
biomes of individual seeds are still rarely investigated. The trans-
mission of various fungal endophytes in the seed and needles of
Pinus monticola, the western white pine, was examined by Ganley
& Newcombe (2006) using a sequence-based methodology. Seven
hundred and fifty surface-sterilized needles yielded the enormous
quantity of 2003 fungal endophytes. Eight hundred surface-
sterilized seeds yielded 16 endophytic isolates [62].
3.2 Epiphytes of Epiphytic microbiota should be distinguished when talking about

Seeds seed microbiomes (i.e., those microbial species that colonize seed
surfaces and may or may not become internalized within seed
tissues and transmitted either vertically or horizontally). Seed epi-
phytes are fairly diverse [63]. The endophytic microbiota may
frequently arise from different seed tissues or environmental
sources than those of the epiphytic microbiota, which makes this
distinction rather artificial given that endophytes can become
epiphytes and vice versa. In contrast to those associated with

the seed coat, which are expected to be far more diverse and
transported horizontally, microorganisms associated with the
embryo and endosperm are more likely to be transmitted vertically
[58, 59]. It is evident from research on the endophytic and
epiphytic seed microbiome that maternally transmitted microbes
have a significant impact on how the core microbiome of
plants develops [63]. Additional evidence comes from the next-
generation sequencing of seeds, which shows that most fresh seeds
contain extremely few bacterial and fungal operational taxonomic
units [60]. Microbiome of plant further regulates the overall plant
defense theory.
Note Representative examples of plant seed germination/sprout-

ing for isolation of endophytes and epiphytes are given in Table 2.
4 Green Extraction and Isolation of Bioactive Components
The idea underlying plant seed survival and longevity is explained

by the whole defense mechanism of plants. The main areas to be
researched in order to comprehend the significance of plant seeds as
a bioresource for the extraction of nutraceutical and bioactive
compounds are antimicrobial biopeptides, enzymes, and
microflora-based defense systems involving prebiotics, probiotics,
and postbiotics associated with seeds. It is the nature’s special
manner of using these parts to carry out the defense mechanism.
Nature uses eco-friendly extraction and isolation processes. The
procedure greatly enhances ecological homeostasis while preserv-
ing the overall health of ecosystem. This is what is referred to as an
in situ green extraction system, and it is used to carry out the
defensive mechanism either horizontally or vertically to ensure
the survival of seedlings as well as the expression of this defense-
related genetic makeup throughout the life cycle of plant. Mother
nature can teach us a lot that will benefit the future of humanity.
Growing new generations using seeds has been the method used
throughout the plant era to start new generations.
In addition, during germination and fermentation, beneficial
components are activated in situ and the life cycle of a healthy plant
continues, seeds play the role of protection from pathogens by
activating a variety of defensive components. Along with defense
components, nutritional components are also increased or acti-
vated. Concepts based on a similar intentional activation process
in- vitro could be a practical strategy to employ the components
from seeds with potential for functional and dietary benefits.
Table 2
Representative examples of plant seed germination/sprouting for isolation of endophytes, epiphytes,
amino acids, enzymes, probiotics, prebiotics, with their classification and bioactivities
Sr.
no. Plant seed Bioactive component Function Reference
1. Fruits and seeds- Endophytes: Lactococcus, Anti-oomycete activity [56, 100]
Cucurbitaceae family Pantoea, Pediococcus
(examples: melon
(Cucumis melo),
cucumber (Cucumis
sativus)
Water melon (Citrullus Activity against
lanatus) t Podosphaera fuliginea
responsible for powdery
mildew on cucurbits
Squash and pumpkin Diverse nutrient
(Cucurbita sp.) acquisition and growth
promotion activities to
the hosts
Seeds belonging to Phyla These microbes may lead to
(Firmicutes, novel seed inoculants to
Proteobacteria) and one assist sustainable food
class within each phyla production
(Bacilli, γ-proteobacteria,
respectively)
2. The asymptomatic wheat Endophytes: Dominant Growth and disease [101]
[Triticum aestivum (L.) bacterial genus identified resistance of wheat plants
cv.] Heixiaomai NO.76) are Erwinia and (antibacterial and
seeds (sterilized sprouted Rhizobiales) and antifungal)
seeds) dominant fungal genus
identified is Emericella
3. Seeds of Brassica (B. juncea Epiphytes: Total epiphytic Antimicrobial biocontrol [102]
L. Czern., B. rapa L., microbial load of 106– agent
B. napus L.) and Triticum 108 bacterial genomes g-
1
(T. aestivum L., seeds was observed.
T. turgidum L. subsp. Examples: Pantoea
durum (Desf.) Husn.) agglomerans
4. Soybean (Glycine max), Enzymes proteases A protease is an enzyme that [74]
Lentil (Lens esculenta), catalyzes proteolysis,
Black gram (Vigna breaking down proteins
mungo), Green gram, into smaller polypeptides
(Vigna radiata) Bengal or single amino acids, and
gram, (Cicer arietinum) spurring the formation of
Groundnut (Arachis new protein products
hypogaea), Pea bean
(Phaseolus vulgaris)
(continued)
Table 2
(continued)
Sr.
5. Sword bean seeds Enzyme α-amylase α-Amylase is an enzyme that [103]
(Canavalia gladiata hydrolyses α bonds of
(Jacq.) DC.) large, α-linked
polysaccharides, such as
starch and glycogen,
yielding shorter chains
thereof
6. Germinating oil seeds Enzyme lipase Lipases are versatile enzyme [104]
(Brassica napus L.) that catalyzes the
hydrolysis of ester
linkages, primarily in
neutral lipids such as
triglycerides
7. Pigeon pea seeds Amino acids Nutritional quality and [105]
(Cajanus cajan) bioactivity of seeds
increases. Germination
helps in increase in
essential and non-
essential amino acids,
digestibility of crude
proteins increases by
conversion to amino
acids
8. Lentils (Lens culinaris), Probiotics: Lactobacillus Beneficial effects on human [61]
Mung beans, (Vigna and Bifidobacterium physiology
radiata), Peanut
(Arachis hypogeal)
9. Faba bean (Vicia faba L.), Prebiotics Serves as food for gut flora, [106]
Lentil (Lens culinaris), Raffinose family undergo anerobic
Common bean (Phaseolus oligosaccharides fermentation in large
vulgaris), and Cowpea account for 67.3, 63.2, intestine
(Vigna sinensis) 53.0, and 51.0% of the
total soluble sugars in
cowpea, faba bean,
lentil, and common
bean, respectively
Other oligosacchrides like
Verbascose in faba bean
and stachyose in the
other three legumes
4.1 Germination vs. Germination is the process by which a seed transforms into a new
Sprouting plant after achieving the conditions necessary to end its dormant
state. The only seeds that germinate are those that have an embryo
in them. The process of germination causes a seed to grow into a
seedling, which subsequently forms the plumule and radicle. The
post-germinative growth of the seedling is thought to occur after
seed germination, which is regarded as the beginning of the first
developmental phase in the life cycle of higher plants. When the
environment is right, a seed will begin to germinate in response to
factors like light, temperature, soil elements and well-understood
chemical pathways. The mature seed resumes growth during the
intricate process of germination, switching from a maturation- to
germination-driven programme of development and subsequent
seedling growth. By definition, a seed’s germination process begins
with the absorption of water and is finished when the radicle
emerges from the surrounding structures [64].
When a monocotyledonous plant seed germinates, the coleo-
rhiza is the first component to emerge from the seed coat, however
when a dicotyledonous plant seed does the same, the radicle
emerges first. The pace at which seeds are absorbing water deter-
mines how quickly they are germinating in both groups. Phase I of
the process begins with a dry seed rapidly absorbing water until all
of the seed tissues are saturated. Phase II is then followed by a
modest intake of water, but phase III is followed by a significant
uptake of water that is associated to the completion of germination.
Phase II is the most significant and is connected to a number of
cellular and physiological processes, including DNA repair and the
translation of both newly generated and stored mRNAs. Both
enhanced metabolic and cellular activity define Phase II. In order
to generate seedlings, embryo cells must decide whether to re-enter
the cell cycle or remain arrested during the germination stage.
When a seed germinates, the quiescent seed’s stalled cell cycle is
released [64].
Understanding the notion of germination of the particular seed
will help you extract functional and neutraceuticals components
from the seeds while taking the same procedure emulating natural
seed germination process into consideration. By optimizing the
many factors related to the process completion, components
could be activated, extracted, and isolated. Sprouting may be
viewed as a commercial green extraction method that could be
optimized for component isolation.
“Sprouts” (Regulation (EC) No 208/2013) are “the product
obtained from the germination of seeds and their development in
water or another medium, harvested before the development of
true leaves and which is intended to be eaten whole, including the
seed” [65, 66].
The American Association of Cereal Chemists (AACC) and

United States Department of Agriculture (USDA) have agreed on
the following definition of “sprouted grains”:
“Malted or sprouted grains that retain all of the original bran,
germ, and endosperm shall be regarded whole grains provided
sprout growth does not exceed kernel length and nutrient
values have not been depleted. It is important to designate
these grains as whole grains that have been sprouted or malted”
[66, 67].
The aforementioned descriptions of germination and sprouting
help us to realize that, during the sprouting process, seeds are
soaked and transformed into digestible forms so they may be used
as food sources, while during germination, a plant grows out of a
seed structure. The fundamental difference between sprouting and
germination is that the latter refers to the process by which seeds
are purposefully compelled to sprout or germinate in order to suit
commercial needs.
4.1.1 Understanding the Understanding the natural germination process of seed is required
Germination Process to for the optimization of sprouting for commercial use. Most agri-
Optimize Extraction by cultural seeds need water, a suitable temperature, and a good
Sprouting Process gaseous atmosphere for germination. Dormancy is a significant
element in the emergence of weeds but has minimal effect on the
seedling emergence of the majority of commercial crops. Addi-
tional germination-promoting elements for weed seeds to consider
are sunshine and nitrate [68]. Let us have a look at various factors
impacting the germination/sprouting process.
Effect of Water Uptake/ Three phases of water intake by the seed are typically observed: a
Imbibition rapid initial phase, a lag phase with little additional uptake, and
finally a second phase of rapid water uptake linked to radicle emer-
gence. Although metabolism begins before seeds achieve their
maximum moisture content, imbibition is recognized as a physical
process and is associated with the first stage of water uptake. Initial
water intake is propelled by matric pressures brought on by the
hydration of protein and starch bodies, cell walls, and other cellular
components. There is an increasing dependence on osmotic poten-
tial, which is defined by the concentration of dissolved solutes, as
the physiological range of water levels is approached. The viability
of seeds and the success of seedling emergence can both be signifi-
cantly harmed by the rate of early water intake. Rapid ingestion can
harm both directly and indirectly through a favorable interaction
with chilling injury. The quality of the seed coat and other factors of
seed vigor directly influence how much harm has been done. By
modulating permeability, the seed coat and other tissues can also
play a significant regulatory role in water intake. Germination is
often documented when radicle growth is first noticed since germi-

nation is strictly terminated when it begins at the conclusion of the
lag period of imbibition. In most species, desiccation tolerance is
gradually lost during the growth of the radicle after germination,
making the beginning of growth an important phase in the process
from seeding to seedling emergence. Each seed in the population
will go through this crucial stage at a different time [68].
Effect of Temperature and Temperature and water potential together have a significant impact
Water Potential Threshold on the percentage of seeds that will germinate, germination time,
and spread of timings within the seed population.
(a) Temperature: Although seeds can sprout at a variety of tem-
peratures, the maximum percentage of germination is often
reduced at the extremes of the temperature range. Because of
this, various seeds within a population may have varied thresh-
olds for high and low temperatures. Germination rate, which
is the reciprocal of germination time for each individual seed in
the population, rises from a base temperature to an ideal
temperature, after which it declines to a ceiling temperature
that represents the upper limit of its tolerance [68].
(b) Water: It has been demonstrated that the pace of development
toward 50% germination is linearly proportional to water
potential, much like with temperature. A scale similar to ther-
mal time called hydro time can be used to describe how seeds
react to various water potentials [68].
Effect of Oxygen on One of the limiting elements in seed germination is oxygen since
Germination germination necessitates the metabolism of storage chemicals,
which depends on respiration. Oleg A. Kuznetsov and
K.H. Hasenstein studied the germination of flax seeds in relation
to oxygen requirements in their 2003 investigations. In trials with
controlled atmospheres, as the oxygen concentration in the atmo-
sphere was dropped, the length of roots and percentage of germi-
nation fell. Seeds absorbed water but did not germinate after 2 days
in environments with less than 5% oxygen. At 10% oxygen, germi-
nation was nearly as high as that of the controls (21% O2), however
the root length was decreased to less than 50%. The seeds grew
when the temperature was 27 °C, which is ideal for the growth of
flax seedlings.
At constant temperature, the root length grows linearly. A
steady oxygen supply and an even number of seeds per chamber
affected the germination rate, demonstrating the importance of
oxygen for the best possible seed germination [69, 70].
Effect of Light on Effect of light on seed germination and seedling form of succulent
Germination species from Mexico were both shown in studies by Joel Flores et al.
in 2015. According to previous research, the adult plant height and
seed mass of cactus plants are related to the amount of light

required for seed germination. Twelve species and two varieties of
one species from the Southern Chihuahuan Desert were subjected
to germination experiments with and without light in order to
better understand the seed photosensitivity of desert species from
the Asparagaceae (subfamily Agavoideae) and Cactaceae. The
photoblastic neutrality of all species was assumed. Eleven species
showed comparable seed germination in both light and darkness,
and three taxa (Mammillaria compressa and the two types of Fer-
ocactus latispinus) demonstrated more germination in the presence
of light than in the absence of it. Higher seed mass reduced depen-
dence on light, making it a crucial element. These results provide
credence to the idea that tiny seed mass and light requirements have
co-evolved as a means of ensuring germination [71]. Effect of light
on seed germination of eight Wetland Carex Species, published by
Kettering et al. in 2006, found that seeds of Carex brevior and
Carex stipata grew more than 25% faster in continuous darkness.
The eight species showed a wide range of germination responses
after being exposed to various durations of white light. For about
50% of germination, Carex brevior needed about 15 min of white
light, whereas C. hystericina, C. comosa, C. granularis, and
C. vulpinoidea needed about 8 h. Red light has taken over the
function of white light in all species. All species, with the exception
of C. stipata, had their induction of germination upon exposure to
white or red light reversed by far-red light [72].
4.1.2 Extraction by The study highlights of Kamala Golla and coresearchers 2016 are
Sprouting considered here for representing isolation of antimicrobial peptides
using sprouting technique. The presence of short peptides with
Sprouting for Isolation of antimicrobial peptides was extensively examined in 50 distinct
Antimicrobial Peptides types of germinating seeds. Proteins were extracted using both
liquid nitrogen and phosphate buffer (PBS) treatments after
selected seeds were germinated on brown sheets over a period of
time. Small peptides of less than 10 kDa were formed by 5 kDa flow
through, and the same was validated by SDS-PAGE (sodium dode-
cyl sulfate–polyacrylamide gel electrophoresis). Staphylococcus
aureus, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas
aeruginosa were the four clinical isolates against which the short
peptides that had been extracted were tested for antibacterial effi-
cacy. Staphylococcus aureus (MTCC 9542), Escherichia coli (MTCC
1698), Klebsiella pneumoniae (MTCC 10309), and Pseudomonas
aeruginosa (MTCC6458) were the reference/standard organisms
employed in this investigation. All cultures were subcultured on
nutrient agar at regular intervals and preserved at -20 °C and 4 °C,
respectively, by suspending them in 10% glycerol. Using 1% mercu-
ric chloride, the surfaces of jowar, paddy, millets, foxtail millets, red
gram, green gram, black gram, ground nut, pea, field bean, and
wheat seeds were sterilized. Depending on the seed variety, the
seeds were soaked in distilled water for 6–12 h before being placed
on sterile brown paper and allowed to germinate for 2, 4, 6, 8,
10, 12, 14, or 16 days, respectively, at 26 °C. A 10% moisture
reduction was achieved by drying ungerminated and germinated
seeds at 600 °C. Before usage, these samples were stored in tightly
packed polyethylene bags at 40 °C. Using a pre-chilled mortar and
pestle and a phosphate buffer pH 7.5, dried seeds were pulverized.
The supernatant underwent additional salt precipitation before
being cutoff, separated, and utilized to estimate total protein and
antibacterial activity. The sample was stored at -20 in a freezer after
being centrifuged for 15 min at 10,000 rpm. After partial purifica-
tion using the ammonium sulphate precipitation technique, sam-
ples were collected for antimicrobial tests. According to the
findings, germinating seeds of soya, barley, maize, jowar, and
wheat had more antimicrobial peptides that act on gram-positive
and gram-negative bacteria [73].
Note Representative examples of plant seed antimicrobial peptides

(AMP’s) isolated using germination process with their classification
and bioactivities is given in Table 1.
Sprouting for Isolation of Akhtaruzzaman et al. [74] reported the isolation and characteriza-
Enzymes tion of protease enzyme from leguminous seeds using sprouting/
germination process. According to the mentioned study, legumi-
nous seeds can be a source of proteases for use in industry. Seven
different types of leguminous seeds—soybean, lentil, black gram,
green gram, Bengal gram, groundnut, and pea bean—were used in
the study to identify and characterize the proteases. Temperature
and pH were found to affect protease activity. Maximum specific
activity was seen in the pH profile of proteases between 7.5 and 9.0.
The seeds were cleaned separately and immersed in distilled water
for overnight germination at room temperature. Due to their low
fat content, all seeds were then ground using an electric homoge-
nizer without the use of acetone, with the exception of soybean and
groundnut. To remove the fat, cold acetone was used to homoge-
nize soybean and groundnut. The homogenates were then finely
pulverized and blended for 3 h with chilled 10 mM Tris–HCl buffer
at pH 8.0 containing 2 M NaCl in a pre-chilled mortar. The
extracted mixtures were put through gauge filters, and the filtrates
were centrifuged for 10 minutes at 10,000 rpm below 4 °C. The
estimated extracellular protein content and further purifications
were done using the collected supernatant. For overnight precipi-
tation, the collected supernatants were saturated with 50% solid
ammonium sulphate. After precipitation, they underwent a 30-min
centrifugation at 10,000 rpm at 4 °C. The precipitate that had been
collected was dissolved in 10 nm Tris–HCl buffer (pH 8), dialyzed
against the same buffer, and then centrifuged at 5000 rpm for
10 min. For the characterization and testing of a particular activity

of enzyme, the supernatant was employed as crude enzyme. Assay
of protease activity procedure for the leguminous seeds using
hemoglobin and casein as substrate were performed. The research-
ers concluded that leguminous seeds could be a source of proteases
for industrial purposes [74].
Sprouting for Isolation of The purpose of this study reported by Sulieman, M.A., 2008, was
Amino Acids to investigate how sprouting affected the cultivars’ chemical, com-
positional, energy, and amino acid contents. Three varieties of
Sudanese lentils—Rubatab, Nadi, and Selaim—were sprouted
over 3 and 6 days. The dried and ground seeds were sprouted. A
determination was made regarding how sprouting altered the prox-
imate makeup and amino acid content. While food energy and
Nitrogen Free Extract (NFE) dropped during sprouting. The
amino acid content of the seeds after sprouting was observed to
change, and it was found that there was a little variance between
cultivars. For the Selaim cultivar, sprouting for 3 days increased the
proportion of all essential amino acids, with the exception of
methionine, and decreasing amino acid content was shown when
the sprouting duration was extended to 6 days, as was the case for
histidine, lysine, and arginine. This outcome was likewise seen for
the Rubatab cultivar, except for methionine and lysine, where the
amount of essential amino acids rose as a result of sprouting. The
essential and non-essential amino acids were enhanced in the Nadi
cultivar after 3 days of sprouting. All cultivars of lentils generally
had low levels of sulphur amino acids like methionine and cystine.
Each cultivar’s bulk was divided into three equal pieces, the first
of which served as a control (unsprouted seeds), the second of
which was given 3 days to sprout, and the third of which was
given 6 days to do so.
The seeds were steeped in distilled water for 2 h at room
temperature prior to sprouting. In sterile petri dishes lined with
damp filter paper, sprouting was done for 3 and 6 days at 4 °C.
Samples were dried at room temperature at the conclusion of each
sprouting phase and crushed to pass through a 0.4 screen for a
future chemical analysis. For further study, the unsprouted seed
control groups were crushed and stored at 4 °C. The amount of
total nitrogen, crude fiber, and ash in a sample of unsprouted and
sprouted seeds were examined. The study found that the proximate
composition and food energy values of lentil underwent a notable
alteration as a result of germination. Before germination, lentil had
a low concentration of amino acids containing sulphur. Essential
and non-essential amino acid levels increased as a result of
germination [75].
Sprouting for Isolation of According to a research by Manju Pathak (2013), Bifidobacterium

Probiotics Strains sp. and Lactobacillus sp. were found in the seeds of lentil (Lens
culinaris), mung bean (Vigna radiata), and peanut (Arachis hypo-
geal), and their populations grow as the seeds germinate. For pea-
nuts, the highest bacterial colony count was found in seeds that had
been soaked for 8 h, whereas for mung beans and lentils, seeds that
had been soaked for 8 h and then germinated for 24 h had the
greatest results. It was concluded that these seeds can serve as a
source of Lactobacillus and Bifidobacterium, and the process of
germination can be used to improve them. The seeds were cleaned
three times before being immersed in RO (Reverse Osmosis) water
for 8 h. After 8 h of soaking, seeds were retained in a damp muslin
cloth for a further 24 and 48 h of germination. The raw seed
samples (R) were taken without soaking, the 8-h sample was
taken after soaking in water for 8 h, the 24-h germinated seed
sample was collected after germination for 24 h after soaking for
8 h, and the 48-h germinated seed sample was collected after
germination for 48 h after soaking for 8 h. Appropriate moisture
and 28 °C temperature were maintained during germination. After
screening, the optimal dilution of 1.25 g of sample seeds was taken
for investigations. Each sample was spread on MRS agar (De Man,
Rogosa and Sharpe agar) medium which was used as the growth
medium. Every sample was applied to the MRS agar medium,
which served as the growth medium and had a pH of 6.2 ± 0.2 at
25 °C. Colonies were counted each day while the plates were
incubated at 37 °C on days 1, 2, and 3. The method used to
determine the number of viable cells on each plate was colony
counts. Genus-specific PCR (Polymerase chain reaction), a catalase
test for lactobacillus, a lysozyme resistance test for bifidobacterium,
and other tests were carried out [76]. The outcomes show signs of
endophyte activation during the germination phase.
Note Representative examples of plant seed germination/sprout-

ing for isolation of amino acids, enzymes, probiotics, prebiotics,
with their classification and bioactivities are given in Table 2.
4.2 Fermentation as An environmentally friendly extraction method is fermentation.

Green Extraction The metabolic process of fermentation involves the action of
Process enzymes to cause chemical alterations in organic substrates. The
metabolic system that controls homeostasis in both the human
body and the natural world is based on fermentation, and ecologi-
cal equilibrium is reliant on this concept. Without using high heat,
ultrasonic wave, or other radiation sources for extraction, which
typically damages a number of bioactive ingredients, fermentation
followed by maceration improves the leaching of plant secondary
metabolites from the matrix. Additionally, different extraction
techniques require the use of chemical solvents that can vary in
polarity and include ethanol, ethyl acetate, chloroform, petroleum
ether, n-hexane, etc. However, aqueous (water) solvent is primarily

needed for fermentation [77]. Since no external chemical solvents
need to be used, a gradient of successively created alcohol or acids
during fermentation aids in a better extraction of active chemicals.
Therefore, compared to other extraction techniques, fermentation
has been seen as an environmentally friendly procedure. Addition-
ally, the fermentation process eliminates unwanted carbohydrates
from the herbal ingredient, improving the formulation’s bio-
availability. The bacteria function as probiotics in fermentation
and improve the bioavailability of existing secondary metabolites.
Different enzymes produced by various micro-species (bacteria,
fungus, and yeast) for the breakdown of the cell matrix play a
significant role as fermentation initiators creating particular bypro-
ducts. One of the oldest medical systems in India, Ayurveda makes
extensive use of both single and multi-herbal medications and
formulations, which are described in numerous Ayurvedic texts
and the Ayurvedic Formulary of India (AFI). By using various
extraction techniques, these formulations transport the active com-
ponents of herbs into menstruum fluids. “Sandhana kalpana”
(Asava and Aristha) is a special Ayurvedic dosage form that involves
fermentation [77–80].
Fermentation has been used to produce food for a very long
time. Edible seeds are essential to the human diet and have numer-
ous health advantages, such as some beans and cereal grains. Edible
seeds and their products are frequently fermented using a variety of
microorganisms, such as lactic acid bacteria, molds, and yeasts,
which are known as generally recognized as safe (GRAS) microbes.
New bioactivities and altered bioactive components can both result
from fermentation. Overall, fermented edible seeds and their pro-
ducts have more bioactive components, especially natural phenolics
and gamma-aminobutyric acid, and they have a variety of bioactiv-
ities, including antioxidant and anticancer effects. As a result, they
can be suggested as a significant component of the human diet or
developed into functional foods to aid in the prevention of specific
chronic diseases [81].
The by-products or metabolites produced as a result of inte-
gration of prebiotics with probiotics in the fermentation process
today are found to be taking stand as postbiotics. The fermentation
end product contains prebiotics, probiotics, vitamins, minerals,
short chain fatty acids, cell lysis products, etc., that could act as
treatment strategies in case of dysbiosis (human flora disturbance)
conditions. Moreover, these combination products of fermentation
could also make the alternatives for the development of prophylac-
tic approaches in the form of nutritional postbiotic health drinks,
powders, etc., considering the homeostasis balance of the body.
4.2.1 Preliminary Guide There are two types of fermentation, naturally occurring and inocu-
to Fermentation lated, depending on the source of the bacteria used in the process.
Procedures According to the amount of water in the system, fermentation can
also be classified as either solid-state (SSF) or liquid-state fermen-
tation (LSF) [82–84]. Before fermentation, edible seeds frequently
require pretreatment, such as soaking, cracking, grinding, sifting,
and boiling. [84] These processing techniques are frequently put
together in combination. Prior to natural fermentation, seeds can-
not be cooked or autoclaved since doing so will partially or totally
eliminate any bacteria already present on the seeds. The most
frequent bacteria utilized in edible seed fermentation as starter
culture and inoculum are lactic acid bacteria (LAB), including
Lactobacillus (Lb.) acidophilus, Lb. brevis, Lb. bulgaricus, Lb. casei,
Lb. fermentum, Lb. johnsonii, Lb. paracasei, Lb. plantarum,
Lb. reuteri, Lb. rhamnosus, Lb. rossiae, Lb. Streptococcus (Sc.) ther-
mophilus, Bifidobacterium (Bb.) animalis, Bb. infantis, Lactococcus
(Lc.) lactis, and Weissella (W.) paramesenteroides [81–84]. In addi-
tion, Bacillus (B.) subtilis has also been commonly used to ferment
edible seeds [83]. Additionally, cultures of fungi (molds) used for
fermentation are Aspergillus (A.) oryzae [85], A. awamori, A. sojae,
A. niger, Agrocybe (Ac.) cylindracea, Cordyceps (C.) militaris,
Coprinus (Cr.) cinereus, Grifola (G.) frondosa, Ganoderma (Gd.)
austral, Gd. neo-japonicum, etc. Cultures of yeasts, such as Issatch-
enkia (I.) orientalis, Saccharomyces (S.) cerevisiae, and S. boularidii,
etc., have also been employed to ferment edible seeds [81, 86]. The
amount of inoculum in the starter culture is essential for the fer-
mentation process. Inoculation of 1% to 10% (bacteria (mL)/sam-
ple (mL or g)) of the starting culture (108 cfu/mL) has been used
regularly in SSF and LSF of edible seeds, and their products, with
106 to 107 cfu/mL LAB in original samples, have been used for
LAB fermentation. In SSF of edible seeds for B. subtilis and fungal
fermentation, inoculation of 5% (bacteria (mL)/sample (mL)) of
the starting culture (105/g sample) has been employed most fre-
quently [81, 83]. To carry out the fermentation effectively, the
inoculum needs to be optimized. The fermentation efficiency is
influenced by a number of variables, including fermentation tem-
perature, duration, humidity, and other circumstances [81]. Other
factors controlling the fermentation process which are reported to
be optimized are temperature, humidity, stirring/shaking speed,
aerobic or anaerobic conditions, pH, fermentation time, etc. [81].
Living creatures are profoundly impacted by temperature
variations. The sensitivity of enzyme-catalyzed reactions to minute
temperature variations is very high. As a result, the environment
temperature frequently affects the metabolism of poikilotherms
organisms whose internal body temperature is influenced by it
[87]. Every degree of temperature count. Temperature is key to
fermentation success. The optimum temperature range for yeast
fermentation is between 32 °C and 35 °C. Every degree above this
range depresses fermentation. Yeast can usually tolerate short-term

fluctuations in temperature [88]. However, operating above ideal
temperatures for longer stretches of time can significantly have
impact on fermentation and secondary metabolite production.
Humidity control is one of the important parameters to be consid-
ered during solid state fermentation process. The study reported by
Taimı́ Carrasco et. Al, 2003, shows the requirement of humidity as
indicator of fermentation success. Humidity is one of the determi-
nant indicators in the dynamic of the solid-state fermentation.
Impact of humidity on actions of enzymes has been predicted in
the study which ultimately found to have impact on the production
the secondary metabolites required during the process [89]. Agita-
tion plays an important mixing and shearing role in fermentation
processes. It not only improves mass and oxygen transfer between
the different phases, but also maintains homogeneous chemical and
physical conditions in the medium by continuous mixing. On the
other side, agitation can cause shear forces, which influence micro-
organisms in several ways, such as changes in morphology, variation
in growth and metabolite formation and even causing damage to
cell structures. Aeration determines the oxygenation of the fermen-
tation process, and also contributes to mixing of the fermentation
broth, especially where mechanical agitation speeds are low. Aera-
tion not only supplies the necessary oxygen for cell growth, but also
eliminates exhaust gas generated during the fermentation process.
However, higher aeration rate results in a reduction in the volume
of fermentation broth. Oxygen supply is necessary for growth of
microorganisms in aerobic fermentation, but some microorganisms
may be affected by oxygen toxicity at excessive oxygen concentra-
tion. So agitation optimization based on type of fermentation is the
ultimate requirement [90]. So as the supportive information, the
following data could be considered for fermentation optimization
process.
Natural fermentation has been reported to control the temper-
ature at 30, 37, or 42 °C which is probably associated with the main
microbes carried by different seeds. In addition, the temperature
commonly controlled at 37 °C for LAB fermentation while fermen-
tation using B. subtilis and fungi has mostly been employed at 30 °C
probably due to the optimum growth at this temperature. For
fermentation time, several hours to several days have been reported,
while 48 and/or 96 h are most commonly used for edible seed
fermentation. In addition, it is better to control the fermentation
humidity at 90% to 95% if possible which can provide a relatively
moist air condition for the growth of microbes. SSF of edible seeds
and bean milk fermentation are generally performed quiescently,
while LSF of edible seeds is commonly carried out by continuous
shaking/stirring, with a speed of 200 to 450 rpm which can accel-
erate the growth of microbes, increase the interaction between
microbes and substrates, and enhance the efficiency of fermenta-

tion. Similarly, adding sugars (1–2%) to the fermentation system,
such as glucose or sucrose can provide an extra energy source to
accelerate the growth of microbes. Moreover, fermentation can be
performed under aerobic or anaerobic conditions, dependent on
the species of microbes involved. For example, it is better to per-
form LAB fermentation in an anaerobic or microaerophilic envi-
ronment. Overall, the fermentation condition is critical for the
efficiency of fermentation and needs to be optimized for ferment-
ing different products [81, 91].
4.2.2 Fermentation for Fermentation is the most prevalent postbiotic source in the food
Isolation of Postbiotics industry. The presence of postbiotics can be found naturally in
several milk-based and other products like kefir, kombucha, yogurt,
and pickled vegetables, etc. [91]. Postbiotics are functional bioac-
tive substances that are produced during fermentation in a matrix
and can be used to improve health. Postbiotics can be thought of as
a catch-all name for all synonyms and related terms of various
components of microbial fermentation. As a result, postbiotics
can consist of a wide range of components, such as metabolites,
short-chain fatty acids (SCFAs), microbial cell fractions, functional
proteins, extracellular polysaccharides (EPS), cell lysates, teichoic
acid, muropeptides derived from peptidoglycans, and pili-type
structures, among others. The use of postbiotics may enable active
bacteria to become more potent or transform them into useful
components. In addition, postbiotics get around the technical
problems of colonization effectiveness and maintaining the
microbes in the product at a high dose. As a result, it is easier to
deliver the active substances where they are needed in the gut, the
shelf life is increased, and perhaps packing and transportation are
also made simpler [92]. The structure and operation of the com-
mensal human gut microbiome can be influenced by postbiotics
produced during fermentation. They also aid in inhibiting possible
pathogens while giving the local microbial population the sub-
strates it needs to produce SCFAs. The following are some poten-
tial advantages of fermented foods and beverages: Kefir’s organic
acids, bacteriocins, carbon dioxide, hydrogen peroxide, ethanol,
and diacetyl all have antimicrobial properties. Kombucha’s low
pH and high acetic acid concentration also inhibit the growth of
pathogens. Meanwhile, conjugated linoleic acid in sauerkraut may
have potential health benefits. Similar types of chemicals are pro-
duced during grain fermentation along with proteolytic activity by
lactic acid bacteria, which transforms wheat proteins into bioactive
peptides (postbiotics). Vitamin B12 is produced during the fer-
mentation of soybeans. Fruit fermentation with Lactobacillus plan-
tarum produces phenolic compounds and a number of organic
acids [93].
Studies conducted in 2017 by Sasithorn Sirilun et al. showed

the advantages of fermented soybeans mediated by lactic acid bac-
teria. By acting as an antioxidant and increasing the quantity of
isoflavones in their aglycone forms, lactic acid bacteria-mediated
fermentation improved the fermented soy broth quality. Addition-
ally, it stopped the development of coliforms in fermented soybean.
East Asian nations consume large quantities of fermented soybean
products, which are important sources of bioactive chemicals.
Examples include cheonggukjang (Japanese natto), doenjang (soy
paste), ganjang (soy sauce), and douchi. A range of new com-
pounds, the majority of which have health benefits, are produced
when cooked soybeans are fermented with bacteria (Bacillus spp.)
and fungi (Aspergillus spp. and Rhizopus spp.) [94].
Note Representative examples of seed fermentation for isolation

of prebiotics, probiotics, and postbiotics are given in Table 3.
5 Experimental Case Report from Our Own Laboratories
5.1 Experiments with Sprouted flaxseeds were explored for prebiotic and postbiotic prop-
Sprouted Flaxseeds erties: It was an attempt to develop, In- vitro biorelevant media and
(Linum usitatissimum, time simulation probiotic proliferation methodology to determine
in the Family Linaceae) prebiotic potentials of flaxseed powder. The research ultimately
came up with excellent findings for prebiotic and probiotic poten-
tials of flaxseed. Prebiotic potential of flaxseed powder was tested
using Bacillus coagulans SNZ 1969 marketed probiotic wherein it
was found that flaxseeds act as excellent prebiotic supplement for
the growth of probiotics, similarly at the outset it was also observed
that endophytes from the seeds of flaxseeds colonize in the presence
of MRS agar media, provided strict sterile conditions were main-
tained to avoid environment contamination. It was concluded that
fermented flaxseed powder could be effective postbiotic supple-
ment which could be explored further in postbiotic supplement
development [95].
5.2 Experiments with In our another unpublished research on comparative evaluation of

Sprouted Ragi (Finger prebiotic and probiotic benefits of sprouted and non-sprouted ragi
Millet) Seeds (Eleusine seeds, it was observed that sprouted ragi seeds grains comparatively
coracana in the Family have excellent prebiotic properties. Sprouted ragi seeds and
Poaceae) sprouted fermented ragi seeds also showed excellent prebiotic and
probiotic potentials. It was also observed that endophytes from the
seeds of Ragi seeds colonize in the presence of MRS agar media,
provided strict sterile conditions were maintained to avoid environ-
ment contamination. Which could further be explored for identifi-
cation and isolation of probiotics and postbiotic components from
the sprouted and sprouted fermented seeds.
Table 3
Representative examples of seed fermentation for isolation of prebiotics, probiotics, and postbiotics
Sr.
1. Hemp seeds (Cannabis Prebiotic potential Ability to support [107]
sativa) probiotics growth was
Probiotics used for observed. Increase in the
fermentation are content of some bioactive
(Lactobacillus compounds like presence
fermentum, of different terpenes that
Lb. plantarum, and inhibit the growth of
Bifidobacterium enteropathogens and
bifidum) high levels of short chain
fatty acids like acetate,
propionate and butyrate
produced during
fermentation that
support the growth of
probiotics
2. Artocarpus integer’s seed Prebiotic potential A. integer extract was found [108]
Probiotics used for to support the growth of
fermentation are probiotics such as
Lactobacillus L. acidophilus and
acidophilus DSM L. casei. The results of the
20079, Lactobacillus present study indicated
casei DSM 20011, and that A. integer extract
Escherichia coli DSM was comparable to the
1103 commercial prebiotics
inulin
3. Red and white rice seeds Probiotic: Lactic acid Gut flora heath booster [109]
bacteria (LAB) stimulate cell-mediated
immunity
4. Fermented finger millet Five potential probiotic Potential in terms of health [110]
flour: Three varieties LAB strains (lactic acid benefits
namely Ravi, Raavana, bacteria) were isolated:
and Oshadha R17 (L. plantarum),
RV02 (L. fermentum)
and RV19 (L. lactis sub
species lactis), RV28
(E. faecium), and O24
(P. acidilactici)
5. Fermented beverage using Postbiotics: Decrease in Enhance the bioavailability [111]
red rice (Oryza sativa antinutrient phytic acid, of minerals, digestibility,
var. Indica, Tapol), increase in phosphorous, and sensory properties of
barley (Hordeum vulgare increase in fibers the final products
L.), and buckwheat (prebiotics), etc.
(Fagopyrum
esculentum),
fermentation culture:
Lactic acid bacteria
(continued)
Table 3
(continued)
Sr.
6. Fermented soybean meal Postbiotics: Product reach Acts as probiotic [112, 113]
using bacillus strains in probiotics, digestible supplement as gut flora
(Bacillus subtilis TP6 peptides, polyglutamic booster and help to
strain) acid, short chain fatty improve innate
acids like lactic acid, immunity, protein
non-reducing content is high, and
oligosacchrides like digestion and absorption
Raffinose, Stachyose, rates are also improved by
etc., isoflavones, low-molecularization of
lipopeptides, protein the proteins.
hydrolysates, and Polyglutamic acid helps
enzymes to reduce body fat
5.2.1 Materials Used Probiotic supplement—Bacillus coagulans SNZ 1969, (Sporlac

sachets, Sanzyme Biologics Private Limited) Ragi Seeds. The herbal
character of plant specimen of ragi was affirmed by a taxonomist at
the Department of Botany, Botanical Survey of India, Pune. It was
validated to be Eleusine coracana (L.) Gaertn. belonging to family
Poaceae. A voucher specimen number No. BSI/WRC/100-1/
Tech./2020 was obtained.
5.2.2 Sprouting A total of 250 g of ragi seeds was taken and cleaned to eliminate
Procedure unfamiliar particles from it. The cleaned seeds were then soaked
into adequate measure of water for 8 h at room temperature. After
8 h, the excess of water from seeds was eliminated utilizing filtra-
tions and the seeds were half dried. The seeds were then kept for
germination for around 24 h. The developed seeds were further
kept for drying. The sprouts on the seeds were shed and cleaned
physically. The seeds were then crushed to get the ragi powder,
passed through sieve number #100 to get uniform particle size.
5.2.3 Fermentation Eleusine coracana sprouted seed powder (30 g) obtained as per the
Procedure above-mentioned procedure was taken, and to it was added to 1 g
of Bacillus coagulans SNZ 1969 powder and 1 g of citric acid
powder. Forty milliliters of water was added to the mixture and
the mixture was placed for 24 h at room temperature. After 24 h of
fermentation, wet mass was extruded through the extruder. The
extrudates are shaped into small spherical granules. The wet gran-
ules were allowed to dry. Methodology: Probiotic proliferation study
of sprouted, non-sprouted, and fermented products was performed
as reported in our own research publication [95].
5.2.4 Research Sprouted ragi seed powder has been proved to have excellent
Highlights prebiotic activity in comparison to non-sprouted powder. Sprouted
and non-sprouted ragi seed powder shows self-probiotic potential.
Sprouted powder support the probiotic spore and culture growth
in the presence of antibiotic as compare to non-sprouted powder.
Similarly, combined run real time simulated biorelevant media
study with and without enzymes and antibiotic (Azithromycin)
also depicted the prominent growth of the probiotic spore pow-
ders. The study concludes that germination of seed increases prebi-
otic and probiotic potential of seed. Overall results indicated that
ragi seed sprouting and fermentation increase the extraction of
prebiotic components and probiotic endophyte activation. Results
helps to conclude that sprouting and fermentation are the natural
in situ green extraction techniques which could be explored well for
health benefits. Table 4 indicates some representative images for
the ragi seed sprouted powder prebiotic as well as probiotic
potentials.
6 Conclusion
Understanding this interaction and developing ways to model the

outcome is essential for developing effective crop establishment
practices as well as beneficial utilization of the plant seeds com-
ponents for maintenance of human health. Nature takes care of
ecological homeostasis through utilization of prebiotics, probio-
tics, and postbiotics. The simplest lesson to be taken from nature is
degraded seeds when they are in soil, in moist conditions, ferment,
gets converted to postbiotics which flourish the soil microflora.
Seeds, the complete package of defense system containing antimi-
crobial biopeptides. The AMP’s get activated during germination
and fights against the pathogens in the zone around the seeds in the
soil. Seeds endophytes and epiphytes take care of vertical and
horizontal innate immune systems throughout the plant life cycle.
These are nature’s unique in situ extraction procedures to activate
the components at the respective required locations. Activation and
utilization of enzymes, vitamins, minerals, prebiotics, postbiotics,
probiotics, etc., occur in nature without harming the ecosystem
balance. Nature makes use of these processes using safe parameters,
solvent like water, temperature, humidity, etc. Enzymes act as a
catalyst for the completion of biochemical reactions. In situ gener-
ation of acidic and basic components acts as buffering agents to
provide respective pH for the completion of the process. Interac-
tions of prebiotics and probiotics during fermentation lead to the
formation of metabolites like short chain fatty acids and alcohol,
which act as natural solvent systems for activation and extraction of
the required components and completion of many reactions. The
Table 4
Prebiotic and probiotic potentials of Ragi seed (Eleusine coracana) sprouted powder
Sr.
no. Observations Images
1. Environmental negative controls: No growth observed in MRS broth
tube as well as MRS agar plate maintained throughout the study, it
indicates maintenance of strict sterile conditions during the
experimentation
2. Negative growth controls with sterile distilled water: No growth after

24 h incubation in tubes as well as after plating and incubation on
MRS agar media (indicates that water does not act carrier for
contamination during studies)
3. Positive growth control: Both MRS broth and MRS agar media
support the growth of Bacillus coagulans probiotic spores
considered for studies
4. Viability of probiotic spores in sterile distilled water: Sterile water with

probiotic spores does not support growth in water but spores
remain viable after 24 h as growth is observed after plating and
incubation further on MRS agar media
5. Prebiotic potentials of sprouted powder: Probiotic spore in the

presence of ragi sprouted powder shows comparatively increased
growth, indicates the supportive prebiotic nature of the ragi seed
sprouted powder(without addition of MRS broth for first 24 h
media)
6. Probiotic potentials of sprouted powder: Sterile water with sprouted

ragi seed powder (with no prebiotic spores added externally) after
incubation for 24 h and plating on MRS agar media showed the
growth indicating the activation of the endophytic microflora
associated with seeds
beneficial interaction of these components during fermentation

gives rise to many health-benefiting constituents. To date many
plant seeds are unexplore for their potential as prebiotics, endo-
phytes, epiphytes, postbiotics, antimicrobial biopeptides, synbio-
tics, nutritional comonents and much more. Further it is imrpotant
to learn and understand what and how nature do these processes.
Nature maintains ecological homeostasis in an optimized manner.
Let us learn to optimize the procedures the way nature does.
Acknowledgments
The authors are thankful to the management, MET’s Institute of

Pharmacy, Bhujbal Knowledge City, Adgaon, Nashik, Maharashtra,
India, for providing the necessary research facilities. The authors
also extend their gratitude toward Savitribai Phule Pune University,
Pune, for supporting the work. Heartfelt thanks to the Institute of
Nutrition, Mahidol University, Thailand, for the collaborative
handshake for research activities.
References
1. World Health Statistics 2022. In: www.who. application and research progress in multiple
int. https://www.who.int/news/item/20-0 fields. Front Microbiol 11. https://doi.org/
5-2022-world-health-statistics-2022 10.3389/fmicb.2020.582779
2. Ecological Balance. In: wwf.panda.org. 11. Kaur AP, Bhardwaj S, Dhanjal DS et al (2021)
https://wwf.panda.org/discover/knowl Plant prebiotics and their role in the amelio-
edge_hub/teacher_resources/webfieldtrips/ ration of diseases. Biomol Ther 11:440.
ecological_balance/. Accessed 22 Sept 2021 https://doi.org/10.3390/biom11030440
3. Study reveals how human brains have evolved 12. Liang L, Chen L, Liu G et al (2022) Optimi-
to be smarter than other animals | Imperial zation of germination and ultrasonic-assisted
News | Imperial College London. In: Imperial extraction for the enhancement of
News. https://www.imperial.ac.uk/news/23 γ-aminobutyric acid in pumpkin seed. Food
9331/study-reveals-human-brains-have- Sci Nutr 10:2101–2110. https://doi.org/10.
evolved/. Accessed 23 Nov 2022 1002/fsn3.2826
4. (2019) Overview of Plant Defenses. In: Over- 13. Ohanenye IC, Tsopmo A, Ejike CECC, Ude-
view of Plant Defenses. https://www.apsnet. nigwe CC (2020) Germination as a biopro-
org/edcenter/disimpactmngmnt/topc/ cess for enhancing the quality and nutritional
Pages/OverviewOfPlantDiseases.aspx prospects of legume proteins. Trends Food
5. Kaur S, Samota MK, Choudhary M et al Sci Technol 101:213–222. https://doi.org/
(2022) How do plants defend themselves 10.1016/j.tifs.2020.05.003
against pathogens-biochemical mechanisms 14. Varsha KK, Narisetty V, Brar KK et al (2022)
and genetic interventions. Physiol Mol Biol Bioactive metabolites in functional and fer-
Plants 28:485–504. https://doi.org/10. mented foods and their role as immunity
1007/s12298-022-01146-y booster and anti-viral innate mechanisms. J
6. Mithöfer A, Maffei ME (2017) General Food Sci Technol. https://doi.org/10.
mechanisms of plant defense and plant toxins. 1007/s13197-022-05528-8
Plant Toxins:3–24. https://doi.org/10. 15. US EPA (2016) How communities have
1007/978-94-007-6464-4_21 defined zero waste | US EPA. In: US EPA.
7. List of edible seeds. ht tps://www. https://www.epa.gov/transforming-waste-
basicknowledge101.com/pdf/health/ tool/how-communities-have-defined-zero-
seedsediblelist.pdf waste
8. Adhikari SR, Baral SD, Pokhrel K (2020) 16. Ernest SKM (2008) Homeostasis. In: Scien-
Non-edible seeds for the production of bio- ceDirect. https://www.sciencedirect.com/sci
diesel. Himal Biodivers:36–38. https://doi. ence/article/pii/B9780080454054005073.
org/10.3126/hebids.v8i1.40239 Accessed 24 Nov 2022
9. Kainat S, Arshad MS, Khalid W et al (2022) 17. (2022) Maintenance of homeostasis in eco-
Sustainable novel extraction of bioactive com- logical systems – population dynamics. In:
pounds from fruits and vegetables waste for Ecology Center. https://www.ecologycenter.
functional foods: a review. Int J Food Prop us/population-dynamics-2/maintenance-of-
25:2457–2476. https://doi.org/10.1080/ homeostasis-in-ecological-systems.html
10942912.2022.2144884 18. Billman GE (2020) Homeostasis: the under-
10. Huan Y, Kong Q, Mou H, Yi H (2020) Anti- appreciated and far too often ignored central
microbial peptides: classification, design, organizing principle of physiology. Front
Physiol 11. https://doi.org/10.3389/fphys. 31. de Oliveira CA, Moreira Gomes V (2011)

2020.00200 Plant defensins and defensin-like peptides -
19. Corvalan C, Hales S, Mcmichael A et al biological activities and biotechnological
(2005) A report of the Millennium Ecosystem applications. Curr Pharm Des 17:4270–
Assessment Core Writing Team Extended 4 2 9 3 . h t t p s : // d o i . o r g / 1 0 . 2 1 7 4 /
Writing Team ECOSYSTEMS AND 138161211798999447
HUMAN WELL-BEING 32. Lay FT, Brugliera F, Anderson MA (2003)
20. Rosbakh S, Phartyal SS, Chen S-C, Poschlod Isolation and properties of floral defensins
P (2022) Functional seed ecology: from single from ornamental tobacco and petunia. Plant
traits to plant distribution patterns, commu- Physiol 131:1283–1293. https://doi.org/
nity assembly and ecosystem processes. Fron- 10.1104/pp.102.016626
tiers Media SA 33. Lay FT, Poon S, McKenna JA et al (2014) The
21. Dalling JW, Davis AS, Arnold AE et al (2020) C-terminal propeptide of a plant defensin
Extending plant defense theory to seeds. confers cytoprotective and subcellular target-
Annu Rev Ecol Evol Syst 51:123–141. ing functions. BMC Plant Biol 14. https://
https://doi.org/10.1146/annurev-ecolsys- doi.org/10.1186/1471-2229-14-41
012120-115156 34. Sher Khan R, Iqbal A, Malak R et al (2019)
22. Fuerst EP, Okubara PA, Anderson JV, Morris Plant defensins: types, mechanism of action
CF (2014) Polyphenol oxidase as a biochemi- and prospects of genetic engineering for
cal seed defense mechanism. Front Plant Sci enhanced disease resistance in plants. 3 Bio-
5. https://doi.org/10.3389/fpls.2014. tech 9. https://doi.org/10.1007/s13205-
00689 019-1725-5
23. Dalling JW, Davis AS, Schutte BJ, Elizabeth 35. Terras FR, Eggermont K, Kovaleva V et al
Arnold A (2010) Seed survival in soil: inter- (1995) Small cysteine-rich antifungal proteins
acting effects of predation, dormancy and the from radish: their role in host defense. Plant
soil microbial community. J Ecol 99:89–95. Cell 7:573–588. https://doi.org/10.1105/
https://doi.org/10.1111/j.1365-2745. tpc.7.5.573
2010.01739.x 36. Aist JR (1976) Papillae and related wound
24. Zalamea P-C, Dalling JW, Sarmiento C et al plugs of plant cells. Annu Rev Phytopathol
(2018) Dormancy-defense syndromes and 14:145–163. https://doi.org/10.1146/
tradeoffs between physical and chemical annurev.py.14.090176.001045
defenses in seeds of pioneer species. Ecology 37. Bradley DJ, Kjellbom P, Lamb CJ (1992)
99:1988–1998 Elicitor- and wound-induced oxidative cross-
25. Jogaiah S, Abdelrahman M (2019) Bioactive linking of a proline-rich plant cell wall protein:
molecules in plant defense. Springer, Cham a novel, rapid defense response. Cell 70:21–
26. Frick KM, Kamphuis LG, Siddique KHM et al 30. https://doi.org/10.1016/0092-8674
(2017) Quinolizidine alkaloid biosynthesis in (92)90530-p
lupins and prospects for grain quality 38. Höng K, Austerlitz T, Bohlmann T, Bohl-
improvement. Front Plant Sci 8. https://doi. mann H (2021) The thionin family of antimi-
org/10.3389/fpls.2017.00087 crobial peptides. PLoS One 16:e0254549.
27. Li J, Hu S, Jian W et al (2021) Plant antimi- https://doi.org/10.1371/journal.pone.
crobial peptides: structures, functions, and 0254549
applications. Bot Stud 62:5. https://doi. 39. Chandrashekhara N-RS, Deepak S et al
org/10.1186/s40529-021-00312-x (2010) Thionins (PR protein-13) mediate
28. Nawrot R, Barylski J, Nowicki G et al (2014) pearl millet downy mildew disease resistance.
Plant antimicrobial peptides. Folia Microbiol Arch Phytopathol Plant Protect 43:1356–
59:181–196. https://doi.org/10.1007/ 1 3 6 6 . h t t p s : // d o i . o r g / 1 0 . 1 0 8 0 /
s12223-013-0280-4 03235400802476393
29. Barbosa Pelegrini P, del Sarto RP, Silva ON 40. Bohlmann H, Broekaert W (1994) The role
et al (2011) Antibacterial peptides from of thionins in plant protection. Crit Rev Plant
plants: what they are and how they probably Sci 13:1–16. https://doi.org/10.1080/
work. Biochem Res Int 2011:1–9. https:// 07352689409701905
doi.org/10.1155/2011/250349 41. Carrasco L, Vazquez D, Hernandez-Lucas C
30. Stotz HU, Thomson J, Wang Y (2009) Plant et al (1981) Thionins: plant peptides that
defensins. Plant Signal Behav 4:1010–1012. modify membrane permeability in cultured
https://doi.org/10.4161/psb.4.11.9755 mammalian cells. Eur J Biochem 116:185–
1 8 9 . h t t p s : // d o i . o r g / 1 0 . 1 1 1 1 / j . antifungal activity and sensitization of plant

1432-1033.1981.tb05317.x pathogenic fungi to tebuconazole by
42. Okada T, Yoshizumi H (1973) The mode of WAMP-2-derived peptides. Int J Mol Sci 21:
action of toxic protein in wheat and barley on 7 9 1 2 . h t t p s : // d o i . o r g / 1 0 . 3 3 9 0 /
brewing yeast. Agric Biol Chem 37:2289– ijms21217912
2294. https://doi.org/10.1080/00021369. 53. Slavokhotova AA, Shelenkov AA, Andreev
1973.10861004 YA, Odintsova TI (2017) Hevein-like antimi-
43. Gao H, Ma K, Ji G et al (2022) Lipid transfer crobial peptides of plants. Biochem Mosc 82:
proteins involved in plant–pathogen interac- 1659–1674. https://doi.org/10.1134/
tions and their molecular mechanisms. Mol s0006297917130065
Plant Pathol. https://doi.org/10.1111/ 54. Yokoyama S, Iida Y, Kawasaki Y et al (2009)
mpp.13264 The chitin-binding capability of Cy-AMP1
44. Finkina EI, Melnikova DN, Bogdanov IV, from cycad is essential to antifungal activity. J
Ovchinnikova TV (2016) Lipid transfer pro- Pept Sci 15:492–497. https://doi.org/10.
teins as components of the plant innate 1002/psc.1147
immune system: structure, functions, and 55. Loo S, Tay SV, Kam A et al (2021) Anti-
applications. Acta Nat 8:47–61. https://doi. fungal hevein-like peptides biosynthesized
org/10.32607/20758251-2016-8-2-47-61 from quinoa cleavable hololectins. Molecules
45. Wang X, Zhou W, Lu Z et al (2015) A lipid 2 6 : 5 90 9 . h t t p s : // do i . o r g / 1 0. 33 9 0/
transfer protein, OsLTPL36, is essential for molecules26195909
seed development and seed quality in rice. 56. Khalaf EM, Raizada MN (2018) Bacterial
Plant Sci 239:200–208. https://doi.org/10. seed endophytes of domesticated cucurbits
1016/j.plantsci.2015.07.016 antagonize fungal and oomycete pathogens
46. Gould A, Camarero JA (2017) Cyclotides: including powdery mildew. Front Microbiol
overview and biotechnological applications. 9. https://doi.org/10.3389/fmicb.2018.
Chembiochem 18:1350–1363. https://doi. 00042
org/10.1002/cbic.201700153 57. Aamir M, Rai KK, Zehra A et al (2020) Fun-
47. Slazak B, Haugmo T, Badyra B, Göransson U gal endophytes: classification, diversity, eco-
(2020) The life cycle of cyclotides: biosynthe- logical role, and their relevance in sustainable
sis and turnover in plant cells. Plant Cell Rep agriculture. Microb Endophytes:291–323.
39:1359–1367. https://doi.org/10.1007/ https://doi.org/10.1016/b978-0-12-
s00299-020-02569-1 818734-0.00012-7
48. Su T, Han M, Cao D, Xu M (2020) Molecular 58. Nelson EB (2017) The seed microbiome: ori-
and biological properties of snakins: the fore- gins, interactions, and impacts. Plant Soil 422:
most cysteine-rich plant host defense pep- 7–34. https://doi.org/10.1007/s11104-
tides. J Fungi 6:220. https://doi.org/10. 017-3289-7
3390/jof6040220 59. Barret M, Guimbaud J-F, Darrasse A, Jacques
49. Deng M, Peng J, Zhang J et al (2021) The M-A (2016) Plant microbiota affects seed
cysteine-rich peptide snakin-2 negatively reg- transmission of phytopathogenic microorgan-
ulates tubers sprouting through modulating isms. Mol Plant Pathol 17:791–795. https://
lignin biosynthesis and H2O2 accumulation doi.org/10.1111/mpp.12382
in potato. Int J Mol Sci 22:2287. https://doi. 60. Ridout ME, Schroeder KL, Hunter SS et al
org/10.3390/ijms22052287 (2019) Priority effects of wheat seed endo-
50. Oliveira-Lima M, Benko-Iseppon A, Neto J phytes on a rhizosphere symbiosis. Symbiosis
et al (2017) Snakin: structure, roles and appli- 78:19–31. https://doi.org/10.1007/
cations of a plant antimicrobial peptide. Curr s13199-019-00606-6
Protein Pept Sci 18:368–374. https://doi. 61. Sarmiento C, Zalamea P-C, Dalling JW et al
o r g / 1 0 . 2 1 7 4 / (2017) Soilborne fungi have host affinity and
1389203717666160619183140 host-specific effects on seed germination and
51. Garcı́a AN, Ayub ND, Fox AR et al (2014) survival in a lowland tropical forest. Proc Natl
Alfalfa snakin-1 prevents fungal colonization Acad Sci 114:11458–11463. https://doi.
and probably coevolved with rhizobia. BMC org/10.1073/pnas.1706324114
Plant Biol 14. https://doi.org/10.1186/ 62. Ganley RJ, Newcombe G (2006) Fungal
s12870-014-0248-9 endophytes in seeds and needles of Pinus
52. Odintsova T, Shcherbakova L, Slezina M et al monticola. Mycol Res 110:318–327.
(2020) Hevein-like antimicrobial peptides https://doi.org/10.1016/j.mycres.2005.
Wamps: structure–function relationship in 10.005
63. Newcombe G, Harding A, Ridout M, Busby https://doi.org/10.5829/idosi.wasj.2013.

PE (2018) A hypothetical bottleneck in the 26.02.1120
plant microbiome. Front Microbiol 77. Vidhu A (2020) Fermentation: a green herbal
9. https://doi.org/10.3389/fmicb.2018. extraction process for polyphenols. Biomed J
01645 Sci Tech Res 31. https://doi.org/10.26717/
64. Wolny E, Betekhtin A, Rojek M et al (2018) bjstr.2020.31.005137
Germination and the early stages of seedling 78. The Ayurvedic Pharmacopoeia of India.
development in Brachypodium distachyon. Part I, Volume –IX. Government of India
Int J Mol Sci 19:2916. https://doi.org/10. Ministry of AYUSH 2016. Published by Phar-
3390/ijms19102916 macopoeia Commission for Indian Medicine
65. EUR-Lex – 32013R0208 – EN – & Homoeopathy Ghaziabad. सत्यमे व जयते
EUR-Lex. In: eur-lex.europa.eu. https:// First Edition
eur-lex.europa.eu/eli/reg_impl/2013/208/ 79. Chaudhary A, Singh N, Dalvi M, Wele A
oj (2011) A progressive review of Sandhana kal-
66. Benincasa P, Falcinelli B, Lutts S et al (2019) pana (biomedical fermentation): an advanced
Sprouted grains: a comprehensive review. innovative dosage form of Ayurveda. Ayu 32:
Nutrients 11. https://doi.org/10.3390/ 408–417. https://doi.org/10.4103/
nu11020421 0974-8520.93925
67. Sprouted Grains. In: encyclopedia.pub. 80. Das C, Ghosh G, Das D (2017) Ayurvedic
https://encyclopedia.pub/entry/15085 liquid dosage form Asava and arista: an over-
68. Benech-Arnold R, Sánchez R, Bewley J view. Indian J Pharmaceut Educ Res 51:169–
(2004) Handbook of seed physiology applica- 176. https://doi.org/10.5530/ijper.51.2.22
tions to agriculture ”T 81. Gan R-Y, Li H-B, Gunaratne A et al (2017)
69. Kuznetsov OA, Hasenstein KH (2003) Oxy- Effects of fermented edible seeds and their
gen requirement of germinating flax seeds. products on human health: bioactive compo-
Adv Space Res 31:2211–2214. https://doi. nents and bioactivities. Compr Rev Food Sci
org/10.1016/s0273-1177(03)00246-1 Food Saf 16:489–531. https://doi.org/10.
70. Shull CA (1914) The role of oxygen in germi- 1111/1541-4337.12257
nation. Bot Gaz 57:64–69. https://doi.org/ 82. Zhang N, Li D, Zhang X et al (2015) Solid-
10.1086/331218 state fermentation of whole oats to yield a
71. Flores J, González-Salvatierra C, Jurado E synbiotic food rich in lactic acid bacteria and
(2016) Effect of light on seed germination prebiotics. Food Funct 6:2620–2625.
and seedling shape of succulent species from https://doi.org/10.1039/c5fo00411j
Mexico. J Plant Ecol 9:174–179. https://doi. 83. Torino MI, Limón RI, Martı́nez-Villaluenga
org/10.1093/jpe/rtv046 C et al (2013) Antioxidant and antihyperten-
72. Kettenring KM, Gardner G, Galatowitsch SM sive properties of liquid and solid state fer-
(2006) Effect of light on seed germination of mented lentils. Food Chem 136:1030–1037.
eight wetland Carex species. Ann Bot 98:869– https://doi.org/10.1016/j.foodchem.2012.
874. https://doi.org/10.1093/aob/mcl170 09.015
73. Golla K, Vutukuru S, Usha J et al (2016) 84. Di Stefano E, Tsopmo A, Oliviero T et al
Screening of small peptides from various ger- (2019) Bioprocessing of common pulses
minating seeds having antimicrobial activity. changed seed microstructures, and improved
IOSR J Pharm Biol Sci (IOSR-JPBS) 11:52– dipeptidyl peptidase-IV and α-glucosidase
6 0 . h t t p s : // d o i . o r g / 1 0 . 9 7 9 0 / inhibitory activities. Sci Rep 9. https://doi.
3008-11115260 org/10.1038/s41598-019-51547-5
74. Akhtaruzzaman M, Mozumder R, Jamal R 85. Onuma K, Kanda Y, Suzuki Ikeda S et al
et al (2012) Isolation and characterization (2015) Fermented brown rice and rice bran
protease enzyme from leguminous seeds. with Aspergillus oryzae (FBRA) prevents
Agric Sci Res J 2(8):434–440 inflammation-related carcinogenesis in mice,
through inhibition of inflammatory cell infil-
75. Sulieman MA, Sulieman MA, Eltayeb MM tration. Nutrients 7:10237–10250. https://
et al (2008) Effect of sprouting on chemical doi.org/10.3390/nu7125531
composition and amino acid content of Suda-
nese lentil cultivars. J Appl Sci 8:2337–2340. 86. Dueñas M, Fernández D, Hernández T et al
https://doi.org/10.3923/jas.2008.2337. (2004) Bioactive phenolic compounds of
2340 cowpeas (Vigna sinensisL). Modifications by
fermentation with natural microflora and with
76. Pathak M (2013) Germinating seeds: source Lactobacillus plantarumATCC 14917. J Sci
of probiotics. World Appl Sci J 26:224–231.
Food Agric 85:297–304. https://doi.org/ antifungal and cytotoxic activity. Antibiotics

10.1002/jsfa.1924 1 0 : 1 6 6 . h t t p s : // d o i . o r g / 1 0 . 3 3 9 0 /
87. Effect of Temperature on Fermentation. In: antibiotics10020166
Vernier. https://www.vernier.com/experi 98. Tsuboi S, Osafune T, Tsugeki R et al (1992)
ment/bwv-16b_effect-of-temperature-on- Nonspecific lipid transfer protein in castor
fermentation/ bean cotyledon cells: subcellular localization
88. Every Degree Counts (2013). https://www. and a possible role in lipid metabolism 1. J
lbds.com/wp-content/uploads/2013/06/ Biochem 111:500–508. https://doi.org/10.
L B D S M a s c o m a _ 1093/oxfordjournals.jbchem.a123787
ThermostabilityDocument.pdf 99. Finkina EI, Balandin SV, Serebryakova MV
89. Carrasco T, Valiño E, Ibarra A et al (2003, et al (2007) Purification and primary struc-
2003) Negative effect of humidity on solid ture of novel lipid transfer proteins from ger-
state fermentation of sugar cane bagasse. minated lentil (Lens culinaris) seeds. Biochem
Cuban J Agric Sci 37(1) https://agris.fao. Mosc 72:430–438. https://doi.org/10.
org/agris-search/search.do?recordID= 1134/s0006297907040104
CU20040021792 100. Khalaf EM, Raizada MN (2016) Taxonomic
90. Zhou Y, Han L-R, He H-W et al (2018) and functional diversity of cultured seed asso-
Effects of agitation, aeration and temperature ciated microbes of the cucurbit family. BMC
on production of a novel glycoprotein GP-1 Microbiol 16. https://doi.org/10.1186/
by Streptomyces kanasenisi ZX01 and scale- s12866-016-0743-2
up based on volumetric oxygen transfer coef- 101. Huang Y, Kuang Z, Wang W, Cao L (2016)
ficient. Molecules (Basel, Switzerland) 23: Exploring potential bacterial and fungal bio-
1 2 5 . h t t p s : // d o i . o r g / 1 0 . 3 3 9 0 / control agents transmitted from seeds to
molecules23010125 sprouts of wheat. Biol Control 98:27–33.
91. Thorakkattu P, Khanashyam AC, Shah K et al https://doi.org/10.1016/j.biocontrol.
(2022) Postbiotics: current trends in food and 2016.02.013
pharmaceutical industry. Foods 11:3094. 102. Links MG, Demeke T, Gr€a fenhan T et al
https://doi.org/10.3390/foods11193094 (2014) Simultaneous profiling of seed-
92. Wegh, Geerlings, Knol et al (2019) Postbio- associated bacteria and fungi reveals antago-
tics and their potential applications in early life nistic interactions between microorganisms
nutrition and beyond. Int J Mol Sci 20:4673. within a shared epiphytic microbiome on Tri-
https://doi.org/10.3390/ijms20194673 ticum and Brassica seeds. New Phytol 202:
93. Discover the World of Postbiotics – Today’s 542–553. https://doi.org/10.1111/nph.
Dietitian Magazine. www.todaysdietitian. 12693
com. https://www.todaysdietitian.com/ 103. Posoongnoen S, Thummavongsa T (2020)
newarchives/JJ20p20.shtml Purification and characterization of thermo-
94. Sirilun S, Sivamaruthi BS, Kesika P et al stable α-amylase from germinating sword
(2017) Lactic acid bacteria mediated fermen- bean (Canavalia gladiata (Jacq.) DC.)
ted soybean as a potent nutraceutical candi- seeds. Plant Biotechnol 37:31–38. https://
date. Asian Pac J Trop Biomed 7:930–936. doi.org/10.5511/plantbiotechnology.19.
https://doi.org/10.1016/j.apjtb.2017. 1209b
09.007 104. Sana NK, Hossin I, Haque EM, Shaha RK
95. Deokar G, Shinde P, Kshirsagar S (2022) (2004) Identification, purification and char-
In-vitro biorelevant media and time simula- acterization of lipase from germinating oil
tion probiotic proliferation methodology to seeds (Brassica napus L.). Pak J Biol Sci 7:
determine prebiotic potentials of flaxseed 246–252. https://doi.org/10.3923/pjbs.
powder. Bioact Carbohydr Diet Fibre 28: 2004.246.252
100335. https://doi.org/10.1016/j.bcdf. 105. Ohanenye IC, Sun X, Sarteshnizi RA, Ude-
2022.100335 nigwe CC (2021) Germination alters the
96. Finkina EI, Shramova EI, Tagaev AA, Ovchin- microstructure, in vitro protein digestibility,
nikova TV (2008) A novel defensin from the α-glucosidase and dipeptidyl peptidase-IV
lentil Lens culinaris seeds. Biochem Biophys inhibitory activities of bioaccessible fraction
Res Commun 371:860–865. https://doi. of pigeon pea (Cajanus cajan) seeds. Legume
org/10.1016/j.bbrc.2008.04.161 Sci. https://doi.org/10.1002/leg3.79
97. Barashkova AS, Sadykova VS, Salo VA et al 106. Abdel-Gawad A (1993) Effect of domestic
(2021) Nigellothionins from black cumin processing on oligosaccharide content of
(Nigella sativa L.) seeds demonstrate strong some dry legume seeds. Food Chem 46:25–
31. https://doi.org/10.1016/0308-8146 its probiotic attributes and bioactive proper-

(93)90070-v ties. Ann Microbiol 69:79–92. https://doi.
107. Nissen L, di Carlo E, Gianotti A (2020) Pre- org/10.1007/s13213-018-1399-y
biotic potential of hemp blended drinks fer- 111. Cardinali F, Osimani A, Milanović V et al
mented by probiotics. Food Res Int 131: (2021) Innovative fermented beverages
109029. https://doi.org/10.1016/j. made with red rice, barley, and buckwheat.
foodres.2020.109029 Foods 10:613. https://doi.org/10.3390/
108. Wong J-C-J, Hii S-L, Koh C-C (2021) Isola- foods10030613
tion of prebiotics from Artocarpus integer’s 112. Seo SH, Cho S-J, Hong Y-H et al. Method
seed. Int J Food Sci 2021:1–11. https://doi. for preparing a fermented soybean meal using
org/10.1155/2021/9940078 bacillus strains. https://patents.google.com/
109. Jeygowri N, Parahitiyawa N, Jeyatilake S et al patent/WO2011031020A2/en. Accessed
(2015) Study on isolation of potentially pro- 24 Nov 2022
biotic Lactobacillus species from fermented 113. Gopikrishna T, Suresh Kumar HK,
rice. Trop Agric Res 26:428. https://doi. Perumal K, Elangovan E (2021) Impact of
org/10.4038/tar.v26i3.8106 Bacillus in fermented soybean foods on
110. Divisekera DMW, Samarasekera JKRR, Het- human health. Ann Microbiol 71. https://
tiarachchi C et al (2018) Lactic acid bacteria doi.org/10.1186/s13213-021-01641-9
isolated from fermented flour of finger millet,
Chapter 15
Essential Oils: Sustainable Extraction Techniques

and Nutraceuticals Perspectives
Olusegun Abayomi Olalere, Chee-Yuen Gan, Abiola Ezekiel Taiwo,
Oladayo Adeyi, and Funmilayo Grace Olaiya
Abstract
Essential oils in their unadulterated form, can be primarily classified into two fundamental chemical
constituents, namely: hydrocarbons, oxygenated and terpenoidal bioactive compounds. The biochemical
characteristics of essential oils exhibit significant variations contingent upon the specific extraction methods
employed. While traditional techniques such as cold pressing, hydro-distillation, and maceration have long
been prevalent, they are not without their drawbacks, such as lower yield, the potential for degradation of
thermolabile compounds, and concerns regarding the environmental impact of solvent usage. In the pursuit
of sustainable and effective extraction, modern methodologies have risen to prominence, including
microwave-assisted, supercritical, and ultrasonic extraction techniques. These innovative approaches have
circumvented the inherent limitations of conventional methods, offering novel possibilities for harnessing
the full potential of essential oils. This chapter offers a brief review of both classical and contemporary
extraction techniques, shedding light on their influence over the biochemical properties of essential oils.
Furthermore, it delves into the promising perspectives of utilizing these oils in for nutraceutical applica-
tions, underscoring their potential for enhancing human well-being.
Key words Essential oil, Extraction, Biochemical activities, Conventional, Classical
1 Introduction
Essential oils can dissolve well in polar solvents including benzene,

toluene, acetone, ethanol, and methanol, despite being volatile
hydrophobic liquids with a comparatively lower density than
water [1]. Due to the existence of a complex mixture of bioactive
substances, the bioactivity, flavor, smell, and components of essen-
tial oils are well known [2]. They often originate from plants like
leaves, exfoliate, twigs, flowers, petals, and pods. There are around
3000 essential oils recognized today, with over 300 of them being
commercially important, primarily in the pharmaceutical, culinary,
domestic, and cosmetic industries [3]. The increasing interest in
the study of essential oils is attributed to their diverse biological
373
374 Olusegun Abayomi Olalere et al.
functions, which encompass anti-inflammatory, antibacterial, anti-

fungal, anticarcinogenic, antioxidant, antiviral, and antimuta-
genic properties [4]. The most extensively explored biological
activity in essential oil research is the antioxidant activity consider-
ing that several biological molecules are affected by oxidation and
thereby prompting many diseases such as cardiovascular and neu-
rological disorders [5].
In recent times, many studies have elucidated the antioxidant
properties of various essential oils in the hope of discovering safe,
natural antioxidants. One such study was established by Shaaban
et al., [6], who investigated the antioxidant activities of the essential
oil from 25 spices including thyme, chamomile, clove leaf, eucalyp-
tus cinnamon leaf, and basil. Tian et al. [7] reported that the
antioxidant activities of essential oils from Egyptian corn silk and
Curcuma zedoaria. Some group of bioactive compounds such as
terpenoids, terpinene, terpinolene, 1,8-cineole, and terpenes has
also been studied and found to boost essential oil’s antioxidant
activities [8]. Also, a wide variety of these plants oils has been
found to exhibit antibacterial properties; as different spices and
herbs, for example, have been traditionally employed as preserva-
tives in food to kill bacteria [9]. Take, for instance, Nandina
Domestica Thunb, which has been reported to be an effective pre-
servative for different food products [10]. Bioactive constituents
with antibacterial activities against the H. pylori from most essential
oils include carvacrol, sabinene, nerol, and isoeugenol
[11, 12]. Badekova et al. [13] reported the antibacterial activities
of thymol and carvacrol against E. coli, which suppressed patho-
genic bacterial strains: key components of essential oils from oreg-
ano and thyme.
In addition, synthetic antiviral drugs have been largely used for
the treatment of viral diseases in humans such as the HSV (herpes
simplex virus) [14]. The use of synthetic drugs is not without their
attentive side effects, which necessitates the application of plant-
based essential oil for the treatment of many viral diseases. Reichl-
ing [15] investigated the effectiveness of lemongrass essential oils
which was reported to provide a significant anti-HSV-1 effect more
than all the drugs that have been previously used. Furthermore,
essential oils have a long history of use against inflammation
because of their strong anti-inflammatory bioactive constituents.
Inflammation has generally been linked to various diseases such as
hypertension, cancer, and stroke [16]. Ogidi et al. [17] investigated
the anti-inflammatory qualities of a pale, clear, cold-pressed Aloe
vera essential oil with great potential as a carrier medium in aroma-
therapy. Sánchez et al. [18] experimented on diabetic rats and cases
of cutaneous ischemia with Aloe vera oil reported to promote
wound healing. In another study, oil extracted from Aloe vera
plant has been used to treat carrageenan-induced edema in rat
paw, which had anti-inflammatory characteristics and suppressed
Essential Oils: Sustainable Extraction Techniques and Nutraceuticals. . . 375
cyclooxygenase activity [19]. The result obtained shows that Aloe

vera oil demonstrated the strongest lipoxygenase inhibitory activity
(up to 96%) with a concentration of 0.5 g/mL. Other oils such as
thyme oil (86%) and bergamot oil (85%) were not lesser in their
effectiveness [19]. Chandel et al. [20] also reported that chamomile
oil had modest lipoxygenase inhibitory action at 0.5 g/mL, while it
had significant lipoxygenase stimulating activity at 5 g/mL (123%).
Also, some essential oils have been proven to have a wide reach
of fungicidal activities on post-harvest infections. The antifungal
characteristics of essential oils are often maximized in the vapor
phase for the storage of food [21]. However, because the food item
may still decay in the vapor phase, more investigation is needed
[21]. Hossain et al. [22] identified the resistance of carvacrol and
thymol against food-borne fungi such as Aspergillus flavus, Asper-
gillus parasiticus, and Aspergillus niger. Essential oils from plant-
based materials reduce free radicals, activates antioxidant enzymatic
cells, and prevent the permeation of mutagens [23]. An aromatic
plant-derived compound such as terpinene, and terpineol, was
reported with such activity. Only a few studies have been conducted
on the antimutagenicity of DNA repair by phenolic and terpenic
compounds found in essential oils [24]. The quality characteristics
and biological activities of essential oil are largely dependent on the
extraction technique utilized.
2 Effects of Conventional Extraction on Biochemical Activities of Essential Oil
Due to the obvious wide range of extraction processes, the bio-

chemical activities of essential oil components are highly diverse.
The most common extraction procedures are cold pressing, hydro-
distillation, and maceration, each with advantages and disadvan-
tages. Take instance, cold pressing (expression) is solely employed
in the extraction of citrus oils as it is primarily utilized to recover
essential oils from citrus fruits. Expression is a four-step procedure
that includes scarification, pressing, centrifugation, and filtration.
In this process, the citrus skins are scrubbed (scarified) to rupture
the microcellular structure holding the essential oils, which is
thereafter pressed to extract the oily substance from the sample
[25] (Fig. 1). The essential oil on the sample surface is then
separated into layers using centrifugal pump and thereafter filtered
to get pure essential oil.
One significant advantage that cold pressing has over other
methods is their high degree of purity and higher biological activ-
ities of the oily extracts. In addition, the cold-pressed oil keeps the
oil’s original flavor and color while also preserving the oil’s biologi-
cally active components. However, the drawback of cold pressing
(expression) is that the sample is not generally extracted at optimal
conditions, resulting in a low extraction yield. Furthermore, most
Fig. 1 Schematic diagram of a typical cold press extraction of citrus peel [26]
plant samples are unsuitable for cold pressing since they cannot
sustain high mechanical pressure [25]. Furthermore, there is fluc-
tuation or inconsistent moisture content in cold pressing, which
might affect the biochemical activities of the essential oil as
reported by Çakaloğlu et al. [25].
Furthermore, the earliest and easiest technique of extracting
essential oils is the hydro-distillation technique, which begins with
the immersion of the plant sample straight into the extracting
solvent (water) within the reactor and then boiling the entire
mixture. This extraction technique is said to be a one-of-a-kind
approach for extracting oil from plant parts such as tough nuts,
wood, seeds, and hard surface powders. It is commonly utilized for
the extraction of oil that contains hydrophobic matter with a high
boiling point. Because the oils are covered in water, this technique
allows essential oils to be extracted at a controlled temperature
without being overheated [27]. The capacity to separate plant
components under 100 °C is the major benefit of this extraction
process [28]. The hydro-distillation set-up consists of a heater
source, a reactor, a condensation chamber that converts vapor
from the reactor into liquid, and a decanter to capture the conden-
sate and separate the water and essential oils mixture (Fig. 2).
water clevenger
water
wa
Essential oil
te
r
water
wa
steam containing
te
r
essential oil
Mixture of
sample and water
Heating mantle
Fig. 2 Schematic diagram of a hydro-distillation apparatus [29]
To recover essential oils by hydro-distillation method, the plant

sample is usually loaded and a considerable amount of extracting
solvent (water) is poured and heated to a boiling temperature;
conversely, steam is normally introduced. The essential oil is
released from the cellulosic oil glands by the impact of high pres-
sure. The water-oil vapor combination is condensed by evaporative
cooling water. Distillate runs from the condensing compartment
into a splitter, where the essential oil separates efficiently from the
condensate. The advantage of this approach is the utilization of the
extracting solvent (water) and the ease with which it can be set up
[28]. This is put up before the dehydration of the plant material and
works best when the material is dry. Compared to other extraction
methods, it is a superior alternative due to the ease of use and
accessibility of its ancillary equipment [30]. Unfortunately, there
are some inherent disadvantages to using this approach. There are a
number of drawbacks associated with extracting biological compo-
nents, including excessive solvent use, poor output yield, liquid
contamination, costly extraction, and an extended process time.
Maceration is another traditional method of essential oil extrac-
tion in which various carrier oils are used as solvents to extract the
bioactive essential oil. The essential oil obtained through the mac-
eration process is also called infused or macerated oils [31]. This
technique is superior to distillation because it recovers higher
molecular compounds from the plant samples [31–33]. The proce-
dure involved loading into a closed vessel of a finely divided plant
material and solvents (menstruum). The mixture of the plant sam-
ple and the solvent are kept for 7 days and intermittently stirred
using a magnetic stirrer. The mixture is then pressurized to collect
the fluid from the waste of the plant (marc). Then, filtering of the
resulting liquid mixture is carried out to remove the infused oil as
presented in Fig. 3.
Fig. 3 Schematic diagram of maceration extraction of infused oils [34]
Past and recent studies had modified the use of the maceration
method for the recovery of essential oil from plant sources. Notable
among them are Kowalskia and Wawrzykowskib [35] who
employed an ultrasound-assisted maceration technique to extract
essential oil from thyme (Thymus vulgaris L.) dried leaves. Kowalski
et al. [36] reported the use of maceration techniques as a prelimi-
nary extraction process before ultrasonic processing of essential oil
from peppermint leaves, marjoram herb, and chamomile flowers.
Mariane et al. [37] investigated the recovery of olive oil from
Brazilian pink pepper using different stages of the maceration
process. Soares et al. [38] incorporated the ultrasonic and macera-
tion process for the extraction of enhancing flavoring of rosemary
and basil extra virgin olive oil. Unfortunately, the use of maceration
for extraction of essential oil has many limitations which include a
longer duration for extraction which could take days for comple-
tions [39]. Higher solvent consumption and a low degree of other
drawbacks affect the effectiveness of maceration extraction of essen-
tial oil [39].These limitations reduce the quality characteristics and
hence the biochemical activities of their essential oils. Traditional
extraction methods often take a long time, which means that some
of the plant material’s bioactive components will inevitably
degrade. Examples of these conventional approaches are listed in
Table 1.
3 Effects of Classical Extraction on Biochemical Activities of Essential Oil
Conventional procedures have been enhanced by new techniques

such as microwave-assisted, supercritical, and ultrasonic essential oil
extractions. Microwave-assisted extraction for example has more
capabilities than the conventional solvent extraction mentioned in
Table 1
Conventional methods of extracting essential oils from various plant sources
Conventional
Sample Parts of plant extraction methods References
Pequi (Caryocar brasiliense) Fruit Cold pressing [40]
Rice bran Husk Cold pressing [41]
Cannabis sativa L. hemp Leaves Cold pressing [42]
Fennel Leaves Cold pressing [43]
Prunus serotine Seeds Cold pressing [44]
Clove Buds Cold pressing [45]
Hemp Seeds Cold pressing [46]
Rosmarinus officinalis Whole plant Hydrodistillation [47]
and Origanum compactum
Lamiaceae (Mint) Leaves Hydrodistillation [48]
Kumquat Peels Hydrodistillation, ultrasonic, [49]
microwave extraction
Schinus molle Leaves and fruits Hydrodistillation, fractional [27]
hydrodistillation,
and steam distillation
Bitter orange Peel wastes Hydro-distillation [50]
O. basilicum L. Leaves Hydrodistillation [51]
O. vulgare L. subspecies hirtum Aerial parts Hydro-distillation [52]
Litsea cubeba (Lour.) Pers. Fruits Hydro-distillation [53]
Aquilaria malaccensis Leaves Hydro-distillation [29]
Thymus serpyllum L. herb Leaves Maceration [54]
Brazilian pink pepper Fruit Maceration [37]
Rosemary and basil Leaves Maceration [38]
Orange peels Peels Maceration [55]
the previous section. In microwave-assisted extraction, conduction

and convection occur at a pace that is so fast that it is frequently
neglected or thought to be inconsequential since it occurs in a
matter of seconds [56]. Microwave heating is usually through
electromagnetic radiation with heat and mass transfer rate uni-
formly spread throughout the heating reactor; unlike the conven-
tional approach where the heat transfer is not homogenous from
the elevated temperatures to the lowest part [57]. The ability to
extract pure essential oils free of undesirable impurities utilizing
microwaves has been shown in several research studies [58]. This

opens up the possibility of shortening the extraction process,
reducing energy consumption, using less solvent, increasing bioac-
tive selectivity, and improving extraction yields [33, 59]. Ionic con-
duction and dipole rotation are the fundamental mechanisms at
work in microwave extraction [60]. These two characteristics may
coexist, with ionic conduction acting as a formidable obstruction to
ion transport when present. This causes a temperature differential
in the extraction media and also creates impedance. Meanwhile, the
biological dipole moment is readjusted into the electric field by
dipolar rotation [61]. Even as dipole revolves around its axis, an
ionic flux is consequently present. In the microwave cavity, the
electrical field produces an ionic current in the medium, which
initiates the separation process. Because of their electromagnetic
characteristics, microwaves’ electric field is an orthogonal orienta-
tion to the magnetic fields [62]. So under the effect of the highly
dynamic electrical field, the solvent provides resistance for its ion
flux. Desai and Parikh [63] argued that the amount of turbulence in
the passage of solvent ions into the plant tissue diminishes as the
dipole rotation lowers, thereby reducing the thermal energy created
in the media [64]. The pressure gradient is subsequently created,
culminating in the transference of mass and energy into the reactor.
This implies that solvents migrate from one zone to another to
cause resistance in the media and hence to isolate bioactive chemi-
cals from the constituents from the plant material [65]. However,
uneven pressure gradients in the reacting vessels are typical of
traditional extraction methods [66]. The appropriate selection of
optimized extraction parameters is critical to the essential oil yield
via electromagnetic-based microwave technology. One such factor
is the microwave irradiation time. When the compounds of interest
are stable to heat, a prolonged extraction time is necessary for the
material with a greater dielectric constant such as ethanol, metha-
nol, and water [67]. The shorter duration of extraction is one of the
merits of microwave technology over the conventional methods of
extraction. This helps in the preservation of thermo-labile consti-
tuents and hence a better biochemical quality of the essential oil
extracted.
Above the critical temperature and pressure for a liquid or gas,
another conventional extraction process known as supercritical
fluid is constantly in use. Liquid and gas phases blend into one
another and disappear altogether in the supercritical region. The
diffusivity and density of supercritical fluids (SFs) are intermediate
between those of liquids and gases. Different SFs have different
solvating powers because its density varies with pressure and tem-
perature, unlike liquids [68]. To isolate individual substances from
a complex combination, this phenomenon is relied upon. Several
different kinds of hydrocarbons, including those with four or more
carbon atoms, nitrous oxide, sulphur hexafluoride, and fluorinated
hydrocarbons, have been studied as potential SFE solvents. Carbon

dioxide (CO2) is one of the most used SFE solvents since it is
non-toxic, abundant, and cheap. In this way, supercritical processes
may be carried out at pressures as low as 1 bar and temperatures as
low as 20 °C. By use of these phenomena, it is possible to separate
individual substances from a complex combination. Alkanes with
four or more atoms, nitrous oxide, fluorinated gases, and fluori-
nated hydrocarbons have all been tested for in SFE solvents. On the
other hand, carbon dioxide (CO2) is the most often used SFE
solvent since it is non-toxic, readily available, and inexpensive
[69]. It paves the way for supercritical operations to take place at
ambient or almost ambient pressures and temperatures. Density,
diffusivity, depressurization, viscosity, and critical temperature are
all crucial characteristics of supercritical fluids. The solubility of a
supercritical fluid is proportional to its density, which in turn
depends on its pressure and temperature. Once the density of the
fluid is known, its solvating ability may be calculated with ease.
Because the diffusion rates are so high, the extraction times are
much shorter than they would be with liquid solvents. This is
crucial because extraction rates are ultimately limited by the rate
at which analyte molecules diffuse from the solid phase into the
liquid phase [68]. Moreover, depressurization can be used to
remove SFEs like carbon dioxide (CO2) and nitrous oxide (N2O)
from analytes because they are gaseous at ambient temperature and
pressure. Supercritical fluids have far lower viscosities than liquids
(often by an order of magnitude), resulting in better flow proper-
ties [68]. This allows supercritical fluids to have direct access into
the plant matrix quite more rapidly than traditional solvents. The
majority of chemicals employed in analytical supercritical fluid
extraction are non-toxic, inert, and generally affordable. Fluids
with low critical temperatures such as CO2 and N2O can be used
to supercritically extract thermally sensitive compounds.
Essential oil from aromatic plants with strong biochemical
activity may now be extracted using the supercritical fluid extrac-
tion (SFE) technique rather than the time-consuming and tedious
traditional methods. Effective and rapid extraction may be achieved
using this technique without the need of high heat, tedious
cleanup, or potentially harmful organic solvents. Most studies on
the SFE of EOs look at how varying factors like temperature,
pressure, fluid flow rate, sample size, modifiers, and fractionation
affect extraction yield. The extraction yield, the amount of time and
resources saved, and the accuracy of the data from future studies
may all be greatly improved by adjusting these settings. Typical
studies on the SFE of EOs look at how changing factors like
temperature, pressure, fluid flow rate, sample size, modifiers, and
fractionation affect the amount of extract that can be extracted.
Estimating how temperature affects individual EOs may be chal-
lenging. The greater the temperature, the less dense the fluid,
which enhances the solubility of the EO, and hence explains

the observed occurrence. So, the extraction yield is determined by
the equilibrium between the density of SC-CO2 and the volatility of
the EOs at the specified circumstances, and this equilibrium shifts at
various temperatures. Furthermore, as many EO constituents are
thermo-labile, higher temperatures may accelerate their degrada-
tion. SFE has the advantage of producing a high yield at low
pressure; this makes it a viable option for extracting EOs; however,
in order to fully comprehend a solute’s behavioral patterns in SFE,
it is important to take into account four characteristics: the solute’s
cutoff point pressure; the solute’s optimum solubility pressure; the
solute’s fractionation pressure; and the solute’s physicochemical
properties[68]. When greater pressures are applied, however,
mass transfer and EO release are both enhanced from the plant
matrix. Therefore, increasing the solvent power results in a decline
in extraction selectivity and an increase in pressure. Separating the
EO from the other co-extracted components requires a fraction-
ation system with at least two separators when high pressures are
applied.
The particle size, surface area, shape, and porosity of the plant
matrix all have a significant role in the SFE yield, and all have an
effect on the quality of the extracts. Limiting the particle size of a
solid matrix increases surface area, decreases resistance to mass
transfer, and improves extraction efficiency, all of which contribute
to a shorter processing time. If the plant matrix is reduced too
much, the solute may be re-adsorbed on its surface, so delaying the
extraction process, and the pressure in the extractor may decrease
[68]. Extraction efficiency is also affected by the rate at which the
SF moves through the plant cells. By decreasing the flow rate, we
may lower the linear velocity. Mass transfer resistance restricts the
injection of analytes into a fluid at low flow rates, and unsaturated
SC-CO2 is introduced to the extraction vessel. As the flow rate of
the fluid rises, the mass transfer resistance decreases until the fluid
being removed is saturated, at which point equilibrium is estab-
lished and maximum yield is achieved. Due to a decrease in resi-
dence time as flow rate rises, the system deviates from equilibrium
and unsaturated fluid exits the extraction vessel, even while the
mass transfer rate remains constant. This is because the leaves that
can be extracted are skipped through if too much moisture enters
their cells [70].
Furthermore, ultrasonic-assisted extraction (UAE) is increas-
ing in popularity in the food and pharmaceutical sectors as a
non-thermal extraction method for its many benefits [71]. This
innovation was made to fix issues with both older and newer
extraction methods. In comparison to conventional methods,
UAE yields a more desirable essential oil quality profile, shorter
extraction times, lower energy usage, fewer contaminants, and
fewer or no solvent requirements [49]. UAE is a simple, effective,
and cheap technology when contrasted to other innovative extrac-

tion methods for essential oil recovery, such as supercritical fluid
extraction (SFE) and microwave-assisted extraction (MAE)
[71]. The flow of ultrasonic waves causes cell disturbances and a
large contact surface area between the extracting solvent and sam-
ple material, enhancing mass and heat transfer, which is believed to
be how UAE achieves its great efficacy in essential oil extraction
[72]. The ultrasonic parameters (such as frequency and amplitude),
product parameters (including viscosity and surface tension), and
environmental factors (including temperature and pressure) all play
significant roles in the formation of cavitation during UAE
[73]. The classification of UAE technologies in food industries
include low intensity-high frequency ( f > 100 kHz) and high
intensity-low frequency (20 kHz < f < 100 kHz) ultrasound
[74]. In the laboratory, UAE can be performed by immersing the
plant material in water or other solvents (e.g., methanol or ethanol)
and allowed to receive ultrasonic treatment at the same time [75].
Generally, process variables of significant importance contri-
buting to the extractability of essential oil from plant materials
include ultrasonic intensity/energy, solvent type, temperature,
ultrasonic time, sample—solvent ratio, and ultrasonic power. Cavi-
tation intensity is determined by the amount of ultrasonic energy
supplied per unit volume of the plant samples. Moreover, there are
lesser cavitation effects when an elevated ultrasonic wave is deliv-
ered to a bigger sample volume. Addressing both intensity and
power density can be explained by considering the amount of
energy applied to a given volume of sample (in joules per milliliter
or watts per gram). Most studies investigate described this in terms
of intensity (W/cm2), but many additionally reported it in terms of
power density (W/mL) as well. These variables are sometimes
underestimated when replicating results from ultrasound, even
though they’re crucial for replicating sonication results from ultra-
sound. Moreover, ultrasonic cavitation and its thresholds are
affected by the solvent’s viscosity, surface tension, and vapor pres-
sure. These three variables raise the cavitation threshold and sample
resistance to device displacement when they are increased. It fol-
lows that to form cavities, the intensity of the oscillations must be
increased. Due to decreased solvent vapor pressure, cavitation bub-
bles rupture more abruptly. This depends on the kind and strength
of the interactions between a solute and its solvent as well as
between a solvent and its solute. Oils being non-polar, non-polar
solvents are the most effective at extracting oil. The effectiveness of
oil extraction gradually decreases the polarity of a solvent. Solvents
such as n-hexane and petroleum ether are both non-polar with the
maximum oil extraction rate. Van der Waals forces are indeed very
weak in such solvents, with inherent lower volatility and boiling
temperatures. Van der Waals forces are also the only forces that exist
within the non-polar solute molecules. In addition, due to
cavitation energy, as extraction time increases, cellular membranes

are ruptured, thereby increasing the contact area between the
ruptured cell walls and the extracting solvent, allowing for a greater
amount of oil to permeate into the solvent. A 40-min increase in
the UAE time can increase oil extraction yield from 23.46 to
26.71% at a fixed solvent-to-solid ratio [76]. Extraction efficiency
declines with increasing extraction time because of equilibrium
between the solid sample and solvents. Temperature is one of the
most important factors in the ultrasonic extraction of essential oil.
Cavitation bubbles increase with temperature rise and this results in
a rise in contact surface area between extracting solvent and the
cellulosic cell walls, as well as a corresponding drop in viscosity.
Mass transfer improves extraction efficiency because the solvent
with lower viscosity is better able to penetrate easily into the sample
matrix. Marhamati et al. [76] reported that an increase in tempera-
ture from 40 to 50 °C, even when the solvent-to-solid ratio was
kept the same, increased the oil extraction from 18 to 20%.
In summary, classical extraction methods, including steam dis-
tillation, solvent extraction, microwave-assisted extraction, and
ultrasonic extraction, play a pivotal role in obtaining essential oils.
However, it’s essential to recognize that these methods can exert
significant influences on the biochemical activities of essential oils,
leading to several noteworthy consequences. Firstly, these extrac-
tion techniques have the potential to bring about alterations in the
chemical composition of essential oils. The application of heat or
solvents during the extraction process can induce modifications in
the oil’s chemical profile, consequently affecting its aroma and
therapeutic attributes. This change in composition can be both
beneficial or detrimental, depending on the specific compounds
involved. Furthermore, the classical extraction methods can impact
the yield and purity of essential oils. Some valuable constituents
may be lost or degraded during the extraction process, while
unwanted contaminants or solvent residues may be introduced,
potentially compromising the oil’s overall quality. Additionally,
the changes in the chemical profile can have a direct bearing on
the therapeutic efficacy of essential oils. Certain bioactive com-
pounds responsible for the oils’ beneficial properties may be either
diminished or enhanced, which, in turn, affects their potential
health benefits. Moreover, the stability of essential oils may be
jeopardized during classical extraction. Exposure to heat or chemi-
cal solvents can lead to oxidative degradation, thereby reducing the
oil’s shelf life and diminishing its overall shelf-stability. The aroma
and flavor of essential oils, integral to their various applications, are
not exempt from the impacts of classical extraction. Some com-
pounds contributing to the characteristic scent and taste of the oil
may undergo alterations or loss during the extraction process,
potentially affecting the sensory qualities of the oil. Lastly, the use
of chemical solvents in classical extraction methods can raise safety
concerns. Residues of these solvents must be diligently removed to

ensure the oil’s suitability for therapeutic, culinary, or other appli-
cations. Failure to do so can render the oil unsafe for use. To
address these concerns and mitigate the potential drawbacks, alter-
native extraction techniques, such as CO2 extraction or cold press-
ing, are increasingly employed. These methods are known to
preserve the biochemical activities and overall quality of essential
oils to a greater extent, ensuring that the end-product maintains its
intended attributes and benefits.
4 Conclusion
The extraction of essential oils (EOs) is gaining more attention than

ever before, because of their numerous benefits. Moreover, due to
the sensitivity of several of their biochemical constituents; extrac-
tion in extreme conditions is not feasible. Classical extraction meth-
ods which allow for lower temperatures and shorter processing
periods have been described as enhanced technologies for extract-
ing high-quality essential oils with minimal component losses.
These methods have significant advantages over other conventional
extraction methods in terms of preserving the main biochemical
constituents of the oil. Many of essential oils’ qualities, like anti-
bacterial, antioxidant, and anti-inflammatory action, are deter-
mined by their constituents and the method of extracting them.
This review therefore critically examined the mechanism and advan-
tages of conventional and non-conventional extraction methods.
This would surely help academics and business professionals choose
the best extraction techniques to get the most out of their materials
while maintaining the highest grade biochemical properties.
References
1. González-Burgos E, Carretero ME, Gómez- strategies into lipid-based delivery systems.
Serranillos MP (2011) Sideritis spp.: uses, Pharmaceutics 13(3):1–35. https://doi.org/
chemical composition and pharmacological 10.3390/pharmaceutics13030327
activities – a review. J Ethnopharmacol 5. Laothaweerungsawat N, Sirithunyalug J,
135(2):209–225. https://doi.org/10.1016/ Chaiyana W. Molecules chemical compositions
J.JEP.2011.03.014 and anti-skin-ageing activities of Origanum
2. Lages LZ et al (2021) Microbiological and vulgare L. essential oil from tropical and medi-
sensory evaluation of meat sausage using terranean region. https://doi.org/10.3390/
thyme (Thymus vulgaris, L.) essential oil and molecules25051101
powdered beet juice (Beta vulgaris L., early 6. Shaaban HAE, El-Ghorab AH, Shibamoto T
wonder cultivar). LWT 148:111794. https:// (2012) Journal of essential oil research bioac-
doi.org/10.1016/J.LWT.2021.111794 tivity of essential oils and their volatile aroma
3. Yangilar F (2021) Biological activities, health components: review bioactivity of essential oils
benefits, extraction methods, food applications and their volatile aroma components: review.
and beneficial effects of essential oils https://doi.org/10.1080/10412905.2012.
4. Cimino C et al (2021) Essential oils: pharma- 659528
ceutical applications and encapsulation
7. Tian J, Chen H, Chen S, Xing L, Wang Y, 17. Olusola Ogidi C, Elizabeth Ojo A, Benjamin
Wang (2013) Comparative studies on the con- Ajayi-Moses O, Modupe Aladejana O, Abike
stituents, antioxidant and anticancer activities Thonda O, Juliet Akinyele B. Synergistic anti-
of extracts from different varieties of corn silk. fungal evaluation of over-the-counter antifun-
Food Funct 4(10):1526–1534 gal creams with turmeric essential oil or Aloe
8. Micalizzi G et al. Development of a novel vera gel against pathogenic fungi. https://doi.
microwave distillation technique for the isola- org/10.1186/s12906-021-03205-5
tion of Cannabis Sativa L. essential oil and gas 18. Sánchez M, González-Burgos E, Iglesias I,
chromatography analyses for the comprehen- Gómez-Serranillos MP (2020) Pharmacologi-
sive characterization of terpenes and terpe- cal update properties of Aloe vera and its major
noids, including their enantio-distribution active constituents. Molecules 25(6):1324
9. El-Saber Batiha G et al (2021) Application of 19. Dahariya S, Prashanth N, Divakar K, Baadhe
natural antimicrobials in food preservation: RR (2020) Aloe vera and its biological activ-
recent views. Food Control 126:108066. ities. World J Curr Med Pharmaceut Res 3
https://doi.org/10.1016/J.FOODCONT. (August):11–13
2021.108066 20. Chandel P, Rawal RK, Kaur R (2018) Natural
10. Bajpai SC, Rahman VK, Kang A (2008) Chem- products and their derivatives as
ical composition and inhibitory parameters of cyclooxygenase-2 inhibitors. Fut Med Chem
essential oil and extracts of Nandina domestica 10(20):2471–2492
Thunb. to control food-borne pathogenic and 21. Střelková T, Nemes B, Kovács A, Novotný D,
spoilage bacteria. Int J Food Microbiol 125(2): Božik M, Klouček P (2021) Inhibition of fun-
117–122 gal strains isolated from cereal grains via vapor
11. Rajkovic K, Pekmezovic M, Barac A, phase of essential oils. Molecules 26(5).
Nikodinovic-Runic J, Arsenijević VA (2015) h t t p s : // d o i . o r g / 1 0 . 3 3 9 0 /
Inhibitory effect of thyme and cinnamon essen- molecules26051313
tial oils on Aspergillus flavus: optimization and 22. Hossain F, Follett P, Dang Vu K, Harich M,
activity prediction model development. Ind Salmieri S, Lacroix M (2016) Evidence for syn-
Crop Prod 65(8):7–13. https://doi.org/10. ergistic activity of plant-derived essential oils
1016/j.indcrop.2014.11.039 against fungal pathogens of food. Food Micro-
12. Korona-Glowniak I, Glowniak-Lipa A, biol 53:24–30
Ludwiczuk A, Baj T, Malm A. Molecules the 23. Laranjo LT, Klaric JA, Pearlman LR, Lovett ST
in vitro activity of essential oils against helico- (2019) Stimulation of replication template-
bacter pylori growth and urease activity. switching by DNA-protein crosslinks. Genes
h t t p s : // d o i . o r g / 1 0 . 3 3 9 0 / 10(1):14
molecules25030586 24. Amkiss S, Dalouh A, Idaomar M (2021)
13. Badekova KZ, Atazhanova GA, Kacergius T, Chemical composition, genotoxicity and anti-
Akhmetova SB, Smagulov MK (2021) Compo- genotoxicity study of Artemisia herba-alba
sition and screening of Origanum vulgare using the eye and wing SMART assay of Dro-
essential oil for antimicrobial activity. J Taibah sophila melanogaster. Arab J Chem 14(3):
Univ Med Sci. https://doi.org/10.1016/J. 1 0 29 7 6 . h t t p s : // do i . o r g / 1 0 . 1 01 6 / J .
JTUMED.2021.05.009 ARABJC.2020.102976
14. Awad SM, Ali SM, Mansour YE, Fatahala SS 25. Çakaloğlu B, Özyurt VH, Ötleş S (2018) Cold
(2021) Synthesis and evaluation of some uracil press in oil extraction. A review. Ukr Food J
nucleosides as promising anti-herpes simplex 7(4):640–654. https://doi.org/10.24263/
virus 1 agents. Molecules 26(10). https://doi. 2304-974x-2018-7-4-9
org/10.3390/molecules26102988 26. Panjaitan R, Mahfud M, Cahyati ED, Puja-
15. Reichling J (2021) Antiviral and virucidal ningtyas L (2021) The study of parameters of
properties of essential oils and isolated essential oil extraction from black pepper seed
compounds – a scientific approach. Planta using microwave hydrodistillation by model-
Med 10(1055):1382–2898. https://doi.org/ ing. IOP Conf Ser Earth Environ Sci 749(1).
10.1055/a-1382-2898 https://doi.org/10.1088/1755-1315/749/
16. Schiffrin EL, Ferreira NS, Tostes RC, Paradis P 1/012032
(2021) Aldosterone, inflammation, immune 27. Volpini-Klein AFN et al (2021) Chemical com-
system, and hypertension. Am J Hypertens position of essential oils from leaves and fruits
34(1):15. https://doi.org/10.1093/ajh/ of Schinus molle obtained by different extrac-
hpaa137 tion methods (hydrodistillation, fractional
hydrodistillation and steam distillation) and 37. Bittencourt Fagundes M et al (2020) Charac-
seasonal variations. J Essent Oil-Bear Plants terization of olive oil flavored with Brazilian
24(2):228–242. https://doi.org/10.1080/ pink pepper (Schinus terebinthifolius Raddi)
0972060X.2021.1914739 in different maceration processes. Food Res
28. Aziz ZAA et al (2018) Essential oils: extraction Int 137:109593. https://doi.org/10.1016/J.
techniques, pharmaceutical and therapeutic FOODRES.2020.109593
potential – a review. Curr Drug Metab 38. Soares VP et al (2020) Ultrasound assisted
19(13):1100–1110. https://doi.org/10. maceration for improving the aromatization
2174/1389200219666180723144850 of extra-virgin olive oil with rosemary and
29. Samadi M, Abidin ZZ, Yunus R, Awang Biak basil. Food Res Int 135:109305. https://doi.
DR, Yoshida H, Lok EH (2017) Assessing the org/10.1016/J.FOODRES.2020.109305
kinetic model of hydro-distillation and chemi- 39. Olalere OA, Abdurahman NH, Alara OR,
cal composition of Aquilaria malaccensis leaves Habeeb OA (2017) Parametric optimization
essential oil. Chin J Chem Eng 25(2): of microwave reflux extraction of spice oleo-
216–222. https://doi.org/10.1016/j.cjche. resin from white pepper (Piper nigrum). J
2016.09.006 Anal Sci Technol 8(1):8. https://doi.org/10.
30. Olalere OA, Abdurahman HN, Yunus RM 1186/s40543-017-0118-9
(2017) Comparative study of pulsed micro- 40. Johner JCF, Hatami T, Meireles MAA (2018)
wave and hydrodistillation extraction of piper- Developing a supercritical fluid extraction
ine oil from black pepper. IIUM Eng J 18(2): method assisted by cold pressing for extraction
87–93 of Pequi (Caryocar brasiliense). J Supercrit
31. Bhadra P, Parida S (2021) Extraction methods Fluids 137:34–39. https://doi.org/10.1016/
of essential oils from aromatic plants in aroma- J.SUPFLU.2018.03.005
therapy and its benefits, no. 978-81-940943- 41. Wongwaiwech D, Weerawatanakorn M,
7-1 Tharatha S, Ho CT (2019) Comparative
32. Olalere CYG, Adedeji PA, Olalere ME, Aljbour study on amount of nutraceuticals in
OA (2021) Multi-objective Deng’s grey inci- by-products from solvent and cold pressing
dence analysis, orthogonal optimization, and methods of rice bran oil processing. J Food
artificial neural network modelling in hot- Drug Anal 27(1):71–82. https://doi.org/10.
maceration-assisted extraction of African 1016/J.JFDA.2018.06.006
cucumber leaves (Momordica balsamina). Can 42. Aladić K et al (2014) Cold pressing and super-
J Chem Eng. https://doi.org/10.1002/cjce. critical CO2 extraction of hemp (Cannabis
24138 sativa) seed oil. Chem Biochem Eng Q 28(4):
33. Olalere OA, Abdurahman NH, Yunus RBM, 481–490. https://doi.org/10.15255/
Alara OR (2017) Multi-response optimization CABEQ.2013.1895
and neural network modeling for parameter 43. Hatami T, Johner JCF, Meireles MAA (2018)
precision in heat reflux extraction of spice Extraction and fractionation of fennel using
oleoresins from two pepper cultivars (Piper supercritical fluid extraction assisted by cold
nigrum). J King Saud Univ Sci. https://doi. pressing. Ind Crop Prod 123:661–666.
org/10.1016/j.jksus.2017.09.010 https://doi.org/10.1016/J.INDCROP.2018.
34. Aromatics ND (2017) A comprehensive guide 07.041
to essential oil extraction methods. [Online]. 44. Alejandra Lu-Martı́nez A, Gabriel Báez-
A v a i l a b l e : h t t p s : // w w w . González J, Castillo-Hernández S, Amaya-
newdirectionsaromatics.com/blog/articles/ Guerra C, Rodrı́guez-Rodrı́guez J, Garcı́a-
how-essential-oils-are-made.html#maceration. Márquez E. Studied of Prunus serotine oil
Accessed 11 July 2021 extracted by cold pressing and antioxidant
35. Kowalski R, Wawrzykowski J (2009) Effect of effect of P. longiflora essential oil. J Food Sci
ultrasound-assisted maceration on the quality Technol 58. https://doi.org/10.1007/
of oil from the leaves of thyme Thymus vulgaris s13197-020-04653-6
L. effect of UAM on the quality of Thymus 45. Hatami T, Johner JCF, Zabot GL, Meireles
vulgaris leaf oil. Res Article Flavour Fragr J MAA (2019) Supercritical fluid extraction
24:69–74. https://doi.org/10.1002/ffj.1918 assisted by cold pressing from clove buds:
36. Kowalski R, Kowalska G, Jamroz J, extraction performance, volatile oil composi-
Nawrocka A, Metyk D (2015) Effect of the tion, and economic evaluation. J Supercrit
ultrasound-assisted preliminary maceration on Fluids 144:39–47. https://doi.org/10.1016/
the efficiency of the essential oil distillation J.SUPFLU.2018.10.003
from selected herbal raw materials. Ultrason 46. Faugno S et al (2019) Can agronomic practices
Sonochem 24:214–220. https://doi.org/10. and cold-pressing extraction parameters affect
1016/J.ULTSONCH.2014.12.008 phenols and polyphenols content in
hempseed oils? Ind Crop Prod 130:511–519. notes: characterization and effect of ultrasound
https://doi.org/10.1016/J.INDCROP.2018. in the maceration of orange peels. Food Res Int
12.084 133(March):109165. https://doi.org/10.
47. El Kharraf S et al (2021) Hydrodistillation and 1016/j.foodres.2020.109165
simultaneous hydrodistillation-steam distilla- 56. Olalere OA, Gan CY (2022) Biopharmaceuti-
tion of Rosmarinus officinalis and Origanum cal application of microwave technology and
compactum: antioxidant, anti-inflammatory, the scalability concerns. Jundishapur J Nat
and antibacterial effect of the essential oils. Pharmaceut Prod 17(1):e121619. https://
Ind Crop Prod 168:113591. https://doi.org/ doi.org/10.5812/jjnpp.121619
10.1016/J.INDCROP.2021.113591 57. Abdurahman NH (2016) A comparative review
48. Rezaei K, Hashemi NB, Sahraee S (2021) Use of conventional and microwave assisted extrac-
of hydrodistillation as a green technology to tion in capsaicin isolation from chili pepper.
obtain essential oils from several medicinal Aust J Basic Appl Sci 10(June):263–275
plants belonging to lamiaceae (mint) family. 58. Olalere OA, Gan CY, Adeyi O, Taiwo AE,
Phytopharmaceuticals Olaiya FG (2022) Upscalability and techno-
49. Yu F, Wan N, Zheng Q, Li Y, Yang M, Wu Z economic perspectives of nonconventional
(2021) Effects of ultrasound and microwave extraction techniques of essential oils. Jun-
pretreatments on hydrodistillation extraction dishapur J Nat Pharmaceut Prod 6:e122792.
of essential oils from Kumquat peel. Food Sci https://doi.org/10.5812/jjnpp-122792
Nutr 9(5):2372–2380. https://doi.org/10. 59. Olalere OA, Abdurahman HN, Mohd R, Alara
1002/fsn3.2073 OR, Ahmad MM, Zaki YH (2018) Parameter
50. Mahshid Heydari MR, Rostami O, study, antioxidant activities, morphological and
Mohammadi R, Banavi P, Farhoodi M, Sarlak functional characteristics in microwave extrac-
Z (2021) Hydrodistillation ultrasound-assisted tion of medicinal oleoresins from black and
green extraction of essential oil from bitter white pepper. J Taibah Univ Sci 0(0):1–8.
orange peel wastes: optimization for quantita- https://doi.org/10.1080/16583655.2018.
tive, phenolic, and antioxidant properties. J 1515323
Food Process Preserv 45(7):e15585 60. Olalere OA, Gan C-Y, Abdurahman HN,
51. Chenni M, El Abed D, Neggaz S, Adeyi O, Ahmad MM (2020) Holistic
Rakotomanomana N, Fernandez X, Chemat F approach to microwave-reflux extraction and
(2020) Solvent free microwave extraction fol- thermo-analytical fingerprints of under-
lowed by encapsulation of O. basilicum utilized Artocarpus heterophyllus seed wastes.
L. essential oil for insecticide purpose. J Stored Heliyon 6(8). https://doi.org/10.1016/j.
Prod Res 86:101575. https://doi.org/10. heliyon.2020.e04770
1016/J.JSPR.2020.101575 61. Alara OR, Olalere OA (2016) Natural Pro-
52. Drinić Z, Pljevljakušić D, Živković J, ducts Chemistry & Research. A critical over-
Bigović D, Šavikin K (2020) Microwave- view on the extraction of bioactive compounds
assisted extraction of O. vulgare L. spp. hirtum from Phaleria macrocarpa (Thymelaceae). Nat
essential oil: comparison with conventional Prod Chem Res 4(5). https://doi.org/10.
hydro-distillation. Food Bioprod Process 120: 4172/2329-6836.1000232
158–165. https://doi.org/10.1016/J.FBP. 62. Olalere OA, Gan CY (2021) Microwave-
2020.01.011 assisted extraction of phenolic compounds
53. Guo Y et al (2021) Deep eutectic solvent- from Euphorbia hirta leaf and characterization
homogenate based microwave-assisted hydro- of its morphology and thermal stability. Sep Sci
distillation of essential oil from Litsea cubeba Technol (Philadelphia) 56(11):1853–1865.
(Lour.) Pers. fruits and its chemical composi- https://doi.org/10.1080/01496395.2020.
tion and biological activity. J Chromatogr A 1795678
1646:462089. https://doi.org/10.1016/J. 63. Shah KA, Bhatt DR, Desai MA, Jadeja GC,
CHROMA.2021.462089 Parikh JK (2017) Extraction of essential oil
54. Jovanović AA et al (2017) Optimization of the from patchouli leaves using hydrodistillation:
extraction process of polyphenols from Thy- parametric studies and optimization. Indian J
mus serpyllum L. herb using maceration, Chem Technol
heat- and ultrasound-assisted techniques. Sep 64. Anwar J et al (2015) Microwave chemistry:
Purif Technol 179:369–380. https://doi.org/ effect of ions on dielectric heating in micro-
10.1016/J.SEPPUR.2017.01.055 wave ovens. Arab J Chem 8(1):100–104.
55. Bruna-Maynou FJ, Castro R, Rodrı́guez- https://doi.org/10.1016/j.arabjc.2011.
Dodero MC, Barroso CG, Durán-Guerrero E 01.014
(2020) Flavored Sherry vinegar with citric
65. Olalere OA, Gan C-Y (2020) Intensification of ultrasound-enhanced subcritical water extrac-
microwave energy parameters and main effect tion of cinnamon bark oil. LWT 147:111673.
analysis of total phenolics recovery from https://doi.org/10.1016/J.LWT.2021.
Euphorbia hirta leaf. J Food Measure Charact 111673
14(2). https://doi.org/10.1007/s11694- 72. Nie J, Chen D, Ye J, Lu Y, Dai Z (2021)
019-00338-7 Optimization and kinetic modeling of
66. Olalere OA, Abdurahman NH, Bin R, ultrasonic-assisted extraction of fucoxanthin
Yunus M, Alara OR, Gan CY (2018) Synergis- from edible brown algae Sargassum fusiforme
tic intermittent heating and energy intensifica- using green solvents. Ultrason Sonochem 77:
tion of scale-up parameters in an optimized 1 0 56 7 1 . h t t p s : // do i . o r g / 1 0 . 1 01 6 / J .
microwave extraction process. Chem Eng Pro- ULTSONCH.2021.105671
cess Process Intensif 132(August):160–168. 73. Li J et al (2010) Optimization of microwave-
https://doi.org/10.1016/j.cep.2018.08.011 assisted extraction of triterpene saponins from
67. Olalere OA, Gan C-Y (2021) Microwave reflux defatted residue of yellow horn (Xanthoceras
extraction – an alternative approach for sorbifolia Bunge.) kernel and evaluation of its
phenolic-rich oleoresins extraction from func- antioxidant activity. Innov Food Sci Emerg
tional plants. Green Sustain Process Chem Technol 11(4):637–643. https://doi.org/10.
Environ Eng Sci:661–678. https://doi.org/ 1016/j.ifset.2010.06.004
10.1016/B978-0-12-819848-3.00016-5 74. Adeyi O (2021) Techno-economic and uncer-
68. Yousefi M, Rahimi-Nasrabadi M, Pourmorta- tainty analyses of heat- and ultrasound-assisted
zavi SM, Wysokowski M, Jesionowski T, extraction technologies for the production of
Ehrlich H, Mirsadeghi S (2019) Supercritical crude anthocyanins powder from Hibiscus sab-
fluid extraction of essential oils. TrAC Trends dariffa calyx. https://doi.org/10.1080/
Anal Chem 118:182–193 23311916.2021.1947015
69. Taiwo A, Madzimbamuto T, Ojumu T (2020) 75. Milić PS, Rajković KM, Stamenković OS, Velj-
Development of an integrated process for the ković VB (2013) Kinetic modeling and optimi-
production and recovery of some selected bio- zation of maceration and ultrasound-extraction
products from lignocellulosic materials. In: of resinoid from the aerial parts of white lady’s
Valorization of biomass to value-added com- bedstraw (Galium mollugo L.). Ultrason Sono-
modities. Springer, p 34 chem 20(1):525–534. https://doi.org/10.
70. Keskinen KI, Kinnunen A, Nyström L, Aitta- 1016/j.ultsonch.2012.07.017
maa J (2002) Efficient approximate method for 76. Marhamati M, Kheirati Kakhaki Z, Rezaei M
packed column separation performance (2020) Advance in ultrasound-assisted extrac-
simulation. In: Proceedings of the international tion of edible oils: a review. J Nutr Fast Health
conference on distillation & absorption 8(4 (Special Issue on Food Safety)):220–230.
(on CD), p 68 https://doi.org/10.22038/jnfh.2020.51138.
71. Guo J, Yang R, Gong YS, Hu K, Hu Y, Song 1288
FL (2021) Optimization and evaluation of the
Chapter 16
Green and Clean Extraction Technologies for Novel

Nutraceuticals
Insha Arshad, Gulden Gosken, Mujahid Farid, Mudassar Zafar,
and Muhammad Zubair
Abstract
Phytochemicals extracted from leaves, fruits, roots, and seeds of plants exhibit high nutraceutical, antioxi-
dant, and antimicrobial potential. Such valuable components can be obtained from natural materials via
extraction, an important parameter in analytical chemistry. Since conventional extractions methods pose
severe environmental threats and have a negative impact on energy economy, moving toward green
strategies is important. Green technologies are ecofriendly and give maximum yield, and the product
obtained are pure and less toxic. Ultrasound, microwave, supercritical fluid, subcritical fluid, pressurized
liquid, enzymatic hydrolysis, radio frequency, electroosmotic dewatering, cold plasma treatment, high-
pressure processing, electrotechnology, ionic liquid, accelerated solvent extraction, and hydrotropic extrac-
tion are some of the methods used in clean and green technologies to extract biologically active components
from plants. This chapter also discusses nonthermal extraction technologies . This chapter on clean and
green technologies, processes, and protocols will provide collective and advanced knowledge to research
community in the food and nutraceutical sectors.
Key words Extraction, Nutraceuticals, Conventional techniques, Toxicity, Green technology
1 Introduction
It is possible to consider extraction as initial step in the develop-

ment of analytical procedure and creation of subsequent pro-
ducts. The phenomenal development of green technology has had
a profound effect on the recovery of natural compounds intended
to be used in food industry. Deep eutectic solvents and ionic
solvents are examples of green solvents that may be used in con-
junction with green technology to recover natural components
without producing toxic effluents and effects. In addition to the
ecologically advantageous features, health and safety concerns of
green procedures are taken into account [1]. A high level of selec-
391
392 Insha Arshad et al.
tivity is required to extract high concentrations of certain com-

pounds or to prevent the extraction of unwanted compounds
during plant extraction process [2].
Technologies that support sustainable development and assist
in the reduction of negative environmental impact are collectively
referred to as “green technologies.” The main advantages of green
technology are sustainability, economic viability, and social equity.
Today, the planet earth and its environment have been permanently
and irreparably harmed. The globe is being moved towards an
ecological landslide by our current course of conduct, and if this
occurs, disaster will be unavoidable. The use of green technologies
is a strategy toward protecting the planet. So, both the advantages
and disadvantages have to be taken into account to further develop
these technologies. Green technology makes use of naturally
replenishable resources. One of the newest green technologies is
green nanotechnology, which employs green chemistry and engi-
neering. One of the main factors contributing to environmental
pollution is the disposal of chemical wastes, which can be overcome
by using green technology. This method is environmentally friendly
and, therefore, can successfully change trash production and pat-
tern. The anticipated sector poised for growth, driven by advance-
ment, encompass green energy, organic farming, eco-friendly
textile, green building construction, and the production of related
goods and materials to sustain environmentally conscious business.
According to Soni (2015), they have no harmful environmental
consequences. The majority of classical methods for solvent extrac-
tion of nutraceuticals depend on the right solvents coupled with
heat and/or agitation to make the target chemicals more extract-
able to enhance mass transfer [3]. The majority of phyto-
constituents are susceptible to heat degradation, as traditional
extraction methods frequently involve prolonged extraction
time [4].
2 Supercritical Fluid Extraction (SFE)
A state of matter of a substance at a temperature and pressure above

its critical point, where neither a liquid nor a gas phase exists, is
referred to as a supercritical fluid (SFE) [5]. In supercritical fluid
extraction, fluids with both liquid and gaseous characteristics at
pressures and temperatures over their critical points are used is
based on the solvating properties of supercritical fluid (SF), which
can be produced by using pressure and temperature above a chemi-
cal, mixture, or element’s critical point [6]. SFE provides substan-
tial operating advantages due to a variety of physicochemical
characteristics of supercritical solvents such as density, diffusivity,
viscosity, and dielectric constant. Supercritical fluids have greater
diffusion properties than any other liquids due to their low viscosity
Green and Clean Extraction Technologies for Novel Nutraceuticals 393
and relatively high pressure, and they may diffuse swiftly through
solid materials, resulting in faster extraction rates. The capacity of a
supercritical fluid to modify its density by altering its pressure
and/or temperature is one of its most crucial characteristics.
Changing the extraction pressure might alter the solvent strength
of the fluid, given that density and solubility are related [7]. This
technology’s expanding industrial utilization is primarily attribut-
able to its selectivity, facility, and separation capacity. According to
Ahmad, Masoodi, Rather, Wani, and Gull (2019), the SFE enables
the extraction of a large number of nutraceutical compounds, many
of which are impossible or impractical to extract using conventional
methods or whose purification requires high resolution columns,
which are not always available on the national market, thereby
making their use very expensive [8]. Supercritical extraction is
often used to separate nonpolar bioactive components (carotenoids
and lipids) since the solvents used in this technique are nonpolar.
One alternative is the extraction of polar compounds like flavonoids
using modifiers like ethanol, methanol, water, and acetone
[9, 10]. Less viscosity and a shorter extraction time lead to an
improvement in the diffusion and mass transfer of the crucial fluid
[1, 11]. Table 1 shows the extraction of various bioactive com-
pounds by supercritical extraction. Factors such as temperature,
pressure, and other inherently changeable features, as well as
some extrinsic factors like the properties of the supercritical fluid,
all affect SFE: interactions with specific analyzers, the sample
matrix, and various environmental factors [6].
Table 1
Extraction of phytocompounds via supercritical fluid extraction methods
Food plant Compositions Conditions References

Rosemarinus Carnosic acid, rosmarinic acid, CO2, ethanol: water [2]
officinalis L. carotenoids, and chlorophylls (50:50 v/v), 25 °C
Castanea sativa Antioxidant and polyphenols 15% ethanol, 60 °C, 350 bar [12]
Rubus idaeus L. Oil 0.4 kg CO2/h, 40 °C, [13]
35 MPa
Beta vulgaris subsp. Amino acids CO2, ethanol, 184 bar, 43 °C, [14]
Saccharifera 76 min
Terminalia catappa Oil CO2, 60 °C, 300 bar [15]
Daucus carota Oil CO2, ethanol, 70 °C, 400 bar [16]
Saccharum L. Amino acid CO2, methanol, 316 bar, 50 ° [17]
C, 76 min
2.1 Working SFE comprises a solvent pump or compressor and a modifier

Principle (or co-solvent) pump, which requires an organic solvent or water.
An extraction chamber or reactor with a heat jacket holds the feed
material. When materials ranging from micrograms to grams are to
be extracted, the capacity of the extraction reactor of an SFE system
is designed to be 50–300 mL, especially for SFE systems in labora-
tory setting. However, when larger volumes of extracts are
required, the capacity of the reactor is increased to hundreds of
liters, especially in industrial-scale SFE operations [18]. It is neces-
sary to have a pressure regulator and a fractionation/collection
vessel (or) flash tank. In this method, dried and ground feed
material is added to the extraction chamber to produce a stable
bed. A cylinder of liquid CO2 (purity 99.998%) is compressed to
working pressure and then cooked to supercritical temperatures in a
temperature-controlled environment. The SC-CO2 is injected into
the extraction reactor and then allowed to diffuse across the feed
material’s fixed bed. The solubilized components are then removed
from the extraction chamber and placed in a separator at a lower
pressure. After that, in the flash tank, the SC-CO2 and solute
precipitate are separated. The SC-CO2 may then be cooled, com-
pacted, recycled, or released into the atmosphere [18]. Figure 1
shows that the extraction vessel contains a high-pressure chamber
where the reaction takes place and a pumping system that pres-
surizes the solvent and delivers to the extraction vessel. The next is a
reliable source of high-purity carbon dioxide. The temperature
control system and pressure control system maintain precise control
over solubility of target compounds [18].
Fig. 1 Flow design of supercritical fluid extraction technique

2.2 Subcritical Fluid Subcritical fluid extraction is an ecologically friendly and perfect
Extraction method that is used in selective extraction operations, in the treat-
ment of agro-food waste, and in industries to manufacture safe and
high-quality products [19]. According to Cravotto et al. (2022)
subcritical, near-critical, or pressurized hot water are all terms used
to describe this technique, in which water is subjected to adequate
pressures (generally 10 to 100 bar) between 100 °C and a critical
temperature of 374 °C (often between 100 and 250 °C)
[20]. Researchers have documented the utilization of supercritical
fluid and their liquid counterparts as appropriate solvents. This
approch not only yields the highest amount of value added nutra-
ceuticals but also represent a sustainable and environmentally
friendly processing method. It serves as a substitution for hazard-
ous organic solvents with more ecologically sound alterna-
tive [21]. In contrast to “supercritical” fluids, “subcritical” fluids,
also known as pressurized liquids like water and ethanol above their
boiling points but below critical temperatures, show good solvency
properties for the extraction of a variety of biological moieties
present in matrices made from agricultural sources. Some of the
drawbacks of supercritical fluids, such as poor affinity of carbon
dioxide for polar solutes and high capitalization costs of the pro-
cess, are alleviated by the use of subcritical fluids. Subcritical water
has been used to extract essential oils with effectiveness [22]. The
use of water being ecologically friendly, the ability of extracting
more rapidly with less amount of solvents, and the ability to pro-
duce higher yields make subcritical water superior to standard
organic solvents (Fig. 2) [19]. Solubility and mass transfer are the
two most important variables in subcritical solvent extraction [23].
High Selectivity
Advantages of
Low viscosity Subcritical fluid High Diffusivity
High Solvent Power
Fig. 2 Different major advantages of subcritical extraction technique

With values that fall between methanol and ethanol, the dielec-
tric constant of water decreases from 80 to about 30 due to the
weakening of hydrogen bonds, which makes the efficient extraction
of moderately polar and nonpolar target compounds possible.
Some advantages of subcritical fluids are summarized in Fig. 2 [20].
2.3 Working Strong hydrogen bonds of water gives it a unique characteristics.

Principle and The characteristics of water are affected by variations in tempera-
Mechanism ture and pressure [24]. Under the required pressure, water will boil
and reach its critical temperature at 100 and 374 °C, respectively.
Hot water that is sufficiently under pressure to maintain liquid
condition at this critical temperature is referred to as subcritical
water [25]. Subcritical water extraction (SWE) has an amazing
ability to change dielectric constants across a broad range by vary-
ing the temperature and pressure. SWE also results in mass transfer
by convection and diffusion. The energy delivered by subcritical
water can disrupt the relationship between cohesive and dispersive
forces by reducing the temperature. While subcritical water can
disrupt cohesive (solute-solute) and adhesive (solute-matrix) inter-
actions by lowering the activation energy required for the desorp-
tion process, elevated pressure can aid in extraction by forcing water
to enter the matrix (pores), which is impossible under normal
pressure [25]. In general, the SWE extraction technique comprises
four steps that follow one another. In the first stage, the solute is
first extracted from the surface at various active sites in the sample
matrix under highly increased conditions of temperature and pres-
sure. The main goal of the second phase is to spread the extracts
throughout the matrix. The sample matrix dictates the third step,
and solutes may be partitioned into the extraction fluid from the
sample matrix. The sample solution is then eluted and extracted
from the extractor using chromatography. Table 2 shows the
extraction of nutraceuticals by using subcritical fluid extraction in
different conditions. The extraction mechanism was modeled ther-
modynamically by Holgate and Tester in 1994. In this paradigm,
chemicals are extracted in two steps from their matrix. Similar to
front-elution chromatography, the compounds must first be des-
orbed from their original binding site in the sample matrix before
being ejected from the sample. Enhancing surface equilibrium
damage and solubility and mass transfer effects might increase the
effectiveness of subcritical water extraction [25, 26].
2.4 Modes of This extraction method has two primary modes: static extraction
Extraction and dynamic extraction. In static extraction, to retain water in a
liquid form, subcritical water is combined with the sample to be
extracted in an extraction vessel and heated to an appropriate
temperature under light pressure. Once extraction is completed,
the extractant is collected for chromatographic examination. This
extraction method is comparable to rapid solvent extraction and its
efficiency typically falls short of dynamic extraction’s efficiency.
Table 2
Extraction of different compounds by using subcritical fluid extraction technique
Food plants Compositions Conditions References

Aquilaria Essential oils 100–175 °C, 1–3 mL/min, 2–9 Mpa, 30 min [28]
malaccensis
Camellia oleifera Free fatty 60–160 °C, 2–7 Mpa, 5–60 min, 1:3–1:25 g/mL [29]
acids
Terminalia chebula Polyphenols 120–220 °C, 2–4 mL/min, 4 Mpa [30]
Piper betle Essential oil 50–250 °C, 2 Mpa, 10–90 min, 0.25–1 mm, [31]
1–4 mL/min
Nelumbo nucifera Polyphenols 100–180 °C, 5–25 min, 1:20–1:60 g/mL, 1–5‰ [32]
NaHSO3
Citrus hystrix Essential oil 120–180 °C, 5–20 g/mL, 5–30 min [33]
Camellia sinensis Flavonoid 40–160 °C, 5 min, 5 Mpa, 1–10 mm, pH 3.0–7.0 [34]
Fig. 3 Flow design of subcritical extraction technique
Following the placement of the samples in the extraction vessel, a

pump continually pumps water into the extractor while the extrac-
tion is carried out at a fixed or gradient temperature. Constant flow
or pressure can both be set for the pump. In addition to accelerat-
ing mass transfer and reducing extraction time, dynamic SWE
allows for customizable continuous extraction. The SBWE system
might get blocked as a result of dynamic SBWE, on the other
hand [27].
Figure 3 shows the subcritical fluid extraction – its extraction
vessel is similar to that of supercritical fluid extraction; pump is used
to deliver solvent; and pressure ranges from 2 to 20 psi. Tempera-
ture and pressure control system maintain the temperature and
pressure below the critical point providing the flexiblity in choosing
the solvents [26].
3 Pressurized Liquid Extraction (PLE)
It is considered one of the advanced extraction technologies over

the conventional methods [35]. This technique was first intro-
duced in 1995 by Dionex Corporation at Picton conference and
described it as an accelerated solvent extraction [36]. In this
method, liquid solvents are used at increased temperature and
pressure [35] and below their critical point. High pressure main-
tains the solvents in their liquid state, which increases the output of
the process as compared to the atmospheric temperature [36], also
known as “accelerated solvent extraction [37], pressurized fluid
extraction (PFE), pressurized liquid extraction (PLE), pressurized
hot solvent extraction (PHSE), high-pressure solvent extraction
(HPSE), high-pressure, high-temperature solvent extraction
(HPHTSE), and sub critical solvent extraction” [5, 38]. The
increase in condition correspond to changes in mass transfer and
solubilities compared to normal condition [36] due to increase in
the dielectric constant (used to evaluate the interaction between
solute and solvent) of the water in the extraction method [38].
Water is readily nontoxic, recyclable food-grade solvent and is
easily available. This method is rapid as compared to the other
conventional methods [39] and yields is pure extracts [40]. Due
to high pressure, it reduces the effect of light- and air-induced
degradation [39], and organic or aqueous solvents either alone or
in combination are used by static or dynamic mode. In the dynamic
mode, the flow of solvent through the sample is continuous, and in
static method, the flow is controlled for a specified time at constant
temperature and pressure [38]. Because of the need for rapid, less
hazardous, and more environmentally friendly extraction technol-
ogies, PLE has gained popularity, particularly in the pharmaceutical
and food sectors. PLE applications for the extraction of pollutants
from various foods have been developed [35].
3.1 Working Handling liquid sample requires their conversion into the solid due
Principle to the absence of commercial kits, achieved by adding adsorbent or
adsorbents. The efficiency of the system is influenced by thermody-
namic and kinetic parameters, with three interconnected processes
playing a crucial role: mass transfer, matrix effect and
solubility [35].
3.2 Factors Factors such as the duration of extraction, temperature, solvent

utilized, and pressure all affect the performance of PLE process.
However, the nature of the matrix, the unique characteristics of the
target molecules, and their positioning within the matrix all affect
the effectiveness of the process. Therefore, it is essential to compre-
hend and establish the influence of these factors on the extraction
process in order to obtain high yields and highly pure extracts
Fig. 4 Flow designs of pressurized liquid extraction (a dynamic; b static)
[41]. This process is constrained by the amount of time, flow rate,

temperature, and pressure management [35]. Hydromatrix is used
to adsorb water from the matrix since moisture in the sample matrix
lowers extraction efficiency, which ultimately increases the
extraction.
3.3 Instrumentation The instrumentation depends upon whether it is dynamic or static

mode. The basic instruments of the pressurized liquid extraction
are reservoirs containing solvent, purging gas source, valves to
restrict the flow, pump, oven in which the extraction coil is placed,
and a collecting vial.
Figure 4 shows the flow design of PLE. Extraction cell is where
the sample and solvent are combined, which is made up of stainless
steel or other materials that can withstand high pressure and tem-
perature. Pressure control system maintains pressure of several
thousand psi and temperature of up to 200 °C or higher. The
solvent delivery system typically includes pumps or syringe driver
that delivers the solvent at a controlled flow rate. The sample center
design may vary and the choice depends upon the nature of
sample [35].
The high-pressure pump is then linked to the solvent reservoir.
After the operation is finished, the pump helps with the extraction
by introducing the solvent into the system. Extraction takes place in
cell, where sample is added to the stainless cell first, then a filter
paper is added, and if necessary, a dispersion agent as well. The cell
is then mechanically or manually put inside the oven. Various valves
and restrictors are needed to control the extraction pressure.
Finally, the endpoint of the extraction system is connected to the
collecting vial. The instrumentation may be more or less sophisti-
cated depending on the process requirements. For example, a
solvent controller may be necessary when there are several solvent
Table 3
Extraction of different compounds via pressurized fluid extraction
Food plant Compounds recovered Extraction condition References

Vaccinium Phenolic compounds Ethanol, 10.3 MPa, 50 °C, 45 min [39]
vitisidaea L.
Vaccinium sect. Anthocyanins, Acetone/water/acetic acid (70:29.5: [42]
Cyanococcus Rydb. polyphenols, 0.5), 20 °C, 500 MPa
flavonoids
Capsicum annum L. Oil n-hexane, 370 MPa, 50 °C, 5.7 min [43]
Solanum lycopersicum Polyphenols Methanol 50%, hydrochloric acid, 45 ° [40]
C, 5 min, 600 MPa
Allium sativum Melanoidins Water, 300 MPa, 25 °C, 5 min [40]
Satureja montana Bioactive phenolic Ethanol (35%), 348 MPa, 20 min, [44]
compounds 20–25 °C
Ficus carica Bioactive phenolic Water, ethanol 600 MPa, 18–19 min [45]
compounds
reservoirs accessible, or an inert gas (often nitrogen) circuit may

help flush out solvent from the lines after extraction. Additionally,
the collecting vial can be placed in a cooling bath to reduce the
temperature of the extracting matter and decrease thermal damage.
Dynamic PLE also involves solvent preheating coils, a pressure
restrictor (back pressure regulator), or a micro metering valve
rather than a static open/close valve as in static PLE [35]. Addition-
ally, it requires a little more complex high-pressure pump to control
the solvent flow rate. The static extraction process depends heavily
on the temperature and extraction duration. The solubility of the
analyte in the extraction solvent and the distribution of the target
component between water and the extraction solvent both affect
the extraction efficiency. Due to the restricted supply of the extrac-
tion fluid, full extraction may not be feasible in the static extraction
mode [36]. In Table 3, extraction of different nutraceuticals by
pressurized liquid extraction is elaborated, in which extraction
conditions vary in each of extraction [1].
4 Ultrasound-Assisted Extraction
The range of an ultrasound frequency falls between 20 kHz and

100 MHz. Cavitation is an extraction method aided by ultrasound
that involves the development, growth, and deflation of bubbles
[46]. Ultrasound-assisted extraction (UAE) is a rapid and “easy-to-
use” technique that can quickly and consistently extract numerous
classes of food components from a variety of plant and food
matrices [47]. This “green” form of processing also conserves

water and energy, enables by-product recycling through
bio-refining, and yields a product that is both safe and of high
quality [48]. Yet, process variables, e.g., time, temperature, ultra-
sonic power, and solvent, affect how effectively these processes
work. To determine the ideal extraction conditions that produce
the best results in terms of target chemical recovery, proper experi-
mental designs and optimization approaches are necessary
[49]. Additionally, since mechanical stirrers cannot be utilized in
supercritical fluid extraction, this method offers a novel approach to
provide agitation [50].
4.1 Working The primary technique employed in sonication is acoustic turbu-

Principle lence. The molecules of the medium undergo a series of contraction
and coalescence as ultrasound passes through them. In a liquid
medium, such alternating pressure changes lead to the production
and eventual rupture of bubbles. Acoustic cavitation is a phenome-
non that occurs when micro bubbles in ultrasonic liquids develop,
grow, and then implosively collapse [51].
Figure 5 shows the ultrasound-assisted extraction. Ultrasonic
transducer is the key component that generates high frequency
sound waves and moves through the extraction solvent and sample;
the power control system controls the intensity of the waves applied
to the sample; the sample holder holds the sample; and the temper-
ature and pressure control maintain the temporary size, tempera-
ture and pressure [4].
The primary technique employed in ultrasound-assisted extrac-
tion is acoustic cavitation. The collapsing cavitation bubbles and
sound waves may be responsible for the fragmentation, localized
erosion, pore formation, shear force, enhanced absorption, and
swelling index in the plant’s cellular matrix. Shockwaves are formed
by cavitation bubbles that rupture, and cellular structure is dis-
turbed by fast particle collisions. Due to rapid fragmentation, the
Fig. 5 Operational design of ultrasound-assisted extraction

bioactive component dissolves in the solvent due to an increase in

surface area, a decrease in particle size, and rapid mass transfer rates
in the solid matrix’s border layer. Erosion occurs in plant tissues as a
result of the localized damage caused by ultrasonography. The
implosion of cavitation bubbles on the tissues of plants may be
the source of this damage. More solvent comes into touch with
the eroded region, thereby increasing extraction yield. Bioactive
substances that are already present in the cell are released as a result
of sonoporation, a phenomenon brought on by the creation of
holes in the cellular membrane during cavitation. Additionally,
the formation and dissolution of cavitation bubbles cause turbu-
lence and shear stress in the fluid, which help break down cell walls
and release the bioactive material. Ultrasound improves the
pomace’s capacity to absorb water, as well as the bioactive com-
pounds’ diffusivity and accessibility to the solvent being employed
for separation. Additionally, ultrasound boosts the extraction rate
and elevates the swelling index of the plant tissue matrix, which
encourages solute desorption and diffusion. The greater extraction
yield in the UAE is the result of a combination of variables rather
than one method [52]. Direct application is accomplished by
immersing ultrasonic probes in the sample and executing direct
ultrasonication over the solution with no additional barrier than
the solution itself. Indirect application is often carried out using an
ultra-sonication bath, in which the ultrasonic wave must first cross
the liquid within the ultrasonic instrument and then the sample
container’s wall. Different examples of nutraceuticals extracted by
UAE are shown in Table 4 [4].
Table 4
Extraction of different compounds via ultrasound-assisted extraction
Food plants Compounds Extraction condition References

Beta vulgaris Betanin and phenolic B-cyclodextrin (5%) solution, ultrasonic [53]
compounds bath, 28 kHz, 80 W, 30 min
Hibiscus Anthocyanins natural food Ethanol (39.1%), 26.1 min, 296.6 W, [49]
sabdariffa colorant 20 kHz, 30–35 °C, probe model
calyx
Rubia Natural food colorant, Ethanol (30%), 55 °C, 400 W, 20 min, [54]
sylvatica anthocyanins, and phenolics bath model
Curcuma Curcuminoids Deep eutectic solvent choline chloride: [55]
longa L. lactic acid, 1:1
Pisum Protein isolates Water at pH 9.6, 750 W, 13.5 min, 33.7% [50]
sativum amplitude, probe model
4.2 Factors Affecting Factors that impact extraction efficiency alone or in combination
the Extraction were investigated. They are as follows:
I. The type of tissue being removed and where in respect to
tissue structures the components are to be removed.
II. The tissue is prepped before being extracted.
III. The characteristics of the components being extracted.
IV. Ultrasonic impacts, which primarily cause superficial tissue
disruption increasing surface mass transfer, enhancing intra-
particle diffusion, loading substrate into the extraction
chamber; improved extractable component yield.
V. Higher extraction rates, particularly early in the extraction
cycle, result in considerable time savings and higher proces-
sing throughput [4].
5 Microwave-Assisted Extraction (MAE)
The low-impact approach known as microwave-assisted extraction

(MAE) employs microwave wavelengths between 1 mm and 1 m
and frequencies between 0.3 GHz (1 m) and 300 GHz (1 mm).
The sample can be penetrated by microwaves, which can then
interact with polar components [56]. According to Silva et al.
(2021) and Alara, Abdurahman, Ali, and Zain (2021), MAE is a
straightforward, rapid, and inexpensive method with certain key
benefits including quick heating, low solvent requirements, and
clean operations [57, 58]. A target chemical can be extracted
from a number of source materials using the MAE process. Due
to its natural ability to swiftly heat the sample-solvent mixture,
MAE is a user-friendly technique that shortens extraction times
and speeds up the extraction process. MAE provides a number of
benefits over conventional techniques, one of which is a drastically
shortened extraction time [59]. According to T. Wu et al. (2012),
MAE heating is based on the direct interaction of microwaves with
molecules through ionic conduction and dipole rotation
[60]. Both are in charge of the simultaneous heating of several
items. Ionic conduction [4] is the electrophoretic movement of
ions caused by a fluctuating electric field. At less aggressive extrac-
tion conditions, an electromagnetic field is applied directly to the
sample, increasing cell breakdowns and the release of chemicals into
the solvent while minimizing harm to sensitive components
[61]. Microwave heating only works on liquids or dielectric materi-
als with persistent dipoles. Different solvents heat up in microwaves
in a manner that is strongly influenced by the dissipation factor, a
measurement of the solvent’s capacity to absorb microwave energy
and transmit it on to the surrounding molecules as heat [4].
5.1 Types of MAE MAE vessels are classified into two types, closed and open. Both
systems have four common components, which are listed below: a
microwave generator that works by using magnetron to generate
microwave radiation; a waveguide through which microwave
spreads to microwave cavity; a circulator that allows the microwave
to change ahead; and an applicator to perform the test [62].
5.2 Instrumentation Mono- or multimode microwave oven cavities are created. The
and Mechanism of monomode cavity, which stimulates just one mode of resonance,
Microwave Extraction can produce a frequency. The sample may be positioned at the
electrical field’s maximum since it is known how the field is
distributed. The size of the multimode cavity raises the possibility
that the incident wave will affect several modes of resonance. The
field can be homogenized by the superimposition of modes. Sys-
tems such as rotating plates are utilized for homogenization
[63]. Modern techniques like MAE warm the solvent with micro-
wave energy and release the plant extractable compositions into an
aqueous phase. Although mass transfer for convention and MAE
takes place in the same direction, they are different because energy
is lost volumetrically during the MAE process inside the illumina-
tion medium. The power of the microwave and the material’s
dielectric loss factor both affect how quickly temperature rises
during microwave heating. Veggi et al. (2013) state that numerous
interactions must occur during the solid-solvent extraction phase of
the MAE process [64]:
• The solid matrix’s contact with the solvent
• Component breakdown or solubilization
• Moving the solute away from the solid matrix
• The solute’s transition from the solid’s surface to the bulk
solution after being extracted
• The extract’s motion in relation to the solid
• Disposal of the extract and solids after separation
As a result, the solvent effectively diffuses into the solid matrix
and the solute disintegrates up until it reaches a concentration that
is constrained by the solid’s physical features [65]. Stability, trans-
formation, and dissemination are the three steps that help compen-
sate the extraction process. Solubilization and partition control the
way the substrate is taken from the outer surface of the subatomic
particle during the equilibrium phase. There are transitional stages
before dissemination. At the solid-liquid interface, mass transfer
encounters resistance; at this point, convection and diffusion are
the dominant modes of mass transfer. The solute must get past the
interactions holding it to the matrix in the final stage in order to
enter the extraction solvent [64, 66]. The closed MAE system is
widely employed for extraction under harsh conditions, such as
high extraction temperatures. Today, the operator has a variety of

options for controlling the extraction process with safe-to-use
MAE equipment intended for laboratory usage. A magnetron
tube, an oven with the extraction vessels set on a turntable, moni-
toring devices for temperature and pressure management, and a
number of electrical power components are used in commercial
systems for closed-vessel MAE. The extraction vessel is filled with
the sample first, then the solvent is added, and lastly the extraction
vessel is sealed. A pre-extraction procedure is started after heating
the solvent using microwave radiation. Heating typically lasts
around 2 min. After that, the sample is subjected to radiation and
extracted for a predetermined period of time often between 10 min
30 min (static extraction stage). After the extraction is finished, the
samples are allowed to cool to a temperature that can be controlled.
Before analysis, there could have been a need for an internal stan-
dard and/or a clean step. Because it can achieve a higher heat
without destroying the volatile component and the fumes remain
in the vessel, less solvent is required [67, 68].
5.2.1 Focused A Soxhlet apparatus with variable heating power was used to per-
Microwave-Assisted form FMAE at 2450 MHz and atmospheric pressure. A quartz
Extraction System (FMAE) extraction tank containing solvent is filled with powdered and
air-dried sample matrix. Once the container had reached room
temperature, the extracts were centrifuged, and the supernatant is
collected and dried by vacuum evaporation. This can manage a
large amount of sample material and an additional reagent may be
added at any time during the process of extraction [67, 68]. Table 5
shows the extraction of various nutraceuticals by MAE at different
conditions. Figure 6 shows the microwave-assisted extraction’s
instruments; the power control system and frequency control sys-
tem adjust the intensity of microwave energy applied to the sample.
The sample holder material should be transparent and distant to
thermal and chemical degradation and should be transparent [67].
5.2.2 Dynamic MAE A dynamic MAE system developed by Ericsson and Colmsjo in
2000 generated extract with yields comparable to those of Soxhlet
extraction but in a lot less time. The dynamic microwave extractor
comprises the solvent supply system, microwave oven, extraction
cell, temperature set point controller with type K thermocouple,
fluorescence detector, and fused-silica restrictor [67].
6 Hydrotropic Extraction
There are many techniques for extracting nutraceuticals from

plants. However, the utilization of hydrotropic extraction for
obtaining natural products for medicinal use is currently becoming
popular due to its potential applications. Both the solubility and
Table 5
Extraction of different compounds via microwave-assisted extraction
Food plants Compounds Extraction conditions References

Humulus Polyphenols Ethanol, 75 °C, 400 W, 1 min [69]
lupulus L.
Arthrospira Natural pigments, Protic ionic liquids (hydroxyethyl ammonium [70]
platensis phycobiliproteins acetate and 2-hydroxyethylammonium
Gomont formate), 62 W, pH 7.0, 2.0 min
Daucus Carotenoids Flaxseed oil, 165 W, 9.39 min [71]
carota L.
Ficus racemose Phenolic Water 3.5 pH, 360.55 W, 30 s [72]
components
Garcinia Antioxidant-rich Ethanol (71%), 300 W, 2.24 min [73]
mangostana xanthone
Plukenetia Phenolic Ethanol (63.0%), 1500 W, 110 s [74]
volubilis compounds
Origanum Essential oil Water, 600 W, 20 min [75]
vulgare
Fig. 6 Flow designs of focused (a) and dynamic (b) microwave-assisted extraction
surface permeability of the solvent are important factors in the

extraction of phytoconstituents. Because of solubility consider-
ation, many phytoconstituents are often not extracted in the stan-
dard extraction process. Hydrotropic extraction is used for the
recovery of naturally occurring secondary metabolites [76]. In sim-
ple terms, hydrotropic extraction involves chemical extraction
through the solubilization of plant matrix with hydrotropic agent.
The term hydrotropy is the solubilization of organic compounds in
water by using hydrotropes or hydrotropic agents. The hydrotropes
are basically amphiphilic molecules which increase the solubility of

organic molecules in aqueous solutions by meditating the interac-
tions between hydrophilic and hydrophobic molecules. Hydro-
tropes can take on a variety of shapes, but their two most
prevalent molecular features are an ionic moiety and a saturated
hydrocarbon ring [77]. It is a molecular phenomenon caused by
poorly soluble solutes becoming much more miscible in an aqueous
solution when a hydrotropic agent is added. This extraction process
is very feasible, and it follows the principles of “green extractions”
being nontoxic and reusable.
6.1 Hydrotropic The term “hydrotropy” was first utilized by Carl A. Neuberg in
Agents 1916 [79]. He used the term “hydrotropic agents” for anionic
organic salts, which increase the miscibility of less-soluble solutes
in aqueous solutions at high concentrations. These compounds are
amphiphilic molecules with a short or branched alkyl chains
attached to ionic or polar groups for hydrophilicity while sulfate,
sulfonate and carbonate group confer hydrophobic characteristics
[78]. Most commonly used hydrophytes are xylene, polyhydroxy
benzene, toluene sodium salts of lower alkanols, aromatic acid
derivatives, sodium alkyl benzene sulfonates, etc. Some neutral or
cationic aromatic derivatives are also used as hydrotropic agents,
but these are rarely available [80]. As hydrophytes have both
hydrophobic and hydrophilic moieties, they are compared to sur-
factants, but the presence of short chain hydrophobic moiety dis-
tinguishes them from surfactants. Therefore, they are sometimes
called short chain surfactants or salting agents. Some hydrotropic
agents are used for selective extraction of nonpolar phytofragments,
which are water insoluble by cell permeabilization [81]. The struc-
tures of some hydrotropes like sodium toluene sulfonate, sodium
xylene sulfonate (SXS), sodium benzene sulfonate, sodium cymene
sulfonate, and sodium cumene sulfonate are shown in Fig. 7.
6.2 General Many theories have been proposed for describing the mechanism of
Mechanism of Action action of hydrotropic agents. The extensive study led to various
perspectives, and their finding were summarized in three
approaches regarding the mechanism.
1. In the first approach, hydrophyte and solutes forcefully interact
to form hydrophyte-solute complexes [80]. The phospholipid
bilayer of plant cell wall is destructed by hydrophytes, after
which the hydrophytes penetrate the inner structure of the
plant cell. When immersed in aqueous medium, the effect of
hydrophytes on cork cells is very minimal. The cork cell wall
contains cellulose and suberine lamellas. Suberine lamellas
make cork cells impermeable to water. Hydrotropic solution
opens these water-impermeable suberine lamellae and then
breaks down the mature cork cells. The cork cell layer is dis-
turbed by hydrotropy, and the aqueous solution penetrates the
Fig. 7 Structures of different hydrotropes
cell wall. When the hydrotropes solution enters the cell cyto-
plasm, the cells swell, releasing them from closely coupled
structures and hydrotropic solution cause precipitation of sol-
ute.. Therefore, the instantaneous recovery of dissolved solutes
from solutions diluted with aqueous solution is viable [82].
2. In the second mode of action, hydrophytes are considered as
“structure breakers and structure makers” as they alter the
solute structure by inserting themselves into water
structure [83].
3. The most widely accepted mechanism for hydrotropic extrac-
tions is the ability of hydrophytes to act as micelle structures at
a particular concentration. When diluted with distilled water,
the hydrotropic solutions cause the bioactive chemicals to pre-
cipitate out of the solution, making it possible to obtain the
extracted solute with ease [84].
6.3 Advantage and Advantages of hydrotropic extraction are its nontoxicity and easy
Disadvantages accessibility. Hydrotropic solvents have some characteristics that
would appeal to modern researchers’ tastes. Hydrotropic solvents
have made a noteworthy mark as a smart choice for phytochemical
extraction, thanks to their toxic-free, chemically inert, affordable,
accessible, temperature- and pH-independent, and high selectivity
properties. In addition to having a high extraction efficiency, pure-
ness, and quality of the crystalline product, this environmentally
friendly solvent can be recycled numerous times with a comparable
extraction efficiency [78]. Some disadvantages regarding this
technique are its poor recycling ability. Water dilution during recov-
ery and reuse creates a large amount of aqueous hydrotropic solu-
tion that needs to be reconcentrated for recycling
[85]. Additionally, the majority of the investigations concluded
that high hydrotropes concentrations are necessary to accomplish
high phytochemical solubilization, which necessitates the con-
sumption of a sizable quantity of hydrotropes salts in order to
create highly concentrated hydrotropic solvents. Most importantly,
there is uncertainty over how efficiently a hydrotropic agent will
interact with the intended solute.
6.4 Extractions Some food materials extracted by using hydrotropic extraction

process, which are used in pharmaceuticals, are described in the
following sections.
1. Vanillin from Vanilla
Vanillin (C8H8O3) from vanilla beans is extracted hydrochemi-
cally using hydrotropic solvents. One of the best-known flavor
components in vanilla beans is the phenol vanillin (4-hydroxy-3-
methoxybenzaldehyde) [86]. By employing organic solvents such
as ethanol, methanol, acetonitrile, acetone, chloroform, and hex-
ane, vanillin is traditionally extracted from vanilla beans [87]. Since
vanilla is widely used in the food, beverage, cosmetics, and pharma-
ceutical industries, it is crucial to determine the amount of vanillin
present in vanilla extract [88]. Nicotinamide, a non aromatic
hydrotropic compound frequently used in pharmaceutical industry,
follow other hydrotropes such as resorcinol and citric acid in terms
of its ability to enhance vanillin solubility in water [77, 89]. Vanillin
exhibits increased solubilty up to 2.6mol/L of nicotinamide, repre-
senting the maximum hydrotropes concentration. The primary
mechanism based on which nicotinamide works is stacking aggre-
gation, which involves the formation of complex molecules with
the solute by binding to its hydrophobic area. But according to a
different theory, nicotinamide causes solubility by dismantling
water’s self-associated structure, which is known to function as a
chaotropic [83].
2. Piperine from Black Pepper
Piperine is selectively extracted from Piper nigrum (Black Pep-
pers) by cell permeable mobility using hydrotropes like sodium
alkylbenzene sulfonates and sodium butyl mono-glycol-sulfate.
Increased extraction rates of aqueous hydrotropes solutions were
suggested to be due to hydrotropic molecules entering the cellular
structures, thereby resulting in cellular permeable mobility. In
order to speed up the extraction of piperine, hydrotrope molecules
adhere to a cell wall and disrupt its structure as well as the bilayer
cell membrane. The recovered piperine is 90% pure and largely free
of oleoresins [90, 91].
3. Limonene from Citrus

Bioactive limonenes are extracted from Citrus aurantium [78]
seeds hydrotopically. Potentially bioactive substances called limo-
noids are found only in citrus fruits and vegetables. Research works
have focused on utilizing aqueous hydrotropic solutions, a novel
method, for removing limonoid aglycones from the seeds of sour
orange (Citrus auratium L.). Hydrotropic concentration, extrac-
tion temperature, and percentage of input material loaded all had
an impact on extraction efficiency. The Box-Behnken experiment
design is used to study two hydrotropes, sodium salicylate (Na-Sal)
and sodium cumene sulfonate (Na-CuS). Data is subjected to
response surface analysis [63] to examine how parameters affected
the effectiveness of the extraction process. Limonene, a prominent
limonoid aglycone, was isolated and measured for process improve-
ment. Maximum limonene yield was produced by both hydro-
tropes at 2.0 M concentration, 45 °C extraction temperature, and
10% solid loading. Na-CuS produced a maximum limonene output
of 0.65 mg/g seeds, whereas Na-Sal produced only 0.46 mg/g
seeds. When bioactive substances are extracted with this method,
the amount of organic solvents used can be drastically reduced
while maintaining environmental friendliness.
4. Curcuminoids from Turmeric
Turmeric (Curcuma longa), which belongs to Zingiberaceae
family, has powerful anticancer properties and is well known for its
many medical advantages such as antibacterial, anti-inflammatory,
antiparasitic, and antifungal properties [92]. Hydrotropes have
been shown to directly impact cell structure, increasing the accessi-
bility of curcuminoids either by breaking the cell wall or by dissol-
ving components of the cell membrane or wall. The most effective
hydrotrope for extracting curcuminoids is NaCS [82]. Due to
presence of both solubilized curcuminoids and hydrotropic in the
recovered precipitate, it exhibited a gradually decreasing concen-
tration of solubilized curcuminoids at concentration exceeding 1.0
mol/dm3, akin to piperine.
5. Citral from Lemongrass
The major element of lemongrass oil is citral (3,7-dimethyl-
2,6-octadienal), which is extracted from the leaves of Cymbopogon
flexuosus (Steud.) Wats. It possesses sedative, antidepressant, anti-
viral, antifungal, and antitumor properties and is commonly used as
a lemon flavoring and scenting ingredient. Maximum citral yields
were reported in a 0.25-mm piece of plant material at 1.75 M
hydrotropic concentration, 5% solid loading, and 30 °C using a
hydrotropy solubilization technique combining NaSal and NaCS
(sodium cumenesulfonate). Citral is extracted more efficiently by
using NaSal than by NaCS.
6. Electro-osmotic Dewatering
In recent era, there is a great interest in electro-regulated food-
processing techniques and their use in the extraction of nutraceu-
ticals. In electro-osmotic dewatering, the moisture of food is
removed at low electrical field and is controlled by the electrical
resistance present in the system to maintain specific conditions.
Electro-osmotic dewatering is basically a drying method and has
recently become quite popular in the nutraceutical business. To
extract moisture from plant materials and food, electro-osmotic
technique employs an electric field of approximately 5–30 volts.
This process is better than the traditional heat drying methods since
it can reduce carbon emissions by about 80%. Furthermore, energy
consumption is reduced up to two-thirds in this technique as
compared to traditional thermal processes. The basic principle
followed by this technique is osmotic dehydration, as it is a drying
process, and enhanced mass transfer. An electrochemical double
layer is formed on the interface of aqueous suspension due to the
applied mechanical pressure. This mechanical dewatering leads to
the drying of substrate [93]. Since electro-osmotic dehydration is
frequently used to concentrate fruit fragments and achieve
improved jam properties on difficult-to-dewater substrates, it
would be excellent to employ it to treat high-sugar fruits and
vegetable by-products that contain labile antioxidant and surface
colorants. Additionally, if the substance is elastic and well grained, it
is too delicate to treat and too viscous to pump, making water
removal more difficult. Prior to industrial deployment, this issue
of electro-osmotic dewatering should be overcome [94].
6.5 Design and Direct current (DC) is used to impart an external electrical field to a
Working Principle semisolid material sandwiched between two electrodes, resulting in
electrical dehydration (ED). When dewatering proceeds downhill
in a bed of semisolid material where the initial water content is
uniform across the bed, the water content in a section of the
material close to the top electrode opposing the drainage surface
decreases regionally. The higher electrode and the dewatered mate-
rial now have a greater electrical contact resistance. To effectively
apply electro-osmotic dewatering to various types of materials, it is
essential to increase the dewatering rate, decrease the final water
content, and utilize the least amount of energy feasible to remove
the water. Electrical dehydration (ED) differs from mechanical
dewatering in that it does not employ fluid pressure, compressive
forces, or centrifugal forces. The process of electro-osmotic dewa-
tering is elaborated in Fig. 8. It offers several advantages over
mechanical procedures, particularly in effectively dewatering colloi-
dal particles, gelatinous substances and solid-liquid mixture based
on biological components, which mechanical techniques may
struggle to adequately adress.
Fig. 8 Process of electro-osmotic dewatering [95]
Fig. 9 Systematic diagram of electro-osmotic process [97]
The application of an electric field causes physical and electro-

chemical reactions in addition to the movement of water molecules
that have a negative impact on the effectiveness of the electro-
osmotic dewatering (Fig. 9) [78, 96].
1. Development of crack as a result of negative pore water pres-
sure and severe drying at the anode
2. Severe variations of pH at both electrodes
3. Development of bubbles at the electrodes as a result of water
electrolysis, which reduces solute-electrode contact
Solvent
Composition
Extraction
Solvent to
time and
Feed Ratio
Cycle
Factors
Effecting
Microwave
Temperature
Power
Fig. 10 Factors effecting the extraction of nutraceuticals
6.6 Factors Some of the factors affecting electro-osmotic dewatering of sub-

strates are given below and also described in Fig. 10 [98]:
1. The amount of material that needs to be dewatered
2. The mixture of substrate and zeta potential of water
3. The pressure used (pressure and electro-osmosis combined
have been shown to be more cost-effective than electro-
osmosis alone)
4. The kind of membrane employed
After retting process, electro-osmosis proves to be a rapid
method for dewatering fibrous plant with high moisture level.
Another post-retting procedure that uses less energy than lengthy
hot air drying is electro-osmosis followed by microwave drying.
Electrochemical based method have been employed in various
sludge treatments, ranging from laboratory to pilot and full scale
experiment [99]. The advantages of electro-osmotic technique
have led to its commercialization since it concurrently achieves
sludge dewatering, odor control, and pathogen removal. Addi-
tional field study and research in the extraction processes would
provide technical input on how to lower startup and operating costs
and set up minimal processing.
References
1. Picot-Allain C et al (2021) Conventional ver- 15. Santos OV et al (2021) CO2 supercritical fluid
sus green extraction techniques – a comparative extraction of pulp and nut oils from Terminalia
perspective. Curr Opin Food Sci 40:144–156 catappa fruits: thermogravimetric behavior,
2. Lefebvre T, Destandau E, Lesellier E (2021) spectroscopic and fatty acid profiles. Food Res
Sequential extraction of carnosic acid, rosmari- Int 139:109814
nic acid and pigments (carotenoids and chlor- 16. Priyanka, Khanam S (2020) Selection of suit-
ophylls) from Rosemary by online supercritical able model for the supercritical fluid extraction
fluid extraction-supercritical fluid chromatog- of carrot seed oil: a parametric study. LWT
raphy. J Chromatogr A 1639:461709 119:108815
3. Soni GD (2015) Advantages of green technol- 17. de Melo MMR, Silvestre AJD, Silva CM
ogy. Soc Issues Environ Probl 3(9):1–5 (2014) Supercritical fluid extraction of vegeta-
4. Shams KA et al (2015) Green technology: eco- ble matrices: applications, trends and future
nomically and environmentally innovative perspectives of a convincing green technology.
methods for extraction of medicinal & aro- J Supercrit Fluids 92:115–176
matic plants (MAP) in Egypt. J Chem Pharm 18. Ishwarya SP, Nisha P (2021) Headway in
Res 7(5):1050–1074 supercritical extraction of fragrances and colors
5. Ahangari H et al (2021) Supercritical fluid 19. Kheirkhah H, Baroutian S, Quek SY (2019)
extraction of seed oils – a short review of cur- Evaluation of bioactive compounds extracted
rent trends. Trends Food Sci Technol 111: from Hayward kiwifruit pomace by subcritical
249–260 water extraction. Food Bioprod Process 115:
6. Sharif KM et al (2014) Experimental design of 143–153
supercritical fluid extraction – a review. J Food 20. Cravotto C et al (2022) Bioactive antioxidant
Eng 124:105–116 compounds from chestnut peels through semi-
7. da Silva RPFF, Rocha-Santos TAP, Duarte AC industrial subcritical water extraction. Antiox-
(2016) Supercritical fluid extraction of bioac- idants 11(5):988
tive compounds. TrAC Trends Anal Chem 76: 21. Martinez JL (2007) Supercritical fluid extrac-
40–51 tion of nutraceuticals and bioactive com-
8. Ahmad T et al (2019) Supercritical fluid extrac- pounds. CRC Press
tion: a review. J Biol Chem Chron 5(1): 22. Srinivas K et al (2009) Optimization of subcrit-
114–122 ical fluid extraction of bioactive compounds
9. Herrero M et al (2013) Compressed fluids for using Hansen solubility parameters. J Food
the extraction of bioactive compounds. TrAC Sci 74(6):E342–E354
Trends Anal Chem 43:67–83 23. Simeonov E, Tsibranska I, Minchev A (1999)
10. Soquetta MB, Terra LDM, Bastos CP (2018) Solid–liquid extraction from plants – experi-
Green technologies for the extraction of bioac- mental kinetics and modelling. Chem Eng J
tive compounds in fruits and vegetables. CyTA 73(3):255–259
J Food 16(1):400–412 24. Teo CC et al (2010) Pressurized hot water
11. Uwineza PA, Waśkiewicz A (2020) Recent extraction (PHWE). J Chromatogr A
advances in supercritical fluid extraction of nat- 1217(16):2484–2494
ural bioactive compounds from natural plant 25. Zhang J et al (2020) Recent advances in the
materials. Molecules 25. https://doi.org/10. extraction of bioactive compounds with sub-
3390/molecules25173847 critical water: a review. Trends Food Sci Tech-
12. Pinto D et al (2020) Valorisation of underex- nol 95:183–195
ploited Castanea sativa shells bioactive com- 26. Noor NQIM et al (2021) Application of green
pounds recovered by supercritical fluid technology in gelatin extraction: a review. PRO
extraction with CO2: a response surface meth- 9(12):2227
odology approach. J CO2 Util 40:101194 27. Cheng Y et al (2021) Subcritical water extrac-
13. Pavlić B et al (2020) Supercritical fluid extraction of natural products. Molecules 26(13):
tion of raspberry seed oil: experiments and 4004
modelling. J Supercrit Fluids 157:104687 28. Samadi M et al (2020) Towards higher oil yield
14. Varaee M et al (2019) Supercritical fluid extrac- and quality of essential oil extracted from Aqui-
tion of free amino acids from sugar beet and laria malaccensis wood via the subcritical tech-
sugar cane molasses. J Supercrit Fluids 144:48– nique. Molecules 25(17):3872
55
29. Liang S et al (2018) Gel polymer electrolytes 42. Briones-Labarca V, Giovagnoli-Vicuña C,

for lithium ion batteries: fabrication, character- Chacana-Ojeda M (2019) High pressure
ization and performance. Solid State Ionics extraction increases the antioxidant potential
318:2–18 and in vitro bio-accessibility of bioactive com-
30. Rangsriwong P et al (2009) Subcritical water pounds from discarded blueberries. CyTA J
extraction of polyphenolic compounds from Food 17(1):622–631
Terminalia chebula Retz. fruits. Sep Purif 43. Ma Y et al (2019) Comparison of the com-
Technol 66(1):51–56 pounds and characteristics of pepper seed oil
31. Musa TA et al (2014) Determination of 4-allyl by pressure-assisted, ultrasound-assisted and
resorcinol and chavibetol from Piper betle conventional solvent extraction. Innovative
leaves by subcritical water extraction combined Food Sci Emerg Technol 54:78–86
with high-performance liquid chromatogra- 44. Moreira SA et al (2020) Effect of high hydro-
phy. Food Anal Methods 7(4):893–901 static pressure extraction on biological activities
32. Yan Z et al (2019) Subcritical water extraction, and phenolics composition of winter Savory
identification and antiproliferation ability on leaf extracts. Antioxidants 9(9):841
HepG2 of polyphenols from lotus seed epicarp. 45. Alexandre EMC et al (2017) High-pressure
Ind Crop Prod 129:472–479 assisted extraction of bioactive compounds
33. Halim NA et al (2020) Screening of factors from industrial fermented fig by-product. J
influencing the yield of Citrus hystrix leaves Food Sci Technol 54(8):2519–2531
essential oil extracted via pressurized hot 46. Azmir J et al (2013) Techniques for extraction
water extraction based on resolution V frac- of bioactive compounds from plant materials: a
tional factorial design. J Food Process Eng review. J Food Eng 117(4):426–436
43(11):e13531 47. Turrini F et al (2019) Traditional or hydro-
34. Kim S-W, Ko M-J, Chung M-S (2019) Extrac- diffusion and gravity microwave coupled with
tion of the flavonol quercetin from onion waste ultrasound as green technologies for the valo-
by combined treatment with intense pulsed rization of pomegranate external peels. Food
light and subcritical water extraction. J Clean Bioprod Process 117:30–37
Prod 231:1192–1199 48. Chemat F et al (2017) Review of green food
35. Alvarez-Rivera G et al (2020) Chapter 13 – processing techniques. Preservation, transfor-
Pressurized liquid extraction. In: Poole CF mation, and extraction. Innovative Food Sci
(ed) Liquid-phase extraction. Elsevier, pp Emerg Technol 41:357–377
375–398 49. Pinela J et al (2019) Optimization of heat- and
36. Mustafa A, Turner C (2011) Pressurized liquid ultrasound-assisted extraction of anthocyanins
extraction as a green approach in food and from Hibiscus sabdariffa calyces for natural
herbal plants extraction: a review. Anal Chim food colorants. Food Chem 275:309–321
Acta 703(1):8–18 50. Perera CO, Alzahrani MAJ (2021) Ultrasound
37. Ghasemi M et al (2013) Copper- as a pre-treatment for extraction of bioactive
phthalocyanine and nickel nanoparticles as compounds and food safety: a review. LWT
novel cathode catalysts in microbial fuel cells. 142:111114
Int J Hydrog Energy 38(22):9533–9540 51. Tiwari BK (2015) Ultrasound: a clean, green
38. Carabias-Martı́nez R et al (2005) Pressurized extraction technology. TrAC Trends Anal
liquid extraction in the analysis of food and Chem 71:100–109
biological samples. J Chromatogr A 1089(1): 52. Kumar K, Srivastav S, Sharanagat VS (2021)
1–17 Ultrasound assisted extraction (UAE) of bioac-
39. Kitrytė V et al (2020) Zero waste biorefining of tive compounds from fruit and vegetable pro-
lingonberry (Vaccinium vitisidaea L.) pomace cessing by-products: a review. Ultrason
into functional ingredients by consecutive high Sonochem 70:105325
pressure and enzyme assisted extractions with 53. Tutunchi P et al (2019) Extraction of red beet
green solvents. Food Chem 322:126767 extract with β-cyclodextrin-enhanced ultra-
40. Grassino AN et al (2020) Insight into high- sound assisted extraction: a strategy for
hydrostatic pressure extraction of polyphenols enhancing the extraction efficacy of bioactive
from tomato peel waste. Plant Foods Hum compounds and their stability in food models.
Nutr 75(3):427–433 Food Chem 297:124994
41. Osorio-Tobón JF et al (2013) Recent applica- 54. Chen X-Q et al (2020) Ultrasound-assisted
tions of pressurized fluid extraction: curcumi- extraction of total anthocyanins from Rubia
noids extraction with pressurized liquids. Food sylvatica Nakai fruit and radical scavenging
Public Health 3(6):289–303
activity of the extract. Ind Crop Prod 150: Ingredients extraction by physicochemical
112420 methods in food. Academic, pp 523–560
55. Patil SS, Pathak A, Rathod VK (2021) Optimi- 67. Chaturvedi AK (2018) Extraction of neutra-
zation and kinetic study of ultrasound assisted ceuticals from plants by microwave assisted
deep eutectic solvent based extraction: a extraction. Syst Rev Pharm 9(1):31–35
greener route for extraction of curcuminoids 68. Mandal V, Mohan Y, Hemalatha S (2007)
from Curcuma longa. Ultrason Sonochem 70: Microwave assisted extraction – an innovative
105267 and promising extraction tool for medicinal
56. Pinto D et al (2021) Microwave-assisted plant research. Pharmacogn Rev 1(1):7–18
extraction as a green technology approach to 69. Carbone K et al (2020) Exploring the potential
recover polyphenols from Castanea sativa of microwaves and ultrasounds in the green
shells. ACS Food Sci Technol 1(2):229–241 extraction of bioactive compounds from
57. Silva AM et al (2021) Salicornia ramosissima Humulus lupulus for the food and pharmaceu-
bioactive composition and safety: eco-friendly tical industry. Ind Crop Prod 156:112888
extractions approach (microwave-assisted 70. Rodrigues RDP et al (2020) Application of
extraction vs. conventional maceration). Appl protic ionic liquids in the microwave-assisted
Sci 11(11):4744 extraction of phycobiliproteins from Arthros-
58. Alara OR et al (2021) Microwave-assisted pira platensis with antioxidant activity. Sep
extraction of phenolic compounds from Carica Purif Technol 252:117448
papaya leaves: an optimization study and LC- 71. Elik A, Yanık DK, Göğüş F (2020) Microwave-
QTOF-MS analysis. Fut Foods 3:100035 assisted extraction of carotenoids from carrot
59. Parthasarathi S et al (2013) A comparative juice processing waste using flaxseed oil as a
study on conventional and microwave-assisted solvent. LWT 123:109100
extraction for microencapsulation of Garcinia 72. Tatakuri M et al (2020) P-90 does the postop-
fruit extract. Food Bioprod Process 91(2): erative course of events influence 2-year mor-
103–110 tality in patients undergoing hyperthermic
60. Wu T et al (2012) Optimization of microwave- intraperitoneal chemotherapy? An evaluation
assisted extraction of phenolics from potato by a novel scoring system. Ann Oncol 31:S119
and its downstream waste using orthogonal 73. Mohammad NA et al (2019) Optimization of
array design. Food Chem 133(4):1292–1298 the antioxidant-rich xanthone extract from
61. Figueroa JG et al (2021) Functional ingredient mangosteen (Garcinia mangostana L.) pericarp
from avocado peel: microwave-assisted extrac- via microwave-assisted extraction. Heliyon
tion, characterization and potential applica- 5(10):e02571
tions for the food industry. Food Chem 352: 74. Sanchez-Reinoso Z et al (2020) Microwave-
129300 assisted extraction of phenolic compounds
62. Bagade SB, Patil M (2021) Recent advances in from Sacha Inchi shell: optimization, physico-
microwave assisted extraction of bioactive com- chemical properties and evaluation of their
pounds from complex herbal samples: a review. antioxidant activity. Chem Eng Process Process
Crit Rev Anal Chem 51(2):138–149 Intensif 153:107922
63. Routray W, Orsat V (2012) Microwave-assisted 75. Drinić Z et al (2020) Microwave-assisted extrac-
extraction of flavonoids: a review. Food Bio- tion of O. vulgare L. spp. hirtum essential oil:
process Technol 5(2):409–424 comparison with conventional hydro-
64. Veggi PC, Martinez J, Meireles MAA (2012) distillation. Food Bioprod Process 120:158–
Fundamentals of microwave extraction. In: 165
Microwave-assisted extraction for bioactive 76. Hartati I (2010) Hydrotropic extraction of
compounds. Springer, pp 15–52 theobromine from Cocoa bean shell. Majalah
65. Chemat F, Cravotto G (2012) Microwave- Ilmiah Momentum 6(2)
assisted extraction for bioactive compounds: 77. Hodgdon TK, Kaler EW (2007) Hydrotropic
theory and practice, vol 4. Springer solutions. Curr Opin Colloid Interface Sci
66. Muhamad II et al (2017) Chapter 14 – Extrac- 12(3):121–128
tion technologies and solvents of phytocom- 78. Nagarajan J et al (2016) Extraction of phyto-
pounds from plant materials: physicochemical chemicals using hydrotropic solvents. Sep Sci
characterization and identification of ingredi- Technol 51(7):1151–1165
ents and bioactive compounds from plant 79. Neuberg C (1916) Hydrotropy. Biochem Z
extract using various instrumentations. In: 76(1):107–108
Grumezescu AM, Holban AM (eds)
80. Vemula VR, Lagishetty V, Lingala S (2010) 91. Raman G, Gaikar VG (2002) Extraction of
Solubility enhancement techniques. Int J Phar- piperine from Piper nigrum (black pepper) by
maceut Sci Rev Res 5(1):41–51 hydrotropic solubilization. Ind Eng Chem Res
81. Mangal A et al (2012) Extraction of herbal 41(12):2966–2976
drugs by using hydrotropy solublization phe- 92. Kuttan R et al (1985) Potential anticancer
nomenon. Int Res J Pharmaceut Appl Sci 2(1): activity of turmeric (Curcuma longa). Cancer
63–74 Lett 29(2):197–202
82. Dandekar DV, Gaikar V (2003) Hydrotropic 93. Citeau M, Larue O, Vorobiev E (2011) Effect
extraction of curcuminoids from turmeric. Sep of electrolytes content on the electro-osmotic
Sci Technol 38(5):1185–1215 dewatering of agro-industrial sludge. In: Food
83. Kim JY et al (2010) Hydrotropic solubilization process engineering in a changing world. Pro-
of poorly water-soluble drugs. J Pharm Sci ceedings of the 11th International Congress on
99(9):3953–3965 Engineering and Food
84. Kumar VS, Raja C, Jayakumar C (2014) A 94. Galanakis CM (2013) Emerging technologies
review on solubility enhancement using hydro- for the production of nutraceuticals from agri-
tropic phenomena. Int J Pharm Pharm Sci cultural by-products: a viewpoint of opportu-
6(6):1–7 nities and challenges. Food Bioprod Process
85. Gaulkar SU, Gaikar VG (2004) Precipitation of 91(4):575–579
piperine from hydrotropic solutions: study of 95. Mujumdar AS, Yoshida H (2009) Electro-
crystal nucleation and growth kinetics from osmotic dewatering (EOD) of
batch experiments. Sep Sci Technol 39(14): bio-materials. In: Electrotechnologies for
3431–3452 extraction from food plants and biomaterials.
86. Gu F et al (2012) Optimization of enzymatic Springer, pp 121–154
process for vanillin extraction using response 96. Burnotte F, Lefebvre G, Grondin G (2004) A
surface methodology. Molecules 17(8): case record of electroosmotic consolidation of
8753–8761 soft clay with improved soil electrode contact.
87. Jadhav D et al (2009) Extraction of vanillin Can Geotech J 41(6):1038–1053
from vanilla pods: a comparison study of con- 97. Martin L, Alizadeh V, Meegoda J (2019)
ventional soxhlet and ultrasound assisted Electro-osmosis treatment techniques and
extraction. J Food Eng 93(4):421–426 their effect on dewatering of soils, sediments,
88. Sinha AK, Sharma UK, Sharma N (2008) A and sludge: a review. Soils Found 59(2):
comprehensive review on vanilla flavor: extrac- 407–418
tion, isolation and quantification of vanillin and 98. Nair GR, Rho D, Raghavan GV (2013) Appli-
others constituents. Int J Food Sci Nutr 59(4): cation of electro-technologies in processing of
299–326 flax fiber. Fibers 1(2):21–35
89. Masilamani D, Morais AB, Gandhi NN (2013) 99. Zeng Q et al (2022) Emerging
Extraction of vanillin through hydrotropy. electrochemistry-based process for sludge
Asian J Chem 25(1):231–236 treatment and resources recovery: a review.
90. Rathod SS, Rathod VK (2014) Extraction of Water Res 209:117939
piperine from Piper longum using ultrasound.
Ind Crop Prod 58:259–264
Chapter 17
Optimization of Nutraceuticals Extraction

Shanza Malik, Ayesha Jabeen, Farooq Anwar, Muhammad Adnan Ayub,
Muhammad Nadeem Zafar, and Muhammad Zubair
Abstract
Optimization is the selection of the most efficient system that can resolve and help in attaining the objective
functions by designing or operating various optimized procedures. In this chapter, optimized extraction of
nutraceuticals is discussed. With an increasing demand for nutraceuticals, there is now greater focus on
nutrapharmaceutical industry that produces safer natural products/extracts in a sustainable manner by cost-
effective and eco-friendly green extraction routes. So the strategies that need to be devised for this purpose
include the evaluation and optimization of various extraction variables (particle size, material/solvent ratio,
extraction time and cycle, type of extraction technique, extraction conditions like temperature, agitation
rate, etc.) that aim to control the stability of the bioactives and achieve the sustainable quality of the end-use
nutraceutical products. In this chapter, we have focused on the optimized extraction of nutraceuticals along
with the recent technological trends that are applicable toward the recovering of best possible levels of such
high-value components.
Key words Optimized extraction, High-value components, Bioactives stability, Green extraction,
Sustainable quality, Healthy products, Nutrapharmaceuticals
1 Introduction
Plants have always served human beings as a source of food and folk
medicine, as well as they offer a platform for isolation of lead
molecules for development of modern drugs. However, in consis-
tent with the notion “let your food be your medicine,” currently
there is a renewed interest on the use of plants as a source of food
and medicine. In line with the concept of optimal nutrition, cur-
rently, the science of functional food and nutraceuticals is an
emerging area in food science [1–3]. In fact, a huge number and
kind of plants, especially herbs, spices, food crops, and medicinal
species have been explored for isolation of a broad range of bioac-
tives and high-value phytochemicals with biological and nutraceu-
tical potential [2–5].
419
420 Shanza Malik et al.
Nutraceuticals are products isolated and/or made from various

food sources that offer additional health advantages beyond their
basic nutritional benefits. They might consist of dietary supple-
ments, nutritious foods, and drinks supplemented with bioactive
ingredients like probiotics, vitamins, minerals, antioxidants, and
herbal extracts. The ability of nutraceuticals to promote general
health and treat and/or prevent specific health disorders underlies
their significance.
In terms of developing and sustaining excellent health, nutra-
ceuticals can be extremely helpful. They offer vital nutrients and
bioactive substances that support numerous biological processes
and aid in preventing nutritional deficits. They aid in the improve-
ment of general health by providing particular beneficial
components [6].
A long list of nutraceuticals with therapeutic effects and
disease-preventing qualities have been developed that may help
lower the chance of chronic diseases. For instance, the antioxidants
in fruits and vegetables work to combat dangerous free radicals,
potentially lowering the prevalence of oxidative stress–related dis-
eases like cancer, aging, inflammation, heart disease, and neurolog-
ical disorders [7].
By providing nutrient support, particularly when specific needs
for nutrients cannot be satisfied through regular diet alone, nutra-
ceuticals can supplement or enhance standard dietary practices.
They fill in nutritional gaps by assuring proper intake of minerals,
vitamins, and other crucial nutrients. They are practical and effi-
cient. In line with the development of nutrigenomics, nutraceuti-
cals also offer the advantage of personalized nutrition by addressing
individual health needs and genetic variations. [8] As scientific
advancements continue, the development of tailored nutraceutical
products based on genetic profiling and biomarker analysis may
become more prevalent, providing customized health solutions.
While nutraceuticals hold promise, it’s essential to note that their
efficacy and safety can vary. It’s important to choose reputable
products to ensure optimal benefits and minimize potential risks
[8, 9].
There are various nutraceuticals that are being used as dietary
supplements (as shown in Fig. 1) and provide a significant amount
of nutrition along with their medicinal benefits. Nutraceuticals are
frequently utilized in medicine as a complimentary treatment to
traditional medicine. To boost treatment outcomes and enhance
patient well-being generally, they may be used in combination with
approved medications or therapies. For example, omega-3 fatty
acid supplements are regularly utilized as an additional therapy for
the control of cardiovascular disease [6, 10].
Nutraceuticals’ ability to encourage optimal aging and
lengthen lifespan is being recognized more and more in the aging
and longevity fields. Resveratrol, a substance found in red grapes,
Optimization of Nutraceuticals Extraction 421
Dietary
supplements
(co-enzyme Q ,
Carnitine)
Terpenoids
Functional
(carotenoids,
foods
modo-terpenes,
(probiotics,
Phyto-steroids)
pre-biotics) Nutraceuticals
(nutrition &
pharmaceuticals)
Polyphenols
(anthocyanins, Micronutrients
flavones, (gallic acid)
stilbenes,
isoflavones)
Micronutrients
(vitamins,
minerals)
Fig. 1 Application of nutraceuticals as dietary fibers [6]
has shown antiaging properties and is thought to activate genes

related to lifespan, possibly postponing age-related degenerative
processes. The process of getting bioactive substances from sources
that are natural, such as trees, fruits, herbal remedies, and marine
life, for use as food supplements or food products that function is
referred to as nutraceutical extraction. There are numerous recog-
nized techniques for extracting nutraceuticals, each with unique
risks and advantages [11].
In order to extract high-quality and bioactive chemicals from a
variety of natural sources, nutraceutical extraction methods must be
optimized. To improve the extraction effectiveness, production
yield, as well as preservation of the nutraceuticals, several
approaches have been developed. Solvent extraction is one of the
often-used technique that uses water, ethanol, or methanol to
extract bioactive chemicals. Utilizing cutting-edge green technol-
ogies like microwave-assisted extraction [12] and ultrasound-
assisted extraction (UAE), which make use of the physical and
thermal impacts to increase extraction efficiency, is another strategy.
Additionally, for the extraction of sensitive chemicals and its envi-
ronmental friendliness have made SFE (supercritical fluid extrac-
tion) employing carbon dioxide as the solvent popular.
Furthermore, enzyme-assisted extraction (EAE), a potential

method that uses enzymes to dissolve cell walls and release valuable
substances, has also come to light. To achieve highest extraction
efficiency, parameters such as solvent concentration, extraction
duration, temperature, or particle size need to be optimized. In
this regard, statistical techniques like response surface methodology
(RSM) and modeling based on artificial intelligence can help the
optimization process [13].
These are only a few methods utilized for the extraction of
nutraceuticals. The type of target chemicals, the attributes of the
source material, and the intended extract properties all influence
the selection of extraction process. For choosing the best extraction
technique, it’s important to consider parameters like efficiency,
selection of solvent and material, cost effectiveness, and the impact
on environment into account.
2 Importance of Optimizing the Extraction Process of Nutraceuticals
Nutraceutical extraction procedure needs to be optimized for a

number of reasons, including the potential to improve product
quality. The quality and chemical structure of nutraceutical goods
are directly impacted by the extraction process. Higher quantities of
bioactive substances can be obtained by reducing the presence of
undesirable components and by improving the extraction process.
As a result, the finished product has enhanced uniformity, purity,
and potency. The pace and degree at which a nutrient or bioactive
chemical is taken up and used by the human system is referred to as
improved bioavailability. Because of their complex structures and
poor solubility when dissolved in water or lipids, several nutraceu-
ticals can exhibit low bioavailability. By enhancing the release of
active chemicals and enhancing their solubility, optimization of the
extraction process can increase bioavailability and maximize the
absorption and bioactivity in the body [14].
The cost of producing a nutraceutical can be considerably
impacted by the extraction method. Optimization enables more
effective use of raw materials, lower energy use, and less waste
production. Nutraceutical producers can achieve better yields,
lower manufacturing costs, and eventually deliver the products at
a more affordable price by expediting the extraction procedure
[8]. Extraction processes that use organic solvents or consume a
lot of energy have the potential to harm the environment. In the
concept of green extraction includes, less hazardous solvents, less
energy, and ecologically friendly alternatives are the goals of opti-
mization strategies [15]. Ecofriendly extraction methods can help
the nutraceutical industry promote sustainability initiatives and
reduce its environmental impact [16].
By streamlining the extraction process, scalability and consis-

tent product quality are guaranteed. Without compromising the
efficacy or purity of the nutraceuticals, it enables the development
of extraction procedures that are standardized and repeatable on a
larger scale. Consistency is necessary for clinical studies, complying
with regulations and meeting customer expectations.
By making improvements in extraction procedures promotes
continued investigation and development in the nutraceutical
industry. Researchers might discover new opportunities for extract-
ing bioactive molecules from various sources by investigating novel
extraction strategies like enzyme-assisted extraction, supercritical
fluid extraction, and the microwave-assisted extraction. This
upgrades the variety in health-promoting items, consumers can
choose from and results in the discovery of new nutraceutical
ingredients.
Therefore, it is crucial to optimize the extraction procedure for
nutraceuticals to ensure product quality, improve bioavailability,
cut costs, promote sustainability, ensure consistency, and spur inno-
vation in the industry. Together, these advantages help the nutra-
ceutical business develop and grow, which eventually benefits both
producers and customers [17].
2.1 Selection of Choosing the source material for nutraceutical extraction is quite
Source Material challenging and critical. The yield and makeup of bioactive chemi-
cals can be influenced by elements like plant species, the portion of
the plant that is used (leaves, roots, seeds, etc.), and the maturity
stage. The main source materials used for nutraceutical extraction
are shown in Fig. 2.
Additionally, the geographical origin of the starting material
and the variation in chemical structure within the identical species
can have an impact on the success of the extraction. Numerous
aspects, including the presence of bioactive chemicals, accessibility,
sustainability, safety, and legal and regulatory considerations,
should be taken into account when choosing the material sources
for extracting nutraceuticals [10].
Here are some typical sources that are frequently utilized to
extract nutraceuticals:
(a) Plants: Numerous dietary supplements, such as vegetables,
fruits, herbal remedies, and medicinal plants, are obtained
from plants. Examples include Ginkgo biloba extract, curcumin
from turmeric, resveratrol from grapes, and the caffeine in
green tea extracts derived from Camellia sinensis.
(b) Marine sources: Marine life like algae, seaweed, and some fish
can be great sources of dietary supplements. Fish oil–derived
fatty acids known as omega-3 (eicosapentaenoic acid/EPA
and docosahexaenoic acid/DHA) are frequently used as
nutraceutical supplements.
Fig. 2 The main sources used for nutraceutical extractions [18]
(c) Fungi: Some varieties of mushrooms, like shiitake (Lentinula

edodes) and reishi (Ganoderma lucidum), are recognized for
their potential as nutraceuticals. They include bioactive sub-
stances like triterpenoids and polysaccharides.
(d) Animal sources: Several nutraceuticals can be made using
materials that come from animals. Collagen peptides from
bovine or fish sources, for instance, are utilized for healthy
skin, joint support, among other advantages.
(e) Microorganisms: Some microorganisms, including yeast and
probiotic bacteria, are sources of nutraceuticals. Active yeast
or bacterial cultures are known as probiotics, and they have
several health advantages, especially for the gut.
(f) By-products and waste products: By-products and waste pro-
ducts from agriculture can potentially be used as valuable
sources for nutraceuticals. For instance, winemaking leftovers
can be used to make grape seed extract [19].
Make sure that the source materials you use are of high quality
and pure. The amount of bioactive compounds and overall stan-
dard of raw material can be affected by cultivation or sourcing
practices, processing procedures, and storage conditions. In order

to guarantee the safety and effectiveness of the extracted nutraceu-
ticals, compliance with regulatory requirements and good
manufacturing procedures (GMP) are also essential.
3 Factors Involved in Nutraceuticals Extraction
The process of separation and isolation of bioactive components

from various natural sources such as trees, marine life, or micro-
organisms is known as nutraceutical extraction. Extraction of bio-
active compounds involves stable circumstances and conditions.
Hence, various factors affect the extraction process and yield of
nutraceuticals, and some factors are described below and shown in
Fig. 3. These factors affect the efficiency of any extraction process as
these parameters are involved in almost all of the extraction tech-
niques. So in order to obtain better yield of nutraceuticals, we need
to optimize the extraction processes and minimize the effect of
these factors on the process.
(i) Solvent Selection
The selection, efficacy, and safety level of the extraction process
can be affected by the solvent choice, which is crucial. Water,
methanol, ethanol, and their mixtures are employed as common
solvents. The effectiveness of the extraction process and the kinds
of phytochemicals and other bioactives extracted can be influenced
by the solvent’s polarity, boiling point, and toxicity. Additionally,
cosolvents’ presence, pH changes, and temperature changes can
affect the yield or stability of the isolated bioactive
components [18].
Fig. 3 Factors affecting the extraction efficiency of plant bioactive compounds

[20]
The kind of nutraceutical ingredient, its ability to dissolve, and

the desired effectiveness of extraction all play a role in the solvent
choice for nutraceutical extraction. Here are some typical solvents
and their properties for extracting nutraceuticals:
(a) Water: Water is the universal solvent and is frequently used to
extract water-soluble nutraceuticals, including certain poly-
phenols, vitamins, and minerals. Although it is a risk-free and
economical choice, it might not be appropriate for extracting a
lipophilic (fat-soluble) chemicals and many other organic
compounds.
(b) Ethanol: Due to its adaptability and capacity to separate both
hydrophilic and lipophilic molecules, ethanol is a commonly
utilized solvent for nutraceutical extraction. Extracting flavo-
noids, polyphenols, and certain oils that are essential is where
it succeeds. Although ethanol is generally safe and enlisted in
biosolvents, nevertheless, a larger quantity of such solvents
should be handled carefully as it is flammable.
(c) Methanol: Particularly for compounds derived from plants,

methanol is a typical solvent. It is efficiently used for extracting
a variety of nutraceuticals and possesses solvency characteris-
tics that are similar to those of ethanol. Methanol must be
handled carefully and under ventilated conditions because it is
highly flammable and poisonous.
(d) Hexane: It is a nonpolar solvent and is typically employed for
the extraction of lipophilic compounds such as fats, carote-
noids, and other lipid-soluble nutraceuticals. Although it has
good extraction efficiency, due to its combustibility and
potential health risks, it needs to be used with caution.
(e) Supercritical fluids: As a matter of green extractions, supercrit-
ical fluids such as supercritical carbon dioxide (CO2) are gain-
ing popularity in the extraction of nutraceuticals,. They have
the benefit of being risk-free, ecofriendly, and very selective in
extracting particular molecules. For extracting delicate sub-
stances including essential oils and heat-sensitive nutraceuti-
cals, supercritical CO2 is especially successful.
It’s crucial to remember that while choosing solvent, factors
such as the particular nutraceuticals of concern, their solubility
traits, safety issues, and legal requirements should be taken into
account. To maximize extraction efficiency and reduce nutraceuti-
cal degradation, the extraction parameters (temperature, pressure,
and extraction time) are to be adjusted [21].
(ii) Selection of Extraction Techniques
Nutraceutical extraction uses both traditional procedures (like
maceration and Soxhlet extraction) and cutting-edge technologies
(like ultrasound-assisted extraction and supercritical fluid
extraction). The type of bioactive chemicals, their physical and

chemical characteristics, and the desired extracting parameters
(yield, selectivity, consumption of energy, etc.) all influence the
technique chosen. With regard to cost, selectivity, and efficiency,
each method has pros and cons. Pressure, temperature, extraction
duration, and agitation are additional variables that affect the
extraction process [22].
(iii) Process Parameters
The efficacy and purity of nutraceutical extraction can be
affected by a number of process variables, including extraction
duration, temperatures, solid-to-liquid ratio, and agitation. To
optimize the extraction yield and maintain the integrity and
biological activity of the target molecules, these parameters must
be optimized.
(iv) Particle Size
The extraction efficiency is influenced by the raw material’s
particle size. Increased surface areas during solvent interactions
are provided by smaller particle sizes, which boost extraction
rates. Therefore, prior to extraction, the raw material is frequently
pulverized or ground into smaller pieces.
(v) Extraction Conditions
Parameters like temperature, pressure, time, and solvent-to-
solid ratio influence the extraction process. Optimal conditions
need to be determined for each specific raw material and target
compound. These conditions should promote efficient extraction
while minimizing the degradation of sensitive bioactive
compounds.
(vi) pH and Ionic Strength
The pH and ionic strength of the extraction medium can affect
the solubility and stability of bioactive compounds. Adjusting the
pH or adding salts can enhance or inhibit the extraction of certain
compounds.
(vii) Pretreatment Techniques
Preprocessing techniques like blanching, drying, freeze-drying,
or enzymatic treatments may be applied to the raw material to
enhance extraction efficiency by breaking down cell walls, deacti-
vating enzymes, or concentrating the target compounds [21].
(viii) Pre- and Post-treatment
The effectiveness and stability of bioactive ingredient extraction
can be impacted by pre- and post-treatment procedures such as
drying, crushing, particle size reduction, and storage conditions.
The efficacy and biological activity of the isolated nutraceuticals

must be maintained by proper handling and processing of the
source material [23].
(ix) Retention and Storage
The durability and bioactivity of these isolated chemicals must
be maintained by appropriate storage practices and preservation
procedures. To avoid deterioration and loss of potency, factors
like light exposure, temperature, oxygen, or moisture content
must be taken into account.
(x) Quality Assurance
To maintain constant product quality and safety throughout
the extraction process, regular quality control procedures should be
put in place. This entails performing contaminant detection tests,
calculating the amount of bioactive substances, and evaluating the
overall purity of the product [24].
4 Techniques for Nutraceutical Extraction
In order to improve the production and quality of the required

bioactive components, it is necessary to optimize the process of
extraction of nutraceutical substances. Following are a few popular
optimal extraction techniques for nutraceuticals, along with
reading recommendations [15]:
(a) Solvent Extraction: One popular technique for extracting
nutraceuticals is solvent extraction. To extract particular mole-
cules, a variety of solvents like methanol, ethanol, and water
can be utilized. Solvent concentration, extraction duration,
temperature, and solvent to sample ratio are all optimization
parameters.
(b) Supercritical Fluid Extraction (SFE): This method extracts
nutraceuticals using supercritical fluids like carbon dioxide
(CO2). In order to create supercritical conditions, pressure
and temperature must be controlled. Selectivity, gentle extrac-
tion conditions, and little solvent residue are just a few benefits
of SFE [25, 26]:
(c) Microwave-Assisted Extraction (MAE): MAE method boosts
the extraction process by using microwave energy. It delivers
quick extraction, lower solvent use, and better extraction
effectiveness. Extraction time, power, liquid or solvent type,
and sample-to-solvent ratio are variables that should be
optimized [27].
(d) Ultrasound-Assisted Extraction (UAE): UAE breaks down cell
walls and improves mass transfer during extraction by using
high-frequency ultrasound pulses. It is a safe and effective

process that can speed up the extraction process and increase
the amount of nutraceuticals produced [28].
(e) Pressurized Liquid Extraction [29]: PLE, often referred to as
accelerated solvent extraction (ASE), increases extraction effi-
ciency by using high temperatures and pressures. It increases
yield while decreasing extracting time and solvent usage.
5 Approaches for Optimized Nutraceutical Extractions
The optimization of general extraction mechanisms takes into

account a number of variables, including picking the best extraction
technique, fine-tuning the extraction’s variables, and improving the
yield as well as the purity of the molecules being extracted. The
following are some essential steps for optimizing nutraceutical
extractions.
(a) Selection of Extraction Method
With the growing importance of natural medicine, it is very
important to select the most efficient and economical technique. As
the bioactive components are present in a meagre amount in natural
medicines, it is obligatory to increase their yield by developing or
choosing methods that provide optimized yield under optimized
conditions [28]. Therefore, the selection of extraction technique is
a very important step. Ultrasound-assisted extraction, solvent
extraction, microwave-assisted extraction, and supercritical fluid
extraction are a few extraction methods that can be applied. Most
of these techniques are successfully applied for extracting various
nutraceuticals at laboratory level, and among these, supercritical
fluid extraction is also being used at industrial scale [13, 15].
The method chosen will rely on elements like the source mate-
rial and the type of target chemicals.
(b) Optimization of Extraction Parameters
The effectiveness of the extraction process and the quality and
bioactivity of the extracted materials can be greatly impacted by
different variables such as extraction duration, solvent type, tem-
perature, pH, agitation solvent-to-sample ratio, and nature of
extracts. To get the desired result, these factors have to be opti-
mized using an experimental approach and statistical methods. For
instance, the response surface technique methodology [30]
involves the use of experimental data and statistical analysis to
determine the optimal conditions and yield. This technique works
for experimental design and test its validity by ANOVA weather the
design is feasible and adequate for desired extraction process
[15, 30].
(c) Selection of Target Species

Nutraceuticals are used as both nutritious agents and medicines
treating wide range of diseases such as allergy, inflammation, cancer,
and diabetes. To extract these compounds from their natural
sources, we need to first define the type of nutraceutical that is to
be extracted and then identify and optimize the method of extrac-
tion. The type of technique utilized depends upon the compound
to be extracted; for instance, tocopherols and free fatty acids are
sensitive to heat [13], so ultrasound-assisted or sonication-assisted
extractions could be used to optimize the product yield.
(d) Choice of Solvents
The effectiveness and selectivity of a specific molecule extrac-
tion can be considerably impacted by the solvent chosen. To find
the most effective solvent for extraction the desired bioactive che-
micals, a variety of solvents with various polarity and characteristics
can be tried. To increase the extraction efficiency, solvent combina-
tions or modified solvents can be utilized. For example, for extract-
ing nutraceuticals from milk thistle, various solvents such as
acetone, ethanol, acetonitrile, or methanol can be used [31]. The
yield can be optimized by knowing which solvent gives the opti-
mum results for the desired extracts.
(e) Quality Control and Standardization
To ensure consistent quality of the nutraceuticals obtained, it is
important to establish quality control measures and standardize the
extraction process and nutraceuticals doses and usages. This
includes testing the extracts for their bioactive compound content,
purity, stability, and potential contaminants. These controls provide
much more assurance to the product obtained. Qualitative as well
as quantitative evaluations of the composition’s overall profile and
primary distinctive chemicals should be included in quality control.
Because the purity levels of samples used in animal tests and clinical
studies directly affect the accuracy of the research results, it is crucial
to both ensure the safety of herbal products for consumers and
accurately analyze the efficacy of nutraceutical extracts [32]. The
structural formulas and natural sources of commonly known nutra-
ceuticals are given in Table 1.
(f) Innovative Extraction Techniques
Researchers are continually experimenting with new methods
to improve the extraction of nutraceuticals [5, 15, 26]. This may
entail developing extraction technology, such as using sophisticated
reactors, membranes, or enzymes, along with investigating
environmentally friendly and sustainable extraction techniques.
Table 1
Different types of nutraceuticals and their sources [29]
Nutraceutical
compounds Chemical structure Sources
Lycopene Plant sources,
tomato
Vitamin D3 Marine
organism,
fishes
Resveratrol Fruits, grapes
B-carotene Plants, carrots
Curcumin Turmeric
Numerous novel techniques including supercritical fluid extraction,

accelerated solvent extraction, microwave-assisted extraction
ultrasound-assisted extraction, and sonication-assisted extraction
have been established for effective extraction of nutraceuticals as
these techniques play a role in shortening the extraction time,
increasing the yield of extraction, diminishing the solvent intake,
and enriching the quality of products [13].
6 Methods for Optimizing Nutraceutical Extractions
Nutraceutical extraction yield can be optimized using several meth-

ods. Some of these commonly employed techniques are described
here briefly.
1. Response Surface Methodology
RSM involves the use of statistical analysis and mathematical
techniques for the optimization of experimental designs. This tech-
nique is utilized to determine the optimized parameters for extract-
ing nutraceuticals from natural sources, especially food sources. In
the context of nutraceutical extractions, RSM helps in determining
the relationship between multiple input variables (such as extrac-
tion time, temperature, solvent ratio, and pH) and the desired
output response (such as yield, concentration of target compounds,
antioxidant activity). Researchers can effectively investigate how
these variables affect the extraction technique and pinpoint the
ideal set of conditions that optimizes the intended result by using
RSM. The flowchart of response surface methodology is illustrated
in Fig. 4 [30, 33, 34].
Fig. 4 Flowchart of RSM scheme [35]

The steps to optimize the extraction conditions are as follows:

I. Design of experiment: RSM calls for the planning and execu-
tion of a series of tests based on a particular experimental
design, for instance the central composite design (CCD) and
a Box-Behnken design. By using these approaches, the
parameter space can be explored effectively while requiring
fewer tests.
II. Data collection: The experiments are performed according to
the designed plan, and data is collected on the responses of
interest. The responses could be the yield of bioactive com-
pounds, the concentration of specific compounds, or any
other relevant parameters.
III. Regression analysis: The collected data is then subjected to
regression analysis to fit mathematical models that describe
the relationship between the input variables and the response
[35]. The models can be linear, quadratic, or higher-order
polynomials, depending on the complexity of the system and
the experimental design.
IV. Model validation: Models must be validated after develop-
ment using additional experiments or statistical methods like
analysis of variance (ANOVA). By doing this, the models are
made sure to accurately describe the system and be suitable
for optimization.
V. Optimization: The ideal conditions for the extraction proce-
dure are then chosen using the verified models. Numerical
optimization techniques that seek to maximize the response
or reach particular goal values can be used to undertake
optimization.
Researchers can learn more about the effects of different extrac-
tion parameters and improve the extraction process to increase the
yield, concentration, or bioactivity of the compounds of interest by
applying RSM to nutraceutical extractions. With this strategy,
resources can be used more effectively, and the need for costly
experimentation is diminished.
Various techniques make use of response surface optimization
to optimize the yield of extraction. In recent studies, this technique
was used to optimize the antioxidant extractions from the roasted
rice germ–flavored herbal tea. This type of herbal tea is very com-
monly used in Asia as these are known for their gentle aroma and
fragrances along with many health benefits. These herbal teas con-
tain bioactive compounds that possess anti-inflammatory, antibi-
otic, antiaging, anticarcinogenic, and antidepressant effects [36].
As response surface methodology (RSM) used to study the
ideal process parameters while analyzing the antioxidant capacity,
total polyphenol content, and attributed like in roasted rice germ
flavored herbal tea. Five central point replicates of a full factorial

approach on three distinct levels with two variables [37] were used
to evaluate the impact of time and temperature on the extraction
process [38].
2. Ultrasound-Assisted Extraction
This technique, also termed sonication-assisted extraction, uses
sound waves that expand and compress while traveling through
medium unlike electromagnetic radiations. One of the promising
techniques for obtaining plant bioactive chemicals is ultrasound-
assisted extraction. This has great performance low solvent and
time consumption, suitability for thermos-sensitive chemicals, and
acceptance as a green extraction process, and ultrasonic-assisted
extraction [37]. The time, energy, and solvent requirements for
conventional extraction have some restrictions. Bioactive compo-
nents can be extracted using ultrasound-aided extraction (UAE) in
a remarkably short duration of time at low temperature with little
need for energy or solvent. UAE is a non-thermal extraction
method and is more effective in maintaining the functionality of
bioactive chemicals [35].
There are two main types of ultrasound-assisted extractions
which are closed ultrasonic built-in with an ultrasonic horn trans-
ducer or baths extractors. To achieve an efficient and successful
ultrasound-assisted extraction, it is vital to consider plant para-
meters such as moisture content, size of particles, and the extrac-
tion solvent [39]. Additionally, a number of variables, including
frequency, pressure, temperature, and sonication time, affect how
well ultrasound works [13].
Procedure
The general steps involved in this procedure are briefly described
below and shown in Fig. 5 [40]:
I. Preparation of sample: The first step is the preparation of
the sample material. It involves cleaning and drying the raw
material to remove impurities and moisture. The sample
may also need to be ground or chopped into smaller pieces
to increase the surface area for better extraction.
II. Solvent selection: Next, an appropriate solvent is selected
based on the target compounds to be extracted. The solvent
should have good solubility for the bioactive components
and be safe for human consumption. Common solvents
used in UAE include water, ethanol, methanol, and their
mixtures.
III. Sample-solvent mixing: The prepared sample material is
then mixed with the selected solvent to create a homoge-
nous mixture. The ratio of sample to solvent can vary
Fig. 5 Flowchart of ultrasound-assisted extraction
depending on the characteristics of the sample and the

desired extraction efficiency. It is important to ensure
good contact between the solvent and the sample material.
IV. Ultrasound extraction: The sample-solvent mixture is trans-
ferred to an extraction vessel or container. The extraction
vessel is usually made of a material that can withstand the
ultrasound waves, such as glass or stainless steel. The vessel
is then placed in an ultrasound bath or probe system.
V. Ultrasound application: Ultrasound waves are applied to

the extraction vessel containing the sample-solvent mixture,
which create cavitation bubbles in the solvent, leading to
the formation and collapse of microbubbles. These micro-
bubbles generate localized high temperatures and pressures,
causing the cell walls of the sample material to rupture and
facilitating the release of bioactive compounds.
VI. Extraction parameters: Several parameters need to be opti-
mized to achieve efficient extraction. These include the
ultrasound frequency, power, extraction time, and temper-
ature. The parameters may vary depending on the charac-
teristics of the sample and the target compounds. Generally,
a frequency range of 20–100 kHz is used, with power levels
between 20 and 500 W.
VII. Filtration and separation: After the ultrasound treatment,
the extract is filtered to remove solid particles and plant
debris. Filtration methods such as vacuum or gravity filtra-
tion are commonly used. The filtrate contains the desired
bioactive compounds extracted from the sample.
VIII. Concentration and purification: The obtained extract may
undergo further concentration and purification steps, such
as solvent evaporation, liquid-liquid extraction, or chro-
matographic techniques, depending on the nature of the
target compounds and the desired purity level.
IX. Analysis and characterization: The final step involves the
analysis and characterization of the extracted compounds
to determine their quantity, purity, and potential nutraceu-
tical properties. Analytical techniques such as high-perfor-
mance liquid chromatography (HPLC), gas
chromatography (GC), mass spectrometry [36], and
nuclear magnetic resonance (NMR) can be employed for
compound identification and quantification.
6.1 Optimization of The optimization of ultrasound-assisted extraction (UAE) involves

Ultrasound-Assisted adjusting various parameters to maximize the extraction efficiency
Extraction and yield of target compounds. Here are some key factors to
consider when optimizing UAE:
(a) Ultrasound frequency: The frequency of ultrasound waves can
significantly impact the extraction efficiency. Different fre-
quencies, typically ranging from 20 kHz to 100 kHz, may
have different effects on specific compounds or sample matri-
ces. Experimentation with different frequencies can help iden-
tify the optimal frequency for a particular extraction.
(b) Ultrasound power: The power level of ultrasound waves affects
the intensity of cavitation and, therefore, the extraction effi-
ciency. Higher power levels generally lead to more cavitation
and potentially higher yields, but excessively high power can

cause degradation or denaturation of heat-sensitive com-
pounds. Optimizing the power level is crucial to strike a
balance between extraction efficiency and compound integrity.
(c) Extraction time: The duration of ultrasound application
impacts the extraction efficiency. Longer extraction times can
enhance the release of target compounds from the sample
matrix. However, there is an optimal extraction time beyond
which additional extraction does not significantly increase the
yield. It is essential to determine the appropriate extraction
time through experimentation and monitoring of compound
extraction kinetics.
(d) Temperature control: Ultrasound-assisted extraction can gen-
erate heat due to the cavitation process. Controlling and
monitoring the extraction temperature are crucial to prevent
thermal degradation of the target compounds. Cooling sys-
tems or periodic cooling intervals during extraction can help
maintain the desired temperature range.
The extraction of important molecules is now frequently done
by using ultrasonography. As the biological activity of the sub-
stances obtained using this green chemistry strategy is evaluated
in the current paper, and the results showed that of the examined
plants, S. nigra flowers and A. linearis leaf extracts exhibited good
total polyphenolic quantity and antioxidant capabilities. The
extracts may serve as powerful sources of physiologically active
compounds [41].
3. Microwave-Assisted Extraction
This technique, one of the novel and optimized procedures
used for extraction purposes, uses microwaves ranging from 0.3
to 300 GHz, and these waves could easily penetrate through bio-
materials and produces heat by interacting with polar molecules
such as water present within the natural compounds [13]. The
shorter duration of extraction is one of the key benefits of this
method. This is mostly due to the superior heating performance
of this method compared to conventional heating technique.
Microwave heating directly heats the solution, whereas conven-
tional heating requires a certain amount of time to warm the vessel
before the heat is delivered to the solution. This minimizes the
temperature gradient and quickens the rate of heating. Addition-
ally, MAE enables the potential of running several samples and a
large reduction in the consumption of organic solvent [42].
The first step in the extraction procedure is to load the sample
inside the extraction vessel. The solvent is then added and the vessel
is then sealed. Microwave radiation is used for heating the solvent
to the desired temperatures, followed by a preextraction procedure.
Usually, the heating process lasts below 2 min. After receiving more
radiation, the sample is removed for a certain period of time, often
between 10 and 30 min. The samples are given time to cool back to
a temperature that is safe to handle while the extraction is finished
[42, 43].
6.2 Optimization of The optimization depends on different variables on which extrac-

MAE tion yield depends. The results could be improved greatly by using
experimental designs and by using response surface methodology
for statistical analysis. For instance, in Microwave-assisted extraction
(MAE) variables could be optimized by the use of a face-centered
central composite design a experimental design used for optimiza-
tion of response surface methodology for optimum recoveries of
total flavonoid content (TFC) and antioxidants (DPPH and ABTS)
of the extracts obtained from leaf Vernonia amygdalina [44].
Similarly another example containing optimization of micro-
wave assisted extraction is to extract phenolic compounds found in
licorice roots using response surface methodology [12]. RSM
reduces the number of unnecessary experiments while enabling
the user to pinpoint the ideal circumstances for a chosen answer.
The relation between various variables (independent variables) and
one or more of the responses (dependent variables) can be studied
using RSM, which is a set of statistical and mathematical techniques
[45]. With great repeatability, microwave-aided extraction can be
finished in minutes as opposed to hours, using less energy and
solvent.
There are some extraction parameters by arranging them
through proper experimental design we could obtain optimized
conditions, these parameters are Extraction Parameters:
(a) Microwave power: This power level determines the intensity of
the microwave energy applied. Higher power levels can lead to
faster extraction, but excessive power may cause degradation
or loss of heat-sensitive compounds. Optimization is required
to find the balance.
(b) Extraction time: The duration of microwave exposure affects
the extraction efficiency. Longer extraction times may increase
the yield, but there is a limit beyond which further extraction
becomes insignificant or detrimental.
(c) Microwave frequency: The most common frequency used for
MAE is 2.45 GHz, as it is readily absorbed by polar molecules.
However, different frequencies may be utilized for specific
applications.
(d) Sample-to-solvent ratio: The ratio of sample to solvent can
impact the extraction efficiency. It should be optimized to
ensure sufficient contact between the sample and the solvent
for effective extraction.
Recently, optimized microwave-assisted extraction has

emerged as an effective extraction technique due to the applications
of response surface methodology. As such this MAE technique has
been in use for about a century but recently the optimization of this
procedure leads to open up new windows of Especially, the short
time of extraction and improvement in yields are major benefits that
makes this technique a novel and fitting in the principle of green
extraction. Reduced solvent usage, shorter extraction periods, and
higher sample throughput are the main advantages. There is a need
for further cleanup when the extraction is finished, even though
careful process development it may produce some extraction selec-
tivity [42]. The future of this technique could be even brighter if
more suitable experimental design is sought along with providing
optimal conditions.
4. Enzyme-Assisted Extraction
Enzyme-assisted extraction is one of the optimized techniques
that lead to enhanced release of bioactive components [26, 33,
34]. By disrupting plant cells and extracting bioactive compound
through the cell wall, it is possible to maximize the release of these
compounds by utilizing enzyme preparations either alone or in
combinations. However, food industry is not currently taking
advantage of biotechnological applications of enzymes to the fullest
extent [46].
Irrespective of the extraction techniques used, there are always
some barriers that hinder the optimal yield of the extract, which
includes the structure of the cells from which the bioactive com-
pound is to be extracted. Cell components that provide cell support
hinder the extraction of the target metabolites. Therefore, enzyme-
assisted extractions provide lesser resis tance caused by these natural
barriers in extraction [47, 48]. As enzymes are specific in nature,
they target the specific portion, and during these extractions, con-
ditions such as temperature, cell structure, and pH are highly
maintained. The enzymes used in EAE catalyzes the C-H bond
present in molecules such as water, which in result disintegrates the
cell structure and allows the permeability of the materials through
cell. This treatment is also used as pretreatment for other
techniques [49].
6.3 Optimization of Enzyme-assisted extractions are known for their optimized results
EAE and conditions. Several studies have shown recently that EAE
provides optimal yield when used with response surface methodol-
ogy with different experimental designs [26, 33, 34]. For instance,
by using EAE with response surface methodology and by optimiz-
ing variables such as temperature, pH, and solvents, a maximum of
proteins was extracted from sugar beets as enzymes are very specific
in nature [50].
One of the bioactive chemicals now available that has consider-

able potential for use in pharmaceutical and medical treatments for
humans is chondroitin sulfate, which is derived from various natural
materials. But commercialization of chondroitin sulfate is challeng-
ing because it is present in very low levels in raw materials and the
raw materials are expensive. In a recent study, this compound was
extracted from Bohadschia argus through enzyme-assisted extrac-
tion. Experimental conditions were optimized with response sur-
face methodology using Box–Behnken design (BBD). And by
using this model, the experimental yield was noticeably
increased [51].
Following are some of the factors to be monitored to optimize
the experimental yield of extracts obtained through enzyme-
assisted extraction:
(a) Enzyme selection: Different enzymes have different specificities
and activities toward different types of compounds. Optimiza-
tion involves selecting the most suitable enzyme for the target
nutraceuticals. For example, cellulases may be used for the
extraction of cellulose-based compounds, while proteases can
be employed for protein extraction.
(b) Enzyme concentration: The concentration of the enzyme used
in the extraction process significantly impacts its activity. Opti-
mization involves determining the optimal enzyme concentra-
tion by testing various concentrations and measuring the
extraction yield. Too low enzyme concentration may result
in incomplete extraction, while too high concentration may
lead to unnecessary enzyme costs.
(c) Pretreatment techniques: Pretreatment of the sample material
can enhance the accessibility of target compounds to the
enzymes. Optimization involves exploring different pretreat-
ment techniques such as grinding, blanching, freezing, or
steam treatment to disrupt the sample matrix and facilitate
enzyme penetration and contact with the compounds of
interest.
(d) Enzyme immobilization: Enzyme immobilization techniques
can improve the stability and reusability of enzymes during
extraction. Optimization involves selecting the appropriate
immobilization method and optimizing the immobilization
conditions to enhance enzyme activity and prolong its
lifespan.
Enzyme-assisted nutraceutical extraction can be optimized to
maximize extraction efficiency, experimental yield, and quality by
paying close attention to these parameters and using optimization
approaches, leading to improved nutraceutical extraction and
improved extraction procedures.
An emerging method that appears to hold promise for more

effective exploitation of natural resources is enzyme-assisted extrac-
tion. Being a sustainable and environmentally friendly technology,
it offers an appealing substitute for traditional extraction methods
[49]. Therefore, it has been concluded that EAE is a quick and
effective choice for extraction that offers a number of noteworthy
advantages that may be stated as follows: moderate circumstances
that not only retain the bioactive components of interest but also
use less energy and solvent while being more effective than other
standard extraction techniques [52].
A comparative analysis of different extraction techniques for
nutraceuticals extractions is given in Table 2. This table depicts the
overview of techniques along with their advantages and
disadvantages.
7 Case Studies
7.1 Extraction of The red stigmas of Crocus sativus L. are used to make saffron, the
Saffron most expensive spice in the entire world. Saffron contains roughly
10% moisture, 12% protein, 5% fat, 5% minerals, 5% crude fiber, and
63% carbohydrates, containing starch, reducing sugars, gum, pec-
tin, pentosans, and dextrin (w/w%). Saffron has also been shown to
contain traces of thiamin, riboflavin, and fatty acids such as palmitic,
linoleic, stearic, oleic, and linolenic acids [54].
The three main bioactives of saffron—crocin, picrocrocin, and
safranal—are extracted in an optimized way by using response
surface process. The extraction process variables include the tem-
perature (5–85 °C), extraction time (2–7 h), and ethanol concen-
tration (0–100%). The three bioactives (picrocrocin, safranal, and
crocin) were detected spectrophotometrically with highest absor-
bance values at 257, 330, and 440 nm, respectively. The final data
were fitted using four models: linear, linear squares, linear interac-
tions, and full quadratic. As anticipated, the whole quadratic model
had the highest R2 values for the picrocrocin, safranal, and crocin
concentrations, respectively, at 83.91%, 86.60%, and 92.42%. Our
findings showed that short durations, high temperatures, and mod-
erate ethanol concentrations had the greatest influence on the
chemical extraction efficiency. An ethanol content of 33.33%,
extraction period of 2 h, and extraction temperature of 85.0 °C,
respectively, were observed. Under these conditions, the observed
empirical values E1 for picrocrocin, safranal, and crocin were
1190.47, 474.02, and 2311.68 , whereas the theoretical values
were 1237.27, 652.08, and 2821.23 [55].
7.1.1 Application of Bee pollen, a product produced by honeybees, is a blend of plant

Response Surface pollen, nectar, and substances secreted by bees. It consists of vari-
Methodology ous bioactive components, including proteins, amino acids,
442
Table 2
Comparison of different extraction processes [53]
Solvent
Extraction Sample vol.
Techniques Description time (min) size (g) (mL) Cost Advantages Disadvantages
Shanza Malik et al.
Solvent Solvent is heated in conventional oven 6–8 1–20 10–100 Moderate Rapid and easy handling High solvent consumption,
extraction and passed by sample thermal degradation,
long treatment time
Microwave- Immersion of the sample in solvent and 3–30 1–10 10–40 Moderate Rapid, easy to handle moderate solvent Extraction solvent must
assisted microwave energy is submitted consumption absorb microwave energy
extraction Filtration step required
Supercritical A high-pressure vessel is filled with 10–60 1–5 30–60 High Rapid, low solvent consumption, Many parameters to
fluid sample and crossed continuously by concentration of extracts, no filtration optimize
extraction the supercritical fluid necessary, possible high selectivity
Ultrasound- Immersion of the sample in solvent and 10–60 1–30 50–200 Low Easy to use Large amount of solvent
assisted submission to ultrasound using a US consumption, filtration
extraction probe or US bath step required
Pulsed electric Pulses of high electric voltages are 5–10 10–50 10–100 High Rapid and non-thermal process Mechanism not well known
field applied to the sample placed in and process
extraction between two electrodes intensification is difficult
Pressurized Heat of the sample by a conventional 10–20 1–30 15–60 High Rapid, no filtration necessary, low solvent Possible degradation of
solvent oven and crossed by the extraction consumption thermolabile analytes
extraction solvent under pressure
High Sample is pressurized (100–1000 MPa) 1–30 10–20 10–50 High Rapid, green technology, high selectivity, High cost equipment
hydrostatic through a pressure transmitter liquid high extraction yield, no degradation of
pressure target molecules
extraction
enzymes, coenzymes, carbohydrates, lipids, fatty acids, phenolic

compounds, essential vitamins, and essential minerals. On average,
bee pollen contains 22.7% protein, which includes vital amino acids
like tryptophan, phenylalanine, methionine, leucine, lysine, threo-
nine, histidine, isoleucine, and valine [56]. Moreover, it contains a
significant amount of beneficial phytochemicals, such as rutin,
resveratrol, quercetin, protocatechuic acid, phlorizin, p-coumaric
acid, myricetin, luteolin, kaempferol, isorhamnetin, gallic acid,
ethyl gallate, chlorogenic acid, catechin, caffeic acid,
2,5-dihydroxybenzoic acid, trans ferulic acid, and salicylic acid
[57]. Additionally, bee pollen is a source of organic acids like oxalic
acid, tartaric acid, malic acid, citric acid, succinic acid, acetic acid,
lactic acid, and gluconic acid. Due to its rich nutritional profile, bee
pollen has been consumed as a functional food or food supplement
for centuries and is utilized in various products. With the growing
demand for nutritious foods, it is important to assess the quality
and safety of bee pollen to ensure consumer well-being.
7.1.2 Extraction Due to their many benefits, green extraction techniques and sol-
Parameters vents have recently attracted growing interest. This study looked at
how different bioactive compounds may be extracted from bee
pollen by using ultrasonic methodology and polar aprotic solvents
(DESs). In this regard, response surface methodology was used to
examine the overall effects of process variables on individual amino
acids, organic acids, and phenolic compounds. These variables
include molar ratio of the DES (1, 1.5, and 2), sonication duration
(15, 30, and 45 min), and ultrasonic power (90, 135, and 180 W)
(RSM). A molar ratio of 2, a sonication duration of 45 minutes, and
an ultrasonic power of 180 were discovered to be the ideal para-
meters. The control group was composed of extracts produced by
the maceration process using ethanol as a solvent. The total indi-
vidual amino acid and total individual organic acid levels utilizing
DESs were greater than those in the control group. Additionally,
when utilizing DESs as opposed to controls, substances like myr-
icetin, kaempferol, and quercetin were extracted at higher amounts.
Antimicrobial activity testing revealed that the DES groups had a
wide range of antibacterial activities against each and every one of
the bacterial species tested. However, this inhibitory effect was
incredibly weak in yeast-like fungi samples. The study for the
extraction of saffron is the first to assess how DESs affect bee
pollen’s ability to extract beneficial compounds. The outcomes
demonstrate the applicability of this novel and environmentally
friendly extraction method and solvent (ultrasonic extraction/
DES) [58].
7.2 Extraction from Ziziphus lotus are pulpy fruits that are valued for their particular
Fruit of Ziziphus lotus flavor, nutritional value, and therapeutic uses and are consumed as
food all over the world. The abundance of bioactive chemicals in
this fruit is thought to be responsible for its useful characteristics.
Unfortunately, despite ideal extraction conditions, the extraction of
these chemicals and their underlying phytochemical characteriza-
tion has been rarely studied. In this study, Z. lotus fruit pulp extracts
were obtained employing heat-assisted extraction method and
response methodology. These extracts obtained were rich in bene-
ficial biocompounds in terms of their compositional and nutraceu-
tical potential [59].
7.2.1 Extraction The optimal conditions were noted to be as follows: time, 71 min;
Parameters temperature, about 50 °C; solid to solvent ratio, 1:60 (g/mL); and
ethanol concentration, 50%. This gave 48.62% output, includ-
ing 106.64 milligrams of Gallic acid equiv [31]/gram dry matter
of reducing ability with the Folin-Ciocalteu (FCR) reagent, and
49.65 mg of quercetin equiv (QE)/g DM of total flavonoid (TAA).
A total of 38 substances were discovered utilizing LC-ESI-MS/MS
analysis using these parameters. Results also revealed that the opti-
mized pulp extracts from Z. lotus fruit exhibited good antibacterial
activity. The pulp can be utilized to extract bioactive chemicals that
can be employed as ingredients in functional foods and nutraceu-
ticals, and this study offers crucial information on their potential
application [60].
7.3 Extraction from The Rubus genus has an extensive record of medicinal use with
Rubus ellipticus notable therapeutic effects in treating the liver and kidney meri-
dians. In China, its roots and bark are used to relieve lower back
pain, enhance vision, and prevent uterine, cervical, and colon can-
cer. The genus has a number of species that are employed as anti-
microbials, anticonvulsants, muscle relaxants, radical scavengers
and used in the treatments of ulcers, gastrointestinal issues, diabe-
tes, and inflammation. Although the potential of nutraceutical and
functional food derived from Rubus ellipticus fruit is well estab-
lished, there are no comprehensive research works on the optimi-
zation of extraction procedures for increasing yield. Plackett-
Burman (PBD) and Central Composite Design were used in the
current work to extract bioactive chemicals (CCD).
7.3.1 Extraction The extraction of bioactive chemicals was strongly affected by

Parameters factors including the solvent to sample ratio, concentration of
methanol, and extraction temperature under linear, quadratic, and
interaction effects (p 0.05 and p 0.001, respectively). The bioactive
compound and antioxidant predicted values were found to be close
to the experimental value with the lowest coefficient of variation.
Additionally, a non-significant lack of fit and a high coefficient of
determination (R2) were identified in the regression analysis.
Under ideal extraction conditions, high-performance liquid chro-

matography with a photodiode detector (HPLC- PDA) examina-
tion found seven bioactive chemicals, with catechin having the
highest concentration (27.67 mg/g DW). In contrast to previous
studies and past publications on the species, the results demon-
strated a 35–99% improvement in yield. The improved procedure
can be scaled up further to maximize the species advantages in the
manufacture of nutraceuticals and energy supplements on an indus-
trial scale [61].
References
1. Mishra AP et al (2018) Satyrium nepalense, a 11. Durazzo A, Lucarini M, Santini A (2020)
high altitude medicinal orchid of Indian Hima- Nutraceuticals in human health. Foods
layan region: chemical profile and biological 9. https://doi.org/10.3390/foods9030370
activities of tuber extracts. Cell Mol Biol 12. Karami Z et al (2015) Optimization of micro-
64(8):35–43 wave assisted extraction (MAE) and soxhlet
2. Anwar F et al (2019) Mentha: a genus rich in extraction of phenolic compound from licorice
vital nutra-pharmaceuticals – a review. Phyt- root. J Food Sci Technol 52(6):3242–3253
other Res 33(10):2548–2570 13. Wang L, Weller CL (2006) Recent advances in
3. Gull T et al (2015) Capparis species: a potential extraction of nutraceuticals from plants. Trends
source of bioactives and high-value compo- Food Sci Technol 17(6):300–312
nents: a review. Ind Crop Prod 67:81–96 14. Kumar Chellappan D et al (2012) Nutraceuti-
4. Ahmad N et al (2022) Wild olive fruits: phe- cals and their medicinal importance. Int J
nolics profiling, antioxidants, antimicrobial, Health Allied Sci 1:47–53
thrombolytic and haemolytic activities. Arab J 15. Chemat F, Vian MA, Cravotto G (2012) Green
Chem 15(12):104241 extraction of natural products: concept and
5. Ahmad N et al (2021) Ultrasonic-assisted principles. Int J Mol Sci 13(7):8615–8627
extraction as a green route for hydrolysis of 16. Chopra AS et al (2022) The current use and
bound phenolics in selected wild fruits: detec- evolving landscape of nutraceuticals. Pharma-
tion and systematic characterization using GC– col Res 175:106001
MS–TIC method. Process Biochem 111:79– 17. Fongang Fotsing Yannick S et al (2021)
85 Extraction of bioactive compounds from
6. Nasri H et al (2014) New concepts in nutra- medicinal plants and herbs. In: Hany AE-S
ceuticals as alternative for pharmaceuticals. Int (ed) Natural medicinal plants. IntechOpen,
J Prev Med 5(12):1487–1499 Rijeka, p 9
7. Barve KH, Kulkarni YA, Gaikwad AB (2016) 18. Kaur, S., et al., Potential health benefits of
Chapter 7 – Nutraceuticals as therapeutic nutraceuticals for human health 2019.
agents for inflammation. In: Watson RR, Pre- p. 193–209
edy VR (eds) Fruits, vegetables, and herbs. 19. Kumar K et al (2017) Food waste: a potential
Academic, pp 121–147 bioresource for extraction of nutraceuticals and
8. Subbiah MR (2007) Nutrigenetics and nutra- bioactive compounds. Bioresour Bioprocess
ceuticals: the next wave riding on personalized 4(1):18
medicine. Transl Res 149(2):55–61 20. Al Ubeed H et al (2022) A comprehensive
9. Farhud D, Yeganeh MZ (2010) Nutrigenomics review on the techniques for extraction of bio-
and nutrigenetics. Iran J Public Health 39(4):1 active compounds from medicinal cannabis.
10. Mishra AP et al (2022) The role of nutraceu- Molecules 27:604
ticals as food and medicine, types and 21. Puri V et al (2022) A comprehensive review on
sources. In: Egbuna C, Sawicka B, Khan J nutraceuticals: therapy support and formula-
(eds) Food and agricultural Byproducts as tion challenges. Nutrients 14. https://doi.
important source of valuable nutraceuticals. org/10.3390/nu14214637
Springer, Cham, pp 1–18
22. Mandal SC, Mandal V, Das AK (2015) response surface methodology. Curr Top
Chapter 6 – Classification of extraction Nutraceut Res 17(4)
methods. In: Mandal SC, Mandal V, Das AK 35. Abu Bakar FI et al (2020) Optimization of
(eds) Essentials of botanical extraction. Aca- extraction conditions of phytochemical com-
demic, Boston, pp 83–136 pounds and anti-gout activity of Euphorbia
23. Don R (2011) Validation of nutraceutical pro- hirta L.(Ara Tanah) using response surface
cess equipment. In: Handbook of nutraceuti- methodology and liquid chromatography-
cals. CRC Press mass spectrometry (LC-MS) analysis. Evid
24. Shang H et al (2018) Extraction condition Based Complement Alternat Med 2020
optimization and effects of drying methods 36. Uysal S et al (2017) Optimization of the
on physicochemical properties and antioxidant extraction process of antioxidants from loquat
activities of polysaccharides from Astragalus leaves using response surface methodology. J
cicer L. Sci Rep 8(1):3359 Food Process Preserv 41(5):e13185
25. Saba I, Anwar F (2018) Effect of harvesting 37. Yusoff IM et al (2022) A review of ultrasound-
regions on physico-chemical and biological assisted extraction for plant bioactive com-
attributes of supercritical fluid-extracted spear- pounds: phenolics, flavonoids, thymols, sapo-
mint (Mentha spicata L.) leaves essential oil. J nins and proteins. Food Res Int 157:111268
Essent Oil Bear Plants 21(2):400–419 38. Rittisak S et al (2022) Response surface opti-
26. Mushtaq M et al (2015) Enzyme-assisted mization for antioxidant extraction and attri-
supercritical fluid extraction of phenolic anti- butes liking from roasted rice germ flavored
oxidants from pomegranate peel. J Supercrit herbal tea. PRO 10(1):125
Fluids 104:122–131 39. Ellington E et al (2003) Supercritical carbon
27. Gomez L, Tiwari B, Garcia-Vaquero M (2020) dioxide extraction of colchicine and related
Chapter 9 – Emerging extraction techniques: alkaloids from seeds of Colchicum autumnale
microwave-assisted extraction. In: Torres MD, L. Phytochem Anal Int J Plant Chem Biochem
Kraan S, Dominguez H (eds) Sustainable sea- Tech 14(3):164–169
weed technologies. Elsevier, pp 207–224 40. Şahin S, Şamlı R (2013) Optimization of olive
28. Zhang Q-W, Lin L-G, Ye W-C (2018) Techni- leaf extract obtained by ultrasound-assisted
ques for extraction and isolation of natural extraction with response surface methodology.
products: a comprehensive review. Chin Med Ultrason Sonochem 20(1):595–602
13(1):20 41. Gadjalova A, Mihaylova D (2019) Ultrasound-
29. Hoti G et al (2022) Nutraceutical concepts and assisted extraction of medicinal plants and eval-
dextrin-based delivery systems. Int J Mol Sci uation of their biological activity. Food Res
23. https://doi.org/10.3390/ijms23084102 3(5):530–536
30. Messadi N et al (2023) Optimization of extrac- 42. Chaturvedi AK (2018) Extraction of neutra-
tion parameters and characterization of Tuni- ceuticals from plants by microwave assisted
sian date extract: A scientific approach toward extraction. Syst Rev Pharm 9(1):31–35
their utilization. Sugar Tech 25(2):460–472 43. Wang Z et al (2006) Rapid analysis of the
31. Wallace SN, Carrier DJ, Clausen EC (2003) essential oils from dried Illicium verum
Extraction of nutraceuticals from milk Hook. f. and Zingiber officinale Rosc. by
thistle. In: Davison BH et al (eds) Biotechnol- improved solvent-free microwave extraction
ogy for fuels and chemicals: the twenty-fourth with three types of microwave-absorption
symposium. Humana Press, Totowa, pp medium. Anal Bioanal Chem 386:1863–1868
891–903 44. Alara OR, Abdurahman NH, Olalere OA
32. Zeng J et al (2011) Standardization and quality (2018) Optimization of microwave-assisted
control of herbal extracts and products. In: extraction of flavonoids and antioxidants from
Traditional herbal medicine research methods: Vernonia amygdalina leaf using response sur-
identification, analysis, bioassay, and pharma- face methodology. Food Bioprod Process 107:
ceutical and clinical studies, pp 377–427 36–48
33. Qadir R et al (2022) Enzyme-assisted extrac- 45. Rai, A.K., et al., Optimization of acid hydroly-
tion of Phenolics from Capparis spinosa fruit: sis conditions of delimed tannery fleshings by
modeling and optimization of the process by response surface method. 2009
RSM and ANN. ACS Omega 7(37): 46. Puri M, Sharma D, Barrow CJ (2012) Enzyme-
33031–33038 assisted extraction of bioactives from plants.
34. Qadir R et al (2019) Enzyme-assisted extrac- Trends Biotechnol 30(1):37–44
tion for optimized recovery of phenolic bioac-
tives from Peganum hermala leaves using
47. Sun T et al (2018) Carotenoid metabolism in carotenoids encapsulated in amorphous poly-

plants: the role of plastids. Mol Plant 11(1): mer matrices. Food Chem 71(2):199–206
58–74 55. Sarfarazi M, Jafari SM, Rajabzadeh G (2015)
48. Rodrı́guez De Luna SL, Ramı́rez-Garza R, Extraction optimization of saffron nutraceuti-
Serna Saldı́var SO (2020) Environmentally cals through response surface methodology.
friendly methods for flavonoid extraction Food Anal Methods 8(9):2273–2285
from plant material: impact of their operating 56. Khalifa SAM et al (2021) Bee pollen: current
conditions on yield and antioxidant properties. status and therapeutic potential. Nutrients
Sci World J 2020 13(6)
49. Łubek-Nguyen A, Ziemichód W, Olech M 57. Bayram NE et al (2021) Phenolic and free
(2022) Application of enzyme-assisted extrac- amino acid profiles of bee bread and bee pollen
tion for the recovery of natural bioactive com- with the same botanical origin – similarities and
pounds for nutraceutical and pharmaceutical differences. Arab J Chem 14(3):103004
applications. Appl Sci 12(7):3232 58. Çelik S et al (2022) Optimization of ultrasonic
50. Akyüz A, Ersus S (2021) Optimization of extraction of nutraceutical and pharmaceutical
enzyme assisted extraction of protein from the compounds from bee pollen with deep eutectic
sugar beet (Beta vulgaris L.) leaves for alterna- solvents using response surface methodology.
tive plant protein concentrate production. Foods 11(22):3652
Food Chem 335:127673 59. Abdoul-Azize S (2016) Potential benefits of
51. Ruensodsai T et al (2021) Optimization of jujube (Zizyphus lotus L.) bioactive com-
enzyme assisted extraction of chondroitin Sul- pounds for nutrition and health. J Nutr
fate from Bohadschia argus by response surface Metab 2016:2867470
methodology. E3S Web Conf 302:02012 60. Abcha I et al (2021) Optimization of extrac-
52. Alavarsa-Cascales D et al (2022) Optimization tion parameters, characterization and assess-
of an enzyme-assisted extraction method for ment of bioactive properties of Ziziphus lotus
the anthocyanins present in Açai (Euterpe oler- fruit pulp for nutraceutical potential. Eur Food
acea Mart.). Agronomy 12(10):2327 Res Technol 247
53. Suwal S, Marciniak A (2019) Technologies for 61. Kewlani P et al (2022) Optimization of
the Extraction, separation and purification of ultrasonic-assisted extraction for bioactive
polyphenols – a review. Nepal J Biotechnol 6: compounds in Rubus ellipticus fruits: an
74–91 important source for nutraceutical and func-
54. Selim K, Tsimidou M, Biliaderis CG (2000) tional foods. Sustain Chem Pharm:25
Kinetic studies of degradation of saffron
Chapter 18
Computational Approach and Its Application

in the Nutraceutical Industry
Prabina Bhattarai, Sampurna Rai, Pankaj Koirala,
Abstract
In recent years, application of the computational approach, a predictive tool, in food and nutritional
sciences has outweighed classical analytical methods. The sophisticated informatics approaches such as
chemo-informatics and bioinformatics methods are integrated with the food industry for the extraction and
identification of bioactive compounds. Model simulation of the molecule and the assessment of the
dynamics are frequently used to study the structure and function of food carbohydrates, lipids, and other
small molecules. Beyond the simulation, current advancement in algorithms has solidified machine learn-
ing’s ability to predict the availability of the functional component and its biomolecular activity. The chapter
summarizes the joint applications of bioinformatics and simulation methods in the extraction and discovery
of bioactive and nutraceutical components, in particular, the selection of analytical procedure, activity
prediction, docking, and physicochemical properties.
Key words Bioactive compounds, Nutraceuticals, Chemo-informatics, Bioinformatics
1 Introduction
The number of studies on nutraceuticals, cosmeceuticals, and func-

tional foods has grown exponentially. This research shows that
nutraceuticals and functional foods provide anti-oxidative, repro-
ductive, renal, gastrointestinal, and many other health benefits.
These health benefits are attributed to several bioactive com-
pounds, such as peptides, phytochemicals, essential oils, polysac-
charides, and other bioactive metabolites. However, the
effectiveness of food that confers health benefits has always been
speculated with skeptical remarks. This has resulted from inconsis-
tencies in the evidence of their beneficial effects. The inconsistency
with the evidence on its effectiveness could be due to a gap between
its clinically controlled and aftermarket effectiveness. In this case,
the main constituent of the gap is the variables acting upon the
449
450 Prabina Bhattarai et al.
biological mode of action, which must be subdued with a compu-

tational approach. This approach not only delivers the probable
biological confounding factors for the mode of action, but also
applies to discovering significant novel bioactive compounds or
novel functions of the existing ones [1].
The combined application of bioinformatics and chemoinfor-
matic can amplify the observation of fundamental mechanisms on
the mode of action, target dynamics, and possible toxicity of the
bioactive compounds. Bioinformatics and cheminformatics, which
utilize computer applications, exploit databases to identify and
interpret chemical and biological data. Furthermore, the interpre-
tation of chemical and biological data is made more accessible by
the development of an artificial intelligence (AI) approach that can
efficiently extract information regarding large-scale molecular
interactions. In this chapter, we will discuss the use of different
food science databases that assist in the chemoinformatic, bioinfor-
matics, and artificial intelligence processes. Additionally, we will
elaborate on the application of chemoinformatic, bioinformatics,
and AI in food science and nutraceutical research.
2 Database in Food Science
Chemical space is a framework for studying the influence of struc-

ture on the biological behavior of a compound. Different types of
molecular and visual representations of chemical spaces aid in
exploring the structure-property and structure-activity relation-
ships (SPR and SAR) and linking the character to the specific
spaces, which can help predict the intersection of specific chemical
and biological spaces [2]. The framework places molecules in the
mathematical dimension based on their physicochemical and che-
moinformatic descriptors. Descriptors are the numerical linear
notation that represents the space. Maximizing the descriptor
representing biological activity can build a qualitative/quantitative
structure-activity relationship (QSAR) matrix and enable complex
multivariate analysis, which is the basis for the computational
approach [3]. The chemical space’s reliability is mostly dependent
on the qualitative and quantitative aspects of the descriptors used to
define the mathematical dimension. Chemical space has been
reported to contain at least 106 organic molecules below 500 Da.
These molecules could be explored by constructing a chemical
space as a database and then navigating the database using molecu-
lar descriptors [4].
Databases are fundamental to discovering new bioactive com-
pounds and pursuing their mode of action, relevancy of therapeutic
claims, and other impact variables. The in silico approach requires
data set representing structure, qualitative information, activity,
physicochemical properties, functional molecular fragment,
Computational Approach and Its Application in the Nutraceutical Industry 451
toxicity, and other physiological interaction to cast out the algo-

rithms [5]. The major benefit of the open-source data/program
remains to include researchers from a wide economic background,
which is crucial in fueling the progression of open science. The
other advantages can be noted for its ability to harbor data from all
spectrums and act as a source of data to cross evaluate and aid in
strengthening the finding’s validity and reliability [6]. The virtual
chemical database has been advanced to harboring millions of
compounds which has significantly accelerated the pursuit of
novel organic molecules for the betterment of health [7]. The
natural bioactive compounds, which are the product of plants,
animals, and microorganisms’ resilience towards harsh ambiance
and other hostile elements, have maximum biologically relevant
scaffolds [8]. Hence, they have bioactivity against the diverse tar-
get, and chemical space is massive to accommodate all the diverse
biologically relevant information—as opposed to synthetic
compounds [9].
Modern medicine constantly pursues novel bioactive com-
pounds from natural sources, be it plant, animal, or microorgan-
isms. The exploration benefits from the advancement in silico
capability, providing direct estimation and additional support to
the in vitro assay. The virtual databases contain structural informa-
tion of the compound, which is the basis for the application of
cheminformatics. The chemical space of the nature-based com-
pound comprises massive information on its bioactivity [10]. The
data library gives an edge to discover the unexplored action of the
compound or open the door of modifying the compound by
stereoselective transformation or adhesion of a new functional
group [11].
The computational approach (docking, QSAR modeling, and
pharmacophore-based modeling) eases the identification of com-
paratively high bioactive compounds and prediction of an efficient
process for extraction, purification, and estimate ADME (absorp-
tion, distribution, metabolism, excretion) along with toxicological
properties [12]. In addition, computational analysis’s complexity is
stacked upon the nature-based bioactive compound’s complex chi-
rality [10]. The undefined chiral centers require vigorous screening
of all the probable stereochemical configurations from the plethora
of complex configurations. Thus, to render reliable prediction out-
comes, the computational approach must recruit datasets from the
different databases (Tables 1 and 2) with precise structures with
well-defined stereochemistry [12].
Generated Databases (GDB) is the largest database featuring
166.4 billion molecules, including both bioactive (aromatic and
planar) and non-aromatic 3D-shaped molecules, which are signifi-
cant for drug discovery. Unlike other databases-which focus on the
combination of the building blocks in novel drugs-like compound
discovery, it prioritizes by lensing into the chemical diversity and
Table 1
Database for chemicals
No. of
Database Availability compound Link Reference
Generated databases- Free 166.4 billion https://gdb.unibe.ch/downloads/ [13]
17
Zinc 20 Free 1.4 billion https://zinc20.docking.org/ [14]
PubChem Free 112 million https://pubchem.ncbi.nlm.nih.gov/ [15]
ChemIDplus Free 4,00,000 https://chem.nlm.nih.gov/ [16]
chemidplus/
Table 2
Database for bioactive compound
No. of
Database Specification molecules Link Reference
Carotenoids Carotenoid structure, structural isomers 1204 http://carotenoiddb. [20]
database and stereoisomers, chemical jp/
fingerprint
ChEMBL Bioactive compounds with drug-like 2.3 https://www.ebi.ac. [21]
properties million uk/chembl/
PADFrag Biological-functional molecular 5919 http://chemyang. [22]
fragments ccnu.edu.cn/ccb/
database/
PADFrag/
FooDB Physicochemical data, sensory 28,000 https://foodb.ca/ [23]
information (color, flavor, aroma), its
concentration effects on other
attributes of food, physiological
effect, and impacts on human health
Ambinter Structure, biological target 38 million https://www. [24]
ambinter.com/
BIOFAC Compounds from the plants, fungi, and 400 https://biofacquim. [25]
QUIM propolis of Mexican origin herokuapp.com/
NPASS Connects natural products with the 35,032 https://bidd.group/ [5]
biological targets NPASS/
BioPhytMol Anti-mycobacterial phytochemical 2582 https://ab-openlab. [26]
information csir.res.in/
biophytmol/
BIOPEP- Bioactive peptides, quantitative 4540 https://biochemia. [27]
UWM parameters of bioactive fragments, uwm.edu.pl/
SMILES code biopep-uwm/
AHTPDB Antihypertensive peptides 6000 https://webs.iiitd.edu. [28]
in/raghava/
ahtpdb/
deep into the first principle of organic chemistry. GDB has subsets
and focuses on the specific spectrum of the compound, such as
FDB17b on the fragment, GDBMedChem on medicinal chemistry
[17], GDBChEMBL on ChEMBL-like molecule [18], and GDB4c
on novel 3D-shaped molecules with quaternary centers [13].
ZINC 20 is a free database hosted by Irwin and Shoichet
laboratories of the University of California, which harbors informa-
tion (docking, 2D, and 3D models) of more than 509 million
purchasable compounds and analogs of over 750 million com-
pounds with a constant update of billions of new molecules [14].
PubChem is the largest chemical database comprising informa-
tion such as 3D structures, biological assay descriptions, and results
of more than 112 million compounds, 298 million substances, and
301 million bioactivities. Compared to other databases, it is more
user-friendly due to its simple and alternative views via dedicated
web pages and presents data related to genes and diseases associated
with the pathway, biological activity, protein, and patent [15].
ChemIDplus is a free web-based search system with more than
4,00,000 chemical records containing over 3,00,000 chemical
structures. This chemical record aids in the identification of the
compounds by providing structure and nomenclature authority
files from the National Library of Medicine (NLM), US states,
and its federal agencies, along with other scientific sites. ChemID-
plus have two versions, ChemIDplus Lite and ChemIDplus
Advanced, on which the latter has additional information regarding
molecular formula, classification code, chemical structure, physical
property, toxicity, and locator code searching [16]. It has been
scheduled to move to PubChem in December 2022 [19].
Carotenoids Database is a repository for 1204 natural carote-
noids from 722 sources of organisms. It contains data regarding the
classification of carotenoid structure, structural isomers, and
stereoisomers, chemical fingerprint which describes the carote-
noids’ chemical substructure, and the details regarding modifica-
tion and eases prediction of the biological function of provitamin A,
membrane stabilizers, allelochemicals, odorous substances, anti-
proliferative activity against the cancerous cell, and reverses multi-
drug resistance (MDR) activity against cancer cell [20].
ChEMBL is a database that harbors information curated from
the primary medicinal chemistry research literature and multiple
other sources. In addition, it contains information regarding the
compound’s bioactivity, molecules, target, and other drug data. Its
web services are built on a RESTful architecture which allows users
to access data programmatically and is also available via other
sources, such as PubChem BioAssay and BindingDB [21].
PADFrag database, which is for a bioactive molecular frag-
ment, holds 1652 FDA-approved drugs, 1259 agricultural chemi-
cals, and 5919 molecules generated from the mentioned drugs and
chemicals. This database consists of physicochemical properties, 3D
structures, and target information of the specific molecules which

has two versions, drug2fragment mode and fragment 2drug mode.
The first mode contains information about the FDA-approved
drug/pesticide and all the fragments. In contrast, the latter mode
contains information about the fragments from FDA drugs/pesti-
cides with their respective FDA-approved drugs/pesticides [22].
FooDB is a class of database comprising more than 28,000
compounds in over 1000 unprocessed food’s elaborative informa-
tion about the biochemical, compositional, and physiological
makeup. It is classified into Food Browse and Compound Browse;
in the first classification, foods are classified as per their chemical
composition, whereas chemicals are grouped as per their food
source in the latter. This includes data regarding the compound’s
nomenclature, structural data, physicochemical data, sensory infor-
mation (color, flavor, aroma), its concentration effects on other
food attributes, physiological effects, and impacts on the human
health database [23].
Ambinter comprises over 38 million compounds’ information
for QSAR, docking, and virtual screening. It is a paid database
managed by Greenpharma, which assists in custom synthesis, che-
moinformatic, and molecular modeling of the compound [24].
BIOFAQUIM contains data on more than 400 compounds
from plants, fungi, and propolis of Mexican origin. The species
are selected by taking into account their geographical location
and the information is curated accordingly [25].
NPASS is a database that provides 446,552 quantitative activity
records, 222,092 target pairs and 288,002 species pairs, 35,032
natural products with 5863 targets (2946 proteins, 1352 microbial
species, and 1227 cell lines) from 25,041 species [5].
BioPhytMol database contains more than 2582 Phyto molecules
from 188 plant families, which are primarily directed towards the
anti-mycobacterial against 25 target mycobacteria. It is experimen-
tally verified and curated with experimental assay preparation,
including details of the compound’s bioactivity, and assists in gen-
erating analogous compounds for target-specific inhibitors [26].
BIOPEP-UWM is a free database comprising proteins, biologi-
cally active peptides derived from food, sensory peptides, amino
acids, and allergenic proteins. The data are also crowd-sourced
from the user. The users can submit new peptide sequences in the
database, and the curators verify and upload them for the public. It
currently holds 739 proteins, 4540 bioactive peptides, 136 aller-
genic proteins with epitopes, and 533 sensory peptides and amino
acids [27].
AHTPDB is a manually curated database from several research
articles and peptide repositories of antihypertensive peptides
(AHTPs) that have been experimentally validated. The database
comprises 1700 unique peptide sequences with 6000 sequences
originating from 35 types of plants (soybean) and animal sources
(pork, fish, chicken, milk, egg, etc). Besides these, the database
withholds information regarding structure (tertiary and second-
ary), inhibitory concentration (IC50), toxicity, bitterness value,
source, purification method, and log value of inhibitory concentra-
tion (pIC50) information [28].
A number of deserted databases are not maintained anymore or
might be maintained poorly. In some cases, the wrong interpreta-
tion might occur due to the lack of a definite standard for stereo-
chemistry and aromaticity, leading to new versions of the same
molecules. The current misrepresentation of “Publish or perish”
has adversely flourished among researchers, guild-leading to the
spawn of databases that have a dire chance of getting maintained
after a certain time of publication.
3 Biomedical Informatics
3.1 Chemoinformatic Chemoinformatic is an in silico technology employed in research

relating to chemistry. It adopts an integrated approach to study and
understand the function of chemical systems using available ligand
resources such as pharmacophore modeling, quantitative structure-
activity relationship (QSAR), docking, and molecular dynamics
(MD) simulations. Cheminformatics has found several significant
uses in the pharmaceutical industry, including agricultural, environ-
mental, and food chemistries [29].
3.1.1 Quantitative QSAR is a chemoinformatic technique that links the relationship

Structure-Activity between the chemical structure and biological activity of the com-
Relationship (QSAR) pounds. It plays a role in designing and screening new molecules
and predicting the activity and mechanism of biologically active
compounds. Compared to conventional techniques, QSAR screen-
ing is more feasible and convenient for identifying various chemical
structures [30]. Moreover, it contributes to quantifying the stabil-
ity, potency, and toxicity of known bioactive compounds [30].
3.1.2 Application The application of the QSAR technique involves the development
of QSAR of a QSAR model, where a set of numerical descriptors related to
the structure of interest serves as independent variables, while the
targeted biological activities are the dependent variables. Then, the
relationship between the dependent and independent variable is
built using multiple linear regression (MLR), partial least square
(PLS) regression, support vector machine (SVC), artificial neural
network (ANN), etc. [31, 32]. For instance, there are many amino
acid descriptors, such as hydrophobicity, bulkiness/molecular size
and electronic property of amino acids, isotropic surface area, and
electronic charge index [32, 33]. However, the properties of amino
acids described by a single parameter are more complicated as they
have less explanatory power and neglect the relationship between
different descriptors [34]. Therefore, studies use the integration of

different amino acid descriptors to predict different properties of
the target peptides [35]. To date, many QSAR studies have inves-
tigated ACE inhibitory or antimicrobial peptides (AMPs), antioxi-
dant, antimicrobial, renin, and DPP-IV inhibition, as well as the
organoleptic properties of the peptides [36].
3.1.3 Antioxidants Antioxidant peptides play a significant role in preventing and treat-
ing various diseases due to their capacity to scavenge free radicals
and prevent oxidative stress [37]. It is essential to determine the
dynamics between human physiology and bioactivity of antioxi-
dants. QSAR has the potential to explore the dynamics and forecast
the interaction between food-derived antioxidant and physiology.
Specifically, QSAR study is focused on dipeptides and tripeptides as
they are absorbed intact from the intestinal lumen to produce
biological effects at the tissue level [38]. Dent et al. 2019 con-
ducted a QSAR study on two datasets of antioxidant tripeptides
where the first dataset contained 214 artificially designed tripep-
tides, and the second dataset contained 72 Beta-lactoglobulin tri-
peptides. The study used 16 amino acid descriptors to conduct
model population analysis (MPA), which improves prediction abil-
ity and interpretability by forming multi-model clusters [39] with
higher cross validated coefficient of determination.
3.1.4 ACE-Inhibitory Several studies have been conducted on identifying and isolating
Peptides several ACE-inhibitory peptides derived from food. Application of
QSAR modeling has identified the novel ACE inhibitory peptides
derived from Qula casein hydrolysates using a two-enzyme combi-
nation. The QSAR model involved amino acid descriptors as pre-
dictors and log transformed IC50 values as the dependent variable.
Amino acid descriptors include five z-scale descriptors where z1
represented lipophilic properties, z2 represented steric properties,
z3 represents electronic properties, and z4 and z5 were related to
other properties like electronegativity, the heat of formation, and
electrophilicity. Finally, the QSAR model was analyzed by SIMCA-
P software (Umetrics, Umeå, Sweden) using partial least squares
regression (PLS). The study also concluded that the peptides pro-
duced by utilizing thermolysin + alcalase exhibited stronger ACE
inhibitory function [40].
3.1.5 Phytochemical Phytochemical peptides can be defined as a chemical peptide pro-

Peptides duced by plants that may provide favorable health benefits to
reduce the risk of major chronic diseases. Urease inhibitor is one
of the phytochemical peptides studied for its ability to treat the
Helicobacter pylori infection. Chopdar et al., developed a QSAR
model using CORAL SEA 17 software to predict the urease inhi-
bitors. The model utilized SMILES and GRAPH descriptors as the
independent variable to predict pIC50 from the NPACT database.

The utilization of both SMILES and molecular graphs descriptors
is known to result in a hybrid descriptor developing a QSAR model
with better statistical quality [40].
3.1.6 Molecular Docking The molecular docking process includes predicting the molecular
orientation of a ligand within a receptor and calculating their
complementarity interaction (binding affinity) using a scoring func-
tion. Molecular docking allows for studying the behavior of pep-
tides in the binding site of target proteins. It is also called a
structure-based method that allows it to determine the structure-
activity relationship of peptides. For example, once the peptides
have been sequenced and their bioactivity has been determined
through in vitro and in vivo assays, they undergo structural prepa-
ration for docking. Next, the receptor-ligand complex structures
are prepared using docking simulation software. The final analysis is
done to predict the binding modes and affinities of a ligand, i.e.,
bioactive peptides [41].
3.1.7 Application of Molecular Interactions

Molecular Docking Studying food enzyme interaction and toxins is essential in food
processing and producing novel foods. Studies have utilized molec-
ular docking to study the binding mechanism between a commonly
consumed protein, ovalbumin (OVA), and a malachite (MG-dye).
They found that there are hydrophobic and van der Waals interac-
tions between ovalbumin and a food additive, indicating that the
mixture of these two components may lead to the formation of a
toxic compound (OVA-MG) [42]. Another study showed the
health-promoting properties of broccoli through the interaction
of an enzyme myrosinase with glucosinolates to produce isothio-
cyanates at a neutral pH. The study also identified competitive
inhibitors (amygdaline and arbutin) of an enzyme myrosinase that
prevents the formation of undesirable compounds at low pH
[43]. Besides, the effect of different nutrients and enzymes on
trypsin and alpha-amylase inhibitors was determined based on the
molecular docking technique [44].
Angiotensin-I-Converting Enzyme (ACE) Inhibitory Peptides

A molecular docking technique has been utilized to determine the
molecular mechanism of novel ACE-inhibitory peptides generated
from Larimichthys crocea titin. Firstly, titin protein was hydrolyzed
using the ExPASy PeptideCutter program to identify peptides. This
program predicts potential cleavage sites in titin protein sequences.
The generated peptides were then compared with known
ACE-inhibitory peptides of the BIOPEP-UWM database and the
antihypertensive inhibiting peptide database (AHTPDB). Further-
more, molecular docking was done to identify the potential
molecular mechanism of the identified tripeptides (WAR and

WQR) using the SwissDock webserver. SwissDock webserver pre-
dicted their binding site and confirmed their interaction with the
ACE active sites [45].
Molecular docking methods have some limitations, such as
using mathematical scoring functions, which might not correlate
with actual experimental binding affinities, and the limited sam-
pling of both ligand and receptor conformations [46]. Molecular
dynamic simulation is another in silico technique that calculates the
motion and equilibrium of each molecule and provides protein-
ligand information at excellent temporal resolution. MDS can be
applied to limit the number of possible ligands obtained with
molecular docking and filter the false-positive molecule that
shows high affinity during the complex formation [47]. In contrast
to molecular docking, MDS can precisely control the experiment’s
physical conditions, from the temperature and ions around the
peptides to the properties of the solvent [41].
3.2 Bioinformatics Bioinformatics is a broad scientific domain mainly concerned with

applying computational resources to biological data. Due to its low
cost and high throughput, bioinformatics has been used in the food
industry to improve the quality and functionality of food sources.
For example, bioinformatics technologies provide information on
the conformation of bioactive peptides (BAPs), predict potential
activities, illustrate molecular interaction mechanisms, and improve
peptide properties, which helps study BAPs for human medicinal
use [48].
3.2.1 Allergens The use of bioinformatics ranges from developing novel nutraceu-
ticals to detecting the toxic compounds in food, including aller-
gens. Regarding the detection of allergens, bioinformatics
facilitates the efficient detection of allergens from the massive
amount of data, predicting information about the allergen, and
can validate the traditional strategies by guaranteeing the reliability
of the conclusions. The bioinformatics approach can determine
cross-reactive allergens by determining the degree of homology of
different allergens within the epitome to maintain immunoglobulin
E (IgE) binding [49]. Bioinformatics was utilized in identifying
latent allergic proteins from chickpeas by collecting known allergen
sequences in the Fabaceae family from the database WHO/IUIS
(http://allergen.org) and Basic Local Alignment Search Tool
(BLAST). The study found that out of seven potential allergens
from chickpeas, four had cross-reactivity with the allergens in the
databases [50]. Another study utilized the proteomics and
METLIN database to identify hazardous allergens (glutaredoxin
and oleosin-B2) in bee pollen. Bee pollen is utilized for its
nutritional properties. Therefore, the presence of hazardous aller-

gens within bee pollen indicates the need for developing the process
to remove those allergens [51].
3.2.2 Bioactive Peptides In silico approach, prior to wet laboratory analysis, hydrolysis of
proteins allows focusing on a small number of peptides for predict-
ing the types and potency of peptides with the selected combination
of protein and enzyme(s) [52]. Bioinformatic approaches narrow
down the number of enzyme combinations for protein hydrolysis
and predict bioactivity or interaction with specific molecules and
receptors by homology-based searches [53]. The co-ordinate of the
cleavage point in the sequence could be based on the specialized
databases such as UniProt Knowledgebase [52]. Furthermore, with
the large set of databases, it is feasible to characterize peptides for
their theoretical physicochemical, specific bioactivity, and sensory
properties [54], and reject peptides with undesirable properties.
For example, Panjaitan, Gomez [55] did a study on optimiza-
tion of enzyme combination using in silico in order to render
tryptic peptides and determined their bioactive property. The resul-
tant peptides sequence was identified using mass spectrometry and
a sequence similarity search was done with BLAST, which revealed
that the aligned with tryptic peptide sequences from Epinephelus
coioides sharing 70% identity to the protein sequences from Epine-
phelus lanceolatus (database). Further, the BIOPEP analysis
revealed that the pepsin and mixed proteases (pepsin, trypsin, and
chymotrypsin A; pepsin, trypsin, and chymotrypsin C) exhibited
better production of ACEI peptides compared to other proteases.
In this way, the computational method is convenient for biotech-
nologists as it can efficiently identify peptide types and determine
appropriate protease combinations that can theoretically produce
optimal bioactive peptides.
3.2.3 By-products The utilization of food by-products is another growing field in the
food industry, which is blooming due to the development of bioin-
formatics tools. The combined application of in silico and ex vivo
has demonstrated the potential bioactivity (ACE inhibition, renin
inhibition, ACE inhibition,) of the peptides from by-products like
pigeon peas waste [56]. Senadheera et al. did a study on sea cucum-
ber where they utilized in silico techniques for generating antioxi-
dant and ACEI peptides by processing the by-products of sea
cucumber. The study utilized mass spectrometry to identified pep-
tides and was virtually screened by the PepRank tool. Then, in
silico, proteolysis was simulated with digestive enzymes using the
BIOPEP-UWMTM database tool. After simulated digestion, Tox-
inPred software evaluated the peptides for toxicity and found that
peptides resistant to the in silico digestion were non-toxic [57].
4 Artificial Intelligence (AI) Approaches in Bioactive Extraction and Nutraceuticals
The term “AI” refers to a computer program in which a machine is

programmed to think and behave as a human. Big data from
different databases is extracted, patterns are developed with the
help of different algorithms, which enables AI to do as humans
do. In an AI-based computational approach, machines can predict
using various algorithms and AI models. Prior to diving into AI, it’s
important to understand machine learning (ML). ML is a subset of
AI that leverages data to generate knowledge for AI systems on its
own. For ML, raw data are encoded into the machine along with
numerous algorithms, such as SVM, regression, decision trees, and
so on, which support machine learning without explicit program-
ming. However, in AI approach, the big data sets are extracted and
utilized to form patterns for AI systems with the help of neural
networks (the heart of deep learning algorithms). With a neural
network, machines could categorize and arrange coded data in a
way that resembled the functioning of the human brain [58].
The application of computational approaches in the field of
applied life-science research, such as protein sequence prediction,
target identification, molecular dynamic simulation, modeling, and
redesign, dates back a long time. However, as AI, ML, and deep
learning have advanced, there has been a significant increase in the
accuracy and precision of their results when compared to the tradi-
tional approach. AI-based molecular and/or deep docking, for
example, has enabled structural-based virtual screening of large
molecular libraries, understanding molecular interaction, and
molecular design with unprecedent accuracy [59, 60]. In the food
and nutraceutical industries, they have adopted AI-guided models
for modeling for optimal extraction, qualitative and quantitative
prediction, process simulation, and release modeling of bioactive
and functional compounds. Integrated systems, such as the
adaptive-neurofuzzy inference system (ANFIS), stepwise linear
regression (SWLR), and multilayer perceptron (MLP) models
that consist of neural networks, are employed to predict the outputs
(nutrients, bioactive molecules, and ligands). In respect of accuracy
and efficiency, the application of these AI-based models for the
prediction of bioactive compounds is superior to that of a tradi-
tional predictive approach (a regression model) [61].
4.1 Machine ML is data-driven and performs on the basis of various numerical

Learning (ML) and statistical models, based on which predictive models can be
developed in in silico applications for extraction scale-up, opti-
mized processing, and understanding the physiological mechan-
isms of the bioactive compounds. Previously standardized
statistical techniques for determining factors such as solute-to-sol-
vent ratio, physical properties, and structural complexity in the
chemical composition are still in practice in the food and nutraceu-

tical industries. These traditional statistical tools are tedious and of
low efficiency in the age of automation, so industries are attracted
towards machine learning and other computational approaches to
produce more efficient and rigorous results. Particularly, the accu-
racy of applying machine learning and conventional statistical tech-
niques in classifying samples (fermented and unfermented rooibos)
based on the phenolic content and antioxidant activities revealed
that ML yields data results nearly identical to those of conventional
statistical analytical techniques [62]. When ML is applied for bio-
active extraction, the selection of the right machine learning algo-
rithm is crucial and depends on several different criteria.
Different sophisticated analytical machines are guided by a
number of machine learning algorithms since decades. Broadly,
ML algorithms are categorized into four different types: super-
vised, semi-supervised, reinforcement, and unsupervised. In the
case of supervised methods, datasets are labeled prior employing
new data sets to analyze. The most common supervised algorithms
in food science applications are the naive bayes classifier algorithm,
SVM, regression (linear and logistic), and random forest. These
algorithms can be employed to analyze data that is predictable,
recurring, or that falls within the range of the training data sets
[63]. For instance, to predict the relationship between the input
parameters (ethanol concentration, solvent/solid ratio, and tem-
perature) and the output response (total flavonoids and extraction
yield) for optimal extraction of flavonoids from celery seed using
response surface methodology governed by different algorithms, a
supervised algorithm (a multiple linear regression algorithm) was
the best model for predictive optimization [64]. Contrarily, the
unsupervised approach groups the data into clusters based on its
characteristics and then uses dimensionality reduction to determine
the most dominantly featured data. The large, unclassified data sets
are more appropriate to analyze using unsupervised methods. For
example, the demonstration of the extracting reaction kinetics and
degradation mechanisms of the phenolic resin under different con-
ditions and the reduction in the variability of the molecular
dynamic simulation were extracted using unsupervised ML. When
the data was subjected to unsupervised ML techniques such as
non-negative matrix factorization (NMF), principal component
analysis (PCA), and non-negative tensor factorization (NTF), the
dimensionality of the data was reduced in order to determine
dominantly featured data [65].
4.1.1 ML Supported In the development of drugs or bioactive compounds, the most

Virtual Screening (VS) time-consuming steps are identification of the most potent com-
pound, parameter optimization for extraction, appropriate delivery,
and validation of the effectiveness of those bioactive compounds in
a clinical setting. Virtual screening has recently been discovered to
be the best alternative to traditional screening. VS is an in silico

approach used in bioactive compounds identification, toxicity eval-
uation, and understanding pharmacokinetics. Virtual screening
employs the use of a database to evaluate how closely an object
and a specific molecule resemble one another to discover novel,
physiologically active compounds with high efficiency and high
throughput. Virtual screening in protein-based nutraceutical
industries could be applied to screen the 3D structure of the target
protein and bioactive peptide via docking, identification of novel
bioactive peptides, and interactions of peptides and ligands
[66]. When the bioactive peptide and bioactivity were screened
using VS, it was discovered that the major flavonoids were 3,4-di-
OMe luteolin and acacetin, while salicylic acid and melilotic acid
were the key phenolic acids contributing to the observed antidia-
betic effect. QSAR, used to identify the critical functional groups
required for protein-phenolic molecular interactions, validates the
role of pearl millet phenolics in inhibiting carbolytic enzymes and
regulating GLUT [67].
Furthermore, structural-based virtual screening (SBVS) pre-
dicts the best interaction mode between two molecules to form a
stable complex. It uses scoring functions to estimate the force of
non-covalent interactions between a ligand and molecular target
[68]. Researchers have employed machine learning approach in
scoring functions to improve SBVS algorithms. To find a new
bioactive peptide, numerical descriptive vectors (NDVs) for peptide
sequences were developed. These techniques use quantitative
structure-activity relationship (QSAR) analysis to predict
angiotensin-converting enzyme (ACE) inhibitor dipeptides,
bitter-tasting dipeptides, and nonameric binding peptides of the
human leukocyte antigens [69]. Lastly, the effectiveness of the VS
can be justified by the research outcomes of Wang, Niu [70], where
VS of a food-derived antihypertensive peptide using a ML strategy
with the eXtreme Gradient Boosting (XGBoost) algorithm predicts
antihypertensive peptide with high efficiency and throughput.
4.2 Artificial Neural Deep learning is a subset of machine learning that depends on a
Network (ANNs) and common algorithm with the important function of automatic
Deep Learning information extraction from raw data. Deep learning is a data-
hungry algorithm, and supervised learning datasets require a large
amount of labeled data. The use of more complex network archi-
tectures in DL expands on the use of deep artificial neural networks
(ANN) in ML. It utilizes the deep ANN made up of several layers of
nonlinear modules to enhance multilayer representation
[71]. Deep learning algorithms perform with accuracy and preci-
sion in ADMET property prediction, target prediction, virtual
screening, and chemical synthesis. Deep learning has been used
for high-performance test screening (HTS), quantitative structural
analysis (QSR), and other purposes. In recent years, de novo
molecule synthesis, which uses sequence data to generate molecules

with desired attributes, has indeed made significant use of deep
learning. Therefore, deep learning is useful for comprehending the
variety and chemistry of natural components in the nutraceuticals
and bioactive industries [63]. In analytical chemistry, ANN-based
modeling is more accurate compared to RSM for extraction opti-
mization because of excellent representation of the nonlinear,
including quadratic equations, relationships. This can be observed
from the study conducted by [72], who utilized the back propaga-
tion (BP) algorithm and a feed-forward multilayer perceptron
(MLP) type architecture of an ANN model to construct the predic-
tive mathematical model with the conditions of the four parameters
(water content, time, temperature, and solid-liquid ratio) and alka-
loids’ yields as outputs. The ANN model provided more precise
predictions because it had a lower MSE value and a higher R2 value.
The utilization and interaction of numerous fully connected or
convolutional hidden layers defines platforms, like deep neural net-
works (DNNs) and convolutional deep neural networks (CNNs),
which have shown potential application in the nutraceutical indus-
try. DNNs are artificial neural networks that include numerous
hidden layers and the ability to simulate complex non-linear inter-
actions. DNNs can still perform even in the absence of a large
training data set. In biological and pharmaceutical chemistry, neural
networks are recently being used to predict ligand binding sites and
optimize molecular properties and structure generation. Ligand-
binding activity from QSAR modeling, predicting binding sites,
and ADMET properties of small molecules can be obtained accu-
rately using DNN [73]. This indicates the possible application in
nutraceutical and bioactive compounds too. For example, predic-
tion of the flavonoid level within the leaves of the velvet apple using
the DNN algorithm was carried out by Qasthari and Saputro
[74]. Velvet apple leaf hyperspectral images were taken between
400 and 1000 nm wavelengths. Laboratory data on flavonoids was
labeled in the cropped leaf image, where flavonoids compound was
prediction with an R2 performance of 70.47% using an unopti-
mized and shallow DL model. Moreover, deep neural networks
have been used to classify Caco-2 binary penetrability. For instance,
Shin, Jang [75] used 663 chemical substances to construct an
in vitro Caco-2-specific data model, where the overfitting and
nonlinear activation issues were resolved via dropout regulariza-
tion. The results show that high-level DNN features outperform
handwrought features in predicting structurally varied chemical
compound cell permeability in Caco-2 cellular lines. CNN, on the
other hand, is being applied to improve the secondary metabolites
of plant. Non-invasive hyperspectral imaging can identify changes
in secondary metabolism, and relevant wavelengths of imaging can
be reduced by integrating deep learning [76]. Those secondary
plant metabolites are connected to spectral information that is
significant for the classification of healthy food sources. The devel-

opment of a deep learning-based regression approach can be
applied to determine the chemical compositions of the fruits.
Zhang 2020 designed convolutional neural networks (CNN) to
predict the chemical composition in dry black goji berries and
determined it using hyperspectral imaging. In terms of modeling
and feature extraction, a deep learning-based regression model like
partial least squares and least-squares support vector machines for
modeling and principal component analysis and the wavelength
transform for feature determination were used, respectively [77].
Lastly, integrated applications of the AI, DL, and ML can aid in
molecular recognition, identification of bioactivity of phytochem-
icals, proteins, and peptides, molecular docking, and interaction
between bioactive compounds and other molecules [78–80], which
can enhance efficiency in the food and nutraceuticals industries. It
also improves functional activity by accurately predicting active
components and target interaction, target validation, and the active
site of the target. Application of AI, ML, and DL assists in produc-
tion maximization of bioactive and nutraceuticals by providing
insight into important factors that influence the multifaceted pro-
cess of extraction and processing [80, 81]. Beside these, ML and
DL help in the prediction of the toxicity of different phytochem-
icals and peptides with high precision using either deep neural
networks, ANNs, or other algorithms to establish a QSAR model
[68, 82]. ML, DL, and AI are now applied in the signal processing
and analysis of nano- and macromolecules for automatic structural
verification and prediction using high-resolution analytical equip-
ment like NMR [83].
5 Future Perspectives
In the last decade, application of the computational approach has

led to robust food and nutrition research. This advancement can be
observed in the current application of specialized “in silico” groups
supporting the hit and lead identification processes concurrently in
large nutraceutical companies. They are being used to predict novel
bioactives such as peptides, as well as discovery procedures in
conjunction with a wide range of experimental techniques to vali-
date properties such as bioactivity evaluation, toxicity, and so
on. The foundation of a computational approach includes virtual
screening, the design of the screening collection, data extraction
and analysis from databases with different algorithms, and data
profiling. In the bioactive compound and nutraceutical industries,
AI approaches such as ML and DL are being significantly utilized.
Several computational researchers concluded that AI performed
better than conventional statistical techniques. Regardless of a
number of limitations and obstacles in AI, the technology will
soon revolutionize research and innovations in the fields of medical

and food science. AI and data science may enable the development
of the “virtual human” in the near future. If this is the case,
bioactive and phytochemical clinical trials will be more extensive
and effective. In a relatively short period of time, the wide variety of
compounds can be evaluated for potential bioactivity under opti-
mum conditions with increased efficiency, omitting the possible
toxicity. Thus, the use of various computational approaches aids
in the prediction and elucidation of molecule interactions,
structure-property relationships, potential bioactivity, and many
other phenomena.
References
1. Carpio LE et al (2021) Computational strate- of complex and diverse compounds. Nat Prod
gies for the discovery of biological functions of Rep 31(1):6–14
health foods, nutraceuticals and cosmeceuti- 12. Chen Y, de Bruyn Kops C, Kirchmair J (2017)
cals: a review. Mol Divers 25(3):1425–1438 Data resources for the computer-guided dis-
2. Medina-Franco JL et al (2022) Progress on covery of bioactive natural products. J Chem
open chemoinformatic tools for expanding Inf Model 57(9):2099–2111
and exploring the chemical space. J Comput 13. Meier K et al (2020) The generated databases
Aided Mol Des 36(5):341–354 (GDBs) as a source of 3D-shaped building
3. Arús-Pous J et al (2019) Exploring chemical blocks for use in medicinal chemistry and
space with machine learning. CHIMIA Int J drug discovery. Chimia (Aarau) 74(4):
Chem 73(12):1018–1023 241–246
4. Liu Y et al (2021) Exploring and mapping 14. Irwin JJ et al (2020) ZINC20—a free
chemical space with molecular assembly trees. ultralarge-scale chemical database for ligand
Sci Adv 7(39):eabj2465 discovery. J Chem Inf Model 60(12):
5. Zeng X et al (2018) NPASS: natural product 6065–6073
activity and species source database for natural 15. Kim S et al (2021) PubChem in 2021: new
product research, discovery and tool develop- data content and improved web interfaces.
ment. Nucleic Acids Res 46(D1):D1217– Nucleic Acids Res 49(D1):D1388–D1395
D1222 16. ChemIDplus U (2014) National Library of
6. Willems H, De Cesco S, Svensson F (2020) Medicine
Computational chemistry on a budget: sup- 17. Awale M et al (2019) Medicinal chemistry
porting drug discovery with limited resources: aware database GDBMedChem. Mol Inform
miniperspective. J Med Chem 63(18): 38(8–9):1900031
10158–10169 18. Bühlmann S, Reymond JL (2020) ChEMBL-
7. Walters WP (2019) Virtual chemical libraries. J likeness score and database GDBChEMBL.
Med Chem 62(3):1116–1124 Front Chem 8:46
8. Rodrigues T et al (2016) Counting on natural 19. ChemIDplus U (2022). Available from:
products for drug design. Nat Chem 8(6): https://chem.nlm.nih.gov/chemidplus/
531–541 20. Yabuzaki J (2017) Carotenoids Database:
9. Harvey AL, Edrada-Ebel R, Quinn RJ (2015) structures, chemical fingerprints and distribu-
The re-emergence of natural products for drug tion among organisms. Database (Oxford)
discovery in the genomics era. Nat Rev Drug 2017(1):bax004
Discov 14(2):111–129 21. Davies M et al (2015) ChEMBL web services:
10. Stratton CF, Newman DJ, Tan DS (2015) streamlining access to drug discovery data and
Cheminformatic comparison of approved utilities. Nucleic Acids Res 43(W1):W612–
drugs from natural product versus synthetic W620
origins. Bioorg Med Chem Lett 25(21): 22. Yang J-F et al (2018) PADFrag: a database
4802–4807 built for the exploration of bioactive fragment
11. Morrison KC, Hergenrother PJ (2013) Natu- space for drug discovery. J Chem Inf Model
ral products as starting points for the synthesis 58(9):1725–1730
23. FooDB. Available from: https://foodb.ca/ healthy gut in the adult human? Nutr Res Rev
24. Ambinter. Available from: https://www. 27(2):308–329
ambinter.com/ 39. Deng B et al (2019) Quantitative structure-
25. Pilón-Jiménez BA et al (2019) BIOFAC- activity relationship study of antioxidant tri-
QUIM: a Mexican compound database of nat- peptides based on model population analysis.
ural products. Biomol Ther 9(1):31 Int J Mol Sci 20(4):995
26. Sharma A et al (2014) BioPhytMol: a drug 40. Chopdar KS et al (2022) Monte-Carlo
discovery community resource on anti- method-based QSAR model to discover phyto-
mycobacterial phytomolecules and plant chemical urease inhibitors using SMILES and
extracts. J Cheminform 6(1):46 GRAPH descriptors. J Biomol Struct Dyn
27. Minkiewicz P, Iwaniak A, Darewicz M (2019) 40(11):5090–5099
BIOPEP-UWM database of bioactive peptides: 41. Vidal-Limon A, Aguilar-Toalá JE, Liceaga AM
current opportunities. Int J Mol Sci 20(23): (2022) Integration of molecular docking anal-
5978 ysis and molecular dynamics simulations for
28. Kumar R et al (2015) AHTPDB: a comprehen- studying food proteins and bioactive peptides.
sive platform for analysis and presentation of J Agric Food Chem 70(4):934–943
antihypertensive peptides. Nucleic Acids Res 42. Rajamanikandan R, Selva Sharma A, Ilanche-
43(Database issue):D956–D962 lian M (2019) New insights into the binding
29. Peña-Castillo A et al (2018) Chemoinformatics interaction of food protein ovalbumin with
in food science. In: Applied chemoinformatics: malachite green dye by hybrid spectroscopic
achievements and future opportunities. Wiley, and molecular docking analysis. J Biomol
Weinheim, pp 501–525 Struct Dyn 37(16):4292–4300
30. Neves BJ et al (2018) QSAR-based virtual 43. Román J, Castillo A, Mahn A (2018) Molecu-
screening: advances and applications in drug lar docking of potential inhibitors of broccoli
discovery. Front Pharmacol 9:1275 myrosinase. Molecules 23(6):1313
31. Iwaniak A et al (2015) Chemometrics and che- 44. Li H et al (2021) Proteinaceous α-amylase
minformatics in the analysis of biologically inhibitors: purification, detection methods,
active peptides from food sources. J Funct types and mechanisms. Int J Food Prop
Foods 16:334–351 24(1):277–290
32. Mei H et al (2005) Support vector machine 45. Fan Y et al (2020) Identification and molecular
applied in QSAR modelling. Chin Sci Bull mechanism of angiotensin-converting enzyme
50(20):2291–2296 inhibitory peptides from Larimichthys crocea
titin. Food Sci Human Wellness 9(3):257–263
33. Collantes ER, Dunn WJ III (1995) Amino acid
side chain descriptors for quantitative 46. Pinzi L, Rastelli G (2019) Molecular docking:
structure-activity relationship studies of pep- shifting paradigms in drug discovery. Int J Mol
tide analogs. J Med Chem 38(14):2705–2713 Sci 20(18):4331
34. Venkatarajan MS, Braun W (2001) New quan- 47. Hollingsworth SA, Dror RO (2018) Molecular
titative descriptors of amino acids based on dynamics simulation for all. Neuron 99(6):
multidimensional scaling of a large number of 1129–1143
physical–chemical properties. Mol Model 48. Gauthier J et al (2019) A brief history of bioin-
Annu 7(12):445–453 formatics. Brief Bioinform 20(6):1981–1996
35. Xu B, Chung HY (2019) Quantitative 49. McClain S (2017) Bioinformatic screening and
structure–activity relationship study of bitter detection of allergen cross-reactive IgE-bind-
di-, tri-and tetrapeptides using integrated ing epitopes. Mol Nutr Food Res 61(8):
descriptors. Molecules 24(15):2846 1600676
36. Peredo-Lovillo A et al (2021) Conventional 50. Kulkarni A, Ananthanarayan L, Raman K
and in silico approaches to select promising (2013) Identification of putative and potential
food-derived bioactive peptides: a review. cross-reactive chickpea (Cicer arietinum) aller-
Food Chem X 13:100183 gens through an in silico approach. Comput
37. Bashir KMI et al (2018) Antioxidant properties Biol Chem 47:149–155
of Scomber japonicus hydrolysates prepared by 51. Yin S et al (2022) A combined proteomic and
enzymatic hydrolysis. J Aquat Food Prod Tech- metabolomic strategy for allergens characteri-
nol 27(1):107–121 zation in natural and fermented Brassica napus
38. Miner-Williams WM, Stevens BR, Moughan PJ bee pollen. Front Nutr 9:822033
(2014) Are intact peptides absorbed from the 52. Peredo-Lovillo A et al (2022) Conventional
and in silico approaches to select promising
food-derived bioactive peptides: a review. Food 66. Lammi C et al (2021) A heuristic, computer-
Chem X 13:100183 driven and top-down approach to identify
53. Daroit DJ, Brandelli A (2021) In vivo bioactiv- novel bioactive peptides: a proof-of-principle
ities of food protein-derived peptides–a current on angiotensin I converting enzyme inhibitory
review. Curr Opin Food Sci 39:120–129 peptides. Food Res Int 150:110753
54. Udenigwe CC (2014) Bioinformatics 67. Krishnan V et al (2022) Polyphenol-enriched
approaches, prospects and challenges of food extract from pearl millet (Pennisetum glau-
bioactive peptide research. Trends Food Sci cum) inhibits key enzymes involved in post
Technol 36(2):137–143 prandial hyper glycemia (α-amylase,
55. Panjaitan FCA, Gomez HLR, Chang YW α-glucosidase) and regulates hepatic glucose
(2018) In silico analysis of bioactive peptides uptake. Biocatal Agric Biotechnol 43:102411
released from giant grouper (Epinephelus lan- 68. Maia EHB et al (2020) Structure-based virtual
ceolatus) roe proteins identified by proteomics screening: from classical to artificial intelli-
approach. Molecules 23(11):2910 gence. Front Chem 8:343
56. Tapal A et al (2019) Nutraceutical protein iso- 69. Mahmoodi-Reihani M, Abbasitabar F, Zare-
late from pigeon pea (Cajanus cajan) milling Shahabadi V (2020) In silico rational design
waste by-product: functional aspects and and virtual screening of bioactive peptides
digestibility. Food Funct 10(5):2710–2719 based on QSAR modeling. ACS Omega
57. Senadheera TRL et al (2022) In silico analysis 5(11):5951–5958
of bioactive peptides produced from underuti- 70. Wang L et al (2021) A novel machine learning
lized sea cucumber by-products-a bioinformat- strategy for the prediction of antihypertensive
ics approach. Mar Drugs 20(10):610 peptides derived from food with high effi-
58. Gupta R et al (2021) Artificial intelligence to ciency. Foods 10(3):550
deep learning: machine intelligence approach 71. Zhou L et al (2019) Application of deep
for drug discovery. Mol Divers 25(3): learning in food: a review. Compr Rev Food
1315–1360 Sci Food Saf 18(6):1793–1811
59. Gentile F et al (2022) Artificial intelligence– 72. Jiang Z-M et al (2019) Green and efficient
enabled virtual screening of ultra-large chemi- extraction of different types of bioactive alka-
cal libraries with deep docking. Nat Protoc loids using deep eutectic solvents. Microchem J
17(3):672–697 145:345–353
60. Schauperl M, Denny RA (2022) AI-based pro- 73. Panteleev J, Gao H, Jia L (2018) Recent appli-
tein structure prediction in drug discovery: cations of machine learning in medicinal chem-
impacts and challenges. J Chem Inf Model istry. Bioorg Med Chem Lett 28(17):
62(13):3142–3156 2807–2815
61. Metekia WA et al (2022) Artificial intelligence- 74. Qasthari ET, Saputro AH (2019) Prediction
based approaches for modeling the effects of system for flavonoid content on Bisbul leave
spirulina growth mediums on total phenolic (Diospyros discolor Willd.) using deep neural
compounds. Saudi J Biol Sci 29(2):1111–1117 networks regression. In: The 4th International
62. Hussein EA et al (2021) Comparison of phe- Seminar on Sensors, Instrumentation, Mea-
nolic content and antioxidant activity for fer- surement and Metrology (ISSIMM),
mented and unfermented rooibos samples November 14, 2019
extracted with water and methanol. Plan The- 75. Shin M et al (2018) Predicting the absorption
ory 11(1):16 potential of chemical compounds through a
63. Zameer R et al (2022) Role of transcriptomics deep learning approach. IEEE/ACM Trans
and artificial intelligence approaches for the Comput Biol Bioinform 15(2):432–440
selection of bioactive compounds. In: Drug 76. Brugger A et al (2021) Spectral signatures in
design using machine learning. Wiley, Hobo- the UV range can be combined with secondary
ken, pp 283–317 plant metabolites by deep learning to charac-
64. Nguyen Thu H et al (2021) Optimization of terize barley–powdery mildew interaction.
extraction conditions of flavonoids from celery Plant Pathol 70(7):1572–1582
seed using response surface methodology. J 77. Zhang C et al (2020) Developing deep
Food Meas Charact 15(1):134–143 learning based regression approaches for deter-
65. Bhesania AS et al (2022) In-depth analysis of mination of chemical compositions in dry black
reaction kinetics parameters of phenolic resin goji berries (Lycium ruthenicum Murr.) using
using molecular dynamics and unsupervised near-infrared hyperspectral imaging. Food
machine learning approach. Comput Mater Chem 319:126536
Sci 206:111215
78. Ho Thanh Lam L et al (2020) Machine 81. Garcı́a-Pérez P et al (2020) Combining medic-
learning model for identifying antioxidant pro- inal plant in vitro culture with machine learning
teins using features calculated from primary technologies for maximizing the production of
sequences. Biology 9(10):325 phenolic compounds. Antioxidants (Basel)
79. Srisongkram T et al (2022) Machine learning 9(3):210
and in vitro chemical screening of potential 82. Douali L (2022) Machine learning for the pre-
α-amylase and α-glucosidase inhibitors from diction of phenols cytotoxicity. Int J Adv Intell
Thai indigenous plants. Nutrients 14(2):267 Inform 8(1):58–68
80. Nguyen TK et al (2022) Deep learning model 83. Cobas C (2020) NMR signal processing, pre-
to identify potential acetylcholinesterase inhi- diction, and structure verification with machine
bitors: a case study of isolated compounds from learning techniques. Magn Reson Chem 58(6):
Pongamia pinnata (L.) Pierre. Nat Prod Com- 512–519
mun 17(7):1934578X221117310
INDEX
A 233, 235, 237, 241–243, 246, 247, 255–271,

283–293, 301, 314, 318, 320, 322, 324–325,
Acids.......................................7, 9, 14, 16, 23, 25, 30–36,
374, 398, 402, 405, 412, 421, 436, 437, 439,
46–54, 57, 60–62, 66, 67, 83, 85, 89, 91, 96, 97,
440, 444, 450–465
106, 116, 139–143, 145, 156, 165–168, 174,
Assessment..............................................26, 38, 142, 144,
177, 185, 187, 189, 193, 212, 214, 218, 229,
156, 191, 204, 218, 285
236–238, 241, 242, 244, 245, 248, 259,
Assurance .............................................................. 428, 430
261–263, 265, 266, 278, 282, 285–287, 290,
291, 300, 302–313, 316, 319, 322, 324, 334, B
338–341, 344, 345, 349, 350, 356–360,
362–365, 393, 397, 400, 402, 407, 409, 420, Benefits ...................................................2, 15, 34, 46, 50,
423, 430, 440, 443, 444, 454–456, 462 59, 61, 62, 69, 105, 106, 117, 119, 166, 167,
Activity ....................................... 2, 12–16, 34, 36, 47–57, 169, 170, 173, 183, 189, 191, 194, 202,
59–62, 68, 69, 79, 89, 93, 95, 104, 113, 117, 216–218, 224, 239, 240, 242, 244, 245, 248,
119–121, 123, 135, 144, 145, 161, 167, 169, 257, 259, 260, 282, 304–313, 334, 335, 348,
173, 174, 183–188, 190, 191, 195, 212–214, 360, 362, 363, 365, 376, 382, 385, 403, 410,
216, 236, 237, 243, 244, 246, 248, 255, 256, 420, 423, 426, 428, 433, 437, 439, 443, 449,
259, 262, 263, 268, 271, 282, 286–290, 300, 451, 456
301, 304, 305, 309–313, 319, 323, 334, Bioactive ...............................................1, 22, 45, 79, 103,
339–341, 343–346, 349, 351, 355, 356, 360, 129, 153, 173, 202, 224, 255, 276, 300, 334,
365, 367, 374–385, 427, 428, 432, 437, 440, 373, 393, 419, 449
443, 450, 453–455, 458, 461, 463, 464 Bioactivity .............................................13, 14, 28, 29, 36,
Analysis ....................................................... 22, 28, 30, 32, 46, 47, 49–62, 65, 115, 141–146, 188, 262, 263,
36, 91, 108, 145, 146, 154, 177, 178, 189, 312, 324, 325, 339, 340, 349, 350, 355, 357,
259–262, 265, 275, 284, 285, 288, 356, 405, 373, 374, 422, 428, 433, 451, 453, 454, 456,
410, 420, 429, 432, 433, 436, 437, 441, 444, 457, 459, 462–465
451, 457, 459, 463, 464 Bioassays .........................................................28, 342, 453
Analytical .................................................. 30–33, 88, 108, Bioavailability ....................................................13, 61, 64,
153, 169, 260, 261, 263, 276, 282–284, 293, 145–147, 262, 263, 324, 358, 363, 422, 423
381, 391, 436, 461, 463, 464 Biological .........................................................1, 2, 12, 15,
Antimicrobial................................... 2, 14, 15, 34, 49, 51, 16, 23, 30, 34–37, 54, 55, 57, 60–62, 69, 79, 93,
52, 54, 61, 62, 120, 121, 123, 129, 130, 173, 117, 119, 120, 139, 167, 169, 183, 187, 188,
174, 243, 244, 246, 248, 256, 258, 334, 202, 205, 206, 209, 216, 223, 225, 236, 237,
337–346, 348, 349, 354–355, 360, 365, 366, 242, 246, 247, 261, 263, 278, 284, 288, 290,
443, 444, 456 300, 310–312, 336, 339, 344, 374, 375, 377,
Antioxidants .........................................2, 21, 45, 80, 104, 380, 395, 419, 420, 427, 428, 437, 449, 450,
129, 176, 205, 232, 256, 282, 304, 358, 374, 452, 453, 455, 456, 458, 463
393, 420, 456 Biomaterials ......................................................8, 230, 437
Application .....................................2, 3, 9, 13–16, 21–38, Bioprocessing ................................................................ 237
46–69, 90, 95, 96, 104–107, 112–123, 132, Byproducts.................................................... 9, 13, 15, 16,
138–143, 146, 147, 153, 154, 163–170, 173, 23, 34, 51, 55, 57, 61, 62, 69, 212, 301–304, 311,
175, 176, 180, 184, 187, 191, 195, 203–206, 315, 316, 319, 321, 322, 325, 344, 358, 411, 459
208–210, 212, 213, 216, 218, 219, 225, 226,
Methods and Protocols in Food Science , https://doi.org/10.1007/978-1-0716-3601-5,
© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer
Nature 2024
469
BIOACTIVE EXTRACTION AND APPLICATION IN FOOD AND NUTRACEUTICAL INDUSTRIES
470 Index
C 300, 304, 311, 336, 340, 341, 343, 344, 347,
349, 358, 374, 420, 453, 456
Carbon ...................................................... 7, 8, 10, 31, 49, Distribution .....................................................25, 81, 144,
50, 95, 130, 132, 144, 156, 160, 161, 163, 164, 154, 166, 167, 169, 183, 209–211, 258, 263,
166, 236, 243, 264, 265, 279, 283, 287, 289, 300, 400, 451
316, 317, 321, 322, 360, 380, 381, 394, 395, Drug ....................................... 7, 25, 32, 34, 55, 57, 106,
411, 421, 426, 428 119, 121, 145, 164, 174, 177–179, 195, 196,
Carotenoids ............................................ 2, 13–16, 34, 50, 217, 285, 312, 374, 451, 453, 454, 461
53, 57, 59, 62, 80, 86, 89, 96, 105, 106, 117, 118,
134, 140, 143, 144, 147, 167, 174, 175, 177, E
183, 185, 190, 194, 203, 213, 217, 233, 234,
237, 243, 256–260, 283, 289, 300, 302–304, Economy.........................................................12, 204, 325
306, 307, 310, 311, 316, 319, 321–323, 393, Edible................................................. 51, 53, 61, 69, 113,
406, 426, 452, 453 115, 119, 164, 288, 289, 299, 310, 334, 358–360
Catalysts ............................................................9, 164, 365 Efficiency .................................................. 3–9, 11, 25, 48,
Chain ......................................................9, 51, 52, 54, 57, 69, 80, 86, 87, 89–93, 95, 96, 105, 106, 120, 122,
92, 163, 232, 240, 257, 299, 310, 325, 350, 357, 130, 134–137, 141, 142, 160, 162, 176, 177,
358, 363–365, 407 182, 183, 185, 188, 190, 193, 203, 204, 216,
Channels ...................................................... 206, 277, 339 217, 224, 230, 231, 245, 258, 261, 265–267,
Chromatographic ..............................................12, 22, 26, 276, 278–280, 282, 291, 293, 314, 318, 320,
28, 29, 283, 396, 436 323, 359, 361, 382, 384, 396, 398–400, 403,
Circular ................................................................. 204, 344 408, 410, 421, 422, 424–427, 429, 430,
Coatings.........................14, 31, 147, 165, 292, 293, 344 435–437, 440, 460–462, 464, 465
Commercialization ............................................... 413, 440 Emerging ...................................................... 2, 5, 7, 9, 69,
Comparison ..........................................31, 49, 51, 91, 92, 147, 255, 258–260, 269, 284, 313–315,
105, 113, 131, 136–138, 144, 145, 160, 204, 318–325, 419, 441
212, 216, 282, 365, 382, 442 Emulsions ................................... 13–15, 65, 66, 167, 191
Compliance.................................................................... 425 Encapsulation .................................................13, 146, 147
Compounds ..........................................1, 21, 45, 79, 103, Entry .............................................................................. 343
129, 155, 173, 202, 223, 255, 276, 301, 337, Environmental............................................ 4, 6, 7, 13, 16,
374, 391, 424, 449 46, 91, 105, 129, 137, 138, 155–158, 163, 169,
Consumer .......................................... 1, 13, 34, 164, 204, 170, 184, 185, 189, 194, 204, 236, 261, 262,
218, 256, 259, 270, 423, 430, 443 278, 282–284, 300, 301, 304, 322, 335, 337,
Control ....................................................... 16, 33, 66–68, 343, 347, 366, 383, 392, 393, 410, 421, 422, 455
107, 110, 191, 218, 224, 226, 246, 257, 265, Enzyme ..................................................... 6, 9, 22, 32, 33,
280, 313, 316, 335, 336, 344, 345, 347, 353, 54, 60, 66, 117, 134, 135, 138, 139, 143, 147,
356, 357, 360, 366, 394, 397, 399–401, 404, 166, 174, 175, 177, 180, 182–191, 193–195,
405, 413, 420, 428, 430, 437, 443, 458 197, 203, 206, 218, 236, 239, 245, 246, 248,
Cost-effectiveness.................................... 5, 239, 313, 422 256, 266, 269, 270, 303, 304, 309, 312, 323,
324, 334, 337, 348–350, 355–358, 360, 364,
D 365, 422, 427, 430, 439, 440, 457, 459, 462
Evaluation................... 65, 182, 262, 263, 362, 462, 463
Deep eutectic solvent (DES) ...................... 137, 162, 402 Extraction ..................................................... 2, 22, 46, 79,
Delivery........................................... 13–14, 106, 399, 461 103, 129, 153, 174, 202, 224, 256, 275, 301,
Demand ..................................................5, 15, 16, 34, 36, 334, 375, 391, 421, 451
69, 79, 158, 175, 177, 182, 204, 218, 219, 224,
237, 242, 246, 255, 270, 337, 443 F
Design....................................................... 16, 89, 91, 170,
207–211, 227, 228, 277, 278, 282, 288, 293, Fatty ............................................. 7, 9, 14, 16, 25, 33–35,
394, 397, 399, 401, 406, 410–412, 429, 47–50, 52, 54, 60, 61, 66, 67, 97, 140, 143, 167,
432–434, 437, 439, 440, 444, 460, 463 177, 212, 238, 241, 242, 245, 248, 278, 291,
Disease .................................................... 2, 14, 16, 33–37, 303, 304, 309, 310, 324, 340, 358, 360,
45, 55, 59, 61–63, 106, 117, 174–176, 195, 196, 363–365, 397, 420, 423, 430, 440, 443
232, 242, 244, 246, 247, 255–257, 260, 263, Films ................................................. 14, 61, 69, 113, 181
Index 471
Flavonoids .......................................................2, 9, 14, 25, 300, 303–313, 333–336, 348, 358, 360, 362,
33, 34, 45, 62, 83, 85, 94, 96, 105, 117, 118, 120, 363, 365, 366, 391, 420, 424, 426, 433, 449,
129, 138–141, 143, 145, 174, 175, 177, 180, 451, 452, 454, 456
185, 186, 193, 196, 212, 213, 215, 217, 234, High-performance liquid chromatography
236, 237, 244, 256, 257, 259, 260, 262, 263, (HPLC)........................................... 12, 26, 29, 38,
266, 268, 286, 288, 302–309, 312, 315, 316, 143, 145, 156, 262, 264, 265, 267, 277, 282, 436
319, 322, 338, 393, 397, 400, 426, 437, 444,
461–463 I
Fluid.............................................................. 7, 10, 11, 22,
Impact....................................................... 7, 8, 22, 24, 33,
50, 95, 104, 106, 111, 121–123, 129–147, 156, 34, 47–49, 55, 67, 87, 89, 91, 105, 134, 136, 144,
159–162, 169, 178, 182, 196, 203, 217, 226, 155–157, 162, 163, 167, 185–188, 202, 203,
228, 245, 265, 275, 276, 278, 282, 285–287,
208, 212, 218, 224, 229, 230, 235, 241, 246,
292–293, 301, 316, 318, 321, 322, 358, 377, 256, 257, 260, 282, 300, 301, 322, 338, 343,
380–383, 392–398, 400–402, 410, 421, 423, 346–348, 353, 360, 377, 392, 403, 410, 412,
426, 428, 429, 431, 442
421–423, 434, 436, 437, 440, 450, 452, 454
Food.............................................................. 2, 21, 50, 96, Innovation ................................................... 382, 423, 465
103, 146, 153, 173, 201, 223, 255, 278, 299, Interactions.............................................9, 11, 15, 57, 82,
336, 374, 391, 419, 449
87–89, 132, 136, 138, 145, 180, 185, 186, 229,
Footprint ....................................................................... 282 257, 261, 276, 335, 337, 340, 346, 347, 352,
Form ............................................................. 8, 16, 34, 36, 360, 365, 366, 383, 393, 396, 398, 403, 404,
48, 51, 56, 88, 91, 109, 123, 130, 139, 143, 161, 407, 427, 440, 444, 450, 451, 456–460, 462–465
163, 166, 168, 178, 180, 182, 185, 187, 189,
In vivo ........................................12, 28, 38, 57, 120, 121,
205, 209, 225, 231, 256, 265, 266, 271, 281, 143, 145, 262, 263, 457
289, 292, 309, 310, 334, 336, 351–353, 358,
362, 383, 396, 401, 460, 462 L
Formulation................................................ 13, 14, 26, 36,
38, 69, 79, 145, 259, 263, 304, 312, 324, 358 Labeling ..........................................................22, 461, 462
Fourier transform infrared spectroscopy Liquid ...................................................5, 31, 48, 81, 105,
(FTIR)............................... 12, 26, 30, 31, 93, 182 130, 154, 182, 201, 224, 261, 276, 304, 354,
Functional................................................. 1, 2, 13–16, 31, 373, 392, 427, 463
33–35, 45, 46, 51, 56, 61, 69, 105, 115, 119, 129, Literature ...................................................... 38, 118, 119,
130, 135, 144, 167, 181, 184, 188, 217, 219, 190, 319, 321, 325, 453
242, 246, 248, 257, 259, 260, 263, 264,
M
269–271, 301, 302, 304, 310, 324, 339, 343,
348, 351, 358, 360, 419, 443, 444, 449–451, Marine source.........................46, 47, 50–64, 68, 69, 423
460, 462, 464 Market.................................. 61, 117, 177, 182, 218, 393
Future ........................................ 16, 36–38, 69, 147, 163, Mass ............................................. 6–8, 11, 14, 29–32, 38,
169, 195, 196, 218–219, 264, 277, 325, 348, 47, 48, 81, 89, 91, 105, 108, 109, 111, 123,
356, 381, 439, 463–465 130–136, 139, 141, 143, 158, 185, 188, 202,
224, 226, 230, 232, 237, 261, 267, 270, 276,
G 279, 281, 282, 284, 288, 317–319, 322–324,
Gas chromatography (GC) ................................... 12, 108, 354, 360, 364, 379, 380, 382–384, 392, 393,
143, 275, 282, 284, 436 395–398, 402–404, 411, 428, 436, 459
Good manufacturing practice (GMP) ......................... 425 Mechanisms ..................................... 8, 33, 34, 36, 56, 57,
Green technology................................................ 105, 119, 59, 81–82, 86, 92, 93, 107–112, 155, 156, 162,
134, 137, 201–219, 255, 266, 321, 391, 392, 169, 170, 189, 191, 195, 205–207, 218, 219,
421, 442 227, 237, 242, 246, 269, 277–279, 293,
334–342, 344, 348, 380, 385, 396, 404–405,
H 407–409, 429, 442, 450, 455, 457, 458, 460, 461
Methods................................................2, 22, 46, 80, 104,
Health ....................................................... 1, 2, 13, 15, 16,
130, 153, 176, 202, 224, 256, 277, 301, 334,
24, 32–34, 38, 46, 50, 55, 60–63, 69, 105, 113,
375, 392, 421, 455
117, 145, 155, 156, 163, 185, 187, 189, 195,
219, 224, 236, 242, 246, 255–257, 259, 285,
472 Index
Microbial ................................................54, 57, 140, 205, Polyphenols .....................................................2, 9, 13, 14,
206, 224, 236, 239–241, 259, 263, 265, 301, 16, 24, 25, 30, 49, 51, 80, 86–88, 94, 105, 115,
337, 339, 346–349, 360, 454 120, 137, 139–141, 143, 146, 155, 175, 177,
Microencapsulation .............................146, 147, 292, 293 183–188, 196, 197, 203, 207, 208, 212–215,
Microwave .................................................... 8, 22, 48, 49, 217, 231, 233, 235, 240, 242–244, 257, 258,
80–82, 86–93, 95, 96, 104, 122, 134, 138, 142, 261, 264, 268, 270, 282, 284, 286–288,
147, 179, 217, 256, 267, 268, 270, 315, 302–305, 307–309, 312, 315, 319, 322–324,
319–321, 323, 379, 380, 403–405, 413, 428, 393, 397, 400, 406, 426, 433
437, 442 Positioning .................................................................... 398
Prebiotics ............................................334, 348–350, 357,
N 358, 362, 363, 365, 366
Preservation .................................................. 67, 113, 117,
Nanoencapsulation.......................................146, 292–293
Nanoparticles....................................................36, 65, 283 137, 184, 203, 204, 206, 216, 224, 237, 239,
Novel ............................................. 6, 8, 9, 11, 22, 46, 50, 245, 259, 380, 421, 428
Pressurised liquid ..............................................10, 11, 28,
55, 92, 129–147, 174, 182, 194, 202–204, 218,
224, 232, 236, 245, 248, 255–270, 276, 282, 104, 217, 261, 264, 268, 275–293, 316, 318,
283, 287, 301, 310, 312, 313, 318, 324, 346, 323, 324, 395, 399, 429
Pressurized.................. 10, 131, 140, 318, 377, 394, 400
391–413, 423, 431, 437, 439, 443, 450, 451,
456–458, 462, 463 Prevention ..........................................16, 33–37, 59, 202,
Novel solvent........................................................ 153–170 246, 255, 256, 271, 311, 358
Nutraceutical ................................................ 9, 13, 15, 16, Probiotics.........................................................33, 68, 263,
334, 348–350, 357, 358, 362–366, 420, 424
21–38, 45–48, 50–69, 80, 88, 96, 117, 129–147,
153, 167, 169, 170, 176, 180, 181, 202–204, Processing ................................................... 3, 7, 9, 13, 14,
219, 225, 232, 237, 241–248, 259–265, 270, 46, 49, 50, 55, 80, 105, 113, 122, 166, 167, 183,
187, 190, 191, 202–210, 216, 218, 219, 223,
271, 288, 292, 304, 324, 325, 333–366,
373–385, 391–413, 419–445, 449–465 224, 228, 237, 239–241, 245, 246, 248, 260,
Nutritional .................................................... 1, 14, 15, 61, 264, 266, 269, 289, 299–304, 318, 321, 322,
325, 336, 340, 343, 359, 378, 382, 385, 395,
65–68, 173, 181, 217, 218, 223, 224, 231, 235,
237, 248, 256, 260, 261, 302, 310, 312, 324, 401, 403, 411, 413, 425, 428, 457, 459, 460, 464
348, 350, 358, 366, 420, 443, 444, 458 Product .................................................1, 22, 61, 79, 103,
142, 156, 173, 203, 225, 256, 278, 301, 335,
O 374, 391, 420, 451
Omega-3......................14, 16, 33–35, 50, 167, 420, 423 Q

Optimization ................................................ 9, 11, 48, 69,
87, 88, 97, 111, 140, 141, 190, 258, 281, 322, Quality ........................................... 6, 7, 9, 14–16, 22, 24,
352, 360, 401, 419–445, 459, 461, 463 25, 28, 34, 36, 38, 46, 47, 49, 66–69, 93, 95, 115,
123, 130, 135, 138, 142, 144, 158, 164, 174,
P 178, 180, 181, 183, 185, 188–190, 193,
202–205, 223, 224, 234, 237, 239–241, 245,
Packaging................................................... 13–15, 69, 181 246, 248, 256, 258, 265, 266, 269, 270, 291,
Patents .................................................................. 242, 453 292, 336, 340, 350, 352, 362, 374, 375, 378,
Pharmaceutical ..................................................1, 2, 9, 12, 380, 382, 385, 401, 408, 422–424, 428–431,
15, 16, 22, 38, 46, 69, 96, 103–105, 112, 113, 440, 443, 457, 458
115, 117, 119–121, 123, 137, 140, 141, 143,
146, 153, 168, 173, 175–177, 184, 188, 190, R
194, 195, 217, 246, 248, 259, 267, 270, 278,
Recovery .......................................... 9, 13, 46, 48, 49, 59,
283, 285, 304, 310, 311, 324, 373, 382, 398,
409, 440, 455, 463 79, 80, 87, 96, 106–108, 117, 130, 134, 136,
Pharmacokinetics ................................................. 145, 462 137, 140, 156, 164–166, 175, 187, 189, 191,
194, 203, 236, 242, 245, 248, 258, 269, 277,
Phytochemicals......................................... 2, 9, 12, 13, 15,
25, 26, 29, 30, 32–35, 45, 46, 62, 95, 105, 112, 278, 281–283, 285–291, 318, 319, 321, 323,
113, 130, 131, 137–141, 179, 183, 188, 191, 378, 383, 391, 401, 406, 408, 409
234, 259, 260, 288, 304, 310, 408, 409, 419,
424, 443, 444, 449, 452, 456, 464, 465
Index 473
Reduction ...................................... 2, 7–9, 14, 28, 50, 62, Standardization ..................................... 38, 286–288, 430
109, 112, 119, 146, 202, 240, 241, 256, 355, Strategies.................................................11, 22, 144, 147,
360, 427, 437, 461 162, 165, 204, 212, 216, 219, 260, 334, 337,
Regulations........................................................9, 90, 107, 348, 358, 392, 421–423, 433, 437, 458, 462
246, 256, 334, 336, 340, 423 Structure ...............................................1, 5, 7, 14, 28, 30,
Residues ........................................... 3, 13, 14, 50, 55, 57, 31, 34, 38, 47, 56, 57, 61, 88, 109, 118, 120, 132,
144, 146, 154, 161, 177, 183, 187, 204, 237, 138, 139, 163, 180, 182, 187, 190, 191, 211,
264, 284–287, 300, 315, 317–319, 322, 325, 241, 257, 264, 266, 281, 282, 289, 313, 338,
338–341, 343, 344, 428 351, 352, 360, 375, 401, 403, 407–410, 422,
Risk .................................................. 2, 13, 14, 34, 35, 55, 423, 431, 439, 450–453, 455, 457, 462, 463
56, 61, 62, 86, 105, 117, 162, 163, 175, 218, Sub critical solvent ........................................................ 398
219, 246, 256, 277, 304, 311–313, 420, 421, Super critical solvent ........................................7, 136, 392
426, 456 Sustainable ................................................ 6, 8, 11, 13, 15,
16, 34, 96, 147, 154, 167, 203, 204, 212, 216,
S 237, 248, 256, 301, 325, 349, 373–385, 392,
Safety analysis ........................................... 14, 15, 38, 145, 395, 430, 441
146, 154, 189, 322, 420, 423, 424, 428, 443
T
Scientific..................................................2, 12, 22, 33, 36,
38, 82, 104, 169, 176, 184, 218, 224, 270, 288, Therapeutic.................................... 15, 34, 36, 38, 60, 61,
420, 453, 458 143, 146, 180, 188, 210, 246, 260, 420, 444, 450
Screening ......................................... 12, 26, 32, 236, 264, Toxicity .......................................... 27, 34, 36, 38, 54, 59,
281, 357, 451, 454, 455, 460–463 105, 106, 143, 146, 156, 157, 163, 164, 182,
Segmentation ................................................................ 210 183, 196, 257, 317, 324, 340, 360, 424, 450,
Sensory .............................................61, 64–68, 144, 181, 453, 455, 459, 462–465
195, 223, 282, 363, 452, 454, 459 Traceability ........................................................... 141, 164
Shelf-life ...................................................... 13–15, 50, 66, Trends .................................... 46, 88, 169, 255, 259, 324
113, 147, 176, 239, 241, 322, 346, 360
Solvents.................................................3, 22, 46, 79, 104, U
130, 153, 176, 202, 224, 256, 275, 313, 357, Ultrasound......................................... 7, 22, 47, 104, 134,
373, 391, 421, 458 182, 203, 267, 289, 319, 378, 400, 421
Sources..................................................1, 21, 46, 95, 103,
UV-visible (UV-Vis) ................................. 29, 30, 93, 265
140, 177, 217, 232, 259, 286, 300, 347, 376,
394, 420, 451 W
Spectrometry ............................................ 31, 32, 38, 143,
185, 260, 265, 284, 288, 436, 459 Waste..................................................... 6, 7, 9, 11–14, 16,
Spectroscopic.............................................................29, 30 49, 50, 83, 85, 91, 105, 113, 115, 116, 139, 142,
Stability ........................................6, 9, 11, 14, 36, 47, 48, 153, 159, 160, 164–166, 174, 181, 185, 189,
50, 61, 64–68, 91, 119, 129, 137, 143, 146, 147, 214, 224, 236–238, 259, 266, 277, 292,
168, 184, 187, 193, 194, 212, 218, 263, 316, 299–305, 310–325, 334, 336, 377, 379, 392,
319, 321, 335, 336, 344, 404, 424, 427, 430, 395, 422, 424, 459
440, 455

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Methods and Protocols

Tanmay Sarkar · Siddhartha Pati Editors

For further volumes:

ISSN 2662-950X ISSN 2662-9518 (electronic)

Paper in this product is recyclable.

University of Campinas Anderson S. Sant’Ana

Chapter 10 explores pressurized liquid extraction, which utilizes high-pressure solvents

Malda, India Tanmay Sarkar

Preface to the series. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v

1 Technologies for Extraction of Bioactive Compounds

12 Pressurized Liquid Extraction for the Isolation of Bioactive Compounds . . . . . . 275

OLADAYO ADEYI • Department of Chemical Engineering, Michael Okpara University of

KHAOULA ELHADEF • Laboratory of Microbial Biotechnology and Engineering Enzymes

NILESH PRAKASH NIRMAL • Institute of Nutrition, Mahidol University, Salaya, Nakhon

SOUMEIA ZEGHOUD • Department of Process Engineering and Petrochemical, Faculty of

Technologies for Extraction of Bioactive Compounds and Its

Key words Bioactive, Health promotion, Extraction, Application

Consumers’ attitude toward a healthy and well-balanced diet is

fruits, vegetables, whole grains, millets, etc. provide health benefits

Plant derived bioactive

Conventional extraction Advanced extraction

Application of bioactiove compounds

Fig. 1 Schematic representation of bioactive compounds and their applications

2 Extraction of Bioactive Compounds

Extraction is one of the most important unit operations followed in

Conventional extraction techniques Advanced extraction techniques

Conventional Extraction Advanced Extraction

Pulsed electric field

Fig. 2 Conventional and advanced extraction methods

growing demand for emerging extraction techniques that have

3 Conventional Extraction Techniques

Conventional or classical techniques such as maceration, Soxhlet

3.2 Percolation Percolation is a more efficient extraction method than maceration

4 Advanced Extraction Techniques

The attention toward novel approaches for the extraction and

identification of the process [13]. Owing to the high yield, reduced

4.2 Ultrasound- Ultrasound-assisted extraction (UAE) is extensively used in the

phase. Ultrasonic treatment causes mechanical impact on the cell

4.3 Microwave- Microwave-assisted extraction has been accepted as an effective

rates, increased extract yield, short processing time, energy saving,

4.5 Enzyme-Assisted Enzyme-assisted extraction is a novel approach for extracting bio

molecular compounds such as phenols and polyphenols, oils and

extraction techniques. It is also referred as pressurized solvent

4.7 Combination of The combination of different extraction techniques is a desirable

ultrasound-assisted enzymatic extraction (UAEE), microwave-

5 Purification of the Bioactive Compounds

Plant materials are a multi-component mixture that contains

6 Extraction of Bioactive Compounds from Agro-industrial Waste

The huge quantity of waste produced from agricultural and food

proteins, polysaccharides, fibers, lipids, carbohydrates, peptides,

7 Nano Emulsion as Potential Delivery Systems for Bioactive Compounds

Nanotechnology has found enormous applications in different sec-

Source Residue Bioactive components Bioactivity References

Lipophilic bioactive compounds such as carotenoids, omega-3

8 Application of Bioactive Compounds

8.3 Pharmaceuticals Phytochemicals are biologically active, naturally occurring chemical

their biological activity for conventional medicines in the preven-

9 Conclusion and Future Perspective

Bioactive compounds are recognized due to their pharmacological