Egbuna, Chukwuebuka_ Ifemeje, Jonathan Chinenye_ Kumar, Shashank_ Udedi, Stanley Chidi - Phytochemistry, Volume 3 - Marine Sources, Industrial Applications, and Recent Advances-Apple Academic Press_CR

PHYTOCHEMISTRY
Volume 3
Marine Sources, Industrial Applications,
and Recent Advances
PHYTOCHEMISTRY
Volume 3
Marine Sources, Industrial Applications,
and Recent Advances
Edited by
Chukwuebuka Egbuna
Jonathan Chinenye Ifemeje, PhD
Shashank Kumar, PhD
Nadia Sharif, PhD
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Phytochemistry / edited by Chukwuebuka Egbuna, Jonathan Chinenye Ifemeje, PhD,

Shashank Kumar, PhD, Nadia Sharif, PhD.
Includes bibliographical references and indexes.
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1. Botanical chemistry. I. Egbuna, Chukwuebuka, editor II. Ifemeje, Jonathan Chinenye, editor
III. Kumar, Shashank, editor IV. Sharif, Nadia, editor
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ABOUT THE EDITORS
Chukwuebuka Egbuna
Chukwuebuka Egbuna is a chartered chemist, a chemical analyst, and an
academic researcher. He is a member of the Institute of Chartered Chemists
of Nigeria (ICCON), the Nigerian Society of Biochemistry and Molecular
Biology (NSBMB), the Royal Society of Chemistry (RSC), United Kingdom,
and the Society of Quality Assurance (SQA), USA. He has been engaged in
a number of roles at New Divine Favor Pharmaceutical Industry Limited,
Akuzor Nkpor, Anambra State, Nigeria, and Chukwuemeka Odumegwu
Ojukwu University (COOU), Nigeria. He has attended series of conferences
and workshops and has collaboratively worked and published quite a number
of research articles in the domain of phytochemistry. He has edited books
with top publishers such as Springer Nature and Elsevier. He is a reviewer
and an editorial board member for various journals, including serving as a
website administrator for the Tropical Journal of Applied Natural Sciences
(TJANS), a journal of the faculty of Natural Sciences, COOU. His primary
research interests are in phytochemistry, food and medicinal chemistry,
analytical chemistry, and nutrition and toxicology. He obtained his BSc
and MSc degrees in biochemistry at Chukwuemeka Odumegwu Ojukwu
University.
Jonathan Chinenye Ifemeje, PhD

Jonathan Chinenye Ifemeje, PhD, is an Associate Professor in the Department
of Biochemistry, Faculty of Natural Sciences, Chukwuemeka Odumegwu
Ojukwu University, Nigeria. He obtained his PhD in applied biochemistry
from Nnamdi Azikiwe University, Awka, Nigeria, and his MSc degree in
nutrition and toxicology from the University of Port-Harcourt, Nigeria.
He has to his credits over 40 publications in both local and international
journals. Dr. Ifemeje is currently the Coordinator, Students Industrial
Work Experience Scheme (SIWES), COOU, and has served as an external
examiner for various institutions. He is the Managing Editor of the Tropical
Journal of Applied Natural Sciences and is serving as a reviewer and an
editorial board member for various journals. He has worked extensively in
the area of phytochemistry, nutrition, and toxicology. He is a member of
various institutes, including the Institute of Chartered Chemists of Nigeria
vi About the Editors
(ICCON), the Nigerian Society of Biochemistry and Molecular Biology

(NSBMB), and the Society of Quality Assurance (SQA).
Shashank Kumar, PhD

Shashank Kumar, PhD, is working as Assistant Professor at the Center for
Biochemistry and Microbial Sciences, Central University of Punjab, Bathinda,
India. He obtained his BSc, MSc, and PhD Biochemistry from the Department
of Biochemistry, University of Allahabad, India. He worked as Postdoctoral
Fellow at the Department of Biochemistry, King George’s Medical University,
Lucknow, India. Dr. Kumar has about 60 published scientific papers/reviews/
editorial articles/book chapters in various national and international peer-
reviewed journals and has been cited more than 1200 times. He has edited
several books on topics such as the “concepts in cell signaling,” “carbohydrate
metabolism: theory and practical approach,” and so forth. He has expertise
in the areas of free radical biology, cancer biology, characterization of plant
natural products, xenobiotic metabolism, and microbiology. He is familiar
with many biochemical techniques such as spectrophotometry, enzyme-linked
immunosorbent assay, electrophoresis, polymerase chain reaction, real-time
polymerase chain reaction, flow cytometry, thin-layer chromatography,
high-performance liquid chromatography, liquid chromatography–mass
spectrometry, cell culture, and microbiological techniques. He has presented
his research findings at more than 25 national/international conferences and
attended about 30 workshops at different universities and medical colleges
throughout the India. Dr. Kumar is a life time member of Italo-Latin American
Society of Ethnomedicine, and the Indian Sciences Congress Association, and
member of the Asian Council of Science Editors, Dubai, UAE, and Publication
Integrity and Ethics, London. He has been awarded the Junior/Senior and
Research Associate Fellowships formulated and funded by various Indian
agencies, such as Indian Council of Medical Research, University Grants
Commission, Council of Scientific and Industrial Research India. Dr. Kumar
laboratory has been funded by the University Grant Commission, India, and
the Department of Science and Technology, India, for working on effects of
various phytochemicals on cancer cell signaling pathway inhibition.
Nadia Sharif, PhD

Nadia Sharif, PhD, is currently working as a Visiting Lecturer at the Biotech-
nology Department of Lahore College for Women University, Lahore,
Pakistan. She obtained her MSc degree in Zoology from Bahauddin Zakariya
About the Editors vii
University, Multan, Pakistan, and did her PhD in Biotechnology, Department

of Lahore College for Women University, Lahore, Pakistan. She has been
awarded the Indigenous and International Fellowship (IRSIP) by the Higher
Education Commission, Pakistan. She has visited the Chemical and Biochem-
ical Engineering Department of The Johns Hopkins University, USA, for her
PhD research. She has about 20 published scientific papers/reviews/book
chapters in various national/international peer-reviewed journals. Dr Nadia
Sharif is serving as a reviewer to the British Microbiology Research Journal,
African Journal of Microbiology Research and Natural Product Journal. She
has expertise in areas of phycology, industrial biotechnology, microbiology,
molecular biology, biostatistics, and biochemistry. She is familiar with many
biochemical techniques such as spectrophotometry, electrophoresis, PCR,
ASE, TLC, HPLC, GC-MS, cell culture, and microbiological techniques. She
has a great interest in working for the sustainable use of natural resources.
She has presented her research findings in more than ten national conferences
and attended about 30 workshops at the majority of major universities and
colleges throughout Pakistan.
CONTENTS
Contributors.................................................................................................xiii
Abbreviations.............................................................................................. xvii
Foreword...................................................................................................... xxi
Preface....................................................................................................... xxiii
PART I: Marine Sources of Secondary Metabolites............................... 1

1. Phytochemicals of Marine Origins.................................................................3
Nadia Sharif, Sana Nayab, Syeda Nazish Arshad, Neelma Munir, and Shagufta Naz
2. Marine Sponge Alkaloids: A Source of Novel Anticancer Agents..............35

Musarat Amina and Hanan M. Al-Yousef
3. Marine Antioxidants and Assay Methods....................................................65

Onyeka Kingsley Nwosu and David Okechukwu Okeke
4. Extraction of Marine Phytochemicals: Methods and Techniques.............89

Nadia Sharif, Neelma Munir, and Shagufta Naz
PART II: Industrial and Medicinal Applications of

Phytochemicals............................................................................... 105
5. Biotechnology Approach to the Production of Phytochemicals:
An Introduction............................................................................................107
Hameed Shah, Andrew G. Mtewa, Chukwuebuka Egbuna, Anywar Godwin,
and Duncan C. Sesaazi
6. Secondary Metabolites Accumulation and Production Through
In Vitro Cultures...........................................................................................131
Hussien M. Daffalla and Azza Migdam Elsheikh
7. Practical Processes Involved in the Production of Phytochemicals by

Plant Tissue Cultures...................................................................................187
Hanan M. Al-Yousef
8. Medicinal and Industrial Applications of Bromelain...............................205

Ramesh Kumar and Abhay K. Pandey
x Contents
9. Cysteine Proteases from Plants and Their Applications..........................221

Juan Abreu Payrol, Walter David Obregón, Juliana Cotabarren,
T. B. Mutsauri, and Inidia Rubio Vargas
10. Phytotherapy and Encapsulation...............................................................241
Şaban Keskin, Merve Keskin, and Sevgi Kolaylı
11. Effective Processing Methods for Fruits and Vegetables..........................253

Maria Aslam, Sidra Khalid, and Hafsa Kamran
PART III: Environmental Concerns and Eco-Friendly

Control Measures........................................................................... 265
12. Effects of Environmental Factors on the Accumulation of
Phytochemicals in Plants.............................................................................267
Sechene Stanley Gololo
13. Effects of Environment on the Chemical Constituents and

Biological Characteristics of Some Medicinal Plants...............................279
Vinesh Kumar and Yogita Sharma
14. Phytochelatins and Heavy Metal Tolerance in Plants..............................293

Maria Catherine B. Otero and Genevieve D. Tupas
15. Phytochemical Biopesticides.......................................................................303

Olumayowa Vincent Oriyomi
16. A Sustainable Approach in Integrated Pest Management:

Role of Phytomolecules as Biopesticide......................................................325
Rakesh Kumar Gupta, Prem Prakash Kushwaha, and Shashank Kumar
17. Essential Oils in Pest Control and Disease Management.........................341

Arvind Saroj, Atul Kumar Srivastava, Ashish Kumar Nayak,
C. S. Chanotiya, and A. Samad
18. Inhibition of Mild Steel Corrosion in Acidic Media by

Phytochemicals.............................................................................................369
Basu Maan Daas
PART IV: Recent Advances................................................................... 391

19. Novel Terpenoids as Anticancer Stem Cell Agents....................................393
Rebati Malik, Santosh Kumar Maurya, Prem Prakash Kushwaha,
Pushpendra Singh, and Shashank Kumar
Contents xi
20. Evaluation of the Phytohemagglutinins Activities of Echinacea

Species in Ontogenesis.................................................................................421
Sergey V. Pospelov
Index...............................................................................................................441
CONTRIBUTORS
Musarat Amina
Department of Pharmacognosy, College of Pharmacy, King Saud University, P. O. Box-2457,
Riyadh 11451, Saudi Arabia
Hanan M. Al-Yousef
Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia
Syeda Nazish Arshad

Department of Chemistry, Lahore College for Women University, Lahore, Pakistan
Maria Aslam
University Institute of Diet and Nutritional Sciences, Faculty of Allied Health Sciences,
University of Lahore, Pakistan
C. S. Chanotiya
Department of Analytical Chemistry, Central Institute of Medicinal and Aromatic Plants, Lucknow, India
Juliana Cotabarren
Centro de Investigación de Proteínas Vegetales (CIPROVE), Departamento de Ciencias Biológicas,
Facultad de Ciencias Exactas, Universidad Nacional de La Plata, 47 y 115 s/N, B1900AVW,
La Plata, Argentina
Basu Maan Daas

Department of Chemistry, Netaji Subhash Mahavidyalaya, Udaipur, Tripura, India
Hussien M. Daffalla
National Centre for Research, Commission for Biotechnology and Genetic Engineering,
Mohamed Nageeb St. No. 61, 11111 Khartoum, Khartoum, Sudan
Chukwuebuka Egbuna
Department of Biochemistry, Faculty of Natural Sciences, Chukwuemeka Odumegwu
Ojukwu University, Anambra State 431124, Nigeria
Azza Migdam Elsheikh

National Centre for Research, Commission for Biotechnology and Genetic Engineering,
Mohamed Nageeb St. No. 61, 11111 Khartoum, Khartoum, Sudan
Anywar Godwin
Makerere University, Department of Plant Sciences, Microbiology & Biotechnology, P. O. Box 7062,
Kampala Uganda
Sechene Stanley Gololo

Department of Biochemistry, School of Science and Technology, Sefako Makgatho Health
Sciences University, Ga-Rankuwa, Pretoria, South Africa
Rakesh Kumar Gupta

School of Environment and Earth Science, Environmental Science and Technology,
Central University of Punjab, Bathinda, Punjab, 151001, India
Hafsa Kamran
xiv Contributors
Merve Keskin
Department of Chemistry, Faculty of Science, Karadeniz Technical University, Trabzon, Turkey
Şaban Keskin
Department of Chemistry, Faculty of Science and Literature, Bilecik Şeyh Edebali University,
Bilecik, Turkey
Sidra Khalid
Sevgi Kolaylı
Department of Chemistry, Faculty of Science, Karadeniz Technical University, Trabzon, Turkey
Ramesh Kumar
Department of Biochemistry, University of Allahabad, Allahabad 211002, India
Shashank Kumar
School of Basic and Applied Sciences, Department of Biochemistry and Microbial Sciences,
Vinesh Kumar
Department of Sciences, Kids’ Science Academy, Roorkee, Uttarakhand, India
Prem Prakash Kushwaha

Rebati Malik
Santosh Kumar Maurya

Andrew G. Mtewa
Department of Chemistry, Institute of Technology, Malawi University of Science and Technology, Malawi
Department of Pharmacology and Therapeutics, Mbarara University of Science and Technology, Uganda
Neelma Munir
Department of Biotechnology, Lahore College for Women University, Lahore Pakistan
T. B. Mutsauri
Departamento de Farmacia, Instituto de Farmacia y Alimentos, Universidad de La Habana,
Cuba. Calle 222, entre 23 y 29, # 2317, La Coronela, La Lisa, La Habana, Cuba
Sana Nayab
Department of Chemistry, Lahore College for Women University, Lahore, Pakistan
Ashish Kumar Nayak

Department of Microbial Genomics and Diagnostic Laboratory, Regional Plant resource center,
Bhubaneshwar, India
Shagufta Naz
Onyeka Kingsley Nwosu

National Biosafety Management Agency (NBMA), Abuja, Nigeria
Contributors xv
Walter David Obregón

Centro de Investigación de Proteínas Vegetales (CIPROVE), Departamento de Ciencias Biológicas, Facultad
de Ciencias Exactas, Universidad Nacional de La Plata, 47 y 115 s/N, B1900AVW, La Plata, Argentina
David Okechukwu Okeke

Department of Applied Biochemistry, Nnamdi Azikiwe University, Awka, Nigeria
Olumayowa Vincent Oriyomi
Institute of Ecology and Environmental Studies, Obafemi Awolowo University, Ile- Ife, Osun State, Nigeria
Maria Catherine B. Otero

College of Medicine Research Center, Davao Medical School Foundation, Inc., Davao City, Philippines
Abhay K. Pandey
Department of Biochemistry, University of Allahabad, Allahabad 211002, India
Juan Abreu Payrol

Sergey V. Pospelov
Department of Agriculture & Agrochemistry, Faculty Agrotechnology & Ecology, Poltava State
Agrarian Academy, 1/3 Skovorody St., Poltava, 36003, Ukraine
A. Samad
Department of plant pathology, Central Institute of Medicinal and Aromatic Plants, Lucknow India
Arvind Saroj
Department of plant pathology, Central Institute of Medicinal and Aromatic Plants, Lucknow, India
Duncan C. Sesaazi
Department of Pharmacology and Therapeutics, Mbarara University of Science and Technology, Uganda
Hameed Shah
CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for
Nanoscience and Technology, Beijing, China
University of Chinese Academy of Science, Beijing 100049, China
Nadia Sharif
Yogita Sharma
Department of Sciences, Kids’ Science Academy, Roorkee, Uttarakhand, India
Pushpendra Singh
National Institute of Pathology, New Delhi, India
Atul Kumar Srivastava

Department of plant pathology, Central Institute of Medicinal and Aromatic Plants, Lucknow India
Genevieve D. Tupas
College of Medicine, Department of Pharmacology, Davao Medical School Foundation, Inc.,
Davao City, Philippines
Inidia Rubio Vargas

ABBREVIATIONS
4-HC 4-Hydroperoxycyclophosphamide
ACE Angiotensin-I-converting enzyme
ACGs Annonaceous acetogenins
AGPs Arabinogalactan-proteins
ALDH1 Aldehyde dehydrogenase 1
AMPs Antimicrobial peptides
AP-1 Activator protein 1
ASE Accelerated solvent extraction
ATP Adenosine triphosphate
ATRA All-trans-retinoic acid
Bcl-2 B-cell lymphoma 2
BT Biotransformation
CAA Cellular antioxidant activity
CA Caffeic acid
CE-MS Capillary electrophoresis coupled with mass spectrometry
CP Clonal propagation
CSC Cell suspension culture
CSCs Cancer stem cells
CS Chitosan
CVS Cell volume after sedimentation
DDT Dichlorodiphenyltrichloroethane
DMAPP Dimethylallyl pyrophosphate
DW Dry weight
EGCG Epigallocatechin-3-gallate
EOs Essential oils
EPA Environmental Protection Agency
ESTs Expressed sequence tags
FD Freeze drying
FW Fresh weight
GAGs Glycosaminoglycans
GC Gas chromatography
GFC Gel filtration chromatography
GS GSH synthetase
xviii Abbreviations
GSH Glutathione
GST Glutathione S-transferase
hGSH Homoglutathione
HIV Human immunodeficiency virus
HMB-PP (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate
HMG-CoA 3-hydroxy-3-methylglutaryl-CoA
hPC Homophytochelatins
HPLC-DAD High-Performance Liquid Chromatography-Diode-Array
Detection
HPLC High-performance liquid chromatography
HUVEC Human umbilical vein endothelial cell
IAA Indole-3-acetic acid
IBA Indole-3-butyric acid
IgE Immunoglobulin E
IPC Immobilized plant cells
IPP Isopentenyl pyrophosphate
LC Liquid chromatography
MAE Microwave-assisted extraction
MALDI Matrix-assisted laser desorption ionization
MALDI-TOF Matrix-assisted laser desorption/ionization time-of-flight
MAPK Mitogen-activated protein kinase
MD Maximum diameter
MDR Multidrug resistance
MEP Methylerythritol phosphate
MMP Metalloproteinase
MP Micropropagation
MS Mass spectrometry
MS MURASHIGE and Skoog
MT Metallothioneins
MVA Mevalonic acid
NAA 1-Naphthaleneacetic acid
NADPH Nicotinamide adenine dinucleotide phosphate
NAFDAC National Agency for Food and Drug Administration and
Control
NBT Nitroblue tetrazolium
NMR Nuclear magnetic resonance
NPs Nanoparticles
NSKE Neem seed kernel extract
Abbreviations xix
PAL Phenylalanine ammonia lyase

PCC Plant cell culture
PCD programmed cell death
PCS Phytochelatin synthase
PCs Phytochelatins
PCV Packed cell volume
PEF Pulsed-electric field extraction
PEs Phorbol esters
PFE Pressurized liquid extraction
PGE2 Prostaglandin E2
PGRs Plant growth regulators
PHWE Pressurized hot water extraction
PLE Pressurized liquid extraction
PMS Phenazine methosulphate
PPO Polyphenol oxidase
PTC Plant tissue culture
PUFAs Polyunsaturated fatty acids
QqQ-MS Triple Quadrupole-Mass Spectrometers
RA Retinoic acid
RARs Retinoic acid receptors
ROS Reactive oxygen species
RP-HPLC Reversed-phase high-performance liquid chromatography
rpm Revolutions per minute
RXRs Retinoid X receptors
SD Spray drying
SEg Somatic embryogenesis
SFC Supercritical fluid chromatography
SFD SPRAY FD
SFE Supercritical fluid extraction
SOD Superoxide dismutase
T-DNA Transfer DNA
Ti Tumor-inducing
TRAIL Tumor necrosis factor-related apoptosis-inducing ligand
UAE Ultrasound-assisted extraction
UV Ultraviolet
WIPO World Intellectual Property Organization
γECS γ-EC synthetase
FOREWORD
I am pleased to write the foreword for this book, Phytochemistry, Volume 3:

Marine Sources, Industrial Applications, and Recent Advances. The scope of
the volume is multifaceted with deep scientific content. The volume covers
trending topics from emerging areas of phytochemistry, including marine
phytochemistry, production of phytochemicals through in vitro culture,
phytochemical biopesticides, and other recent advances. Though there are a
few books in this category, the organization of this book particularly makes it
exceptional. I commend the editors and the chapter contributors for the good
work.
I do believe that everyone with related interest will find this book very
useful.
—Prof. Chukwunenye C. Anene
Dean Faculty of Natural Sciences,
Former Ag. Vice Chancellor
Chukwuemeka Odumegwu Ojukwu University, Nigeria
PREFACE
This book, Phytochemistry, Volume 3: Marine Sources, Industrial Applica-

tions, and Recent Advances, is a comprehensive book on phytochemistry
written by professionals from key institutions. The contributors are authors
of the finest academic traditions. The chapters were drawn carefully and
integrated sequentially to aid flow, consistency, and continuity. This book is
designed to be useful to researchers, teachers, students, phytochemists, plant
biochemists, food and medicinal chemists, marine chemists, nutritionists,
analytical chemists, industrialists, and plant biotechnologists.
The chapters were grouped into four parts: Part I: Marine Sources of
Secondary Metabolites; Part II: Industrial and Medicinal Applications of
Phytochemicals; Part III: Environmental Concerns and Eco-Friendly Control
Measures; and Part IV: Recent Advances.
In Part I, Chapter 1, Sharif et al. presents marine phytochemistry and
fundamentals. In Chapter 2, Musarat and Hanan discusses marine sponge
alkaloids with its potentials as anticancer agents. Chapter 3, by Nwosu and
Okeke, presents marine antioxidants and assay methods. In Chapter 4, Sharif
et al. details the various extraction and identification protocols.
In Part II, Chapter 5, Shah et al. presents an introduction on the biotech-
nological approach in the in vitro production of phytochemicals. Chapter 6
by Daffalla and Elsheikh provides a comprehensive detail on the in vitro
production of phytochemicals. Hanan presents the practical steps involved
in the production of phytochemicals by plant tissue culture in Chapter 7. In
Chapter 8, Kumar and Pandey presents the medicinal and industrial applica-
tions of bromelain. Chapter 9 by Payrol et al. is a detailed study on the
cysteine proteases from plants and their applications. Keskin et al. presents
phytotherapy and encapsulation in Chapter 10. Chapter 11 is a review of the
effective processing methods for fruits and vegetables.
In Part III, Chapter 12, Gololo presents the effects of environmental
factors on the accumulation of phytochemicals in plants. Kumar and Sharma
in Chapter 13 further emphasize the effects of the environmental factors on
the chemical constituents and the biological characteristics of some medic-
inal plants. Chapter 14 by Otero and Tupas is a review of phytochelatins and
heavy metal tolerance in plants as regards to phytoremediation. Chapter 15
by Oriyomi details the phytochemical biopesticides. In Chapter 16, Gupta
xxiv Preface
et al. discusses the sustainable approach to integrated pest management.

Saroj et al. in Chapter 17 presents the role of essential oils in pest control
and disease management. In Chapter 18, Daas presents the potentials of
phytochemicals in serving as mild steel eco-friendly corrosion inhibitors.
In Part IV, Chapter 19, Malik et al. presents findings of novel terpenoids
as an anticancer stem cell agents. Chapter 20 by Pospelov is a comprehensive
study of the phytohemagglutinins activities of Echinacea species in ontogen-
esis. The author presents novel findings spanning many years of research
including a new technique for conducting mass analysis for determining the
hemagglutination activity of Echinacea extracts.
As highlighted above, this book contains trending chapters written by
professionals. I recommend this book to researchers, students, and everyone
with interest in phytochemistry. My sincere appreciation goes to the chapter
contributors for their great contributions, patience, and cooperation during
the editorial process. I will remain grateful to the volunteer reviewers and my
co-editors. A special thanks goes to my family for their support and patience
during the editorial process. To the management of Apple Academic Press, I
will remain thankful for your guidance and support. To the readers, I appre-
ciate your thoughtfulness and would welcome reviews about the book with
an open heart. Thank you.
—Chukwuebuka Egbuna
MNSBMB, MICCON; AMRSC
Department of Biochemistry, Faculty of Natural Sciences,
Chukwuemeka Odumegwu Ojukwu University,
Anambra State- 431,124, Nigeria
E-mail: [email protected]
PART I
Marine Sources of Secondary Metabolites
CHAPTER 1
PHYTOCHEMICALS OF
MARINE ORIGINS
NADIA SHARIF1*, SANA NAYAB2, SYEDA NAZISH ARSHAD2,
NEELMA MUNIR1, and SHAGUFTA NAZ1
1
Department of Biotechnology, Lahore College for Women University,
Lahore, Pakistan, Tel.: +923237501948
2
Department of Chemistry, Lahore College for Women University,
Lahore, Pakistan
Corresponding author. E-mail: [email protected]
*
ORCID: https://orcid.org/0000-0002-8125-9270
*
ABSTRACT
Oceans are consecrated with a variety of plants and animals. Marine plants
consist of microscopic algae to towering underwater kelp forests and undu-
lating seagrass beds. They possess a stock of several types of natural products
as they are developed in a chemically rich environment. These compounds
are also biomedically important and are obtained from aquatic life by using
sophisticated extraction techniques. Due to their pharmacological activities,
these compounds are frequently used in the treatment of lethal diseases such
as cancer, arthritis, and acquired immunodeficiency syndrome. This chapter
throws light on the recent advances and outcomes in the field of marine
phytochemistry.
1.1 INTRODUCTION
The aquatic region has a wide range of temperature underneath freezing

temperatures in Antarctic waters to around 350°C in deep hydrothermal
vents, wide pressure range, oligotrophic to a eutrophic range of nutrients,
4 Phytochemistry, Volume 3
and far-reaching photic and non-photic regions. From microorganisms to

mammals, this wide-ranging variability has expedited a lot of speciation at
all phylogenetic levels. Although the marine biodiversity is far more than
terrestrial organisms, looking up in the making for the use of marine herbal
products as pharmaceutical marketers is yet in its beginning due to the priva-
tion of ethnomedical antiquity and the problems that are related to the assort-
ment of marine organisms (Faulkner, 1992). The progress of new secluded
operating machines and new diving techniques has resulted in the collection
of marine samples in recent times, from shallow to deep waters from which
about 5000 novel compounds have been derived. The marine plants grow
within the reach of sunlight for photosynthesis and nearby ice and salt water
surface. Algae that are the plentiful type of marine plants are essential for
a balanced ecosystem as well as for maintaining food chain (Teagle et al.,
2017). Water is the requirement of life. Earlier plants including algae were
designed in physiques of saline water that covers the prehistoric Earth. In
the Silurian period (about 441–410 million years past), some aquatic plants
started to grow on land; however, a lot of plants endured merely water based.
The importance of marine plants can be evaluated as they are fundamental
organisms to provide the nourishment in the food web and without their
existence, it was not possible for the marine animals to evolve or survive
(Carpenter, 2014).
Marine plants are comprised of microscopic algae to towering under-
water kelp forests and undulating seagrass beds. Regardless of this diversity,
the majority of marine plants play an enormous role in marine ecosystems.
The very base of the marine food web is formed by tiny algae that are only
visible when they multiply into large blooms. Bull kelp and giant kelp like
large and multicellular algae in marine environment provide the protection
and food to numerous marine species. Many marine animals are harbored
by the marine flowering plants or seagrasses, such as eelgrass or surfgrass.
Depending on the tolerance to light, air, waves, and predators grazing,
different marine plants thrive in different environments. The evolutionary
history of the marine plants is traced from the fossils found in the marine
sediments formed by algae. The plate tectonics involving changes in ocean
shape and continents movement has greatly affected the distribution of
marine plants. Marine plants included small unicellular organisms to big
convoluted systems. Two major types of marine plants included the algae/
seaweeds and seagrasses. Among these, algae/seaweeds are simple forms of
plants and are often microscopic, whereas seagrasses represent members of
the more complex plants (Rich and Maier, 2015).
Phytochemicals of Marine Origins 5
As sunlight is required for the manufacturing of food in all marine

plants, therefore, they generally grow in nearby water planes. Nutrients are
also collected from elements that fluxes wash up from sea floors. Marine
plants can acclimatize to a particular state, like inadequate light and the
caves underwater. Several are phosphorescent, producing chemical lights.
Phytoplanktons are the minutest marine plants. They are single-celled and
make the foundation of the marine food chain. Bacillariophyta diatoms
are glossy infinitesimal cells which are recurrently associated together in
chains. A small number of marine plants (angiosperms); though grow in
the tropical coasts, are frequently accrued flowering marine plants. Green
algae (Chlorophyta) are the utmost marine plant. Chlorophyll makes these
algae to have bright green coloring. As the leaves of algae calcify, the layers
are added to ocean deposits. Botanists consider that 200,000 algae species
occur, although just 36,000 are known. Red algae (Rhodophyta), supplied
with pigment phycoerythrin, are the major type of marine plants and the
most diverse. Some red algae stick to corals, hence making reefs. Both
green and red algae species favor cold to warm water. Contrary, brown algae
(Phaeophyta), tinted with the pigment fucoxanthin, are generally present in
temperate or cold water, and also some species living in tropics. On reefs,
brown algae commonly are the prevailing organisms. Blue-green bacteria or
cyanobacteria are principally microscopic constituents which alter nitrogen
from the atmosphere into forms that furthermost marine plants can consume
(Morrissey et al., 2016).
Marine biotechnology is the study which includes the procedures
to create the partial or full alteration of products, to mend animals or
plants, or to advance microorganisms for precise consumptions. Through
molecular and biotechnological procedures, humans have been capable to
explicate numerous natural means (Jha and Zi-Rong, 2004). The marine
environs might comprise 80% of the world’s animal and plant species.
Currently, various bioactive complexes have been hauling out from many
marine animals and marine organisms (Haefner, 2003). The exploration of
different metabolites as of marine organisms has occasioned the seques-
tration of more or less of 10,000 metabolites, several of which are capable
of pharmacodynamic activities. Natural products have extensively been
supplied as pigments, foods, insecticides, fragrances, medicines, and so
forth. Because of their tranquil openness, land plants have aided the key
foundation of therapeutically valuable goods, particularly for outmoded or
traditional medicine. The ocean provides a diverse living environment for
invertebrates as it covers 70% of the earth’s surface. In terms of chemicals
and biology, the marine environment is rich in diversity. Marine plants are
a unique source of chemical compounds that may have potential industrial

applications in pharmacy, medicine, agriculture, cosmetics, and research.
The reservoir of bioactive natural compounds that are provided by the
marine environment is exceptional as a lot of them show the chemical and
structural features that are not present in land natural products. Marine
organisms have evolved biochemical and physiological contrivances
that comprise the bioactive compounds for the applications in commu-
nication, predation protection, infection, reproduction, and competition
(Halvorson, 1998).
Hence in near future, the marine natural products are going to play a
major role in drug discovery. Till now, approximately 7000 natural products
of marine origin have been extracted in which algae comprise 25%, sponges
33%, coelenterates 18%, while 24% includes bryozoans, echinoderms,
mollusks, and ascidians (Kijjoa and Sawangwong, 2004).
1.2 DISCOVERY OF MARINE PHYTOCHEMICALS
Four steps of drug discovery from marine plants are the identification of drug
target, validation of target, identification of lead compounds, and optimiza-
tion (see Volume 1 and 2 of this book for more information). Completely new
therapeutic approaches have been opened up by the marine natural products.
Their advantages include the use of marine drug availability, identification,
and appreciation of unique biochemical pathways. These have contributed
to research tools in biochemistry and molecular cell biology (Grabley and
Sattler, 2003).
Saponins, flavonoids, alkaloids, anthraquinones, and volatile oils are
the various chemical components of marine organisms. For the identifica-
tion of phytochemicals, rapid and reproducible analytical techniques have
been developed and these techniques include the high-performance liquid
chromatography (HPLC) combined with different detectors like diode
array detector, refractive index detector, a mass spectrometric detector,
and evaporative light scattering detector. A significant step to check or
control for crude drugs quality as well as to elucidate their therapeutic
mechanism is by analyzing and screening the bioactive compounds. The
compounds of marine origin discovered initially were noxious or were not
operative in the treatment of pharmaceutical tenacities diseases. Instead,
they are beneficial as agrochemicals, cosmetic ingredients, or biological
tools (Fenical, 1997).
A lot of drugs are derived from aquatic organisms. One example is a

neurotoxin isolated from a snail, with potency 10,000 times more than
morphine. For the treatment of herpes virus and cancer, two compounds
have been isolated from sponge and also an antibiotic has been derived from
fungi. Nevertheless, there are numerous other compounds that are isolated
from a marine source and presently are in clinical trials, and it is probable
that several additional might progress to the treatment.
The conventional methods employed for extraction, isolation, and iden-
tification of phytochemicals were discussed in details in Chapter 4 of this
volume.
1.3 UNIQUENESS OF MARINE PHYTOCHEMICALS
The marine vegetation comprises of microflora (cyanobacteria, bacteria,

fungi, and Actinobacteria), microalgae, macroalgae (seaweeds), and flowering
plants (mangroves and other halophytes). Inhabiting nearly 71% of globally,
the sea is opulent in biodiversity, and the microalgae and microflora unaided
organize higher than 90% of marine biomass. This huge aquatic floral reserve
will compromise a pronounced opportunity for finding the novel medica-
tions. It cannot be deprived of that with 3.5 billion years of presence on land
and knowledge in biosynthesis; the aquatic microflora lingers environment’s
greatest cradle of elements. The aquatic entities produce unique compounds
to endure risky differences in salinity, pressure, temperature, and so forth,
prevalent in their environs, and the compounds formed are inimitable in
variety, organizational, and efficient structures (Kathiresan et al., 2008).
The efforts for the extraction of medicines from the ocean came into
limelight lately in the 1960s. Nevertheless, the efficient analysis initiated in
the mid-1970s, through the period of 1977–1987, brought about 2500 novel
metabolites from diverse aquatic organisms. Researchers have noticeably
established the aquatic environs as the tremendous basis of unique compounds,
not present in land cradles. Until now, more than 10,000 compounds are
extracted from aquatic organisms, though much more new complexes are
revealed each year. Nearly 300 exclusive rights on bioactive aquatic natural
products were acquired amid 1969 and 1999 (Kathiresan et al., 2008).
Certain aquatic organisms are demonstrated to be the persuasive foundations
of treatments. These are generally invertebrates that comprise sponges, sea
fans, sea hares, sea corals, nudibranchs, tunicates, and bryozoans. Currently,
it is assumed that contagious floras extant in the invertebrates are liable for
the fabrication of curative complexes. The exploration of aquatic faunal
and floral species is essentially unheeded. Various complexes derivatives

of aquatic entities have anticancer and antioxidant bioactivities yet they are
essentially unknown well.
Since ancient times, the aquatic organisms are cast-off for pharmaceutical
applications in China, India Europe, and Near East. China and Japanese are
utilizing seaweeds for feeding. The seaweeds particularly brown seaweeds
are opulent in iodine which results in a minimum occurrence of glandular
ailments and goiter. Antiquity divulges that maritime states were using
seaweeds as a vermifuge, general anesthetic, and liniment similarly for the
treatment of gout, cough, abrasions goiter, venereal illness, and so forth.
Sterols and associated complexes existent in seaweeds are able to low plasma
blood cholesterol level. Seaweed nutritional filaments accomplish diverse
functions like antitumor antioxidant, antimutagenic, and anticoagulant. The
seaweeds also impart a significant part in alteration of triglyceride breakdown
in the humanoid body. Calcium, sodium, and potassium are accompanying by
low mean systolic pressure and low menace of hypertension. All seaweeds
propose an amazing level of potassium that is alike with our plasma level.
Seaweed extract is remarkably analogous to human blood plasma. Two
Japanese investigators applied an inimitable practice of seaweed compounds
mixing with water to ancillary entire blood while transfusion and this has
been effectively strained in about 100 procedures (Langseth, 1995).
Even though the practice of seaweeds as a source of drugs is not as
common as it was previously, seaweed polymer excerpt used in medicine,
pharmacy, and biochemistry is well established. Efforts are in process for
the introduction of jelly capsules that are made of seaweed alginic acid and
secrete insulin so that diabetic patient gets freed from the injection. The
lozenge concentrates fortification on the patient’s immune system and white
blood cells. Seaweed exudates like carrageenan isolated from red seaweed
or algin isolated from brown seaweed are opulent sources of soluble fibers
(Langseth, 1995).
Numerous aquatic plants are able to produce bioactive materials that
exhibit antibacterial, antifungal, antiviral activities, and also antialgal activi-
ties (Li and Hu, 2005). The fermentation of an Eisenia bicyclis broth with
Candida utilis YM-1 in the course of 1 day enriched the phenolic content
such as dieckol, phlorofucofuroeckol A, eckol, and dioxinodehydroeckol
and activity of inhibition against methicillin-resistant Staphylococcus
aureus and foodborne pathogenic bacteria. Cell wall and storage of green,
brown, and red seaweed polysaccharides, together with ulvans, alginates,
fucans, laminarin, and carrageenans are able to elicit resistance retorts in
plants against pathogens.
1.4 CLASSES OF PHYTOCHEMICALS
Marine organisms are a rich source of biologically active and therapeuti-

cally compelling compounds. Polysaccharides and polyphenols are the
utmost set of complexes that are valid for biological activities such as
anticancer and antioxidant activity. There are above 40,000 diverse species
of phytoplankton, 680 species of aquatic algae, including Chlorophyta,
Phaeophyta, and Rhodophyta generally known as green, brown, and red
seaweeds, respectively, and 71 mangrove plant species recognized globally
in the aquatic biotope. They offer amino acids, vital fatty acids, vitamins,
bioflavonoids, ionic trace minerals, enzymes, and other compounds.
1.4.1 ALKALOIDS
The term alkaloid was formally suggested by Meissner in 1819 to describe

“alkali-like” complexes originated in plants; nevertheless, it was not indeed
distinct (Swan, 1967). They include the major collection of secondary
chemical components prepared generally of ammonia complexes, consisting
essentially of nitrogen bases manufactured from amino acid with numerous
radicals substituting one or more of the hydrogen atoms in the peptide ring,
utmost encompassing oxygen (see Chapter 2, Volume 1 for more infor-
mation). The complexes have elementary possessions and are alkaline in
response, altering the red litmus paper blue. In actual, one or more nitrogen
atoms that are contemporaneous in an alkaloid characteristically has 1, 2, or
3° amines, subsidize to the alkalinity of the alkaloid. The degree of alkalinity
of alkaloid differs significantly, contingent on the organization of the mole-
cule, and occurrence and position of the functional groups. The mainstream
of alkaloids occurs in solid form like atropine, certain as liquids containing
hydrogen, nitrogen, and carbon. Maximum alkaloids are generously solvable
in alcohol and yet they are thriftily soluble in water, their salts are generally
decipherable. The alkaloids solutions are strongly nasty. These nitrogenous
compounds work in the defense of plants against pathogens and herbivores
and are broadly oppressed as drugs, amphetamines, tranquilizers, and toxins
because of their powerful activities. In natural surroundings, the alkaloids
occur in huge quantities in the roots and seeds of plants and frequently in
amalgamation with vegetable acids. Alkaloids have drug solicitations as
analgesics and central nervous system stimulants (Madziga et al., 2010).
Above 12,000 alkaloids are recognized to occur in around 20% of plant
species and only a few have been subjugated for therapeutic determinations.
The term alkaloid ends with the suffix –ine and alkaloids of plants origin
in clinical use contain the anesthetics morphine and codeine, the muscle
relaxant (+)-tubocurarine, the antibiotics berberine and sanguinarine, the
vinblastine anticancer agent, the anti-arrhythmic ajmaline, the pupil dilator
atropine, and the sedative scopolamine. Other significant plant originated
alkaloids include the addictive amphetamines caffeine, ergotamine, nicotine,
ephedrine, and cocaine. Amino acids turn as antecedents for the biosynthesis
of alkaloids with lysine and ornithine generally utilized as preparatory
constituents. As the time passed, the delineation has altered to a compound
that has nitrogen atom(s) in a cyclic ring. The term alkaloid includes halo-
genated cyclic nitrogen-containing elements and various biological amines.
The latter is not present in land plants and is particular to aquatic organisms;
aquatic algae are included in them. Regarding alkaloids chemistry and its
anticancer activities, numerous studies are available in terrestrial plants;
however, insufficient data is available in marine plants (Guven et al., 2010).
The first alkaloid is morphine that was isolated from a terrestrial plant in
1805, whereas in 1969, hordenine as the first marine alkaloid was extracted.
Today, about 2000 alkaloids are recognized. Their occurrence is rare in
marine algae and abundant in terrestrial plants.
Among numerous kinds of compounds acquired from plants, convention-
ally the alkaloids have been of attention because of their pronounced biological
activities in humans and animals. Well-known examples of anticancer alkaloids
are taxol that is available clinically since 1994 from the western yew, Taxus
brevifolia, and camptothecin and derivatives of Camptotheca acuminata pres-
ently are in clinical trials. Marine algae alkaloids are divided into three groups,
first are a group of indole and halogenated indole alkaloids, the second group
are phenylethylamine alkaloids, and rest alkaloids are included in the third
group. In structure, most of the marine algae alkaloids are related to indole
and phenylethylamine groups. The phytoconstituents and activities of these
alkaloids were not completely examined. In comparison with terrestrial plants,
marine plant-derived alkaloids are rare (Gross et al., 2006).
A huge and progressively developing set of secondary metabolites is
included in marine indole alkaloids. The biological activities of these varied
alkaloids make numerous complexes of this group striking as the starting
points for the advancement of pharmaceutical products. Numerous marine-
derived indoles remained attractive for different biological activities such
as antimicrobial, antioxidant, cytotoxic, and antineoplastic. Furthermore, it
acts on human receptors and enzymes. Pyridoacridine also represents a large
family of alkaloids possessing inimitable marine nitrogenous composites.
Different marine organisms such as sponges (see Chapter 2 and 3 of this
volume for details), ascidians, anemones, prosobranch mollusk, and tunicates

are evidenced for their presence. A great interest in accordance with pyrido-
acridines is raised due to their substantial biological activities. Anticancer
activity possessing alkaloids are also extracted from coastal mangroves
(Kathiresan and Duraisamy, 2005). Another alkaloid is Rhizophrine, which
is a most important component of Rhizophora mucronata and Rhizophora
stylosa leaves. Likewise, the acanthicifolin presence in Acanthus illicifolius,
brugine, an alkaloid with sulfur, in Bruguiera sexangula, and benzoquinones
in Aegiceras corniculatum and Kandelia kandel are reported.
Indole alkaloids are either consequential from direct indole precursor or
from tryptophan. In microorganisms and plants, the indole precursor is made
from chorismate via anthranilate and indole-3-glycerol-phosphate. The even-
tual phase of the tryptophan biosynthesis is rescindable and thus free indole
can also be made in this catabolic procedure. An anonymous algicolous
fungus sampled from the surface of the marine Rhodophyta alga Gracilaria
verrucosa led to the extraction of Nb-acetyltryptamine. 2-methylsulfinyl-
3-methylthio-4,5,6-tribromoindole, 3-methylsulfinyl-2,4,6-tribromoindole, and
4,6-dibromo-2,3-di(methylsulfinyl)indole were extracted from the Formosan
red alga Laurencia brongniartii. 2,3,4,6-tetrabromo-1-methyl-1H-indole was
sequestered from the marine red alga Laurencia decumbens. From the marine
red alga Laurencia similis, 3,5,6-tribromo-1H-indole, 3,5,6-tribromo-1-methyl-
1H-indole, 2,3,6-tribromo-1H-indole, 3,5-dibromo-1-methyl-1H-indole, and
2,5-dibromo-1-methyl-1H-indole were extracted (Li et al., 2010).
Granulatamides A and B were sequestered from the soft coral Eunicella
granulate and it exhibited cytotoxic activity alongside the tumor cell lines
DU-145, LNCaP, SK-OV-3, IGROV, IGROV-ET, SK-BR3, SK-MEL-28,
HMEC1, A549, K562, PANC1, HT-29, LOVO, LOVO-DOX, HeLa, and
HeLa-APL (GI50—concentration which possessions a growth inhibition
of 50%—1.7–13.8 μm). N-acetylated aplicyanins B, D, and F are effective
antimitotic as well as cytotoxic activities alongside the tumor cell lines
HT-29 (GI50 0.39, 0.33, and 0.47 μm, correspondingly), A549 (GI50 0.66,
0.63, and 1.31 μm, correspondingly), and MDA-MB-231 (GI50 0.42, 0.41,
and 0.81 μm, correspondingly), while aplicyanin E exhibit merely cytotoxic
effects (GI50 values 7.96–8.70 μm) (Reyes et al., 2008).
1.4.2 POLYPHENOLS
The terms phenolics, polyphenolics, phenols, or polyphenol are imparted to

extracts that are natural constituents and impart color to plant fruits. Through
the action of phenylalanine ammonia lyase (PAL) action, the phenolics are
produced from phenylalanine in plants. They are essential for plants and
have many roles. They are applied to the rule over human pathogenic perma-
nency as they fight against the herbivores and pathogens via plant defense
mechanisms. Their three classes include (a) flavonoids: polyphenols that are
comprised catechins, flavones, flavanones, and xanthones, (b) non-flavonoid
polyphenolics, and (c) most common phenolic acid of marine plants that is
caffeic acid proceeded by chlorogenic acid that is responsible for allergic
dermatitis in humans (Kar, 2007). As natural antioxidants, phenolics are used
as nutraceuticals, are applied for the treatment of heart ailment, and help
fight against cancer and inflammations. Hesperidin, naringin, chlorogenic,
rutin, and flavones are examples of some other phenols. Phenolics of marine
origin are considered as a significant group of natural antioxidants. Phenolics
are made up of one or more aromatic rings and have one or more hydroxyl
groups. Chemically, polyphenols classes are coumestans and isoflavones
like isoflavonoids, flavones, flavanones, flavanols, flavonols, flavanonols,
anthocyanins like flavonoids hydroxycinnamic acids and hydroxybenzoic
acids like phenolic acids, tannins and proanthocyanidins like phenolic poly-
mers, lignin, and stilbenes. Structurally, several thousand polyphenolics are
reported as the secondary metabolites of marine plants. Their sources include
fruits, vegetables, cereals, and leguminous plants. Many factors affect their
concentration such as technological, environmental, and genetic factors.
Polyphenols provide protection against pathogens and ultraviolet radia-
tion (Lee and Lip, 2003). Phenols are also involved in plants growth, repro-
duction, and pigmentation. Phlorotannins group of phenolic compounds
are abundant in brown algae and is present in lower quantity in red algae.
They constitute the cell wall but are also important for therapeutic proper-
ties and secondary ecological functions. They are important against human
immunodeficiency virus (HIV), inflammatory, aging, cancer, diabetes,
bacterial, and allergic infections. Additionally, to protect against UV radia-
tion, they are involved in protective mechanism against biotic factor and
play an integral role in algae reproduction (Frankel, 1998). Because of the
health benefits discussed above, this group of compounds is receiving an
increasing concern by the food manufacturers and consumers. Antioxidants
from natural sources need to be explored further for their great potential.
The sustenance of significant omega-3 polyunsaturated fatty acids (PUFAs)
like eicosapentaenoic acid (C20:5 n-3) and docosahexaenoic acid (C22:6
n-3) have enormous beneficial effects on human health. Nevertheless, lipid
oxidation is the possibility that might affect the seafood having the great
quantities of PUFAs (Boyd et al., 1993). Lipid oxidation has a lot of harmful
effects and to overcome the possible toxicity and food safety concerns instead
of synthetic antioxidants, there is an increase in adaptation for natural anti-
oxidants (Maqsood and Benjakul, 2013). For natural antioxidants, the food
industry is focusing more towards the phenolic compounds of the marine
source. Different marine phytochemicals including phenolic compounds are
reported to retard the oxidation of lipids in a different kind of seafood.
As stated above, the polyphenols of plants are composed of one or more
phenolic rings and are formed as a result of plants secondary metabolism.
In the terrestrial environment, vegetables, fruits, tea, beer, wine, chocolate,
and coffee are made up of phenolic compounds. Moreover, in recent years,
they are also extracted and applied to food as colorants and to enhance the
shelf life of food. Though the sources from where these compounds are
extracted affect their activities, this is credited to the varied factors like the
environment in which it grows, its genotype or variety, temperature, light,
growth time, environment, type of soil and their dispensation, and posthar-
vest storage. Phenolic contents and antioxidant activities or both and other
biological activities and quantity of active compounds are all affected by
the listed factors. The antimicrobial, antioxidant, and scavenging activities
of polyphenols are comprehensively disseminated in plants (Bors et al.,
1990). The high amount of polyphenols like flavonoids, lignin, epicatechin,
phenolic acids gallic acid, anthocyanidins, tannins, catechin, and epigal-
locatechin are observed in seagrass, seaweed, and mangrove like marine
plants. The health and nutritional benefits of these polyphenol composites
are great like they help act against oxidation, bacteria, cancer, inflammation,
viruses, and human platelet aggregation. It is described that the augmented
nutritional consumption of natural antioxidants and the abridged coronary
heart disease, cancer transience likewise extended life expectancy. More-
over, polyphenols with high antioxidant activity and natural metal chelators
inhibit diverse toxic metal ion-induced organ dysfunctions (Xu et al., 2006).
The prior research proposes that polyphenols can restore α-tocopherol by
reducing the α-tocopheroxyl radical. The association of anticarcinogenic and
antioxidant activity is confirmed in a chemically swayed mouse carcinoma
system with low-molecular-weight polyphenols (Kakegawa et al., 1985).
The aquatic red algae, for example, Osmundea pinnatifida, has been widely
known for the antileishmanial, antioxidant, antimicrobial, and antifungal
activities (Shibata et al., 2002). In addition, scutellarein 4′-methyl ether has
antiallergic, anticytotoxic, and anticancer bioactivities in vivo and in vitro
(Yuan et al., 2005).
Marine and land polyphenols are comparative in a few regards, however
very specific in their compound configurations. Land polyphenols are
constructed in light of flavonoids or gallic acids. Marine polyphenols, phlo-

rotannins of algae, are just recognized in brown algae and are restricted to
phloroglucinol polymers (Heo et al., 2005). These are usually distributed
in plant vegetation and are high molecular weight phenol compounds.
Tannins are dissolvable in water and alcohols are originated in the stem,
root, bark, and external layers of plant tissue. Tannins have a distinctive
highlight to tan, that is, to change over things into leather. They are acidic
in response and the acidic response is ascribed to the nearness of phenolics
or carboxylic group (Kar, 2007). Tannins are composed of hydrolyzable
tannins yet condensed tannins. The hydrolyzable tannins are known as gallo-
tannins and ellagitannins. On heating, they make pyrogallic acid. Co-joint
examples concerning hydrolyzable tannins include the aflavins, daidzein,
and genistein yet glycitein. In Ayurveda, formulations primarily grounded
over tannin-rich flora have been utilized for the remedy concerning ailments
like diarrhea, rhinorrhea, and leucorrhoea. Six phlorotannins were identified
from the brown seaweeds E. bicyclis and Eclonia kurome through HPLC
analysis, these include phloroglucinol, phloroglucinol tetramer (MW 478),
eckol, phlorofucofuroeckol A, dieckol, 8,8′-bieckol. The crude phlorotannins
extracts showed inhibitory effects on HAase (Nakamura et al., 1996). As
compared to an anti-allergic drug (disodium cromoglycate), half-maximal
inhibition (IC50) values of crude phlorotannins of E. bicyclis and E. kurome,
two land polyphenols (catechin, epigallocatechin gallate), prevent four times
resiliently (Fukuyama et al., 1989).
Edible seaweeds retain comprehensive potential bioactive constituents:
polyphenols and phlorotannins (Zhao et al., 2007). Edible seaweed like
Palmaria palmate possesses antioxidant activities and inhibits the cancer
cell propagation. Brown seaweed Ecklonia cava enzyme hydrolysis
produces a big quantity of complexes with increased biological activities in
comparison to water and organic extract counterparts. Phloroglucinol and
its polymers named eckol (a trimer), phlorofucofuroeckol A (a pentamer),
dieckol, and 8,8′-bieckol (hexamers) separated from E. bicyclis show
antioxidant activity. The phlorotannins isolated from E. kurome perform as
antiplasmin inhibitor; though, additional phlorotannins bioactivities, from
the physiological viewpoint of humans, are still obscure (Setty et al., 1976).
1.4.3 SAPONINS
The term saponins is coined from a plant Saponaria vaccaria (Quillaja sapo-
naria). Two main types of saponins comprise triterpene saponins and steroid
saponin. Saponins are harmful and cause a lot of illness, like by causing
hemolysis; they are responsible for cattle poisoning and mucous membrane
irritation (Kar, 2007). Saponins are also medicinally useful as they possess
both anticancer and hypolipidemic activities. The two noteworthy sorts
of steroidal saponins are diosgenin and hecogenin. Steroidal saponins are
utilized as a part of the commercial production of sex hormones for clinical
use. For instance, progesterone is gotten from diosgenin. The most copious
beginning material for the production of progesterone is diosgenin secluded
from Dioscorea species, once provided from Mexico, and now from China.
Other steroidal hormones, for example, cortisone and hydrocortisone, can
be set up from the starting material hecogenin, which can be detached from
sisal leaves discovered broadly in East Africa (Sarker and Nahar, 2007).
The uses of saponins are as natural detergents, well known to primitive
people as fish poisons. The intriguing pharmacological properties related
to the Chinese medication “giwieng” are viewed as a panacea and other
fascinating biological activities, for example, spermicidal (Netz and Opatz,
2015), molluscicidal, antimicrobial, anti-inflammatory, and cytotoxic activi-
ties. Pharmacologically significant steroidal saponins such as sapogenisis
and sapogenins are produced by Avicennia officinalis. Modified terpenes
that are Liomonds are known insect antifeedant and growth regulators (Yim
et al., 2005).
1.4.4 POLYSACCHARIDES
Throughout the most recent couple of years, restorative and pharmaceutical

industries have demonstrated an expanded concern for marine polysaccha-
rides. Polysaccharides or glycans are a group of constituents with the most
widely recognized components of monosaccharide like D-glucose, D-fructose,
D-galactose, L-galactose, D-mannose, L-arabinose, and D-xylose. Certain
monosaccharide byproducts present in polysaccharides incorporate the
amino sugars (D-glucosamine and D-galactosamine) and also their products
(N-acetylneuraminic corrosive and N-acetylmuramic corrosive) and simple
sugar acids (glucuronic and iduronic acids). Carrageenans, agar, and alginate
are algae-derived polysaccharides. Agar provides the prime support to algae
cell walls; it is an unbranched polysaccharide and it is extracted from the
genera Gelidium and Gracilaria. Galactose sugar molecules are involved in
its constitution. Carrageenans are polysaccharides of galactan with rotating
1,3- and 1,4-connected galactose buildups, which fill spaces between the
cellulosic plant structures of seaweeds (Matsuda et al., 2003). The active
constituents retained in algae polysaccharides are principally sulfated ones.

Through the tumoricidal activities of natural killer cells and macrophages,
the sulfated polysaccharides are involved in enhancing the innate immune
response (Berry et al., 2004). In order to present the antigen to helper T-cells
and to produce TNF-alpha and interleukin-1 beta kind of cytokines, the
antigen-presenting cells move in and out of tumor tissues. Therefore, T-helper
cells advance the action of cytotoxic T-cell, which has a strong cytotoxic
impact on tumor cells. Sulfated polysaccharides can improve the versatile
invulnerable reaction by advancing such a process. It is also reported that
the sulfated polysaccharides diagnose number cell adhesion systems. They
augment the proliferative response of T-lymphocytes by binding CD2, CD3,
and CD4 in T-lymphocytes (Aisa et al., 2005). A sulfated polysaccharide
(B-1) extracted from marine Pseudomonas sp. Culture filtrate persuades
human leukemic cells apoptosis (Berteau and Mulloy, 2003). PI-88, a sulfated
oligosaccharide, persuades pancreatic islet carcinoma apoptosis, whereas
sulfated glycosaminoglycans (GAGs) restrict the transcription functionality
and consequently persuade the murine melanoma cells apoptosis.
Fucoidan is a derivative of the cell wall of the rhodophytes and it is
the sulfated polysaccharides representative (Sadir et al., 2001). Fucoidan
imparts human lymphoma HS-Sultan cell lines apoptosis and causes the
caspase-3 stimulation and extracellular signal-regulated kinase pathway
downregulation (Richard et al., 2006). They are capable of diverse biological
activities, going from comparatively unpretentious perfunctory sustenance
roles to added convoluted special effects on cellular functions binding
proteins like adhesion proteins (Li et al., 2008), growth factors, cytokines
(Zemani et al., 2005), and diverse enzymes, comprising coagulation prote-
ases. Consequently, they contribute as GAGs in cell adhesion, proliferation,
differentiation, and migration. Fucoidan is involved in modulating clinically
pertinent spectacles like angiogenesis, atherosclerosis, and tumor metastasis
(Sweeney et al., 2000). For as far back as decade, fucoidans disengaged
from various species have been widely contemplated because of their
differed biological activities like cell reinforcement, restorative potential in
surgery, gastric defensive impacts, blood lipids decreasing, anticomplemen-
tary activity against renalpathy, hepatopathy, and uropathy, and its action as
antivirus, anticoagulant, antitumor, antithrombotic, and immunomodulatory
agent. Contrasted and other sulfated polysaccharides, fucoidans, have been
progressively examined lately to build up the medications or food utilitarian.
The characteristics of fucoidan, like its sulfation, molecular weight, confor-
mation of its sugar residues, and type are dependent upon the species of
seaweed (Matou et al., 2002).
Sulfation is perilous for fucoidan in vivo activity. Particularly, desulfated

fucoidan fails to stimulate in vitro angiogenesis or the in vivo induction
of immature CD34+ cell mobilization. The protamine presence causes the
abolishing of native fucoidan mobilization (Carte, 1996). The principal
sulfation configuration consists of a trisulfated disaccharide recurrence
analogous to the one present in heparin. Nevertheless, heparin has no
influence on in vitro angiogenesis induction through human umbilical vein
endothelial cell (HUVEC) and is not involved in immature CD34+ cell
mobilization. Moreover, pentosan sulfate, heparan sulfate, and chondroitin
sulfate constrain in vitro angiogenesis and show anticoagulant activities.
Fucoidan can disturb heparan sulfate development dynamic/cytokine build-
ings and can be ancillary for cell-surface heparan sulfates in balancing out
the development dynamic/development dynamic receptor collaboration.
Fucoidan can intercede development factor-initiated endothelial progenitor
cell (EPC) separation by connecting with a “receptor” that advances endo-
thelial cell attachment, movement, expansion, and separation, coordinates
with a development dynamic receptor and transduces the intracellular signs
obligatory to actuate the angiogenic phenotype. Such alleged fucoidan
receptor may comprehend a sugar-binding domain that collaborates with the
fucoidan starch backbone.
1.4.5 CHLOROPHYLL
Chlorophylls are responsible for the giving the premise to organisms on

land, the oxygen in the air, and impart colors to plants. These colors have
been streamlined amid advancement for the proficient gathering of daylight
and the consequent vitality transduction into great vitality mixes such as
adenosine triphosphate (ATP) and Nicotinamide adenine dinucleotide
phosphate (NADPH). Synthetically, chlorophylls are cyclic tetrapyrroles
(Mochizuki et al., 2010). Chlorophylls have specifically a few chattels that
evidently condense them vital for photosynthesis (Grimm et al., 2006).
Chlorophylls intensely absorb visible and near-infrared light, which is vital
for light harvesting, and they possess long-lived excited states which avert
damage to the transitorily stowed energy into heat.
Chlorophyll and its resultants are additionally utilized broadly in phar-
macological items. Chlorophyll is stated as to quicken twisted recuperating
by over 25% in a few investigations. Subsequently, chlorophyll empowers
tissue development, keeps the headway of microscopic organisms, and
accelerates the injury recuperating process. Chlorophyll is comparative in
substance structure to hemoglobin and, all things considered, is anticipated

to animate tissue development in a comparable manner by the help of a
fast CO2 and O2 interchange (Ma and Dolphin, 1999). Due to this property,
chlorophyll is utilized not just in the treatment of ulcers and oral sepsis yet
additionally in proctology.
A chronic ulcer is a critical medical issue in the public arena, with exten-
sive phases essential for its cure (Ma and Dolphin, 1999). Chlorophyll’s
capacity to expand the rate of mending is an achievement for ulcer sufferers.
The use of balms comprising chlorophyll derivatives was present to dispose
of torment following a few days as well as to enhance the presence of the
influenced tissues (Louda et al., 2008). The release from the ulcer and its
feature odor additionally enhanced altogether following a couple of days
of chlorophyll action (Louda et al., 2008). Analogous assets, which form
chlorophyll a key compound in the treatment of ulcers, likewise form its key
in the treatment of post-agent wounds from rectal surgery.
1.4.6 AMINES/PEPTIDES
The species and amount of marine bioactive peptides are more than that
of land bioactive peptides; though marine life forms are presented to more
extraordinary conditions than that terrestrial which influence the marine
bioactive peptides to have huge distinctive amino acid conformations and
successions from terrestrial bioactive peptides. In addition, marine bioactive
peptides can be acquired from different marine plants, animals, and lower
life forms. Each is one of a kind as a group; marine bioactive peptides have
preferred bioactivity in a few zones over land bioactive peptides thinking
about their incredible ordered assorted variety and extraordinary qualities
(Wang et al., 2017).
The estimation of cell reinforcement movement is an essential screening
strategy. Some compound techniques are utilized, including decreasing force,
hydroxyl radical rummaging action, superoxide anion radicals searching
action, rummaging responsive oxygen species, and restraint of lipid
peroxidation. Notwithstanding the wide utilization of these substance cell
antioxidant action tests, none of them consider the bioavailability, take-up,
and mechanism of the cancer prevention agent mixes (Liu and Finley, 2005).
Lately, cell culture models give a method that is savvy and moderately quick
and can clarify metabolic issues (Wolfe and Liu, 2007). One approach is to
utilize the cell cellular antioxidant status measured by the methyl thiazolyl
tetrazolium test and ensure HepG2 cells against H2O2-incited cytotoxicity.
Nonetheless, since the convergence of H2O2 is not clear, this technique

ought to have a preparatory test. Likewise, cellular antioxidant activity
(CAA) is identified with HepG2 cells. The CAA test is deliberated a higher
indicator of in vivo activity as compared to in vitro assays since it includes
the acquaintance of the antioxidants to the intricacy of biological substrates
beneath the physical settings (Frankel and Meyer, 2000). Unquestionably,
the best antioxidant agent is applied for animal models and human examina-
tions, though they are not suitable for the preliminary examinations as are
time-taking and expensive (Liu and Finley, 2005). As such, despite the fact
that there is an extraordinary variety of strategies utilized for antioxidant
agent testing, there are no endorsed institutionalized techniques.
Hypertension is a standout amongst the most widely recognized cardio-
vascular illness around the world (Wilson et al., 2011). Around 75% of
hypertensive ailment, 54% of strokes, 25% of other cardiovascular mala-
dies, and 47% of ischemic coronary illness were attributable worldwide to
hypertension (Lawes et al., 2008). Among the procedures identified with
hypertension and in the control of circulatory blood pressure, the angio-
tensin-I-converting enzyme (ACE) assumes a vital part. ACE can catalyze
the transformation of angiotensin I to angiotensin II, and angiotensin II is
an intense vasoconstrictor that builds peripheral vascular protection and
subsequently lifts arterial pressure (Skrbic and Igic, 2009).
In this way, in the advancement of medications to control hypertension,
ACE inhibitors and angiotensin receptor blockers are currently utilized
clinically for the cure of different cardiovascular maladies. Be that as it may,
the engineered medications, for example, enalapril, captopril, and lisinopril
(Elavarasan et al., 2016), are accepted to have certain symptoms such as
angioneurotic edema, skin rash, or loss of taste. Because of these unfavorable
symptoms, there is a pattern towards empowering the advancement of normal
ACE inhibitors. Lately, normally happening peptides with ACE inhibitory
action were acquired from different marine life forms, for example, green
algae, sea cucumber (Acaudina molpadioides), tuna, sole (Limanda aspera),
blue mussels (Mytilus edulis) (Je et al., 2005), jumbo squid (Dosidicus
gigas), oysters (Crassostrea gigas), and shrimp. The disclosure of the far-
reaching dispersion of antimicrobial peptides (AMPs) in the course of recent
years has given bits of knowledge into the intrinsic resistance frameworks
that allow multicellular life forms (Zasloff, 2002). Advancing through the
positive choice, the AMPs are considered as profoundly substantial immune
effectors (El-Gamal et al., 2013).
Because of their unique living condition, properties and structure,
currently, much consideration has been paid to marine-determined bioactive
peptides. The marine plants are in close contact with organisms and give a
tremendous source of AMPs. Furthermore, vast seawater harbors have 106
bacterial and 103 fungal cells for each milliliter, and most marine life forms
have particular populaces of microorganisms on their surfaces or inside
the bounds of their tissues. Researchers have isolated AMPs from Atlantic
cod (Gadus morhua), mud crab (Scylla paramamosain), oyster (C. gigas),
yellow catfish (Pelteobagrus fulvidraco) (Su, 2011), sponge (Trichoderma
sp.), and marine snail (Cenchritis muricatus), and the AMPs from marine
life forms are inexpensive, safe, and natural, having high bioactivity. These
marine organism-derived peptides showed other bioactivities as well like
anticoagulation, immunomodulation, antitumor activity, appetite suppres-
sion, calcium binding, cardiovascular protection, neuroprotection, and
antidiabetic activity (Cheung et al., 2015).
1.4.7 ESSENTIAL OILS
Essential oils are the volatile and odorous products of numerous animals
and plant species. Essential oils keep a potential of evaporation upon air
exposure at room temperature and hence also denoted as volatile oils or
eerie oils. Essential oils enhance the aroma of some species and contribute
the essences or odoriferous constituents of the aromatic plants (Martinez
et al., 2008).
Essential oils are stashed unswervingly through the plant protoplasm or
by the hydrolysis of several glycosides and structures like the plant structures
that are directly allied with the excretion of essential oils such as Lamiaceae,
for example, Lavandula angustifolia glandular hairs, oil tubes of Apiaceae,
for example, Foeniculum vulgare and Pimpinella anisum, Piperaceae, for
example, Piper nigrum—black pepper modified parenchymal cells, and pine
oil passages of Schizogenous. Different parts of plants like stem, leaves,
roots, flowers, and rhizomes are linked to the essential oils. More than 200
different chemical components are associated with flavor and odor of the
essential oils, contributed by the trace constituents (Firn, 2010).
Different methods for the preparation of essential oils include direct
steam distillation, expression, and extraction via enzymatic hydrolysis. In
direct steam distillation process, the plant part is boiled in a distillation
flask; it passes the volatile oil and steam through the water condenser and
consequently collects the extracted oil in Florentine flask. Selection of the
distillation process among direct, steam and water, and water distillation
depends upon the source of the extraction material. Extrusion or expression
of volatile oils is achieved through the sponge process, scarification, rasping,

or by a mechanical method. While using the sponge method, first the washed
plant material is cut into two halves and squeezed; oozed volatile oil is
collected through the sponge and then squeezed in the vessel and the floating
oil is separated. The scarification apparatus is an Ecuelle a Piquer that is a
large funnel made of copper and has a tinned inner layer. To penetrate the
epidermis, various pointed metal needles are present. In rasping, initially
the peel of the plant part is removed and then oil is extracted while pressing
through which oil is oozed. Turbid appearance is later on changed and oil
separates from the fluid that is further decanted and filtered. Heavy-duty
centrifugal devices are used in the mechanical process of essential oil
extraction. The yield of oil has now increased remarkably with the advent of
modern mechanical devices.
1.5 MARINE SOURCES AS CRADLES OF METABOLITES
Nature has been a cradle of therapeutic agents since a long time and a striking
number of contemporary drugs were extracted from plants; several centered
on their utilization in traditional treatment (see Volume 2 of this book).
Moreover, researchers have also revealed that the plant aids a phytoreme-
diation mediator (Sajn et al., 2005) as a biosorbent for toxic metals (Malik,
2007), for power alcohol and biogas making as a fertilizer and medicinal
plants. Similarly, in its traditional form and at the squat invasion, it obliges
as fish food, where herbivorous fishes are kept and cultivated combined with
other non-predatory species to endorse the fish progression (Ling, 1960).
Numerous reports are available on the antibacterial, antifungal, anti-
viral, anti-inflammatory, antidiabetic, antioxidant, and cytotoxic activities
of seaweeds (Mohammed et al., 2013). The genus Sargassum is broadly
disseminated in the clement and tropical oceans of the world. They are well
known for their biological activities and secondary metabolites (Deepa
et al., 2009). The terpenoid components of sargassum are responsible for
vasodilatory effects, cell toxicity, acetylcholine-esterase inhibition, and
antioxidant activity. Seaweed inhabits in the clear tropical and intertidal
zones. Constitutional analysis of marine algae is not done much. Most of
the products of the seaweed industry are obtained from one Gracilaria/
Gracilariopsis species, three Gelidium species, and two kelps. Additionally,
there are economically important seaweeds and their significance is evalu-
ated by assessing their potential for drug development (Carte, 1996). On
the other hand, novel cultivation techniques are explored through extensive
laboratory researches resulting in new cultivation initiatives. It is confirmed

by different studies that Sphaerococcus coronopifolius is a good source of
antibacterial compounds (Ireland et al., 1993); Ulva lactuca shows anti-
inflammatory modules; and Portieria hornemannii has potential as an anti-
tumor agent (Ali et al., 2002). A unique compound sphingosine with antiviral
activity is extracted from Ulva fasciata. Stypoldione, as a potent cytotoxic
metabolite that is involved in the prevention of mitotic spindle formation
and microtubule polymerization inhibition, has been sequestered from the
tropical brown alga, Stypopodium zonale (Xu et al., 2002). P. hornemannii is
recognized as a unique cradle of cytotoxic penta-halogenated monoterpene,
halomon, which showed one of the best differential cytotoxicity airings
steered by the National Cancer Institute, USA. Halomon was designated for
the development of preclinical treatment; subsequently, it showed toxicity to
renal colon brain and tumor cell lines. An inhibitor of adenosine kinase in
the form of the iodinated nucleoside is extracted from Hypnea valitiae, and
it helped to study nucleotide regulation and metabolism other than studying
the adenosine receptors (Carte, 1996).
Codium iyengarii algae inhabitant Arabian Sea Karachi coast found rich
with two new steroidal glycosides, iyengarosides A and B, and iyengadione
that are effective against numerous bacteria (Blunt et al., 2004). Two new
bioactive sterols were also isolated from Sargassum carpophyllum in the
South China Sea. Some of their functions include cytotoxicity against cancer
cell lines and morphological abnormalities in plant pathogenic fungus
Pyricularia oryzae. Stigmast sterol of Sargassum polycystum is sampled
from North China Sea. Algae among the marine organism are a rich source
of components of worth importance. Algae are involved in converting the
PUFAs in arachidonic acids that are involved in homeostasis in mammalian
systems as well as help coping different diseases like cancer, ulcers, asthma,
psoriasis, arteriosclerosis, and heart diseases (Perry et al., 1988).
1.6 MECHANISM OF ACTION OF PHYTOCHEMICALS
Diverse mechanisms of action of phytochemicals have been recommended.

They might obstruct microorganisms, affect certain metabolic procedures,
or might temper gene expression as well as signal transduction paths.
Phytochemicals might moreover be applied as chemopreventive or chemo-
therapeutic mediators with chemoprevention denoting to the practice of
mediators to constrain, converse, or impede tumorigenesis. As molecular
mechanisms may be communal to chemoprevention and cancer therapy, so
the chemopreventive phytochemicals are applicable to cancer therapy (for

more details, see Volume 1 and 2 of this book). A different mode of actions
is exhibited by the essential oils and extracts of plants, like the intrusion
of the cell membrane phospholipids that resulted in cellular constituent’s
loss and increase in permeability. Certain detailed modes of actions are
deliberated here.
1.6.1 ANTIOXIDANTS
Reactive oxygen species (singlet oxygen, superoxide, peroxyl radicals,

hydroxyl radicals, and peroxynite) cause the oxidative stress that leads to
cellular destruction. Antioxidants provide protection to cells against the
damaging effects of these reactive oxygen species (Mattson and Cheng,
2006). A number of diseases like atherosclerosis, carcinogenesis, cerebral and
cardiac ischemia, diabetic pregnancy, neurodegenerative disorders, aging,
rheumatic disorder, and DNA damage are treated with natural antioxidants,
and hence, they provide protection and maintenance against degenerative
and chronic diseases (Jayasri et al., 2009).
Antioxidants scavenge the “free-oxygen radicals” to give a relatively
“stable radical.” In the form of metastable chemical species, the free
radicals apt to snare electrons from the instantaneous environs molecules.
These radicals uncertainty does not scavenge efficiently in time and might
harm vital biomolecules such as DNA, mitochondria, lipids, and proteins,
comprising all membranes leading to abnormalities and disease conditions
(Uddin et al., 2008). Therefore, free radicals convoluted in a lot of diseases
like atherosclerosis, tumor inflammation, diabetes, rheumatoid arthritis,
gastrointestinal ulcerogenesis, hemorrhagic shock, cystic fibrosis, infertility,
asthma, early senescence cardiovascular disorders, parkinsonism, Alzheim-
er’s-like neurodegenerative diseases, and acquired immunodeficiency
syndrome. Insufficient amount of antioxidants is produced by the human
body which plays an important role in the prevention of oxidative stress. The
body has the natural antioxidant mechanisms like catalases and glutathione
that remove the free radicals produced in the body (Sen, 1995). Hence, this
insufficiency should be fulfilled in the form of new phytochemicals search,
finding efficient methods for extraction and determining phytochemicals
basic structures and mode of action.
Plants free radicals scavenging molecules with great antioxidant activity
include terpenoids, flavonoids, vitamins, and phenols. In the human body,
free radicals are scavenged by plants-, vegetables-, and fruits-derived
flavonols, phenolics, carotenoid, ascorbic acid, and vitamin E. Substantial

antioxidant assets have been detailed in phytochemicals that are essential to
decrease the incidence of fetal diseases (Hertog et al., 1993; Anderson et al.,
2001). Numerous dietary polyphenolic components derivative of plants
are further active antioxidants in vitro as compared to vitamins E or C and
therefore may subsidize considerably in in vivo protective effects (Jayasri
et al., 2009). Antioxidants are often added to foods to avert the oxidation
radical chain reactions; they terminate the reaction or delay the oxidation
process through inhibition of initiation and propagation step. Food industries
are focusing and are providing funds for the commercialization of natural
antioxidants instead of synthetic compounds because of the safety concerns
of synthetic compounds. Due to safety concerns of synthetic compounds,
food industries have focused on finding natural antioxidants to replace
synthetic compounds. Additionally, the consumer concern is providing a
spur to explore natural sources of antioxidants.
1.6.2 ANTICARCINOGENESIS
Among diverse phytochemicals, the polyphenols are potent carcinogen inhib-

itors (Liu, 2004). Phenolic acids usually considerably abate the development
of the specific cancer-promoting nitrosamines from the dietary nitrites and
nitrates. For the treatment of colon cancer, glucosinolates exert a significant
protective support in the metabolism of carcinogens. Isothiocyanates and
the indole-3-carbinols interfere emphatically, preventing the procarcinogen
activation and thus phase II enzymes are activated. Glutathione S-transferase
and NADPH quinone reductase are such enzymes which prevent the change
in the structure of nucleic acids by especially detoxifying the electrophilic
metabolites. A potent phase II enzyme inducer is sulforaphane. It is respon-
sible for the particular cell-cycle arrest. It also causes the cancer cells apop-
tosis and the apoptosis of the neoplasm (cancer) cells. A significant inhibitor
of breast cancer is d-D-gluconolactone that is produced by sulforaphane.
Indole-3-carbinol especially prevents the human papillomavirus that causes
uterine cancer. In prostate cancer cells, it downregulates the CDK6 and
upregulates p21 and p27; it also blocks the estrogen receptors especially
existing in the breast cancer cells. d-D-gluconolactone causes the G1 cell-
cycle arrest as well as prostate and breast cancer apoptosis and increases
p53 expression in cells that are treated with benzopyrene. The depression
of B-cell lymphoma 2 (Bel-2) signaling pathways and Akt, NF-kappaB, and
Mitogen-activated protein kinase (MAPK) is caused by d-D-gluconolactone
at large extent. Neoplasm development in prostate glands, breast, and colon

are blocked by the phytosterols. Though the exact mechanism is yet to be
discovered, still it is assumed that in the neoplasm development, the ensuing
cell-membrane transfer is caused by them and hence reduces the inflamma-
tion expressively.
1.6.3 ANTIMICROBIAL ACTIVITY
Plants primary and secondary metabolites that protect them against protozoa,
bacteria, fungi, and pathogenic bacteria are now applied to help human
diseases (Nascimento et al., 2000). Phytochemicals like phenolic acids help
to lower dental caries and urinary tract infections through minimizing the
chances of adherence. Plants can also apply the bactericidal or bacteriostatic
activity against microorganisms (Jakhetia et al., 2010). It is noteworthy that
results of the antimicrobial action of the similar plant fragment confirmed
most of the time mottled from study to study. The probable reason might be
the sample type, habitat, extraction, and activity testing protocol. Same plant
constituents’ concentration varies in different geographical regions, topo-
graphical factors differences, plant age, and soil composition. Therefore, the
technical procedures must noticeably be recognized and sufficiently applied
and stated.
1.6.4 ANTI-ULCER
Marine phytochemicals have been reported as potent antiulcer agents. They

constrain the growths of Helicobacter pylori in vitro and also its urease
activity. The efficacy of various excerpts in a liquid medium and at low pH
increases their effectiveness even in the human stomach. They also exert an
inhibition effect on the kidney and intestine Na+/K+ ATPase activity and on
alanine transport in rat jejunum (Jakhetia et al., 2010).
1.6.5 ANTIDIABETIC
A phytoconstituent extract “cinnamaldehyde” displays substantial antihy-

perglycemic effect that results in lowering the triglyceride and total choles-
terol levels, simultaneously, causing an increase in high-density lipoproteins
(HDL) cholesterol in streptozotocin (STZ)-induced diabetic rats. Thus,
cinnamaldehyde could be used as a natural oral agent, with hypolipidemic

and hypoglycemic effects. Currently, it is found that the polyphenols
having procyanidin type-A polymers are probable to upsurge the quantity
of thrombotic thrombocytopenic purpura, insulin resistance, and glucose
transporter-4 in 3T3-L1 adipocytes (Jakhetia et al., 2010).
1.6.6 ANTI-INFLAMMATORY
Essential oils from marine phytochemicals have a tremendous anti-

inflammatory action and cytotoxicity against HepG2 (human hepatocellular
liver carcinoma cell line) cells. It suppresses the nitric oxide production by
lipopolysaccharide-stimulated macrophages (Jakhetia et al., 2010).
1.6.7 MULTIFUNCTIONAL TARGETS
A lot of molecular targets of nutritional phytochemicals are reported from

cell growth pathways, cell cycle proteins, pro- and antiapoptotic proteins,
protein kinases, cell adhesion molecules, and transcription factors to
metastasis. It is possible to overcome cancer drug resistance because of multi-
target nature of phytochemicals as the multi-faceted mechanism of action
perhaps hampers the cancer cell’s aptitude to progress resistance against
phytochemicals. Phytochemicals cause the modulation of inflammation,
redox signaling of transcription factors, and redox-sensitive transcription
factors (Surh, 2003). For example, nitric oxide (NO), a signaling molecule
of significance in inflammation, is modulated through plant polyphenols and
other botanical extracts (Shanmugam et al., 2008). Several phytochemicals
are categorized as phytoestrogens that aid health-promoting properties,
signifying phytochemicals as marketed nutraceuticals (Moutsatsou, 2007).
1.7 APPLICATIONS OF MARINE BIOACTIVE COMPOUNDS
Recent trends in useful sustenance and dietary supplements have verified

that the bioactive molecules shed a most important therapeutic role between
ethnical human sicknesses. Nutritionists and biomedical researchers are
cooperating to find new bioactive molecules that have expanded intensity
and restorative advantages. A lot of world biota consists of marine life
that is providing secondary metabolites and bioactive compounds of
wide application. These bioactive molecules are involved in anti-fibrotic,

antiviral, anti-inflammatory, antiparasitic, anticancer, and antibiotic activi-
ties. Additionally, they may cause the activation and inhibition of certain
enzymes, and can also act on transporters and sequestrants as modulating a
range of physiological pathways (Suleria et al., 2015).
1.7.1 MEDICINE
Many compounds are available in marine plants that provide protection

against various diseases. In addition to classified phytochemicals of marine
organisms, some compounds, their activities, and finding related to their
medicinal benefit are given below. The marine polyphenols, phlorotannins
that are phloroglucinol polymers (1,3,5-trihydroxybenzene), are extracted
from Phaeophyceae group of algae. They are different from terrestrial
phlorotannins and are derived from phloroglucinol (Sanjeewa et al., 2016).
Applications of algal phlorotannins include anti-melanogenic, anti-skin
aging, antioxidant, anti-inflammatory, hepatoprotective, and anti-allergic
activity. Among brown alga-derived phlorotannins, phloroglucinol, eckol,
dieckol (Heo et al., 2009), diphlorethohydroxycarmalol (Kim et al., 2015),
and octaphlorethol A were described as effective tyrosinase inhibitors.
Phlorotannins of three types such as “phloroglucinol, eckol, and dieckol”
were quarantined from E. cava by Heo et al. (2009). The anti-melanogenic
activity of eckol and dieckol was determined by the inhibition of mushroom
tyrosinase and melanin synthesis in B16F10 cells.
The Phaeophyceae algae are opulent cradles of meroterpenoid
compounds, which are useful for human health. Particularly, the Sargassum
genus is reported for containing a high amount of meroterpenoids. Algal
meroterpenoids have anti-inflammatory, antioxidant, antiaging, anti-
atherosclerotic, anti-adipogenic, antidiabetic, anticarcinogenic, and neuro-
protective activities. Fucoidans, a fucose-rich sulfated polysaccharide, is
principally present in aquatic brown algae and echinoderms (Fitton et al.,
2015). Fucoidans inhibit the activity of tyrosinase, elastase, and matrix
metalloproteases (Senni et al., 2006). Protein hydrolysates of peptides and
proteins from marine dispensation wastes exhibit different activities like
calcium binding, antimicrobial, HIV inhibition of protease, and suppression
of appetite actions. Osteoporosis, dental caries, hypertension, and anemia
can be prevented by components which bind and solubilize minerals such as
calcium (Korhonen and Pihlanto, 2006).
1.7.2 FOOD INDUSTRY
In the recent years, the consumer interest is increased in the relationship

between diet and health. There is presently more acknowledge that when
joined with a healthy life, useful sustenance may make a positive imitation
of well-being and prosperity. Today, consumers need foods to enhance health,
increase life, and/or decrease the danger of disease and then lengthen the
threshold of health. In particular, some bioactive of interests are proteinaceous
in characteristic (or origin) yet consist of amino acids, proteins, and peptides.
Furthermore, as the opulent cradles of protein, some marine organisms are best
starting materials for the technology of protein-derived bioactive peptides. The
focus on seaweed as much a cause over bioactive nitrogenous complexes has
currently been appraised (Harnedy and FitzGerald, 2012). Bioactive peptides
are particular fragments of proteins that notwithstanding are wellsprings of
amino acids nitrogen having probable biological capacities inside the body;
these incorporate opioid, antithrombotic, immunomodulatory, antibacterial,
and antihypertension actions. Moreover, certain of these peptides may
additionally show multifunctional properties (Meisel, 2004).
KEYWORDS
•• antimicrobial
•• antioxidant
•• phytochemicals
•• ocean
•• seaweed
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CHAPTER 2
MARINE SPONGE ALKALOIDS:

A SOURCE OF NOVEL ANTICANCER
AGENTS
MUSARAT AMINA* and HANAN M. AL-YOUSEF
Department of Pharmacognosy, College of Pharmacy,
King Saud University, Riyadh, Saudi Arabia. Tel.: 011-805320
Corresponding author. E-mail: [email protected].
*
ORCID: https://orcid.org/0000-0003-4545-253X.
*
ABSTRACT
The marine environment has always been a source of novel classes of biologi-
cally active compounds. Primitive organisms are the source of a vast range
of secondary metabolites which enable them to flourish in a harsh marine
environment, and in particular huge wealth of unique and rare novel classes
of metabolites produced by marine sponges has continuously attracted the
attention of researchers who are trying to develop new drugs. The present
chapter describes the research on novel anticancer alkaloids obtained
from marine sponges. More than 110 isolated novel antitumor cytotoxic
compounds confirmed activity in vitro cancer cell lines bioassay and are of
current interest to national cancer institutes for further in vivo evaluation.
This chapter describes the structure, origin cytotoxic, and anticancer evalu-
ation of sponge-derived alkaloids.
2.1 INTRODUCTION
The search for natural bioactive compounds from primitive marine

organisms is still an emerging field in comparison to long traditional
terrestrial pharmacognosy. However, in the last two or three decades,
isolation of fascinating array of unique metabolites with pronounced

biological activities has attracted the tremendous attention of scientists
(Newman and Cragg, 2007). Most of the pharmacologically active
compounds, predominantly alkaloids and terpenoids were obtained from
marine sponges and are considered as an important producer of novel
diverse marine metabolites. Around 5300 different new and natural
compounds have been reported from sponges every year with promising
biological activities associated with them which include anticancer,
antiviral, antibacterial, antimalarial, antifungal, immunosuppressive,
anthelminthic, anti-inflammatory, and muscle relaxants activities
(Molinski et al., 2009). In addition, uncommon nucleosides, marine
sponges also produce other classes of alkaloids, sterols, terpenes, amino
acid derivatives including cyclic peptides, fatty acids, peroxides, and so
forth. Several sponge-derived constituents are already in preclinical and
clinical trials as agents against cancer, microbial infections, inflammation,
and other diseases (Newman and Cragg, 2004). However, in many cases,
development of drug is severely hampered due to a limited supply of the
respective compounds as they are often present only in little amounts in
the sponge tissue.
The word cancer is burdened globally and about 8.8 million victims of
malignant tumors are claimed per year, which is causing one in six deaths
worldwide (WHO, online available). Current two major issues with anti-
cancer therapy are safety and low efficacy. Therefore, the need for identifica-
tion of new anticancer strategies with a better pharmacotoxicological profile,
to be used individually or in combination with conventional chemotherapy,
is required. Natural constituents in this context could play a vital role as
they are less toxic in comparison to conventional chemotherapy drugs, more
effective, easily available, and inexpensive (Amin et al., 2009). Natural
compounds are usually considered better and safe profile drugs than tradi-
tional anticancer agents and their ability to prevent cancer formation and
development is through interaction with multiple cell signaling pathways
(Amin et al., 2009).
In this chapter, we describe the novel anticancer alkaloids isolated from
marine sponges. The structures, origin, and pharmacological activities asso-
ciated with an isolated marine sponge are reviewed (Fig. 2.1), with the main
emphasis on the anticancer potential of poised isolated constituents: imid-
azole, indoles, pyridines, pyrrolesquinolones, isoquinolines, guanidines, and
other structural families.
Marine Sponge Alkaloids 37
FIGURE 2.1 Alkaloids isolated from marine sponges.

FIGURE 2.1 (Continued)









2.2 MARINE SPONGE ALKALOIDS AND THEIR ANTICANCER

POTENTIALS
2.2.1 CYTOTOXIC IMIDAZOLES ALKALOIDS
Naamidines are the biologically active imidazole alkaloids, commonly

found in Leucetta, Clathrina, and Leucosolenia genera of the marine
sponges (Carmely et al. 1989; Ciminiello et al., 1990; Ralifo et al., 2007).
Structurally, they are comprised of central imidazole ring to which one or
two modified benzyl groups are attached at the N-3 or C-4, C-5 positions,
and most of the alkaloids belonging to this group contain a hydantoin or
hydantoin derivative at the N-6 position. These alkaloids are reported for
various biological properties such as cytotoxic, leukotriene B4 receptor
antagonist, and antimicrobial activities (Plubrukarn et al., 1997; De Guzman
et al., 1999). Carmel et al. (1989) reported a series of 2-aminoimidazole
alkaloids called naamidines (e.g., 1) from Leucetta chagosensis which is
a marine sponge. Two imidazole alkaloids, naamidines H (2) and I (3),
were extracted from L. chagosensis (marine sponge) collected from North
Sulawesi, Indonesia and showed cytotoxicity against HeLa cells with IC50
values of 5.6 and 15 μg/mL, respectively (Tsukamoto et al., 2007). Two
new imidazole alkaloids, methyldorimidazole (4), preclathridine B (5) along
with known compounds naamine E (6) and leucettamine C (7) were isolated
by L. chagosensis (Hassan et al., 2009). The isolated compounds showed
cytotoxicity against P388 at 2–10 μg/mL (Y. Kashman, personal communi-
cation). Girolline (8), obtained from Pseudaxinyssa cantharella, was found
active against P388 at 0.001–1 μg/mL and the activity was further confirmed
in vivo in mice (P388 at 1 mg/kg doses (Ahond et al., 1988). The sponge
Leucetta from Saipan and Guam was the source of pyronaamide (9) and
found to inhibit activities of KB cells (MIC 5 µg/mL) (Akee et al., 1990).
Nortopsentins A (10), B (11), and C (12), having a characteristic 2,4-bis
(3-indolyl)imidazole skeleton were obtained from Spongsorites ruetzleri,
a deepwater marine sponge. Nortopsentins A, B, and C showed in vitro
cytotoxicity against P388 cell: IC50 (mg/mL) 7.6, 7.8 and 1.7, respectively
(Sakemi and Sun, 1991; Sun et al., 1991; Kawasaki et al., 1996).
2.2.2 ANTITUMOR INDOLE ALKALOIDS FROM MARINE

SPONGES
Manzamines are one of the most complex alkaloidal structures. Manzamine

A (13) is found in the Haliclona sp., Pellina sp., Xestospongia sp., and Pachy-
pellina sp. from marine sponges and reported to possess various biological
properties such as antimicrobial, anti-inflammatory, and anticancer activities
(Radwan et al., 2012). Manzamine A (13) was extracted from a Haliclona sp.
of sponge from Okinawa as its hydrochloride salt with an IC50 of 0.07 μg/mL
against P388 cells in vitro (Sakai et al., 1986) and enhanced the sensitivity to
tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-apoptosis
inducers in human adenocarcinoma pancreatic cells (Guzman et al., 2011).
The same sponge resulted in the disclosure of manzamines B (14) and C
(15) (Sakai et al., 1987), while as manzamines E (16) and F (17) were later
isolated from genus Xestospongia (Ichiba et al., 1988). Manzamines A–F
showed cytotoxicity against P388 cells in vitro: IC50 (μg/mL) 0.07, 0.06,
0.03, 0.05, and 0.05, respectively. A marine sponge crude extract designated
PAL93-055 collected from Palau resulted in the isolation of two new isomeric
manzamines, namely N-Methyl-epi-manzamine D (18) and epi-manzamine
D (19). These two isolated isomers (18, 19) showed cytotoxic effects against
B16F10 and HeLa cell lines with highest potency (IC50: 0.1 mg/mL) for
N-methyl-epi-manzamine D (18) for B16F10 cell line and were consistent
with previously reported manzamine alkaloids (Zhou et al., 2000).
Dragmacidin (20), obtained from deepwater sponge, was found to be
toxic to P388 cells (IC50: 15 μg/mL) as well as to KCT-8 human colon, A549
human lung, and MD-AMB human mammary cells, all with IC50 of 1–10 μg/
mL (Kohmoto et al., 1988). Another closely related compound dragmacidon
A (21) was later reported by Morris and Andersen (1989), which exhibited
cytotoxicity against Ll210 cells with ED50, 10 μg/mL similar to dragmacidin.
Topsentins, bisindole alkaloids were discovered almost at the same
time by two different groups. Bartik et al. (1987) in Belgium were the first
to report topsentins A (22), B1 (23), and B2 (24) from Topsentia genitrix
sponge and their toxicity effects to fish at 15–20 mg/L (Braekman et al.,
1987). Subsequently, Tsujii and Rinehart (1988) also isolated topsentins A
(designated deoxytopsentin), B1 (designated, topsentin), and B2 (designated,
bromotopsentin) along with a new metabolite dihydrodeoxybromo topsentin
(25) from family Halichondriidae, a deepwater sponge. Also, in vitro and
very weak in vivo activities against P388 (IC50: 12.0, 2.0, 7.0, 4.0 μg/mL)
and tumor-initiating cell, TIC132 at 75 mg/kg, TIC126 at 75 mg/kg, respec-
tively were reported.
Fascaplysin (26) is an unusual pentacyclic-fused planar alkaloid isolated
from Fascaplysinopsis bergquist sp. sponge (Roll et al., 1988). Recently, three
new members of fascaplysin, namely 3-bromofascaplysin (27), 14-bromore-
ticulatine (28), 14-bromoreticulatate (29) along with reticulatate (30), were
isolated from the sponge Fascaplysinopsis reticulate and two collections of
the tunicate Didemnum sp. (Segraves et al., 2003). These isolates (26–30)
were tested for in vitro solid tumor selectivity against a panel of human and
murine tumor cells. Among all tested compounds, fascaplysin (26) exhibited
murine solid tumor selectivity and was found to be most cytotoxic. Beside
this, fascaplysin (26) possesses a vast range of biological activities including
antimicrobial, p56 tyrosine kinase, HIV-1-RT, antimalarial, potency to
various cancer cell lines (Hörmann et al., 2001; Kirsch et al., 2000; Schmidt
and Faulkner, 1996; Jimenez et al., 1991a and 1991b). It induces apoptosis,
cell-cycle arrest, and reactive oxygen specie generation (Hamilton, 2014).
Moreover, fascaplysin also exhibited synergistic cytotoxicity with the
topoisomerase I inhibitors in competent to camptothecin, 10-hydroxycamp-

tothecin, and topotecan on chemoresistant NCI-H417 small cell lung cancer,
but the mechanism of action is still unknown (Hamilton, 2014).
Trachycladindoles A-G (31–37), a 2-amino-4, 5-dihydroimidazole
bearing alkaloids have been isolated from Trachycladus laevispirulifer
(Southern Australian marine sponge). They displayed a substitution-depen-
dent pattern cytotoxicity against human cancer cell lines A549, MDA-MB-
231, and HT-29 (Capon et al., 2008).
2.2.3 CYTOTOXIC PYRIDINES ALKALOIDS FROM MARINE

SPONGES
Theonelladins are the pyridine skeleton alkaloids. Theonelladins A (38), B

(39), C (40) and D (41) were isolated from the Theonella swinhoe in addi-
tion to swinholide macrolides (Kobayashi et al., 1989). Theonelladins A–D
exhibited in vitro cytotoxicity against L1210 cells: IC50 (μg/mL) 4.7, 1.0,
3.6, and 1.6, respectively, and KB cells: ED50 (μg/mL) 10.0, 3.6, 10, and
5.26, respectively. Also, the theonelladins are reported to be 20 times more
efficient in causing the release of Ca2+ from sarcoplasmic reticulum than
caffeine. Other pyridine-related alkaloids niphatynes A (42) and B (43) have
been isolated from Niphates sp. collected from Fiji, which were cytotoxic
against P388 cells: IC50 (μg/mL) 0.5 (Quinoa and Crews, 1987).
Echinoclathrines A–C (44–46), the new class of pyridine alkaloids
possessing 4-aryl-2-methylpyridine moiety as the main structure element,
were isolated from Echinoclathria sp. from an Okinawan sponge. All these
compounds (44–46) were tested for cytotoxic properties and results showed
that compound 44 was cytotoxic against A-549, HT-29, and P388 cells with
IC50 value of 10 µg/mL. Also, compounds 44 and 45 demonstrated weak
immunosuppressive activity in a mixed lymphocyte reaction assay with IC50
of 7.9 and 9.7 µg/mL, respectively (Kitamura et al., 1999).
The macrocyclic alkaloids haliclamines A (47) and B (48) extracted from a
sponge belonging to genus Haliclona were reported to be active against various
cell assays: P388 cells—IC50 (μg/mL) 0.75, 0.36; L1210 cells: IC50 (μg/mL)
1.5, 0.9; inhibition of cell division of eggs of sea urchin Hemicentrotus pulcher-
rimus: IC50 (μg/mL) 5, 10, respectively (Fusetani et al., 1989). Biogenetically,
these alkaloids are closely related to halitoxin (Schmitz et al., 1978).
The unique bis-3-alkylpyridine alkaloids containing cis-cyclopent[c]
isoxazolidine moiety, pyrinodemins A-D (49–52) and related -alkyl pyridine
alkaloids have been isolated from Amphimedon sp. from the Okinawan
marine sponge (Hirano et al., 2000a). Cytotoxicity evaluation revealed

pyrinodemin (A–D [49–52]). Compounds 50 and 51 demonstrated signifi-
cant cytotoxicity against KB epidermoid carcinoma cells (IC50: 0.5 mg/
mL, each) and murine leukemia L1210 (IC50: 0.07, 0.06, and 0.08 mg/mL,
respectively) in vitro comparable to pyrinodemin A (Hirano et al., 2000a).
Novel tetracyclic alkylpiperidine alkaloids, arenosclerins A (53), B (54),
and C (55) along with haliclonacyclamie E (56) have been isolated from
Arenosclera brasiliensis, a marine sponge (Torres et al., 2002). All these
compounds were tested for cytotoxicity and showed an almost similar range
of cytotoxicity, irrespective of the cancer cell line tested.
Marine sponge Amphimedon sp. collected from Okinawan resulted
in the isolation of two bis-3-alkylpyridine alkaloids, pyrinodemin G, and
pyrinodemin H. The structures of pyrinodemins G and H are composed of
N-methoxy amide moiety and an α,β-unsaturated 3,5-disubstituted δ-lactone
moiety at a junction of two 3-alkylpyridines, respectively. There is a
possibility of multiple structures for pyrinodemins G and H. Furthermore,
pyrinodemins G and pyrinodemin H showed significant antitumor activity
against P388 murine leukemia cells with IC50 values of 9.6 and 2.5 mg/mL
(Williams et al., 1998).
The pyridoacridines constitute an interesting class of secondary
metabolites from a marine source and many compounds of this series are
reported to possess potent cytotoxic activity (Molinski, 1993; Ding et al.,
1999). Amphimedine (57) was the first pyridoacridine alkaloid isolated from
marine organism Amphimedon sp. in 1983 (Schmitz et al., 1983). A few
years later, De Guzman et al. reported the isolation of neoamphimedine (58)
and deoxyamphimedine (59), two structurally related compounds, from two
tropical Xestospongia sponges (Tasdemir et al., 2001). Neoamphimedine
(58) showed significant antineoplastic activity tumors in athymic mice. The
results showed that neoamphimedine was as effective as 9-aminocamptoth-
ecinin in HCT-116 tumors-bearing mice and as effective asetoposide in KB
tumors-bearing mice. However, amphimedine (57) neither showed any potent
antineoplastic activity nor induced any catenation or DNA aggregation in
vitro. Therefore, the results suggested that neoamphimedine (58) possesses
two-mediated mechanism of anticancer and cytotoxicity potential (Marshalla
et al., 2003). Pyridoacridine alkaloids, labuanine A (60), (9-aminobenzo[b]
pyrido[4,3,2-de] [1,10] phenanthrolin-8(8H)-one (61)), (9-hydroxybenzo[b]
pyrido[4,3,2-de] [1,10]phenanthrolin-8(8H)-one) (62) and biemnadin (63),
were extracted from Biemna fortis from an Indonesian marine sponge (Aoki
et al., 1969). The isolated pyridoacridine alkaloids induced multipolar neuri-
togenesis in more than 50% of cells at 0.03–3 µM concentration. Compound
(60), exhibited the strongest neuritogenic activity and induced increase in

acetylcholinesterase, arrested cell cycle at the G2/M phase and a neuronal
marker in Neuro 2A. Whereas compounds (61), (62), and (63) induced
neurite outgrowth in more than 50% of cells at 1–3 µM concentration. The
huge difference in neuritogenic activity between compounds (61) and (62) is
due to the presence of amino group at C-9 (Fuente et al., 2001).
2.2.4 CYTOTOXIC PYRROLES ALKALOIDS FROM MARINE

SPONGES
A novel bisquinolinylpyrrole named as halitulin (64) has been isolated

from the Haliclona tulearensis, a marine sponge, and has shown potent
cytotoxicity against panel of cell lines, such as murine leukemia P388 (IC50:
0.025 mg/mL), human colon carcinoma HT-29 (IC50: 0.012 mg/mL), human
melanoma MEL-28 (IC50: 0.025 mg/mL), and human lung carcinoma A-549
(IC50: 0.012 mg/mL) (Kashman et al., 1999). Wright and Thompson in
1987 reported imidazolyl pyrroloazepines alkaloid (65) from Teichaxinella
morchella and Ptilocaulis walpersi sponges and showed mild cytotoxicity
against L1210 cells (IC50: 10.0 μg/mL).
2.2.5 ANTITUMOR QUINOLINES AND ISOQUINOLINES

ALKALOIDS
An unidentified species from Indonesian marine sponge of genus Xesto-

spongia sp. has resulted in isolation of four new novel (66–69) alkaloidal
compounds from aaptamine class in addition to known aaptamine (70),
isoaaptamine (71), demethyl(oxy)aaptamine (72) and its dimethylketal (73)
(Calcul et al., 2003; Shen et al., 1999). These compounds were screened for
antitumor against KB cells and all the tested compounds (70–73) showed
significant cytotoxic activity on KB cells (ID50 mg/mL, 3.7, 0.5, 1.8, 3.5,
new four compounds 66–69 = > 10). In another study, antitumor activity
screening of compound (isoaaptamine, demethyl(oxy)aaptamine) against
Ehrlich ascites tumors in mice showed 95% of inhibition in mice pretreated
with compounds (71) and (72) at 25 μg/mL (Fedoreev et al., 1988). These
compounds were also reported from genus Suberites of the marine sponges.
Compounds (71) and (72) were also reported from the Aaptos aaptos
sponge and showed toxic effects on HeLa cells with ED50: 2 and 0.87 μg/
mL, respectively (Nakamura et al. 1987). Shubina et al. (2010) reported
2,3-dihydro-2,3-dioxoaaptamine (74), 6-(N-morpholinyl)-4,5-dihydro-5-

oxodemethyl(oxy)aaptamine (75), and 3-(methylamino)demethyl(oxy)
aaptamine (76) from the Vietnamese sponge Aaptos sp. All the isolated
compounds were tested for apoptosis-inducing activity and compound (76)
showed promising apoptosis-inducing activity (40% of early and 56% of late
apoptosis at 208 μM concentration).
Four new dimeric aaptamine alkaloids, suberitines A (77), B (78), C (79),
and D (80) together with two known monomers have been isolated from
marine sponge Aaptossu beritoids, collected from Xisha Island in South
China. Cytotoxic evaluation of suberitine A–D (77–80) against P388, K565,
and HeLa cell lines, suberitines B and D showed potent cytotoxicites against
selected P388 cell lines with IC50 values of 1.8 and 3.5 μM, respectively (Liu
et al., 2012).
Isobatzellines A–D (81–84) are the pyrroloquinoline alkaloids that were
reported to be obtained from Batzella sp. of the Caribbean sponge (Sun et al.,
1990). Significant cytotoxicity has been reported for these isolated Isobatzel-
lines A–D; in addition to antifungal activity against Candida albicans. Renierol
(85) isolated from the Xestospongia caycedoia Fijian sponge and inhibited the
growth of L1210 cell with IC50 value 3 μg/mL (McKee and Ireland, 1987).
2.2.6 CYTOTOXIC DISCORHABDINS/PRIANOSINS ALKALOIDS
Discorhabdins and prianosins are unusual but closely related fused pyrro-
lophenanthroline sulfur-containing alkaloids, which were independently
reported by two groups of researchers. These natural discorhabdin alkaloids
possess a unique structure with core tetracyclic pyrroloiminoquinone
skeleton bound to spirocyclohexanone at the C-6 position and few contain
sulfur-containing substituents at the carbon-6 position (Hu et al., 2011). The
first reported compound was discorhabdin C (88) by Perry et al. in 1986.
Few years later, the same group reported remaining discorhabdins, that is,
discorhabdins A (86), B (87), D (89) (Perry et al., 1988a and 1988b). The other
sources for these discorhabdins reported were Latrunculia brevis, Prianos
sp., and Haplosclerida. Cytotoxicity results for this class of compounds
showed that compounds (86), (87), and (88) were inactive in vivo P388 cell
lines (TIC < 12%, toxic dose 2 mg/kg), while discorhabdin D (89) was found
active in vivo against P388 cell lines (TIC132% at 20 mg/kg dose). Thus,
the results showed that only discorhabdin D showed modest in vivo activity
against P388 cell line: TIC at 20 mg/kg. Kobayashi et al. in 1987 isolated
prianosin A (90) from Prianos melanos, an Okinawan sponge. Structurally
prianosin A is a non-protonated form of discorhabdin A. Other prianosins

B–D (91–93) were reported by Cheng et al. in 1988. Cytotoxicity screening
of prianosins A–D (91–93) against Ll210, L5178Y cell lines and results
showed that only prianosin D (93), induced Ca2+ release from sarcoplasmic
reticulum with a potency 10 times that of caffeine.
2.2.7 POLYCYCLIC AROMATIC ALKALOIDS: ACRIDINE

ALKALOIDS
During the last few years, polycyclic aromatic alkaloid obtained from
sponges and ascidians, containing a tetracyclic moiety in common, includes
the acridine ring system. Amphimedine was the first described compound of
this series and has been isolated from Amphimedon sp. from a gum sponge.
Amphimedine showed in vitro activity against P388 cells with ED50 value of
0.4 μg/mL but proved inactive in vivo (Schmitz et al., 1983).
Dercitin (94), obtained from various species of Dercitus, a deepwater
sponge, which was found active against P388 in vitro with an IC50 value of
0.54 μg/mL as well as in vivo with TIC 170 at 5 mg/kg and against human
cancer cell lines HCT-8, A-549, and T47D in vitro with an IC50 value of
1.0 μg/mL. Mechanism of action studies revealed that dercitin disrupts the
DNA, RNA, and protein synthesis in the P388 system by binding to DNA
and inhibiting nucleic acid synthesis. It is an effective inhibitor of DNA Nick
translation at concentrations that disrupt the superhelical density of DNA. It
relaxes covalently closed supercoiled ΦX174DNA, indicating intercalation
as the mode of binding (Burres et al., 1989). Dercitin was slightly active
against B16 tumors in mice in vivo (TIC125 at 1.25 mg/kg). In addition,
dercitin is reported to possess immunosuppressive and antiviral activity. The
remaining members of dercitin family, namely nordercitin (95), dercitamine
(96), dercitamide (97), and cyclodercitin (98) have been isolated from
two species of family Pachastrellidae, a deepwater sponge (Gunawardana
et al., 1989; see Gunawardana et al., 1992). These compounds (nordercitin,
dercitamine, dercitamide, and cyclodercitin) showed cytotoxic activity
against P388 cells with IC50 (μM) 4.79, 26.7, 12.0, and 1.9, respectively, in
addition to immunosuppressive activity.
Cyclodercitin (98) easily undergoes oxidation on exposure to air to give
cyclodercitin (∆13) (99), which is also cytotoxic. Cytotoxic motuporamines
A–C (100–102) are the first example of macrocyclic alkaloids reported
from the Xestospongia exigua, a marine sponge (Williams et al., 1998). X.
exigua are extracted by bioassay-guided fractionation resulted in isolation of
mixture of motuporamines A (100), B (101), and C (102) along with known

members of 3-alkyl piperidine alkaloidal series xestospongin/araguspongine
and petrosin. The motuporamines having spermidine-like substructure
represent a new class of cytotoxic alkaloids derived from sponge which are
biogenetically formed of basic building blocks, that is, ammonia, acrolein,
and a long-chain dialdehyde involving a pathway of 3-alkylpiperidine
alkaloids isolated from marine sponges in the order Haplosclerida. The
underivatized mixture of motuporamines demonstrated modes of cytotox-
icity against a panel of human solid tumor cancer cell lines in vitro with an
IC50 value of 0.6 mg/mL (Baldwin and Whitehead., 1992; Andersen et al.,
1996). Thorectandramine, a novel quaternary hexacyclic alkaloid, has been
isolated from genus Thorectandra from a Palauan sponge (Charan et al.,
2002). The compound showed in vitro cytotoxicity against MALME-3M
(melanoma), OVCAR-3 (ovarian), MCF-7 (breast), and A549 (non-small
lung cell cancer) four human tumor cell lines (Charan et al., 2002).
Plakinidines are acridine alkaloids with slight variation in the acridine
moiety from the Fiji sponge and a Plakortis sp. assayed using an antiparasite
bioassay, which led to the isolation of plakinidines A (103) and B (104)
(Inman et al., 1990). Subsequently, plakinidine A was reported to inhibit
reverse transcriptase activity at 1 μg/mL. In the same year West et al. (1990)
described the isolation of plakinidines A (103), in addition to plakinidines
B (104) and C (105) from the same source and reported cytotoxicity against
L1210 cells with IC50 values of 0.7, 0.1, and 0.3 µg/mL respectively.
2.2.8 CYTOTOXIC GUANIDINES ALKALOIDS
Ptilomycalin A (106), a novel guanidine polycyclic alkaloid, obtained from

the Caribbean sponge from Ptilocaulis spiculifer (Kashman et al., 1989) and
Hemimycale sp. from a Red Sea sponge, which showed toxic effects on P388
cells (IC50: 0.1 μg/mL) as well as antifungal activity against C. albicans and
antiviral activity against herpes simplex virus (HSV) at 0.2 μg/mL.
Guzii et al. (2010) reported a novel polycyclic guanidine alkaloid monan-
chocidin (107) from marine sponge Monanhora pulchra that induced cell
death in human leukemia THP-1, human cervix epitheloid carcinoma HeLa,
and mouse epidermal JB6C141 cells with IC50 value of (μM) 5.1, 11.8, and
12.3, respectively. Monanchocidin also induces 60% of early apoptosis in
THP-1 cells at 3.0 μM. Urupocidins A and B, bisguanidine alkaloids with
an unprecedented skeleton derived from polyketide precursors and having
N-alkyl-N-hydroxylguanidine moiety, were obtained from the same sponge,
that is, M. pulchra by Makarieva et al. (2014) and urupocidin A showed the
increase in the production of nitric oxide in murine macrophages through
inducing inducible nitric oxide synthase expression. Recently, eight new
rare guanidine alkaloids, namely monanchocidin A (108), monanchocidin B
(109), monanchomycalin C (110), ptilomycalin A (106), monanchomycalin
B (111), normonanchocidin D (112), urupocidin A (113), and pulchranin A
(114) have been isolated from M. pulchra. All of these constituents showed
cytotoxic properties and are able to prevent epidermal growth factor-induced
neoplastic transformation of JB6 P+ Cl4 1 cells in vitro. Moreover, the
current study suggests that these marine guanidine alkaloids hold potential
to eliminate human cancer cells and prevent cancer cell formation and
spreading (Dyshlovoy et al., 2016).
2.2.9 BROMINE CONTAINING ALKALOIDS
Ma’edamines A–B (115–116) are the bromotyrosine alkaloids, possessing

unique 2(1H) pyrazinone moiety sandwich between two bromotyrosine units
and were extracted from Suberea sp. from the Okinawan marine sponge.
Biogenetically, ma’edamines A and B may be originated by 11, 12-dehydro
form of aplysamine-2 or purpuramine H through formation of a six-membered
ring and dehydroxylation. Cytotoxic screening of ma’edamines A (115) and B
(116) showed cytotoxicity against L1210 murine leukemia cells (IC50: 4.3 and
3.9 mg/mL, respectively), and KB epidermoid carcinoma cells (IC50: 5.2 and
4.5 mg/mL, respectively) in vitro. Furthermore, compound (115) exhibited in
vitro inhibitory activity against c-erbB-2 kinase (IC50: 6.7 mg/mL), whereas
compound (116) proved to be inactive (IC50 > 10 mg/mL) (Hirano et al., 2000b).
Matemone (117), a bromine-containing oxindole alkaloid along with
known 6-bromoindole-3-carbaldehyde was isolated from Iotrochota
purpurea sponge collected from the Indian Ocean, exhibited mild cytotox-
icity against growth of three cell lines: pancreatic cancer MIA PaCa-2 cell
line, lung cancer NSCLC-N6 L16 strain18, and prostate cancer DU145 cell
line with IC50 (mg/mL) 24, 30, and 27 respectively (Carletti et al., 2000).
A bromopyrrole longamide (118) was obtained originally from Agelas
longissima from a Caribbean sponge and later was also isolated from Japa-
nese marine sponge from Homaxinella sp. (Umeyama et al., 1998). The
compound showed cytotoxicity against lymphocytic leukemia P388 cells,
in addition to antibacterial activity (MIC 60 mg/mL). Longamide B methyl
ester in racemic form was isolated from isolated from as Homaxinella sp. and
showed weak in vitro cytotoxic activity against lymphocytic leukemia P388
cells (30 mg/mL) (Umeyama et al., 1998). The corresponding longamide B

(119) acid in racemic form has been isolated from the Agelas dispar from the
Caribbean marine sponge showed modest antibiotic activity against several
strains of Gram-positive bacteria (MIC 50 mg/mL). Hanishin (120), the ethyl
ester of longamide B has been isolated from highly polymorphic extracts of
Acanthella carteri sponge and showed cytotoxicity toward human non-small
lung carcinoma NSCLC-N6 cells (9.7 mg/mL) (Mancini et al., 1997).
2.2.10 CYTOTOXIC PYRIMIDINE ALKALOIDS
Variolins A–D (121–124) is a group of heterocyclic alkaloidal compounds

obtained from Kirkpatrickia variolosa, an Antarctic marine sponge, and are
composed of tricyclic skeleton, which has no precedents in either terrestrial
or marine natural products, a pyrido[3′, 2′:4, 5]pyrrolo[1,2-c] pyrimidine,
substituted at position 5. Biological screening of these compounds exhibited
interesting antiviral and antitumor activity against murine leukemia P388 cells
(Perry et al., 1994; Trimurtulu et al., 1994). Variolin B showed potent cytotoxic
and cytostatic effects against various human leukemia K-562, U-937, MOLT-4
cell lines and Jurkatt, human ovarian carcinoma OVCAR-3, SKOV-3 cell
lines, and human intestinal carcinoma LoVo cell line. Variolin B was equally
effective against LoVo carcinoma and its multidrug resistant variant LoVo/
Dx overexpressing Pgp. The concentration of variolin B in the nM range for
1 h cause G1 cell arrest and a decrease in progression rate of S-phase cells to
G2, while as concentrations in mM range for a little time induced blockade in
G2 phase. Studies revealed that variolin B is a strong activator of apoptosis
assessed by biochemical and morphological methods (Erba et al., 2002).
2.2.11 ALKALOIDS DERIVED FROM SPONGES IN CLINICAL/

PRECLINICAL TRIAL
Sponges have proven to be an interesting source of a large array of bioactive

alkaloids, and few of them reported in the literature can potentially compete
with anticancer alkaloids isolated from terrestrial plants, and are in clinical
or preclinical trials. Soni et al. (2000) discovered the new potential use of
fascaplysin, a marine sponge-derived natural product. While investigating
for inhibitors of cyclin-dependent protein kinases and key enzymes involved
in the mammalian cell cycle, they observed that fascaplysin selectively
inhibited Cdk4 kinase in vitro with an IC50 0.35 μM. Molecular modeling
studies showed that fascaplysin binds to the ATP binding pocket of Cdk4
by interacting through a bidentate hydrogen bond/acceptor pair (Soni et al.,
2000). The fact that fascaplysin caused G1 arrest not only in normal human
fibroblasts but also in both human colon carcinoma and osteogenic sarcoma
cell lines make this marine chemical an interesting candidate for the further
study of cellular processes regulated by Cdk4 kinase in mammalian cells.
Manzamine is another series of sponge which was studied by Zhou et al.
(2000) in novel antiangiogenesis, while Hirano et al. (2000b) showed that
ma’edamine A had inhibitory activity against the c-erbB-2 kinase in vitro.
All these compounds were tested in cytotoxicity assays that most commonly
consisted of panels of either human or murine tumor cell lines. In a few
reports, cytotoxicity studies were very extensive and included the National
Cancer Institute 60-tumor cell line screen (Calcabrini et al., 2017). These
novel compounds exhibited potent cytotoxic activity, defined as an IC50 of
4.0 g/mL, and would appear as possible candidates for studying mechanism
of action. This would help to determine if the reported cytotoxicity was the
result of a pharmacologic rather than a toxic effect on the tumor cell used for
the reported investigation.
2.3 CONCLUSION
Marine sponges have been clearly established as a source of novel alkaloids

possessing promising anticancer and cytotoxic properties that can be used for
selecting hits in various pharmacotherapeutic areas. At least three leads are
already in clinical trials or appear to be destined for such testing. These attained
leads and the promise of uncovering new biochemical techniques that may
be crucial for studying disease mechanisms provide a continuing impetus for
further exploration of marine sponges as a potential source of anticancer drugs.
KEYWORDS
•• sponge
•• marine environment
•• alkaloids
•• anticancer
•• secondary metabolites
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CHAPTER 3
MARINE ANTIOXIDANTS AND

ASSAY METHODS
ONYEKA KINGSLEY NWOSU1,* and DAVID OKECHUKWU OKEKE2
1
National Biosafety Management Agency (NBMA), Abuja, Nigeria,
Tel.: +2348065002754
Department of Applied Biochemistry, Nnamdi Azikiwe University,
2
Awka, Nigeria
*
*
ABSTRACT
The marine organisms are now an emphasis of natural products drug

discovery research because of its relatively unexplored biodiversity
compared to terrestrial environment. Research has been developed using
many biotechnological tools to harness and to produce new marine essential
compounds with the aim of increasing the availability and chemical diversity
of marine organisms’ essential constituents. Indeed the bioprospecting
marine organisms for secondary metabolite production are a promising area
in marine biotechnology. Most of these marine organisms are great sources
of natural antioxidants and nutritional compounds. This chapter examines
the concepts of marine organisms, the antioxidants of the marine organisms
(algae and sponges), and determination of antioxidant activities.
3.1 INTRODUCTION
The marine environment is a good source of chemical and biological active

compounds. The organisms that occupy the marine environment are said to
be potential sources of interesting compounds for uses towards economic
development like the food industry, neutraceuticals, pharmaceutical industry,

cosmetic industry, and so many other industrially important compounds
(Marine Biotechnology, 2003). There are enormous marine-based products
within the marine environment which can increasingly promote the exploi-
tation of the vast diversity of the marine life. The marine ecosystem still
remains an untapped reservoir of biologically active compounds which have
considerable capabilities to provide chemical constituents towards the devel-
opment of new conventional and ethnopharmacological drugs and as well as
supply food ingredients towards the progress of new functional foods and
nutraceuticals. However, production of ample amount of persistent quality
and inexpensive marine products has been the foremost impediment for
marine biotechnologists, notwithstanding the boundless potential of marine
organisms (Slevan et al., 2012).
Research has been developed using many biotechnological tools to
harness and to produce new marine essential compounds with the aim of
increasing the incessant availability and chemical diversity of marine organ-
isms’ essential constituents. Certainly, the hunting of marine organisms
for the production of secondary metabolite is an auspicious area in marine
biotechnology. Most of these marine organisms are the richest sources of
natural antioxidants and nutritional compounds (Balakrishan et al., 2014).
The antioxidants usually produced by the marine environment include flavo-
noids, benzoic acid, gallic acid, and cinnamic acid (Al-Saif et al., 2014).
Marine environment can thereby be considered as producers of substances
that are useful for the treatment of human diseases since antioxidants appli-
cation serves as an efficient method in avoiding or minimizing or reducing
the formation of toxic oxidation products, lipid peroxidation in food prod-
ucts, and maintaining nutritional quality. Among the demerits of the marine
environment as benefits to human health is that secondary metabolites from
their ecosystem are often produced only in trace amounts in such that large
quantity of sources must be collected to be able to attain adequate amounts
of the target compound.
3.2 MARINE ORGANISMS
The marine environment contains more than 200,000 described species of

invertebrates and algae, however, it is estimated that this number is but a small
percentage of the entire number of species that have yet to be discovered and
described. Conservatives estimates suggest that marine environment subsur-
face bacteria could constitute as much as 10% of the total living biomass
Marine Antioxidants and Assay Methods 67
carbon in the biosphere. However, thousands of chemical compounds have

been isolated from a relatively small number of these species that have been
studied to date.
Marine plants, animals, and microbes produce compounds that have
potential as pharmaceuticals due to the secondary metabolites they contain.
The marine organisms became a focus of natural products drug discovery
research due to its relatively unexplored biodiversity when compared to
terrestrial environment. The thought of marine organisms as the basis of
natural products for pharmaceuticals and therapeutics was introduced by the
first work of Bergmann in the 1950s (Bergmann and Feeney, 1951) which led
to the only two marine-derived pharmaceuticals that are clinically available
today; anticancer drug, Ara-C and antiviral drug, Ara-A. Among the vast
array of marine organisms with exclusive biological properties, the most
studied and characterized are algae (seaweeds) and sponges.
3.2.1 ALGAE
Marine algae are chlorophyll-containing organisms made up of a cell or

grouped together in colonies or as organisms with many cells, occasion-
ally interacting together as simple tissues (Lordan et al., 2011). The
marine algae, therefore, would be classified as unicellular or multicellular
vegetative organisms that do not have true roots or stems and greatly vary in
morphology and size up to 70 m long, 3 - 10 µm in length and growing up
to 50 cm/day (El Gamal, 2010). Marine algae are a heterogeneous group of
plants with long fossil history which can be classified into two main groups
according to their size. They are:
i) Macroalgae: They occupy the littoral zone, which included green

algae, brown algae, and red algae. They are mainly considered by
biologists as the seaweeds. They possess several characteristics
that are used to categorize them including the nature of their chlo-
rophyll, the presence, and absence of flagella and their cell wall
chemistry.
ii) Microalgae: They are known as phytoplankton and dominate in both
benthic and littoral habitats and also throughout the ocean waters.
The phytoplankton comprises organisms such as diatoms (bacillari-
ophyta), dinoflagellates (dinophyta), blue-green algae (cyanophyta),
yellow-brown and green flagellates (chlorophyta, prasinophyta,
prymnesiophyta, chrysophyta, cryptophyta, and rhaphidiophyta). As
photosynthetic organisms, this group plays a key role in the produc-

tivity of oceans and constitutes the basis of the marine food chain
(Bold and Wyne, 1985).
3.2.1.1 MACROALGAE (SEAWEEDS)
Macroalgae or seaweeds are a source of biologically active phytochemicals,

which comprise of fatty acids, polysaccharides, carotenoids, phycobilins,
vitamins, sterols, phycocyanins, and tocopherol among others (Lordan
et al., 2011). The nutritional and chemical composition of the macroalgae
depends on many factors which include species, water temperature,
geographical origin, seasonal, environmental, and physiological varia-
tions, and processing methods (Mabeau and Fleurence, 1993). The high
antioxidant content is one of the major nutritive features of macroalgae.
Seaweeds are well known for containing reactive antioxidant molecules
such as glutathione (GSH) when fresh, as well as secondary metabolites
like carotenoids (ά- and β-carotene, fucoxanthin, astaxanthin), catechins,
and tocopherols (Yuan et al., 2005). Among macroalgae, natural anti-
oxidants, terpenoids, phlorotannins, polyphenols, phenolic acids, antho-
cyanins, hydroxycinnamic acid derivatives, and flavonoids are important
(Bandoniene and Murkovic, 2002). Flavonoids have effective antiviral,
anti-allergic, and have free radical scavenging abilities and also offer
protection against cardiovascular mortality. Table 3.1 shows some algae
proven to possess some antioxidants.
TABLE 3.1 Algae Sources of Nutritional Antioxidants

Antioxdants Specie of algae References
Vitamin C Ulva sp., Monostroma undulatum, Undaria MacArtain et al. (2007),
pinnatifida, Ascophyllum nodosum, Laminaria Bocanegra et al. (2009),
digitata, Porphyra umbilicalis, Palmaria Brown et al. (1997)
palmata, Thalassiosira pseudonana,
Chaetoceros muelleri, Gracilaria changgi
Vitamin E Ulva rigida, A. nodosum, Dunaliella Taboada et al. (2010),
tertiolecta, U. pinnatifida, L. digitata, P. MacArtain et al. (2007),
umbilicalis, P. palmata Carballo-Cárdenas et al.
(2003)
Carotenoids Porphyridium cruentum Rebolloso-Fuentes et al.
(2000)
TABLE 3.1 (Continued)
Antioxdants Specie of algae References

α-carotene Chlorella pyrenoidosa, Dunaliella salina Inbaraj et al. (2006), Hu
et al. (2008)
β-carotene A. nodosum, C. pyrenoidosa, Chlorella Inbaraj et al. (2006), Cha
vulgaris, Chlorococcum, D. salina, Fucus et al. (2010), Yuan et al.
serratus, Fucus vesiculosus, G. changgi, (2002), Hu et al. (2008),
Haematococcus pluvialis, L. digitata, Okai et al. (1996), Plaza
Laminaria saccharina, Pelvetia canaliculata, et al. (2010)
Phormidium sp., Porphyra tenera,
Synechocystis sp.
Antheraxan- D. salina, L. digitata, L. saccharina Yokthongwattana et al.
thin (2005)
Lutein C. pyrenoidosa, C. vulgaris, Chlorella Del Campo et al. (2007),
zofingiensis, Chlorococcum, D. salina, H. Inbaraj et al. (2006), Cha
pluvialis, Muriellopsis sp., Phormidium sp., et al. (2010), Yuan et al.
P. tenera, Scenedesmus almeriensis. (2002), Hu et al. (2008),
Okai et al. (1996), Rodri-
guez-meizoso et al. (2008)
Zeaxanthin L. saccharina, P. canaliculata, Phormidium Inbaraj et al. (2006), Hu
sp., A. nodosum, C. pyrenoidosa, D. salina, et al. (2008), Plaza et al.
F. serratus, F. vesiculosus, H. pluvialis, (2010), Rodriguez-meizoso
Himanthalia elongata, L. digitata, L. et al. (2008)
saccharina, P. canaliculata, Synechocystis sp.
α-tocopherol P. cruentum, Laminaria ochroleuca, Durmaz et al. (2007),
Saccorhiza polychides, Himanthalia elongate Sánchez-Machado et al.
(2002)
γ-tocopherol Tetraselmis suecica, P. cruentum Carballo-Cárdenas et al.
(2003), Durmaz et al.
(2007)
Pheophytin a C. vulgaris, P. cruentum Cha et al. (2010),
Rebolloso-Fuentes et al.
(2000)
Pheophytin b C. vulgaris, P. cruentum Cha et al. (2010),
Rebolloso-Fuentes et al.
(2000)
Polyphenols Fucus sp., H. pluvialis, Laminaria sp., Bocanegra et al. (2009),
Porphyra sp., Spongiochloris spongiosa, Klejdus et al. (2009),
Undaria sp. Bocanegra et al. (2009)
Astaxanthin C. vulgaris, Chlorococcum sp. Mendes et al. (1995), Li
and Chen (2001)
Source: Adapted from ref Lordan et al. (2011). https://creativecommons.org/licenses/by/3.0/
3.2.1.2 MICROALGAE
There are over 50,000 different species of microalgae of which only a few
have been characterized (Bhakuni and Rawat, 2005). This group of microor-
ganisms is exceptionally diverse and represents a major unexploited resource
of valued bioactive compounds and biochemicals such as pigments, antioxi-
dants, fatty acids, and vitamins (Mata et al., 2010). Microalgal production
of carotenoids, such as β-carotene and astaxanthin, has been an attractive
area of research as they are valuable bioactive ingredients that can present at
relatively high concentrations in algal cells. Moreover, larger quantities of
carotenoids can be produced when cultivated algae are induced by control-
ling certain environmental growth conditions. The strains of microalgae
that are recently being studied for use as natural producers of commercial
carotenoids include Dunaliella salina, Sarcina maxima, Chlorella protothe-
coides, Chlorella vulgaris, and Haematococcus pluvialis.
D. salina has been found to be the most appropriate organism for the mass
production of β-carotene as it can produce β-carotene in the range of 14% of its
dry weight (Metting, 1996). β-carotene has strong antioxidant properties which
help to mediate the harmful effects of free radicals implicated in numerous
life-threatening diseases, including many forms of coronary heart disease,
premature aging, cancer, and arthritis. The antioxidant qualities of β-carotene
can also assist the body in suppressing the effects of premature aging caused
by ultraviolet (UV) rays (Dembitsky and Maoka, 2007). Momentous amounts
of xanthophylls, especially zeaxanthin, which possesses distinctive biological
properties with potential for disease prevention can be accumulated by D.
salina (Yokthongwattana et al., 2005). β-carotene derived from microalgae
is more biologically active than synthetically produced β-carotene and can be
marked as a “natural” food additive (Rasmussen and Morrissey, 2007). Natural
β-carotene also contains numerous carotenoids and essential nutrients that are
not present in the synthetic form and can be consumed in greater quantities as
the body tissues regulate its use (Olson and Krinsky, 1995).
Carotenoids and chlorophylls are also antioxidants that can be produced
in both closed and open-culture systems by another unicellular alga known
as “Haematococcus” (Rasmussen and Morrissey, 2007). H. pluvialis has the
capacity to accumulate large quantities (1.5–3% of dry weight) of the high-
value carotenoid, astaxanthin. H. pluvialis has been considered as a dietary
supplement in the United States and it has also been approved in several
European countries for human consumption (Mata et al., 2010). With up to
10 times antioxidant activity stronger than other carotenoids, astaxanthin
provides defensive activity against inflammation, cancer, and UV light. The
health benefits of astaxanthin alongside with its strong coloring characteris-

tics make it a prospective ingredient for use in the cosmetics, nutraceutical,
and food and feed industries.
Antioxidants vitamins A, C, E, biotin, and folic acid are also found to be
in high concentrations in many microalgae and are largely rich in chloro-
phylls (Spolaore et al., 2006). Microalgae are rich sources of nutritious and
biologically active compounds, however, not only is it their huge variety that
marks these microorganisms fascinating but also the likelihood of growing
them in diverse conditions and using them as natural reactors, results in an
enhancement of certain bioactive compounds. Though, prior to this, the algal
material must be analyzed for the presence of toxic compounds.
3.2.2 SPONGES
Sponges are among the first classes of marine invertebrates to be studied

by natural products chemists. Sponges are multicellular, undifferentiated
organisms equipped with an adept water-filtration system. Although techni-
cally considered animals, physiologically sponges are among the simplest
metazoans. Possessing no differentiated tissue to form digestive, nervous,
and circulatory systems, they are composed primarily of a porous network
of channels and chambers through which water moves. Following phago-
cytosis through apertures at the inhalant surface (ostia), food particles are
filtered through progressively smaller sieve-like channels into the mesohyl
for metabolism. The sieves are designed because indiscrete intake of water
and particles by non-selective filter feeders result in ingestion of many
particles which are of no biochemical value to the sponge. In order to retain
sufficient nutrients for metabolism while simultaneously keeping waste
levels from overwhelming the filtering system, the sponge must constantly
excrete particles from its aqueous environment. Materials that cannot be
used for energy are eliminated through apertures (oscules) in the exhalant
surface. The entire sponge biomass is involved primarily in maintaining its
low-pressure water-pumping system (Bergquist, 1978).
There have been misconceptions about sponges among taxonomists by
lack of consistent defining characteristics of sponge morphology. Amorphous
shapes, color variability within a species, and both flexibility in size and
pigment intensity depending on the degree of light exposure create classifi-
cation challenges. However, advancements in biochemical and histological
tests aid classification of collected sponges, and other researchers have begun
exploring the interesting technique of chemotaxonomy—identification by
differential metabolite production. Chemotaxonomy has been used to distin-

guish sponges down to the genera, but cannot be used to determine classifica-
tion at the species level (Verpoorte, 1998). There are three classes of sponges:
i) Demospongiae: This class includes 95% of identified species and

spans a wide range of habitats from intertidal depths to marine
trenches in fresh or brackish water.
ii) Hexactinellida: This class is characterized by six-membered hexactine
siliceous spicules. This rather unusual class lacks a mesohyl matrix
and is morphologically the most distinct from other poriferan classes.
iii) Calcarea: This class is composed of calcium carbonate-based skeletal
materials. Crystalline calciferous spicules may grow individually or
as one mass.
3.2.2.1 ANTIOXIDANTS FROM SPONGES
Sponges produce bioactive metabolites as part of their defensive system and

they rarely develop a synergistic relationship with both algae and microorgan-
isms. The symbionts to an extent are the true source of secondary metabolites
found in sponges. Biotic factors such as epifaunal diversity and morphology
alongside abiotic factors like pH, salinity, temperature, and so forth which
are influenced by seasonal changes are responsible for the biosynthesis
of secondary metabolites. Even though many bioactives have been found
in sponges, only a few of these compounds have been commercialized.
Therefore, the study of the chemical ecology of secondary metabolites is a
hopeful value. Interestingly, these secondary metabolites are promises for
developing potent drugs (Bell, 2008). Among these bioactive compounds,
antioxidants are of special interest due to the functions of free radicals in
many ailments including cancer, aging, and atherosclerosis. Marine sponge
extracts from unused by-products such as head and viscera of salmon have
been heavily investigated as cheap sources of natural antioxidants (Wu and
Bechtel, 2008). However, few works have been carried out to investigate
antioxidant properties of marine natural products isolated from sponges.
Extracts of Fascaplysinopsis reticulate, Callyspongia siphonella, Niph-
ates furcate, Callyspongia sp., Callyspongia clavata, and Pseudosaberities
clavatus collected from a coral island of Kish was found to possess high
hydroxyl and DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging
activity (Seradj et al., 2012). Rhabdastrella globostella and Spirastrella
inconstans var. digitata (Dendy) extracts were found to elevate above the
control basal values of the specific activity of almost all the antioxidant-related
enzymes including glutathione peroxidase, glutathione reductase, catalase,
and superoxide dismutase (SOD) in the extract-treated rats (Chairman et al.,
2012). Also, Aurora globostellata was found to possess high-DPPH radical
scavenging activity exhibiting a good antioxidant activity (Sugappriya and
Sudarsanam, 2016). These sponges are said to possess antioxidant activities
due to its possession of high-phenolic and aromatic compounds.
Several studies have demonstrated that numerous bioactive metabolites
originally extracted from sponges, were in fact synthesized or transformed by
bacterial strains. Hence, the sponge-associated bacteria could make up a renew-
able source of biomedical agents. As accumulated evidence suggests, it offers
the likelihood to use the sponge-associated bacteria for the production of biologi-
cally active substances instead of the sponge itself. Because bacteria quickly
produce excess amounts of biomass, biologically active secondary products
can simply be produced in huge amounts on a biotechnological scale without
the necessity to harvest or cultivate the sponge (Donia and Hamman, 2003).
3.3 DETERMINATION OF ANTIOXIDANT ACTIVITY
The interpretation of results obtained from in vitro measurement of the anti-

oxidant activity of a marine organism or of a crude plant extract should be
handled with caution. The selection of the appropriate assay to be used ought
to be based on the intended applications of the antioxidant. The methods to
examine the antioxidant activity of a sample can be divided in principle into
major categories:
1. Measuring its capability to donate an electron or hydrogen atom to a

specific ROS or to an electron acceptor.
2. Testing its ability to remove any source of oxidative initiation, for
example, inhibition of enzymes, absorption of UV radiation, and
chelation of transition metal. Free radicals can also be reduced by
other mechanisms like:
i. Metal chelation by complexing with transition metal ions,
thereby hindering metal-catalyzed initiation and decomposition
of lipid hydroperoxides.
ii. Reducing the rate at the initiation of new radicals.
3. The other mechanisms include oxygen scavenging, singlet oxygen
quenching, and blocking of peroxidant effects by binding certain
proteins containing catalytic metal sites.
Enzymatic and nonenzymatic methods can be used to determine and or

measure the antioxidant activities of marine organisms.
3.3.1 ESTIMATION OF ENZYMATIC ANTIOXIDANTS
The enzymatic antioxidants normally analyzed in the parts marine organism

sample are SOD, catalase, peroxidase, glutathione S-transferases (GSTs)
and polyphenol oxidase (PPO).
3.3.1.1 ESTIMATION OF SUPEROXIDE DISMUTASE
Principle: SOD assay is based on the inhibition of the formation of NADH-

phenazine methosulphate (PMS)-nitroblue tetrazolium (NBT) formazon.
The color formed at the end of the reaction can be sampled into butanol and
measured at 560 nm.
Reagents: Sodium pyrophosphate buffer (0.025 M, pH 8.3); PMS
(186 µM); NBT (300 µM); NADH (780 µM); glacial acetic acid; n-butanol;
potassium phosphate buffer (50 mM, pH 6.4).
Preparation of enzyme sample: The sample (0.5 g) should be ground
with 3.0 mL of potassium phosphate buffer, centrifuged at 2000 g for 10 min,
and the supernatants should be used for the assay.
Assay: The assay mixture should contain 1.2 mL of sodium pyro-
phosphate buffer, 0.1 mL of PMS, 0.3 mL of NBT, 0.2 mL of the enzyme
preparation, and water in a total volume of 2.8 mL. The reaction should
be initiated by the addition of 0.2 mL of NADH. The mixture should be
incubated at 300°C for 90 s and arrested by the addition of 1.0 mL of glacial
acetic acid. The reaction mixture should then be shaken with 4.0 mL of
n-butanol, allowed to stand for 10 min and centrifuged. In the butanol
layer, the intensity of the chromogen should be measured at 560 nm in a
spectrophotometer.
One unit of enzyme activity is defined as the amount of enzyme that gave
50% inhibition of NBT reduction in 1 min.
3.3.1.2 ESTIMATION OF CATALASE
Principle: At 240 nm, the UV absorption of hydrogen peroxide can be

measured. This absorbance decreases when degraded by the enzyme cata-
lase. The enzyme activity can be calculated from the decrease in absorbance.
Reagents: Phosphate buffer: 0.067 M (pH 7.0); hydrogen peroxide

(2 mM) in phosphate buffer.
Preparation of enzyme sample: A 20% homogenate of the sample
should be prepared in phosphate buffer. The homogenate should be centri-
fuged and the supernatant should be used for the enzyme assay.
Assay: H2O2-phosphate buffer (3.0 mL) should be taken in an experimental
cuvette, followed by the quick addition of 40 μL of enzyme sample and mixed
thoroughly. Using a spectrophotometer, the time required for a decrease in
absorbance by 0.05 units should be recorded at 240 nm. The enzyme solution
containing H2O2-free phosphate buffer should serve as the control.
One enzyme unit should be calculated as the amount of enzyme required
to decrease the absorbance at 240 nm by 0.05 units.
3.3.1.3 ESTIMATION OF PEROXIDASE
Principle: In the presence of the hydrogen donor pyrogallol or dianisidine,

peroxidase converts H2O2 to H2O and O2. Spectrophotometrically, the oxida-
tion of pyrogallol or dianisidine to a colored product called purpurogalli can
be followed at 430 nm.
Reagents: Pyrogallol—0.05 M in 0.1 M phosphate buffer (pH 6.5);
H2O2—1% in 0.1 M phosphate buffer (pH 6.5).
Preparation of enzyme sample: A 20% homogenate should be prepared
in 0.1 M phosphate buffer (pH 6.5) from the sample, clarified by centrifuga-
tion and the supernatant should be used for the assay.
Assay: To 3.0 mL of pyrogallol solution, 0.1 mL of the enzyme sample
should be added and the spectrophotometer should be adjusted to read zero
at 430 nm. To the test cuvette, 0.5 mL of H2O2 should be added and mixed. In
the spectrophotometer, the change in absorbance should be recorded every
30 s for up to 3 min. One unit of peroxidase is defined as the change in
absorbance/minute at 430 nm.
3.3.1.4 ESTIMATION OF GLUTATHIONE S-TRANSFERASE
Principle: The principle of GST assay is based by its ability to conjugate

Glutathione (GSH) and 1-chloro-2, 4-dinitrobenzene (CDNB), the extent of
conjugation causing a proportionate change in the absorbance at 340 nm.
Reagents: Glutathione (1 mM); 1-chloro-2,4-dinitrobenzene (CDNB)
(1 mM in ethanol); phosphate buffer (0.1 M, pH 6.5).
Preparation of enzyme sample: The sample (0.5 g) should be homog-

enized with 5.0 mL of phosphate buffer. For the assay, the homogenates
should be centrifuged at 5000 rpm for 10 min and the supernatants should
be used.
Assay: The activity of the enzyme should be determined by observing
the change in absorbance at 340 nm. The reaction mixture contained 0.1 mL
of GSH, 0.1 mL of CDNB and phosphate buffer in a total volume of 2.9 mL.
The reaction should be initiated by the addition of 0.1 mL of the enzyme
sample. The readings were recorded every 15 s at 340 nm against distilled
water blank for a minimum of 3 min in a spectrophotometer. The assay
mixture without the sample should serve as the control to monitor nonspe-
cific binding of the substrates.
GST activity should be calculated using the extinction coefficient of the
product formed (9.6 mM−1 cm−1) and should be expressed as nanomoles of
CDNB conjugated/minute.
3.3.1.5 ESTIMATION OF POLYPHENOL OXIDASE
Principle: Phenol oxidases are copper-comprising proteins that catalyze the

aerobic oxidation of phenolic substrates to quinines, which are autooxidized
to dark brown pigments known as melanin. These can be estimated spectro-
photometrically at 495 nm.
Reagents: Tris-HCl (50 mM, pH 7.2) containing sorbitol (0.4 M) and
NaCl (10 mM); phosphate buffer (0.1 M, pH 6.5); catechol solution (0.01 M).
Preparation of enzyme sample: The enzyme sample should be prepared
by homogenizing 0.5 g of sample tissue in 2.0 mL of the sample on medium
containing tris HCl, sorbitol, and NaCl. At 2000 g the homogenate should
be centrifuged for 10 min and the resultant supernatant should be used for
the assay.
Assay: Phosphate buffer (2.5 mL) and 0.3 mL of catechol solution
should be added in the cuvette and the spectrophotometer should be set
at 495 nm. The enzyme sample (0.2 mL) should be added and the change
observed in the absorbance should be recorded for every 30 s up to 5 min in
a spectrophotometer.
One unit of catechol oxidase or laccase is defined as the amount of enzyme
that transforms 1 μmol of dihydrophenol to 1 μmol of quinone per min.
The activity of PPO can be calculated using the formula:
Enzyme units in the sample = K × (µA/minute),
where, K for catechol oxidase = 0.272; K for laccase = 0.242.
3.3.2 ESTIMATION OF NONENZYMATIC ANTIOXIDANTS
The nonenzymatic antioxidants normally analyzed in samples are ascorbic

acid, α-tocopherol, total carotenoids, lycopene, reduced glutathione, total
phenols, flavonoids, and chlorophyll.
3.3.2.1 ESTIMATION OF ASCORBIC ACID
Principle: Treatment with activated charcoal converts ascorbate into dehy-

droascorbate which reacts with 2,4-dinitrophenylhydrazine to form osazones.
When osazone is dissolved in sulfuric acid, it produces an orange-colored solu-
tion, whose absorbance can be measured spectrophotometrically at 540 nm.
Reagents: TCA (4%); 2,4-dinitrophenyl hydrazine reagent (2%) in 9N
H2SO4; thiourea (10%); sulfuric acid (85%); standard ascorbic acid solution:
100 μg/mL in 4% TCA.
Preparation of sample: Ascorbate should be prepared from 1 g of the
sample using 4% TCA and the volume should be made up to 10 mL with the
same. The supernatant obtained after centrifugation at 2000 rpm for 10 min
should be treated with a pinch of activated charcoal, shaken vigorously
using a cyclomixer, and kept for 5 min. By centrifugation, charcoal particles
should be removed and aliquots should be used for the estimation.
Procedure: Standard ascorbate ranging between 0.2mL - 1.0mL and the
aliquot of the treated supernatant ranging between 0.5mL - 1.0mL should
be taken. The volume should be made up to 2.0 mL with 4% TCA. DNPH
reagent (0.5 mL) should be added to all the tubes, followed by two drops
of 10% thiourea solution. The contents should be mixed and incubated at
370°C for 3 h resulting in the formation of osazone crystals. The crystals
should be dissolved in 2.5 mL of 85% sulfuric acid, in cold. To the blank
alone, DNPH reagent and thiourea should be added after the addition of
sulfuric acid. The tubes should be cooled in ice and the absorbance should
be taken at 540 nm in a spectrophotometer.
A standard graph should be constructed using an electronic calculator set
to the linear regression model. The concentration of ascorbate in the samples
should be calculated and expressed in terms of mg/g of sample.
3.3.2.2 ESTIMATION OF TOCOPHEROL
Principle: The Emmerie–Engel reaction is based on the reduction of ferric

to ferrous ions by tocopherols, which, with 2,2′-dipyridyl, forms a red color.
Tocopherols and carotenes are first sampled with xylene and read at 460 nm
to measure carotenes. A correction is made for this after adding ferric chlo-
ride and read at 520 nm.
Reagents: Absolute alcohol; xylene; 2,2′-dipyridyl (1.2 g/L in
n-propanol); ferric chloride solution (1.2 g/L in ethanol); standard solution
(D, L-α-tocopherol, 10 mg/L in absolute alcohol); sulfuric acid (0.1 N).
Preparation of sample: The sample (2.5 g) should be homogenized in
50 mL of 0.1 N sulfuric acid and allowed to stand overnight. The contents
of the flask should be shaken vigorously and filtered through Whatman No.1
filter paper. Aliquots of the filtrate should be used for the estimation.
Procedure: In three stoppered centrifuge tubes, 1.5 mL of the sample,
1.5 mL of the standard, and 1.5 mL of water should be pipetted out separately.
To all the tubes, 1.5 mL of ethanol and 1.5 mL of xylene should be added,
mixed well, and centrifuged. Xylene (1.0 mL) layer should be transferred
into another stoppered tube. To each tube, 1.0 mL of dipyridyl reagent should
be added and mixed well. The mixture (1.5 mL) should be pipetted out into
a cuvette and the absorbance is taken at 460 nm. Ferric chloride solution
(0.33 mL) should be added to all the tubes and mixed well. The red color devel-
oped should be read exactly after 15 min at 520 nm in a spectrophotometer.
The concentration of tocopherol in the sample should be calculated using
the formula:
Sample Abs 520 − Abs 460 × 0.29 × 0.15

Tocopherols ( µ g ) = .
Standard Abs 520
3.3.2.3 ESTIMATION OF TOTAL CAROTENOIDS AND LYCOPENE
Principle: Total carotenoids and lycopene can be estimated in a sample

using petroleum ether and estimated at 450 nm and 503 nm, respectively.
Reagents: Petroleum ether (40–60°C); anhydrous sodium sulfate;
calcium carbonate; alcoholic potassium hydroxide (12%).
Procedure: The experiment is done in the dark to avoid photolysis of
carotenoids once the saponification is complete. The sample (0.5 g) should
be homogenized and saponified with 2.5 mL of 12% alcoholic potassium
hydroxide in a water bath at 60°C for 30 min. The saponified sample should
be transferred to a separating funnel containing 10–15 mL of petroleum
ether and mixed well. The lower aqueous layer should then be transferred
to another separating funnel and the upper petroleum ether layer containing
the carotenoids should be collected. The procedure should be repeated until
the aqueous layer become colorless. A small amount of anhydrous sodium
sulfate should be added to the petroleum ether sample to remove excess

moisture. The final volume of the petroleum ether sample should be noted.
The absorbance of the yellow color should be read in a spectrophotometer
at 450 nm and 503 nm using petroleum ether as blank. The amount of total
carotenoids and lycopene should be calculated using the formulae:
Abs 450 × Volume of sample × 100 × 4

Amount of total carotenoids =
Weight of the sample
3.12 × Abs 503 × Volume of the sample × 100

Amount of lycopene = .
Weight of the sample
The total carotenoids and lycopene are expressed as mg/g of the sample.
3.3.2.4 ESTIMATION OF REDUCED GLUTATHIONE
Principle: Reduced glutathione in reaction with DTNB (5,5′-dithiobis nitro-

benzoic acid) produces a yellow colored product that absorbs at 412 nm.
Reagents: TCA (5%); phosphate buffer (0.2 M, pH 8.0); DTNB (0.6 mM
in 0.2 M phosphate buffer); standard GSH (10 nmol/mL of 5% TCA)
Preparation of sample: A homogenate should be prepared with 0.5 g
of the sample with 2.5 mL of 5% TCA. The precipitated protein should be
centrifuged at 1000 rpm for 10 min. The supernatant (0.1 mL) should be
used for the estimation of GSH.
Procedure: The supernatant (0.1 mL) should be made up to 1.0 mL with
0.2 M sodium phosphate buffer (pH 8.0). Standard GSH corresponding to
concentrations ranging between 2 and 10 nmol should also be prepared.
About 2 mL of freshly prepared DTNB solution should be added and the
intensity of the yellow color developed should be measured in a spectropho-
tometer at 412 nm after 10 min.
The values are expressed as nanomoles GSH/g sample.
3.3.2.5 ESTIMATION OF TOTAL PHENOLS
Principle: Phenols react with the phosphomolybdic acid in Folin–Ciocal-

teau reagent to produce a blue-colored complex in alkaline medium, which
can be estimated spectrophotometrically at 650 nm.
Reagents: Ethanol (80%); Folin–Ciocalteau reagent (1 N); sodium
carbonate (20%); standard catechol solution (100 μg/mL in water).
Procedure: The sample (0.5 g) should be homogenized in 10X volume

of 80% ethanol. The homogenate should be centrifuged at 10,000 rpm for
20 min. The procedure should be repeated with 80% ethanol. The super-
natants should be pooled and evaporated to dryness. The residue is then
dissolved in a known volume of distilled water. Different aliquots should
pipette out and the volume in each tube should be made up to 3.0 mL with
distilled water. Folin–Ciocalteau reagent (0.5 mL) should be added and the
tubes must be placed in a boiling water bath for exactly 1 min. The tubes
should be cooled and the absorbance is taken at 650 nm in a spectropho-
tometer against a reagent blank. Standard catechol solutions (0.2–1 mL)
corresponding to 2.0–10 μg concentrations should also be treated as above.
The concentration of phenols is expressed as mg/g tissue.
3.3.2.6 ESTIMATION OF FLAVONOIDS
Principle: Flavonoids react with vanillin to produce a colored product,

which can be measured spectrophotometrically.
Reagents: Vanillin reagent (1% in 70% sulfuric acid); catechin standard
(110 μg/mL).
Preparation of sample: The samples (0.5 g) should first be prepared
with methanol:water mixture (2:1) and secondly with the same mixture in the
ratio 1:1. The samples should be shaken well and allowed to stand overnight.
The supernatants should be pooled and the volume should also be measured.
This supernatant should be concentrated and then used for the assay.
Procedure: A known volume of the sample should be pipetted out and
evaporated to dryness. Vanillin reagent (4.0 mL) should be added and the
tubes should be heated in a boiling water bath for 15 min. Varying concentra-
tions of the standard can also be treated in the same manner.
The optical density should be read in a spectrophotometer at 340 nm. A
standard curve is constructed and the concentration of flavonoids in each sample
is well calculated. The values of flavonoids are expressed as mg/g sample.
3.3.2.7 ESTIMATION OF CHLOROPHYLL
Principle: Chlorophyll is prepared in 80% acetone and the absorbance is

measured at 645 and 663 nm. The amount of chlorophyll is calculated using
the absorption coefficient.
Reagent: Acetone (80%, prechilled).
Procedure: Chlorophyll should be prepared from 1 g of the sample using

20 mL of 80% acetone. The supernatant should be transferred to a volumetric
flask after centrifugation at 5000 rpm for 5 min. The solution should be repeated
until the residue become colorless. The volume in the flask should be made up
to 100 mL with 80% acetone. The absorbance of the sample should be taken in a
spectrophotometer at 645 and 663 nm against 80% acetone blank. The amount
of total chlorophyll in the sample should be calculated using the formula:
20.2 ( Abs 645 ) + 8.02 ( Abs 663) × V

Total Chlorophyll = ,
1000 × W
where V = final volume of the sample; W = fresh weight of the leaves. The
values are expressed as mg chlorophyll/g sample.
3.3.3 EVALUATION OF THE RADICAL SCAVENGING EFFECTS
The scavenging effects of samples are usually evaluated against 2,2-

diphenyl-2-picrylhydrazyl (DPPH), 2,2-azino-bis (3-ethylbenzothiazoline-
6-sulphonic acid) (ABTS), hydrogen peroxide, superoxide, nitric oxide, and
hydroxyl radicals.
3.3.3.1 DPPH RADICAL SCAVENGING EFFECTS
The ability of samples to scavenge the DPPH radical are usually tested in
a rapid dot-plot screening and quantified using a spectrophotometric assay.
Principle: DPPH radical reacts with an antioxidant compound that can
donate hydrogen, and gets reduced. DPPH, when acted upon by an antioxi-
dant, is converted into diphenyl picryl hydrazine. This can be identified by
the conversion of purple to light yellow color.
a) Dot-plot rapid assay
Reagents: TLC plates (silica gel 60 F254-Merck); DPPH (0.4 mM) in

methanol.
Procedure: Aliquots of the sample should be spotted carefully on TLC
plates and dried for 3 min. The sheets bearing the dry spots should be placed
upside down for 10 s in a 0.4 mM DPPH solution and should be layer-dried.
The stained silica layer should reveal a purple background with yellow spots,
which should show radical scavenging capacity.
b) DPPH spectrophotometric assay
Reagents: DPPH—2,2-diphenyl-2-picrylhydrazyl hydrate (0.3 mM in

methanol); methanol.
Procedure: The marine sample (20 μL) should be added to 0.5 mL
of methanolic solution of DPPH and 0.48 mL of methanol. The mixture
should be allowed to react at room temperature for 30 min. Methanol
should serve as the blank and DPPH in methanol without the sample
should serve as the positive control. After 30 min of incubation, the
discoloration of the purple color should be measured at 518 nm in a
spectrophotometer. The radical scavenging activity should be calculated
as follows:
( Absorbance of control − Absorbance of sample )

% Antioxidant activity = × 100 .
( Absorbance of control )
3.3.3.2 ABTS SCAVENGING EFFECTS
The antioxidant effect of the marine sample can be studied using ABTS
(2,2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid) radical cation
decolorization assay.
Reagent: ABTS solution (7 mM with 2.45 mM ammonium persulfate).
Procedure: ABTS radical cations (ABTS+) are produced by reacting
ABTS solution (7 mM) with 2.45 mM ammonium persulphate. The
mixture should be allowed to stand in the dark at room temperature for
12–16 h before use. Aliquots (0.5 mL) of the three different samples should
be added to 0.3 mL of ABTS solution and the final volume made up to
1 mL with ethanol. The absorbance should be taken at 745 nm in a spec-
trophotometer and the percent inhibition was calculated using the formula.
( Control − Test ) ×100

Inhibition ( % ) = .
Control
3.3.3.3 HYDROGEN PEROXIDE SCAVENGING EFFECTS
Reagents: Phosphate buffer (0.1 M, pH 7.4); H2O2 (40 mM) in phosphate

buffer.
Procedure: A solution of H2O2 (40 mM) should be prepared in phos-

phate buffer. Marine samples at the concentration of 10 mg/μL should be
added to the H2O2 solution (0.6 mL) and the total volume made up to 3 mL.
The absorbance of the reaction mixture should be recorded at 230 nm in a
spectrophotometer. A blank solution containing phosphate buffer, without
H2O2 should be prepared. The extent of H2O2 scavenging of the sample
should be calculated as:
% Scavenging of Hydrogen Peroxide =

( ( Abs of Blank − Abs of sample 230 ) × 100 )
.
Abs of Blank
3.3.3.4 MEASUREMENT OF SUPEROXIDE SCAVENGING ACTIVITY
Principle: This assay is based on the inhibition of the production of NBT

formazon of the superoxide ion by the sample and is measured spectrophoto-
metrically at 560 nm.
Reagents: EDTA (0.1 M containing 1.5 mg of NaCN); NBT (1.5 mM);
riboflavin (0.12 mM); phosphate buffer (0.067 M, pH 7.6).
Procedure: Superoxide anions should be generated in samples that
should contain in 3.0 mL, 0.02 mL of the marine samples (20 mg), 0.2 mL
of EDTA, 0.1 mL of NBT, 0.05 mL of riboflavin, and 2.64 mL of phosphate
buffer. The control tubes should also be set up where dimethyl sulfoxide
(DMSO) should be added instead of the sample. All the tubes should be
vortexed and the initial optical density measured at 560 nm in a spectro-
photometer. The tubes should be illuminated using a fluorescent lamp for
30 min. The absorbance should be measured again at 560 nm. The difference
in absorbance before and after illumination is indicative of superoxide anion
scavenging activity.
3.3.3.5 MEASUREMENT OF NITRIC OXIDE SCAVENGING ACTIVITY
Principle: Sodium nitroprusside in aqueous solution, at physiological pH,

spontaneously generates nitric oxide, which interacts with oxygen to produce
nitrite ions that are estimated spectrophotometrically at 546 nm.
Reagents: Sodium nitroprusside (100 mM); phosphate buffered saline
(pH 7.4); Griess reagent (1% sulphanilamide, 2% H3PO4 and 0.1% naph-
thylethylenediamine dihydrochloride)
Procedure: The reaction should be initiated by adding 2.0 mL of sodium
nitroprusside, 0.5 mL of PBS, 0.5 mL of the sample (50 mg), and incubated
at 25°C for 30 min. Griess reagent (0.5 mL) should be added and incubated
for another 30 min. Control tubes should be prepared without the samples.
The absorbance should be taken at 546 nm against the reagent blank, in a
spectrophotometer.
3.3.3.6 MEASUREMENT OF HYDROXYL RADICAL SCAVENGING

ACTIVITY
The extent of hydroxyl radical scavenging from Fenton reaction is quanti-

fied using 2′-deoxyribose oxidative degradation.
Principle: The principle of the assay is the quantification of 2′-deoxy-
ribose degradation product, malondialdehyde, by its condensation with
thiobarbituric acid.
Reagents: Deoxyribose (2.8 mM); ferric chloride (0.1 mM); EDTA
(0.1 mM); H2O2 (1 mM); ascorbate (0.1 mM); KH2PO4-KOH buffer
(20 mM, pH 7.4); thiobarbituric acid (1%)
Procedure: The reaction mixture contained 0.1 mL of deoxyribose,
0.1 mL of FeCl3, 0.1 mL of EDTA, 0.1 mL of H2O2, 0.1 mL of ascorbate,
0.1 mL of KH2PO4-KOH buffer and 20 µL of the sample in a final volume
of 1.0 mL. The mixture should be incubated at 37°C for 1 h. At the end of
the incubation period, 1.0 mL of TBA should be added and heated at 95°C
for 20 min to develop the color. After cooling, the Thiobarbituric acid
reactive substances (TBARS) formation should be measured spectropho-
tometrically at 532 nm against an appropriate blank. The hydroxyl radical
scavenging activity should be determined by comparing the absorbance
of the control with that of the sample. The percent TBARS production for
positive control (H2O2) should be fixed at 100% and the relative percent
TBARS should be calculated for the sample treated groups.
KEYWORDS
•• antioxidants
•• marine antioxidants
•• sponge
•• DPPH
•• radical scavenging
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CHAPTER 4
EXTRACTION OF MARINE
PHYTOCHEMICALS:
METHODS AND TECHNIQUES
NADIA SHARIF1,*, NEELMA MUNIR1, and SHAGUFTA NAZ1
1
Department of Biotechnology, Lahore College for Women University,
Lahore 54000, Pakistan, Tel.: +92 3237501948
*
*
ABSTRACT
It is imperative to document proper, specific, practical, and eco-friendly

supportive extraction systems for marine phytochemicals. These procedures
should fulfill the necessities in regard to the extraction and isolation of the
bioactive of interests. Conventional extraction systems are comparatively
time-taking, requiring high solvent volumes and are extensively laborious.
These methods regularly deliver low selectivity and extraction of bioac-
tive compounds from marine plants. The utilization of new best-in-class
extraction methods, for example, pressurized liquid extraction, supercritical
fluid extraction, ultrasound-assisted extraction, accelerated solvent extrac-
tion (ASE®), and microwave-assisted extraction systems have proven a
successful contrasting option to the issues experienced with the utilization
of conventional extraction techniques.
4.1 INTRODUCTION
Considering the immense biodiversity of marine organisms, the utilization of

proper approaches that are able to quickly screen diverse marine metabolites
are of remarkable importance. To outline the isolation technique, a lot of
factors need to be considered. These factors comprise dissolvability, atomic

weight, and heat resistance. At first, a reasonable extraction method ought
to be chosen. The utilization of naturally clean propelled extraction methods
is important for the proper extraction of a bioactive. After extraction, the
bioactive properties of the extract are accessed through the various in vitro
and in vivo assays such as antihypertensive, antimicrobial and antioxidants.
After confirming the biological activities and functional categorization, the
next step is the chemical categorization. Structural confirmation is possible
through analytical techniques that are dependent on temperature, pH, and
solubility (Tejesvi et al., 2008; Grosso et al., 2015).
The extraction of bioactive components from the marine plant is a prag-
matic workout at different conditions with different solvents. The failure or
success of the extraction method relies on the condition of extraction. The
partition of the crude extracts or chromatographic techniques to fractionate
the extract grounded on the acid property, polarity, or atomic size is the next
step. Recrystallization, distillation or sublimation methods are applied to
offer appropriate wholesome structural exploration (Grosso et al., 2015).
The polarity of the compound of interest is of importance for the solvent
choice. Other factors such as molecular affinity between solute and solvent,
cosolvent use, mass transfer, human toxicity, financial feasibility and envi-
ronmental safety must likewise be considered in deciding the solvent for
the extraction of bioactive compounds (Cowan, 1999). More details can be
obtained from Volume 1 of this book.
4.2 CONVENTIONAL EXTRACTION TECHNIQUES
The extraction of bioactive compounds from marine plant samples can be

achieved by numerous conventional extraction techniques. Mostly, these
techniques rely on the application of mixing/heat and extracting the power
of different solvents. The bioactive yield can be influenced by the technique
for molding and extraction. Different solvents can be utilized to separate
distinctive phytoconstituents (Wong and Kitts, 2006). Acid hydrolysis is a
significant chemical modification that is able to bring a significant change in
structural and functional properties (Lee and Jeffries, 2011). It is a preferred
method over other pretreatments due to its effectiveness and low cost (Loow
et al., 2016). Commonly used dilute acids include H2SO4, HNO3, HCl,
H3PO4, and other acids. On the other hand, alkali hydrolysis of different plant
samples is also tried in different experiments but a lot of times it results in
reduced functionality and is of little benefit (Kristinsson and Rasco, 2000).
Extraction of Marine Phytochemicals 91
Enzymatic hydrolysis from animal and plant cradles has been deliberated
extensively and by numerous authors over the past 60 years, and it is still the
most commonly used method for adding value to the target organism. The
preferred commercial enzymes are of bacterial origin, including Alcalase,
Neutrase, and Flavourzyme, as well as from animals and plants, including
trypsin, pepsin, papain, bromelain (see Chapter 8 of this volume), and subtil-
isin. The conventional techniques for the extraction of bioactive compounds
from diverse plants are Soxhlet extraction, maceration, and hydrodistilla-
tion. Soxhlet extractor was first suggested by German chemist Franz Ritter
Von Soxhlet (1879). The Soxhlet extraction has extensively been utilized for
extracting valued bioactive compounds from numerous natural cradles. It is
recognized as a model for the comparison of novel extraction substitutes.
4.2.1 MACERATION
Maceration was utilized as a part of homemade tonic for quite a while.

It turned into a famous and modest approach to get fundamental oils and
bioactive mixes. For small-scale extraction, maceration forms the most part
comprising of a few stages. Right off the bat, pounding of plant materials into
little molecules is utilized to expand the surface zone for legitimate blending
with the dissolvable. Also, in maceration process, fitting a dissolvable named
as menstruum is included in a shut vessel. Thirdly, the fluid is stressed off
however, the marc which is the strong deposit of this extraction procedure is
squeezed to recuperate a huge measure of blocked arrangements. The mate-
rial gets stressed and the press out fluid is blended and isolated and separated
by filtration. Periodic shaking in maceration encourages extraction in two
ways; (a) enhanced dissemination, (b) expel concentrated arrangement from
the example surface for conveying the new dissolvable to the menstruum for
more extraction yield (Azmir et al., 2013).
4.2.2 HYDRODISTILLATION
Hydrodistillation is performed before dehydration of plant materials without

solvents. Steam distillation, direct steam distillation and water distillation
are three different hydrodistillation methods (Vankar, 2004). In hydrodistil-
lation, to start with, the plant materials are pressed into a still compartment;
second, water is included and after that conveyed to boil. On the other
hand, coordinate steam is infused into the plant material. Boiling water and
steam go about as the fundamental compelling components to free bioactive

mixes of plant tissue. Circuitous cooling by water gathers the vapor blend of
water and oil. Consolidated blend flows from the condenser to a separator,
where oil and bioactive mixes isolated from the water (Silva et al., 2005).
Hydrodistillation includes three principles in physicochemical procedures;
decomposition by heat, hydro-diffusion, and hydrolysis. At a high extraction
temperature, some volatile components might be lost. This disadvantage
restrains its utilization for thermolabile compound extraction.
4.3 NONCONVENTIONAL EXTRACTION TECHNIQUES
Though different extraction systems exist, some are known as conventional

and some as nonconventional that are available for the isolation and charac-
terization of marine plant materials. The nonconventional extraction system
seems more natural to me because of diminished utilization of manufactured
and natural chemicals, decreased operational time, and better yield and
nature of concentrate that has been produced in the recent 50 years (Grosso
et al., 2015). To overcome the limitations of conventional extraction methods
and the demand for new extraction systems to augment the inclusive yield
and discernment of bioactive constituents from plant materials has resulted
in advanced extraction techniques introducing techniques such as enzyme
digestion, microwave heating, supercritical fluids and accelerated solvents
extraction systems (Troy et al., 2017). The benefits of nonconventional
extraction methods are reduced consumption of organic solvent, automation
and reduced extraction time. Some of the most promising nonconventional
extraction techniques are ultrasound-assisted extraction, enzyme-assisted
extraction, microwave-assisted extraction (MAE), pulsed-electric field-
assisted extraction, supercritical fluid extraction (SFE) and pressurized
liquid extraction (PLE) (Selvamuthukumaran and Shi, 2017).
4.3.1 ULTRASOUND-ASSISTED EXTRACTION
Ultrasound-assisted extraction (UAE) works by increasing the porousness

of the cell wall of the sample. It causes fast extraction as UAE consider-
ably decreases the time of extraction and raises the yield of extraction, as
it produces cavitation bubbles in the solvent during the expansion phase
(Zou et al., 2013). The local tensile strength of the liquid exceeds because
of the negative pressure exerted by the expansion cycle (Wang, 2011).
Unconstrained arrangement rises in the fluid below its boiling point, that
is, cavitation impact, because of dynamic focusing and increment in the
mechanical pushing, that is, inner contact of the cells (Mukherjee, 2002).
The UAE relies on many components: (a) force, (b) time, (c) dissolvable, (d)
temperature, (e) throb, (f) network (Lavoie and Stevanovic, 2007).
The utilization of ultrasound can be partitioned into two unmistakable classes:
low intensity–high recurrence (100 kHz–1 MHz) and high intensity–low recur-
rence (in the vicinity of 20 and 100 kHz) ultrasound, the latter being the main
case that prompts the interruption of cell dividers and films (Kong et al., 2014).
4.3.2 PULSED-ELECTRIC FIELD EXTRACTION
Pulsed-electric field extraction (PEF) technique is utilized to enhance mass

transfer practices, it disrupts cell membranes. The sample lattice is put
between two anodes in a group or a consistent stream treatment chamber
and presented to repetitive electric frequencies (Hz–kHz) with an extreme
(0.1–80 kV/cm) electric field for brief periods (from a few nanoseconds to a
few milliseconds). A restrained PEF treatment is determined by the applica-
tion of 100–10,000 µs treatment times with field strength of 0.5–1.0 kV/cm
in the range of 1–10 kV/cm and short times (5–100 µs). The utilization of
electric pulses causes the arrangement of reversible or irreversible pores in
the cell films, characterized as electroporation or electro-permeabilization,
which thus helps the fast dissemination of the solvents and the improvement
of the mass exchange of intracellular mixes (Zbinden et al., 2013; Poojary
et al., 2016). The particular analyte’s extraction can be accomplished by
regulating the pore arrangement, which is reliant on the force of the treatment
connected (electric field quality, single pulse length, repetition frequency of
the external electric pulses, duration, intensity and amplitude of particular
energy input) and the cell attributes (i.e., shape, size, direction of introduc-
tion in the electric field). Depending on different factors, either reversible or
irreversible pores are generated in the membranes. Irreversible pores forma-
tion is of great significance for abstraction of bioactive compounds from
marine microorganisms (Zbinden et al., 2013; Raso et al., 2016).
4.3.3 MICROWAVE-ASSISTED EXTRACTION
In the last decade, MAE has been effectively utilized for the extraction of
numerous biologically active compounds from a variety of natural cradles
(Navarro et al., 2007; Perino-Issartier et al., 2011). This technique has

been reported to enhance the extraction yield of bioactive compounds from
various matrices compared to traditional solid-liquid extraction (Kaufmann
and Christen, 2002). MAE involves the extraction of high-value composites
from natural cradles such as functional food ingredients, phytonutrients,
pharmaceutical, and nutraceutical actives from biomass. MAE is an effec-
tive technique that proceeds with irradiation of microwave to hasten the
removal of diverse compounds of natural cradles. It involves the use of
electromagnetic waves with a frequency range of 300 MHz–1000 GHz (Patil
and Shettigar, 2010).
MAE acts upon the quick and discerning centralized heating of mois-
ture in the sample by microwaves. In general, the mechanism of MAE
is through inter- and intramolecular friction, together with collision and
movement of a very large number of charged ions, triggering quick heating
of the reaction system and the subsequent breakdown of cell walls and
membranes (Grosso et al., 2015). Because of centralized heating and main-
tenance of pressure within the sample cells, a fast exchange of extracted
compounds from cells to the extracting solvent takes place. In MAE,
both mass and heat incline slog in a similar direction, whereas it works
in opposite directions in conventional heating. Therefore, a direct heating
to the surface matrix of sample exists in conventional heating and it is
subsequently followed by surface to the core heating through conduction
(Choi et al., 2006).
Though the usage of MAE may vitiate bioactive carbohydrates because
of localized high temperature (Routray and Orsat, 2012), numerous
studies have reported the significant use of MAE for the extraction of
bioactive materials from marine organisms. Several investigators have
applied MAE for fish tissues (Reyes et al., 2009), oysters (Bhattacharya
et al., 2015), and shrimp (Tsiaka et al., 2015). Acid hydrolysis of proteins
for peptide mass mapping and tandem mass spectrometric analysis of
peptides through MAE has also been reported (Zhong et al., 2005).
Furthermore, MAE apposite the degradation of special organisms such
as algae, have cells that are enclosed by a vibrant, intricate, and carbohy-
drate-rich cell wall that requires the breakdown of cell walls principally
significant (Popper et al., 2011). Some investigators have deliberated the
antioxidant activity of sulfated polysaccharides from Brown Seaweed
(Yuan and Macquarrie, 2015) under different algae/water ratios, extrac-
tion times and pressures. These researches indicated that MAE is an
effective technology.
4.3.4 PRESSURIZED LIQUID EXTRACTION
Pressurized liquid extraction (PLE) is also recognized as pressurized fluid

extraction (PFE), accelerated solvent extraction (ASE), high-pressure
solvent extraction or enhanced solvent extraction (Nieto et al., 2010), and
was introduced in 1996 (Richter et al., 1996). PLE involves the application
of pressure at temperatures higher than the normal boiling point of liquids.
Combined high pressures and temperatures are used that utilize a small
amount of solvent and provide faster extraction. When high temperature is
applied, it increases the solubility of the analyte with high mass transfer rate,
decreases the surface tension and viscosity of the solvent, hence improving
extraction yield (Herrero et al., 2006a; Mendiola et al., 2007).
The PLE instrumentation is quite simple; it has a solvent reservoir that is
coupled to a high-pressure pump through which solvent is introduced in the
system. An oven where the extraction cell is placed, a valve is also present to
maintain the pressure inside the system. For the collection of extract, a vial
is placed at the end of the extraction system. Moreover, the framework could
outfit with a condenser gadget for fast cooling of the resultant concentrate.
The extraction time is variable as indicated by the example to be tested (Nieto
et al., 2010). Ever since the early advent of PLE, there has been a great
deal of exertion centered on getting bioactive mixes from marine sources.
Presumably, the fundamental explanation behind the critical improvement
of PLE-based systems is the likelihood of its robotization alongside the less-
ened extraction time and solvents required, as specified beforehand. PLE has
been used, for example, to think carotenoids from Dunaliella salina micro-
algae. The best yields were obtained with ethanol and no essential extraction
temperature and time (160°C and 17.5 min). The examination of compound
depiction by tiptop liquid chromatography (LC) joined with diode display
revelation (High-Performance Liquid Chromatography-Diode-Array
Detection (HPLC-DAD) raised that the concentrates contained, other than
all-trans-b—carotene and isomers, a couple of different minor carotenoids
that seemed to contribute earnestly to the cancer prevention agent action of
concentrates (Breithaupt, 2004; Herrero et al., 2006b).
PLE has been used, for example, to isolate carotenoids from brown
macroalgae, Himanthalia elongata, Eisenia bicyclis and Cystoseira abies-
marina. It was noted that ethanol at high temperatures yields higher fuco-
xanthin than other oxygenated carotenoids. Based on the selected solvent,
PLE is reported as an idyllic system for the separation of bioactive phenolic
complexes from marine algae (Porphyra tenera-nori, Stypocaulon scoparium
and Undaria pinnatifida-wakame (Wang et al., 2010; Lopez et al., 2011).
4.3.5 SUPERCRITICAL FLUID EXTRACTION
SFE is an alternative extraction method which was introduced by Hannay

and Hogarth in 1879. A significant attribute of SFE is the exceedingly
diminished (frequently to zero) work of lethal natural solvents. CO2 is the
dissolvable most regularly used to remove bioactive mixes from common
sources utilizing SFE. In fact, CO2 has a progression of properties for
bioactive extraction: it is fetched effective, its basic conditions are feasible
(30.9°C and 73.8 bar), and it is an ecologically well-disposed dissolvable
that is generally recognized as safe for use in the nutrition industry (Ibanez
et al., 2012).
An interesting application of SFE is the screening of phenols, flavonoids
such as phenolic compounds from marine cradles. An innovative hyphen-
ated practice was advanced for the extraction and fortitude isoflavones from
marine macroalgae (Halopytis incurvus, Chondrus crispus, Sargassum
vulgare, Hypnea spinella, Porphyra sp., U. pinnatifida, and Sargassum
muticum), Spongiochloris spongiosa freshwater algae, and Scenedesmus
and Nostoc like cyanobacteria (Klejdus et al., 2010). Moreover, SC–CO2
extraction use was reported to get the flavonoid antioxidants from Chlorella
vulgaris. Authors compared SC–CO2 extraction at 50°C, 310 bars, utilizing
50% aqueous ethanol as a modifier, with ultrasonic extraction with 50%
aqueous ethanol. It was found that flavonoid contents gained under SFE
conditions were considerably high as compared to UAE. It leads to a high
antioxidant activity but correspondingly improved lung cancer metastasis
inhibition (Wang et al., 2010).
4.3.6 PRESSURIZED HOT WATER EXTRACTION
Pressurized hot water extraction (PHWE), also known as superheated water

extraction, pressurized low polarity water extraction or subcritical water
extraction is a particular use of PLE with H2O as extracting solvent. PHWE
is built on using H2O at temperatures above its atmospheric boiling point but
keeping it as a liquid through pressure. In static PHWE, a batch process occurs
in one or many extraction cycles while the solvent is replaced in between. In
dynamic PHWE, it involves continuous flow and pumping of solvent at a
selected flow rate by the extraction vessel that contains the sample. So it
requires a somewhat more sophisticated HPLC pump in order to control the
water flow rate and a pressure restrictor or a micrometering valve instead of
static open/close valve (Herrero et al., 2006a; Mendiola et al., 2007).
HPLC-DAD, HPLC-Triple Quadrupole-Mass Spectrometers (QqQ-MS),

and gas chromatography (GC)–MS were used to determine the chemical
composition. Short-chain fatty acids turned out to be accountable for the anti-
microbial activity, whereas the antioxidant activity is correlated with vitamin
E, together with simple phenols, carmelization produces, and neoformed
antioxidants isolated while the extraction at high temperatures (Plaza et al.,
2010). The current research stated that PHWE at high temperatures is
involved in extracting new antioxidant compounds as was done for microalga
Haematococcus pluvialis. Different microalgae (C. vulgaris), macroalgae
(S. vulgare, Porphyra spp., C. abies-marina, S. muticum, U. pinnatifida,
and Halopitys incurvus), and plants (rosemary, thyme, and verbena) were
studied for extraction via PLE (Plaza et al., 2010). Phycobiliproteins from
the Spirulina platensis cyanobacteria were extracted through PLE. Capillary
electrophoresis coupled with mass spectrometry (CE-MS) was applied to
monitor and optimize the PLE of proteins from S. platensis. The combina-
tional application of PLE and CE-MS permitted the achievement of extracts
rich in phycobiliproteins in short extraction times (Herrero et al., 2006a).
4.4 ISOLATION AND PURIFICATION TECHNIQUES
In a characteristic technique to discover the marine bioactive, the

compounds primarily extracted from the aquatic organisms, the excerpt is
vetted for a special bioactivity, fractionated, and finally filtered to yield a
single bioactive molecule. In addition, for an effectual process, it is neces-
sary to clearly research methods such as membrane filtration systems, gel
or size exclusion chromatography, ion-exchange column chromatography,
and reversed-phase high-performance liquid chromatography (RP-HPLC)
(Patil and Shettigar, 2010).
4.4.1 MEMBRANE FILTRATION
Membrane filtration can be used at different levels. Ultrafiltration with a high

molecular weight cut-off can be used for the separation of non-hydrolyzed
compounds. Membrane filtration can be operated at normal temperature,
and there are no chemical reactions during the process. Membrane filtration
can provide a high amount of separation compared to other chromatographic
separation, and then, this technology always shows applications for the
separation and recovery of bioactive compounds from diverse raw matrices
(Li et al., 2012).
In recent years, many researchers have used membrane filtration as the

first purification step. An example is the use of cross-flow microfiltration to
make the galacturonic acid content of pectin increase from 68.0–72.2%. The
membrane filtration technology has demonstrated a potential application in
the separation of bioactive products.
4.4.2 GEL FILTRATION
The partially purified extract could be subjected to gel filtration chroma-

tography (GFC) and ion-exchange chromatography, with reversed-phase
C18 HPLC for the final purification step (Nazeer and Kumar, 2011; Zhang
et al., 2012). GFC, likewise called size exclusion chromatography, has been
utilized for more than 40 years for the isolation, desalting, and estimation of
the atomic weight of certain molecules. GFC is the least difficult and mildest
of the majority of the chromatography systems and isolates particles based
on sizes. Its partition system is to channel atoms as per their sizes; some
littler particles enter the pores of the gel and undergo a more drawn out
separation, while bigger atoms indicate considerably shorter maintenance
times. Thus, a critical favorable position of GFC is that elution conditions
can be shifted to suit the sort of test and also the necessities for advanced
cleaning, investigation or capacity without changing the division (Wang
et al., 2017).
Trypsin inhibitor was purified from Yellowfin Tuna (Thunnus albacores)
roe, followed by column chromatography on Sephacryl S-200, Sephadex
G-50, and diethylaminoethyl-cellulose, and it was finally found to have
an apparent molecular weight of 7 × 104 Da. (Wu et al., 2014). Sephadex
is recommended for rapid group separation such as desalting and buffer
exchange and it is widely used in the marine organism purification field
(Bougatef et al., 2010; Hsu, 2010). Thus, GFC is cumbersome, time-
consuming, and costly, its high selectivity and high resolution make this
technology applicable to various separation and purification fields.
4.4.3 LIQUID CHROMATOGRAPHY (LC)
4.4.3.1 HIGH-PERFORMANCE LC
HPLC is an extensively applied method for the separation, identification, and

purification of bioactive compounds (Singh et al., 2014). Analysis involving
HPLC could fully reflect the information of the sample and does not require
the collection of fractions; preparative HPLC needs to consider the purity,
production, production cycle, and operating cost. Furthermore, RP-HPLC
is applied to fractionate samples based on their different assets, particularly
when analyzing the structural and configurational assets of compounds (So
et al., 2016; Song et al., 2016). The main advantages of this technology
include the ease of operation, high resolution, and sensitivity, and it always
uses a short time to get the elution spectra compared to the GFC and IEX,
which each time is essentially about20–30 h long.
In recent years, HPLC is usually combined with qualitative equipment
such as mass spectrometry (MS) and LC followed by tandem mass spec-
trometric recognition, a standard method for the characterization bioactive
molecules (Vijaykrishnaraj and Prabhasankar, 2015), which has revealed
a new era in the physical explication of compounds (Careri and Mangia,
2003); even though this technique is very particular and stout, it is exclusive
and time-consuming (Mann and Jensen, 2003). Electrospray ionization and
matrix-assisted laser desorption ionization (MALDI) have been identified
as imperative tools for bioactive compounds detection and categorization
(Leonil et al., 2000); matrix-assisted laser desorption/ionization time-of-
flight (MALDI-TOF) mass spectrometric analysis is the backbone analysis
for various hydrolysates or semipurified fractions (Singh et al., 2014) of
marine microorganisms and so forth.
4.4.3.2 LIQUID CHROMATOGRAPHY-NUCLEAR MAGNETIC

RESONANCE
The combination of chromatographic separation technique with nuclear

magnetic resonance (NMR) spectroscopy is one of the powerful and
time-saving approaches for the separation and structural interpretation of
unidentified composite and mixes, particularly for the structure analysis of
oxygen and light-sensitive materials. The online liquid chromatography-
NMR (LC-NMR) practice permits the incessant recording of time changes
as they appear in the chromatographic run, automated data procurement,
and LC-NMR advances in speed and sensitivity of detection (Daffre et al.,
2008). The recent introduction of pulsed field gradient technique in high-
resolution NMR as well as three-dimensional technique improves the appli-
cation in structure elucidation and molecular weight information. These new
hyphenated techniques are helpful in the areas of pharmacokinetics, toxicity
elucidations, drug metabolism, and drug discovery progression.
4.4.4 GAS CHROMATOGRAPHY
Coupling capillary column GC with Fourier-transform infrared spectrometer

provides an effective resource for separating and recognizing the components
of various mixtures. GC-MS technique can be directly interfaced with rapid
scan mass spectrometer of different forms. The flow rate from a capillary
column is normally kept low enough that the column output can be served
directly into the ionization chamber of MS. The simplest mass detector in GC is
the ion trap detector. In this technique, ions are created from the eluted sample
by electron effect or chemical ionization and stowed in a radio frequency
field; the ensnared ions are then evicted from the area of storage to an electron
multiplier detector. The discharge is meticulous so that scanning on the basis
of the mass-to-charge ratio is probable. The ions ploy detector is remarkably
dense and less affluent than quadruple instruments. GC-MS instruments have
been used for documentation of hundreds of constituents that are present in the
natural and biological system (Daffre et al., 2008; Nagani et al., 2012).
4.4.5 SUPERCRITICAL FLUID CHROMATOGRAPHY
Supercritical fluid chromatography (SFC), the most technologically advanced

extraction system, is a hybrid of gas, usually CO2 and liquid chromatography
that combines some of the best features of each. The liquid is impelled via
a cylinder that holds the material needed to be extracted. Furthermore, the
extract-laden liquid is driven into a parting chamber where the extract is parted
from the gas and the gas is mended for reuse. This technique is an essential third
kind of column chromatography that is starting to find application in numerous
industrial, administrative and research laboratories. Coupled SFE-SFC and
coupled SFE-GC and SFE-LC systems are available for a critical analysis.
KEYWORDS
•• bioactive compounds
•• conventional extraction
•• enzymatic hydrolysis
•• marine phytochemicals
•• membrane filtration
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PART II
Industrial and Medicinal
Applications of Phytochemicals
CHAPTER 5
BIOTECHNOLOGY APPROACH
TO THE PRODUCTION OF
PHYTOCHEMICALS:
AN INTRODUCTION
HAMEED SHAH1,2, ANDREW G. MTEWA3,4,
CHUKWUEBUKA EGBUNA5,*, ANYWAR GODWIN6, and
DUNCAN C. SESAAZI4
1
CAS Key Laboratory for Biomedical Effects of Nanomaterials and
Nanosafety, National Center for Nanoscience and Technology,
Beijing, China
2
University of Chinese Academy of Science, Beijing 100049, China
3
Department of Chemistry, Institute of Technology, Malawi University
of Science and Technology, Malawi
4
Department of Pharmacology and Therapeutics, Mbarara University
of Science and Technology, Uganda
5
Department of Biochemistry, Faculty of Natural Sciences,
Chukwuemeka Odumegwu Ojukwu University, Anambra State
431124, Nigeria. Tel.: +2347039618485
6
Department of Plant Sciences, Microbiology and Biotechnology,
Makerere University, P. O. Box 7062, Kampala, Uganda
*
Corresponding author. E-mail: [email protected];
[email protected]
*ORCID: https://orcid.org/0000-0001-8382-0693
ABSTRACT
This chapter details the biotechnological approach to the production of

phytochemicals. The production of phytochemicals is usually known to be
natural in plants. Recently, the propagation techniques have taken newer

paths involving the preservation of plant species that saw the advantage of
further manipulation of the growth environment of the plants to suit better
growth patterns. Researchers understood that similar methods can be used to
enhance the production of specific phytochemicals for potential drug leads,
food additives, and fragrances. This first worked with the use of plant parts
being grown on other plants, then in laboratory bioreactor setups and in
modern days, the use of plant cell cultures. Plant cell culturing techniques
have an advantage in that they are clean and allow flexibility in the attain-
ment of the exact phytochemicals so desired. This technique is the best so
far for propagation. However, it is an expensive venture that requires huge
financial investments.
5.1 INTRODUCTION
The dependence of human beings on plants is an open secret which dates

back to time immemorial, and still, human beings are constantly depen-
dent on plants, for their curiosity of novel drugs isolation to treat different
diseases and their use as fuels in certain parts of the world. The phytochem-
ists are well aware of the fact that drugs isolation is a tedious job, which is
more cynical in terms of natural products quality and yield, primarily due to
environmental changes either accidentally in the form of acid rains, storms,
and floods, or natural in the shape of latitude and altitude change of the same
species in different locations of the world, and in some cases with effects
from plants disease and so forth. These problems along with the advance-
ment of knowledge in terms of understanding the plant pathways lead to the
synthesis of different compounds. The effects of different nutritional and
environmental factors on the plants make the scientists able to develop some
artificial techniques to overcome their desire for obtaining some specific
molecules or plants as a whole which could be better utilized for the welfare
of human beings. In order to deal properly with these issues, there emerge
a need to applying the technique of cell and tissue culture, which involve
the in vitro cultivation of a desired plant in a controlled environment by
providing all the requisites in an artificial environment such as the nutri-
tion’s required for its normal growth, including organic and inorganic salts,
obligatory vitamins, plant growth hormones and amino acids and so forth.
A well-established laboratory with the accomplished medium for growth
is in the ominous need for this process to complete. The process makes
researchers able to obtain the desired drugs in a time-independent manner
Biotechnology Approach to the Production of Phytochemicals 109
from the natural environmental cycles. They also obtain the required product
in more yield and without the internal changes of decomposition or other
structural changes due to many factors involved with the growth of the plant
in the natural environment and through the natural cycles. The technique is
also valuable in terms of obtaining the desired drugs without leading to the
deforestation of the plants.
Plant cell and tissue culture is an important area of phytochemistry which
although being nascent, is highly important due to some aspects such as
the source of obtaining different cost-effective phytochemicals, obtaining
the desired products with better quality and quantity under a controlled and
properly observed system, preserving the plant species, minimizing cost and
labor effects and so forth.
Since the beginning of the 20th century, scientists have returned to nature
as a source of potential drugs in pharmaceutical development (Georgiev,
2013; Pant, 2014). Plants constitute the largest part of the natural source
of known natural products (over 80%) including pharmaceuticals in drug
discovery (Smetanska, 2008). Many drugs in the market today came from the
information and materials of indigenous knowledge of plants and their tradi-
tional uses (Pant, 2014). Already, over 25% of modern medicines are derived
indirectly or directly from plants, especially with 60% in cancer therapy and
75% in infectious diseases, but also for drugs used in immunosuppression
therapy and metabolic syndrome-related diseases’ treatment (Georgiev,
2014). The most common and typical way of obtaining phytochemicals and
other such important natural product compounds is through extraction from
the source plants (Ochoa-Villarreal et al., 2016). In the course of the years,
population increase and demand for plant products (medicines, clothing, and
shelter), infrastructural development, and social gratification (illegal trade,
religion, and pleasure) have contributed to the exploitation and depletion
of plants including medicinal plants (De Luca et al., 2012; Chattopadhyay
et al., 2004). Many plant species are still at a threat of depletion from their
natural habitats in regard to the projected population growth of about a third
by 2050 (FAO, 2009) which will have increased social demand for more
plant use and exploitation. With the need for phytochemicals for continuous
drug discovery and development, nutritional, and social needs, techniques
of plant cell culture are providing alternatives for production and harvesting
of bioactive compounds from plants without contributing to plant species’
depletion. The culturing of plant cells is a latent source of important phyto-
chemicals which can be used as pharmaceuticals, nutraceuticals, and food
additives among which are colorants, flavors, and fragrances (Smetanska,
2008; Zhong, 2001). Plant cell culture has been practiced as early as the
1930s with the first patent for phytochemical production from cell culture
filed in 1956 by Pfizer Inc., (Ratledge and Sasson, 1992). As reported by
Smetanska (2008), the progression of the techniques in 1989, saw a phyto-
chemical, Shikonin, being for the first time produced on an industrial scale
by Mitsui Petrochemical Industries Ltd. Plant cell culture has established
its place in commercial applications as well as in biochemistry, genetics,
pharmaceutical, and cell biology research. To date, there are not less than
28,000 patents related to plant cell culture products in various fields of
natural products applications (Ochoa-Villarreal et al., 2016) and this still
remains a cutting age next-generation technology in phytochemical use and
allied natural product compounds.
5.2 HISTORICAL BACKGROUND OF PLANT CELL/TISSUE

CULTURE
The theoretical base for plant tissue culture is provided by the cell theory put
forward by Schleiden in 1838 and Schwann in 1839. Thus, it is the regenera-
tive ability of plant cells, which is readily expressed by plant cells and tissue
culture, through the help of suitable chemical and environmental stimuli,
soon after its separation from parent plant followed by in vitro culture. An
auxiliary step was taken by the German Scientist, Gottlieb Haberlandt, whom
in his address to the German Academy of Sciences in 1902 while discussing
his experiments on cell culture growth, proposed that in future such studies
will bring interesting information about cells in terms of their properties and
potentialities. Unfortunately, he did not get any satisfactory results from his
own culturing experiments, yet he is the pioneer in establishing the concept
of totipotency and hence is known as the father of plant tissue culture. Soon
after Gottlieb Haberlandt experiments and concept of plant tissue culture,
much emphasis was given on this burgeoning field by scientists. In 1904,
Hanning succeeded in culturing coniferous embryo that was successfully
followed by Brown in 1906 with the culturing of Barley embryos. In 1922,
the isolated root tips were cultivated by Kotte which successfully leads to
tomato root tip culturing in 1934 by White. Laibach successfully rescued
embryos from nonavailable seeds of a cross between Linum perenne × L.
austriacum. Full embryo development in the ripening seeds of some species
was achieved by Tukey in 1934. LaRue in 1936, also succeeded to develop
the phenomena of precocious germination, followed by his attempts for
ovary culturing in 1942, and first endospermic tissue culturing of immature
maize in 1949. Loo in 1945 and Ball in 1946 likewise cultured the buds
successfully. This effective achievement encouraged the scientists to further

optimize the conditions for plant cell tissue culture. In 1962, Kanta reported
the in vitro pollination and fertilization of plants, primarily based on the
earlier achievements of Tuleke, who reported the culturing of cell colonies
from Ginko pollen grains in 1953, successfully followed in 1969 by Street
for his reports on the culture effected in the presence of organized and disor-
ganized media and which make him able to study virus under the effects of
these root media as well as to study plant physiologically under the effects of
culture media. In other words, the scientists were aware regarding the role of
media and different nutrient agents present in the media, thus having much
elaborative potential on the applications effects of plant tissue culture.
5.3 NEED FOR PLANT CELL CULTURE
Different extraction methods are used to avail phytochemicals from the

plant matrices sourced from the whole plant, or parts of it. This method
avails phytochemicals that were naturally produced by the plants over some
time in the past. Some of the challenges with relying on natural source plant
produced phytochemicals are as follows:
5.3.1 TYPICAL LOW YIELDS
Active compounds of interest are typically in low concentrations from the

source plant-derived matrices (Ochoa-Villarreal et al., 2016), an undertaking
which usually proves to be economically costly. For example, a 2-kg sample
may yield concentrations in the order of nanograms of a particular compound
of interest, and there is no control to that.
5.3.2 SLOW GROWTH RATES OF SOURCE PLANTS
The natural slow growth of some plants have a reducing effect on the
production potential of phytochemicals. This means the plant may take years
to grow only to produce very low concentrations of desired compounds.
Synthesis of desired phytochemicals provides an alternative route
of availing these compounds for use, especially for simpler structured
molecules. However, multiple chiral centers for many natural compounds
including phytochemicals make total synthesis difficult particularly where

they are region- and/or stereo-specific.
5.4 ADVANTAGES OF CELL CULTURING
There are several advantages of producing and ultimately harvesting phyto-

chemicals from plants using plant cell cultures over conventional methods
of extraction from plant parts or whole plants. This section highlights some
of the advantages which are worth noting and investing for.
5.4.1 CONSERVATION OF PLANT SPECIES
Owing to the depletion of most plant species, the laboratory scientist

should be the last to endanger them more and to contribute to the damage.
Undertaking plant cell cultures ensure that the actual plant is left alive to
blossom whilst science of metabolite harvesting is as well flourishing. The
cells can be multiplied and at the same time is kept alive as viable cell lines
for further studies.
5.4.2 CONTAMINATION FREE
With the detailed care invested in the biotechnology and the bioprocess, yields
are free from insects and microbes (Hussain et al., 2012). It is largely very
difficult to ensure microbe-free phytochemical yields from the conventional
plant part or tissue extraction technology which is to some extent, likely to
affect biochemical analyses due to metabolic activities of the microbes.
5.4.3 HIGH YIELDS AT A REDUCED LABOR COST
Since the cell growth bioprocess is controlled and automated, there will be no
need for laborious activities once the set up is done apart from regular monitoring.
High acceptability: Consumer acceptability for plant cell culture prod-
ucts is higher as consumers get to have surety that the products are no-GMO
(Murthy et al., 2015).
5.4.4 FREE FROM CLIMATIC AND SOIL CONDITIONS

INFLUENCES
Naturally, some phytochemicals may not favorably develop in particular

climatic conditions and soil profiles in which the plant is growing. Cell culture
phytochemical production can be designed to develop any particular type
of metabolites of interest under controlled conditions to or toward targeted
desired yields. This means, cells from any type of plant despite where they
are traditionally found can be grown at any place but in controlled condi-
tions to produce the desired phytochemicals in vitro (Hussain et al., 2012).
5.4.5 ENSURE A STABLE SUPPLY CHAIN
Harvesting phytochemicals and other natural compounds from plant cell

culturing technology act more as a renewable source of plant compounds
with an add-on that one may wish to control production depending on
need, thereby establishing a robust chain of supply for various compounds
(Ochoa-Villarreal et al., 2016).
Pros of plant tissue culture
The use of plant tissue culture offers several benefits, which include the
following:
1. It enables the rapid production of mature plants, in large quantities,

which do not require much space or large vessels for storages (Grout,
2017).
2. Tissue culture greatly enhances the production of plants from recal-
citrant seeds or seeds with very low germination rates.
3. One can significantly shorten the time required for harvesting since
it is no longer necessary to wait for the entire life cycle of seed
development, especially for plants that take longer to mature and
side (Sharma, 2015).
4. Plant tissue culture is an indispensable tool for producing disease-
free plant material.
5. Tissue culture is ideal for the production of facsimile or exact copies
of plants with particularly desirable characteristics; for example, in
this case, is plants which produce standard quantities of particular
compounds of interest
6. It is useful in the mass production of planting material, especially for

commercial purposes.
7. Through plant tissue culture, it is possible to have a continuous
supply of young planting material throughout the year, irrespective
of the season, once an ideal plant tissue culture line is established
(George, 2008; Sharma 2015).
8. Plant tissue culture is an ingenious method of plant propagation
while bypassing the availability of seeds or even the natural pollina-
tors of the plants. It is also useful for plants that are sterile or do not
produce viable seeds.
9. Because plant tissue culture does not require the use or harvesting of
a whole plant or large parts of it, it is ideal for cloning of critically
endangered plants since the mother plant is virtually left intact.
10. With tissue culture through micropropagation, one usually produces
more robust plants. This results in faster growth in comparison to
similar plants produced by conventional methods such as seeds or
cuttings (Sharma, 2015).
11. Plant material needs little attention between subcultures and there is
no labor or materials required for watering, weeding, spraying, and so
forth,; micropropagation is most advantageous when it costs less than
traditional methods of multiplication (George and Debergh, 2008).
Cons of plant tissue culture
Much as plant tissue culture comes with all these advantages, and benefits, it
comes with some challenges. Key among them is the facilities:
1. The resources: Tissue culture requires a highly specialized labora-

tory for it to be carried out. These laboratories are expensive to set
up and run. They require specialized equipment and reagents.
2. The personnel: Tissue culture requires highly trained and skilled
personnel to run the laboratories. This staff requires a lot in terms of
resources need for training them.
3. Once the parent stock is infected, sick, or susceptible to disease, all
the resultant material will possess this undesirable characteristic.
4. Plant tissue culture can result in reduced genetic diversity. This can
come about if the current plant stock has a similar genetic makeup.
Reduced or low genetic diversity reduces the chances of the plants
resisting diseases and pathogens that may attack the plant in future.
5. Not all plants can be successfully cultured by this method because of

lack of knowledge on the right medium or because of the secondary
metabolites produced by some plants, which inhibit the growth of the
explant (an excised portion of a plant from which plant tissue culture
is initiated). There are no a one-size fits all as different plant species
require differences in their protocols such as the type of nutrient
medium among others. As a result, protocols need to be developed
for individual plant species, and this is often a tedious process of trial
and error.
6. It is a highly laborious and technical exercise and if not well
conducted, it can result in contamination of the entire stock.
5.5 BIOTECHNOLOGY APPROACH TO PHYTOCHEMICAL

PRODUCTION
The in vitro biotechnology of plant cell culturing involves engineering

of metabolic pathways and conditions and is vital for today’s and next-
generation production of phytochemicals. These activities are conducted on
plant cells outside (detached from) the plant as opposed to in vivo activities
that are conducted on cells that are well within or surrounded by other plant
cells in a plant. Plant cell secondary metabolism engineering mainly aims
at lowering levels of undesirable compounds, increasing desired phyto-
chemical content and introducing the production of novel compounds into
specific plants (Kirakosyan et al., 2009).
Considerations to invest in an aseptic laboratory environment and appa-
ratus with qualified personnel are required. The first step is to obtain a callus
from any part of the whole plant containing cells that are dividing and is
known to be highly productive. The knowledge of portions of high produc-
tivity helps to maximize the availability of, particularly desired compounds.
The callus’ friable portions are transferred into a premade liquid medium,
made as homogenous as possible, and is then stored under suitable environ-
mental conditions (agitation, air, temperature, light, and others) (Chattopad-
hyay et al., 2004). The following section outlines details in the techniques
used in getting good phytochemical yields from plant cell culturing.
Naturally, plants produce tiny organic molecules involved directly in
metabolic processes of development and growth, called primary metabo-
lites. However, they also produce secondary metabolites, which are rela-
tively more abundant in higher plants for chemical defense against insects,
microorganisms, and higher predators and for attracting pollinators and seed
dispersers (Bhojwani and Dantu, 2013). Among these secondary metabolites

is an overwhelming range of phytochemicals that man has used and is still
using in medicine, nutrition, cosmetics, and neuro-manipulation among
other areas. Science has developed strategies to obtain phytochemicals from
these plant materials.
5.6 ENHANCING PHYTOCHEMICAL PRODUCTION
Plant cell culturing and component harvesting and plant cell biotechnology,
in general, is made more convenient and attractive through the application of
system biology and functional genomics (Kirakosyan et al., 2009). Produc-
tivity in a bioreactor can be enhanced by a proper strategy of cultivation,
timely feeding of appropriate metabolic precursors, and extraction of any
undesired intracellular metabolites (Chattopadhyay et al., 2004).
5.6.1 DELIBERATE ISOLATION OF HIGH YIELDING CELL LINES
The best choice of the parent plant, known to have high levels of the product
desired for the induction of callus is always a good starting point. This gives
a guarantee of getting best yielding cell lines. This process is systematic and
involves actual screening of the wide range of cell clones, among those that
are capable of producing highly. The identification and selection of these
cell lines can be achieved well if the desired product is pigmented. Selection
can be done visually, or by using analytical techniques.
5.6.2 MUTATION STRATEGIES
The involvement of mutation strategies has also been used to get high
yielding cell lines. Here, a large cell population is exposed to a toxic inhibitor
or environmental stress. Cells that are capable of resisting this procedure are
the ones that will grow.
5.6.3 STRESS MODIFICATION
Phytochemical production can also be enhanced by a deliberate manipula-

tion of the culture environment. Many secondary metabolite pathways’
expressions can easily be modified by external factors such as stress factors,

nutrient levels, growth regulators, and light. Many of the constituents of
plant cell culture media are important determinants of growth and yielding
of phytochemicals (Misawa, 1985).
5.6.4 CULTURE ENVIRONMENT OPTIMIZATION
The environment in which cultures are grown has proven to be one of

the most effective factors in determining the yield of phytochemicals.
Temperature is required to be checked, controlled, and specified for each
and every species. For example, lowering the temperature at the cultivation
stage increases total fatty acid content in each cell, dry weight (Toivonen
et al., 1992). Kreis and Reinhard (1992) reported that upon maintaining the
temperature at 19°C, digitoxin was transformed to digoxin most favorably,
but at 32°C, the yielding of purpurea glycoside was favored more in Digitalis
lanata cell cultures.
Light application strongly stimulated anthocyanin accumulation in cell
cultures of Vitis hybrids and Dausus carota (Seitz and Hinderer, 1988). This
process was also found to affect sesquiterpenes’ composition in Matricaria
chamomilla callus cultures (Mulder-Krieger et al., 1988). It is not only
the application of light that positively affects phytochemical yields, light
withdrawal also positively contributes to yields as was the case with callus
cultures of Citrus limon that prompted monoterpenes accumulation (Mulder-
Krieger et al., 1988).
5.6.5 pH OF THE MEDIUM
The pH of the culture medium before autoclaving is usually between 5 and 6

and extremes pHs are avoided. As the culture grows hydrogen power in the
cells changes. Chenopodium rubrum cell culture suspension indicated that an
increase in the medium pH from 4.5 to 6.3 enhanced the cytosolic pH of the cells
by 3.0 units and their vacuolar pH by about 1.3 units (Husemann et al., 1992).
5.7 TYPES OF PLANT CELL CULTURE
Cell culturing basically consists of three main types: primary cell culture,
semicontinuous cell cultures, and continuous cell cultures.
1. Primary cell culture: Cell culturing which involve the direct culturing
of cells separated from the parent plant tissue is called primary
cell culture. Cell culturing basically starts with biopsy of the tissue
isolated from the source (~1 cm3) through dissection from tissue or
organ of the parent organism. Following the dissection, the next step
involves the isolation of single cell suspension which is carried out
by the removal of cell basal membrane or cell basal connections.
The techniques may involve physical interferences, such as cutting
with the help of surgical knives, or chemical digestion methods
such as using certain chemicals or may involve biological digestion
methods such as using enzymes trypsin or collagenase and so forth.
In the next steps, these obtained cell suspensions are further purified
through different methods, including the serial dilutions along with
centrifugations, adherence, antibody coupling to a fluorescent dye,
microdissection, and so forth. After the maximum purification, these
culturing cells are finally transferred to the cell culture vessels, where
the cells being freed from the cellular connections are able to stick to
the culture vessel surface while the nonsticky cells and their residuals
are removed from the cell culture vessels by centrifugation and so
forth, as mentioned in the above lines, which lead to the completion
of this initial step in the cell culturing. Primary cell culturing is a
tedious job, and could be maintained for a limited time because the
cell media is consumed along with the filling up of the space provided
for cell growth. It is very important to note that apart from a lot of
efforts to isolate and culture pure cells in culture vessels, still there
are some other cellular organelles or cells on the cell culture vessels,
such as fibroblasts, and toxins and so forth. In other words, it is not
completely possible to culture pure cell on the culture vessels during
this primary culture, yet, it is still noticeable that this primary culture
grown still greatly resembles the tissue culture obtained from the
parent organism biopsy, and the complete purification involve further
steps, after which the pure cell culture is usually obtained in the
secondary culturing by passaging. Thus, this step has drawbacks of
not pure cell culture and having limited cell growth and proliferation
ability. Then their need further measures to overcome these short-
comings. In the next step, the propagation of the undesired cells could
be stopped by different techniques such as through the introduction
of medium suitable only for the growth of the required cell culture
which may lead to the death of the undesired cells. In the next steps,
providing new cell media for the plant growth could also provide
more nutrition and space for the plant cell culture growth.
2. Semicontinuous cell culture: This cell culture is basically dependent
on the primary cell culture and involves the further growth of the
primary cell culture in a different environment. In semicontinuous
cell culture, the medium is continuously changed for 24 h and the
cells are also usually diluted, thus making room for the culture
growth. However, after a certain point, it becomes unsuitable to
continue the culture due to the building up of increasing number of
competitors, predators, contaminants, and metabolites. Fibroblasts
are still present here, yet the ratio of toxins is almost negligible.
This culture has the advantage of obtaining more cells with
more specificity because here we can introduce some new space and
new rich nutrition medium per the specificity of the cell line we are
desired in the cell culture, as a result of which we get the specific
cells more suitable for their application. The cells in this culture
retain the actual number of chromosome pairs and are thus diploid.
These are clones cell lines and thus even a single cell is capable
to give rise to subsequent generations. Their passaging ability is
between 60 and 100 times, and hence considered more suitable in
virology. These cultures are used primarily in human chickenpox
virus and poliomyelitis virus production.
3. Continuous cell culture: As the name confirms, in these cultures,
the cells are grown in a medium where the nutrition is continuously
supplied and the grown cell culture is removed simultaneously. In
practice, a volume of fresh culture medium is added automatically
at a rate proportional to the growth rate of the algae, as for example,
while an equal volume of culture is removed, thus maintaining the
culture at a maximum growth rate. The process involves the growth
of the culture in a medium with a hole, in which after the addition
of the fresh media, the old media flow out automatically. The culture
has the ability to be well suspended with a maximum concentration
of O2 and CO2. These cell cultures are also known as immortal, or
transformed lines, and are heteroploid with an abnormal number
of chromosomes usually not present in pairs. These cell lines are
capable to pass through thousands of passages. These cells are
obtained by the engineered or spontaneous transformations of the
cells in vitro or by the culturing of tumor cells such as HeLa cells or
RD cell lines. Its early development involves the microbial genera-

tion for industrial and research purpose.
5.8 BASIC REQUIREMENTS OF PLANT CELL/TISSUE CULTURING
Cell culture is basically an art of keeping live the cells of organisms in

an artificial environment by providing all the basic requirements for
their normal growth in terms of their food supplementation along with
the required hormones and so forth. The cell culturing has covered the
areas of vaccine formation, monoclonal antibody, enzyme production,
growth factor preparation, hormone production, and interleukin produc-
tion. The development of regenerative medicine concept is another area of
application of cell culture. These all methods involve the transformation/
differentiation of the cell from the initial stage of the selective product
which mainly depends on the internal cellular program and the outside
environmental factors.
As mentioned in the above lines, special working conditions are neces-
sary for cell culturing involving the handling of the cell by sophisticated
instruments in the well-equipped labs with sterile conditions and proper
planning for the safe contamination of the waste. Cell culturing is achieved
only when proper conditions are maintained for the safety of the laboratory.
An environment suitable for the reproduction of the cells in the desired direc-
tion involves factors such as free space and nutrition support. After the initial
growth cell passaging is the desired action which can be achieved keeping in
mind the factors such as cell concentration, pH of the new environment, and
the time interval between the two passaging actions and so forth. Below are
given a short summary of the requirements for maintaining a cell culture and
the lab in proper conditions.
5.8.1 LAB SAFETY
Lab safety could be categorized as problems related to the working environ-

ment and working material. The working environment involves the flow of
air along with the passage of water and so forth, that is, the environment
of the lab and definitely some surrounding area, which need to be properly
maintained to achieve the best health conditions for the working scientists
as well as for the cultured cells and other materials such as chemicals
and so forth. In the second category of working materials, there include
equipment’s, solvents, and other reagents. Chemicals are to be considered

as the most erudite and important part of the lab in terms of their inactivity
by mishandling along with their corrosive, toxic, mutagenic, and inflam-
mable properties. On the other hand, the proper handling of the instruments
is also an important factor in lab safety perspective as it could lead to serious
accidents. Different infections are the major safety concerns of the related
materials, as the plant cell cultures may contain different viruses, bacteria,
and so forth, which may cause the workers to spread different infections in
their body per their originating organism from which they had been isolated
and they may have these pathogens at the time of handling them. Problems in
the cell culture laboratory could harm the human health and the environment.
Legal regulations should be firmly followed along with work and security
protocols, thoroughly analyzed and procedure well as per the requirements
of the location environment and lab conditions. The staff should also be
well educated in terms of handling the objects inside the lab and following
the rules and regulations. Some important procedures prepared by National
Institutes of Health, World Health Organization, and so forth.
5.8.2 CONSTITUENTS OF MEDIA
Media is an important component of the normal cell growth during plant

culturing. It should have to provide all the components of the cell culture,
which mimics the natural environment that is provided by soil. For example,
it should be able to provide 17 essential elements of carbon (C), oxygen
(O), hydrogen (H), nitrogen (N), potassium (K), calcium (Ca), phosphorus
(P), magnesium (Mg), sulfur (S), iron (Fe), nickel (Ni), chlorine (Cl),
manganese (Mn), zinc (Zn), boron (B), copper (Cu), and molybdenum
(Mo). Some species growth also requires the presence of more elements of
aluminum (Al), cobalt (Co), iodine (I), and sodium (Na). Each plant strain
shows a different tendency in growth toward a specific media. Hence, it
is very important to maintain specific conditions for the growth of a cell
culture in a media.
5.8.3 MACRONUTRIENTS
These are the constituents required in a concentration of more than

0.5 mM/L. The main chemical present in high concentration is the sucrose,
one of the main source of C, O, and H for the plant growth. In common
mediums, it is used in the concentration range of 20,000–30,000 mg/mL.

It acts as a full house for the cell growth along with its role in metabolite
signaling. As per the nature of media, other sources of energy such as
glucose, sorbitol, raffinose, and so forth may also be used in the medium.
Other micronutrients include salts such as Ca3 (PO4)2, Ca (NO3)2, KNO3,
MgSO4.7H2O, NaNO3, NH4H2PO4, NH4NO3, NaH2PO4. H2O, (NH4)2SO4,
CaCl2.2H2O, CaCl2, KCl, KH2PO4, K2SO4, and so forth, the concentration
of which varies per medium.
5.8.4 MICRONUTRIENTS
These are the constituents required in a concentration less than 0.05 mg/L,

and include mainly the metals of iron, molybdenum, copper, zinc, manga-
nese, and boron. These micronutrients are extremely important for the plant
growth, although being in very small quantities. Usually, these elements are
also introduced as salts in the medium, but in case of iron, usually chelating
agents such as EDTA and so forth, which make iron able to be absorbed in
low pH.
5.8.5 VITAMINS
These are the organic supplements added to the plant’s cultures required for
their metabolism, with their role as metabolic cofactors or enzyme cofactors.
For example, thiamine is an important cofactor for carbohydrates metabo-
lism and is added in a concentration of 0.1–5 mg/L to the medium. The
other commonly used vitamins are pantothenic acid (B5), pyridoxine (B6),
nicotinic acid (B3), and myoinositol.
5.8.6 pH
Different types of cells favor different conditions for their growth in terms
of pH effect. Mostly, the cell cultures favor the normal growth at basic pH
between 7.4 and 7.8. However, some cultures, for instance, the epidermal
cells can be grown at acidic media with a pH 5.5; yet, it has not been univer-
sally adopted. Phenol red is commonly used as an indicator of pH change. At
pH 7.4, it embarks red color, while with a decrease in pH to 7.0, it becomes
orange, with a little more decrease to pH 6.5, it then becomes yellow, pink at
basic 7.6, while purple at 7.8.
5.8.7 PLANT HORMONES
These supplementary materials are added frequently to the serum-free media

and are usually not expressed in the general constituents for any media. The
hormones are usually supplemented to the media as organic complex mate-
rial such as orange juice, tomato juice, coconut water, casein hydrolysate,
yeast extract, and so forth.
5.8.8 PLANT GROWTH REGULATORS
Determine the development pathway of plant cells and tissues in the culture
medium. The auxins, cytokinins, and gibberellins are most commonly used
plant growth regulators. These stimulate cell division and regulate the growth
and differentiation of shoots and roots on explants and embryos in liquid or
semisolid liquid cultures. Skoog and Miller studied the effects of two plant
hormones as growth regulators first and found that high concentration of
cytokinin in comparison to auxin lead to better shoots production, while a
reverse lead to better roots production. The organic complex material added
to the media usually act as a source of plant hormones and as the source of
plant growth regulator.
5.9 APPLICATIONS OF PLANT CELL CULTURE
Cell culturing could be applied in different directions if is grown by a proper

procedure and maintained under appropriate conditions. Perhaps the big
advantage of this technique is that new reshaped cells are obtained in a selec-
tive environment, which can be further engineered per our requirements.
5.9.1 AGRICULTURE
Food is the cradle of energy which is primarily obtained from plants.

Owing to ecological pollution, along with the political instability in some
regions and the low economic and instrumental strength of farmers in some
regions, the quality and quantity of food are affecting greatly. Moreover,
bad weathers, the soil infertility, annual nature of plants, and the natural
low production ability of certain plants along with the increasing population
are some extra contributing factors in making the situation worse. In this
scenario, the plant cell culturing is a greater hope to overcome these issues
because the better quantity and quality could be achieved by applying the
artificial environmental, nutritional and genetic modifications in the cells
culturing of the plants. For instance, the annual plants could be and have
been produced throughout the year for their food and other purposes by
providing the artificial environment. Moreover, through genetic engineering,
the genes which are causing diseases in plants have been changed with the
healthy ones in their seeds. Similarly, the genes involved in the high produc-
tion of food could be transferred from one plant species to another through
genetic engineering, and then their planting could lead to better food. In the
scientifically approved environment, the food production is much better than
the plants grown by farmers in their traditional manners.
5.9.2 INDUSTRY
Plant cell tissue culture is also actively contributing to the industry. Even
in the form of agricultural applications, it is contributing a good asset to
the market. Plant tissue culture is being grown both in the developed and
developing countries. The plant cell cultures are established and marketed
to the laboratories to be available to a scientist for their ongoing research.
The plants cultured through plant cell culturing are sold locally and
internationally, and a good financial reward is collected from them. The
plants culturing for the financial purpose include plants for ornamental
purposes, plants culturing in area with high species rate and then their
sale to areas or countries of low or not available species, plants growth for
specific microbes collection and then those microbes sale at national or
international level, plants culturing for specific metabolites production by
pharmaceutical industry are some of the applications of plant cell culturing
in the industry.
5.9.3 MEDICINAL
Secondary metabolites of medicinal importance are produced through this

technique. The production of bioactive metabolites through biotechnological
approaches, specifically plant tissue culture is regarded to be on the same

side with the industrial agricultural production for secondary metabolites.
5.9.3.1 ANTICANCER DRUGS
Taxol is a common anticancerous drug isolated from the inner bark of several
Taxus species. The procedure is very costly and laborious, yet the final yield
is also very low. Moreover, a high proportion of the plants are destroyed
for taxol isolation. Although related procedures, such as total synthesis, or
semisynthetic precursors has also been applied, yet the problems of cost
and the laborious extraction are the issues. To overcome this diseases of
cancer by taxol, plant cell tissue culturing is considered to be the best option.
The preliminary studies carried out during the optimization of the culture
favors the increased production of taxol and related toxoids from the Taxus
sp.; however, still there is need to study factors such as gene involvement,
biosynthetic pathways, their regulation and so forth, and after all finaliza-
tion, the technique could be applied commercially.
5.9.3.2 ANTIDIABETIC
Diabetes is a disease increasing day by day, both in the developed and non-
developed world. Basically consisting of two types, the type 1 is totally
insulin dependent while the type 2 involves primarily usage of some drugs.
Both the insulin and the drugs or secondary metabolites such as quercetin
used in the diabetes treatment are widely cultured. It is also interesting,
that the insulin produced through recombinant deoxyribonucleic acid tech-
nology, involving the use of Escherichia coli and yeast is safer than the
one reported from porcine because the human-derived insulin may contain
disease of the parent organisms. On the other hand, secondary metabolites
such as quercetin and so forth which have anti-diabetic efficiency is been
synthesized by plant cell culture.
5.10 APPLICATIONS OF PLANT TISSUE CULTURE
Plant tissue culture applications involve the aseptic culture of cells, tissues,
organs, and their components under specified chemical and physical condi-
tions in vitro. These applications cut across several fields in the plant sciences
including conservation biology, agriculture, forestry, plant biotechnology,

and horticulture among others (Bhatia and Dahiya, 2015). They include:
1. Providing a useful research tool in phytoremediation studies. Results

from plant tissues culture research on phytoremediation can be used
to predict the responses of plants to environmental pollutants. This
can lead to remodeling of phytoremediation designs and subse-
quently lower of the conventional whole phytoremediation experi-
ments (Doran, 2009).
2. The conservation of endangered plant species that faced with extinc-
tion. The risk is eminent in plants with a complex reproductive
biology and threatened by loss of habitat, small population sizes,
and their long life cycle (Loyola-Vargas and Vázquez-Flota, 2006).
3. Germplasm storage: The ability to store germplasm for prolonged
periods of time especially for plants that are propagated by vegetative
means or have recalcitrant seeds is limited when using conventional
or traditional means. The use of plant tissue culture overcomes this
barrier when applied to other complementary techniques such as
cryopreservation under liquid nitrogen at −196oC, which allows for
the preservation for decades (Grout, 2017).
4. Production of secondary metabolites: When subjected to the right
conditions, the growth and division of cells from an explant can give
rise to a mass of undifferentiated cells known as callus. This can be
maintained on a semisolid medium as or grown as a suspension of
cells in liquid medium. The growing cells of different plant species
can yield many valuable secondary metabolites including elite
compounds such as pharmaceuticals and cosmetics. Cells can also
be grown in a batch culture or grown over longer periods with a
limited, continual harvest (Loyola-Vargas and Vázquez-Flota, 2006;
Grout, 2017).
5. The production of disease or pathogen-free seeds or plants especially
where traditional propagation methods such as the use of cuttings
have fueled the spread of diseases in crops. Bacterial pathogens
will typically infest the vascular tissues that provide transport to
the shoot and root. Viral particles and phytoplasmas, however, are
significantly limited by the absence of vascular tissue in the terminal
tissues of the meristematic dome (Grout, 2017).
6. Large-scale commercial production of plants used for example in
the flower industry and landscaping among others. The regeneration
of in vitro plantlets is the supports production en masse of clonal
populations of field or glass-house established plants. This is of

particular value when dealing with crops that are, conventionally,
vegetatively propagated, for example, fruits, ornamentals and forest
trees (Grout, 2017).
7. Plant tissue culture contributes to crop selection and improvement
by providing a non-seasonal multiplication system for elite and rare,
material. It also provides a selection system that can be used to screen
for potentially valuable crop characteristics. In addition, it also facili-
tates the production of genetically modified plants (Grout, 2017).
8. Research for screening plant cells instead of whole plants for
specific purposes such as yield or production of certain compounds,
herbicide resistance tolerance among others.
9. Chromosome doubling and induction of polyploidy. These include
doubled haploid, tetraploids, and other forms of polyploids. Haploids
in plant breeding programs are a useful way of shortening the time
required to complete backcross programs. This facilitates the reten-
tion of the character under transfer and stabilizes the transferred
genetic materials in a homozygous form. The production of dihap-
loid plants from haploid cultures shortens the time taken to achieve
uniform homozygous lines and varieties. The use of haploids in
breeding programs makes it possible to isolate individual genomes
irrespective of whether they are dominant or recessive (Kumar and
Loh, 2012).
10. It can be used to cross distantly related species by protoplast fusion
and generation of the novel hybrids (Sharma, 2015).
5.11 LARGE SCALE APPLICATIONS OF PLANT CELL CULTURING
Bioreactors are critical for successful yielding of phytochemicals in an

industrial setting (Bhojwani and Dantu, 2013). Care must be taken in the
selection of suitable bioreactors to avoid affecting metabolic pathways. In
this regard, aggregation of cells, aeration, proper mixing, and shear sensi-
tivity should be taken into account (Chattopadhyay et al., 2004).
It should be noted that plant root and tissue cultures have great genetic
stability and at times, better metabolic performances than cell line cultures
but their operating techniques and bioreactor development are highly labo-
rious and require huge investments (Flores and Curtis, 1992). Despite that,
plant cell cultures have a higher industrial application potential than plant
organ and tissue cultures because of closely related know-how invested in
microbial cultures over the years. This does not mean the two methods are
entirely the same, the differences in the growth type and nature of the cells
require the plant cell line to be further developed beyond the microbial cell
culture (Payne et al., 1987).
5.12 CONCLUSION AND FUTURE PERSPECTIVES
It is evident that plant cell culturing is involved in our daily life from research
to the most dependent things of food and medicine. We are taking help from
this technique by enhancing every area of our life related to the plants. It
helps us to grow more plants and trees, both as source of medicine as well
as for our domestic uses of ornamental or other daily uses, it also helps us to
grow more food from plants, along with its support for complicated medi-
cines from plants for the notorious diseases such as cancer and so forth, plant
cell cultures have already had a unique and important role in bioproduction,
bioconversion, or biotransformation, and biosynthetic studies. From future
perspective, it may help in transgenic plant studies, through which it may
help to accelerate the conventional multiplication rate of crops related plants,
which is important to strive drought in some regions of the world, where the
crops are wiped out by diseases, while all the currently employed techniques
will definitely improve both methodologically and technically, which will
bring more future to human life.
Although phytochemical production through in vivo plant culturing is
viable in the laboratory, the feasibility of the bioprocesses and recurrent low
yields of compounds of interest from original materials limit its applica-
tions in industries (Kirakosyan et al., 2009). The other challenge with cell
culture phytochemical production is that the process requires huge capital
expenditure and running costs. It is not easy for low-cost production units to
have these technologies in place.
See Chapter 6 and 7 for more details.
ACKNOWLEDGMENT
Hameed Shah acknowledges financial support from University of Chinese

Academy of Sciences, and The World Academy of Sciences under the CAS-
TWAS President’s fellowship program.
KEYWORDS
•• plant cell culture

•• plant tissue culture
•• plant biotechnology
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CHAPTER 6
SECONDARY METABOLITES
ACCUMULATION AND PRODUCTION
THROUGH IN VITRO CULTURES
HUSSIEN M. DAFFALLA* and AZZA MIGDAM ELSHEIKH
National Centre for Research, Commission for Biotechnology and
Genetic Engineering, Mohamed Nageeb St. No. 61, 11111 Khartoum,
Sudan
*
Mob.: +49918349142
*
ABSTRACT
In vitro plant culture comprises three distinct types, namely cell, tissue,
and organ cultures. Each type of this culture involves three components,
namely plant material, medium and culture conditions. In turn, each of
these components has various characteristics, for example, concentrations,
size, and practices. The utilization of in vitro cultures for the production
of secondary metabolites was comprehensively studied to date. Production
of many pharmaceuticals, nutraceuticals, food additives, and agrochemicals
was successfully realized. However, most of these compounds were still
commercially produced directly from intact plants. The high cost is the main
restraint for in vitro production of phytochemicals. Therefore, reducing the
costs below conventional methods must be the core intention. Cost reduction
has been managed mainly by increasing biomass growth and/or biomaterials
accumulation. These can be performed through the optimization of the three
components of each culture type by manipulating their characteristics. This
chapter addressed various achievements in phytochemicals production by in
vitro cultures.
6.1 INTRODUCTION
Plant secondary metabolites are derived biosynthetics from the primary

metabolites present at a much lower concentration (less than 1% dry weight)
and depend mainly on the physiological and developmental stages of
plants (Crozier et al., 2009; Nandani et al., 2013). Although they have a
slight role in photosynthesis, respiration or growth and development, they
are efficiently used for survival and diversification, hence generated during
a specific developmental period of the plant (Collin, 2001; Crozier et al.,
2009; Nandani et al., 2013). While secondary metabolites have no physio-
biochemical functions, they play major biological roles such as pollination
attractants, recruiting seed dispersers, environmental adaptations, or protec-
tive agents against climatic stress, microorganisms, other plants, and preda-
tors (Alfermann et al., 2003). Various types of secondary metabolites were
accumulated by arid and semiarid plants to tolerate the harsh abiotic stress,
especially water deficiency (Meena and Patni, 2008). During senescence,
leaves show beautiful color which is formed to protect the leaf cells from
photooxidative damage and for effectiveness in nutrient recovery (Winkel-
Shirley, 2002). A further view is that the secondary compounds may be an
expedient storage, in which the stored carbon and nitrogen are recycled
back into the primary metabolism when there is a demand (Collin, 2001).
According to their function, human beings utilized these natural products
for centuries to meet their needs for health, food, agricultural, and daily
necessities. In particular, plant-derived chemicals are valuable sources for a
variety of pharmaceuticals, flavors, aromas, pigments, food additives, dyes,
oils, resins, bio-based fuels, plastics, enzymes, preservatives, fragrances,
cosmetics, and agrochemicals (Zhong, 2001; Rao and Ravishanker 2002;
Alfermann et al., 2003). Plants remain the main exclusive source for many
medications. In recent years, there has been an increasing interest in plant
antioxidants that are used as an unmodified form of natural food preservatives
to replace synthetic substances (Nandani et al., 2013). The WHO reported
that medicinal plants are used by around 80% of the world population for
treating some diseases as intact plants or their extracts (Smetanska, 2008).
Although secondary plant products are very common, this does not
imply that every plant can produce every product. The bioactive compounds
are of more limited occurrence. Many of these commercially valuable
phytochemicals are secondary metabolites not essential for plant growth,
produced in trace amounts, and often accumulate in specialized tissues (Kim
et al., 2002; Alfermann et al., 2003). Some secondary metabolites are found
only in certain species of families, showing the individuality of species or
Secondary Metabolites Accumulation and Production 133
specific growth condition (Zhong, 2001; Nandani et al., 2013). Similarly, the
bioactivities of plants are inimitable to specific plant species or groups as the
mixture of secondary products in a particular plant is taxonomically distinct
(Meena and Patni, 2008). Also, certain plant organs or just one type of cell
could contain only specific secondary compounds (Kim et al., 2002; Rao and
Ravishanker, 2002). This particular organ called the medicinal part in phar-
macognosy is liable for the accumulation or secretion of those compounds.
For instance, flavonoids acting as ultraviolet (UV) protectants are specifi-
cally accumulated in epidermal cells (Liu et al., 2008). The expression of the
genes responsible for the formation of secondary metabolites is extremely
high in the tissue where those metabolites are primarily accumulated.
Moreover, only 10% of medicinal plant species are cultivated (Julsing et al.,
2007) and the rest are still collected from the wild. The chemical structure
of secondary plant products is more complex than that of primary products
(James et al., 2008). Slow plant growth rates and ineffective purification
procedures have an additional prohibition role in obtaining less sufficient
amounts of active compounds (Kim et al., 2002). Efforts to produce large
quantities of physiologically active secondary compounds by organic
synthesis are ongoing. However, nearly all active secondary compounds are
structurally complex, and in many cases, are impossible to synthesize or
the yields are too low (Materska, 2008; Nandani et al., 2013). To overcome
these difficulties, plant tissue culture techniques have been developed for
rapid, large-scale production of cells and their secondary compounds (Rao
and Ravishanker, 2002; James et al., 2008). The advantage of this method
is providing an uninterrupted and reliable supply of secondary metabolites
capable to accumulate a higher concentration of compounds compared to
the whole plants. For example, a 750 L bioreactor containing 600 L suspen-
sion culture of Lithospermum erythrorhizon would yield 1.2 kg of shikonin
within 2 weeks, while the plants covering a whole 1 ha would yield about
9 kg shikonin after 4 years (Alamgir, 2017). By calculation, the production
of 8 bioreactors in 2 weeks was equal to 4 years of a 1 ha field of shikonin
yields. However, few such successful examples on in vitro production of
biomaterials were reported. Yet, in vitro plant culture represents a prospec-
tive alternative for industrial secondary metabolites production, but there are
a number of obstructions in the way that can be classified as physiological
such as slow growth rate, heterogeneity, genetic instability, low metabolite
content, product secretion, and operational such as the requirement for
illumination, mixing, sterilization, and result from biomass growth as shear
sensitivity and wall adhesion (Zhong, 2001). Optimization of operational
and instrumentation systems to a corresponding advanced technology used
in animal and microbial production is compulsory for retrofit existing biore-

actor (Smith, 1995). This chapter summarized the works on the production
of phytochemicals from the three distinct types of in vitro cultures, namely
cell, tissue, and organ cultures in a bioreactor, liquid, and solid systems. The
focus will be on providing examples of cultural systems of each culture type
as useful guidelines.
6.2 ADVANTAGES OF IN VITRO PLANT CULTURES
In vitro plant culture is the main biotechnology technique that has the
potentiality to produce valuable and natural next generation products safe for
human use in medical, food and agricultural applications. Various alkaloids,
saponins, anthraquinones, polyphenols, and terpenes have been obtained from
in vitro cultures of various plant species. Numerous advantages of utilizing
in vitro cultures for synthesis of bioactive secondary metabolites, have been
listed by many authors (Collin, 2001; Chattopadhyay et al., 2002; Rao and
Ravishanker, 2002; Alfermann et al., 2003; Vanisree and Tsay, 2004; James
et al., 2008; Smetanska, 2008; Tan et al., 2010; Kuo et al., 2011; Gaosheng
and Jingming, 2012; Yildiz, 2012). Below is the summary of the advantages:
1. In vitro propagation of a large number of genetically uniform and

pathogen-free clones of a high phytochemical accumulation in a
limited time and space.
2. Completely running under controlled environment autonomously
from the effects of the abiotic or biotic factors.
3. It is possible to select cultivars with a higher phytochemical productivity.
4. Automation of main culturing steps such as cell growth and hence,
regulation of metabolic processes can decrease costs and, simultane-
ously, increase yield.
5. As cells have a higher rate of metabolism and shorter biosynthetic
cycles than intact plants, they can accumulate phytochemicals in
higher concentration per dry weight compared to the whole plants.
6. Extraction from the in vitro tissues is much easier than extraction
from the complex structure of the entire plant tissues.
7. Biotransformation of the exogenously supplied substrates into valu-
able products.
8. Production of novel compounds that are not produced or not been
detected in intact plants.
9. No herbicide and insecticide required during culturing.
6.3 PRODUCTION OF SECONDARY METABOLITES THROUGH

TISSUE CULTURE
Plant cell culture techniques include ordinary undifferentiated cultures

(callus, cell suspension, and protoplast), organ (shoot and root cultures),
and special cultures (hairy root and immobilized cells). The production of
secondary metabolites in plant in vitro cultures is contingent on the degree
of cell proliferation and differentiation (George, 2008). Careful selection
of productive cells and cultural conditions resulted in an accumulation of
several products in higher levels in cultured cells. Notably, every type of
culture has its appropriate external cultural conditions such as temperature,
gas exchange and light as well as medium conditions like pH, nutrients, and
plant growth regulators (PGRs) concentration, which favors cells growth
and accumulation of secondary metabolites. For instance, the production of
tryptophol in the cell culture of Ipomoea purpurea becomes double at pH
6.3 and is inhibited at pH 4.8 (Alamgir, 2017). The endogenous hormones
content interact with the exogenous supplement of PGRs not only to induce
growth of cells but also the formation of the secondary compounds. Each
explant type shows diverse responses, in terms of biomass and phytochem-
ical accretion, to external and internal factors. Praveen et al. (2010) studied
the distribution of withanolide-A in eight organs of Withania somnifera, and
found that the quantity of withanolide-A gradually decreased from young
leaves to the root.
To date, many plant species are under commercial utilization to produce
natural products using in vitro culture (Table 6.1). Some include Panax
ginseng (ginsenoside; 27% of dry weight in 20,000 L), L. erythrorhizon
(shikonin; 20% of dry weight), Coleus blumei (rosmarinic acid; 15%
of dry weight), Taxus spp. (paclitaxel; in 75,000 L), Coptis japonica and
Thalictrum minus (protoberberines; 13.2% of dry weight in 4000 L), and
Morinda citrifolia (anthraquinones; 18% of dry weight) (Smith, 1995;
Collin, 2001; Gaosheng and Jingming, 2012; Alamgir, 2017). On the other
hand, several products other than pharmaceuticals have been commercially
released. Recently, protecting cosmetic supplements from the cell culture of
Saponaria pumila, and dietary supplements consisting of the freeze-dried
biomass of Theobroma cacao cell suspension were trademarked (Geor-
giev, 2015). The commercial production of natural plant-based insecticides
mainly pyrethrum (Chrysanthemum cinerariaefolium), nicotine (Nicotiana
spp.), rotenone (Lonchocarpus utilis), and azadirachtin (Azadirachta
indica) has been gradually increased (George, 2000). Veterinary vaccines
were also produced through in vitro cultures with the first vaccine against
TABLE 6.1 Examples of Secondary Metabolites Production from Cell Culture. 136
Plant species Medium + PGRs (mg/L) Explant Light Culture Products Yield References
used regime period (d)
Arnebia M9 Leaf Dark 10 Naphthoquinone 637.15 mg/L Kumar et al.
euchroma (2011)
Artemisia MS + 0.5 BAP + 0.5 Leaf Continuous 12 Artemisinin 0.06% Lo et al. (2012)
annua 1-Naphthaleneacetic acid
(NAA)
Astragalus MS + NAA 1 + Kin 2 Mesocotyl Continuous 14 Isoquercitrin, 5.3 mg/g, 8.1 mg/g Ionkova (2009)
missouriensis Quercitrin DW
Azadirachta MS+ nd Seed kernel Dark 12 Azadirachtin 2.98 mg/g Prakash and
indica Srivastava (2005)
Barringtonia B5 + 2.0 Leaf 16 h 10 Lycopene 0.69 mg/g DW Behbahani et al.
racemosa 2,4-Dichlorophenoxyacetic (2011)
acid (2,4-D)
Centella MS + 2 2,4-D Leaf 16 h 26 Quercetin, 0.146 mg/g, 0.141 Tan et al. (2010)
asiatica Luteolin, mg/g, 0.056 mg/g
kaempferol DW
C. asiatica MS + 3 2,4-D + 1 kin Leaf 16 h 12 Luteolin 390 μg/g DW Tan et al. (2013)
Cinchona WPM + 15 μM Pic*, 0.5 μM Leaf 12 h 49 Quinine 11% DW Ratnadewi et al.
ledgeriana BA (2013)
Crocus B5 + 2 NAA + 2 Indole-3-acetic Corm Dark 21 Crocin 1.74 mg/g Chen et al. (2003)
sativus acid (IAA) + 0.5 BA
Ficus MS + 10.74 µM NAA/4.65 µM Leaf 16 h 21 Rutin, quercetin, 39.13 mg/g, Ong et al. (2011)
deltoidea Kin 3.92 mg/g,
8.65 mg/g DW
Phytochemistry, Volume 3
Morinda B5 + 2,4-D 5 μmol/L Nd Dark 14 Anthraquinones 40 μmol/g FW Stalman et al.
citrifolia (2003)
Spilanthes MS + 15 μM BA + 5 μM 2,4-D. Leaf 16 h 28 Scopoletin 35.63 µg/g DW Abyari et al.
acmella (2016)
Glehnia B5 + NAA 1 + kin 0.01 Petiole dark 20 Anthocyanins 14% DW Miura et al.
littoralis (1998)
Papaver MS+ 1 2,4-D + 0.1 kin Hypocotyl– 16 h 16 Thebaine 10.76 μg/g DW Farjaminezhad
bracteatum cotyledon et al. (2013)
Psoralea MS+ 1 2,4-D + 0.5 NAA + 1.5 Leaf 16 h 16 Psoralen 9850 μg/g DW Ahmed and Baig
corylifolia BAP (2014)
Lavandula LS + 0.2 2,4-D Nd dark 11 Rosmarinic acid 3348 mg/L Georgiev et al.
vera (2007)
Scrophularia MS + NAA 0.5 + BA 2.0 Leaf dark 17 Acteoside 14.25 μg/g Khanpour-
striata Ardestani et al.
(2015)
Solanum MS +1.0 2,4-D + 0.1 BAP Stem 10 h 28 Solasodine 220.5 mg/g Loc et al. (2014)
hainanense
M. citrifolia MS+ NAA (2.5 µM) +0.5 µM Root 16 h 45 Anthraquinone 16.99 mg/g DW Sreeranjini and
BAP Siril (2013)
Vitis vinifera MS + 0.1 BA Leaf 16 h 14 Phenolics, 71.91 mg/g, Sae-Lee et al.
Resveratrol 277.89 µg/g DW (2014)
Eriobotrya MS + 2.5 BA + 1 NAA Leaf Dark 30 Triterpene 151.54 mg/g Ho et al. (2018)
japonica
TABLE 6.1 (Continued) 138
E. japonica MS+ NAA 0.5 Immature 10 nd Tormentic acid 63.1 mg/g DW Li et al. (2017)
embryo
S. striata MS+ 0.5 NAA + 2.0 BA Stem Dark 29 Acteoside 14.25 µg/g FW Khanpour-
Ardestani et al.
(2015)
Salvia MS+ 0.1 NAA+ 0.2 BA + 0.5 Hypocotyl Continuous 238 Carnosol, 0.05 mg/g, Grzegorczyk
officinalis 2, 4-D Rosmarinic acid 18.57 mg/g DW et al. (2005)
PGRs: plant growth regulators, M9: Fujita et al. (1981), MS: Murashige and Skoog (1962), Pic: picloram, B5: Gamborg et al. (1968), WPM: Lloyd
and McCown (1980), LS: Linsmaier and Skoog (1965), Nd: not determined.
Newcastle disease virus developed using tobacco BY-2 cells but not released
commercially (Georgiev, 2015). Sweeteners used to replace sugar such as
glycyrrhizin, stevioside and thaumatin were produced from cell cultures
of Glycyrrhiza glabra, Stevia rebaudiana, and Thaumatococcus daniellii,
respectively (Rodríguez-Sahagún et al., 2012). The approval for plant
cell-based recombinant therapeutic protein for human use was utilizing
genetically engineered carrot and tobacco cell cultures patently protected
(Georgiev, 2015).
6.3.1 SECONDARY METABOLITES PRODUCTION FROM

UNDIFFERENTIATED CULTURES
Undifferentiated culture is the culture of cells in solid medium (callus),

in liquid medium (cell suspension) or both in liquid or semisolid medium
(protoplasts). Undifferentiated plant cells are genetically heterogeneous that
make it promising to select cell strains with superior metabolites production.
6.3.1.1 CALLUS CULTURE
6.3.1.1.1 Initiation of Callus
Callus is an amorphous and undifferentiated mass of thin-walled parenchyma

cells frequently growing and dividing. Callus culture involves induction of
cell aggregates from proliferating cells, tissues or organ explants on solid or
semisolid media commonly containing auxin alone or high auxin/cytokinin
ratio. Callusing is normally considered as a response of explants to wounds
and appeasers in the form of tissue dedifferentiation. Theoretically, a single
living cell is capable of developing into a whole plant and this phenomenon
is called cellular totipotency. Gautheret and Nobecourt were the first to
induce callus culture from carrot (Daucus carota L.) root tissues with the
aid of indole-3-acetic acid (IAA) in 1939 (Varshney and Anis, 2014). For the
production of a particular compound, it is advantageous to initiate the callus
from the plant part that is known to be a high or a specific producer for a
compound. The age and physiological status of the mother plant could affect
the formation of callus. Callus is mostly classified, according to morphology
and texture, into compact or friable. Compact callus consists of densely
aggregated cells. Friable callus consists of loosely associated cells which
can easily detach. Compact calli are preferred for initiation of organogenesis
or embryogenesis cultures, while the friable calli are used to generate cell
suspension cultures. The auxin/cytokinin ratios are the main controllers in
determining the type of callus. Generally, the chosen appropriate explant
should be fundamentally healthy with vigorous growth. The type of auxin
and cytokinin and the ratio between the two are important for callus forma-
tion (Lee et al., 2011). The size and the shape of the initial explant are
not critical, although proliferation might not occur with explants below a
critical size. In general, fairly large pieces of tissues were favored because
of the large numbers of cells present increased the chance of obtaining a
viable culture. On the other hand, the endogenous content of nutrients and
hormones are higher according to the size of the tissue (Yildiz, 2012). There-
fore, a high surface area/volume ratio was desirable for maximum growth.
The type of explants also affects the nature of cells induced. A single cell
or a uniform group of cells produced homogeneous callus, while explants
excised from an organ consist of many cell types, that is, leaf, root, which
produced heterogeneous callus.
Callus culture protocol from explant to proliferation usually contains
three developmental stages namely, induction, division, and differentiation.
1. Induction stage is where cells prepare to divide and metabolism is

activated.
2. Division stage is characterized by a rapid cell division and an increase
in metabolic activity, cells become dedifferentiated as meristem
materialized, and the cell size reduces due to cell division.
3. Differentiation stage starts with the maturation of some cells, there-
fore, leading to the expression of certain metabolic pathways that
lead to the formation of secondary products. The duration of each
stage is dependent on the physiological status of cells and cultural
conditions. The metabolite biosynthesis varies accordingly.
6.3.1.1.2 Maintenance of Callus Culture
Callus produces an undifferentiated mass of cells, which are aggregate of

heterogeneous or homogenous dedifferentiated cells. Cultures of callus
must be optimized for long-term maintenance of high growth rate, biomass
gain, and secondary metabolites production. The initial cells induced on the
explant are actively growing mass of cells called primary callus. The callus
can be subcultured or subdivided for one of three reasons, biomass produc-
tion, the establishment of cell suspension, or to regenerate organs/whole
plants with each requiring an appropriate hormonal balance. When primary

callus is separated from the remaining explants tissues and subcultured into
a same or different media for biomass growth, the newly formed callus is
called the secondary callus. The biomass enlargement of callus is dependent
on the composition of the subsequent medium, mainly on the concentration
of auxins and cytokinins. The types and the combination ratios of auxin
and cytokinin are imperative for increasing the callus rate of production and
growth (Lee et al., 2011). Both percentage induction and growth of callus
are important in determining the final dry weight produced by an explant.
The higher growth rate is an indicator of biomass gain in a shorter culture
period which is important to diminish the production cost. Moreover,
higher final dry weight is correlated, in some cases, to high accumulation
of secondary products. Maximum stimulation of flavonoids is associated
with good biomass growth achieved (Ionkova, 2009). The establishment
of callus growth curve, therefore, is essential to verify the point (time) of
stationary phase which is the suitable time for subculture process. Scheduled
subculture into a fresh medium with the same compositions is imperative
for long-term maintenance of the callus nature (friable or compact) and to
avoid browning, or for the harvesting of calli with a higher fresh/dry weight
or by-product yields. The growth of callus might show a general growth
pattern of a sigmoid (S-shaped) curve when growth is plotted against time.
Callus curve commonly contains three phases namely: (1) a lag phase with
adaptation and initial slow growth, (2) log phase which is the main period
for rapid and maximum growth, and (3) the stationary phase where growth
is sluggish and lastly, stops. When callus is about to reach the stationary
phase, it must be subdivided and subcultured. Callus induced on Grewia
asiatica explants was stimulated and growth increased when subcultured
periodically (Sharma and Patni, 2013).
The PGRs concentration, cell-to-cell contact, type and age of explant,
and any early differentiation which is present seem to favor the biosynthesis
of secondary products (Parr, 1989). Callus culture was reported to be utilized
for phytochemicals production, commonly flavonoids (Filová, 2014). For
example, callus culture of Maackia amurensis accumulated elevated concen-
trations of isoflavones and pterocarpans approximately four times higher
than the content of the heartwood of intact plants (Fedoreyev et al., 2004).
Other phytochemicals also, for example, ginseng was produced by callus
culture of Ginsenoside, Panax ginseng, which was six times higher than the
whole plant (Alamgir, 2017). Callus tissues of Zataria multiflora produced a
very high level of rosmarinic acid (158.26 mg/g) compared to (12.28 mg/g)
that produced by the propagated shoots (Françoise et al., 2007). Matkowski
(2004) compared the production of four isoflavonoid compounds in the

calli of Pueraria lobate from different explant sources (root, leaf, and stem)
(Table 6.2). The results reveal that the kind of explant affected both the total
amount of isoflavonoids and the quantity of each compound. It was also
found that the age of explant source affected the isoflavonoids content.
Callus culture has several advantages that makes it useful for secondary
metabolites production: (1) Can be induced from a wide range of explant
sources compared to other cultural types (Table 6.2). (2) It has the possibility
to be maintained for longer cultural periods compared to organ cultures.
(3) When established, it is easier to use for the initiation of a distinct type
of culture, from a single cell to the whole plant. (4) Photosynthesis is
inconsequential; can grow under dark condition.
6.3.1.2 CELL SUSPENSION CULTURE
6.3.1.2.1 Initiation of Cell Suspension Culture
Cell suspension culture is induced by suspending an undifferentiated friable

callus into liquid media containing PGR. The callus then separates into cells
which are uniformly bathed in liquid medium under continuous agitation on
horizontal reciprocating or gyratory shakers which favor cells growing in a
disorganized form. However, an organ such as leaf was reportedly used for
direct induction of cells in liquid media bypassing the callus induction stage.
For instance, a rapid growth and mass of cells are released into suspension
as leaf sections of Chenopodium rubrum are kept floated on Murashige and
Skoog (MS) medium with rotary shaking for 4 days under light (Geile and
Wagner, 1980).
After the initial shaking, any large or unspoiled callus can be eliminated
by filtration. The perfect initiation of cell suspension cultures depends
on several factors including plant growth regulators, medium nutrients,
biological factors and physical factors. The size of initial callus inoculum is
determined by the critical initial cell density which is the effective density
that maintains culture growth. In other words, it was the lowest inoculum
density per volume of medium at which a cell culture will grow. Dixon
and Gonzales (1994) assumed that 10% of initial cell density to the total
volume was necessary to initiate suspension culture. They further noted that
decreasing the inoculum size will affect the growth curve pattern, wherein
a small inoculum size leads to a longer lag phase (Dixon, 1985). While
increasing the inoculum size make cells enter the stationary phase earlier
TABLE 6.2 Examples of Secondary Metabolites Production from Callus Cultures.
Plant species Medium + PGRs Explants Light Culture Products Yield References
(mg/L) used period (d)
Ambrosia MS + 10 μM kin, 1 μM Leaf Continuous/ 35 Altamisine, 0.9 mg/L, 2.9 mg/L, Goleniowski and
tenuifolia 2–4, D dark Psilostachynolides B-C, 3.0 mg/L, DW Trippi (1999)
Psilostachynolides
Arachis MS + 4 NAA+ 1 BAP Stem Dark 30 Piceatannol, Resveratrol 5.31 µg/g, Ku et al. (2005)
hypogea 11.97 µg/g
Aronia MS + 2 NAA + 2 BA Bud Continuous 28 Phenolic acids 83.84 mg/100 DW Szopa and Ekiert
melanocarpa (2014)
A. annua MS + 0.5 NAA + 0.5 Seedling 16 h nd Artemisinin 96.8 mg/L Baldi and Dixit
Kin (2008)
Aspidosperma MS + 1 2,4-D + 1.5 Leaf/ 16 h 140 Ramiflorin 0.014% DW Olivira et al.
ramiflorum BAP Hypocotyl (2001)
A. indica MS+ 4 Indole-3-butyric Leaf Dark 42 Azadirachtin 0.0007% DW Allan et al.
acid (IBA)+ 1 BA (1994)
B. racemosa WPM+ 2.0 2,4-D Leaf 16 h 35 Lycopene 1.12 mg/g DW Behbahani et al.
(2011)
Capsicum MS + 2,4-D 2.0 + 0.5 Placenta nd 21 Capsiacin 1.6 mg/g FW Umamaheswari
annuum Kin and Lalitha
(2007)
Cassia senna MS + 16 µM BA Cotyledon 16 h nd Sennosides 0.13% Shrivastava et al.
(2006)
C. asiatica MS + 2,4-D 2.0 + 1.0 Leaf 16 h 21 Asiaticoside 0.88 mg/g DW Kiong et al.,
Kin (2005)
Coscinium MS + 2.0 2,4-D + 2.0 Petiole 12 h 35 Berberine 1.788% DW Khan et al.
fenestratum BAP (2008)

C. sativus B5+ IAA 2 + BA Corm Dark 21 Crocin 0.43 g/L Chen et al.
(2003)
Cynara MS+ 4.4 mm BA+ Leaf Dark 30 Cynarin, Chlorogenic 0.46 mg/g, Trajtemberg
cardunculus 16.1 mM NAA acid 0.26 mg/g DW et al. (2006)
Hypericum MS/B5 + BA 4.0 Apical 16 h 30 Hypericin, 20 μg/g, 180 μg/g Gadzovska et al.
perforatum segment Pseudohypericin DM (2005)
M. citrifolia MS+ NAA 2.5 µM Root 16 h 45 Anthraquinone 20.26 mg/g DW Sreeranjini and
Siril (2013)
Passiflora MS + 2.0 kin + 3.0 Leaf 16 h 30 Orientin 1163 μg/g DW Antognoni et al.
quadrangu- 2,4-D (2007)
laris
Pluchea MS + 1.0 NAA + 0.5 Leaf 16 h 42 Quercetin 0.23 mg/g DW Arya et al.
lanceolata BAP (2008)
P. lanceolata MS + 1.0 NAA + 0.5 Leaf 16 h 42 Quercetin 1.86 mg/g Arya and Patni
BAP (2013)
P. corylifolia MS + IAA 5.71 μM + Root continuous 280 Daidzein, Genistein 2.28%, 0.21% DW Shinde et al.
BAP 4.40 μM (2009)
Pueraria MS + 25 μmol/L 2,4-D Root 16 h 168 Daidzin, Genistein, 8.67, 3.42, 25.61, Matkowski
lobata + 0.5 μmol/L BA 3′-Methoxy-puerarin, 18.35 mg/100 g FW (2004)
Puerarin
Rheum ribes MS + 1.0 IBA + 1.0 BA Hypocotyl 16 h 42 Catechin 18.26 mg/100 g FW Farzami and
Ghorbanli
(2005)
S. officinalis MS + 0.1 NAA + 0.2 Hypocotyl Continuous 357 Carnosol, Rosmarinic 0.06 mg/g, Grzegorczyk
BA + 0.5 2, 4-D acid 15.77 mg/g DW et al. (2005)
Securinega SH+ 0.5 IAA+ 5.0 KIN nd 16 h 28 Securinine, 1.73 mg/g, Raj et al. (2015)
suffruticosa Allosecurinine 3.11 mg/g DW
S. striata MS+ 0.5 NAA + 2.0 Stem Dark 60 Acteoside 0.0016 mg/g FW Khanpour-
BA Ardestani et al.
(2015)
Solanum MS+ NAA 1 + Kin Stem, 16 h 56 Anthocyanin 70 μg/g FW Chaudhary and
melongena 0.25 Node, Mukhopadhyay
Leaf (2012)
Zataria B5 + BAP 0.75 Node 16 h 42 Rosmarininc acid 158.26 mg/g DW Francoise et al.
multiflora (2007)
Zingiber MS + 0.5 2,4-D+ 0.1 Shoot tip Dark 56 Gingerol 30 µg/100 mg FW El-Nabarawy
officinale BA et al. (2015)
PGRs: plant growth regulators, MS: Murashige and Skoog (1962), B5: Gamborg et al. (1968), WPM: Lloyd and McCown (1980), SH (Schenk and
Hildebrandt, 1972), Nd: not determined.
(Mustafa et al., 2011). Higher uptake rates of ammonium, nitrate, and sugars
were observed in the low inoculum-density (50 g FW/L) compared to the
high inoculum-density (100 g FW/L) of Catharanthus roseus cells cultures.
Moreover, the high-inoculum-density cultures produced higher ajmalicine
concentrations compared to low inoculum-density, while catharanthine
production was not affected by inoculum density (Lee and Shuler, 2000).
The physical factors include light, temperature, agitation speed, and culture
flask volume. Agitation conditions provide medium and cell mixing, aeration
and oxygenation to minimize hypoxia, and hence, maintain proliferation.
The high agitation speed (150 revolutions per minute (rpm)) induces
nicotine accumulation in tobacco culture, but when normal speed was
applied (110 rpm), it resulted in a decrease of nicotine synthesis (Alamgir,
2017). The size of the culture flask or bottle is determined by the volume
of the medium. The proportion of liquid-volume relative to the flask size is
essential for sufficient aeration which is considered to be about 20% of the
total volume of the flask (Dixon, 1985). Various types of vessels have been
used for shaking including conical flat-bottom round flasks, and bottle. For
aeration without shaking, various devices can be used which include roller
bottles, magnetic stirrers, and nipple flasks and tumble tubes. The envelope-
shaped culture vessels named “culture bag” and box-shaped named “culture
pack,” made of fluorocarbon polymer film were used for the production of
shikonin by L. erythrorhizon cell cultures (Fukui et al., 1999).
6.3.1.2.2 Maintenance of Cell Suspension Culture
Culturing of cell suspension in shaken flasks is usually named batch

culture; limited by the amount of medium or exhaust carbon source during
the culture period. Final biomass obtained is dependent on the quantity of
the nutrient. However, when the culture medium is replenished during the
culture, the culture system is named continuous culture. The continuous
culture is usually carried out in bioreactors, where a continuous collection of
cell biomass and replacement with the fresh medium are practicable.
The use of cell suspension cultures for secondary metabolite production
is based on the concept of biosynthetic totipotency, where each cell could
retain the complete genetic information for the production of a range of
compounds found in the parent plant (Rao and Ravishankar, 2002).
There are several factors affecting the maintenance of cell cultures such
as PGRs, nutrients, inoculum size, and media volume. Therefore, exten-
sive optimization studies in cultured plant cells are necessary to increase
the cell biomass and production of bioactive compounds. It was found

that an increase in the sucrose concentration in a culture medium could
result in an increase of secondary metabolite production (Collin, 2001).
Among the different sucrose concentrations studied, the high concentra-
tion (5%) resulted in maximum anthraquinone production in M. citrifolia
cell cultures (Sreeranjini and Siril, 2013). At an aggregate size of 250–500
μm, Centella asiatica cells gave the highest luteolin content compared to
<250 μM and 500–750 μM. Production of flavonoid was high with the
inoculum density of 0.3 g/25 mL compared to 0.1 g/25 mL and 0.5–1.5
g/25 mL (Tan et al., 2013).
A fine cell suspension culture can be obtained by filtering of cultures
through pipetting or sieving, DeLong flasks, and the addition of a low
concentration of pectinase (macerozyme) or cellulase. Mazarei et al. (2011)
obtained three types of aggregate on suspension culture of Panicum virgatum.
Filtering the initial suspension through a 210 µM mesh at 2-week intervals
resulted in an ultrafine type culture. Then the culture was allowed to settle for
a while prior to removing 6–8 mL of supernatant and replaced with 12–14 mL
of fresh medium at 10–14 days intervals. The feeding with the fresh medium
is sustained for about 2 months which resulted in a sandy type culture. Over
time, the sandy type cultures become aggregated and release fine cells giving
the supernatant a milky appearance, milky type culture. Pectinase can dissolve
the intercellular pectins by breaking up the aggregates (Dixon, 1985).
The cell suspension cultures have diverse advantages compared to
stationary cultures. 1) By growing in a liquid medium, each cell or aggregate
consistently share the contents of nutrients, aeration, and growth regulators.
In a callus culture, only cells in contact with the medium surface would
directly acquire nutrient. 2) Cell cultivation of a plant offers immense oppor-
tunity for multi-investigation of that plant at physiological, biochemical and
molecular levels. 3) Using continuous culture proffers the possibility of
sustaining the high producer cells to be vigorous and steady in production
for the desired period of time. 4) Cells are a source for selection of higher
producer lines, mutant induction, and protoplasts isolation in large numbers.
5) Cells are relatively fast in growth, dividing every 22–70 h depending
on the nutrient medium and may double within 15 h. 6) in solid media,
the phenolic compounds released by oxidase enzyme from the explant
when excised, will accumulate under the explants and cause browning on
the medium surface. These phytotoxic products prevent explants to grow
further, then turn necrotic and ultimately die. In liquid media, the phenolics
would wash away from the explant and be diluted.
There are many reports of successful production of secondary metabolites

in cell suspension culture. The first patent for the production of metabolites
by cell cultures was reported in 1956 by Pfizer Inc. (Smetanska, 2008). The
first industrial production of a compound by undifferentiated cell cultures of
L. erythrorhizon was shikonin in 1983 by Mitsui Petrochemical Industries
Ltd. (Fujita and Tabata, 1987). Cell suspension culture of Maclura pomifera
showed a high level of metabolite accumulation (0.91%) than stems
(0.26%), leaves (0.32%), and fruits (0.08%) of the parent plant (Monache
et al., 1995). The tormentic acid content of the cell suspension culture of
Eriobotrya japonica was determined to be 18.4 times greater than that of the
leaves (Li et al., 2017).
The analysis of cell growth that was maintained in a batch culture type
(finite medium) tends to develop a sigmoidal pattern of growth (S-shaped
curve) during a given period of time (Jha and Ghosh, 2005; George, 2008;
Santos et al., 2010). This kinetics growth contains five phases namely: (1)
A lag phase, in which cells prepare to divide beginning with the metabolite
mobilization, synthesis of proteins, and specific metabolites; (2) A period
of exponential growth in which the cell division rate increases; (3) Linear
growth in which the cell division is slow but the rate of the cell expansion
increases; (4) Deceleration phase, a period when the rate of cell division
decreases and cell expansion occurs; and (5) A stationary phase when growth
ends and culture becomes inviable, that is, no cell division or increase in
weight occurs.
Each phase of growth is separated from another according to the values
of different measurable growth parameters, such as cell number, total DNA
content and fresh/dry weights. Therefore, the total linked phases, when
plotted, develop the S-shaped growth curve. Similar patterns of growth
might occur in the organ cultures. During the period of stationary growth,
some differentiation of cells may occur in cell cultures but generally, it is
less complete than that which occurs in callus cultures (George, 2008). The
consequences of relatively slow growth are that cell suspensions require long
periods of culture before substantial amounts of biomass and products have
accumulated (Constabel and Tyler, 1994). Cultures cannot be maintained in
the stationary phase for long periods. Cells begin to die as the components
of the medium become exhausted. Subculturing often becomes urgent when
the density of cells, tissue or organs becomes excessive. However, high plant
density may be acceptable as a biotic stress factor that could improve yield
due to the high competition among plants for water and minerals (Yildis,
2012). Nevertheless, subculture is required to increase the volume of a
culture or to increase the weight/number of cells. Likewise, the firm closed
vessels used to maintain aseptic conditions for culturing plant material

leads to the accumulation of toxic metabolites and the exhaustion of the
medium, so subculturing is impressive (Varshney and Anis, 2014). Callus
or suspension cultures are subdivided into new explants for re-culturing on
fresh medium and are performed periodically. Consequently, the importance
of establishing calli and cell growth curve when working with medicinal
and aromatic plants not only helps to determine the correct time for media
exchange and subculturing but also the time of secondary metabolites
production (Santos et al., 2010).
The management of suspension cultures requires a high expenditure for
labor, space, and equipment. Alternatively, the growth of callus on the solid
medium has been appraised, principally when the depiction of large numbers
of individual cultures is required.
6.3.2 SECONDARY METABOLITES PRODUCTION BY ORGAN

CULTURES
Any in vitro or ex vitro plant organ can be used as explant to initiate an in

vitro organ culture. In vitro explants, for organ culture, can be obtained from
other culture types through embryogenic and organogenic redifferentiation,
or direct induction from the node. Roots and, to a lesser extent, shoot is the
most cultured organ for secondary metabolites production. Other organs such
as flowers have also been reported (Matkowski, 2008). Like undifferentiated
cells, when organs are cultured for the production of secondary metabolites,
the aim is to achieve maximum growth rate and biomass gain. Although
most of the valuable secondary metabolites are produced from cell and
callus cultures, important medicinal plants such as Atropa belladonna and
Digitalis lanata fail to accumulate considerable amounts of by-product
when cultured as undifferentiated cells (Gaosheng and Jingming, 2012).
Shoot culture of Aronia melanocarpa produced more phenolic acids than
both its callus culture and intact fruit (Szopa and Ekiert, 2014). Similarly,
saponin is specifically produced in the roots of P. ginseng, and hypericin
and hyperforin are accumulated only in the foliar glands of Hypericum
perforatum (Alamgir, 2017). In vitro differentiated cell cultures are more
convenient for the large-scale production of valuable phytochemicals
compared to undifferentiated cells cultures for several reasons. (1) Organ
cultures generally display a lower sensitivity to shear stress in a bioreactor
(Bourgaud et al., 2001). (2) Differentiated cultures are relatively more stable
in the production of secondary metabolites (Rao and Ravishankar, 2002).
(3) Shoots and roots are more predictable in their behavior (Parr, 1989).
(4) Organ cultures accumulate secondary products with concentrations
that are often analogous to those of the intact plants (Oksman-Caldentey
and Hiltunen, 1996). However, organ cultures in the bioreactor revealed
nonuniform growth of biomass (Bourgaud et al., 2001). Also, organ cultures
tend to grow slowly, and alteration of the yield and profile of a product is
not easy (Oksman-Caldentey and Hiltunen, 1996). Therefore, up to date, the
only commercial example of the use of plant organ cultures for secondary
metabolite production is the cultivation of ginseng roots (Filová, 2014).
6.3.2.1 ROOT CULTURE
Different compounds are reported to be only induced by root systems of

trees. Therefore, production of such compounds required cutting of the tree.
Roots are the medicinal parts used for the extraction of many important
medicinal plants such as P. ginseng, Panax notoginseng, Coptis chinensis,
and Salvia miltiorrhiza (Gaosheng and Jingming, 2012). Moreover, in some
manners, undifferentiated cells are unable to produce a target compound as
it is specific to only root cells. To establish the protocol for root culture
and production of secondary metabolites, there are three steps. Step 1 is
the induction of the adventitious roots. Adventitious roots can be obtained
from the leaf explant, cultivation of root explants, shoots or induced from
callus. Auxin supplement is required, mostly Indole-3-butyric acid (IBA) or
1-Naphthaleneacetic acid (NAA) and to a less extent IAA. MS and B5 media
are the most used medium cultures for induction and growth of roots. Step
2 is the culturing of adventitious roots in a liquid medium. After induction,
adventitious roots can be directly harvested, or for more biomass growth and
accumulation of phytochemicals, inoculated into liquid media and cultured
in shaking flask or bioreactors. When roots are cultured, the aim is to increase
production of secondary metabolites and this can be achieved by either raising
the concentration of the compounds or increasing the total biomass. Both
compounds and root biomass were affected by the medium contents, that is,
sucrose concentration, PGRs types and concentration, and nutrients, as well
as inoculum size and culture period (Table 6.3). For a bioreactor system,
to uphold a continuous culture, growth kinetics was required for medium
replacement or root harvest. While in the batch culture system, the medium is
finite, therefore, establishing a growth curve is imperative in identifying the
suitable day for subculture. Several studies reported that altering a medium
during roots cultures will enhance metabolite accumulation. For example,
TABLE 6.3 Examples of Secondary Metabolites Production from Root Culture.
(mg/L) used regime period (d)
Aloe vera MS+0.3 IBA Leaf Continuous 35 Aloe emodin, 4.42 μg/g, Lee et al. (2013)
Chrysophanol 63.65 μg/g
Andrographis MS+ 2.7 μM NAA Leaf Dark 28 Andrographolide 72.86 mg/g DW Praveen et al. (2009)
paniculata
Bupleurum B5 + 8 IBA Root Dark 56 Saikosaponin-a, 420 mg/L, Kusakari et al. (2000)
Falcatum Saikosaponin-b 350 mg/L
Cephaelis MS+ 1 IAA, 3 Leaf Dark 70 Emetine, cephaeline 0.514%, 1.319% Teshima et al. (1988)
ipecacuanha IAA DW
Datura metel B5 + 12 μM IAA Leaf 16 h 35 Hyoscyamine, 4.35 mg/g, Ajungla et al. (2009)
Scopolamine 0.28 mg/g DW
Echinacea ½ MS + 2.0 IBA Root Dark 28 Total phenol and 65.5 mg/g, Wu et al. (2009)
angustifolia flavonoids 39.2 mg/g DW
H. perforatum l/2 MS + 0.1 kin Root Dark 35 Chlorogenic acid, 0.97 mg/g, Cui et al. (2010)
+ IBA Hypericin 1.389 mg/g
M.citrifolia MS+ 5 IBA Leaf Dark 28 Anthraquinone 251.89 g/L DW Baque et al. (2011)
Panax ginseng SH + 5 IBA Root Dark 28 Ginsenosides 3.5 mg/g DW Marsik et al. (2014)
Peritassa WPM + 4.0 IBA Cotyledons Dark 7 Maytenin 972.11 μg/g DW Paz et al. (2013)
campestris
Plumbago B5 + 0.1 NAA Leaf Dark 24 Plumbagin 1.64 mg/g DW Jaisi et al. (2013)
indica
P. indica B5 + 0.1 NAA Leaf Dark 20 Plumbagin 12.5 mg/g DW Jaisi and
Panichayupakaranant
(2016)
Plumbago B5 + 1.0 NAA + Leaf 16 h 48 Plumbagin 0.129% DW Panichayupakaranant
rosea 0.1 kin and Tewtrakul (2002)
Polygonum MS+ IBA 9.84 μM Leaf Dark 28 Phenolics, 53.87 mg/g, Ho et al. (2018)
multiflorum Flavonoids 27.96 mg/g DW
Prunella MS+ 0.5 NAA Callus nd 49 Phenolics, 0.995 mg/g, Fazal et al. (2014)
vulgaris Flavonoids 6.615 mg/g-DW
P. corylifolia MS+ NAA 0.5 + Leaf 16 h 28 Psoralen 3.73 mg/ml Siva et al. (2015)
IAA 1.0 + IBA 1.5
Raphanus 1/2 MS + 0.5 IBA Root tip 14 h 28 Anthocyanin 250 mg/100 ml Betsui et al. (2004)
sativus
Rhus javanica LS+ 10 − 6 M IAA Leaf Dark 21 Galloylglucoses, 30 mg/g, Taniguchi et al.
Riccionidin A 0.32 mg/g FW (2000)
Talinum MS + 2.0 IBA Leaf Dark 28 Saponin 12.56 mm2/0.01 g Manuhara et al.
paniculatum DW (spot area) (2014)
Withania l/2 MS + 0.5 IBA Leaf 16 h 35 Withanolide-A 8.8 mg/g Praveen and Murthy
somnifera (2010)
PGRs: plant growth regulators, MS: Murashige and Skoog (1962), B5: Gamborg et al. (1968), WPM: Lloyd and McCown (1980), SH (Schenk
and Hildebrandt, 1972), nd: not determined.
root biomass and formation of tanshinone and protocatechuic aldehyde in S.

miltiorrhiza adventitious roots increased with the rise in sucrose to 60 g/L
in MS medium (Guo et al., 2005). In contrast, maximum production of
anthraquinone, phenolics, and flavonoids was achieved in the root culture of
M. citrifolia when sucrose was decreased to 10 g/L (Baque et al., 2011). The
source of nitrogen, that is, nitrate and ammonium nitrogen, in the medium
was reported to affect secondary metabolites accumulation. The maximum
secondary metabolite production was achieved with nitrate as the sole
nitrogen source (Guo et al., 2005). However, increasing NH4+:NO3− ratios to
10:20 in the medium lead to decrease in the pH, and interrupted the mineral
absorption which all resulted in low biomass in Eleutherococcus koreanum
culture (Lee and Paek, 2012). Culture period can affect the root growth as
with time, depletion of auxin and sucrose forms the medium and in the same
time, produce compound accumulated in the medium, which might hamper
root growth. Ishimaru and Shimomura (1995) reported that the concentration
of sangulin H-6 reached a maximum level at week 4 of root cultures of
Sanguisorba officinalis, and then decreased, while the amount of sanguiin
H-11 increased, presuming there is a biosynthetic conversion from sangulin
H-6 into sanguiin H-11. On the other hand, the root inoculum size must be
manipulated. Ho et al. (2018) studied the effect of inoculum density (3–15
g/L) and found that the 5 g/L density resulted in the highest root dry weight
and the highest concentration of bioactive compounds in the root culture
of Polygonum multiflorum. Table 6.3 shows some of these protocols. Step
3 is to enhance accumulation of metabolites with the addition of elicitors,
precursor feeding, or biotransformation.
6.3.2.2 SHOOT CULTURE
Shoot cultures (aerial parts) are usually used for the micropropagation of
medicinal plants. However, many metabolites can be produced directly in
shoot cultures of plant materials. For example, Artemisia annua did not
produce artemisinin in callus or hairy root cultures but produced only in
shoot cultures (Kim et al., 2002). Moreover, in vitro shoot multiplication
of Frangula alnus produced the highest anthraquinone content (Namdeo,
2007). Shoots once established, reveal genetic stability and high capability
for accumulation and production of secondary metabolite (Table 6.4).
However, the problem with shoot cultures is the requirement for exogenous
supply of plant growth regulators.
TABLE 6.4 Examples of Secondary Metabolites Production from Shoot Culture. 154
Plant species Medium + PGRs Explant Light Culture Products Yield References
A. MS + 0.5 NAA + 1 Bud Continuous 28 Hydroxybenzoic 50.66 mg/100 g, Szopa and
melanocarpa BA acid, Salicylic acid, 91.86 mg/100, Ekiert (2014)
Coumaric acid 54.44 mg/100 DW
A. indica 1/2MS + IBA 0.5 Embryo nd 28 Azadirachtin, Nimbin 0.008 mg/g, Srividya et al.
0.003 mg/g DW (1998)
H. perforatum MS/B5 + BA 1.0 Apical 16 h 30 Hypericin, 50 μg/g, 350 μg/g DM Gadzovska
segment Pseudohypericin et al. (2005)
H. perforatum LS + 0.1 NAA + 0.1 Seed Continuous 21 Neochlorogenic acid, 118.81 mg/100 g, Kwiecien
BA 3,4-Dihydroxyphenyl- 129.29 mg/100 g DW ́et al. (2015)
acetic acid,
Myristica MS + 26.85 Callus (leaf) Continuous 35 Myristin, methyl 4.33%, 84.62% Indira Iyer
fragrans NAA + 4.44 BA eugenol et al. (2009)
Ophiorrhiza MS + 5 BA + 0.5 Leaf 12 h 60 Camptothecin 0.065% Vineesh et al.
rugosa NAA (2007)
P. corylifolia MS + 8 µM TDZ Cotyledonary 16 h 28 Daidzein, Genistein 1.23%, 0.38% DW Shinde et al.
nodes (2009)
Rehmannia MS + 1.0 BAP + 0.1 Callus 16 h 35 Catalpol 45 mg/g DW Piątczak et al.
glutinosa IAA (hypocotyl) (2015)
S. officinalis MS + IAA 0.1 + BA Shoot tip Continuous 420 Carnosic acid, Carno- 4.77 mg/g, 0.63 mg/g, Grzegorczyk
0.45 sol, Rosmarinic acid 16.3 mg/g DW et al. (2005)
S. suffruticosa HM + 0.3 mg/L Callus 16 h 28 Securinine, 6.02 mg/g, 3.7 mg/g Raj et al.
NAA + 3. 0 mg/L (cotyledon) Allosecurinine DW (2015)
2iP + 1.0 mg/L BA
Thymus MS + BA4 Seed 16 h 30 Flavonoids 0.64 mg/g FW Karalija and
vulgaris Parić (2011)
PGRs: plant growth regulators, MS: Murashige and Skoog (1962), B5: Gamborg et al. (1968), LS: Linsmaier and Skoog (1965), HM: Huang and
Murashige (1976), Nd: not determined.
6.3.3 SECONDARY METABOLITE PRODUCTION BY

TRANSGENIC ORGANS
Plant cell cultures are the most popular types of in vitro techniques that have
been investigated because they are easier to manipulate especially in a biore-
actor. But the plant cell cultures are subject to somaclonal variations which
may result in the loss of productivity with culture age. Also, many cultured
undifferentiated cells did not produce secondary metabolites. The ordinary
root of many medicinal plants is basically the source of bioactive ingredi-
ents, but generally, roots exhibit slower growth than cultures of plant cells
and are difficult to harvest. Although organ cultures are reported to produce
valuable compounds, they are still hormone-dependent. To overcome this
problem, several alternatives were explored which resulted in the use of
organized or semiorganized tissue of hairy roots, shooty teratomas or crown
galls cultures. These transformed organs have been obtained by genetic
transformation with Agrobacterium rhizogenes or a mutated Agrobacterium
tumefaciens. Production of secondary metabolites from transformed tissue
was reported to be high yielding, stable, and promising.
6.3.3.1 HAIRY ROOT CULTURE
Hairy root cultures can be obtained from various host plants but mainly
from dicotyledonous. The part that can be infected includes leaf, other
organs or even protoplasts. Typically, culturing root explants involves the
exogenous supply of phytohormone with a very slow growth, resulting in
the poor synthesis of secondary metabolite (Rao and Ravishankar, 2002).
The increase of biomass of hairy roots is resultant of the rate of elongation,
lateral branching, and diameter thickening of roots (Oksman-Caldentey and
Hiltunen, 1996).
There are several advantages of using hairy root culture for production
of secondary metabolites. The hairy root phenotype is characterized by
(1) hormone-independence, (2) fast growth (0.1–2.0 g dry weight/L/day)
as unorganized cell suspension, (3) absence of geotropism, (4) extensive
root branching, (5) genetically and biochemically stable, (6) expression of
specific metabolic pathways as normal roots with similar or higher yields,
(7) capability of transforming inert xenobiotics into bioactive metabolites,
(8) maintaining the stability of yields, (9) the possibility of clone selection
for high-yielding stable hairy root lines, and (10) like adventitious roots,
hairy roots secrete metabolites into the liquid medium, making it easy for
collection (Flores et al., 1993; Oksman-Caldentey and Hiltunen, 1996;

Anand, 2010; Alamgir, 2017). Some hairy roots, when exposed to light, turn
green and can be grown photoautotrophically, and these photoautotrophic
roots show much higher levels of total alkaloids than the dark-grown roots
(Flores et al., 1993).
Culturing of hairy roots in bioreactors showed some disadvantages asso-
ciated with medium agitation such as sensitivity to shear stress or related
to space as hairy roots grow actively hence occupy full space in the vessel.
Bourgaud et al. (2001) described different techniques to overcome these
problems such as to protect the roots from agitation by using screens or wire
meshes, and immobilization of hairy roots into a polymer matrix. Moreover,
secondary metabolites produced by hairy roots are limited to those synthe-
sized in roots of the intact plants (Oksman-Caldentey and Hiltunen, 1996).
Table 6.5 is an example of some secondary metabolites produced from the
hairy root and crown gall cultures.
6.3.3.2 CROWN GALL AND SHOOTY TERATOMA CULTURES
The insertion of a tumor-inducing (Ti) plasmid by A. tumefaciens into a plant

causes crown gall disease to appear as unorganized tumor at the part of the
infection. Transfer DNA (T-DNA) encodes the enzyme that regulates the
plant growth hormones (i.e., auxins and cytokinins) and has been detected
in the transformed tissues. Endogenous phytohormones produced can affect
the secondary pathway, therefore, crown gall tissue has been used for the
production of various phytochemicals.
Different types of A. tumefaciens strains (e.g. wild-type or mutant
strains Ti plasmids) used outcomes in different hormonal imbalance after
infection. Consequently, the resultant morphology of crown gall tumors is
formed either by amorphous callus or teratomas containing organized stem
and leaf-like structures, the so-called shooty teratomas (Floryanowicz-
Czekalska and Wysokinska, 2000). The shooty teratomas are formed due to
overexpression of the cytokinin biosynthetic gene, that is, decrease in the
auxin/cytokinin ratio in the transformed crown gall tissue leading to leaf and
shoot formation. Shooty teratoma induction is highly desired as it provides
a possibility to culture shoots independent of hormones for production of
phytochemicals that are synthesized only in the aerial parts of plants. The
number of plant species forming stable fully developed shooty teratomas
is limited. The proper establishment of shooty teratomas requires several
demands such as shoots that must be fully differentiated, rootless and callus
TABLE 6.5 Some Secondary Metabolites Produced from Hairy Root and Crown Galls Cultures.
Plant species Medium Explant Culture type Light Culture Products Yield References
regime period (d)
A. indica MS Leaf, Stem Hairy root Dark 30 Azadirachtin 3.6 μg/g, Sundaram and
2.7 μg/g DW Curry (1996)
Calendula ½ MS Cotyledon, Hairy root Dark 30 Oleanolic acid 8.42 mg/g DW Długosz et al.
officinalis Hypocotyls (2013)
Cannabis B5 Callus Hairy root Dark 35 Cannabinoid 2.0 μg/g DW Farag and Kayser
sativa (2015)
Coleus B5 Leaf Hairy root Dark 84 Forskolin 2.36 mg/g DW Pandey et al.
forskohlii (2014)
Datura ½ B5 nd Hairy root Dark 33 Hyoscyamine 211.2 mg/L Hilton and Rhodes
stramonium (1993)
Portulaca ½ MS Cotyledon Hairy root 16 h 28 Dopamine 1.21 mg/g Moghadam et al.
Oleracea (2014)
T. MS Leaf Hairy root Dark 14 Saponin 71 365 mg/L/g DW Manuhara et al.
paniculatum (2015)
Valeriana MS Leaf Hairy root Dark 48 Valerenic acid 3.02 mg/g DW Torkamani et al.
officinalis (2014)
Salvia 6,7-V Node (Teratoma) cell Dark 16 Tanshinone 2.22 mg/250 mL Chen et al. (1997)
miltiorrhiza suspension
S. 6,7-V Node (Teratoma) cell Dark 12 Cryptotanshinone, 150 mg/L, 20 mg/L, Chen and Chen
miltiorrhiza suspension Tanshinone I, Tanshinone 50 mg/L, 530 mg/L, (1999)
IIA, Rosmarinic acid, 216 mg/L
Lithospermic acid B
PGRs: plant growth regulators, MS: Murashige and Skoog (1962), B5: Gamborg et al. (1968), 6,7-V: Veliky and Martin (1970), Nd: not determined.
free, and capable of growing in a liquid medium without browning, callusing

or hyperhydricity (Subroto et al., 1996). Shooty teratomas can produce
typical shoot-derived metabolites such as mint oil from Mentha citrata
(Spencer et al., 2009) as well as biotransformation of root-derived metabo-
lites such as nicotine and hyoscyamine (Saito, 1993). The shooty teratomas
of C. roseus epicotyls and stem nodal explants displayed a 10-fold yield of
vincristine over the untransformed cultures (Begum et al., 2009). The main
advantage of shooty teratoma cultures over untransformed shoots cultures
is that exogenous growth regulators are not required (Subroto et al., 1996).
The co-culture of shooty teratomas and hairy roots of A. belladonna in a
hormone-free medium produced scopolamine with an amount 3–11 times
higher than that produced by the leaves of the intact plant (Floryanowicz-
Czekalska and Wysokinska, 2000).
6.3.4 BIOREACTOR FOR PRODUCTION OF SECONDARY

METABOLITES
The bioreactor is a physical/thermal system for the maintenance of cells in

the best culture conditions for a fast growth (Ruffoni et al., 2010). It helps:
(1) provide a pilot or large-scale production of biochemicals compared to
shake flasks. Bioreactors vary considerably in the volume size which ranged
between 1 and 5 L lab scales to 75,000 L industrial scale. The production of
secondary metabolites in large bioreactors is preferred over the ordinary in
vitro cultures for several reasons. (2) Bioreactor culture offers the benefits of
using a liquid medium compared to static cultures. (3) Automatic controlling
of the whole production process including optimizing the medium conditions,
such as temperature, pH, dissolved oxygen, carbon dioxide, carbohydrates,
and mineral nutrients. 4) Online measurements of the growth of the culture.
6.3.4.1 BIOREACTOR TYPES
A wide type of bioreactors has been designed so that they can fit the different
types of cultures and can be grouped into three types depending on the
agitation way; mechanically-agitated bioreactors, pneumatically-driven
bioreactors, and non-agitated bioreactors. Mechanical bioreactors or stirred
tank has a mechanical device for stirring medium, including a turbine impeller,
helical ribbon impeller, and vibrating perforated plates. Wave reactors are
mechanically agitated bioreactors, although they have no direct impeller
agitation, they provide a wave motion within the liquid medium that is
caused by swinging of the vessel (Georgiev et al., 2009; Ruffoni et al., 2010).
Pneumatic bioreactors are vessels lacking a stirring device and agitation of
the medium is put to function by air flow. These types of bioreactors can be
alienated, depending on the means of providing the airflow, into two kinds,
airlift and bubble column reactors. In airlift bioreactor, the medium is agitated
and aerated by the introduction of air through the top of the column. While
in the bubbling reactor the medium is agitated and aerated by introducing
air from the bottom of the column. Airlift and bubble column reactors have
been utilized for the cultivation of photoautotrophic or photomixotrophic cell
suspension cultures (Georgiev et al., 2009).
Non-agitated bioreactors were considered the third group of bioreactors
although they lack a means of agitation. However, the air is also mixed with
media prior delivering to the cultures. These include temporary immersion
and nutrient mist bioreactors. In temporary immersion bioreactors, the media
is pumped to the culture section and kept for a short time, and then returned
to the storage tank. In nutrient mist bioreactors, the sterilized mix of air and
media is sprayed above the surface of cultures.
The numerous advantages that have been shown by the different biore-
actor designs can be used as a guide to choose the type that fits the desired
kind and purpose of the in vitro cultures. Helical ribbon impeller was found
to be efficient for mass transfer of medium and gasses, and less damaging to
cells than other used impellers (Smith, 1995). Rotary drum reactors are char-
acterized by sustained suspension homogeneity, low shear stress, and higher
oxygen transfer ability (Chattopadhyay et al., 2002). Stirred tank bioreactors
are commonly used due to large-scale production, use with highly viscous
cultures, high oxygen allocation and good culture mixing (Yue et al., 2014).
Bubble column bioreactors are typified by low capital and operational costs,
low shear stress and uncomplicated system (Ruffoni et al., 2010) due to the
absence of stirring part.
Collectively, the main considerations for choosing a bioreactor should
be adequate oxygen availability, low shear stress to cells, adequate nutrient
supply, and product removal from cells (Yue et al., 2014).
Several modifications and enhancements have been applied to the
bioreactors for better performance such as minimum shear stress, good aera-
tion and adapted impeller blades to ensure no damage to cultures. A new
agitated bioreactor named the centrifugal impeller has been developed for
shear-sensitive in vitro cultures in which a conventional vessel is agitated
by a centrifugal-pump-like impeller (Georgiev et al., 2009). To improve the
homogenization of culture medium, an airlift mesh-draught reactor with
wire helixes was designed for large-scale hairy root culture of Solanum
chrysotrichum (Ruffoni et al., 2010). Increasing blade size has been found to
reduce shear stirred-tank bioreactor. The accomplishment of the optimization
of a bioreactor system for a type of culture indicates that the production of
secondary metabolites is ready for scaling up to a commercial level (Smith,
1995). Cells of Podophyllum hexandrum, when cultured in a 3 L stirred
tank bioreactor, showed an increase of 27% in productivity compared to
shake flask culture (Chattopadhyay et al., 2002). Digitalis purpurea cell line
cultured in airlift bioreactors improved the yield of digitoxin up to 430 mg/L
(Gaosheng and Jingming, 2012).
6.4 ANALYSIS OF CULTURE GROWTH
For the production of secondary metabolites using in vitro cultures, the

measurement of growth parameters is required. Such measurements are
imperative to verify what is needed for the optimization of media contents,
cultural conditions or other components, to maintain a high growth rate and/or
yields. In addition, bioassays of plant growth hormones and comparisons of
genotype performance can be performed by evaluating the growth character-
istics. The detailed analysis of the biochemistry of plant in vitro cultures will
certainly allow for improved manipulation of natural product biosynthesis.
These measurements involve a growth curve for the cultures by plotting
the measured values. When the inoculum material is cultured in the medium,
there is a lag period preceding growth then followed by exponential and
linear growth continuing until a gradual deceleration is reached and finally,
the culture enters a stationary phase. This pattern of growth is the so-called
S-shaped curve.
Measuring growth parameters of batch cultures is imperative to ensure
the reproducibility of production for experiments both in the laboratory and
on industrial scales. Based on different parameters, the culture growth can be
measured by several methods. These methods can be grouped into catego-
ries depending on the way of sampling or measuring a culture, destructive
methods, and nondestructive methods.
6.4.1 DESTRUCTIVE METHODS
These methods involve harvesting of whole or samples of the biomass so

sacrificing the cells. This includes various procedures such as measuring
the fresh and dry weights (DWs), cell number, cell viability, and protein
estimation (Dixon, 1985; Schripsema et al., 1990; Dixon and Gonzales,
1994; Doležel et al., 2007; Mustafa et al., 2011).
6.4.1.1 FRESH WEIGHT (FW) AND DRY WEIGHT (DW)
Fresh weight (FW) and DW are the most common parameters that are
usually measured to monitor the growth of cells per volume cultured.
The measurement of FW is less accurate because of variations in the
adhering water (Schripsema et al., 1990). Measurement of DW is most
frequently used because it is considered to be more precise. DW is
usually used for the preparation of a growth curve, target compound
yield, enzyme activity curve, and gene expression curve (Gaosheng and
Jingming, 2012).
6.4.1.1.1 Organ and Callus Cultures
For FW, the biomass harvested from the culture ensuring no medium is
attached, is placed on a pre-weighed piece of aluminum foil. When the culture
is harvested, the weight must be acquired immediately to reduce variations
caused by water evaporation. To obtain DW, there are different methods
to dry the biomass harvested. (1) The biomass can be air-dried under lab
conditions (25–30°C) by exposing the samples to fresh air until complete
evaporation of the water content. This method is suitable for small biomass
and it takes a long time. (2) Heating in a hot air oven at 40–60°C for 12–24 h
is also reported for thermostable compounds. This is followed by placing
the sample in a desiccator until cooling (15–20 min) and then recording the
DW. The process needs to be repeated until constant weight is reached. 3)
Drying in a freeze dryer is preferred because it is quick, and to guarantee
that no compound will be affected by heating as in the oven method, or due
to microorganism contamination of the cells during the air-drying method.
Freeze-drying retains higher levels of phenolics content in plant samples
than in air-drying (Liu et al., 2008).
Monitoring root growth (normal adventitious or hairy roots) can be
invasive if precise measurements are required. After harvesting, root
numbers (primary and laterals), root length (primary and laterals), total
length (primary + laterals), and root growth unit (cm per root tips) are
obtainable.
6.4.1.1.2 Cell Culture
For acquiring the FW of cell suspension, an appropriate sample volume of

cells is collected on a pre-weighed Whatman filter paper or a nylon fabric
placed in a funnel. The cell is then washed with sterile distilled water to
remove the medium. After draining under vacuum, the cells with the filter are
reweighed immediately before the cell loses fluid. For DW measurement, the
cells and the nylon together with the cells are dried at 45–80°C for 12–24 h,
which are then cooled in a desiccator and weighed to a constant weight (e.g.
10% moisture). The FW and DW can be expressed as per milliliter culture.
The obtained FW and DW values can be used to calculate the growth
efficiency indices such as growth index, relative DW, FW increment, growth
rate, root-growth ratio, growth ratio, and biomass doubling time. These
growth indices can be used to measure the growth of all types of cultures
but mainly they are used for callus and root cultures because they require
tedious work in weighing and calculations. Some of the most used equations
for calculation of growth indices are provided.
1. Growth index % (Gi) = (G1 – G0)/(G0) × 100

where G1 is the FW after specified cultivation period, and G0 is the
FW of the inoculum.
2. The relative DW% (RDW) = DW/FW × 100
3. FW increment (FWi) = FW1 – FW0
where fw1 is the final FWafter every cycle per time and fw0 is the
initial FWat the beginning of each cycle.
4. Growth rate (Gr) = (W1/W0) -1 per unit time
where W1 is the final weight, W0 is the inoculum weight.
5. Average growth rate (AGR) = ln Wf – ln Wi/t
where ln is Naperian (natural) logarithm, Wf = final weight of the
fresh matter, Wi is inoculum weight, t is specific cultivation period.
6. Specific growth rate (μ) = In Xt – In X0/t
where X0 is the initial cell density, Xt is the cell density at time t.
7. Growth ratio (GR) = DWh - DWi/DWi
where DWh is the harvested DW, DWi is the inoculated DW.
8. Doubling time growth (Dt) = ln2/m
where ln2 is a natural logarithm, m is slope of the line.
9. Callus induction frequency (Cif) = PN/TN × 100
where PN is the number of explants producing callus, TN is the total
number of cultured explants.
10. Callus index (CI) =100n × G/N

where n = number of explants initiating callus, G is visual callus
rating of initiated explants, N = total number of explants planted.
Visual callus rating: 1 = 25%, 2 = 50%, 3 = 75%, 4 = 100%
11. Secondary metabolites yield (SMY) = DW (g/L) × content (mg/g
DW).
After the biosynthesis of target compounds, it might be accumulated in

the cells (intracellular) or secreted into liquid media (extracellular) and in
some cases, in both. To determine the yield of a compound, both contents
should be summed to represent the producing ability.
In addition to the abovementioned weight-dependent parameters, cell
suspensions have special measurements of growth reported in various
research works (Hahlbrock, 1975; Sung, 1976; Dixon, 1985; David et al.,
1989; Dixon and Gonzales, 1994; Mustafa et al., 2011). These methods
include packed cell volume, cell counting, or cellular protein content. These
techniques involve tedious preparation of the cells.
6.4.1.1.3 Packed Cell Volume
Cell division leads to a rise in the number of cells which cause an increase
in the proportion of cells per milliliter of suspension and hence can be used
to estimate growth of the cell in cultures. For estimating PCV, a sample (e.g.
10 mL) of uniformly dispersed suspension culture needs to be transferred
into graduated centrifuge tubes (e.g. 15 mL) and centrifuged at 2000 rpm for
5–10 min. The measurement is conducted after cells are allowed to completely
sediment in the tube. PCV was normally expressed as a percentage of the
compacted volume of the cell pellet to the total culture volume. PCV can
be considered as a partly-distractive method because it involves sacrificing
only a sample of the culture.
6.4.1.1.4 Cell Viability
Throughout the period of culture, cell death may occur in suspension cultures
because of, for example, the exhaust of medium and accumulation of toxic
substances. Determining the viability of cells before cell counting is very
important to ensure that data on the number of cells is correct. Cell viability
can be carried out by the examination of protoplasmic streaming and the
presence of an intact nucleus under a microscope. Cell viability can be

determined by stains such as Evans methylene blue (10%). Nonviable cells
become blue in color, while viable cells turn colorless. Cell viability may
also be analyzed by other methods such as fluorescein diacetate, Tetrazolium
test (2,3,5-triphenyl tetrazolium chloride), 3‐[4,5‐dimethylthiazol-2yl]-
2,5-diphenyl tetrazolium bromide, and the mitotic index depending on the
activity of reductase enzyme. The percentage of viability is usually recorded
by taking 10 mL of sample. The measurement of cell concentration by cell
counting has also shown a reasonably good correlation with DW parameter.
6.4.1.1.5 Number of Cells
The increase in the number of cells due to cell division is a measurable indi-
cator of growth through the culture period. However, this can be performed
only with fine cell suspension cultures. Cell suspension cultures containing
large cell aggregates, therefore, must be broken down into individual cell
components before cell counting. Several treatments are used for the disso-
ciation of aggregates into individual cells. The most reported is digesting the
suspension with chromic acid (Cr2O3 0.5H2O) or chromium trioxide (CrO3).
A sample of the cell suspension (e.g. 1 mL) is added to a solution (e.g.
2 mL) of 2.5% chromic acid or 8% chromium trioxide heated to 60–70°C
for 5–15 min. The mixture is cooled and vigorously shaken for 15 min for
effective cell separation.
Hydrolytic enzymes such as cellulase and pectinase are also reported.
The enzyme method can be applied by mixing 1 mL of the cell suspension
with 0.5 mL of 10% cellulase and 0.5 mL of 5% pectinase. The mixture is
then incubated for 30 min at 25°C with rotatory agitation at 100 rpm.
Then the suspension sample is dispersed with hypodermic needles on a
hemocytometer slide. The cell count is measured under a microscope using
a cell counter. Cell counting chamber such as the Sedgewick Rafter cell
or the Neubauer chamber can be used. A sample with fixed volume (e.g.
10 μL) of the suspension is spread over a defined area (e.g. 10 squares).
The number of cells counted within the 10 squares represents the number
of cells in 10 μL. By calculation, cell density is easy to determine per whole
volume of suspension, that is, multiply by 100 for density per milliliter.
The cell number is usually comparable to the DW, while the PCV is usually
comparable to theFW.
Cell viability, by the exclusion of vital stains, can be performed in the same
sample after using enzymes because cells stay viable. Although chromium
trioxide method is quicker and less complicated than the use of enzymes, cell
viability cannot be estimated in the same sample due to cell death.
6.4.1.1.6 Nutrients and metabolite concentrations
Some nutrients/metabolites in the cell suspension culture medium show

correlation with growth in a single culture flask. For example, the total
nitrate and sugar levels in the medium can be used to measure growth.
6.4.1.1.7 Protein content
Protein content can be obtained by extraction of the whole harvested cells

or in a sample of the culture. Cells collected on a Whatman glass fiber filter
are washed twice with boiling 70% ethanol. Acetone is used to dry the mate-
rial prior to the transfer to a solution of 1M NaOH followed by heating at
85°C for 1 h. Then hydrolyzed protein is determined in the filtrate. The total
protein content is expressed per gram of cells. In general, the protein content
per culture increases with the increase in growth, therefore showing the least
values at the stationary phase.
6.4.2 NONDESTRUCTIVE METHODS
These methods include different procedures used to evaluate culture growth

which allows quick estimations while maintaining sterile conditions. These
measurements include several methods such as the culture optical density,
residual electrical conductivity, and pH of the medium (Dixon, 1985;
Schripsema et al., 1990; Dixon and Gonzales, 1994; Doležel et al., 2007;
Mustafa et al., 2011).
6.4.2.1 CELL GROWTH MEASUREMENTS
6.4.2.1.1 Cell Volume after Sedimentation
Cell volume after sedimentation (CVS) involves the culture of cell suspension
in 250 mL Erlenmeyer flasks. For performing the measurement, a CVS
device was designed by Blom et al. (1992) to hold the 250 mL Erlenmeyer
flask kept at an angle of 60°. Then the suspension is allowed to settle for at
least 5 min. The height of the cell suspension from the bottom of the flask
in the 60° position is measured to represent the volume of cells. Difficult to
settle cells of fine and thick consistency of cell. According to Mustafa et al.
(2011), the measurement of suspension cultures with volumes lower than
50 ml is less accurate because of the shape of the Erlenmeyer flask.
6.4.2.1.2 Settled Cell Volume
The total size of cells per milliliter of suspension is a result of the increase in
the number of cells by cell division. Therefore, it can be used to estimate the
growth of the cell in suspension cultures. For determination of SCV, the cells
are transferred to 50 mL Falcon tubes and allowed to settle for 30 min. Then
the volume of the tube which was occupied by the whole suspension was
measured as SCV. To ensure that the measured value is accurate, a second
reading after another 30 min can be done. If the variation between the two
readings is higher than 5%, a third measurement is favored. SCV is usually
comparable to the FW.
6.4.2.1.3 Dissimilation Curve
The dissimilation of sugars by cell culture causes a loss of weight of the

contents of the culture flask. The value obtained can be used to follow the
growth in a single culture flask. The important precaution is that culture
flasks are closed firmly to ensure no evaporation occurs. To obtain the
dissimilation curves, the cumulative loss of weight for every flask will be
calculated. By weighing one culture flask regularly (during and after the
experiment), information can be obtained about the different growth phases,
for example, the length of the lag time, the doubling time, the moment the
stationary phase is reached, the biomass yield and the amount of intracel-
lularly stored carbohydrates. The dissimilation curves can be correlated to
the concentration of sugars in the medium, the DW, and the FW.
6.4.2.1.4 Sidearm-Turbidity Method
Monitoring growth by measurement of turbidity of liquid cultures has been

reported. The method involves measuring the turbidity of cultures grown in
sidearm flasks. Sidewise tilting of the flask fills the sidearm so that turbidity can
be read thoroughly in a photoelectric colorimeter with a blue filter at 400–465 nm.

The turbidimetric measurement of cultures grown in sidearm flasks is a quick,
easy and routine way of recording plant cell growth in batch cultures. The less
accuracy in the measurement of cell concentration by turbidity (optical density)
is because of the large variations in the cell size and cell aggregation. However,
it has shown a reasonably good correlation with DW.
6.4.2.1.5 Electric Conductivity
The electric conductivity of the medium decreases with increase in the

culture growth due to ion uptake by cells; that is, nitrate concentration. The
measurement of this decrease is useful for the assessment of cell growth. This
method offers several advantages over other methods, such as (1) continuous
and online monitoring of cell growth, (2) no sampling is required, (3) it is
economical and efficient, (4) it provides an accurate, reliable, and reproduc-
ible measurement of plant cell growth rate, and (5) it is independent of cell
aggregation, growth morphology, and apparent viscosity.
The medium conductivity can give an impression of growth in a certain
culture but is unsuitable for comparison of different cultures.
6.4.2.2 CALLUS FW
All the mentioned equations and methods provided above for callus growth
measurements are destructive sampling methods, and in addition, require
frequent handling of the calli. Therefore, to overcome these disadvantages,
other methods were widely used including qualitative/quantitative index
(McLean et al., 1992), concentric circles (Fowler and Janick, 1974), and
surface area or volume of the callus as a basis for growth assessment
(Mottley and Keen, 1987). Other methods for callus growth estimation were
also reported using calculated quantities of the average callus diameter, the
elliptical surface and the circular surface, determined from the measured
linear callus dimensions (Berardi et al., 1993).
6.4.2.2.1 Qualitative and Quantitative Index
The callus growth and appearance were rated using numerical values 0, 1, 2,
3, and 4, which represent dead, poor, fair, good, and excellent, respectively
(Pua et al., 1985). A wide range of numerical values can also be used with
a rating scale from 0 up to 9. For example, 0 = no tissue growth, 1 = initial
callus growth from stem ends, 2 = callus arising from one stem end, up to
9 = callus growth 4 times the originally estimated mass.
Also, the score for callus induction as illustrated by Matkowski (2004)
can be used such as: – no callus and poor growth, + good induction but poor
growth, ++ good initiation and moderate growth, +++ best induction and
vigorous growth.
6.4.2.2.2 The Concentric Circles
This method involves using a sequence of concentric circles of various sizes

with a shared center. The concentric circles are drawn on transparent sheets,
therefore, can be put, for example, on a Petri dish containing the callus
to estimate its diameter. Then the correlation coefficient can be obtained
between FW and the diameter. The disadvantage of this method is that calli
always vary in shape which is irregular.
6.4.2.2.3 Callus Area Method
Different nondestructive methods based on the measurements of the callus

area were also reported. These are point-counting method, electronic
planimeter, callus standard width and callus greatest width.
The point-counting method can be done by placing callus pieces on the
surface of 36 plates of NH4-S-RMOP agar medium and pressed slightly into
the medium to obtain a satisfactory contact. This is important to prevent the
calli from becoming disconnected from the medium surface when taking size
measurements. Point counts were obtained by placing a transparent overlay
at random on the base of the Petri dish. The overlay had points traced on
its surface, arranged in a square pattern at intervals of 5.0, 2.5 or 1.0 mm.
A minimum of six points per callus area were used in each case. Only those
points with their centers exactly on or inside the edge of each callus area
were counted as “in” and the areas of individual calli were calculated using
the following formula:
A = N × D2
where A = calculated area, N = no. of “in” points, D = distance between the
points.
6.4.2.2.4 Greatest and Standard Widths
Greatest width is obtained using a ruler to measure the distance between the
two farthest points on each callus surface at a given time. Standard width
was obtained using a ruler to measure the length of a line drawn at random
through each callus surface on the base of each Petri dish at the beginning
of the experiment.
6.4.2.2.5 Electronic Planimeter
For electronic planimeter readings, the callus borders are first carefully
traced onto a plastic transparency. Planimeter area readings are obtained by
tracing along the borders of each callus. Areas were automatically calculated
and displayed.
6.4.2.2.6 Linear Callus Dimensions
From above the Petri dish cover surface, two linear callus dimensions of,
the maximum diameter (MD) and the greatest length at the right angle (PD).
MD and PD are used to calculate the average callus diameter, callus surface
as an ellipse and as a circle.
6.4.2.3 ORGAN CULTURES GROWTH MEASUREMENTS
The growth of shoot and root in cultures can be monitored by special param-
eters. Such measurements can be conducted from out of the culture flask
without losing sterilization. Growth in shoot culture is usually measured
mainly using parameters such as the number of shoots, shoot length, and
so forth. Similarly, root growth (normal adventitious or hairy roots) can be
monitored by estimation of root numbers (primary and laterals), root length
(primary and laterals), total length (primary + laterals) and the morphological
features such as thickness and secondary root formation.
6.5 EXTRACTION AND ANALYSIS OF SECONDARY METABOLITES
The extraction of bioactive compounds from plant materials is the first step
in the utilization of phytochemicals (Dai and Mumper, 2010). Preparation of
plant sample in some cases is important. For example, the flavonoid quercetin
can be obtained from the extraction of the quercetin glycosides followed by
hydrolysis to release the aglycone and subsequent purification (Harwood
et al., 2007). Secondary metabolites can be extracted from fresh, frozen or
dried plant samples. Usually, before extraction, plant samples are preferred
to be firstly air-dried or freeze-dried. The dried materials are then treated by
milling, grinding, and homogenization. Usually, the traditional techniques
such as Soxhlet, maceration, reflux, and hydro-distillation, which have been
used for decades, form the first choice for extraction of phytochemicals.
There are a number of factors that influence extraction of compounds from
the plant matrix such as the chemical nature of the solvent, the sample to
solvent ratio, sample particle size, disruption techniques, temperature as well
as the time of exposure. For example, maceration with alcoholic solvents
and plant solvent ratio of 1.5:10 can yield a greater level of quercetin (Nobre
et al., 2005). Solvent extractions with methanol (particularly), ethanol,
acetone, ethyl acetate, often with different proportions of water, are the most
commonly used procedures of plant extracts. Weak organic acids, such as
formic acid, acetic acid, citric acid, tartaric acid and phosphoric acid, and
low concentrations of strong acids, such as trifluoroacetic acid and hydro-
chloric acid are recommended to minimize peak tailing (Dai and Mumper,
2010). After homogenization and selection of solvent, the extraction should
be performed at temperatures that do not permit degradation of compounds
of interest. This is done simply by allowing mixtures to macerate for a time
(24–48 h) so that the solvent can penetrate all parts of the ruptured cells and
solubilize compounds with similar polarity.
For accurate measurements and reliable quantitative determination of the
phytochemical contents in raw plant materials, chromatographic methods
with appropriate detection are commonly used. High-performance liquid
chromatography (HPLC) currently represents the most popular and reliable
technique for analysis of compounds. Liquid chromatography (LC) of flavo-
noids is usually carried out in the reversed-phase mode, on C8- or C18-bonded
silica columns (Dai and Mumper, 2010) ranging from 100–250 mm in length
and usually with an internal diameter of 3.9–4.6 mm (Liu et al., 2008).
Ultra performance liquid chromatography instruments are based on the use
of small particle size chromatographic columns (less than 2 μm) and offer
substantial resolution enhancement resulting in more efficient separation of
the compounds. This is applicable for monitoring of secondary metabolites
production in vitro which is characterized by a small amount. In addition, it
greatly reduces the analytical time and can withstand high pressure (Liu et al.,
2008). Moreover, it has been known to consume less solvent than HPLC.
Gradient elution is generally performed with a binary solvent system

(Liu et al., 2008). LC is usually performed at room temperature, but
temperatures up to 40°C are sometimes required to reduce the time of
analysis and because thermostated columns give more repeatable elution
times. However, the constant column temperature is recommended for
reproducibility (Dai and Mumper, 2010). If the main aim of the study is
to determine the major flavonoids in a sample, the run time of 0.5–1 h
is usually sufficient to separate the 5–10 compounds of interest (Rijke
et al., 2006).
All flavonoid aglycones contain at least one aromatic ring and, conse-
quently, efficiently absorb UV light. The first maximum, which is found in
the 240–285 nm range, is due to the A-ring and the second maximum, which
is in the 300–550 nm range, is due to the substitution pattern and conjuga-
tion of the C-ring (Rijke et al., 2006). UV detection is the recommended
tool in all LC-based analysis types, and LC with multiple wavelength or
diode–array detection is a reasonable tool for various studies such as
quantification of the main aglycones or for interim subgroup classification.
Flavonoids detection is usually carried out at 250, 265, 290, 350, 370 or
400 nm, and 500–525 nm for anthocyanidins (Rijke et al., 2006; Dai and
Mumper, 2010). The other colored compounds such as carotenoid and
quinoid pigments or UV-absorbing phenolics are commonly estimated by
this technique (Matkowski, 2008). In flavonoid analysis, fluorescence detec-
tion is used only infrequently because few classes of flavonoids have innate
fluorescence, including the isoflavones and flavonoids with OH group in the
3-position, such as 3-hydroxyflavone and catechin. However, the techniques
involve spectrophotometry that is suitable for some groups of compounds
and is inappropriate for other types.
Under the usual reversed-phase conditions, the more polar compounds
are generally eluted first. Thus, diglycosides precede monoglycosides,
which precede aglycones. The elution pattern for flavonoids containing
equivalent substitution patterns is flavanone followed by flavonol and
flavones. This elution pattern holds for both aglycones and glycosides for
isomeric compounds, which differ in the structure of the saccharine attached
at the 7-position, the rutinoside being eluted ahead of the neohesperidoside
(Robards and Antolovich, 1997).
The use of hyphenated techniques such as LC–mass spectrometry and
LC-nuclear magnetic resonance (NMR) or Two-dimensional-NMRis the
best means for structure determination of novel compounds detected only in
in vitro cultures and not in intact plants. (Matkowski, 2008).
6.6 STRATEGIES TO IMPROVE PRODUCTION
The tissue culture cells typically accumulate large amounts of secondary

compounds only under specific conditions. This means that the maximiza-
tion of the production and accumulation of secondary metabolites by plant
tissue culture requires a strategy to enhance biosynthesis and production of
secondary compounds. A number of reported methods (Kim et al., 2002;
Rao and Ravishankar, 2002; James et al., 2008 Kuo et al., 2011; Cai et al.,
2012) based on different environmental and nutritional factors are known
to influence the biosynthetic pathways of secondary metabolites such as:
(1) obtaining efficient cell lines for high growth, (2) selecting high yielding
cell clones, (3) immobilization of cells to enhance yields of extracellular
metabolites, (4) infusion of metabolites to facilitate downstream processing,
(5) relationship with growth phase and subculturing, (6) manipulating the
environment and medium, (7) adsorption of the metabolites to separate the
products from the medium and to overcome feedback inhibition (8) biotrans-
formation, (9) precursor feeding, and (10) elicitation.
6.6.1 SCREENING OF HIGH-GROWTH CELL LINE
Undifferentiated plant cells are genetically heterogeneous that makes it

promising to select cell strains with superior metabolites production. Selec-
tion of a stable cell line and finding the optimum conditions for cell growth
and maintenance, therefore, is fundamental. There are many reports on the
selection of cultured cell strains that give high yields of useful compounds
(Rao and Ravishankar, 2002). Selected cell strains have been obtained by
various selection methods such as cell cloning, cell tolerance to stress agents,
visual selection, fluorescence assay, HPLC technique, radioimmunoassay,
and other bioassays selection. The selected cell lines can be cultivated in the
lab to supply a continuous and reliable source of natural products.
The selected cell lines must be stable in growth and production of targeted
compounds even after subculture or maintenance for a long period as variability
in metabolite accumulation leads to a reduction in productivity. Variability in
culture characteristics has been attributed to genetic changes by mutation or
epigenetic changes due to physiological conditions (Yue et al., 2014).
Cell tolerance to stress and bioassays selection strategies involve
exposure of a number of cells to a toxic or environmental stress, and only
cells that are able to resist the selection procedures will survive (Rao and
Ravishankar, 2002).
6.6.2 CELL IMMOBILIZATION
Immobilization is defined as the confinement of cells in or on a suitable

matrix to enable the flux of the product in an extracellular medium (Anand,
2010). Generally, the cells are added to different types of gels such as agar,
agarose, gelatin, polyacrylamide or calcium alginate (Rodríguez-Sahagún
et al., 2012).
6.6.3 OPTIMIZATION OF MEDIUM AND CULTURE CONDITIONS
Several strategies have been adopted for the enhancement of bioactive

metabolite production in in vitro cultures. The media constituents (source and
concentration) namely, nutrients, carbohydrates, phytohormones, vitamins
have to be optimized for both a maximum biomass gain and the accumula-
tion of the targeted metabolite. The cultural conditions including temperature,
pH, light, and oxygen also gradually affect the final yields. There are several
types of in vitro culture media used widely for the growth of cultures and
production of phytochemicals, such as MS (Murashige and Skoog, 1962), B5
(Gamborg et al., 1968), LS (Linsmaier and Skoog, 1965), SH (Schenk and
Hildebrandt, 1972), and other improved liquid media according to the growth
behavior of plant cells (Yue et al., 2014). The constituents of those media, i.e.,
carbon, nitrogen, phosphate, inorganic mineral and even pH, are well known
in its level and type. A comparative analysis of the growth of Artemisia
annua hairy roots in two medium formulations showed that the B5 medium
was significantly optimum over MS for seven of eight factors studied (Kim
et al., 2002). The higher total triterpene production achieved by culturing E.
japonica cells in MS medium was likened to that of B5 or N6 medium (Ho
et al., 2018). It is commonly known that the levels of inorganic nutrients in
the MS medium are higher than in B5 medium. The in vitro culture is grown
heterotrophically and depends only on sugar as a carbon source (Rao and
Ravishanker, 2002). The sugar types that is, sucrose, glucose, and fructose
and their concentrations have main effects in phytochemicals production.
Rosmarinic acid production was found to increase by five times with increase
of sucrose concentration from 3 to 5% (Alamgir, 2017). However, the critical
factor, which directly affects both biomass production and phytochemicals
accumulation, is the type and concentration of auxin or cytokinin or the
auxin/cytokinin ratio (Lee et al., 2011). For example, NAA was superior to
IAA and 2,4-Dichlorophenoxyacetic acid for the biosynthesis of anthocyanin
in Glehnia littoralis cultures cell (Miura et al., 1998). The phytohormones are
essential for the good stimulation of phytochemicals, except with a hormone-

independent hairy root culture.
Light irradiation condition includes several factors namely, wavelength,
intensity, and photoperiod which are reported to affect both the growth and
production of secondary metabolites by the most grown in vitro cultures. For
example, flavonoids production has been shown to affect positively with the
good irradiation. Increasing light intensity doubles the growth yield of A.
annua hairy roots (Kim et al., 2002). Hairy roots of Acmella oppositifolia,
Datura stramonium, and Lippia dulcis turn green when exposed to light
consequence to developing mature chloroplasts fully capable of photosyn-
thesis (Flores et al., 1993). In addition to light, temperature, CO2, and O2 play
an important role in controlling greening, growth and secondary product
formation in hairy root cultures (Chandra and Chandra, 2011). However,
Miura et al. (1998) established a light-independent system for anthocyanin
production on the culture of G. littoralis, by optimizing CO2 supply. The
light regime for in vitro culture ranges between the ideal 16/8 h photoperiod
to total darkness and continuous light. The pH of the medium is also impor-
tant in regulation of secondary metabolites yield.
6.6.4 BIOTRANSFORMATION, PRECURSOR FEEDING, AND

ELICITATION
Biotransformation is the enzymatic catalysis of new active components,

which is stimulated by substrate feedings. The main enzymes are those
that can undergo reactions such as reduction, oxidation, methylation,
isomerization, acetylation, esterification, and glycosylations (Chandra
and Chandra, 2011). There are two core reasons to choose plant cells for
biotransformation: (1) cells catalyze the reactions stereospecifically, resulting
in pure products, and (2) they can perform regiospecific modifications that
are not easily synthesized by chemical or microorganisms (Smetanska,
2008). An alternative to biological transformation is a combined chemical
and biological approach. This can be performed by using cell cultures to
complete the difficult stages in a synthesis or using enzymes isolated from
the cultured cells and the conversion is achieved in a cell-free environment
(Collin, 2001). Plant in vitro cultures has been reported to transform
exogenously supplied compounds into naturally existent and novel products.
S. rebaudiana cell cultures are used to transform steviol into steviobioside,
a glycoside 300 times sweeter than sucrose (Sasson, 1991). Coffea Arabica
cell culture supplemented with vanillin and capsaicin has resulted in the
production of vanillin glucosides and capsaicin glucoside, respectively

(Smetanska, 2008).
Precursor feeding is based on the theory that any intermediary compound
involved in the biosynthetic pathway of a product, represents an upright
chance to heighten the final yield (Yue et al., 2014). A number of factors are
to be considered when applying the precursor to the cell culture medium,
such as the concentration and the time of addition of the precursor (Chat-
topadhyay et al., 2002). Problems related to feeding include a certain level
of precursors being toxic to plant cells and inhibiting cell growth, in some
cases, they inhibit secondary metabolism accumulation (Yue et al., 2014).
Therefore, for one specific plant species, precursor concentration should be
carefully adjusted, compared and optimized through the feeding experiment
(Gaosheng and Jingming, 2012).
Elicitors are compounds which stimulate any type of physiological
abnormality of the plant when introduced in a suitable concentration (Patel
and Krishnamurthy, 2013). Therefore, the plant accumulates the secondary
compounds as a defense mechanism against the stress induced by the elicitor.
According to their origin and function, the elicitors are divided into abiotic
and biotic. Abiotic elicitors are physical and chemical factors capable of
inducing stress in the plant thus affecting the secondary metabolites accumu-
lation. These include heavy metals, mineral salts and osmotic stress agents
(chemicals elicitors), temperature, light, and UV, (environmental condi-
tions), and wounding (physical elicitors). The biotic elicitors are those which
have a biological origin, either from pathogenic microorganism or plant. The
biotic elicitor may be extracted material (spores and cell wall) or toxins and
compounds produced by an organism (polysaccharides, lipids, and glyco-
proteins). The sources are bacteria, yeast, fungal arthropods, and plants.
Biosynthesis of flavonoids is induced either by light via phytochrome or/
and UV-photoreceptors (abiotic elicitor), or by infection with a phytopatho-
genic organism (biotic elicitor) or compounds (abiotic/biotic elicitor) which
induce the synthesis of antimicrobial compounds in plants (Sasson, 1991).
Methyl jasmonate (MJ) is the most used elicitor in enhancing secondary
metabolites accumulation and production. MJ was reported to maximize the
production of paclitaxel, taxanes, and diterpenes from in vitro cultures of
Taxus sp. (Patel and Krishnamurthy, 2013).
Before applying elicitors to the culture, several precautions and stages
are required to get the intended results. These include elicitor specificity,
the concentration of elicitor, stage and age of culture, elicitor fitting with
the medium compositions, and the duration of elicitor contact. Elicitor treat-
ment at late log phase results in higher biomass yield as well as secondary
metabolite production, whereas during the early phase, it leads to the

immediate increase in secondary metabolite production while lowering the
biomass yield (Chandra and Chandra, 2011). High dosage of elicitor has
been reported to induce a hypersensitive response leading to cell death,
whereas an optimum level was required for secondary metabolite induction
(Patel and Krishnamurthy, 2013).
6.6.5 RELATIONSHIP WITH GROWTH PHASE AND

SUBCULTURING
To enhance the production of secondary metabolites, it is important to under-

stand when in the growth phase a specific product is formed. For example, if
a product is mainly formed during exponential growth, the batch culture can
be used to keep the culture actively growing, for long-term maintainance and
for maximization of the yields (Kim et al., 2002).
6.7 CONCLUSION
Plant in vitro culture is a complex and multiphase process which requires

good management to maximize the production of desired compounds. This
process includes three parts which are explant, medium, culture conditions,
which must be managed through a time course for the highest degree of
accumulation and maximum level of production. Each part can be subdi-
vided into different components that require specific manipulation methods
to achieve the goals of the process. The interaction between the components
of one part and those of other parts during manipulation require careful
management. All these characteristics are those that make in vitro culture
systems attractive for production of secondary metabolites. A very wide
variety of secondary products have now been demonstrated in the literature.
However, for a process to be economically feasible, it has to outperform
the conventional procedures. This can be achieved with the development
and optimization of the process in all in vitro culture types for high daily
productivity (see Chapter 7). As declared above, it requires several strate-
gies varying in the means and time course of application. The modification
of the medium compositions is the primary concern in those strategies. The
selection of cell lines with stability and enhanced levels of product, either in
continuous systems or in batch systems, is similarly important. Organized
cultures have sizeable advantages over undifferentiated cultures which
often tend to show circumscribed commercial production of secondary

metabolites. The introduction of the transformation techniques so as to
produce fast growth and phytohormone-dependent cultures, to regulate the
biosynthetic pathways, is also likely to be a significant step towards making
cell cultures more generally applicable to the commercial production of
secondary metabolites.
KEYWORDS
•• plant cell culture

•• in vitro production of phytochemicals
•• suspension culture
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CHAPTER 7
PRACTICAL PROCESSES INVOLVED

IN THE PRODUCTION OF
PHYTOCHEMICALS BY PLANT TISSUE
CULTURES
HANAN M. AL-YOUSEF
Department of Pharmacognosy, College of Pharmacy, King Saud
University, Riyadh, Saudi Arabia, Tel.: +966555287629,
ORCID: *https://orcid.org/0000-0002-2607-0918
ABSTRACT
Plant tissue culture (PTC) has wide applications in many areas. These appli-
cations are categorized into three; basic research, environmental aspects, and
commercial items. Current research in PTC is highly magnified on commercial
applications like crop development, secondary metabolite induction, and many
strategies for involving genetic interference. Plant biotechnology has a key role
to play in solving problems related to development of farms and fruit trees.
In vitro techniques are being potentially applied to supplement the traditional
methods of vegetative propagation and production of plants. Micropropaga-
tion in vitro techniques have advantages over traditional methods of vegeta-
tive propagation; small spaces needed, high multiplication rate, seasonal-free
dependence under controlled culture condition, and plant-free microbes.
7.1 INTRODUCTION
Plant tissue culture (PTC) refers to the in vitro cultivation of all parts of a
plant under aseptic circumstances. Any PTC techniques must contain many
basic facilities. These include rooms/area for washing and preparation of
media, sterilization, storage, collection of data, controlled incubators, and

so forth. PTCs should be incubated under specific temperature, humidity,
air ventilation, light/dark quality, and for a fixed duration. The PTC tech-
nique requires many organic and inorganic substances for culture media
preparation. Growth room is a critical area where PTCs are maintained under
controlled circumstances to achieve most favorable growth. Plants regen-
erated from in vitro PTCs are transplanted to vermiculite container (pots
containing a yellow or brown mineral which shown as an alteration product
of mica and other minerals and used for insulation or as a moisture-retentive
medium for growing plants). The potted plants are transferred to growth
cabinets or greenhouses and kept for further observations under controlled
circumstances (Bhatia and Dahiya, 2015).
PTC system is used to study basic issues related to differentiation and
growth under ultimate reproducible circumstances. It has also been applied
in various practical applications in many areas such as horticulture, agricul-
ture, and in research centers. It is interesting to know that PTCs are more
economical and safe than classical method. There is upswing evidence that
it might be possible to store precious germplasm of plant in culture media
under low temperature. Recent techniques used such as gene transfer medi-
ated by Agrobacterium and transgenic plants regeneration were established
(Herrera-Estrella et al., 1983; De Block et al., 1984), which have been
proven to be helpful after inducing desirable agronomic traits to transgenic
plants (Shah et al., 1986). The concept “tissue culture” is utilized to include
numerous variations, for example, meristem tissue culture for propagation
of strawberry, orchids, potato, and grape (virus-free plants), cell suspension
culture (CSC), protoplast tissue culture, organs (Gamborg, 2002), also pollen
or anther tissue culture for inducing haploid plants (Guha and Maheshwari,
1968). One of the most common practical application of PTC techniques
include the micropropagation (MP) of medicinal plants, agriculture, horti-
cultural crops, and ornamental by regeneration of plant (Bhatia, 2015).
Therefore, the goal of this chapter is to critically estimate the practical
applications involved with the use of plant cell culture (PCC), organ culture,
and others in vitro as a valuable tool for many researches and also for crop
amelioration by biotechnology.
7.1.1 MAIN LABORATORY SETUP
Laboratories that can handle PTC experiments need glassware and dispos-
able plasticware, reagents/chemicals (which serve as mineral nutrients for
Practical Processes Involved in the Production of Phytochemicals 189
media culture preparation), distilled or deionized water, pH meter, autoclave,

magnetic stirrer, filter sterilization units, weighing balance, oven, refrig-
erator, and so forth. Instrument such as laminar flow, a sterile air ventilation
are required to keep aseptic media, explants and surrounding environment.
Incubation of the cultures can be carried out on shelves fitted with lighting.
Cultures might also be incubated in rotary shakers or growth chambers (for
cell suspensions). For collecting and recording of the data as well as image
capture, cameras and microscopes with photography hyphenates will be
required (Sharma et al., 2015).
7.1.2 PREPARATION OF CELLS/TISSUES FOR CULTURING
Any part of the plant which is enucleated and placed in culture is claimed as
the explant. This might be root, leaf disks, cotyledons, shoot tips, hypocotyls,
axillary buds, and zygotic embryos. The chosen explants must be aseptic,
which always include surface sterilization by using many dilutions (10–30%
v/v) of Clorox or any bleach which have sodium hypochlorite (5.25% w/v)
as the main ingredient. Chemicals such as silver nitrate or alcohol can also
be used for surface sterilization. Prior to inoculating the explants onto the
culture medium, it must be rinsed with autoclaved distilled/deionized water
to get rid of chemical traces and then trimmed to remove the dead cells at
the edges due to harsh chemicals used for sterilizing. There are many surface
sterilization methods which are simplified and modified where the explant
donor tissues, such as floral buds and seed pods, might be directly dipped
into 70% alcohol with light flamed. The anthers or seeds are then enucle-
ated aseptically for culture. Through this procedure, the explants free from
chemical agents leads to a high survival rate of the explants. This procedure
is useful for orchids (intact seed pods or anthers) in closed floral buds of
many species.
Presurface sterilization is very important treatment especially for field-
grown plants such as guava; scions were acquired from chosen field-grown
plants and grafted to seedling rootstocks (Loh and Rao, 1989). Grafted plants
were served in the laboratory for gathering nodal explants. To eliminate
apical predominance and enhance propagation of the axillary buds, sanitary
scion branches were decapitated 5–8 days before excision of the nodular
explants. Surface sterilized of nodular segments were done by using 80%
alcohol followed by Clorox solutions (5 and 3%) prior to successful setup of
cultures. Physiological state and age of the explant donor plant might have
an important influence on the success of plants regeneration. Many studies
have reported that cotyledons (3–6 days old) seedlings of Brassica spp. are
known as the important sources of regenerative explants for adventitious
(transverse) shoot and genetic transformation mediated by Agrobacterium
(Sharma, 1990). In petunia, the leaves are the main sources of explant, the
first entire expanded leaf is chosen. A woody tree species such as mangosteen,
solely young red leaves induced shoot buds in tissue culture. Moreover,
mangosteen leaf segments (3-mm transverse sections) observed a significant
polarity of regeneration with shoot buds driving from the midrib beside the
apical cut end of leaf segments (Goh et al., 1994). Hypocotyls and seedling
roots are also used in many species as the explant. In few of the cereals (rice
and corn) and numerous grasses (Frame et al., 2002), also, in many of the
coniferous trees (Lu et al., 1991), the zygotic embryo is preferred explant for
tissue culture initiation. When an appropriate explant is chosen and prepared
for tissue culture, it should be incubated on a suitable nutrient medium for
growth and differentiation.
7.1.3 NUTRIENT MEDIA
Numerous mineral preparations are available to PTC. The major media

include Murashige and Skoog medium (MSO or MS0 (MS-zero) medium)
(Murashige and Skoog, 1962) and Gamborg’s B5 medium (Gamborg et al.,
1968). PTC media are made up of macro and micronutrients, phytohor-
mones, vitamins, adjuvants like coconut water, and sucrose (2–3% w/v).
The nutrient media is prepared by mixing stock solutions of many chemical
substances or from manufactories mixed powder. Adjuvants like vitamin C,
polyvinyl pyrrolidone, and activated charcoal which might be needed for
few species that show ultimate cases of browning tissue on excision and
release of polyphenolic compounds form the damaged cells. The pH of the
medium culture is adjusted to 5.8 and an appropriate gelling material is
added if a semisolid matrix is preferred. Agar (8–10 g/L) and the Gellangum
such as Phytagel or Gelrite (2–3 g/L) are most common gelling agents used
for PTC media.
The culture medium is sterilized by using autoclave around 20 min.
Cell culture may be in the form of a liquid suspension (small clusters of
cells), or in semisolid nutrient medium (apart from callus culture). If a liquid
nutrient medium is needed it might be similarly prepared but without adding
gelling agent and sterilized by using an autoclave or by filter sterilization
(0.22-µm pore size). Furthermore, heat labile substances of the media like
some phytohormones such as indoleacetic and gibberellic acid as well as
antibiotics, when used, should be filter sterilized and put into an autoclaved
medium that is cooled to about 60°C prior to aliquot the medium to aseptic
culture vessels.
The choice of a suitable nutrient medium for a selected tissue/species is
generally provided by empirical trials. Therefore, the medium ingredients
should be classified into four categories and use three different concentra-
tions for each (low, medium, high) category and prepare many combinations
of the substances. However, one can begin from the standard MS medium
culture and vary the ingredients of the many macro and micronutrients, and
phytohormones and vitamin. Moreover, one of the major substances that
have a potent effect on regeneration is the concentration and type of phyto-
hormones in the culture medium (Skoog and Miller, 1957). In addition, a
high ratio of cytokinin to auxin in the explant preferable shoot regeneration, a
comparatively high auxin to cytokinin ratio preferable root regeneration, and
the intermediate ratio causes callus propagation. Usually, phytohormones
concentration in the medium is higher (normally 10 ± 7–10 ± 5 M), this is due
to the endogenous concentration depending on the efficiency of uptake of the
substance by the explant from the external medium. Thereby, optimization
of the suitable phytohormone concentrations in the medium may also be
empirically defined in the earlier set of investigational experiments. Once an
optimum medium combination is determined, it may be used as an identified
medium for the species/closely related species of plants.
7.2 PRODUCTION OF NATIVE PLANT CONSTITUENTS
In PTC, the rate of cell growth and biosynthesis in cultures initiated from a
very small amount of plant material is quite high and the final product may be
produced in brief period. PCCs are maintained under controlled conditions
both environmental and nutritional which ensure the continuous yields
of metabolites. CSC offers a more effective mechanism of incorporative
precursors into cells that are found in the whole plant. It is possible to cite
some more examples of cell cultures which synthesize comparatively high
amount of natural plant products, but in many other cases PCCs either do not
produce the natural compounds or do so only in very small amounts. This
could be attributed to the following facts:
1. PCC is different from the intact plants in many ways, morphologi-

cally, cytologically, and physiologically and thus lack modifying the
interaction of the intact organism.
2. Inadequate knowledge of the nutrients and other culture nutrition

to support the synthesis of the product in question is another prob-
able reason for the deficient performance of the PCCs in terms of
secondary metabolite production, for example, the yield of alkaloids
in cell, cultures are known to be affected by the growth phase of the
cultures, composition of the medium, incubation conditions such as
light and the genotype of the cell.
3. The genetic and epigenetic instability of the cells are considered the
most serious problems in the production of the natural compounds
by the PCCs.
This requires constant selection of the cells capable of synthesisizing the

compounds to sustain production. If the above problems are solved or mini-
mized, PCCs could become a practical method for the industrial production
of some important natural compounds.
A few but well-established examples of secondary compounds synthe-
sizing in a high amount by PCCs are known.
1. CSCs of Coleus blumei have been reported to accumulate rosmoric

acid up to 15% of the dry weight of the cell which is five times higher
than the alkaloid content in the intact plant (Zenk et al., 1977).
2. Cell and callus cultures of Catharanthusroseus have been reported
to synthesize the relatively high amount of serpentine and ajmalicine
(as high as 1%). Cell cultures derived from high-yielding strains also
produce copious quantities of the alkaloids. Cell cultures from some
low-yielding strain of C. roseus also accumulated elevated level
of the alkaloids. Other reports showed that stem calli of C. roseus
continued to synthesize serpentine even after 12 years of their initia-
tion (Verpoorte et al., 1993).
3. Morinda citrifolia is a commercial source of anthraquinones. PCCs
of the C. citrifolia contained 20 times more anthraquinones content
than the roots (Zafar et al., 1992).
7.2.1 LOW PRODUCTION OF SECONDARY METABOLITES

WAS COUNTERED BY SEVERAL TRYERS
1. Addition of precursors, for example, mevalonic acid increase steroidal

synthesis.
2. Use of Zenk et al. media in cultured cells have been investigated to

increase both biomass and secondary metabolites as for ajmalicine
production in C. roseus.
3. Addition of precursor L-tryptophan to the culture media of Cinchona
ledegriana increases its quinoline alkaloid content.
4. Addition of thiosemicarbazide to CSC of Panax ginseng promote
biosynthesis of saponin and inhibit phytosterol.
5. Addition of colchicine to CSC of Valeriana spp. increases the vale-
potriates (60 folds).
6. Addition of copper sulfate to the CSC of various Solanaceae induced
formation of sesquiterpene.
7.3 SOMATIC EMBRYOGENESIS (SEG)
In somatic embryogenesis (SEg), cells are developed into plants during

embryogenetic stages without gametes fusion. After the first report of SEg
in PTC of carrot, the importance of SEg in joining effective cloning of favor-
able genotypes has been recognized. It is interesting to say that plants regen-
erated via direct SEg are usually more uniform than plants regenerated from
callus tissues. SEg could also generate secondary SEg from their surfaces.
Secondary SEg occurs when primary SEg gives rise to successive cycles of
embryos. Secondary embryogenesis supplied a way to induce considerable
populations of vegetative propagules in a brief period (Lee et al., 1997).
Therefore, secondary embryos might be helpful for recovering many plants
from the genetic transformation, clonal propagation (CP), and produced
mutation. Promoting embryogenic cells might be faced with microprojectile
bombardment (genetic transformation), then the transformed cells might be
chosen and regenerated inside plants. So, cassava (secondary embryos) were
used for the production of mutation in vitro via γ-irradiation and the mutant
plants have altered starch constituent were obtained (Joseph et al., 2004).
7.4 MICROPROPAGATION (MP)
MP may be known as a technique in which any meristematic (vegetative)

part of plant-like shoot bud and tip, and so forth is occurred aseptically
(cultured on sterile media) under controlled circumstances to produce
plantlet which is same copy of its parent plant. MP can be determined as
CP in vitro. The “clone” term was first used by Webber for cultivated plants
that were propagated vegetatively. The term derived from Greek (clone =
twig, broken off like propagules for multiplication). It signifies that plants
developed from meristematic parts are simply transplanted parts of the
identical individual and these plants are typical. This technique of culturing
plants has a wide applied including morphology, biochemistry physiology,
genetic engineering, and molecular biology through SEg, axillary bud, and
adventitious budding (Bonga et al., 1987; Bonga and Aderkas, 1992).
7.4.1 GENERAL TECHNIQUE OF MP
MP is one of the major techniques of PTC. It is the practice of rapidly

multiplying stock plant material to produce many progeny plants by using
advance PTC methods. MP is used to multiply new plants like those that
have been genetically manipulated or breed via classical plant breeding
methods. Moreover, it is used to provide enough plantlets for planting from
a stock plant which has no seeds, or does not well respond to vegetative
reproduction. Interestingly, the use of this method for CP of plants is due
to the success in this field with orchids. During progress in this area, it has
been reported that multiplication of various fruit and ornamental cultivars
is practiced on a commercially practical method of CP. So, CP is the multi-
plication of genetically similar individuals via asexual reproduction. Plant
regeneration may be accomplished by culturing different tissue sections
either via lacking adventitious origin (preformed meristem) such as axillary
bud proliferation approach or through de novo origin (callus and PCCs).
The stimulation of axillary buds, (which are often found in the axil of each
leaf) lead to provide a shoot. This technique exploits the normal ontogenic
path for branch development by lateral meristem. In nature, these buds stay
recumbent for many periods based upon the growth pattern of the plant.
In many species, abstraction of terminal bud is substantial to break the
apical predominance and motivate the axillary bud to grow into a shoot. Due
to perpetually applied of cytokine in a cultured medium, the shoot grown
by the bud, which is found on explants (shoot tip cutting/nodal segment)
develops axillary buds. Then, the shoot is separated and rooted to induce
plants or shoots and are handled as propagules for propagation. The impor-
tance of using axillary bud proliferation from meristem, bud culture, or
shoot tip regeneration in an incipient shoot has been already differentiated
in vivo. So, only root differentiation and elongation are needed to establish
an entire plant. Also, this technique preserves the precise arrangement of
whole layers necessary if a chimeral plant’s genetic part is to be protected.
In an identical chimera, the surface layers of grown meristem are of differing

genetic foundation and it is their contribution arrangement to the plant organ
that produces the desisted properties. If the integrity of the meristem keeps
intact and improvement is normal in vitro, therefore the chimeral pattern
will be protected. However, callus tissue was permitted to form shoot prolif-
eration thereafter and was from the adventitious origin; subsequently, there
might be a risk that the chimeral layers of original explants might not all be
represented in the specially need from in the adventitious shoots (Das and
Mitra, 1990).
PTC is a useful technique for removing viruses from infected plantlets
and for inducing virus-free plant seedlings. Although shoot-tip culture has
been utilized for this purpose, the propagation rate of virus-free plantlets
is low, and it is time-consuming. Many PTC techniques have been estab-
lished to improve the efficiency of propagation, however, all have inherent
defects as practical methods, it also needs period for cultivation, low
propagation rates, and the need of mastering highly versatile skillful tech-
niques. Ayabe and Sumi have established a new PTC method for garlic that
utilizes the stem disc as an explant. These findings show that the stem disc
culture method is of practical use for the MP of garlic plants, especially as
virus-free seed plants induced by shoot-tip culture. Moreover, this method
has improved a new system, epoch making garlic cultivation, in which
seedlings instead of cloves are used for propagation. Due to seedlings
much more easily cultivated by machinery than cloves, this culture system
is functional for the practical cultivation of garlic, especially in large-scale
cultivation (1998).
7.4.2 MP STAGES
Plant MP method aims to induce clones (true identical copies of a plant in

copious quantities). This process is often divided into the following stages
(Durzan, 1988):
7.4.2.1 STAGE 0 PRE-PROPAGATION STAGE
The pre-propagation stage needs suitable maintenance of the parent plants

in the clean enclosed areas free from insect and disease conditions and
with little dust. Glass greenhouse, plastic tunnels, and net covered tunnels,
supplied high-quality explant source plants with low infection level.
Collection of explants for CP should be made after suitable pretreatment of

the parent plants with pesticides and fungicides to decrease contamination
in the in vitro cultures. This develops growth and multiplication rates of
in vitro cultures. The control of contamination starts with the pretreatment
of the parent plants. The selection of explant depends on the methods of
shoot multiplication. All organs of plant namely nodal segment, internodal
segments, shoot and root tip. For axillary bud production, callus culture, SEg
explants nodal segments, internodes, and leaves are collected.
7.4.2.2 STAGE 1 INITIATION OF ASEPTIC CULTURE
Sterilization and establishment of explants (plant organ used to initiate a

culture) were done. Select explant based on the procedure of shoot multipli-
cation to be followed. For MP and callus culture work, the explants of choice
are nodes and internodes/leaves, respectively. For SEg work, the explant of
choice is internodes and leaves.
7.4.2.3 STAGE 2 MULTIPLICATION OF CULTURE
It is an important stage; the rate of multiplication was detected by the

significant success of MP system this can be provided by enhanced axillary
branching via adventitious bud and callusing formation. For enhancement
of axillary branching, the axillary bud found in the axil of each leaf either
grow into a single or cluster of shoots in the presence of cytokinins (BAP
1.0 mg/L) in the culture medium. For adventitious bud formation, the buds
derived from the part other than leaf axils or shoot apex are called adventi-
tious buds. Through callusing, plant cells are totipotent. In PTC, the mass
of differentiated cells are known as callus. This either produced shoot bud
or bipolar structure like SEg. This technique is used when the goal is to
produce change especially in self-pollinating species with a narrow genetic
dependent.
7.4.2.4 STAGE 3 IN VITRO ROOTING OF SHOOTS
Grown shoots lack root system in vitro. For the production of roots, they
were transferred to rooting medium. For rooting half strength MS culture
medium supplemented with 1.0 mg/Ls auxin hormone was used.
7.4.2.5 STAGE 4 HARDENING AND ACCLIMATIZATION OF

TISSUE CULTURE PLANTLETS
This is the final stage and needs appropriate handling of plants. The trans-
plantation from completely controlled circumstances should be gradual.
This process of gradually preparing the plants to grow in the area conditions
is known acclimatization. The plants induced in tissue culture, despite green
in color, cannot prepare enough food for their own survival. Furthermore,
inside the culture vessels humidity is high and therefore the natural protec-
tive covering of cuticle is not fully grown. Thus, immediately after transfer
plants were kept under high humidity. The optimum environment was
supplied to plants in greenhouse.
7.4.3 ADVANTAGES OF MP
MP has many advantages over conventional plant propagation techniques;

the main advantage of MP is the induction of several plants which are clones
of each other. MP can be utilized to produce infection/disease-free plants.
MP also induces rooted plantlets suitable for growth, time-saving for the
grower if cuttings and/or seeds are slow to grow. It has a substantially high-
frequency rate, producing numerous of propagules while traditional tech-
niques may only produce a few a number. Furthermore, it is the only viable
technique of regenerating genetically manipulated cells or cells after fusion
of protoplast. Also, it is helpful in multiplying plants which induce seeds
in uneconomical quantities, or if plants are sterile and do not produce good
seeds or if cannot be stored. MP usually produces robust plants, owing to
accelerated growth when compared to typical plants produced by traditional
methods. Few plants with tiny seeds such as most orchids are effectively
grown from seed in sterile culture. A considerable number of plants may
be produced/m2 and the propagules may be stored longer period and in a
smaller area (Sharma et al., 2015).
7.4.4 DRAWBACKS OF MP
MP is not always the excellent methods of multiplying plants, circumstances

that limit its use are as follows (Verpoorte et al., 1993):
1. It is very expensive and may have an excessive cost more than 70%.
2. A monoculture is produced after MP, owing to a lack of overall infec-

tion/disease resilience (might be vulnerable to the same infections).
3. An infected plant may release infected progeny.
4. Not all plants could be successfully cultured as the proper culture
medium is not known and/or the plants release secondary metabo-
lites that may kill the explant.
7.5 BIOTRANSFORMATION
Biotransformation (BT) is a technique which uses the enzyme located in

the plant callus to transform the externally supplied less active compounds
(substrate) to a more active product by a certain chemical reaction such
as hydroxylation or glycosylation. Two approaches are being followed in
the BT studies using cell cultures. First: cells are supplied with substrate
compounds normally not available to the plant such as synthetic compound
analogs of intermediate or products from other species with the objective of
obtaining compounds unknown in nature. Second: enhancement of produc-
tion of a natural compound is being attempted by feeding nature intermedi-
ates of the plants (precursors of the compound in question) (Sommer and
Brown, 1979).
Plant cells have been shown to glycolysate added substrates. Glyco-
side can be of higher commercial value than the aglycones. For example,
aglycon steviol is not sweet, whereas the glycoside steviobioside can be
used as a sweetening agent. Cell cultures of Stevia rebaudiana and Digi-
talis purpura have been reported to glycolysate steviol to steviobioside,
in addition to stevioside. Other compounds glycolysated by cell cultures
are diphenols, steroids, cardiac aglycons, and cardiac glycosides (Zafar
et al., 1992).
Cell cultures of Digitalis lanata have been reported to increase the
activity of a compound which is digitoxin by its conversation to digoxin.
BT of digitoxin (less active and more toxic) to digoxin by the B-12-hydrox-
ylation reaction is affected by an enzyme located in the cell of D. lanata.
Commercial exploitation of this process would enable utilization of the large
stocks of digitoxin which accumulate as a by-product in the manufacture of
digoxin from D. lanata (Nmila, 2000).
The potentiality to produce anticancer agents are explicated by the BT
of synthetic di-benzylbutanolides to lignans appropriate for conversion to
etoposide including cultures of Podophyllum peltatum by using a semi-
continuous method (Kutney et al., 1993). Interestingly, hydroxylation and
oxidation reactions have also been determined for the BT of podophyllum

lignans in CSCs of Forsythia intermedia (Broomhead and Dewick, 1991).
Some BT are stereospecific and have the possibility for the isolation of opti-
cally active substances from the racemate mixture, so, Nicotinia tabacum
PCCs can specifically hydrolyze the R-configuration forms of monoterpenes
like bornyland isobornyl acetate (Zafar et al., 1992). Various biochemical
transformations by PCCs have been determined, and contain hydroxylation,
isomerization, epoxidations, ester formation and saponification, glycosyl-
ation, methylation, demethylation, and oxidation. For this technique to be
commercially valuable the product must be sufficient in good amounts and
the reaction should be not more than one which is more easily performed by
microbes or by chemical reaction.
7.6 TRANSFORMED HAIRY ROOT CULTURE
Bhatia has been reported that the production of a transgene and its expression
via PTC supported by many genetic materials which is the most crisis point
argument nowadays. Incorporation of genes which induces stress tolerant
plants will improve the production of secondary metabolite (2015). Some
soil bacteria such as Agrobacterium can trigger a transformation of plant cells
by incorporating into their genome t-DNA via the bacterial plasmid. Such
transformed roots, formed by inoculating the host plant, when developed
in a hormone-free medium to give copious roots claimed as “hairy roots
or transformed root.” Elimination of the Agrobacterium leads to enhance
growth of the root profusely. Some plants which normally induce secondary
metabolites, the hairy roots accumulate these metabolites in quantities like
those presented in the intact plant.
Agrobacterium tumefaciens and Agrobacterium rhizogenes are most
commonly used to effect on transformation. With normal roots and cells
cultures, it is possible to use transformed roots to perform biological conver-
sions not associated with the whole plant normally. The rapid hairy roots
growth rate offers the probability of rapid conversions. Ginseng hairy root
cultures have been found to convert digitoxigenin by esterification at C-3
with stearate, myristate, and palmitate into new compounds, and by the
formation of sophorosides and gentiobioside. Parr et al., have studied on the
tropane alkaloids biosynthesis fed on the S-analogue of tropinone (8-thiabi-
cyclo (3.2.1.) octan-3-one) to transform root cultures of Datura stramonium
and gave rise S-analogue of tropine, with 3-O-acetylester (1991).
7.7 IMMOBILIZATION OF PLANT CELLS
Immobilized plant cells (IPC) can be utilized in a comparable way as

immobilized enzymes influence complicated chemical reactions. Petersen
et al have reported on the IPC of D. lanata cells by suspending cells
in a sodium alginate solution, the alginate precipitating and entrapped
cells with calcium chloride (CaCl2) solution become pellet allowing the
product to get hard. These granules were catalyzed by the conversion of
digitoxin to purpurea glycoside A and hydroxylated β-methyldigitoxin
to give rise β-methyldigoxin. Despite the hydroxylating effect of the
entrapped cells was around half that of suspended cells, the pellets have
the advantage which the biocatalyst was reusable for periods extending to
2 months (1987).
A technique which has been established useful for the research on
alkaloid formation in Coffea arabica is to utilize a polypropylene sheeting
membrane of specified porosity, pore size, and thickness on which to cells
immobilized in a 3-mm thick layer; the nutrient medium circulates under the
membrane. C. roseus cells have also been studied and developed methods
have been done for the release of alkaloids sequestrated in the cell vacuoles
without damaging the culture (Zafar et al., 1992).
7.8 ORGAN CULTURE
Organs can be gained in the culture either by using growing points from
intact plants, or sterilized roots or seedlings; or by differentiation obtained
from callus tissue cultures by appropriate hormones. Usually, cultured
organs will be synthesized secondary metabolites which might be either
in poor yield or non-existent in the normal PCC. Therefore, quantities of
cardenolide isolated from D. purpurea and D. lanata cultures increment as
tissue differentiation yields.
Enhanced induction of alkaloids takes place when roots grow from
the tropane alkaloid (Solanaceae) callus cultures. C. roseus leaf cultures
and Rauwolfia serpentina synthesize a diversity of alkaloids. Dimeric
alkaloids have been estimated in organ cultures of C. roseus, postulating
the probability of an efficient induction system for these worthy alkaloids.
The dimers occurred solely in those cultures contained catharanthine and
vindoline. Whereas CSCs of Papaver bracteatum were obtained to synthe-
size sanguinarine and orientalidine, the shoot and root cultures induced
thebaine. In Hyoscyamus muticus, the normal and hairy root cultures
after treatment with jasmonic acid and its methyl ester induces enormous
quantities of conjugated polyamines and methyl putrescin. Never the less,
the increment of tropane alkaloid induction was not remarkable (Bionde
et al., 2000).
7.9 FLOWERING IN VITRO
One of the recent applications of PTC is to accelerate the breeding cycle of

various ornamental species that have extended period such as orchids which
require more than 3 years of vegetative development prior flowering. Under
control conditions, flowering might be observed 5 months after germination
of the seed instead of 3 years needed in field-development of plants, as well
as separation of flower colors was found in in vitro flowers. This reduces
labor costs and maximizes the space needed for orchid breeding. Moreover,
flowers produced in culture might be pollinated with pollen grains collected
from field-developed plants or self-pollinated in vitro trigger the formation
of seed pod in culture (Sim et al., 2007; Hee et al., 2007).
7.10 CONCLUSION
The previous discussion has shown that the PTC promises to be a worthy
tool for physiology, morphogenesis, molecular biology, and cell signaling
research, furthermore, crop development via biotechnology. With the predic-
tion of plant crop yield to be expanded by 2050 due to sustain consumption
of the food and fuel needs with increasing population, it is secure to predict a
firmly improved technology of PTC will be continued to encourage research
as well as to agricultural biotechnology in the next decades.
KEYWORDS
•• plant tissue culture

•• production of phytochemicals
•• biotransformation
•• organ culture
•• micropropagation
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CHAPTER 8
MEDICINAL AND INDUSTRIAL

APPLICATIONS OF BROMELAIN
RAMESH KUMAR and ABHAY K. PANDEY*
Department of Biochemistry, University of Allahabad, Allahabad,
Uttar Pradesh 211002, India, Mob.: +91 98395 21138
*
ABSTRACT
Proteases are a unique class of enzymes which occupy an important position

with respect to their enormous physiological and commercial applications.
Bromelain is a proteolytic enzyme present in pineapple along with closely
related proteinases. Bromelain have found application in the food, animal
feed, and textile industries. It has gained importance as a phytotherapeutic
agent because of its safety and efficacy coupled with lack of unwanted side
effects after oral administration. Numerous therapeutic advantages have been
stated for bromelain including improved absorption of antibiotics and other
drugs, platelet aggregation inhibition, surgical traumas, angina pectoris,
bronchitis, sinusitis, pyelonephritis, and thrombophlebitis. Bromelain also
modulates immune functions and has the potential to remove burn debris,
accelerates wound healing, and acts as an anticancer agent. Bromelain will
earn wide recognition as an antimetastatic drug, platelet aggregation inhibitor,
skin debridement facilitator, along with other therapeutic applications after
advanced clinical trials.
8.1 INTRODUCTION
Bromelain (EC 3.4.22.32) is a proteolytic enzyme derived from the fruit and
stem of pineapple plant (Ananas comosus) in the aqueous extract containing
many closely related proteinases and other compounds exhibiting various
medicinal activities (Onken et al., 2008; Hale et al., 2010). Pineapple, a

tropical fruit, is used as a food item and a medicinal supplement in many
countries. It is native to Central and South America that is grown in many
tropical and subtropical countries namely India, South Africa, Kenya, China,
Hawaii, Malaysia, the Philippines, and Thailand. Several native cultures
such as the Philippines, Hawaii, and so forth, use it as a folk remedy for the
treatment of many diseases (Pavan et al., 2012). The beneficial effects of
bromelain (Fig. 8.1) have been reported in varied health-related studies in
lowering inflammation, swelling, pain, and bruising related to trauma and
surgery. Bromelain has lesser undesirable effects in contrast to nonsteroidal
anti-inflammatory drugs (Lapeyre-Mestre et al., 2013; de la Barrera-Núñez
et al., 2014).
Bromelain primary component is a sulfhydryl proteolytic fraction. It
also contains acid phosphatase, a peroxidase, many protease inhibitors, and
organically bound calcium. It shows stability over a large pH range. There-
fore, it is not necessary to protect protease from stomach acid. Nevertheless,
it may be essential to defend the enzyme from digestion in the gut by acid
proteases. It may be administered with a buffering agent such as bicarbonate
or with water/solution containing nutrients to assist with absorption of fluid
and nutrients (Tochi et al., 2008). Aqueous extract of pineapple contains a
complex mixture of proteases and non-protease components. Bromelain is
used in food processing for meat tenderization. It directly influences pain
mediators such as bradykinin (Omojasola et al., 2008). However, its anal-
gesic properties are associated with its anti-inflammatory properties (Brien
et al., 2004). Edema reabsorption in the blood circulation has been shown
to be promoted by the fibrinolytic action of bromelain (Maurer et al., 2001;
Gaspani et al., 2002). It reduces swelling, bruising, pain, and healing time
after trauma and surgical procedures (Taussig, 1980). Bromelain increases
the prothrombin to thrombin conversion time indirectly and consequently
activating plasmin formation from plasminogen which results in the preven-
tion of fibrin formation (Tochi, 2008). All these cause a reduction in vascular
permeability. Besides, the pro-inflammatory prostaglandin (especially
PGE2) synthesis is inhibited by bromelain (Hale et al., 2005; Maurer, 2001).
Bromelain is active over a pH range of 4.5–9.5. Fruit derived bromelain
contains a lower amount of proteases in comparison with stem bromelain
(White et al., 1988). It has been reported that the beneficial physiological
effects of bromelain are due to multiple factors rather than due to a single
proteolytic fraction (Walsh et al., 2002). Bromelain is also popular as a
health-promoting nutritional supplement. The half-life of bromelain is about
6–9 h. It is absorbed in the human intestine without losing its biological
Medicinal and Industrial Applications of Bromelain 207
activity. The highest concentration of bromelain in the blood was recorded

1 h after its administration (White et al., 1988). The focus of the current
chapter is to present industrial and pharmacological activities of bromelain.
FIGURE 8.1 Pharmacological activities of bromelain.
8.2 INDUSTRIAL APPLICATIONS OF BROMELAIN
Bromelain is a pineapple protease having numerous industrial applications.

It is used mainly in tenderization, foods, detergents, and textile industry.
Bromelain is also an active ingredient of skin products and tooth-whitening
dentifrices.
8.2.1 BAKING INDUSTRY
Gluten is an important functional component of wheat food products, such

as flour. It consists of two major proteins, namely gliadin and glutenin.
Upon hydration gluten forms lattice-like structures and becomes insoluble.
Therefore, it is necessary to degrade gluten to evade resistance during dough
stretching (Walsh, 2002). The utilization of bromelain, a proteolytic enzyme
can improve dough formation, augment solubility, and stop dough shrinkage.
Hence, it permits the even rise of dough during the baking procedure (Kong
et al., 2007). Bromelain has also been used in the production of hypoal-
lergenic flour that is appropriate for utilization by wheat-allergic patients.
The immunoglobulin E-binding epitope, Gln–Gln–Gln–Pro–Pro, is a major
allergen in flour. Thus, the addition of bromelain can help in breaking down
epitope structure by hydrolyzing peptide bonds near pro (proline) residues
(Watanabe et al., 2000).
8.2.2 ANIMAL FEED
Generally, forages provide 90% of food energy and nutrients to ruminant

animals at a low cost. Climatic factors, forage species, cultivars, and preser-
vation methods influence the variations in the nutritional value of forages.
Therefore, evaluation of the soluble nitrogen components in the rumen is
essential to measure the forage quality to maximize its consumption in rumi-
nants diet (Givens et al., 2002). The in situ technique is extensively used to
investigate the protein degradability of forage. Unfortunately, its practica-
bility is restricted by the technical problems and the lack of standardization
(Abdelgadir et al., 2013). The measurement of protease-mediated protein
degradation in the rumen can be an option to substitute the conventional
method, which is highly expensive and time-consuming. The addition of
protease to animal feed can enhance protein availability and decrease the
price of animal feed. The use of bromelain in the evaluation of protein
degradation in cereals, hays, forages, protein concentrates, and silages in
ruminants have been successfully performed (Polaina and MacCabe, 2007).
8.2.3 TEXTILE INDUSTRY
Recently, the utilization of proteases in the silk industry has increased

enormously, particularly the cocoon cooking method which uses strong
alkaline agents and chemicals. However, this conventional technique has a
harmful effect on the silk thread quality. Hence, by using enzyme treatment
an effective cocoon cooking method can be developed which will decrease
softening time coupled with rising production and energy economy. Use of
pineapple extract with 9.8 mM of Na2CO3 has been shown to reduce the
softening time from 20 h to 30 min at 60°C (Singh et al., 2003). Tactile
behavior and wettability of silk has been improved by the pretreatment of
wool and silk with bromelain (Koh et al., 2006). These properties increase
the dye uptake by the silk fibers and wool and simultaneously maintain their
tensile characteristics.
8.3 MEDICINAL USE OF BROMELAIN
Bromelain is an immunomodulator and acts as a detoxifier of the system. It

eliminates gastritis induced by drug and other harmful effects. Hence, it is
suggested for use in adjuvant therapy with antibiotics in chemotherapy. It
has also been shown to help in the management of several ailments during
clinical studies.
8.3.1 EFFECT OF BROMELAIN ON REGULATORS OF

INFLAMMATION
Bromelain has been approved as a pharmaceutical product since 1956 as an

anti-inflammatory agent after surgery and infection. The immunomodula-
tory action of bromelain is brought about by the induction of CD-2-mediated
T-cell stimulation and increases T-lymphocyte multiplication in splenocyte.
However, it has an insignificant effect on purified CD4+ and CD8+ T cells.
On contrary, bromelain downregulates the discharge of pro-inflammatory
cytokines and chemokines produced by the inflamed tissues, that is, IL-2/4/6,
G-CSF, and IFNγ (Onken et al., 2008). Another study reported the release of
inflammatory cytokines was induced in peripheral blood mononuclear cells
sequestered from healthy donors as a result of IFNγ stimulation in addition
to IFNγ release by activated NK cells (Engwerda et al., 2001). These obser-
vations suggested that bromelain exhibited the immunostimulatory effect
only on the healthy immune system against foreign antigens. Upregulation
of the Bax/B-cell lymphoma 2 (Bcl-2) ratio and induction of caspases 3 and
9 as well as inhibition NF-кB and COX-2 expression by bromelain have
been shown to induce apoptosis in mouse skin papilloma in vivo suggesting
the cytotoxic and antitumor effects of bromelain (Kalra et al., 2008). Müller
et al. (2016) have reported that bromelain possesses comparatively better
cytotoxic action against TFK-1 and SZ-1 (human cholangiocarcinoma cell
lines) in vitro than papain. Expression of CD44 surface marker, a marker of
tumor proliferation and migration, is diminished in presence of bromelain. In
glioma cells, bromelain treatment reduces the invasion, migration, and adhe-
sion abilities without showing any adverse effect on the normal surrounding
cells (Mayer et al., 2008).
There is accumulating evidence showing the role of NF-кB signaling in

many types of cancers. Among multiple target genes of NF-кB is Cox-2,
involved in the synthesis of prostaglandin E2 (PGE2), a pro-inflammatory
lipid that also acts as an immunosuppressant and facilitator of cancer
proliferation by promoting conversion of arachidonate into PGE2. Inhibi-
tion of NF-кB, Cox-2, and PGE2 activity has been considered as a potential
treatment of cancer and chronic inflammatory diseases. Bromelain down-
regulates NF-кB and Cox-2 expression. Additionally, in human monocytic
leukemia and murine microglial cell lines, bromelain was shown to inhibit
bacterial endotoxin (LPS)-induced NF-кB activity and the expression of
PGE2 as well as Cox-2 (Kalra et al., 2008). One of the interesting possi-
bilities to investigate is whether bromelain generates cell-permeable peptide
fragments similar to the synthetic NF-кB essential modulator binding
domain peptides that possess NF-кB suppressing capacity. Bromelain can
stimulate the innate immune system by activating neutrophils to produce
ROS. Bromelain, as an ingredient of a polyenzyme preparation, stimulates
the production of ROS and cancer cell killing potential in neutrophils in
vitro. Similar activities are also exhibited in the neutrophils isolated from
healthy volunteers taking same polyenzyme preparation (Brakebusch et al.,
2001). ROS is also involved in intracellular regulation of functional activity
in other cell types. Bromelain capacity to amend the levels of intracellular
ROS would have a direct influence on the signaling pattern in immune and
cancer cells. It is established that activated neutrophils and unwarranted
ROS production bring about DNA damage and cancer pathogenesis. Thus
excessive production of ROS is front-runner of oxidative stress conditions
which are advantageous for cancer (Roessner et al., 2008). Recent reports
have proposed that cancer cells induce the functional activity of neutrophils
together with ROS production. However, in view of overall cytotoxic/anti-
cancer activities of bromelain, it could be predicted that bromelain activity
on ROS production in cells obtained from cancer patient may be focused
towards inhibition of cancer. Further studies are essential to reveal these
mechanisms (Klink et al., 2008).
8.3.2 BROMELAIN RELIEVES OSTEOARTHRITIS
Osteoarthritis is commonly occurring form of arthritis in Western world

and in the United States and its prevalence ranges from 3.2 to 33%
depending on the joint (Lawrence et al., 1998). Comparative study of
diclofenac and a combination of bromelain, trypsin, and rutin in about
100 patients having osteoarthritis of the knee for 6 weeks treatments

resulted in considerable and similar reduction in the pain and inflamma-
tion (Akhtar et al., 2004). As a food supplement, bromelain may act as a
substitute for nonsteroidal anti-inflammatory drugs. It plays a vital role in
the pathogenesis of arthritis (Brien et al., 2004). Bromelain has analgesic
properties which result from its direct influence on pain mediators such as
bradykinin (Kumakura et al., 1988).
8.3.3 COAGULATION REGULATORY ROLE
Oral administration of bromelain significantly lowers adenosine phosphate

induced platelet aggregation ex vivo (Heinicke et al., 1972). In the study on
the effect of bromelain on plasma fibrin (ogen) and blood coagulation, it
displayed twin activity on blood coagulation, that is, at low concentration
it showed a procoagulant effect while at higher concentration anticoagulant
effect was observed (Errasti et al., 2016). Surgeons need to pay atten-
tion while prescribing bromelain to the patients with bleeding disorders.
However, some researchers reported contrary to the above findings that in
healthy volunteers, bromelain does not have a significant effect on the mech-
anism of blood clotting. Clinical study on breast cancer patients and healthy
volunteers with oral bromelain treatment showed increment in the activated
partial thromboplastin time while prothrombin time and plasminogen
remained unaltered (Eckert et al., 1999). In another clinical trial, patients
with edema and inflammation were treated with 40-mg oral bromelain four
times daily for 1 week. No significant therapeutic effect was observed on
prothrombin time, bleeding, and coagulation which suggested that used
dose of bromelain did not affect blood clotting (Cirelli and Smyth, 1963).
Bromelain increases the serum fibrinolytic ability and inhibits the synthesis
of fibrin, and thereby affects blood coagulation. Dose-dependent effect of
bromelain on serum fibrinogen level is observed in rats. At the higher dose
of bromelain, prothrombin time, as well as activated partial thromboplastin
time, is markedly prolonged. Studies have proved that bromelain is an effec-
tive fibrinolytic agent (Taussig and Batkin, 1988).
8.3.4 SKIN WOUND HEALING ACTIVITY
Bromelain topical application to burns and skin wounds has been demon-
strated to be an effective and safe method for necrotic tissue debridement,
an alternative to surgical debridement (Cordts et al., 2016; Krieger

et al., 2012). It also finds support from the reports of other workers
who have proved bromelain local applications to be noninvasive, safe
and effective, and promotes natural wound healing (Koller et al., 2008).
Debridement of necrotic tissue is due to a non-proteolytic component
escharase having a molecular weight of 45,000 Da and is present in
bromelain extract which also helps in healing. Method for isolation of
escharase from pineapple stem is known. Escharase assists in digestion
and separation of devitalized eschar tissue from burn injury (Houck et al.,
1983). In a multicenter, open-label, controlled and randomized clinical
study, patients having deep partial and complete thickness burns were
treated with a bromelain-rich topical agent NexoBrid which was applied
for 4 h or by standard of care. There was reduced time to complete debride-
ment, need for surgery and need for autografting in bromelain treatment
group (Rosenberg et al., 2014).
8.3.5 ANTIMICROBIAL ACTIVITY
Bromelain supplementation protects animals against Escherichia coli and

Vibrio cholerae enterotoxin-mediated diarrhea. Bromelain acting as anti-
adhesion molecule modifies the receptor attachment sites and hence affects
the secretory signaling pathways in the intestine (Chandler and Mynott,
1998). Bromelain efficacy against specific infections has been attributed
to its capability to resist certain effects of specific intestinal pathogens as
well as its synergistic action with antibiotics. It also shows antihelminthic
activity against the intestinal nematodes in vitro (Stepek et al., 2005; Stepek
et al., 2006). On the other hand, in vitro antifungal activity of bromelain in
presence of trypsin against Candida albicans is accounted for stimulation
of phagocytosis and respiratory burst killing. An infectious skin disease
condition, pityriasis lichenoides chronica, has been shown to be resolved
completely by bromelain (Massimiliano et al., 2007). In humans it also
elevates the levels of some antibiotics in blood and urine (Shahid et al., 2002).
Antibiotic therapy combined with bromelain has shown better efficacy than
treatment with antibiotics alone in bronchitis, pneumonia, sinusitis, cuta-
neous Staphylococcus infection, thrombophlebitis, cellulitis, pyelonephritis,
urinary tract infections, and in rectal and perirectal abscesses (Mori et al.,
1972). In children with sepsis, bromelain, trypsin, and rutin combination
as an adjuvant therapy with antibiotics has produced good results (Shahid
et al., 2002). An improvement in protein utilization has been observed in
elderly nursing home patients administered with a blend of bromelain and

Aspergillus niger derived enzymes (Glade et al., 2001). Further combination
of bromelain with sodium bicarbonate, sodium alginate, and essential oils
has been shown to improve dyspeptic symptoms considerably. Success of
bromelain administration as a digestive enzyme for the treatment of exocrine
pancreas insufficiency, pancreatectomy, and intestinal disorders has also
been reported. A mixture of bromelain, ox bile, and pancreatin is effective in
reducing stool fat elimination in pancreatic steatorrhea patients, resulting in
symptomatic improvements in pain, flatulence, and stool frequency (Pelli-
cano et al., 2009).
8.3.6 CELL GROWTH MODULATION BY BROMELAIN
Impaired the cell cycle in normal cells might proceed to uncontrolled cellular
growth and result in transformation to cancer cells. Concerted interaction
of various pathways inside the cells provides protection to their DNA from
ensuing injury due to genomic instability and toxicity (Chobotova et al.,
2010). Checkpoint proteins are critical for monitoring the normal cell cycle
activity. Checkpoint controls are often lost in tumor cells and thus for
cancer chemotherapy, control of cell cycle is used as one of the essential
tactics (Beuth et al., 2005). Bromelain inhibits NF-κB translocation through
G2/M arrest to apoptosis in human epidermoid carcinoma and melanoma
cells. The process of apoptosis is fundamental in the developmental and
homeostatic maintenance of complex biological systems (Báez et al.,
2007). The apoptotic changes are brought about by shrinkage of the cell,
chromatin condensation, and fragmentation of DNA and the activation of
caspases, the cysteine proteases. Generally, apoptosis is achieved by either
mitochondrial pathways (intrinsic) or death receptor pathways (extrinsic).
The mitochondrial pathway is characterized by the upregulation of the
expression of a pro-apoptotic protein, Bcl-2-like protein 4 (Bax) through
p53 acting as a transcription factor. Bax antagonizes an anti-apoptotic
protein Bcl-2 present in the mitochondrial membrane (Snowden et al.,
2001). In case of an increase in Bax/Bcl-2 ratio, the protection provided
by Bcl-2 on the mitochondrial membrane is interrupted. This facilitates
the release of cytochrome c into the cytoplasm and attaches with apoptotic
protease activating factor-1 to form an apoptosome complex. It activates
caspase-9 that causes commencement of the caspase cascade resulting
in the enzyme-mediated destruction of cytoplasmic proteins and DNA
damage and ultimately leads to cell death (Guimarães-Ferreira et al., 2007).
Bromelain upregulates expression of p53 and increases Bax expression

causing the release of cytochrome c in tumor cells which selectively induces
the mitochondrial apoptotic pathway (Tysnes et al., 2001). Furthermore,
bromelain diminishes the activity of Akt and extracellular signal-regulated
kinases, the cell survival regulators and thereby encourages apoptosis
mediated tumors cell death (Mantovani et al., 2008). Bromelain treatment
has been shown to cause matrigel invasion capacities as well as cell growth
inhibition in mouse tumor cell lines in vitro (Juhasz et al., 2008). In addition,
bromelain treatment has been shown to considerably reduce the growth of
gastric carcinoma Kato-III cell lines.
8.3.7 EFFECT ON ANGIOGENESIS AND METASTASIS
High mortality rates associated with cancer results because of the metastatic
migration of cancer cells from the original site. Four interconnected biological
events are essential for tumor metastasis namely cell invasion, cell prolifera-
tion, cell adhesion, and the angiogenesis (Kleef et al., 1996). Interestingly,
the antitumor activity of bromelain is associated with its obstructive effect
on tumor cell metastasis as it potentially hampers the metastatic progression
of tumor at an array of critical points. Bromelain activity is mediated by
inhibition of cell surface adhesion proteins, the key elements responsible for
important pro-cancer events such as cell adhesion, migration, and inflam-
mation. This inhibition is predominantly imposed by suppression of NF-κB
activation. Furthermore, bromelain inhibits the invasiveness of human
cancer cells by suppressing matrix metalloproteinase (MMP)-9 expression
(Philchenkov, 2004; Li et al., 2005) through inhibiting activator protein 1
(AP-1) and NF-κB signaling pathways. A study conducted on bromelain
reported that it primarily inhibits the phosphorylation of NF-κB leading to
a reduction in the c-Jun N-terminal kinases’ phosphorylation and conse-
quently activation of AP-1. A relationship between platelets and tumor cells
are commonly observed in malignancies. Platelet activation and the platelet-
based production of numerous factors enabling angiogenesis is initiated
by tumor cells. In addition, tumor cells have the capability to cover them-
selves with the platelets, making tumor-platelet aggregates which provide
protection to tumor cells from immune recognition. Oral administration of
bromelain has been shown to reduce platelet aggregation and activation in
vitro (Garbin et al., 1994). Moreover, in vitro bromelain treatment is associ-
ated with a reduction in platelet count in healthy volunteers (Gläser and
Hilberg, 2006). Proteolytic activity of the bromelain has been accounted for
inhibition of platelet activation. Thus, bromelain obstructs platelet‑mediated
tumor growth and development, and thwarts the production of tumor-platelet
aggregates by uncovering cancer cells and revealing them to the immune
system (Kalra et al., 2008). The induction of growth of new blood vessel
is a necessary step for tumor development and metastasis so as to arrange
for the metabolic requirements of briskly proliferating malignant cells.
Angiogenesis is regulated by numerous pro-angiogenic genes and signaling
molecules. Anti-angiogenic effect of bromelain has been displayed against
many cancer cell lines (Karlsen et al., 2011). Bromelain regulates a range
of pro-angiogenic growth factors, enzymes, and transcription factors (Wu
et al., 2012). Bromelain also prevents the angiogenic response produced by
FGF-2 arousal in endothelial cells of mouse and reduces the MMP-9 expres-
sion, an enzyme associated with tissue remodeling that is important for the
growth and development of new blood vessels (Wallace, 2002). Further-
more, bromelain treatment has been shown to decrease the levels of COX-2
and VEGF, the angiogenic biomarkers in hepatocellular carcinoma cells,
and caused a decline in tumor neo-capillary density when compared with
untreated cells. Bromelain has also been proven to affect several cellular
adhesion molecules linked with the processes of tumor development and
metastasis (Juhasz et al., 2008).
8.4 CONCLUSION
Bromelain occupies the vital position with respect to its vast medicinal
and industrial applications. It has received growing acceptance among
patients and researchers as a phytotherapeutic drug. Bromelain provides
numerous therapeutic benefits including anticancer, antimicrobial, anti-
inflammatory, antithrombotic, coagulation regulator, and immunomodu-
latory activities.
ACKNOWLEDGMENT
Ramesh Kumar acknowledges financial support from CSIR as Junior

Research Fellow. Authors acknowledge UGC-SAP and DST-FIST Facilities
of the Department of Biochemistry, University of Allahabad, Allahabad for
providing necessary infrastructure.
KEYWORDS
•• bromelain
•• enzyme
•• Ananas comosus
•• industrial applications
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CHAPTER 9
CYSTEINE PROTEASES FROM PLANTS

AND THEIR APPLICATIONS
JUAN ABREU PAYROL1*, WALTER DAVID OBREGÓN2,
JULIANA COTABARREN2, T. B. MUTSAURI1, and
INIDIA RUBIO VARGAS1
1
Departamento de Farmacia, Instituto de Farmacia y Alimentos,
Universidad de La Habana, Calle 222, entre 23 y 29, # 2317,
La Coronela, La Lisa, CP 13600, La Habana, Cuba,
Tel: 5372020930/2716789/2679207
2
Departamento de Ciencias Biológicas, Facultad de Ciencias
Exactas, Centro de Investigación de Proteínas Vegetales (CIPROVE),
Universidad Nacional de La Plata, 47 y 115s/N, B1900AVW, La Plata,
Argentina, Tel: 542214226977/6979/6981, Telefax: 542214226947
*
[email protected]
ABSTRACT
Cysteine proteases are everywhere in nature. In the plants, they perform

multiple functions associated with growth and development. This chapter
addresses the essential aspects associated with the main botanical families
studied by the presence of these enzymes, their classification in clans, fami-
lies and subfamilies, and other subgroups; fundamental structural features
associated with each group and their relationship with the mechanism of
action; the main functions that they perform in plants and applications they
have today in social life, as well as other potentialities that systematic study
can provide. In general remarks, the principal applications and potentiali-
ties of these enzymes are shown, especially of those more studied: papain
and bromelain. The chapter also identified the major sources of information
about cysteine proteases in web databases; also a general bibliography about
the theme supports these comments, useful for those academics interested in
deeper knowledge about these interesting enzymes.
9.1 INTRODUCTION
Due to the various ways in which proteolytic enzymes affect the health and
well-being of humanity, this century has seen a remarkable acceleration on
the pace of research in peptidases, revealed in a number of annual publica-
tions on their study which already exceeded 1885 citations in the PubMed
database of the US National Center of Biotechnology Information, only in
the course of 2017.
The MEROPS database (https://www.ebi.ac.uk/merops/), major web
database on proteolytic enzymes, their inhibitors, and substrates, which
reached 20 years in 2016, increasing the number of peptidase sequences
registered in its systematic updates: 413,834 (August 2013), 523,871 (July
2015), and 912,290 (September 2017). The analysis of complete sequences
of several genomes has shown that approximately 2% of the information
encoded by genes are peptidases, indicating that this is one of the larger
functional groups of proteins (Barrett et al., 2012).
Cysteine proteases of plants are a well-characterized group of proteolytic
enzymes, among which are those in clan CA, a superfamily of papain (family
C1) has been studied. This family includes endopeptidases with different
specificities, aminopeptidases, exopeptidases, and some members without
catalytic activity, but the plant sources produce endopeptidases a widely
applied in medicine and foods, among others fields.
The forecast for the 2022 market for enzymes of plant origin exceeds
USD 41 billion, of which an important part is cysteine plant proteases. Due
to its qualities and wide industrial application, this trend must be maintained.
These arguments reveal the need to advance in the research of this group
of natural products, of wide perspectives and potential impact in branches
important for humanity as health and nutrition.
9.2 CLASSIFICATION/STRUCTURE
In cysteine-type peptidases, the sulfhydryl group of a cysteine residue attacks

the peptide bond, as a nucleophile. Also, a proton donor and general base
are required; in this case, a histidine residue (Azarkan et al., 1996). Among
the plant peptidases, cysteines presently represent around 19.6% of the 912,
Cysteine Proteases from Plants and Their Applications 223
290 sequences annotated in the MEROPS database, the third in abundance

after serine peptidases (36.6%) and metalloproteinases (32.7%) (Rawlings
et al., 2016). Cysteine proteases are grouped into 92 families belonging to 16
different clans (Rawlings et al., 2016). In 2008, 76 families belonging to 13
different clans were recorded(Caffini, 2009), while in 2004 they had scored
40 families and 6 clans (Grudkowska and Zagdanska, 2004), which reveals
a sustained increase in research in this field.
Forty-one families are belonging to the CA clan, which makes it the
most important of all. Another 22 families are part of the CD, CE, CF, CL,
CM, CN, CO, CP, CQ, and CR clans; all share the mechanism of catalysis
that includes the nucleophilic cysteine. Another 18 families are belonging
to clans PA, PB, PC, and PD, which include peptidases with other catalytic
mechanisms (serine and threonine) and 11 remain without assignment to a
clan. The majority, including the main and best-known cysteine proteases,
are found in the CA clan particularly in the C1 family, the papain family,
C01.001, EC 3.4.22.2, clan archetype. The C1 family is subdivided into two
subfamilies such as C1A and C1B. The cysteine peptidases that are grouped
in C1A have been evolutionarily very successful, represent the largest group
of these enzymes in the eukaryotes and are much distributed in the vegetable
kingdom, Including secreted and lysosomal (or vacuolar) enzymes, mainly
endopeptidases. Along with them in this subfamily, there are endopeptidases
of DNA viruses, protozoa, and animals. There are also some exopeptidases
of bacteria, fungi, and animals.
The distinction between exopeptidases and endopeptidases in the
subfamily is unclear. In some members, one or the other activity may predom-
inate, although against some substrates they act in reverse. The sequences
have been assembled in the same clan, considering that the structures whose
crystallographic data are known are similar or because they contain motifs
with similar sequences in the environment of the catalytic residues (Barrett
et al., 2012). There are 40 peptidases of the C1 family that have data of the
three-dimensional structure obtained by X-ray crystallography with different
degrees of resolution, in some cases, forming complexes with inhibitors,
including 12 of plant origin, it is worth mentioning papain, caricain, chymo-
papain, and stem bromelain. In total, 227 enzymes throughout the family
have been completely sequenced(Rawlings et al., 2016). In Figure 9.1, it can
be seen that the simple polypeptide chain of papain forms two structural and
between them the active site cleft. The N-terminal domain widely is a set
of α helices, while the C-terminal domain contains a β-barrel. A long helix
is along the upper center of the molecule and the catalytic cysteine is at the
start of this (Barrett et al., 2012).
FIGURE 9.1 (See color insert.) Structure of papain refined at 1.65 Å resolution.
Source: Adapted from Kamphuis et al. (1984). (The image was prepared from the protein
data bank entry 9PAP.)
9.2.1 STRUCTURAL CHARACTERISTICS OF SUBFAMILY C1A

TYPE PAPAÍNA
Some basic structural characteristic features of the peptidases of C1A

subfamily, particularly those of plant origin, stand out as common elements
in this group (Barrett et al., 2012):
• They are α/β proteins, with the nucleophilic Cys at the beginning of a
helix and the catalytic His at the start of a β-sheet. A typical feature is
the presence of 3 disulfide bonds, sometimes more.
• Many are inhibited by the compound E-64 (Fig. 9.2) irreversibly and
by proteins of the cystatin family, although some cystatins can inhibit
legumain, a CD clan peptidases, because they have a second reac-
tive site and some inhibit also metallopeptidases (Alvarez-Fernandez
et al., 1999; Valente et al., 2001).
• Those that enter in the secretory pathway usually exist as inactive
precursors, with N-terminal propeptides (signal peptides); for
mature (active) enzyme they also function as inhibitors. Propeptides
homologous exist in most members of the C1A family, similar to
that of papain, with 100 or more residues (those of cathepsin B are
shorter and of different sequence) and must act the same, blocking the
active site when joining it in an inverted position to which a substrate
would make it. The papain-type propeptides can be identified by
the presence of the ERFNIN motif, in which some of the following
residues are conserved (numbered according to the propeptide of
papain): Glu64, Arg68, Phe72, Asn75, Asn83, Phe96, Asp98, and

Glu103 (Novinec and Lenarcic, 2013). It is emphasized that the Pro2
residue is frequently conserved in mature peptidases, this can prevent
the attack by aminopeptidases since the Xaa-Pro bond is resistant to
many such enzymes.
FIGURE 9.2 (See color insert.) Structure of E-4.

Source: The image was created with MarvinSketch 17.29.0 from Chemaxon (https://www.
chemaxon.com).
• Most C1A cysteine peptidase propeptides consist of two parts

and share a similar fold. The N-terminal portion is formed by two
α-helices and an extended β-sheet and interacts with a “binding pro-
union loop” located superficially on the mature protease. There is
a long central α-helix of 25–30 residues that contain the ERFNIN
motif. The C-terminal segment links the two domains of the enzyme,
being anchored to the S′ sites by a short α-helix. This arrangement
of the substrate binding site prevents contacts with the catalytic site
of the enzyme. However, the modes of binding of the substrate and
the propeptide occur in opposite directions: the side chains of the
propeptide must use the same binding sites of the substrates, but the
reverse orientation of the peptide chain results in such a position of
the peptide bond that it makes it resistant to rupture (Harrison et al.,
1997; Rzychon et al., 2004).
• Plant peptidases appear to have homologous C-terminal extensions to
papain (Lycopersicon esculentum, Arabidopsis thaliana, and α and β
oryzains of Oryza sativa) (Barrett et al., 2012) (Fig. 9.3).
FIGURE 9.3 (See color insert.) Full protein feature view of papain and cathepsin B from
Trypanosoma brucei showing propeptides length.
Source: Adapted from Kamphuis et al. (1984) and Koopmann et al. (2012). (The image was
prepared from the Protein Data Bank entries 9PAP and 3MOR, using de entries P00784 and
Q6R7Z5 from UniprotKB database. In grey all length sequence, in green signal peptide +
activation peptide (propeptide domain) and peptidase chain (Ec 3.4.22.2 for papain).
• Most of the peptidases of the C1A subfamily and all plants with
known structure are monomers, although they exist in multi-domain
organizations (Barrett et al., 2012) (Fig. 9.4).
FIGURE 9.4 (See color insert.) Structure of multi-domain protease from Crocus sativum
refined at 1.31 angstroms resolution. (The image was prepared from the Protein Data Bank
entry 3U8E. There are three identical units join by weak electrostatic interactions.)
From an evolutionary point of view, several groups can be distinguished

within the enzymes of the C1A subfamily, two main groups are the cathepsin
L (papain type) and the cathepsin B type, presents in Eukaryotes. Enzymes
from the cathepsin B group have a shorter propeptide than cathepsin L group
and the ERFNIN motif is not conserved in these cathepsin B type peptidases
propeptides (Novinec and Lenarcic, 2013).
In the C1A subfamily, cathepsin-L type peptidases are expanded between
plants with modifications, while two other groups, cathepsin X and dipep-
tidyl peptidase I groups are absent (Richau et al., 2012).
9.2.1.1 OTHER PLANTS CYSTEINE PROTEASES IN CLAN CA
Other families in the CA clan contain plant peptidases. The enzymes from
C12 family are structurally very similar to papain. The molecules have
two lobes, one consisting mainly of helices, the other containing a β-barrel
surrounded by helices. The catalytic Cys is at the beginning of one of the
helices and the catalytic His is at the beginning of a β strand. One difference
with papain is that the first strand of the β-barrel precedes the helix in the
sequence carrying the catalytic Cys. The peptidases of this family have no
propeptides and are intracellular. They are peptidases that hydrolyze the
ubiquitin-conjugated glycine link wherever there is a α-peptide or isopeptide
bond, with diverse specificities.
The C19 family is the second group of C-terminal ubiquitin hydrolases.
Their structures are more complicated than those of C12, many are multi-
domain proteins, have a greater variety and are intracellular, but they are
capable of releasing ubiquitin from much larger polyubiquitinated peptides.
9.2.1.2 OTHER PLANTS CYSTEINE PROTEASES
Other clans and families group cysteine proteases of plant origin, but in much
less relevance than the clan CA and especially family C1, subfamily C1A.
Among the families of the CD clan is C13, which includes endopep-
tidases that break asparaginyl bonds. Among them, legumain, which has
been found in a great variety of dicotyledonous plants, in legume seeds and
is responsible for the posttranslational processing of seed proteins before
storage. It is not inhibited by the E - 64.
In the remaining families, cysteine peptidases of plant origin are rare or
have not been found.
9.3 CATALYTIC MECHANISM
A complete review of the catalytic mechanism of clan peptidases CA is cited

in Polgár (2013), which explain the role of residues Cys25, His159, Gln19,
and Asn175 in the catalysis. In mature papain, the catalytic dyad consists of
residues Cys25 and His159, which are respect to the active site in opposite
domains of the cleft. Also, they are functionally important residues Gln19
and Asn175 (Fig. 9.5).
FIGURE 9.5 (See color insert.) Tridimensional structure of papain and position of the
residues involved in catalysis Cys25, His159, Gln19 y Asn175, and Trp177.
Source: Adapted from Kamphuis et al. (1984). (The image was prepared from the Protein
Data Bank entry 9PAP.)
The sulfur atom of Cys25 and the imidazole ring of His159 are in the
same plane, atoms of sulfur and nitrogen are separated by 3.4 Ǻ, the distance
corresponding to a contact van der Waals. The imidazole ring is hydrogen
bonded to the side chain of Asn175 and the hydrogen bridge is protected
from the solvent by the indole ring of Trp177, aiding the convenient orienta-
tion of the imidazole ring in His159.
The thiol group in the active site is deprotonated by histidine, starting
the catalysis of the CA peptidases, with a basic side chain, forming the
thiolate-imidazolium ion pair. Then the thiolate anion of the deprotonated
cysteine nucleophilically attacks the sessile peptide bond over the carbonyl
carbon (Fig. 9.6A). The oxygen atom, negatively charged, allows to form
the first tetrahedral state of transition. Oxyanion is stabilized by hydrogen
bonding with the NH groups of the side chain of Gln19 and the skeleton
of Cys25, which results in the formation of oxyanion hole (Fig. 9.6B), an
electrophilic center that stabilizes the tetrahedral intermediary. Following
rotation of the His159 residue allows proton transfer of the imidazolium
cation to the peptidic nitrogen in the bond to be hydrolyzed and that is
when the break occurs. The newly formed amine substrate is bounded by
a hydrogen bond to His159, while the carboxylic part of the substrate is
linked to Cys25 through a thioester bond, forming the acyl-enzyme inter-
mediate (Fig. 9.6C).
The next reaction step involves the exit of a fragment of the substrate
with an amino-terminal and the attack of a water molecule. The histidine
residue is reconverted to its deprotonated form and imidazole nitrogen
contributes to the polarization of the water molecule, which then attacks
the acyl-enzyme over the carbonyl carbon (Fig. 9.6D), resulting in the
second tetrahedral intermediate (Fig. 9.6E). In the final step, the deacyla-
tion of the thioester leads to the recovery of the carboxyl group in the
hydrolyzed substrate, at the same time of the release of the active enzyme
(Fig. 9.6F) (Harrison et al., 1997; Rzychon et al., 2004). An interesting
feature is that when the active site has accommodated the substrate, the
slot is extended by 1 Å.
FIGURE 9.6 (See color insert.) Schematic representation of the catalytic mechanism of
papain.
chemaxon.com).
Most papain-type peptidases have a broad specificity, and the main deter-
minant is the residue at P2 position in the substrate (Berger and Schechter,
1970; Novinec and Lenarcic, 2013) (Fig. 9.7). They accept in P2 position
residues with a voluminous and hydrophobic side chain not charged,
although there are variations in this behavior.
FIGURE 9.7 (See color insert.) Nomenclature of schechter y berger (1970) for the
specificity of the substratum of a peptidase.
Source: Adapted from Berger and Schechter (1970).
The enzyme cathepsin B readily accepts Arg, which can be explained

by the fact that the residue that lies at the bottom of pocket S2 in papain is
Ser205, but in cathepsin B is Gln.
Among some interesting variations in cysteine peptidases mechanisms
are the Asn 175 residue, which contributes to the orientation of the imidazo-
lium ring of the catalytic His159, is occasionally replaced by Asp in the C12
family and others. Also, the catalytic Cys25 is usually followed by a hydro-
phobic aromatic amino acid, and in all the known ones of the C12, glycine
occupies that position. In Clan CD, the C13 family is of interest for the pres-
ence of cysteine peptidases from vegetable origin, legumains. Experimental
evidence indicates that in the catalytic mechanism of these enzymes does not
exist the ion pair thiolate—imidazolium or is different from the existing one
for the CA clan (Csoma and Polgár, 1984). Papain-type cysteine peptidases
from plants are usually endopeptidases but may exhibit other activities as
result of structural modifications to the folding of papain, which restrict the
access of substrate to the active site in some side of its cleft, probably as an
evolutionary strategy of the organisms in which they exist. This is the cause
of that we find total or partial activity as exopeptidases, carboxypeptidases,

and aminopeptidases (Novinec and Lenarcic, 2013).
Because the intensity of research in cysteine peptidases, the number of
clans, families, and structures that continue to appear is growing. Although
in plants predominate the features described, apparently for evolutionary
reasons; particularly in bacteria and viruses appear not only different folds
but different catalytic entities have been found. It would not be surprising
that novel studies on peptidases of the vegetal kingdom have similar results.
9.4 ROLE IN NATURE
Plant proteases take part in almost all life processes in plants. They are
implied in several physiological processes, including protein degradation,
digestion, maintenance of cells, signaling, differentiation, growth, develop-
ment, apoptosis, maturation, synthesis, degradation of reserve proteins during
the germination of seeds, circadian rhythms, senescence, and programmed
cell death (PCD). They play fundamental roles to regulate the biological
processes, such as recognition of pests and pathogens (Konno et al., 2004)
and in the effective induction of defensive responses and in resistance to
unfavorable conditions that include the attack of herbivores, water, and
environmental stress (García-Lorenzo, 2007; Caffini, 2009).
Many of plant enzymes of the C1 family are essentials in protein
degradation in vacuoles. They appear in fruits, particularly immature; their
activity prevents insects feeding and hydrolyzes endogenous proteins during
the maturation of fruits (Turk et al., 2012).
In the natural selection process some families of plant cysteine proteases
have been diversified in the competition between plants and their pathogens:
plants produce proteases that suppress the growth of fungi, fungi generate
inhibitors specific to these proteases and these are diversified to favor the
immune response (Kaschani et al., 2010).
Perhaps, the most important function is their involvement in proteosomes,
related to various metabolic processes such as hormonal signaling, cell cycle,
embryogenesis, morphogenesis, floral development, and oxidative stress
(Watanabe and Lam, 2005). It is usually considered that these proteases have
cleaning functions (“housekeepers”), eliminating nonfunctional proteins and
recycling the amino acids released in the hydrolysis. However, some appear
to be part of a signal cascade (Sasabe et al., 2000), others seem to act in
presence of predators by different mechanisms: (a) liberating elicitors of the
invader who, when recognized, unleash the defense mechanism; (b) binding
of the elicitor to the protease causing the activation thereof, triggering a

cascade of reactions involving the breakdown of proteins involved in the
defensive process; and (c) binding of the elicitor to the protease could inhibit
its activity, acting on elicitor—protease complex as a signal to unleash the
defensive mechanism (van der Hoorn and Jones, 2004).
Peptidases type cathepsin L represent the majority of the repertoire of
peptidases of the C1 family in plants, the most recognized are the papain
(papaya, Carica papaya L.), bromelains (pineapple, Ananas comosus L.),
and ficain (figs, Ficus sp.), produced by plants in large quantities and very
used for medicinal products, in food industry and for numerous other appli-
cations (González-Rábade et al., 2011).
Papain and bromelain protect plants from parasites, such as fungi or
insects (Konno et al., 2004) (Lopez-Garcia et al., 2012). For other homolo-
gous peptidases also have been described roles in plants immunity (Shindo
et al., 2012). Xylem-specific peptidases implicated in PCD related with
xylogenesis have been found (Avci et al., 2008) and others with general
senescence of plants (Otegui et al., 2005).
Peptidases of type cathepsin L (papain), B, H, and F are mainly respon-
sible for the proteolytic activity during the senescence of the leaves. In
this crucial process, nutrients from leaf are relocated to storage or growing
tissues. Massive protein degradation involves extensive metabolic networks,
different subcellular compartments, and various types of proteases and regu-
lators (Diaz-Mendoza et al., 2014).
9.5 APPLICATIONS OF VEGETABLE CISTEIN PEPTIDASES
Historically, peptidases have had multiple applications, in many cases as

mixtures and not as purified enzymes. In ancient times, fig juice was used to
coagulate the milk (contains ficaina) and rennet from the stomach of cattle to
make cheese (contains chymosin). Industrially they are used to remove hair
from skins and other modifications in the leather industry, to tenderize meat,
clarify beers, enhance the flavors of cheeses, and others foods, as biological
detergents and cleaning liquids for contact lenses, among other applications.
In the area of medicine, they are used to eliminate gastrointestinal parasites,
removal of dead skin of burn patients, determination of blood groups, and
so forth. They are of relevant value in current medical research, molecular
biology, and biotechnology.
The beginning of its great commercial boom was marked in the 80 s of the last
century, global enzyme trade in the middles of 80 was well above the 500 million
annual dollars, at that time an annual growth of 5% was predicted, estimated that
was the real growth that was reaching in subsequent years, for example, in 1997
the total value traded was close to 1500 million dollars (Caffini, 2009).
Towards the end of 1990s, it was reported that the increase in those
years was of the order of 6.5% per year, initially was estimated that for
2009 would reach a figure close to 5100 million dollars; being the proteases
among the enzymes more required, the fundamental reason due to the wide
use of these enzymes in the processing of materials of natural origin, but also
was foresaw the development of novel applications, in particular pharma-
ceuticals, such forecast result are accurate, given the multiple applications
that have gradually reached such enzymes (Caffini, 2009).
On the other hand, these enzymes are capable of activating the target
protease receptors and in this way, they will act as important pharmacological
and toxicological agents (Domsalla and Melzig, 2008). Cysteine peptidases
are usually obtained from fruits such as papaya (C. papaya), kiwi (Actin-
idia chinensis), and pineapple (A. comosus). However, the most come from
microbial sources, although several plant peptidases remain irreplaceable for
some applications, in particular, papain, bromelain, and ficain. Nowadays, the
interest in them grows due to the wide variety of their medical and industrial
applications and research reports. However, the number of plant proteases
isolated and characterized continues relatively very low (Caffini, 2009).
9.5.1 PAPAIN TYPE ENZYMES IN GENERAL
The fruit of green papaya (C. papaya), the leaves and the latex of the bark
are rich in enzymes known as papain, the better well-known and the most
applied.
In the area of foods, the papain-like cysteine peptidases have been used
in the manufacture of chewing gum, toothpaste, meat tenderizers, beer’s
clarification (for its value for foam production and malting of beer as result
of the fermentation of barley or basic cereal), cheese production, preser-
vation of species, food supplements (its positive effect on casein and cow
whey protein degradation in the stomach of children is used), and so forth. In
addition, it is also believed that these enzymes have antifungal, antiviral, and
also antibacterial properties (Berger and Asenjo, 1940; Buttle et al., 2011;
Esti et al., 2013; He et al., 2014).
In December 2017, a review of World Intellectual Property Organization
(WIPO) patents database registers 5143 hits related to papain, 108 of them
in 2017, an excellent number in this group of compounds.
In medicine, they are applied to treat digestive problems, wound, and

burns healing (improve epithelialization and reduce wound size) (Gurung
and Škalko-Basnet, 2009; Singh and Singh, 2012). There are studies in
other directions, such as the removal of kidney stones, hypertension,
urinary tract problems, menstrual pains, analgesia, dysentery, diarrhea,
and fever (Hasimun and Ernasari, 2014) (Berger and Asenjo, 1940; He
et al., 2014).
They seem to have an essential role in the degenerative and invasive
diseases of the immune system (e.g., cathepsin K catalyzes bone degradation
in osteoclasts, its selective inhibition could be valuable in osteoporosis, some
forms of arthritis) (Lecaille et al., 2002). Papaya leaf extracts significantly
inhibit proliferative responses of solid tumor cell lines taken from cervical
carcinoma, in addition, for example, remove adenocarcinomas, hepatocel-
lular carcinomas, lung adenocarcinoma, pancreatic epitheloid carcinoma,
and mesotheliomas (Ikram et al., 2015).
They are applied in many other areas, such as cosmetics, textiles,
detergents, food industry, baking, obtaining of modified proteins (protein
hydrolysates) destined to food industry, manufacture of leather, recovery of
silver, and the treatment of industrial waste with high protein concentra-
tion (such as “tails”of fishmeal industry) (Berger and Asenjo, 1940; Caffini,
2009; Amri and Mamboya, 2012).
Papains (a mixture of papaya peptidases) helps to protect immature
fruits, for its action in the control of diseases of plants produced by parasites
such nematodes and arthropods (Miller and Sands, 1977; Konno et al., 2004;
Stepek et al., 2007).
Cysteine peptidases from papaya are much more likely to succeed in
terms of safety and tolerability than other medications that are used as
anthelmintics because they are part of the current human food (Stepek
et al., 2006).
They are also used in the production of peptides, synthesis of molecules,
and pharmaceutical products industry (Berger and Asenjo, 1940; He et al.,
2014). The relatively recent and novel application in the synthesis of peptides
in nonaqueous media (obtaining of aspartame sweetener) opens a road to
growth (Bordusa, 2002).
9.5.2 OTHER PLANTS CYSTEINE PEPTIDASES
Bromelain is the second phytoprotease in importance, after papain and

the most studied in Bromeliaceae, is found in leaves, stem, and fruits of
pineapple (A. comosus). In 2005, more than 320 patents on their industrial
applications and pharmaceutical companies were published, of them about
130 were American, 95 were Japanese, and almost all the others from
European countries, concentrated in the two decades prior to that date. In
December of 2017, a revision in WIPO patent database records 529 entries,
12 of them in 2017, a notable rythm in last 12 years.
They are much applied in various areas, such as food industry and
cosmetics. Highlight their potential in medicine (Maurer, 2001), mainly,
because of its anti-inflammatory and anticancer properties, in addition to its
ability to induce cellular apoptosis (Arshad et al., 2014).
It has been applied to treat the rheumatoid arthritis, circulatory disor-
ders (bruising, thrombophlebitis, and coagulation), oral, rectal and peri-
rectal inflammation, ulcers, diabetes, angina, bronchitis, sinusitis, wound
healing, surgical traumas, pyelonephritis, relieves pain, and swelling and
to strengthen the absorption of drugs such as antibiotics. It is considered
nontoxic and without side effects, it is possible to use it in doses between 200
and 2000 mg/kg for long periods (Taussig and Batkin, 1988; Maurer, 2001;
Salas et al., 2008; Pavan et al., 2012).
Other therapeutic areas are now actively exploring, some with results
known from traditional medicine (anthelmintic) and its presence in the
market grows continuously (Arshad et al., 2014).
Ficain, from fig (Ficus sp.) is another cysteine protease used in the food
processing (brewery, meat, and so forth.) (Homaei et al., 2014). Unfortu-
nately, it has not been well characterized, partly because of it self-hydrolyzes
(Baeyens-Volant et al., 2015).
Pinguinain has been slightly studied. It is obtained from the fruits of
Bromelia pinguin. It has been successfully tested to clean the devitalized
tissue in bedsores, it can be useful as fibrinolytic, clot breaker, and anti-
inflammatory (Toro-Goyco et al., 1968), although do not appear in scientific
literature studies that endorse them directly. On the other hand, an effec-
tive procedure was developed to separate schistosoma eggs (Schistosoma
mansoni) from tissues of infected animals, as part of a diagnostic procedure
of the infection with this parasite (Toro-Goyco and Rodriguez-Costas, 1976;
Toro-Goyco et al., 1980).
Its capacity as an anthelmintic was evaluated and in the synthesis of
nonaqueous systems, with encouraging results. It is expected that pinguinain
behavior would be similar to the proteases of A. comosus, in particular to
fruit bromelain, with which keeps the greatest similarities in composition
and properties (Payrol et al., 2005, Caffini, 2008).
KEYWORDS
•• plant cysteine proteases

•• clan CA
•• family C1
•• papain
•• bromelain
REFERENCES
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Phytochemistry, Volume 3 A
FIGURE 9.1 Structure of papain refined at 1.65 Å resolution.

Source: Adapted from Kamphuis et al. (1984). (The image was prepared from the protein
data bank entry 9PAP.)
FIGURE 9.2 Structure of E-4.

chemaxon.com).
B Phytochemistry, Volume 3
FIGURE 9.3 Full protein feature view of papain and cathepsin B from Trypanosoma brucei
showing propeptides length.
Source: Adapted from Kamphuis et al. (1984) and Koopmann et al. (2012). (The image was
prepared from the Protein Data Bank entries 9PAP and 3MOR, using de entries P00784 and
Q6R7Z5 from UniprotKB database. In grey all length sequence, in green signal peptide +
activation peptide (propeptide domain) and peptidase chain (Ec 3.4.22.2 for papain).
FIGURE 9.4 Structure of multi-domain protease from Crocus sativum refined at 1.31
angstroms resolution. (The image was prepared from the Protein Data Bank entry 3U8E.
There are three identical units join by weak electrostatic interactions.)
FIGURE 9.5 Tridimensional structure of papain and position of the residues involved in catalysis Cys25, His159, Gln19 y Asn175, and Trp177.
Source: Adapted from Kamphuis et al. (1984). (The image was prepared from the Protein Data Bank entry 9PAP.)
C
D
FIGURE 9.6 Schematic representation of the catalytic mechanism of papain.

Source: The image was created with MarvinSketch 17.29.0 from Chemaxon (https://www.chemaxon.com).
FIGURE 9.7 Nomenclature of schechter y berger (1970) for the specificity of the substratum of a peptidase.
Source: Adapted from Berger and Schechter (1970).
E
F
FIGURE 17.4 Diagrammatic representation of the antifungal mode of action of essential oil (EO).
FIGURE 17.5 Target sites in insects as a possible neurotransmitter-mediated toxic action of essential oils.
G
H Phytochemistry, Volume 3
Bcl2-119034 CXCR4-181183
CHK1-3034821 MTH1-21577087
VEGFR2-159593 Carbonic anhydrase2-3503
FIGURE 19.12 LigPlot of lead terpenoids with Bcl2, VEGFR2, CXCR4, MTH1, CHK1
and Carbonic anhydrase 2 proteins.
FIGURE 20.5 Dynamics of activity of lectins of E. purpurea of the first year of vegetation. I—root system; II—leaf blade; III—leafstalk;
IV—stems; V—not blossoming inflorescences; VI—blossoming inflorescences. Sampling time: 1 June; 2 July; 3 August; 4 September; 5 October.
I
J
FIGURE 20.6 Dynamics of activity of lectins in leaves of E. purpurea of the first year of vegetation. Rosette leaves: I-blade; II-leafstalk; Stem
leaves: III- blade; IV-leafstalk. Sampling time: 1-June; 2-July; 3-August; 4-September; 5-October.
FIGURE 20.8 Dynamics of activity of lectins in leaves of E. purpurea of generative period of ontogenesis. Rosette leaves: I—blades; II—stems;
Stem leaves: III—blade; IV—leafstalk; Sampling time: 1—renewal of vegetation; 2—regrowth; 3—formation of inflorescences; 4—flowering;
5—fruit formation; 6—ripening of fruits.
K
L
FIGURE 20.9 Dynamics of activity of lectins of E. pallida of the first year of vegetation. I—root system; II—leaf blade; III—leafstalk; IV—
stems; V—not blossoming inflorescences; VI—blossoming inflorescences. Sampling time: 1 July; 2 August; 3 September; 4 October.
FIGURE 20.10 Dynamics of activity of lectins in rosette leaves of E. pallida of the second year of vegetation. Sampling time: I—regrowth;
II—formation of inflorescences; III—flowering; IV—fruit formation; V—ripening of fruits.
M
N
FIGURE 20.11 Dynamics of activity of lectins in stem leaves of E. pallida of the second year of vegetation. Sampling time: I–regrowth;
II–formation of inflorescences; III–flowering; IV–fruit formation; V–ripening of fruits.
FIGURE 20.12 Dynamics of activity of lectins in stems and rhizomes of E. pallida of the second year of vegetation. Sampling time: I—regrowth;
II—formation of inflorescences; III—flowering; IV—fruit formation; V—ripening of fruits.
O
P
FIGURE 20.13 Dynamics of activity of lectins in inflorescences of E. pallida of the second year of vegetation. I—date of inflorescences
forming; II—blossoming inflorescences Sampling time: 1 June; 2 July: 3 August.
CHAPTER 10
PHYTOTHERAPY AND
ENCAPSULATION
ŞABAN KESKIN1, MERVE KESKIN2,*, and SEVGI KOLAYLI2
1
Department of Chemistry, Faculty of Science and Literature, Bilecik
Şeyh Edebali University, Bilecik, Turkey
Department of Chemistry, Faculty of Science, Karadeniz Technical
2
University, Trabzon, Turkey, Tel.: +902282141641

*
ORCID: https://orcid.org/0000-0001-9365-334X
*
ABSTRACT
Phytotherapy can be defined as a treatment for preventing from exact

complaint or for assistive application to a modern remedy. Encapsulation
could be defined as coating a phytochemical with a suitable polymeric
material. This technology is widely used in the pharmaceutical industry
since the solubility and stability of a phytochemical could be increased by
this technology. Controlled release properties of phytochemicals could be
improved by this technique as well. Targeting the phytochemicals to the
right place in a body is the main scope of encapsulation. Encapsulation also
contributes for the phytotherapy practitioners to administer a phytochemical
in dose-controlled manner because encapsulation provides a determined
composition for a product. Deciding on the method to achieve better
encapsulation is principally conditioned by the chemical constituents of the
extract and the solvent used for extraction.
The current chapter discusses encapsulation methods used in
phytotherapy. The comparison of encapsulation methods by telling its
advantageous and disadvantageous sides was also discussed. In the present
chapter, one can find the necessity of encapsulation of phytochemical(s)
as well.
10.1 INTRODUCTION
Phytotherapy is a treatment either for preventing from exact complaint

or for assistive application to a modern remedy (Choubey et al., 2013).
Phytotherapeutic applications go back to ancient times. From the beginning
of prehistoric age to date, plants or herbs are used as medicines. It was
estimated that one million plant species were grown all around the world and
only 20,000 of them could be used for phytotherapeutic purposes (Parıldar et
al., 2011). In future, the number of plants will be increased with the increase
of researches being conducted on plants and herbs.
The usage of whole plant or plant part was the main phytotherapeutic appli-
cation till the beginning of 19th century. Phytotherapeutic applications have
shifted to purify the active compound(s) from the plant with the exploration
of extraction techniques and put them into a formulation alone or in combina-
tion. Phytotherapeutic compounds such as saponins, polyphenols, flavonoids,
volatile oils, and so forth, are not enduring after extraction (Arriola et al.,
2016). They may undergo some modification as they are highly susceptible to
pH, humidity, oxygen concentration, and light or they may give side reactions
with active ingredients put in the formulation together. Encapsulation of these
active components could prevent them from these side effects.
As mentioned above, phytochemicals are highly susceptible to envi-
ronmental condition after extraction, the stability of them could be ensured
by encapsulation processes. When encapsulated a phytochemical could
maintain its activity for a long time. This increased shelf life makes the
capsules potential drug or drug additive. In addition to shelf life increasing,
encapsulation also provides a phytotherapist to arrange the concentra-
tion of phytochemicals. This is because one can calculate the amount of
phytochemical(s) in the capsules after encapsulation process. This makes it
easy for the phytotherapy practitioner to estimate and prescribe the amount
of daily intake for the patient. Moreover, encapsulation also provides us
to target a phytochemical to the right place of the body by the ability of
controlled release of the encapsulated compound(s) (Desai and Park, 2005;
Nedovic et al., 2011). Encapsulation can also be used to mask any off-flavors
of a phytochemical (Stojanovic et al., 2012; McClements, 2015).
10.2 ENCAPSULATION AS BIOTECHNOLOGICAL TOOL
Encapsulation, an immobilization technique, is the packaging of solid,

liquid, or gaseous components such as phytochemicals, enzymes, cells, and
Phytotherapy and Encapsulation 243
other substances with a protein, lipid, or carbohydrate-based coating mate-

rial. Macro, micro, or nanoscale capsules could be obtained, depending on
the technique used and process conditions. The material in the capsule is
expressed as core, internal phase, or filler. However, the wall can sometimes
be a shell, a coating, or a membrane. The core could be a crystal, rough
absorbent particle, emulsion, suspension, or suspension of smaller capsules
(Gökmen et al., 2012)
Encapsulation has many underground practices in the fields of pharmacy,
veterinary, biotechnology, food industry, chemical, agriculture, feed, and
medicine. The selection of encapsulation method, an encapsulant or wall
material mainly relies on the properties of phytochemicals. By the help of
current knowledge and technology, varied size of particles can be obtained
based on the various properties of the core, wall material, and encapsula-
tion technique (Gharsallaoui et al., 2007). The morphology of capsules can
be described as mononuclear, poly/multinuclear, matrix, multiwall, and
irregular. Figure 10.1 represents the morphology of capsules (Peanparkdee
et al., 2016).
FIGURE 10.1 Representation of the morphology of capsules.

Source: Adapted from Peanparkdee et al. (2016). Open Access. http://www.agrsci.jp/ras/
article/view/23/48
10.3 ENCAPSULATION TECHNIQUES
Choosing of encapsulation technique mainly depends on the physical and

chemical properties of the core material. There are three classes of encap-
sulation techniques, namely as chemical, physical, and physicochemical
methods (Jyothi et al., 2010). Chemical methods cover in situ polymerization
and liposomes; physical methods range from spray-drying to fluidized bed

coating and physicochemical methods include coacervation and solgel
encapsulation (Gibbs, 1999; Gouin, 2004). The capsules produced by using
these methods separately have different properties and application field
(Fang and Bhandari, 2010).
10.3.1 SPRAY-DRYING METHOD
Spray-drying is an encapsulation method which requires the homogenization

of core material (phytochemical) into an aqueous solution of encapsulating
agent. The homogenate is then converted to a powder form by heating and
spraying. Out of the spray nozzle, the solvent (water) is quickly evaporated
and powder capsules are formed. Obtained capsules are highly enduring
to chemical or microbial deterioration as a result of a reduced amount of
water. This is one of the advantageous properties of spray-drying method.
Reduction of water amount in the capsules also contributes to lower storage
and transportation costs. Hydrocolloids such as gelatin, modified starch,
or dextrin are used as coating materials. The technique is reproducible and
suitable for industrial applications as well. The inlet and outlet temperature
in this method is the most substantial factor limiting its application. It has
been reported that physical and chemical properties of core material can be
effected from the drying temperature either too high or too low (Tan et al.,
2015). It is also possible to lose some of the active components of core
material especially the phenolic compounds (Gharsallaoui et al., 2007).
10.3.2 LIPOSOMES
Liposomes are formed by using nontoxic phospholipids and cholesterol.

The spherically shaped capsules can carry hydrophobic and hydrophilic
molecules. Physicochemical properties like surface charge and size of a
liposome are mainly derived from lipid composition used for the production.
Liposomes are generally used in the pharmaceutical and food industries for
entrapment of an aqueous solution within a phospholipid bilayer. Liposomes
are microscopic pockets formed by phospholipid bilayers surrounding
aqueous compartments. Liposomal entrapment can protect phytochemi-
cals from environmental and chemical stresses, including the presence of
enzymes or reactive chemicals and exposure to extreme pH, temperature,
and high ion concentrations (Gouin, 2004).
10.3.3 COACERVATION/PHASE SEPARATION METHOD
Simple coacervation is the process that occurs when a hydrophilic polymer

is put into two phases. In this method, a polymer rich phase and dilute liquid
phase are formed. Coacervation can be induced by various means like a
change in temperature, pH, and electrolyte balance of the system or addition
of non-solvent. Complex coacervation is the complexation reaction. Two
different polymers oppositely ionized or crosslinking agents like glutaral-
dehyde should be used for complex coacervation. Once the coacervation
process is completed, phase separation could be carried out by using one
of the separation techniques such as filtration or centrifugation. Then the
separated particles are washed and dried (Schmitt et al., 1998).
10.3.4 SOLGEL ENCAPSULATION
Solgel chemistry started with the hydrolysis and condensation of metal

alkoxides. The solgel encapsulation technique can occur in two steps.
First is the generation of a sol by the hydrolysis of alkoxides. The
second step is the synthesis of a gel through polycondensation to create
metal-oxo-metal or metal-hydroxy-metal bonds. Phytochemicals can
be encapsulated by using the solgel technique. Relatively mild process
conditions like temperature and pH make this method advantageous. The
temperature required for the process is often close to room temperature.
So this method minimizes the thermal degradation of phytochemicals.
The controllable particle formation in concern with particle and pore size
also makes this method valuable (Flora and Brennan, 2001; Gupta and
Chaudhury, 2007).
10.3.5 FREEZE-DRYING METHOD
Freeze drying takes place in three stages; freezing, basic drying phase, and
second drying step. The first step is to freeze the mixture of phytochemical
and shell material. The second step is the sublimation of ice known as
lyophilization, under vacuum. Finally, bound water is removed by evapora-
tion under reduced pressure. In some situation preheating could be required
to obtain a uniform mixture of shell and core material depending on the
solubility of shell matrix. Although this processing has some benefits like
decreased heat deterioration, controllable water content of end product but
high operation cost, longer process time, and requiring a freeze dryer makes
the method inapplicable for industrial scale.
10.3.6 EXTRUSION-EMULSION METHOD
Extrusion technique is the oldest and most universal method used in making
capsules with hydrocolloids. In this method, alginate is the most used hydro-
colloid. Encapsulation is performed in two steps by using extrusion process.
The first step is the preparation of hydrocolloid solution in concentration
range 0.6–2%. In this step phytochemical(s) should be homogenized into
hydrocolloid solution. The second step is the addition of the homogenate
to a divalent cation solution mostly Ca2+ solution in concentration range
0.005–1.5 M. Addition of the homogenate into hardening solution can be
performed by passing it through the syringe needle. The shape and the size
of obtained capsules mainly depend on the alginate solution viscosity and
divalent cation concentration as well as the margin between the droplet and
hardening solutions. Being a simple and cheap method, it is also advan-
tageous as there is no damage to phytochemicals, and it does not require
deleterious solvent. The difficulty of large-scale application, large size
distribution, and limited choice of wall material are the disadvantages of this
method (Koç et al., 2010).
In the emulsion technique, a small volume of phytochemical(s)/polymer
slurry (dispersed phase) is added into a large volume of vegetable oil
(continuous phase) like soy, sunflower, corn, and light paraffin oil. Emul-
sions could be obtained by two strategies, namely water in oil (W/O) or oil
in water (O/W). After obtaining an emulsion, cross-linking should be carried
out to form gels. Gelling can be obtained by different mechanisms such as
ionic, enzymatic, and interfacial polymerization. This process can easily be
scaled up and obtained beads are considerably smaller ranging from 25 µm
to 2 mm. The cost of this method is a bit high due to the usage of vegetable
oils, surfactant, and emulsifier (tween 80) (Krasaekoopt et al., 2003).
10.3.7 CO-CRYSTALLIZATION
Co-crystallization is an application of sucrose as a matrix surrounding the

phytochemicals. Nanosucrose between 3 and 30 µm during dragging of
materials between or within sucrose crystals is a spontaneous crystallization
of varying microcrystalline clusters. The amount of saturated sucrose syrup
is mixed with the preset core material. The encapsulated material is then
transferred from the container, emptied and dried to the proper moisture
content. The core material is primarily comprised of crystals formed among
the cracks (Barbosa-Canovas et al., 2005).
10.4 COATING MATERIALS
The composition of the coating material specifies the final product func-
tional properties. So an ideal coating material has the following properties
(Bansode et al., 2010);
• Good rheological properties at high concentration and easy to

encapsulate.
• It should be an emulsion and dispersing property and the emulsion
stability must also be high.
• During the coating process with the core material and during storage,
it should not react in such a way as to disrupt the core material.
• It should be able to coat the core material in a stable manner both
during the processing and storage.
• Must be soluble in the desired solvent.
• It must be cheap.
10.5 APPLICATION FIELDS OF ENCAPSULATION
Encapsulation technology is a widely used in several industries, especially

food and pharmaceutical industries since it can increase solubility, enhance
stability, and improve the controlled release properties of compounds such
as essential oils, antioxidants, enzymes, drugs, and so forth. Therefore, this
section focuses on the applications of encapsulation in these industries.
Table 10.1 represents the industrial applications of encapsulation.
As mentioned before, phytotherapy could be used either for preventing
of exact illness or for assistive application to a modern remedy. In modern
therapy, doctors have the consensus on the usage of phytochemicals in a
dose-controlled manner. That is why scientists are searching for both the
active compound(s) of medicinal plants and better extraction methods for
them. After the extraction, the concentration of phytochemical(s) can be
determined sensitively by using advanced analytical techniques like HPLC,
LC-MS, and GC-MS, and so forth. Although it is possible to determine the
concentration of phytochemical in extracts, still there are some limitation

factors like solvent of the extract for prescribing them to a patient. Encapsu-
lation of phytochemical(s) from the extract is a biotechnological way to solve
this problem. At the end of this chapter, some examples of phytochemical
encapsulation are given.
TABLE 10.1 Some Industrial Applications of Encapsulation.

Pharmaceutical industry Food industry Other industries
Specific drug delivery Functional foods Textile industries
Oral drug delivery Probiotics Fragrance finishing
Transdermal drug delivery Antioxidants Color change materials
Stomach-specific drug delivery Vitamins Fire retardants
Colon-specific drug delivery Dietary fiber
Small intestine-specific drug delivery Food preservatives Cosmetic industries
Bitter taste masking Food colorants Essential oils
Food flavor Polyphenolic compounds
Source: Adapted from Peanparkdee, et al. (2016). Open Acess. http://www.agrsci.jp/ras/
article/view/23/48
Bidens pilosa L. is a South American medicinal plant with proved

antimalaric, hepatoprotective, and antioxidant activities, generally linked
to their secondary metabolites, flavonoids, and polyacetylenes. Increasing
the stability of its phytochemicals, extract of B. pilosa was encapsulated
by using spray-drying method. Obtained capsules were evaluated as stable
for three different stress storage conditions both in open containers and in
sealed sachets (Cortés-Rojas et al., 2016). Although it is not common sense
to introduce phytochemicals to the body by injection since the complex
nature of phytochemicals but an injectable nanoparticulate system was
achieved. mPEG–PLGA–mPEG (PELGE) platform was reported to be used
for the preparation of nanoparticles. Sustained release of co-encapsulated
four active components of Ginkgo biloba extract was managed by Han et al.
(2012). It was stated that the bitter melon (Momordica charantia L.) is a
medicinal fruit mostly used for the handling of diabetes, due to its content
of saponins, phenolics, and flavonoids and its antioxidant capacity. As
mentioned before, extracted phytochemicals are highly susceptible to the
environmental conditions. That is why encapsulation is a highly practical
tool in order to improve the shelf life of phytochemicals extracted from
the bitter melon. Tan et al. reported that water extract of bitter melon was
encapsulated by a spray-drying method by using maltodextrin and gum

arabic as an encapsulating agent. Improved shelf life and better handling
were reported as well (Tan et al., 2015).
It was known that herbal essential oils such as Cuminum cyminum, are
natural antifungal agents consisting of many different volatile compounds.
In a study, ıt was reported that encapsulation by chitosan (CS)–caffeic acid
(CA) system was used to improve antimicrobial activity and stability of
the C. cyminum essential oils against Aspergillus flavus. An encapsulation
efficiency of 85% was reported to be achieved. As revealed through the
release kinetics studies, 78% of the encapsulated oils were released in
1 week. Reported results imply that because the oils are volatile and unstable
due to the environmental factors, capsulated oils significantly showed better
performance. A decreased minimum inhibitory concentration of encapsulated
oils against A. flavus is the evidence of better performance of encapsulated
oils (Zhaveh et al., 2015).
10.5.1 ENHANCING BIOAVAILABILITY OF PHYTOCHEMICALS
Bioavailability could be described as the amount of interested compound(s)

that achieve the bloodstream after oral administration in its native form.
Recently, the numbers of studies on phytochemicals have been growing
around the world since their therapeutic effects and relatively smaller side
effects as compared to the synthetic drugs. However, many phytochemicals
in herbal extracts in spite of their efficacious in vitro activity exhibit poor or
negligible in vivo activity since their poor bioavailability. This could be due
to the low concentration of phytochemical(s) in the extract, the most often
in microgram quantities in a liter of solution resulting in poor absorption.
Weak lipid solubility of most phytochemicals could also contribute to their
poor bioavailability. It is known that a small portion of phytochemical(s)
administered orally can pass the membrane barrier in gastrointestinal (GI)
as unaltered because of insufficient residence time, low permeability, and/
or low solubility. The presence of a deforming agent in the GI tract (pH,
enzymes, the presence of other nutrients, and so forth.), limits the activity
and the potential health benefits of phytochemicals.
As stated before, encapsulation could protect the core material (phyto-
chemicals) through GI track from the deforming agents. Encapsulation also
provides an increased concentration for the core material. The concentration
of phytochemicals in capsules could be hundred or thousand times more
than its counter extract solution. By choosing the suitable encapsulant and
encapsulation techniques it is possible to encapsulate a phytochemical to

increase its bioavailability. It could be achieved by the ability of controlled
release property of a capsule. For instance, alginate capsules pass through
the stomach without any deformation, they release the core material in
intestine since they are completely destroyed (Kang et al., 2009; Gupta and
Kesarwani, 2013; Muttepawar et al., 2014).
KEYWORDS
•• encapsulation
•• phytotherapy
•• extrusion
•• liposomes
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CHAPTER 11
EFFECTIVE PROCESSING METHODS

FOR FRUITS AND VEGETABLES
MARIA ASLAM*, SIDRA KHALID*, and HAFSA KAMRAN
University Institute of Diet and Nutritional Sciences, Faculty of Allied
Health Sciences, University of Lahore, Pakistan
*
[email protected]
ABSTRACT
Fruits and vegetables are rich sources of antioxidants namely vitamin

C, carotenoids, and phenolic compounds; which have health-protective
effects. Diet rich in fruits and vegetables pose low risk of degenerative
diseases such as atherosclerosis, diabetes, cancers and neural disorders
through the minimization of the risk of oxidative stress. Food processing
has effects on fruits and vegetables from harvest, transport, and to storage
in ways that can lead to adversarial variations in the physiological status.
Moreover, the health-endorsing content of fruit and vegetables depends
on their processing history. Vitamin C and polyphenols are more vulner-
able to lose during processing and these health-promoting phytochemicals
including antioxidants in fruits and vegetables should be well-preserved
during storage and handling.
11.1 INTRODUCTION
Some food, especially fruits and vegetables, deteriorate as a result of physi-

ological aging, metabolic alterations, increased respiration rate, and high
production of ethylene gas. These aspects contribute to depigmentation,
loss of firmness, development of off flavors, acidification, and microbial
spoilage significantly. Simultaneously, food processors are using emerging

approaches to process perishable commodities, along with enhanced nutri-
tional and sensorial quality (Pasha et al., 2014). Phytochemicals are bioac-
tive non-nutrient plant compounds present in numerous fruits, vegetables,
grains, and other plant foods (Dembinska-Kiec et al., 2008). Most common
fruits and vegetables including berry crops, many tree fruit crops, and onions
are plentiful in phenolic antioxidants. In Western European diet, apples,
onions, and tea make available the greatest of antioxidants flavonoids by
giving benefits to their content and their rate of consumption. A number
of studies have shown that in fully ripe fruits, pH and colored antioxidants
(e.g., carotenoids and anthocyanins) often reach their highest level. Some
methods such as freezing, cutting, drying, tempering, bleaching, steriliza-
tion, and extraction have been used in the food industries. The benefits
of using ultrasound for food processing, includes more actual mixing and
micromixing, high energy and mass transfer, lessened thermal and concen-
tration gradients, decreased temperature, selective extraction, minimized
apparatus size, quicker reaction to extraction control process, quick start up,
improved production, and exclusion of process steps (Chemat and Khan,
2011). Fruits and vegetables are collected, shifted, and stored in ways that
can carry out physiological stresses that lead to adverse changes in their
optical quality and other chemical outlines. By using an effective method of
processing, fruits and vegetable shelf life can be increased. Steps included in
processing are raw material planning, cleaning, trimming, and peeling, and
later cooking, canning, or freezing.
It is known that lower risks of several degenerative diseases are linked
to a diet rich in fruit and vegetables. These outcomes have formed a new
viewpoint relating to the potential of diet in preventing serious ailments in
the future. Moreover, the health-endorsing content of fruit and vegetables
severely depends on their processing history. Processing is predictable to
affect activity and bioavailability of bioactive compounds (Nicoli et al.,
1999). Antioxidants functions in human nutrition have increased interest,
especially due to their link with health-positive effects for a number of
chronic diseases, which include cardiovascular diseases and certain types
of cancer. Fruits and vegetables are perishable and hard to preserve as
fresh products. Dried fruits and vegetables can be simply stored, shifted at
comparatively lowest cost, have cheap packing costs, and their low water
content delays microbial spoilage. Drying methods using air, freezer, micro-
wave, and solar energy are the most systematically studied among drying
methods (Kamiloglu et al., 2016). Besides increasing the usefulness and
accessibility of processed groceries, handling is also aimed at optimizing
Effective Processing Methods for Fruits and Vegetables 255
nutrient accessibility and to maintain product quality along with the decline
of losses and wastes of products. It should be concluded that the value of the
final products is directly linked to the processing operations and conditions
and remarkably differ from natural non-processed commodities. Among
possible effects of processing on the whole quality of fruit and vegetable
products, the most important are naturally occurring components, develop-
ment of original constituents with an increased antioxidative potential or
prooxidant activity and connections between different compounds (Figiel
and Michalska, 2016).
11.2 PROCESSING METHODS FOR FRUITS AND VEGETABLES
11.2.1 HIGH-PRESSURE PROCESSING
To increase shelf life of the product, high-pressure processing technique is

used, that ranges from 100 to 900 MPa to inactivate microorganisms. The
pressure is applied by two mechanisms (1) direct pressure (2) indirect pressure.
In both cases pressure is maintained, which are extracted from product size
and geometry, hence, decreases the adverse effect on texture, tissue rupture,
and other quality parameters of the products (Table 11.1). The technique is
skillful of preserving flavor, color pigments nutrient, and antioxidant content
(Tewari et al., 2017); however, availability of carotenoids in different types of
vegetables were found slightly altered (McInerney et al., 2007).
11.2.2 POWER ULTRASOUND TECHNIQUE
The use of power ultrasound to aid the freezing of fruits and vegetables is
a comparatively new concept. Cavitation and a sponge effect produced by
sound waves, affect the freezing rate and qualities of the frozen products.
The practice of using ultrasound in the freezing process aids to eliminate
enzymes and microbes and improves the ice crystal nucleation process
(Table 11.1). High freezing rate, quicker crystallization, uniform distribution
of ice crystals, better microstructure, and good product quality are the bene-
fits of ultrasound-assisted freezing over conventional freezing. However,
quite little is recognized about the fundamental thermodynamics, moisture
diffusion, and heat transfer in the ultrasound-assisted freezing process. The
design of proper transducer systems and freezers to suit the requirements
of fruits and vegetables has not been taken into account yet. It seems that
TABLE 11.1 Processing Methods used for Fruits and Vegetables. 256
S/ Processing method Technique Benefits Limitations References
No.
1. High-pressure processing High pressure Preserve color, flavor, Altered availability of Tewariet al. (2017),
nutrients, antioxidant carotenoids McInerney et al.
(2007)
2. Power ultrasound technique Ultrasound rays Eliminates enzymes and Altered physicochemical Islam et al. (2017)
microbes, improves the ice properties of food
crystal nucleation
3. Ozone __ Mycotoxin and pesticide Losses in physical quality Karaca and Velioglu
control (2007)
4. Drying Heat Increase shelf life Decrease heat sensitive nutrients Rodríguez et al.
quality, altered nutritional and (2017), Karam et al.
physical properties (2016)
5. Spray drying Spray Cost effective, maintain Change biologically active Figiel and Michalska
phytochemicals compounds (2016)
6. Thermosonication Conventional Improve the microbial and __ Anaya-Esparza et al.
thermal processing enzymatic inactivation rates, (2017)
shelf life, decrease effect on
the nutritional content
7. Radiation processing Irradiation process Influences the antioxidant Change bioactive compounds Xue et al. (2016)
position
8. Ohmic heating Electrical resis- Less thermal damage Depends on electrical Kaur et al. (2016)
tance heating conductivity of food product
9. Minimally processed, fresh-cut Physical change Increase shelf life Physical damage Varoquaux and Wiley
fruits and vegetables (2017)
10. Minimally processed refriger- Freezing Nutrition security __ Yildiz (2017)
ated fruits and vegetables

there is a vital need to observe the effect of the ultrasound-assisted freezing

process on the physicochemical properties of frozen fruits and vegetables
(Islam et al., 2017).
11.2.3 OZONE
Ozone is one of the powerful oxidants; it is effective against several kinds of

microorganisms on fruits and vegetables. Expected results have been found
in solving the food industry problems like mycotoxin and pesticide remains
by ozone operation. Impulsive decay expects forming dangerous remains
in the treatment medium makes ozone safe in food applications. If not used
properly, ozone can cause some harmful effects on products, for example,
losses in physical quality (Table 11.1). Treatment conditions should be in
detail observed for all kinds of products for actual and safe use of ozone
(Karaca and Velioglu, 2007).
11.2.4 DRYING
Drying allows the procurement of products with a long shelf life by drop-
ping the water activity to a low level for the growth of microorganisms,
enzymatic reactions, and other inactivation reactions to be inhibited.
Despite the benefits of this operation, heat-sensitive products quality is
decreased when high temperatures are in use (Table 11.1). The usage of
low drying temperatures decreases the heat damage but being the drying
time longer, oxidation reactions occur and a lessening of the quality is
also observed. For this reason, alternative techniques are being studied.
Power ultrasound measured a developing and favorable technology in the
food industry. The latent of this machinery depends on its ability to speed
up the mass transfer processes in solid–liquid and solid–gas systems.
The strengthening of the drying practice by the use of power ultrasound
can be accomplished by changing the product behavior during drying,
using pretreatments like soaking in a liquid medium or by applying power
ultrasound in the gaseous medium during the drying process (Rodríguez
et al., 2017).
Drying is a food preservation process in which water removal mini-
mizes many of the moisture-driven worsening reactions impacting the
bioproduct quality. Dried fruits and vegetables and their use in powder form
have enlarged attention in the food industry. During powder processing,
drying and grinding conditions significantly influence the quality features

of biological materials. It not only modifies the nutritional value but also
results in changes in textures, sensory, functional, and physical properties.
These changes are of abundant position and need to be controlled through
retro-engineering methodologies (Karam et al., 2016).
11.2.5 SPRAY DRYING
Spray drying (SD) is a genuine technique in which liquid products are

directly turned into powders. SD is used typically in industry, especially for
pharmaceuticals making and in the processing of dairy and fruit products
(Jain et al., 2012). This technique was set up to be 4–5 times low cost as
likened to freeze drying (FD). Likewise, it is confirmed that application
of SD process for dehydration of chokeberry juice into powders resulted
in better maintenance of total phenolic compounds, total flavonoids, total
monomeric anthocyanin’s (Table 11.1) when likened to FD however, in
the literature there are some point of data connected with connotation of
both drying techniques in positions of the changes of the biologically active
compounds (Figiel and Michalska, 2016).
Spray FD (SFD) is another FD technique that produces distinctively
powdered products even as still including the incomes of conventionally
freeze-dried products. In relations of product structure, quality, and the
retention of volatiles and bioactive compounds, SFD has dormant applica-
tions in high-value products due to its edge over other drying techniques. In
cases where other drying techniques cannot provide these product qualities,
SFD stands out despite the costs and difficulties involved (Ishwarya et al.,
2015). Between the techniques used, FD is considered as the best method
because low-temperatures functional in this process allow the highest reten-
tion of bioactive compounds comparable with the raw material (Figiel and
Michalska, 2016).
11.2.6 THERMOSONICATION
Thermosonication is a unique and workable technique that is working to

replace the conventional thermal processing (Table 11.1). It can improve the
microbial and enzymatic inactivation rates, lengthen product shelf lifespan
and diminish the impact on the nutritional content and general quality of
fruit and vegetable juices (Anaya-Esparza et al., 2017).
11.2.7 RADIATION PROCESSING
Irradiation treatment is a usable storage method in which ionizing or

nonionizing radiation is exposed to food. G-rays, X-rays, or high-energy
electrons are the basis of ionizing radiation, which carries higher energy to
ionize molecules or atoms (Table 11.1). Nonionizing radiation is character-
ized primarily by microwaves, infrared, visible light, and ultraviolet rays
(UV-A, UA-B, and UA-C). Radiation influences the antioxidant position by
changing the gathering of antioxidant bioactive compounds (polyphenols,
vitamin C, flavonoids, and so forth) or the activity of antioxidant-related
enzymes. These effects change due to factors like energy carried by radiation,
the exposure time, the type of product treated, and many other. Ultraviolet is
the most normally used radiation form preservation of fruits and vegetables
(Xue et al., 2016).
11.2.8 OHMIC HEATING
Ohmic heating, as well called electrical resistance heating, joule heating,

or electroconductive heating, is an advanced thermal food processing
technique when electric current is passed due to electrical resistance its
heat is internally generated in a sample. It is a different technique which
delivers quick and smooth heating, resulting in less thermal damage to the
food product (Table 11.1). The recent literature stated that, plant products are
most suitable and often used for ohmic heat processing. More than heating
of fruits and vegetables, the practical electric field under ohmic heating
causes various changes in quality and nutritional factors which consist of
inactivation of enzymes and microorganisms, degradation of heat-sensitive
compounds, changes in cell membranes, viscosity, pH, color, and rheology
(Kaur et al., 2016).
11.2.9 MINIMALLY PROCESSED, FRESH-CUT FRUITS AND

VEGETABLES
Physiological (due to the enzymatic and metabolic activity of the

living plant tissue) and microbial activities (due to the proliferation of
microorganisms) are connected to the nutritional value, phytochemical
quality, sensory quality, safety quality, and spoilage of minimally
processed, fresh-cut fruits and vegetables (Table 11.1). Processing of
fresh fruits and vegetables destroys the inner compartmentalization that

separates enzymes from substrates and removes the natural protection
of the epidermis. Therefore, plant tissues undergo physical damages that
make them much more perishable than when the original product is in
one piece. Furthermore, processing outcomes in a stress reply by the
produce characterized by an bigger respiration rate (wound respiration)
and ethylene production, leading to faster metabolic rates; changes in
metabolic rates and damage of the plant tissue lead to exposure to air,
desiccation (transpiration), and gathering of enzymes with substrates, all
leading to quality degradation. Proper processing and packaging reduce
the changes and quality loss with increased shelf life (Varoquaux and
Wiley, 2017).
11.2.10 MINIMALLY PROCESSED REFRIGERATED FRUITS AND

VEGETABLES
Slightly fresh processed plants foods have fresh-like, living cells and active
enzyme-containing qualities. They are packaged and a little processed and
refrigerated (Table 11.1). All crops are grown, harvested, and transported,
under progressive hygienic and sanitary practices. The raw materials are
washed, cut and spin dried, processed, and packaged in cool factories with
tremendously sanitized conditions. Minimally processed refrigerated fruits
and vegetables food system covers the several activities and performers in
food value chains involved in transforming inputs into outcomes, which
for a sustainable food system should include food and nutrition security,
convenience to the consumer, environmental quality, and human well being
(Yildiz, 2017).
Epidemiological studies have established a positive link between the
intake of fruits and vegetables and prevention of diseases like athero-
sclerosis, cancer, diabetes, arthritis, and also aging. Fruits and vegetables
help enhance good health by preventing diseases. Most studied antioxi-
dants are phenolic flavonoids, lycopene, carotenoids, and glucosinolates.
Antioxidants have also been recommended to have a distinct role as
preservatives. These preservatives are defined by the US Food and Drug
Administration as a substance used to preserve food by delaying decline,
rancidity, or discoloration caused by oxidation. During processing and
storage lipid peroxidation is a deteriorative reaction of foods (Kaur and
Kapoor, 2001).
11.3 EFFECT OF PROCESSING METHODS ON PHYTOCHEMICAL

CONTENT
The role of phytochemicals having antioxidant properties in reducing the

effects of oxidative stress in the development of disease and aging process
is well established and accordingly contribute to the health-protective role
of fruits and vegetables (Alvarez-Suarez et al., 2014). Varying processing
procedures such as dehydration or drying, canning, extrusion, high-hydro-
static pressure, ohmic heating, and pulsed electric field limit the availability
of phytochemicals (Nayak et al., 2015).
Thermal degradation, air exposure due to disturbance of plant tissues and
cells, and enzymatic activity within plants may affect phytochemical content
during food processing. For instance, vitamin C being heat sensitive can
be easily degraded during processing (Francisco et al., 2010). In contrast,
fat-soluble compounds such as β-carotene exhibit normally a comparatively
greater retention during thermal procedures (Svelander et al., 2011). In addi-
tion, the bioavailability of these health-related compounds can be subjective
to processing circumstances due to the formation of certain microstructures
(Parada and Aguilera, 2007). For example, as compared to unprocessed
fruit, in vitro bioavailability of lycopene from tomato can be improved by
mechanical and heat processing (Tibäck et al., 2009). Similarly, increased
bioavailability of carotenes from both tomato and carrot has been observed
by certain treatments to disturb plant tissues and cells, such as high-pressure
homogenization (Svelander et al., 2011). β-cryptoxanthin present in certain
fruits and vegetables including papaya and pumpkin has health-protective
role in the prevention and treatment of certain diseases, especially cancer
(Ma et al., 2011). Studies exploring the effect of drying on β-cryptoxanthin
content discovered that drying usually reduces the β-cryptoxanthin content
present in fruits and vegetables (Table 11.2). FD has been found to be an
effective method in terms of retaining antioxidant compounds in contrast to
the other drying techniques. Due to use of low temperatures, FD is better
able to preserve heat-sensitive antioxidants. In addition, FD results in greater
extraction capacities of antioxidants with the establishment of ice crystals
within the plant matrix in freezing, which may lead to a larger interruption of
cell structure, permitting more solvent infiltration into the matrix. Conversely,
less cell damage is caused by other drying methods, as well as harmful effects
of high-temperature processing, both of which support the loss of antioxi-
dants (Kamiloglu et al., 2016). Thus, food processing methods can be used to
improve the phytonutrients content and bioavailability of final food products
based on fruits and vegetables (Sánchez-Campillo et al., 2012).
TABLE 11.2 Effect of Thermal processing on Phytochemicals.

S/ Phytochemical Food Processing Effect Reference
No. method
1. Lycopene Tomato Heat processing Improved Svelander et al.
bioavailability (2011)
2. Carotenes Tomato, carrot High-pressure Improved Parada and
homogenization bioavailability Aguilera (2007)
3. β-cryptoxanthin Fruits, vegetables; Drying Decreases Kamiloglu
papaya, pumpkin, content et al. (2016)
11.4 CONCLUSION
Fruits and vegetables have disease-preventive effect and processing methods

can damage its disease-protective properties. These naturally occurring
compounds impart bright color to fruits and vegetables and act as antioxidants
in the body by destroying harmful free radicals, which are associated with most
degenerative diseases. Fruits and vegetables should be minimally processed to
retain its phytochemical activity.
KEYWORDS
•• processing method
•• heat processing
•• high-pressure homogenization
•• drying
•• fruits and vegetables
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PART III
Environmental Concerns and
Eco-Friendly Control Measures
CHAPTER 12
EFFECTS OF ENVIRONMENTAL
FACTORS ON THE ACCUMULATION
OF PHYTOCHEMICALS IN PLANTS
SECHENE STANLEY GOLOLO
Department of Biochemistry, School of Science and Technology,
Sefako Makgatho Health Sciences University, Ga-Rankuwa, Pretoria,
South Africa, Tel.: +27 12 521 4372
ABSTRACT
The effects of environmental factors on phytochemical compositions of

medicinal plants highlight some disparities in the manner in which they
respond to different environmental conditions. In this chapter, the effects of
different environmental factors such as geographical locations of different
altitudes, seasonal variation, and different exposure levels to pollution on
the phytochemical composition of selected medicinal plants are discussed.
The accumulation of phytochemicals in medicinal plants as a response to
the influences of different environmental factors appear to differ among
different plants. Thus, the accumulation of phytochemicals in medicinal
plants as a response to environmental factors follows a non-general trend.
12.1 INTRODUCTION
The use of medicinal plants for healthcare purpose is a very old practice that is
as old as humankind. The knowledge of medicinal plants with healing proper-
ties has been passed along generations, which, contributed, to their continuous
usage up to this modern age (Petrovska, 2012). It is estimated that the use of
medicinal plants has tremendously increased with about 80% of the population
worldwide reported to be relying on herbal remedies for their health care needs
over the past decades (Ekor, 2014). The increased reliance or usage of herbal
remedies emanates from several factors that include their perceived and proven
effectiveness, preference over manufactured products, cost-effectiveness, and
perceived safety (Bandaranayake, 2006). Medicinal plants are to be regarded
as plants that possess properties or compounds that can be used for healthcare
purposes or that may be used as leads in the development of important drugs
(WHO, 2008). The compounds that contribute to the medicinal properties of
these plants are known or referred to as phytochemicals.
Phytochemicals, also known as phytoconstituents, are found in different
plant species and are classified based on their chemical and biological prop-
erties. Phytochemicals include compounds such as alkaloids, flavonoids,
phytosterols, saponins, polyphenols, terpenes, lectins, and many others
(Webb, 2013). Phytochemicals are also referred to as secondary metabo-
lites, in the sense that they are not necessarily required for the growth of
the plant. They are synthesized mainly to contribute to the adaptation and
survival of the plants in their interaction with the environment as defenses
against pathogens as well as herbivorous and symbiotic insects (Kennedy
and Wightman, 2011). Furthermore, phytochemicals are defined as chemical
compounds of natural occurrence that induce biological activities, which
are of health benefits to humans and animals. Phytochemicals possess many
inherent biological activities such as anti-inflammation, anticancerous, anti-
microbial, and antioxidation (Graça et al., 2016).
Generally, these chemical compounds protect plants from environmental
hazards such as pollution, stress, drought, ultraviolet exposure and patho-
genic attack. It is this inherent function of phytochemicals in plants, the
function of protecting the plants from hostile environmental conditions that
informs their accumulation depending on the extent of the risk. Phytochemi-
cals accumulate in different plant parts, such as the roots, stems, leaves,
flowers, fruits, and seeds as a response to the growing conditions (Saxena
et al., 2013). It is therefore apparent that the accumulation of phytochemicals
in plant parts is dependent on its interaction with the environment. Environ-
mental conditions that may influence the accumulation of phytochemicals in
plants include altitude, seasonal variation, ecological factors such as soil pH,
soil organic matter, and mineral content, and the geographical conditions
such as climate (Liu et al., 2015). The aim of this review was to establish
whether the accumulation of phytochemicals in plants as influenced by
their interaction with different environmental factors follow a general trend
among medicinal plants and whether that trend is adhered to different phyto-
chemicals based on the available literature.
Effects of Environmental Factors on the Accumulation of Phytochemicals 269
12.2 ENVIRONMENTAL INFLUENCES ON PLANTS

PHYTOCHEMICALS
Environmental conditions may influence the types, the contents, and propor-
tions of phytochemical constituents in plant species. Some constituents are
only synthesized or their accumulation is increased or decreased under certain
environmental conditions (Liu et al., 2016). Therefore, the comparison of
the phytochemical compositions in plants under the influence of different
environmental factors may be either qualitative, quantitative, or both. In this
regard, different studies have determined the presence or amounts of specific
phytochemicals in several plant species under different growing conditions
and locations. Many of such studies have demonstrated significant differ-
ences in the amounts of phytochemicals of one plant or many plants as influ-
enced by different environmental conditions such as geographical locations,
seasonal variations, pollution, and several ecological factors.
12.2.1 EFFECT OF GEOGRAPHICAL LOCATION ON THE

PHYTOCHEMICALS OF MEDICINAL PLANTS
Geographical location may be described in terms of altitude and climatic

conditions. Studies have been conducted on several medicinal plants that
demonstrated that their phytochemical compositions were affected by
geographical location parameters from which they were collected. In the
study by Liu et al., the contents of phytochemicals in the leaves of Potentilla
fruticosa from different growing locations in China were determined, whereby
phytochemical contents were found to be qualitatively and quantitatively
different (2016). The qualitative differences in the extracts of the leaves of
P. fruticosa from different locations were determined by High-performance
liquid chromatography analysis. According to the study, the differences in
the chromatographic profiles of the leaf extracts of the plant species samples
from different locations were displayed by the presence of some peaks in
extracts of samples from some locations that are absent in the extracts of
other samples from different locations. The observed significant differences
were attributed to altitude and temperature patterns of the growing locations.
Quantitatively, altitude was positively correlated with flavonoids, rutin,
and total phenolic contents, while also negatively correlated to total tannin
content. The temperature was positively correlated with flavonoids and rutin
content, while negatively correlated to total phenolic content. In the same
study, highest tannin, flavonoid, and rutin contents were found in leaves
sample from the Kangding, Sichuan province, whereas lowest tannin and
rutin contents were recorded in the leaves from Shangri-la, Yunnan. The
lowest flavonoid content was found in the leaves from Jingyuan. However,
in contrasting observation, the highest total phenolic content was recorded
in leaves samples from Shangri-la, Yunnan province. The provinces differ
in terms of altitudes, thus the different phytochemicals in the same plant
respond differently to the same variable.
The effect of climate change on the phytochemical composition of
Aloe vera was determined in a study by Kumar et al. (2017). In this study,
extracts of samples from highland and semiarid zones showed higher total
phenolic contents than those from tropical zones. Wild bush tea (Athrixia
phylicoides) growing at locations differing in altitude, climate, and edaphic
factors were shown to have differences in the amounts of phytochemicals
by a study conducted by Nchabeleng et al. (2012). The highest polyphenol
content was recorded in bush tea samples from Haenertsburg area, while the
lowest was in samples from the Levubu area. In contrast, the samples of the
same plant species from Levubu showed the highest amounts of tannins and
lower tannins were recorded in samples from Khalavha area. In the same
study, a positive correlation was shown between total polyphenol content
and altitude, whereas climatic conditions, soil macroelements, and soil pH
did not affect total polyphenols and tannins. Thus, the findings of this study
suggest that high amounts of total polyphenols are found in wild bush tea
growing at high altitudes. However, the findings of the same study suggest
that the accumulation of tannins is high at low altitudes in wild bush tea.
The results of the cited studies depict a picture whereby some medicinal
plants have high amounts of different phytochemicals under the influence of
contrasting location factors. For example, a medicinal plant possesses high
amounts of tannins at high altitude location and the same medicinal plant
possesses high amounts of other phytochemicals such as flavonoids at low
altitude location. The findings of these studies also demonstrate the posses-
sion of high amounts of a specific phytochemical group by some medicinal
plants at relatively higher altitudes while the accumulation of similar
phytochemicals is not affected by altitude in other different medicinal plants.
While several studies indicate that geographical location does have an effect
on the phytochemical compositions of medicinal plants, it appears that the
effect is not in a general form. Examples of relative altitudes under which
selected medicinal plants possess high amounts of specific phytochemicals
are presented in Table 12.1. The altitudes are referred to as relative to the
highest altitude of locations in one study was not necessarily the highest in
other studies and the same applies to lower altitudes.
TABLE 12.1 Examples of Relative Altitudes Under which Selected Medicinal Plants have a High Accumulation of Specific Phytochemicals.
Medicinal plants Alkaloids Essential oils Phenols Tannins Flavonoids Saponins References
Potentilla fruticosa – – HA LA HA – Liu et al. (2016)
Aloe vera – – HA LA – – Kumar et al. (2017)
Athrixia phylicoides – – HA LA – – Nchabeleng et al. (2012)
Zanthoxylum alatum – LA – – – – Gupta et al. (2011)
Primula denticulata HA – HA HA – IA Khaleefa et al. (2015)
Sphagnum junghuhnianum – – HA – LA – Majuakim et al. (2014)
Arnica montana – – LA – HA – Clauser et al. (2014)
Matricaria chamomilla – – HA – HA – Ganzera et al. (2008)
HA: relative high altitude; LA: relative low altitude; IA: relative intermediate altitude; (–): not determined.
12.2.2 EFFECT OF SEASONAL VARIATION ON THE

PHYTOCHEMICAL COMPOSITION OF MEDICINAL PLANTS
Seasonal variations are characterized by fluctuations in mean temperatures

and rainfalls. Several studies were conducted to determine the season during
which high accumulation of phytochemicals is obtained in selected medicinal
plants. To cite a few, Ncube et al. reported on the observed effect of seasonal
variation on the phytochemical compositions of Tulbaghia violacea, Hypoxis
hemerocallidea, Drimia robusta, and Merwilla plumbea (2011). The study
showed total phenolic contents to be generally higher in spring in all plants,
whereas tannins and flavonoids were either higher in spring or winter except
for H. hemerocallidea whose tannin levels were higher in autumn. In the
same study, saponins were higher in winter in all plants.
Effect of seasonal changes on the phytochemical quantity was also
observed for the leaves of three medicinal plants from the Limpopo prov-
ince of South Africa, namely Barleria dinteri, Grewia flava, and Jatropha
lagarinthoides (Gololo et al., 2016). The three medicinal plants were found
to have high amounts of alkaloids and tannins during colder seasons in South
Africa (autumn and winter), whereas flavonoids were in high amounts during
warmer seasons (spring and summer). Saponins were in high amounts during
a warmer season in G. flava and J. lagarinthoides and were not significantly
different among seasons in B. dinteri. However, phenols showed a different
trend with high amounts recorded in autumn for G. flava, summer for J.
lagarinthoides and no significant seasonal difference in B. dinteri.
Examples of different seasons during which selected medicinal plants have
a high accumulation of specific phytochemicals are presented in Table 12.2.
As seen in the geographical location, the accumulation of phytochemicals in
medicinal plants is affected by seasonal variation. However, the findings of
the cited studies indicate that some medicinal plants possess higher amounts
of different phytochemicals during particular seasons and that different
medicinal plants possess high amounts of similar phytochemicals during
different seasons. Thus, the effect of seasonal variation on the accumulation
of phytochemicals also does not follow a general trend for medicinal plants.
12.2.3 POLLUTION ON THE PHYTOCHEMICAL COMPOSITION

OF MEDICINAL PLANTS
Pollution may be described as the direct or indirect introduction of

substances, as a result of human activity, into the air, water, or land that
TABLE 12.2 Examples of Different Seasons During which Selected Medicinal Plants have High Accumulation of Specific Phytochemicals.
Medicinal plants Alkaloids Phenols Tannins Flavonoids Saponins References
Tulbaghia violacea – S1 S1/W – W Ncube et al. (2011)
Hypoxis hemerocallidea – S1 A – W
Drimia robusta – S1 S1/W – W
Merwilla plumbia – S1 S1/W – W
Barleria dinteri A/W NSD A/W S1/S2 NSD
Grewia flava A/W A A/W S1/S2 S1/S2 Gololo et al. (2016)
Jatropha lagarinthoides A/W S2 A/W S1/S2 S1/S2
I. montana – S1 – S2 – Roux et al. (2017)
A: autumn; S1: spring; S2: summer, W: winter; NSD: no significant difference; (–): not determined.
may affect living organisms (Trivedy, 1995). The disparities in the phyto-
chemical compositions of selected medicinal plants as a result of expo-
sure to pollutants have been shown through some studies. In this regard,
Ujuwundu et al. investigated the effect of gas flaring on the phytochemical
composition of Treculia Africana and Vigna subterranean (2013). The
findings of such particular study demonstrated that tannins and cyano-
genic glycosides were in higher amounts in samples from polluted areas as
compared to those from unpolluted areas. In a separate study by Olivares,
phenols were found to be in higher amounts in the samples of Tithonia
diversifolia collected at heavy traffic roadside than in light traffic roadsides
(2003). The findings of the study by Rezanejad also reported significantly
higher levels of flavonoids in the pollen grains of Thuja orientalis from
the industrial and agricultural polluted area rather than in samples from an
unpolluted area (2009).
While the findings of the earlier cited studies project a scenario of a
positive correlation between pollution and the accumulation of phenolic
compounds, there are exceptions among medicinal plants as demonstrated
by the findings of the study by Gierlych and Karolewski (2000). In contrast
to the findings of the earlier cited studies, no significant differences were
found in the total phenolic contents of Pinus sylvestris between samples
from polluted and the unpolluted areas (Gierlych and Karolewski, 2000).
Differences in exposure to air pollutants were also found not to influence
flavonoid composition in the saplings of Psidium guajava (Myrtaceae)
both qualitatively and quantitatively (Furlan et al., 2010). Thus, the find-
ings of these studies suggest no generality in the effect of pollution on
the phenolic contents of medicinal plants. The suggestion is supported by
the contrasting findings of the cited studies regarding the accumulation of
phytochemicals in different medicinal plants upon exposure to different
levels of pollution.
12.2.4 NON-GENERALITY IN THE ENVIRONMENTAL

INFLUENCES ON PLANTS PHYTOCHEMICALS
First, the screening of plants used as health remedies for the presence of
specific phytochemicals is important to ascertain their medicinal value.
In addition to screening, quantitative analysis of phytochemicals found in
medicinal plants affords the determination of which plants are good sources
of specific phytochemicals. Since different phytochemicals exert specific
biological activities, the determination of plants from which specific
phytochemicals are found in relative abundance is also of paramount

importance. Such findings will enable the isolation of phytochemicals in
high yields, a limitation that is often encountered in the study of medicinal
plants. As demonstrated through a number of studies, the accumulation of
most phytochemicals is influenced by environmental factors (Ncube et al.,
2011; Nchabeleng et al., 2012; Saxena et al., 2013).
However, the findings of several studies paint a picture that the envi-
ronmental conditions under which certain phytochemicals are found in
abundance in one plant are not necessarily a given scenario for the next
plant. In other words, the response to environmental stimuli in terms of
phytochemical accumulations does not follow a general trend for different
plants. In addition, the environmental conditions under which a specific
phytochemical is found in abundance in a particular plant species are not
necessarily the same for other phytochemicals even in that same particular
plant species. As such, the influence of environmental factors on the phyto-
chemical composition of medicinal plants is mostly both plant species and
phytochemical specific. The study of the phytochemical compositions of
medicinal plants under the influence of different environmental conditions
has immense potential to contribute to the quality assurance of the use of
medicinal plants. It is important to determine the beneficial aspects or the
potential toxicity of specific compounds that are present in medicinal plants
under certain environmental conditions and absent in the same plants under
different conditions.
12.3 CONCLUSIONS
The review of the effect of environmental factors on phytochemical

compositions of selected medicinal plants highlights some disparities
in the manner in which different medicinal plants response to different
environmental factors. Disparities could be seen in the accumulation
of phytochemicals in medicinal plants as a response to differences in
geographical locations, seasonal variation, and different exposure levels
to pollutants. The review, therefore, indicates non-generality in the
accumulation of the phytochemicals of medicinal plants as a response
to environmental factors. Based on these non-generality in the environ-
mental factors-induced phytochemical composition of medicinal plants,
the undertaking of more studies for the determination of environmental
conditions under which important medicinal plants possess high amounts
of phytochemicals is recommended.
KEYWORDS
•• medicinal plants
•• phytochemical accumulation
•• environmental factors
•• seasonal variation
•• pollution
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CHAPTER 13
EFFECTS OF ENVIRONMENT ON THE

CHEMICAL CONSTITUENTS AND
BIOLOGICAL CHARACTERISTICS OF
SOME MEDICINAL PLANTS
VINESH KUMAR* and YOGITA SHARMA
Department of Sciences, Kids’ Science Academy, Roorkee,
Uttarakhand, India, Mob.: +91 9997228095
*
ABSTRACT
Environmental factors have significant effects on the growth and devel-

opment of plants. Climate change is responsible for the variation in
environmental conditions of different areas differently. The environmental
circumstances like frequency and intensity of weather events, such as hurri-
canes, storms, floods, and droughts are life-threatening. These events might
affect the flora and fauna of the place and also affects environmental factors
such as atmosphere, water, temperature, light, fire, nutrients, grazers, and
so forth. Change in environmental conditions has the effect on the habitat
and growth of plants. The environmental factors have significant effects
on the chemical constituents, generally on plant secondary metabolites.
These plant constituents are responsible for biological and pharmaceutical
activities. The results reveal that environment has the significant effects
on the plant phenology, secondary metabolites, and biological properties
of medicinal plants. This chapter describes the effects of environmental
factors on the chemical constituents and biological characteristics of some
medicinal plants.
13.1 INTRODUCTION
Environmental factors are of two types, that is, biotic and abiotic factors.
These factors have significant effects on the growth and development of
organisms. The abiotic components of environment include air, water, soil,
minerals, light, rain, and climatic conditions while biotic components are
all organisms including microorganisms. There is a continuous interaction
between these biotic and abiotic components. Plants are the producer, which
synthesize organic food from inorganic substances by several physiological
processes including photosynthesis.
Medicinal plants are extremely valuable to human beings to cure diseases
and maintain health. Medicinal plants are the backbone of traditional systems
of medicine including herbal therapy, Ayurveda, and aromatherapy; and also
have economic importance (Vinesh and Devendra, 2013). The lifecycle
of the medicinal plant from the dormancy state to germination, vegetative
propagation, growth, flowering, fruit and seed development and again when
it goes into a new dormant state, affected by several environmental factors.
The medicinal plants are very sensitive to their environment. The change
in the environmental conditions of an area may affect the medicinal plant
at any stage of their growth and development. The biogeochemical reac-
tions occur in the plant affected by changes in the environmental conditions.
These changes are also responsible for the nature of secondary metabolites
synthesized by plants.
The wealth of herbs of any country has an influence on the medical
sector as well as on the economics of any country (Vinesh and Devendra,
2013). The urbanization, transportation, industrialization, deforestation, and
agricultural activities are the major contributors responsible for increasing
the level of greenhouse gases in the atmosphere. The greenhouse gases are
responsible for global warming and climate change. The climate change is
responsible for the extreme weather events and environmental conditions
of an area. The frequency and intensity of these extreme weather events
increases which are life-threatening for all organisms including medicinal
plants. The human population is increasing day by day and increasing the
stress on medicinal plants.
Warming directly affects the rate of plant respiration, photosynthesis, and
other biogeochemical processes. The predicted patterns of global climate
change are the major concern in many areas of socioeconomic activities, such
as agriculture, forestry,and so forth, and are a major threat to biodiversity and
ecosystem function. Climate change has effects on the growth and production
Effects of Environment on the Chemical Constituents 281
of Saccharum officinarum and Mangifera indica (Vinesh et al., 2011). Climate

change is a result of the emission of greenhouse gases (e.g., CO2, CH4, and
N2O, etc.) in the past century that will cause atmospheric warming (IPCC,
2007a).
With environmental climatic factors, competition within the species is
also responsible for plant species. The different plant species show different
responses toward environmental factors.
13.2 CONSEQUENCES OF ENVIRONMENT CHANGE
The climate changes are responsible for the change in temperature, precipita-
tion, and season duration of a particular area. These environmental changes
could shorten the ripening period of the medicinal plants. The shortening in
the ripening period may decrease the size, weight, and production of grains
and fruits (Vinesh, 2011). The quick increasing concentration of greenhouse
gases in the troposphere has significant changes in regional and seasonal
climate pattern. These changes can strongly affect the distribution and diver-
sity of plants (Huntley, 1999).
Different plant species will react differently to climate change. Some
species of medicinal plants will stay in place but adapt to new climatic
conditions through assortment. Other species will shift to higher latitudes
or altitudes. Some species of medicinal plants may become extinct
(Keutgen, 1997). The results of some studies showed that range shifts of
individual species are likely to result in changes in community compo-
sition, as a result of local extinction and dispersal/migration (Benning,
2002). Salick states that due to rise in temperatures, some cold adopted
plant species migrate upward until there are no higher areas to inhabit,
at this tolerance point (altitude) these plant species may be faced with
extinction (2009).
The plant autecology depends on temperature, water, atmosphere,
light, and biotic factors (Daubenmire, 1974). Many authors have also
reported different responses to environmental factor by plants with C4
and C3 photosynthetic pathways (Christie and Detling, 1982; Ehleringer,
1985; Cadwell, 1985). If the changes in environmental factors including
temperature, precipitation, and drought are beyond the tolerance level, the
pressure will increase on medicinal plants. These factors can also modify
the secondary metabolites which can affect the medicinal properties of
the species.
13.3 ENVIRONMENTAL FACTORS AND THEIR EFFECTS ON

MEDICINAL PLANTS
The environmental factors which affect the growth, development, and

productivity of medicinal plants are temperature, water, light, atmosphere,
fire, nutrients, drought, and grazers.
13.3.1 TEMPERATURE
Researchers have recognized that temperature regulates the rate of physi-

ological processes and effects the growth and development of medicinal
plants. Each biological process occurs in a certain range of temperature and
has an optimal (minimal and maximum) operating temperature (Larcher,
1980). The temperature response also depends on other environmental
factors such as moisture and radiant energy (Laude, 1974).
According to Cooper and Tainton (1968), the suitable range of tempera-
ture lies between 20 and 25°C for almost all temperate C3 grasses while for
subtropical C4 grasses, the range is in between 30 and 35°C. As noted by
some authors, the increase in temperature with water stress is responsible for
mineral deficiency in plants. The decrease in temperature below the toler-
ance level also affects the plant physiology.
This result in delaying starting of growth of leaves, the opening of
stomata, restricts water movement to roots and decreasing the length of
photosynthesis during spring. In grassland farming of some regions of
the world, winter is a major cause of injury and death of forage plants
(Smith, 1964).
The alternative change in above and below freezing temperature can
cause damage to plant by frost heaving, acclimation, and freezing of plant
cells. Freezing of plant parts due to low temperature is a major cause of plant
death or loss and affect the distribution of plants.
According to Smith (1964), old plants are more sensitive to winter injury
than a younger plant because of insects, diseases, and poor fertility of the
land.
Winterkill (death of plants) due to low temperature has been reported
in rangelands for several shrub species. These include Caenothus velu-
tinus (Stickney, 1965), Purshia tridentata (Jensen and Urness, 1979),
Atriplex canescens (Van Epps, 1975), and Artemisia tridentata (Hanson,
1982).
13.3.2 WATER
Water is an important liquid, which sustains life on earth planet. All living
organisms including plants require water to sustain their life. The lack and
excess of water create stress on plants. The availability of water affects the
growth and development of water (Brown, 1977). He also describes that the
water deficiency occurs in plant when the rate of transpiration is more than
the rate of water absorption.
The prolonged stress of water on the shoot development effects leaf size
and internode length. According to Slatyer (1974), the root growth reduces in
proportion to shoot growth, delaye flowering time, reduces the size, number
and capability, and ceased growth and development followed by death in
severe water scarcity.
According to the study of Feldman (1984), in flooded areas oxygen
supply decreases or get stopped and normal exchanges of gasses from roots
may also get disturbed. The flooded environment also affects the metabolism
and inhibits the plant growth. The tolerance level varies from one plant
species to another species. The morphological and physiological adaptations
also get affected by the flooded environment.
13.3.3 LIGHT
According to Smith (1982), light is an essential environmental factor, for

the existence of higher photoautotrophic plants. The rate of photosynthesis
depends on the intensity and duration of light. Decrease in the amount of
light, effects the plant growth and physiology (Harper, 1977). The amount
of light absorbed depends on several factors including the age, size and
shape of leaves, the angle of incident light, and physiological conditions
(Risser, 1985).
The leaf is the first place of the plant, which accepts and interacts
with light. The growth rate of a plant depends on the leaf surface area.
According to Brougham (1956), the maximum growth rate occurs when
leaves absorb 95% of incoming solar light. The plants in limited light
resources adjust their structure and growth rate in available light (Harper,
1977).
Therefore, light plays an essential role in the growth and development
and is responsible for physiological changes in plants.
13.3.4 ATMOSPHERE
The blankets of gases include nitrogen 78%, oxygen 21%, and carbon
dioxide 0.03%. The carbon dioxide is required for photosynthesis, oxygen
is required for respiration, and nitrogen required for other physiochemical
processes. The toxic pollutants from the atmosphere, hydrosphere, and
lithosphere are also absorbed by the plant (Larcher, 1980). The cloud, wind,
fog, and some other component of the troposphere can influence the plant
growth and development.
13.3.5 FIRE
Medicinal plants are generally found in hilly and mountain areas. Fire
can affect medicinal plants directly or indirectly through heat and
gases. According to Scifres (1980), plant responses to fire are different
with phenology and morphological stages of growth and development.
The fire of forest severely destroys many plants species every year.
The existence of these species is dependent upon the capacity to restore
after losing of aerial stems.
13.3.6 GRAZERS
Grazers especially livestock, wildlife, and insects eat plant leaves and shoot
tissues. This affects the growth and development of plants. The plants show
responses with an increase in leaf replacement potential. This resistance
depends upon the stage of growth as well as on the species of plant (Hyder,
1972). Grazers also affect the soil fertility and influence several other factors
including soil erosion.
13.3.7 COMPETITION
Plants grow with different other plant species and have competition for
resources (Miller, 1980). The growing conditions for different plant species
differ from one species to another. The availability of resources affects the
number and distribution of plant species.
13.4 EFFECTS OF ENVIRONMENT ON CHEMICAL CONSTITUENTS
Climatic factors include moisture, light, temperature, and so on (Yang,

2004). They regulate the processes of water, soil, and heat conditions. These
are the primary factors for the growth and development of herbal medicine
(Yang, 2001).
There are studies which noticed that climate change is causing visible
effects on the distribution, growth, life cycles, and on secondary metabolites.
The plants of high altitudes of the Himalayas, those remains under the snow
cover for a significant period of time and unprotected to the severe environ-
mental situations, clamps a vast potential to work as a medicine for some
chronic illnesses (Kala, 2009).
The result of different studies showed that climate change has significant
effects on natural resources and alter temperature ranges, weather events,
seasonal patterns, and so forth (IPCC, 2007a, b; Root, 2003; Parmesan,
2003). The moments of climate change are now being felt in different areas
across the world and its consequences on seasonal movement in terrestrial
ecosystems are significant and well recognized (Root, 2003; Walther, 2002).
Results suggest that range shifts of individual plant species are probable
to outcome in deviations in community arrangement, as a consequence of
native extinction and migration (Hansen, 2001; Benning, 2002).
In the Indian Himalayan region, predominantly in alpine areas, variations
in temperatures and snow patterns are already affecting the phenology and
distribution of some plant species (Nautiyal, 2004). The study of Khan-
duri (2008), has explored some phenological alterations in more than 650
temperate plant species.
Alpine ecosystems are more sensitive to climate warming since plant
species have altitudinal limits of climatic limits and limitation of resources
(Panigrahy, 2010). Thus, alpine plant species have various morphological
and physiological modifications to adept against adverse climatic changes.
13.5 EFFECT OF ENVIRONMENT ON SECONDARY CHEMICAL

PRODUCTION
Climate change influences weather patterns, temperature and result in loss

of habitat, the shift of species distribution, altered species composition and
has effects on growth and development of plant species. There has been
little concern on medicinal plants and their secondary metabolite production.
The changes in environmental conditions may directly change plant fitness
(Galen and Stanton, 1991; Wookey, 1993), as well as change the reproduc-
tive success of plants species and flowering phenology (Hughes, 2000).
The studies showed that change in environment could alter the chemical
composition to change itself for the survival in high altitude region.
Predominantly, the temperature stress can alter secondary plant metabolites
and many other compounds that plants synthesis, which is basically respon-
sible for medicinal activity (Salick, 2009; Zobayed, 2005). Normally when
plants are under stress, secondary metabolite production may rise because
growth is often self-conscious more than photosynthesis, and the carbon
fixed not assigned to growth is in its place assigned to secondary metabolites
(Mooney 1991).
Numerous studies have observed the effects of increased temperatures
on plant’s secondary metabolite production, but most of these studies have
conflicting results (Jochum, 2007). Some reveal that secondary metabo-
lites production increases with increase in temperatures (Litvak, 2002),
while others report that these decrease with increase in the temperature
(Snow, 2003).
According to Chaturvedi (2007), the increase in temperature and the CO2
level will change growth cycles of alpine plants and active chemical consti-
tutes of the plants as a result of physiological changes (Chaturvedi, 2007).
The change in the secondary metabolites may affect the medicinal value and
activities of the plant. For a long-term supply of quality raw material for
curing illnesses, studies on the effects of environment on plant secondary
metabolic production and composition become important.
Medicinal plants are the rich sources of many biologically active
compounds including phenolic compounds, tannins, steroids, and flavo-
noids. Phenolic constituents and other secondary plant metabolites show
a chemical interface between environment and plants. The synthesis and
concentration of plant metabolites depend upon several environmental
factors (Gobbo-Neto, 2007).
The tannin concentration has been found higher during spring and
summer in species of lotus (Gobrehiwot, 2001). The same results were seen
in Alnus rubra (Gonazlez, 2002). Bruno (2011) observed that the concentra-
tion of metabolites in the leaves of plant Lafoensia pacari influences by
temperature, micronutrients, and other environmental factors. The concen-
tration of phenolic compounds in plant tissues depend upon the surrounding
temperature. It was founded that at low temperature, the concentration of
phenolic compounds was maximum (Padda, 2008). He also suggested that
high concentration of phenolic compounds at low temperature is due to the
increased activity of phenylalanine ammonia lyase enzyme.
Júlio et al. (2006) found a close relationship between the rainfall level
and tannins concentrations in plants Myracrodruon urundeuva and Anade-
nanthera colubrina.
According to Hatano et al. (1986), the ecological factors are responsible
for qualitative and quantitative changes in the level of tannins. The environ-
mental factors can change the biosynthetic pathways. In many species, the
concentration of phenolic compounds increases at high temperature.
According to Gebrehiwot (2002), the change in the concentration of
tannin occurs with changing in the seasons. Similar results were observed
in the A. rubra. The study of Coley (2002) showed that the plants growing
in fertile soil have a high concentration of total phenolic compounds and
tannins in comparing the plants growth in poor soil. The concentration of
phenol and tannin is high in Ceratonia siliqua in water stress.
The environmental conditions also affect the size of plant leaves, rhizome,
and stem. Plants resist biological, physical, and chemical environmental
stresses by regulating the accumulation of secondary metabolites in long
periods of adaption to the environment. Ecological factors are the dominant
factors affecting the secondary metabolites of the plants (Ncube, 2012).
Environmental conditions show a key part in describing the function and
distribution of plants, in combination with other factors. There is already
evidence that plant species are shifting their ranges in altitude and latitude
as a response to changing regional climates. The timing of phenological
events such as flowering is often related to environmental variables such
as temperature. Changing environments are therefore expected to lead to
changes in life cycle events, and these have been recorded for many species
of plants (Parmesan and Yohe, 2003).
The weather or environmental conditions have a significant effect on
phytochemical constituents, concentration, moisture contents, ash contents,
and biological characteristics of the plants. India has tremendous causes
to be worried about the impacts of climate change on plants especially
medicinal herbs. In the future, research should be carried out on others
plants worldwide.
Medicinal plants are the rich source of phytochemicals with interesting
biological and medicinal properties (Gibson, 1998). These phytochemicals
protect plants from disease and repair damaged cells. These plant metabo-
lites protect plants cells and tissues from environmental hazards such as
drought, ultraviolet exposure, pollution, and pathogenic attack (Gibson,
1998; Mathai, 2000).
About 4000 phytochemicals have been listed and are grouped by
physical characteristics and chemical characteristics with their protective
functions (Meagher, 1999). These phytochemicals are present in vegetables,

stem, leaves, flowers, fruits, legumes, nuts, whole grains, spices, herbs, and
seeds (Mathai, 2000; Costa, 1999). The environmental factors including soil
nutrients, foliar nutrients, and climate affect the leaf of Myrcia tomentosa
which has essential oil composition (Leonardo et al. 1980).
13.6 EFFECT OF ENVIRONMENT ON BIOLOGICAL ACTIVITIES
The primary chemical constituents include vitamins, proteins, sugars, amino

acids, chlorophylls, and so forth. Secondary metabolites include flavonoids,
alkaloids, lignans, terpenes, plant steroids, saponins, phenolics, curcumin,
glucosides, and flavonoids (Hahn, 1998). The biological activities of these
medicinal plants depend upon the nature and concentration of phytochemicals
plant metabolites. These phytochemicals are also known as secondary
plant metabolites and have biological properties including antimicrobial
effect, antioxidant activity, decreasing of platelet aggregation, modulation
of detoxification enzymes, stimulations of the immune system, anticancer
property, and modulation of hormone metabolism. Plants synthesize these
chemicals to protect themselves and can also be used by human to protect
against diseases (Narasinga, 2003). The amounts of these phytochemicals
depend upon the variety, processing, nutrients, and environmental conditions
(King, 1999). Phytochemicals may also be used as food supplements
(American Cancer Society, 2000). The change in the nature and concentration
of secondary metabolites may alter the biological and medicinal properties of
medicinal plants.
13.7 CONCLUSION
Environmental factors have significant effects on the growth and develop-

ment of plants. Medicinal plants are extremely valuable to human beings
to cure diseases and maintain health. Medicinal plants are the backbone of
the traditional system of medicine and also have economic importance. The
environmental factors which affect the growth, development, and produc-
tivity are temperature, water, light, atmosphere, fire, nutrients, drought,
and grazers. The change in the environmental conditions of an area may
affect the medicinal plant at any stage of their growth and development.
The biogeochemical reactions occur in the plant affected by changes in the
environmental conditions. These changes are also responsible for the nature
of secondary metabolites synthesized by plants. Medicinal plants are the rich

source of phytochemicals with interesting biological and medicinal proper-
ties. The change in the nature and concentration of secondary metabolites
may alter the biological and medicinal properties of herbs.
KEYWORDS
•• environment
•• climate change
•• fauna and flora
•• medicinal plants
•• metabolites
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CHAPTER 14
PHYTOCHELATINS AND HEAVY

METAL TOLERANCE IN PLANTS
MARIA CATHERINE B. OTERO1 and GENEVIEVE D. TUPAS2,*
1
College of Medicine Research Center, Davao Medical School
Foundation, Inc., Davao City, Philippines.
2
Department of Pharmacology, College of Medicine,
Davao Medical School Foundation Inc., Davao City, Philippines,
Tel.: +639175351638
*
ABSTRACT
Heavy metal tolerance is an adaptation of plants to the terrestrial environment.

Since most plants rely on the soil for nutrients and water, mechanisms to neutralize
the effects of leached heavy metals on plant metabolism evolved. Chelation by
heavy metal-binding ligands is the most common way plants respond to heavy
metal toxicity. Phytochelatins (PCs) are cysteine-rich peptide ligands found
in plants and yeast that bind and sequester toxic metal ions into vacuoles to
detoxify the plant cells. Mounting evidence suggests that numerous plants are
able to produce PCs. However, constitutively expressed PC synthase (PCs)-like
genes were also reported in certain animals, but there is no evidence of animal
gene products with PC functions, suggesting roles other than PC biosynthesis.
PCs, glutathione, and O-acetyl-L-serine influence hyperaccumulation of heavy
metals in certain plants. The ability of plants to clean up heavy metals from the
soil and water contributes to phytoremediation in metal contaminated areas.
14.1 INTRODUCTION
There are several ways by which plants respond and adapt to heavy metal
toxicity. These include exclusion, immobilization, activation of the general
stress response in plants, and chelation and sequestration of metal ions

(Cobbett, 2000). Among these responses, the most common is chelation of
metal-binding ligands, and in certain cases, the localization of the resulting
ligand–metal complex in the vacuole.
Metal-binding ligands include organic acids such as citrate and malate,
and the peptides ligands (Cobbett, 2000). PCs are analogous to metallothio-
neins (MT), a class of metal-binding proteins, in terms of functions, but
differ entirely in the structure and biosynthesis. MTs are expressed by the
cell, while PCs are synthesized enzymatically and are not encoded in the
plant genome. Bacteria and other prokaryotic cells produce MTs similar to
mammalian MTs, but not PCs. PCs offer an advantage over MTs in metal
binding since PCs also possess strong reactive oxygen species (ROS) scav-
enging activities (Tsuji et al., 2002).
There are two general characteristics of PCs. First, PCs are synthesized
only when the plant cells are exposed to metal ions such as Cu2+, Pb2+, Zn2+,
and Ag+ and are thus heavy metal-inducible. Second, PCs are capable of
binding to more than one type of metal or metalloid, hence, it is not very
specific to a particular heavy metal.
14.1.1 PHYTOCHELATIN (PC) STRUCTURE
PCs are cysteine-rich peptides found in plants and yeast that bind and
sequester toxic metal ions. The term was coined by Grill et al. (1985)
when they found peptides with (γ-Glu-Cys)n-Gly general structure in plants
exposed to Cd2+, with variations only in the number of γ-Glu-Cys (γ-EC)
repeats, denoted by n (Grill et al., 1985). PCs generally have the range 2–5,
but studies have shown that the number of γ-EC repeats can go as high as 11
(Cobbett, 2000).
Table 14.1 list the general structure of PC and its variants. PC-like
peptides have been reported in certain yeast species, where the glycine at
the C-terminal was replaced by des-glycine. Other PC variants have been
characterized as well, and differ from PC because the terminal glycine is
replaced by either alanine, glutamate, serine, or glutamine. Homophyto-
chelatins derived from homoglutathione were reported in several plants
from the family Fabaceae and have the general formula of (γ-Glu-Cys)
n
-Ala. Iso-phytochelatins found in maize have glutamate instead of the
terminal glycine, while those found in horseradish substituted the terminal
glycine with glutamine. Hydroxymethyl-phytochelatins in plants from the
Gramineae family have serine instead of the terminal glycine (Inouhe, 2005).
Phytochelatins and Heavy Metal Tolerance in Plants 295
TABLE 14.1 General Structure of Phytochelatins and their Variants.

Type General structure
Phytochelatin (γ-Glu-Cys)n-Gly
Homophytochelatin (γ-Glu-Cys)n-Ala
Iso-phytochelatin (γ-Glu-Cys)n-Glu or
(γ-Glu-Cys)n-Gln
Hydroxymethyl-phytochelatin (γ-Glu-Cys)n-Ser
14.1.2 FUNCTIONS OF PCs
PCs relieve heavy metal stress and detoxify the plant cells in the presence
of glutathione (GSH). Many other cellular processes are mediated by GSH,
and elevated GSH levels alone do not always predict enhanced heavy metal
tolerance (Xiang et al., 2001). In cyanobacteria, which are the ancestors of
chloroplasts in plants, antioxidant activities are mainly carried out by GSH
(Yadav, 2010).
PC and PC-related peptides have been reported in plants and yeast.
Mounting evidence suggests that numerous plants are able to produce PCs.
Based on expressed sequence tags, it is clear that phytochelatin synthase
(PCS) genes can be found in all higher plants (angiosperms, gymnosperms,
and bryophytes). PCS genes were also found to be expressed constitutively,
suggesting roles other than PC biosynthesis (Yadav, 2010). PCS-like genes
were also reported in the nematodes, roundworms, and schistosomes
(Inouhe, 2005), but there is no evidence of animal gene products with PC
functions (Cobbett, 2000).
14.1.3 PC BIOSYNTHESIS
GSH and PC biosynthesis overlap because GSH acts as a direct substrate in

PC formation. PCs are non-translationally formed from the reduced form
of GSH by transpeptidation, which is catalyzed by the PCS (Yadav, 2010).
The steps in PC biosynthesis are shown in Figure 14.1. First, the
γ-glutamylcysteinesynthetase (γ-EC synthetase, γECS) mediates the
formation of the γ-glutamylcysteine from cysteine and glutamate. The
γ-ECS acts as the rate-limiting step and its activity is enhanced by Cd2+.
The next step is catalyzed by GSH synthetase, which adds a glycine to the
γ-EC to form GSH. Lastly, the PCS (formerly called the γ-glutamylcysteine
dipeptidyl transpeptidase) sequentially adds the γ-EC units of the GSH to

form PCs (Cobbett, 2000; Inouhe, 2005). Cd2+ was found to be the best
activator of PCS, followed by Ag+, Bi3+, Pb2+, Zn2+, Cu2+, Hg+, and Au+. PC
biosynthesis is regulated through a negative feedback mechanism, where
the PC produced chelates the activating metal ions to terminate the reac-
tion (Cobbett, 2000).
FIGURE 14.1 Glutathione-dependent synthesis of phytochelatins.
14.2 HEAVY METAL TOLERANCE IN PLANTS
The soil is the major sink of heavy metals that may come from various
industrial and agricultural applications and the natural geochemical cycle
(Wuana and Okieimen, 2011), and in order to survive, plants must respond to
this environmental stress. The most common heavy metals in contaminated
soils include arsenic (As), lead (Pb), zinc, (Zn), chromium (Cr), cadmium
(Cd), mercury (Hg), nickel (Ni), and copper (Cu) (Evanko and David, 1997).
Although normally found in low levels, anthropogenic activities catalyze
the accumulation of heavy metals in soils. These activities include, but are
not limited to, sewage sludge, use of fertilizers, emissions from machines
and factories, metal mining, and metal smelting (Yadav, 2010). Because
heavy metals are resistant to chemical or microbial degradation, they can
persist in soils for a very long time, only changing their chemical forms and
bioavailability (Kirpichtchikova et al., 2006; Adriano, 2003). Most plants
rely on the soil for nutrients and water and so mechanisms to neutralize the
effects of leached heavy metals on plant metabolism evolved. Thus, heavy
metal tolerance is postulated to be an adaptation of plants to the terrestrial
environment (Inouhe, 2005).
14.2.1 EFFECT OF HEAVY METALS ON PLANTS
Certain heavy metals and metalloids are essential for plants and animals.
Copper, molybdenum, manganese, iron, zinc, and nickel are required for
optimal growth and development of higher plants. Arsenic, lead, cadmium,
and tin are also necessary, but in very low concentrations (Alloway, 2013).
Once the concentrations of these heavy metals go above the threshold and
accumulate in the cells, they can lead to toxicity in the plant cells since
metals cannot be metabolized.
In general, once the plant is exposed to excess heavy metals, the cells
experience oxidative stress and ROS are generated. Excess ROS in the cells
can induce oxidation and consequent modification of membrane lipids,
proteins and cellular amino acids, and DNA (Yadav, 2010). When not
resolved, the ionic homeostasis will be disturbed and cellular damage can
ensue (Singh et al., 2011; Demiral and Turkan, 2005). Indirect consequences
of increased heavy metal concentration in the plant include the substitution
of essential nutrients, effects on growth of beneficial microorganisms, and
decreased soil fertility (Alloway, 2013). Taken together, both the direct and
indirect consequences of high heavy metal concentration in plants will result
in decreased plant growth and eventually, plant death (Fig. 14.2).
FIGURE 14.2 Consequences of heavy metal stress in plants.
Here we discuss the effects of elevated levels of specific heavy metals in

the development of plants.
14.2.1.1 EFFECT OF EXCESS CADMIUM (CD)
Plants growing beyond the permissible limit of 100-mg cadmium level

for every 1 kg of soil already show signs and symptoms of cellular injury
such as browning of root tips, chlorosis, inhibition of growth, and death

(Mohanpuria et al., 2007). Excess Cd interferes with transport, utilization,
as well as uptake of water and Mg, K, Ca, and K by plants. It also alters the
activity of the plant enzymes in the root and shoot, such as Fe(III) reductase,
nitrate reductase, and even ATPases. Additionally, lipid peroxidation of the
cell membrane is induced and chlorophyll biosynthesis is inhibited (Asati
et al., 2016).
14.2.1.2 EFFECT OF EXCESS LEAD (PB)
One of the most abundant heavy metal in the soil is lead. Its effects on plants
in excess concentrations may include morphology, plant growth, and photo-
synthesis disruption. Lead toxicity can result in interference with enzyme
activity. This eventually causes inhibition of stem and root elongation,
inhibition of leaf expansion, and induction of abnormal morphology, such
as radial thickening and lignifications. In corn, lead was found to reduce
the percentage of germination, suppress plant growth, reduce biomass, and
decrease in protein content of plant cells (Asati et al., 2016).
14.2.1.3 EFFECTS OF EXCESS COPPER (CU)
Copper is an essential micronutrient with important roles in ATP synthesis

and carbon fixation (Pichhode and Nikhil, 2015). It is also required in
various proteins in the electron transport chain and the photosystems. In
excess amounts, however, copper can result in leaf chlorosis and growth
retardation. Seed production and biomass are reduced, as well as root growth
(Yadav, 2010). Copper mostly accumulates in the roots. In tomato, the
enzyme activities of guaiacol peroxidases, catalase, and polyphenol oxidase
increased in response to increasing Cu. At higher concentrations of Cu,
enzyme activities decreased, suggesting that other detoxification systems
are already in place (Martins and Mourato, 2006).
14.2.1.4 EFFECTS OF EXCESS ARSENIC (AS)
Inorganic As in the form of arsenate or arsenite is easily taken up by plants.

Arsenic competes directly with phosphate for the root plasmalemma uptake
(Yadav, 2010). Besides transport, other phosphate-dependent pathways in
metabolism can also be affected. Once As is translocated to the shoots, it

can inhibit biomass accumulation and expansion and overall plant growth.
Arsenic can also negatively affect plant reproduction by reducing fruit
production and fertility (Garg and Singla, 2011). Certain plant species are
able to tolerate increased As in the soil. In grasses, for example, As toler-
ance develops when the high-affinity phosphate/arsenic uptake system is
suppressed. In this way, the influx of As is reduced so that the plant is detoxi-
fied through constitutive mechanisms (Meharg, 1994).
14.2.2 MECHANISMS OF HEAVY METAL TOLERANCE
Tolerance to various heavy metals can be achieved in several ways. One is to

sequester the heavy metals into vacuoles, which was documented in plants
and yeast. Sequestration of PCs to the vacuole was observed in tobacco
plants in response to excess cadmium, where almost all of the Cd and low
and high molecular weight PC–Cd complexes were localized in the vacuole.
The transport of these complexes to the vacuolar membrane is through an
ATP-dependent, protein gradient-independent transporter (Cobbett, 2000).
Another mechanism is to pump the heavy metals out of the cell membrane,
as in the proton gradient-dependent Cd/H+ antiporter. Last is to chelate the
toxic metals and bind them to thiol compounds that are found in the cytosol
like GSH (Yadav, 2010).
Genetic manipulations in PC synthesis and GSH-related genes in plants
have allowed investigations about tolerance in various heavy metals. Mishra
et al. (2006) have shown that Pb tolerance is also mediated by PCs in coon-
tail plants. However, overproduction of PCs may deplete GSH, and cause
oxidative stress. Inorganic arsenic also induced PC synthesis in vitro and in
intact land plants.
14.3 METAL HYPERACCUMULATION AND PHYTOREMEDIATION
14.3.1 HYPERACCUMULATOR PLANTS
There are over 400 species of plants that can hyperaccumulate trace metals,
metalloids, and nonmetals in their shoots and more than 100 of these are
heavy metal hyperaccumulators (Yadav, 2010). The plant family Brassicaceae
accounts for the most number of metal hyperaccumulators, with 87 species
across 11 genera showing activity against Ni, Zn, Cd, and Pb (Prasad and
Freitas, 2003). Five fern species from the family Pteridaceae have been
found to be arsenic hyperaccumulators: Pteris vittata, Pteris cretica, Pteris
longifolia, Pteris umbrosa, and Pityrogramma calomelanos (Meharg, 2002).
The brake fern, P. vittata, can carry more than 20,000 mg/kg per dry weight
of its fronds.
This ability of certain plants to store a large amount of metals coming
from its environment highlights their potential in biogeochemical pros-
pecting and phytoremediation. However, leached metals coming from
exudates of hyperaccumulator plants can have a detrimental effect on the
growth of neighboring plants (Prasad and Freitas, 2003). The safety of
human consumption of these special plants is also an important issue that
must not be overlooked and should be regulated.
14.3.2 PHYTOREMEDIATION
Hyperaccumulation of metals in certain plant species has been taken advan-

tage in plant-based remediation of contaminated sites or phytoremediation.
There are four mechanisms involved in phytoremediation in water, sediment,
or soil: rhizofiltration, phytostabilization, phytovolatilization, and phytoex-
traction (Prasad and Freitas, 2003). These mechanisms depend largely on
the bioavailability of the metals in the target site. But because of the slow
speed at which contaminated sites can be cleaned by hyperaccumulators,
whether genetically engineered or naturally occurring, phytoremediation is
considered as a long-term solution.
14.4 BIOMARKERS FOR METAL HYPERACCUMULATION
In addition to PC and GSH, the levels of the cysteine intermediate O-acetyl-

L-serine had been strongly correlated to metal hyperaccumulation in certain
plants (Yadav, 2010). Hyperaccumulator plants possess long-distance
transport systems for metals through the transpiration stream. This apoplastic
route carries heavy metals from the roots to the shoots. This ability to suck
out metals from the soil and water is said to contribute to phytoremediation
in metal contaminated areas (Inouhe, 2005). Overexpression of the GSH1
gene was found to be a promising strategy in producing transgenic plants
that have highly efficient Cd phytoremediation capacities (Yadav, 2010).
PCs and PC-related peptides can potentially be used as biochemical
indicators of heavy metal contamination. Roots and shoots are sensitive
to various metals, as influenced by the rapid changes in the levels of PCs,

GSH, and cysteine. These changes can be harnessed to quantify heavy
metal contamination in habitats. (Inouhe, 2005). But other less understood
mechanisms for metal detoxification may affect such biochemical changes,
interpretation must be made with caution.
KEYWORDS
•• phytochelatins
•• heavy metal tolerance
•• phytoremediation
•• hyperaccumulation
•• plants
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CHAPTER 15
PHYTOCHEMICAL BIOPESTICIDES
OLUMAYOWA VINCENT ORIYOMI
Institute of Ecology and Environmental Studies, Obafemi Awolowo
University, Ile-Ife, Osun State, Nigeria, Tel.: +2347031245211,
E-mail: [email protected].
ORCID: http://orcid.org/0000-0002-2438-6740.
ABSTRACT
Alarming pest infestation leading to food shortage and the corresponding

food demand resulting in the indiscriminate application of synthetic pesti-
cides has raised global concerns on food security and environmental safety.
Phytochemical biopesticides have been used as repellents, antifeedants,
fungicides, herbicides, nematicides, molluscicides, and insecticides among
others. Phytochemicals form an interesting group of bioactive compounds
which are generally slow in action as plant protectants but safer on the
environment and humans as compared to synthetic pesticides. Plant species
which belong to families of Solanaceae, Labiatae, Euphorbiaceae, Astera-
ceae, Meliaceae, and Fabaceae are peculiar among over 2000 plants with
secondary metabolites having insecticidal properties. Currently, drawing
interests are species of the Araceae family. Plant metabolites (phytochemi-
cals) with most important insecticidal activities are alkaloids, flavonoids,
phenols, steroids, and terpenoids. This chapter underlines the unique proper-
ties and prospects of phytochemical biopesticides as potential alternatives to
synthetic pesticides in the control of agricultural pests.
15.1 INTRODUCTION
World’s population has been estimated to surge by 38.57% from 6.134

billion to 8.5 billion inhabitants by 2025 with a significant growth rate in
the developing countries (Agrios, 1997). This will inevitably require an

additional agricultural production to meet the impending monstrous popula-
tion growth. Economic history provides us with ample of evidences that
agricultural revolution is a veritable ingredient for economic growth espe-
cially in developing nations (Izuchukwu, 2011). Agriculture is among the
major pillars capable of sustaining any economy. Two-third of today’s world
population depend on agriculture for livelihood, but nowadays, growth and
production of agricultural crops are hampered daily by pests among several
other limiting factors (FAO, 2014).
Insect pest is any insect in the wrong place. They are considered pests
whenever they are in competition with humans for resources and are present
in significant numbers (William and Robert, 1994). Agricultural pest species
that damage crops are estimated at 67,000 by Kumar (2015). Insect pests
inflict damage on humans, farm animals, and crops. Also, pests damage
important agricultural foods as well as their products in godowns, bins, and
stores, causing huge physical deterioration and economic losses to stored
foods (Oonagh and Paul, 2009). In the world, food availability is adversely
affected by insects and pests during growth and post-harvest periods
(Kulkarni et al., 2009). Annually, one-fifth of the world’s total crop produc-
tion is damaged by herbivorous insect pests. About 67,000 agricultural pest
population has been estimated to be involved in crop damage.
One major factor responsible for pest multiplication is the creation of
altered habitats particularly in the tropical and sub-tropical zones, where
favorable climate provides a viable environmental atmosphere for a broad
range of insect activities and survival (Kfir, 1997). The artificial agroecosys-
tems created fulfill man’s needs by providing selected crops with large sizes,
high yield, and nutritious value clustered in a confined area. The system
satisfies man’s demands for agricultural crop production and in the process
provides a suitable environment for herbivorous insects (Kerin, 1994). Pests
are capable of developing to biotypes capable of beginning new lives and
adjust to new situations, for instance, they can become resistant to numerous
control measures and agents, thereby escalating the problem of pest infesta-
tion. In underdeveloped countries, the menace of pest infestation is further
complicated with a rapid annual increase in human population (2.5–3.0%) in
comparison to a 1% increase in food production (William and Robert, 1994).
Efforts to salvage problems caused by pest infestation have caused the use of
pesticides to rise over the years.
Pesticides are defined as natural or synthetic agents capable of killing
an unwanted plant or animal pests. They are referred to as substances or
mixture of substances intended for preventing, destroying, or controlling
Phytochemical Biopesticides 305
pests including vectors of human or animal diseases, harmful species of

plants or animals which interfere with production/processing, storage and
marketing of food, agricultural commodities, wood and wood products, or
animal feeds, or agricultural product that may be administered to animals
to control pests inside or outside of their bodies (FAO, 1989). A pesticide is
also defined as chemical formulated to combat activities of pests and disease
vectors on domestic animals, agricultural crops, and human beings (WHO/
UNEP, 1990). The term pesticide includes chemicals applied as growth
regulators, defoliants, desiccants, fruit-thinning agents, or chemical agents
which are used for preventing premature fall of fruits or substances applied
to crops either before or after harvest to prevent deterioration during storage
and transport or on animals to control pest infestation and growth retarda-
tion. All the definitions stated above show that chemical pesticides (organic)
are toxic agents which are used to eliminate or at least reduce insect pest
population in other to ensure food availability and security.
Insecticides such as miticides, avicides, defoliants, bactericides, algae-
cides, piscicides, desiccants, nematicides, herbicides, rodenticides, fungi-
cides, and so on are common examples of pesticides. Unknowingly to some,
chemical pesticides have only recently become popular; it was the naturally
occurring compounds or extracts that were used as pesticides in ancient
times. The earliest pesticides were salt, sulfurous rock, extracts of tobacco,
red pepper, and the like. The Napoleonic army was rumored to have applied
ground chrysanthemums to combat lice with limited effectiveness. Petro-
leum oils and other heavy metals were used as natural agents to control pests
in the 1940s. They were then replaced for use by heavy synthetic pesticides;
the most famous of the synthetic pesticides was dichlorodiphenyltrichloro-
ethane (DDT, C14H9Cl5). It is noteworthy that pesticides exclude chemicals
used as fertilizers, plant and animal nutrients, food additives, and animal
drugs. Generally, chemical pesticides are highly toxic, very persistent in
the environment, and have residues which are harmful and can contaminate
crops, food commodities and also pollute soil and groundwater. According
to World Health Organization, the human death toll of about 20,000 people
is attributed annually to pesticide poisoning in the third world countries
(Casida and Quistad, 2000). Synthetic pesticides adversely affect nontarget
organisms such as pollinators, fish, birds, and animals. The indiscriminate
and excessive use of synthetic chemicals has resulted in increased resistance
to pests. Phytochemicals/biopesticides, on the other hand, are less toxic, least
persistent, environment-friendly, and safe to humans and nontarget organ-
isms (Walia et al., 2017). There are interests on phytochemical pesticide due
to concerns on a rampant supply of synthetic pesticide and its irreversible
impact on the environment and human health at large. Also, its presence in
the food web is quite worrisome (Anupam et al., 2012).
According to reports although with developments still in infancy but
fast gaining huge attention is the modern approach to pest control involving
nanotechnology (science that uses materials having 10−9 m size) application.
The technology uses nanoparticles (NPs) like mesoporous silica NPs, porous
hollow silica NPs, silica NPs, and so on, as nanobiopesticides in biological
pest management (Popat et al., 2012). In this process, materials prepared
from noble metals such as gold, silver, and platinum or metal oxidase mate-
rials (TiO2, ZnO, AgO, MgO), ceramics, semiconductors, silicates, magnetic
materials, lipids, polymers, emulsions, dendrites, and quantum dots are used
as NPs. The technology encapsulates bioactive compounds with NPs to form
nanobiopesticides used to control pests. NPs possess insecticidal property
due to their numerous unique characteristics such as:
1. Extraordinary strength
2. High chemical reactivity
3. High electrical conductivity and optical properties
4. Distinct physical, chemical, and biological properties associated
with high atomic strength
5. Specific maintenance of size and shape
6. Size–depth qualities
7. High surface-to-volume ratio
8. High stability
9. An ordered layer of particles arrangement due to hydrogen bonding,
dipole forces, hydrophilic and hydrophobic interaction, surface
tension, and gravity
10. Slow release and high efficiency on host plant capable of deterring
insect attack
11. Higher mobility and lower toxicity
12. High pest specificity and effectiveness
13. High self-assemblage stability, specificity, encapsulation, and biocom-
patibility (Wang et al., 2009)
15.2 PHYTOCHEMICAL PESTICIDES
Phytochemicals (botanicals) are secondary metabolites which majorly

perform the role of defense mechanism and help plants withstand continuous
selection pressure from herbivorous predators and other environmental
factors (Sarkar and Kshirsagar, 2014). Phytochemical pesticides are natu-

rally slow-acting agents or crop protectants; they are usually safer to the
environment as well as to humans than the conventional pesticides with
minimal residual effects. Moreover, plant botanicals contain mixtures of
biologically active substances which are able to prevent resistance buildup
in pests and pathogens. Numerous investigations have established that
biological activity is usually distributed in various parts of a plant though
the degree of lethality of the active components may vary in these parts
(Rajapake and Ratnaseka, 2008). The use of plant botanicals has now been
more recommended as a capable substitute for plant protection with minimal
negative effects (Isman, 2006). Literature revealed that about 2121 plant
species possess pest management properties, out of these, 1005 plant species
exhibit insecticidal properties. Among this population, 384 species exhibit
antifeedant properties, 297 species show repellent properties while 27
species possess attractant properties and 31 species have growth-inhibiting
properties (Gopalakrishnan et al., 2011). At present, the world’s botanical
pesticides are still at a meager 1% (Anupam et al., 2012).
15.3 ADVANTAGES OF PHYTOCHEMICAL PESTICIDE OVER

SYNTHETIC PESTICIDE
Following are the advantages of phytochemical pesticide over synthetic

pesticide:
1. Promotes sustainable agriculture: It does not cause a negative effect

on crop plants, soil health, and environment. It presents a relatively
low risk to beneficial predators and parasites (nontarget organisms).
2. Reduces crop losses: Several plant diseases/plant pathogens can
be effectively managed by reducing disease incidence and related
losses in the crop plants.
3. Eco-friendly: It does not cause ecological imbalance and fits in suit-
ably in any agro-ecosystem. It is safer to humans and the environ-
ment than conventional pesticide as it presents no residue problems
and does not persist in the environment.
4. Biodegradable: It rapidly degrades under the exposure of sunlight.
It has limited field persistence and a short shelf life. It breaks down
rapidly in the environment.
5. Organic farming: It suitably fits in the organic farming system as it
is eco-friendly in nature.
6. Cheaper: It is cheaper when compared to synthetic pesticides.

7. Integrated pest management: It is easily included in the framework
of integrated pest management routine.
8. Specificity in action: It has a narrow target range and a very specific
mode of action.
9. Favorable application period: It has a relatively critical application
time and suppresses, rather than eliminate pest population
10. Easy Metabolism: it is easily metabolized by animals receiving sub-
lethal doses;
11. Easy Formulation: it has a broad spectrum of activity, and is easy to
process and use.
15.4 LIMITATIONS OF PHYTOCHEMICAL BIOPESTICIDES
Following are the limitations of phytochemical biopesticides:
1. Phytochemical biopesticides are easily degraded in the field under

high temperature, light, moisture, and pH. Their degradability
reduces the availability of their active components.
2. As a result of earlier limitation, frequent application of botanicals is
needed. This implies that cost of application will increase. Addition
of cheap local materials such as gum arabic, egg white, and so forth,
or commercial products and protectants of ultraviolet (UV) rays
(e.g., soap water and antioxidants) to extend the residual efficacy of
sprayed botanical solution have been suggested (Gahukar, 2010).
3. Operating rules for registration of phytochemical biopesticides are
quite stringent and procedures involved are time consuming. The
technical formalities prescribed for chemical pesticides are also
prescribed for plant products. These procedures need be changed in
the future to encourage easy formulations and registration of newer
botanicals.
4. Presently, there is an inadequate awareness of the importance and
value of patents (regulations provided by government agencies) on
the use of phytochemical biopesticides (Gahukar, 2010).
5. Interdisciplinary expertise required for extraction, isolation, separa-
tion, purification, structural elucidation, bioassay, regulatory, and
other related aspects is still inadequate.
6. Stabilizers, potentiators, UV screens, antioxidants, and other instru-
ments are required to increase stability, shelf life, and residual life
of products; these will help to improve bioefficacy in botanicals in

other to suppress resistance development in insects. However, these
materials are scares and expensive.
7. Some plant species are indigenous only to a certain region(s). Some
plants form a community of biodiversity which are valuable assets
for the local people. Most times, plants having insecticidal properties
are only abundant in few climatic zones and government protected
areas which are un(der) exploited not only for traditional prepara-
tions but also for commercial formulations of botanicals. Such plant
species are protected by forest rules and regulations against exploi-
tation. These stringent biodiversity protection laws have included
many plants in the red list and thus, provision of botanicals has
greatly been impeded.
8. Processes involved in isolation, synthesis, and formulation, struc-
tural elucidation, bioassay, and others of phytochemicals are long
and expensive (Jaglan et al., 1997).
15.5 SELECTION CRITERIA FOR PLANTS SOURCES OF

BIOPESTICIDES
Dimetry (2014) prescribed the following criteria for consideration in

selecting a plant for botanicals:
1. It should be a perennial plant.

2. The plant should have a broad distribution and be available in large
numbers in nature or possibly be grown by agricultural procedures
like tissue cultures and genetic engineering. Plant must be easy to
replicate or regenerate.
3. Plant parts to be used should be removable: leaves, flowers, fruits,
and so forth.
4. Harvesting does not mean total destruction of the plant (always
avoid the use of roots or barks).
5. Plants should require small space to thrive and survive. It must be
easy to manage and require little water and fertilization.
6. The plants could have additional uses (e.g., medicinal use).
7. The plants should not possess high economic value.
8. Their biologically active components should be effective at a low
concentration.
15.6 EFFICACY OF SELECTED PHYTOCHEMICAL BIOPESTICIDES
Researchers have reported the efficacy of several botanicals extracted from

plants. Examined below are some of these plants and their phytochemical
constituents:
15.6.1 NEEM
Neem tree, scientifically known as Azadirachta indica is a good source of

phytochemicals and more than 300 bioactive secondary compounds have
been isolated from neem. Isolated bioactive chemicals include ketones,
limonoids, phenolics, steroids, carotenoids, and enzymes (Rafiq et al., 2012).
The chemistry, environmental behavior, and pesticidal effects of neem and
neem products have been reported widely (Veitch et al., 2008). Laboratory
tests indicated that neem-extract-based insecticides are effective against pest
species with agricultural and environmental interests (Koul, 1999). Limonoid,
known as tetranortriterpenoid, forms the major group of neem’s secondary
compounds such as azadirachtin, nimbin, nimbidin, salannin, and so forth
(Govidachari et al., 1995). Reports have established that azadirachtin and
its compounds possess high insecticidal activity. Neem has many biological
properties such as low persistency in nature, low toxicity against nontarget
organisms, and systemic action. Azadirachtin has been studied more than any
other plant for botanicals and has received greater attention for biopesticide
formulation than others because of its bioefficacy against insects (Prakash
et al., 2002). Azadirachtin showed antifeedant effects on insects especially
those belonging to Lepidoptera order have been found to be most susceptible
to azadirachtin as compared to other orders like Coleoptera, Hemiptera,
and Homoptera including caterpillars, pink bollworms, leafworms, beetles,
gypsy moths, mushroom flies, and so forth, (Mordue and Blackwell, 1993).
Azadirachtin influences hormonal coordination in insects which modifies the
metamorphosis. It seems to act as “ecdysone blocker” that blocks the release
of vital hormones, thereby inhibiting molting in insects and metamorphosis
in general (Ascher, 1993). Azadirachtin limits feeding in insects leading to
death (Thomson, 1992). Generally, the effects of azadirachtin depend on
both dosage and time of application which prevent ecdysis and apolysis that
lead to death during molting.
Natural neem has expressed notable anti-insecticidal activity against
noctuid moths, leafworm (Spodoptera litura), Peridroma saucia,
Oncopeltus fasciatus, leafhopper, Jacobiasca lybica, and whitefly (Sharma
et al., 2003a, b). This broad range insecticidal activity is aided by neem’s
phago and oviposition deterrent, repellent, antifeedant, growth depressant,
molting depressant, and sterilant properties. Products of neem prolong larval
developmental times and prevent larval maturation (Mordue and Blackwell,
1993). Reports of broad insecticidal properties of neem on Plutella
xylostella, Pieris brassicae, Spodoptera littoralis, and other pests are also
well documented (Hasan and Ansari, 2011). High insecticidal activity was
also demonstrated by extracts from neem seed kernel under field conditions
against insect species of Orthoptera, Lepidoptera, and Coleoptera.
Bioactive products of neem include azadirachtin, nimbinene, salannin, and
nimbin. They act as systemic and contact poisons against pests (Koul et al.,
1996). Insects differ markedly in their behavioral/physiological responses
to azadirachtin/related compounds/neem extracts. Observed differences
in physiological responses and toxicity to neem have been observed in S.
littoralis (Lepidoptera), O. fasciatus (Hemiptera), Schistocerca gregaria
(Orthoptera) (Aerts and Mordue, 1997). Lepidopteran insects show higher
sensitivity to azadirachtin than the Orthopoda. Summation of neem’s
antifeedant and toxicity properties increase its efficacy insects. Malformation
of S. littoralis at various developmental stages by azadirachtins has been
confirmed by many authors (Nathan and Kalaivani, 2006). Inter-genus
variation in term of effectiveness was also shown by different compounds of
neem against Lepidopteran members, Heliothis virescens and Helicoverpa
armigera (Blaney et al., 1990). Reports of interfamily variations of
azadirachtin on Noctuidae members have been well documented. Inhibition
of S. litura and Actebia fennica were less than other species (Isman, 1993).
Nathan et al. (2005) reported growth and antifeedant inhibition activities of
various neem products (azadirachtin, deacetylgedunin, salannin, gedunin,
17-hydroxyazadiradione, and deacetylnimbin) against the rice leaf folder
and legume pod borer H. armigera. Apart from major tetranortriterpenoids
(nimbin and salannin) of neem seeds, photo-oxidation products of
tetranortriterpenoids such as nimbinolide, isonimbinolide, salanninolide,
and isosalanninolide have also demonstrated anti-insect effects against
S. littoralis (Jarvis et al., 1997). Extracts from neem seed kernel have a
profound effect on rice leaf folder and sorghum shootfly Atherigona soccata.
The effect of neem seed kernel extract (NSKE) was reported for potato
tuber moth Phthorimaea operculella, H. armigera, and Lampides boeticus
(Irulandi and Balasubramanian, 2000). Azadirachtin is the major active
constituent of neem extracts, the effects of neem extracts could be due to the
sum or synergy of azadirachtin and other terpenoids present in it (Martinez
and Van Emden, 2001). The activity of azadirachtin in neem extracts is high,
but non-azadirachtin compounds (e.g., 6-ß-hydroxygedunin or different

volatiles from neem) isolated from A. indica also seem to be involved in the
bioefficacy of neem (Koul et al., 2003).
Purified neem compound/formulation exhibits instability and produces
negative effects on natural enemies, its role as a broad-spectrum insecticide
at very low concentrations with residual properties and reduced resistance to
pests has made these compounds important in insect pest management and
commercialization in countries such as the United States, Canada, Mexico,
European Union, and New Zealand and in Asian countries, such as India
and China. Neem products are the most commercially important of plant
species used in insect pest management (Schmutterer, 2002). Examples of
commercially available neem products are the Wash Away Louse, Neemix,
Neemazal, Tre-san, Neem Gold, Neem-EC, Margosan-O, Thai Neem 111,
MiteStop, Picksan Louse Stop, and Neem Excel (Yule and Srinivasan,
2013). Their efficacies have been shown against various pests and insects.
15.6.2 ANNONA
Researches have shown annonaceae to be potent elicitors of insecticidal

activities. All plant species in this family have toxic compounds such as
flavonoids, acetogenins, and alkaloids that confer them insecticidal effects
(Grzybowski et al., 2012). In the genus of annona, two species are known
to have outstanding insecticidal properties; Annona muricata and Annona
squamosa. The bioactive effects of Annonaceous acetogenins (ACGs)
have been confirmed against species of insects like Myzus persicae, spider
mites, mosquito larvae, striped cucumber beetles, melon aphids, Colorado
potato beetles, Mexican bean beetles, bean leaf beetles, European corn
borers, blowfly larvae, and free-living nematodes (Cólom et al., 2008).
Extracts from annonaceous plants have been studied in several groups of
agriculturally important insects. There is a similarity in the biological activity
of acetogenins to that of limonoid azadirachtin isolated from A. indica
(Mordue and Nisbet, 2000). Recent reports revealed ACGs are effective
against store pests of grains. Extracts of A. squamosa and Annona mucosa
have exhibited toxicity against Tribolium castaneum and Sitophilus zeamais,
respectively (Ribeiro et al., 2013). Isoquinoline alkaloids are also associated
with plant protection against herbivorous insect pests. Isoquinoline interacts
with neural signal transduction and interfere with neuroreceptors through
enzymes involved in neurotransmission and ion-gated channels (Wink,
2000). A. muricata and A. squamosa have been applied as bioinsecticides
to control Culex quinquefasciatus and Aedes albopictus mosquitoes in

Madagascar (Ravaomanarivo et al., 2014). Upon application of the extracts,
significant insecticidal effects were observed with the extracts of the two
plants compared to induced mortality by deltamethrin, an insecticide of
reference. Research has revealed that seeds of A. squamosal and A. muricata
contain a great amount of acetogenins (Das et al., 2007). This group of
compounds is referred to as mitochondrial complex I inhibitor. Few alkaloids
are associated with A. muricata which not only affect mortality of pupal and
adult stages of mosquitoes but also reduce reproductive rate of their adults by
decreasing their fecundity and egg hatchability (Kaushik and Saini, 2009).
Annona products are till date not available commercially and studies have
not addressed the commercial use of these products.
15.6.3 PONGAMIA
Pongomia is among the numerous chemicals used to protect plants from

pests in modern agriculture (Stepanycheva et al., 2014). It is one of the most
interesting subjects of study in recent years with widespread synanthropic
and insecticidal activities (Pavela, 2009). The oil of pongamia contains
5–6% flavonoids. Karanjin, a furanoflavonoid compound, is the main
constituent of pongomia flavonoid, known for its insecticidal properties
(Kumar and Singh, 2002). Isolation of this flavonoid compound from oil
and de-fatted oil cakes has been reported by several authors (Susarla et al.,
2012). Insecticidal property of Karanjin has been enhanced by structural
modifications which convert Karanjin to karanj ketone, karanj ketone oxime
esters, and karanj ketone oxime N-O-nonanoate. This modified insecticidal
property has been tested on aphid and had a remarkable effect (Lipaphis
erysimi) (Mondal et al., 2010). In 2014, Stepanycheva et al. revealed that
Pongomia pinnata oil-based formulated products had aphicidal activity
for M. persicae adults and larvae. They were found to possess prolonged
action and exert negative effects on pest offsprings. Several compounds of
pongamia, either oil or extract from methanol/aqueous/chloroform/acetone
solvents are biologically active against insect pests. Pongamia compounds
act as oviposition deterrents, insecticides, repellents, antifeedants, and
larvicides (Kumar and Singh, 2002). The extract of P. pinnata is toxic against
H. armigera, S. litura, and pests of stored grains (Reena et al., 2012). Karanj
oil and Karanj leaf extract have demonstrated insecticidal activities against
mustard aphid; L. erysimi (Singh, 2007). Karanj extract is a constituent of
commercial insecticidal formulations such as Salotrap, Plexin, Karrich,
RD Repelin, and RD9 Repelin used for controlling insect pests. PONEEM
is another regulated insecticide formulated from pongam and neem oils.
However, compared with neem products, reduced efficacy of Karanj extract
in aqueous solutions is a limiting factor for their wide spread acceptability
and applications.
15.6.4 JATROPHA
Jatropha curcas, referred to as physic nut, is a tropical plant which belongs

to the class of Euphorbiacea native to North America, Africa, and Asia.
J. curcas is a tropical ubiquitous plant often used for fencing by farmers
(Jide-Ojo et al., 2013). The use of Jatropha in pest control has been reported
by a plethora of researchers around the world (Nash, 2005). Extracts of
Jatropha possess insecticidal or molluscicidal/anthelminthic activities on
agricultural and nonagricultural pests (Musa and Olaniran, 2015). Jatropha
consist 47% fat and various antinutritional factors such as saponin, phytate,
trypsin inhibitor, and cyanogenic glycosides (Rakshit et al., 2008). Many
terpenoids are present as secondary metabolites in Jatropha. To date, 65
forms of terpenes from Jatropha have been formulated (Devappa et al., 2011).
Phorbol esters (PEs) are the major toxic constituents of Jatropha. Structural
property, biological activity, toxicity, medicinal properties, phytochemistry,
and pharmacological properties of PEs in Jatropha on animals have been fully
investigated by researchers (Sabandar et al., 2013). The toxic constituents of
PEs present in extracts of J. curcas possess molluscicidal, insecticidal, and
fungicidal properties (Liu et al., 1997). The PEs contained in Jatropha oil
were effectively toxic against insect pests of field crops and stored grains such
as Callosobruchus maculatus, C. chinensis, Clavigralla tomentosicollis, S.
zeamais, Rhyzopertha dominica, T. castaneum, Oryzaephilus surinamensis,
L. erysimi, Pieris. rapae, P. operculella, Tetranychus urticae, O. fasciatus,
Coptotermes vastator, Amrasca biguttula, Aphis gossypii, and Aphis fabae
(Adebowale and Adedire, 2006; Silva et al., 2012) by oviposition deterrent,
antifeedant, ovicidal, and anti-birth depressants. The PE-enriched fraction
of Jatropha has been tested against many insect pests such as Spodoptera.
frugiperda, C. maculatus, Corcyra cephalonica, Busseola fusca, Sesamia
calamistis, H. armigera, and Manduca sexta (Khani et al., 2012). Extract
from Jatropha gossypiifolia showed toxic effects on Lepidopteran pests:
Ostrinia nubilalis, B. fusca, and S. nonagrioides (Valencia et al., 2006).
Furthermore, PE fraction has shown antifeedant and insecticidal effects
against S. frugiperda and Spodoptera exigua (Bullangpoti et al., 2012).
15.6.5 WEEDS AS ANTI-INSECTS
Here, it is worth considering the importance of weeds as botanical insecticides.

Lantana is an invasive species in the tropical and subtropical zones of the
world. Lantana exhibits insecticidal properties on aphids, mites, potato tuber
moths, and Spilosoma obliqua (Suliman et al., 2003). Parthenium and cyperus
were successfully used to minimize Epilachna beetle, diamond back moth,
and cabbage head caterpillar in vegetables (Prijono et al., 1997). Calotropis
was also reportedly active on rice plant hoppers (Prakash et al., 2008).
15.6.6 MEDICINAL HERBS AS PLANT PROTECTANTS
There are reports on the use of medicinally grouped herbs in the control of
agriculturally important pests. Herbs such as Gynandropsis gynandra, Vitex,
Ocimum, Catharanthus roseus, and Euphorbia royleana were effective against
caterpillars, H. armigera, Maconellicoccus hirsutus, mustard aphid, and
Epilachna beetle (Prakash et al., 2008). Also, Epilachna beetle was controlled
by herbs such as Strychnos nux-vomica and Solanum xanthocarpum (Chitra
et al., 1991). Plants like Vernonia amygdalina and bitter gourd known for their
bitter tastes have shown efficacy against flea beetle on okra and coffee leaves
(Leucoptera coffeella) (Onunkun, 2012). Passiflora mollissima referred to as
banana passion fruit has been used in food industry to control pests.
15.6.7 SPICES AND CONDIMENTS AS PLANT PROTECTANTS
Reports on domestic botanicals from condiments and spices such as peppers

and garlic as sources of botanical pesticides have been well documented
(Antonious et al., 2007). Powdered chili pepper, for example, deterred onion
fly, Delia antique, and Earias insulana. Garlic is effective against soft-
bodied insects such as aphids, cumin (Nigella sativa) and Epilachna beetle.
15.6.8 ESSENTIAL OILS
Essential oils (EOs) are mixtures of volatile organic compounds produced as

secondary metabolites in plants majorly for defense against pests and patho-
gens. EOs are majorly extracted from plant families such as Lamiaceae,
Myrtaceae, Lauraceae, and Asteraceae. EOs just like other plant botanicals
have antifeedant, fumigant, toxicity, repellent, reproduction retardant, and
growth-reducing effects on pests (Singh and Singh, 1991). EOs are neuro-
toxic and interfere with neuromodulator octopamine (Enan, 2005) or iono-
tropic receptors like GABA-gated ion channels. Zoubiri and Baaliouamer
(2014) have reported about 230 plants and EOs with active compounds
showing insecticidal activities.
15.6.9 BOTANICAL PESTICIDES FROM HERBAL COMPOST
Together with insecticides derived from leaf and seed extracts of plants,
composts from plants have severally been used in insect pest control
(Gopalakrishnan et al., 2011). Bio-washes of crude extracts of Annona,
Jatropha, and Pongamia vermin composts have killed H. armigera and S.
litura. Compost from maize stover was effective in the control of whiteflies,
Zonocerus variegatus, Podagrica sp. and B. tabaci with high efficacy of
60–80% control. Organic composts, especially the one from maize stover,
can be used as an insecticide in organic farms to raise okra. Foliar application
of organic composts is effective for controlling pest infestation of Telfairia
occidentalis (Alao et al., 2011).
15.6.10 MISCELLANEOUS
Plantago, flowers of male Zea mays, mahua, Psoralea corylifolia, Linden-

bergia grandiflora, and velvet bean (Mucuna cochinchinensis) have all
demonstrated degree of insecticidal effects against insect pests of various
agricultural crops. Also reported were extracts of tea, Solanum nigrum,
cassava, Solanum incanum, sweet potato, Mexican marigold, Mexican tea,
blackjack (Bidens pilosa), thorn apple, papaya, and aloe for exhibiting insec-
ticidal effects (Alves et al., 2011).
15.7 SYNERGISM
Biological activities of botanicals enhance combinations of biomolecules. An

extensive study was carried out on formulation of PONEEM from pongam and
neem oils. Phytochemicals present in PONEEM karanjin and azadirachtin have
been used to control Scirtothrips dorsalis in chili thrips. They were efficient as
feeding deterrent (Packiam and Ignacimuthu, 2013). The phytopesticides was
also examined against S. litura and H. armigera. The study examined different
concentrations of the phyto-mixture on oviposition-deterrent activities of the

pests (Packiam et al., 2012). A formulation with pongam oil and Thymus
vulgaris/Foeniculum vulgare showed lower LC50 values against diamondback
moth (P. xylostella) than pongam oil alone indicates the synergism between
the botanicals (Pavela, 2012). Combined extract from Acacia arabica,
Bacillus thuringiensis subsp. kurstaki (Btk), Nicotiana tabacum, A. squamosa,
Eucalyptus globulus, Datura stramonium, Ipomoea carnea, Lantana camara,
and P. pinnata was formulated by Rajguru et al. (2011). They investigated
the efficacy of the formulated mixture and its synergistic activity against S.
litura larvae and reported the mixture to be possessing high mortality showing
that high effectiveness of the fortified extracts was due to synergistic action.
Bioactive extracts from N. tabacum A. arabica, and D. stramonium have given
great results compatible with Btk. Mixture of extracts from neem seed kernel
and Murraya koenigii leaf was also found to be effective against adult beetles
(Radha and Susheela, 2014). The formulated mixture acted as antifeedant with
varying degree of toxicity on cowpea weevil as:
NSKE + M. koenigii leaf extract > neem > M. koenigii.
The use of recombinant DNA technology to enhance the efficacy of

biopesticides has been reported by researchers. According to reports, under-
standing of genes from microorganisms and crop plants has enabled isola-
tion of effective genes against pests and diseases to be controlled (Kumar,
2013). In other cases, fusion proteins are used to design next-generation
biopesticides. DNA technology makes use of toxins (though not toxic to
higher animals) combined with carrier protein to control pests, the toxins
are toxic to insect pests when consumed orally, that is more effective when
ingested by predators.
15.8 FUTURE PROSPECTS OF BOTANICAL INSECTICIDES
Evidently, botanical insecticides have plenty benefits over the commonly

applied synthetic pesticides with many deleterious effects on humans,
animals, environments, and economically important microorganisms. Eco-
friendly botanical insecticides probably cannot be expected to completely
replace synthetic insecticides within a short time. However, with increasing
campaign and advancements in botanical insecticides, the use of botanical
insecticides, having numerous benefits will soon outface synthetic pesticides
application. Benefits of using botanical insecticides include environment
and health safety coupled with the protection of agricultural crops as against
synthetic insecticides associated with risk of integrating hazardous residues
in food crops. Also, the prospect of botanical insecticides will be appreci-
ated in human and animal protection against medically important vectors or
insects (Pavela, 2012).
The future looks bright for botanicals with negative reports on synthetic
pesticides associated with environmental risks resulting from their indis-
criminate application. These negativities on synthetic pesticides have shifted
interest toward botanical pesticides as alternative agents in pest management.
Phytochemicals will in the future play vital roles in pest control both in the
industrialized and developing countries. Countries rich in viable biodiversity
should quickly bioprospect their flora to document their botanicals in order
to prevent future biopiracy and establish their sovereignty on botanical pesti-
cides developed from their plants (Dimetry, 2014). Application of botanical
biopesticides may require knowledge of insect pests on which the botanicals
would be successfully be applied. Also, a correct time of application is
essential to ensure the efficacy of the biopesticide (Kumar, 2015). A better
understanding of the mode of action of the biopesticides, their effects, regula-
tory issues that arise on their adoption may help to raise their profile among
the public and policy-makers. With this promising future, farmers, research
institutes, governments, and all concerned agencies are encouraged to start
growing economically important plants reported to be rich in phytochemical
biopesticides. Botanical trees may be the next global oil to be explored.
15.9 REGISTRATION AND REGULATIONS OF BOTANICALS
Pesticide registration is the process through which Environmental Protection

Agency (EPA) examines the ingredients of a pesticide/botanical, the site or
crop on which it is to be used, the amount, frequency, timing of use, and
storage and disposal instructions of the plant botanical. Before any botanical
can be registered, marketed or used, specific agencies, bodies or authorized
ministries empowered by the government of any interested country must
ascertain the safety of such botanical for use. A botanical cannot legally
be used, sold, or distributed if not registered with EPA in the country. EPA
makes online resources, such as the Pesticide Registration Manual (blue
book) available to assist applicants through the registration process (USEPA,
2014). In the United States, for instance, The Federal Insecticide, Fungicide,
and Rodenticide Act requires that EPA evaluates proposed pesticide to assure
that its use does not pose risks to the health of humans, environment, biota,
and nontarget organisms. This evaluation involves an extensive review of

health and safety information of the impending botanical. In Nigeria, the
National Agency for Food and Drug Administration and Control (NAFDAC)
empowered by Sections 5 and 29 of NAFDAC laws. NAFDAC was itself
established by Decree No. 15 of 1993, now encapsulated in the NAFDAC
Act, Cap NI Laws of the Federation of Nigeria. NAFDAC mandates include
evaluation of pesticides to ensure safe and effective pesticides are avail-
able for use by the public. Act No. 19 of 1993 (as amended) specifies that
no processed food, drug, cosmetic, drug product, medical device, or water
shall be manufactured, advertised, imported, exported, sold, or distributed
in Nigeria unless registered in accordance with the provisions of Decree
No.15 of 1993. Arising from the law, the Agency has also set various regula-
tions in motion such as Pesticides Registration Regulations, Chemical, and
Chemical Products (control, monitoring) Regulations. The latter is still in
draft form awaiting approval. Pesticide residues in Nigeria are analyzed and
monitored in an International Atomic Energy Agency-accredited laboratory
at the NAFDAC Central Laboratory Complex, Oshodi, Lagos.
Pesticides registration is overseen by many units under NAFDAC. The
registration of pesticides and agrochemicals is supervised by Veterinary
Drugs and Pesticides Unit under Registration and Regulatory Affairs Direc-
torate of NAFDAC. The Chemical Import Control and Chemical Monitoring
units of the Narcotics and Controlled Substance Directorate are responsible
for controlling agrochemicals and pesticides. NAFDAC has made provisions
for a functional database which contains all registered regulated products in
Nigeria made available at www.nafdacregistry.net. About 354 pesticides and
agrochemicals had been registered in Nigeria as in August 2008. Nigeria is
also in support of ECOWAS regional structure and procedures for pesticide
registration through the West Africa Committee on Pesticide Registration
ratified by ECOWAS council of ministers.
KEYWORDS
•• synthetic pesticides
•• biopesticides
•• future prospects
•• botanicals
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CHAPTER 16
A SUSTAINABLE APPROACH IN
INTEGRATED PEST MANAGEMENT:
ROLE OF PHYTOMOLECULES AS
BIOPESTICIDE
RAKESH KUMAR GUPTA1, PREM PRAKASH KUSHWAHA2, and
SHASHANK KUMAR2,*
1
School of Environment and Earth Science, Environmental Science
and Technology, Central University of Punjab, Bathinda,
Punjab 151001, India, Mob.: +919335647413
2
Department of Biochemistry and Microbial Sciences, School of Basic
and Applied Sciences, Central University of Punjab, Bathinda,
Punjab 151001, India
*
Corresponding author. E-mail: [email protected],
[email protected]
*ORCID: https://orcid.org/0000-0002-9622-0512
ABSTRACT
Though the use of pesticides has offered significant economic benefits by

enhancing the food production and the prevention of vector-borne diseases,
evidence suggests that their use has adversely affected the health of human
populations and the environment. In the search for eco-friendly solutions to
control the insect pest and environmental management, the great interest in
plants and their chemo-biodiversity as a potential source of phytochemicals
for biopesticides has increased over the time. Plants are a rich source of
bioactive molecules for biopesticides. These substances have been exploited
as commercial products and may play an important role in an integrated
pest management as a benefit of humankind and suitable for environmental
protection. For a successful application of phytochemical-based biopesti-

cides limited a broad range of criteria such as biological, environmental,
commercial, and regulatory must be satisfied. Plant products are compatible
with several other biopesticides and synthetic pesticides. They are therefore
recommended for large-scale application in pest control and sustainable
environment. There is thus an urgent need to organize sustainable natural
sources, develop quality control, adopt standardization strategies, and
modify regulatory mechanisms.
16.1 INTRODUCTION
An estimate of the global crop losses due to pest has declined from 13.6%
in post-green revolution era to 10.8% toward the beginning of this century
while in India follows the same trend from 23.3 to 15.7% at present (Dhaliwal
et al., 2015). Thus, the worldwide food production is adversely affected by
these pests during the crop growth, harvest, and storage (Kulkarni et al.,
2009). Although the synthetic chemical method has effective tools in modern
crop management to control the pest and diseases. The extensive use of
pesticides effectively control insect pest and led to increase the agricultural
output. Ideally, a pesticide must be destructive to the targeted pests, but
not to nontarget species, including man (Aktar et al., 2009). However, the
synthetic pesticides are highly toxic, recalcitrant, and longtime persistent in
the environment. It contaminated crops, food commodities, and their toxic
residues accumulated in food, water, air, and soil. Unfortunately, the wide-
spread use of these chemical has led to many negative consequences (Pavela,
2008). Besides, the use of plant-based biopesticides offers an attractive
alternative to manage the insect pests and diseases in an eco-friendly way.
Because phytochemical-based biopesticides are natural, renewable, readily
biodegradable, non-pollutive, nontoxic, easily available, and relatively cost-
effective. Besides these attributes high specificity to target pests, and pose
no or less hazard to the environment or to human health, slow resistance
development, and less residual activity environment. Owing to the above
attributes, the role of biopesticides is considered as a potent and reliable tool
in integrated pest management programme (IPM) to manage insect pests.
Thus, these increase attention to search out plants-based natural insecticidal
products (Pirali-Kheirabadi and da Silva, 2010).
It may contribute potential substitute to currently used insect control agents.
Plants constitute a rich source of plant-based bioactive chemicals (Qin et al.,
2010). Plant-derived insecticides contain natural active biological compound
A Sustainable Approach in Integrated Pest Management 327
which act as a deterrents or repellents (Islam et al., 2009), fumigants (Choi

et al., 2006), insecticides (Tang et al., 2007), antifeedants (González-Coloma
et al., 2006), and may the influence some other biological parameters such
as growth rate (Nathan et al., 2008), life duration, and reproduction (Isikber
et al., 2006). These compounds belong to various groups of chemicals such
as alkaloids, rotenoids and pyrethrins, steroids, terpenoids, phenolics, and
essential oils have been reported for their insecticidal properties (Shaalan
et al., 2005). Most of them are secondary metabolites that are known for their
insecticidal properties (Lopez et al., 2008) and in many cases plants have
been used as home remedies to kill or repel insects (Kim et al., 2010). The
novel research on the interactions between plants and insects pest has clearly
shown the potential use of plant-based metabolites for this purpose (Kamaraj
et al., 2010).
To investigate the suitable alternatives to traditional insecticides,
phytochemical-based biopesticides have been widely scrutinized. With the
objective of contributing to these studies, there were many plants species that
have been tested and evaluated against a diverse group of insect pests in the
laboratory as well as field conditions, particularly for insecticidal activity.
Research on insecticidal activity of various plants and plant materials
against different insect has been conducted and has yielded positive results
(Khaliq et al., 2014; Radha, 2014; Yohannes, 2014). This has revealed in
terms of toxicity, mortality, inhibition, suppression of oviposition repellency
and reduces the reproduction potentiality. This chapter focuses on the
plant-based pesticide product and its activity against the economically
important insect pests. The aim of this chapter is to contribute an overview
of the plant’s origin biological active compounds that have been reported to
possess insecticidal activity against insect pest.
In this regard, plants being considered as an alternative source of
insect control agents owing to constitute a rich source of plant-driven
bioactive chemicals (Kim et al., 2003). Yet only a few phytochemicals are
currently used in agriculture, and there are few promises for the commer-
cial advancement of new biopesticides products. Several factors appear
to limit the success of plant-driven biopesticide, most notably regulatory
barriers and the availability of competing products of microbial-based
biofertilizers that are cost-effective and relatively safe compared with
their predecessors. The significance of these plant genetic resources, as
potential insecticides become evident when looking at the context of an
increasing number of insects, showing resistance against conventional
insecticides (Adeyemi, 2010).
However, threat and problems allied with the use of chemicals lead
to increasingly rigorous environmental regulation of pesticides (Pavela
et al., 2010). There is thus a need to develop safer, more eco-friendly, and
efficient substitute that has the potential to replace conventional pesticides
and are suitable to use (Tapondjou et al., 2005). In this context, screening
of plant-based natural products has received much attention of researchers
around the world (Kebede et al., 2010). Keeping all these facts in mind, an
emerging challenge in the new millennium is to produce more and more
food from keeping the environment safe and sustainable IPM.
16.2 CONCEPT OF BIOPESTICIDES FROM PLANTS-BASED GREEN

COMPOUND
Phytochemicals (botanicals) are plant-based natural occurring products of

biological active compound as potential sources of new green biopesticides
over the conventional synthetic pesticides (Table 16.1). They are also
called natural insecticides or biopesticides. The term phytochemical-based
biopesticide embraced a wide diversity of biologically active chemical.
In general, these active ingredients extracts/isolated from different parts
of the plants such as leaves, roots, barks, fruits, seeds (Altemimi et al.,
2017). These botanicals products from plants classified as either primary
or secondary plant metabolites. There are several medicinal and aromatic
plants that have the ability to synthesise and produce numerous secondary
metabolites which are known for their insecticidal properties (Lopez et al.,
2008). These compounds belong to various groups of chemicals such as
alkaloids, rotenoids and pyrethrins, steroids, terpenoids, phenolics, and
essential oils have been reported for their insecticidal properties (Shaalan
et al., 2005). These plant-based active constituents act in various way
including insecticides (kill to adults, ova, and larvae), insect repellents,
antifeedants, molluscicides, fungicides, and phytotoxins (Okwute, 2012).
Few of the secondary metabolites alter the behavior and life cycle of insect’s
pests which are called as semiochemicals. In addition, currently, essential
oil which is a mixture of volatile compounds accumulated in seeds, flowers,
and leaves are among the best-known substances evaluated against insects
(Pitasawat et al., 2007). Various conventional use of phytochemicals have
been reported such as nicotine from Nicotiana tabacum, rotenone from
Lonchocarpus sp., derris dust from Derris elliptica, and pyrethrum from
Chrysanthemum cinerariiaefolium (El-Wakeil, 2013).
TABLE 16.1 Phytochemical-based Insecticide Used to Control Different Insect Pests.
Plant Scientific name Family Active principle Plant parts used Insect pests
Neem Azadirachta indica Meliaceae Azadirachtin Seeds and leaves Aphids, whiteflies, leafhoppers, psyllids, scales,
mites and thrips, armyworms, cutworms, stem
borers, bollworms, leaf miners, lepidoptera
Pyrethrum Chrysanthemum Asteraceae Pyrethrin Dried flowers Caterpillars, aphids, bugs, cabbage worms,
cinerariifolium beetles, leafhoppers, spider mites
Sabadilla Schoenocaulon officinale Liliaceae Cevadine and Seeds Grasshoppers, codling moths, armyworms,
vertridine cabbage loopers, squash bugs, aphids
Ryanodine Ryania speciosa Flacourtaceae Ryanoids Woody stems Codling moths, potato aphids, onion thrips, corn
earworms
Tobacco Nicotiana tobaccum and Solanaceae Nicotine Plants leaves Aphids, thrips, caterpillars
Nicotiana rustica
Rotenone Derris eliptica and Fabaceae Rotenone, related Roots Bugs, aphids, potato beetles, spider mites,
Lonchocarpus spp. isoflavones carpenter ants
16.3 CONVENTIONAL BOTANICALS: AZADIRACHTIN,

NICOTINE, ROTENONES, SABADILLA, AND PYRETHRUM AND
THEIR MODE OF ACTION
16.3.1 AZADIRACHTIN: MODE OF ACTION
Neem, Azadirachta indica is evergreen, tall, and fast-growing plants

belonging to Meliaceae family, native to Indian subcontinent (Bhattacharyya
et al., 2007; Lokanadhan et al., 2012). It has medicinal and pesticidal
properties that have been used from the ancient time at least before 2500
years (Bhattacharyya et al., 2007). Various part of neem plants such as the
seeds, bark, and leaves contain active compounds with justifying medicinal
properties such as antiseptic, anti-inflammatory, antiviral, antipyretic,
antifungal uses, and anti-ulcer. At present, neem plant is recognized as a
natural and eco-friendly product which has much to offer in solving global
agricultural, environmental, and public health problems. There are hundreds
of active compound that have driven from neem tree used to manufacture a
number of valuable products. Azadirachtin is the main bioactive ingredient
used to manufacture biopesticides. Neem oil and seed extracts and other
active ingredients known to possess as insect growth regulators with the
addition to germicidal and antibacterial properties which are used to protect
and control bacteria, fungi, nematode, and different kinds of other insect
pests. Neem insecticides and their other product are very helpful in plant
protection and management (Lokanadhan et al., 2012).
The active compound of neem acts at different levels and in various ways
(Lokanadhan et al., 2012). Azadirachtin, a well-studied compound acts as
antifeedant, antiperistaltic, oviposition deterrent, growth regulator, and affect
juvenile hormone (Bhattacharyya et al., 2007; Lokanadhan et al., 2012). It has
been reported that azadirachtin significantly affects the Lepidopteran group of
pest as antifeedants (<1–50 ppm, depending upon species) while Hemiptera,
Homoptera, and Coleoptera, are less affected to azadirachtin behaviorally with
up to 100% antifeedants. Azadirachtin active compound affects the synthesis
and release of ecdysteroids (molting hormone). These lead to the disruption
of the endocrine hormonal system controlling growth and mounting process.
This regulates the release of eclosion hormone which controls the motor
programme of molting. It has an insecticidal effect on severely reduced growth
which causes abnormal and delayed molts resulting in death before the molt.
It targets the reproductive organ led to alteration to ecdysteroid and juvenile
hormone resulting in the reduction of viable egg and their progeny. Other
physiological effect such as cellular processes through affecting the blockage
of cell division, inhibition of the digestive enzyme, protein synthesis, and

losses of muscle tone in muscles cells. (Lokanadhan et al., 2012).
16.3.2 NICOTINE: MODE OF ACTION
Nicotine and nornicotine an alkaloid obtained from N. tabacum and

Nicotiana rustica belong to the members of the Solanaceae (Walia et al.,
2014; Ononuju et al., 2016). It is well-known botanical insecticide. Other
nicotine analogs compound are nornicotine and anabasine obtained from the
plants belong to the family Chenopodiaceae have also possessed insecticidal
properties (Ononuju et al., 2016). Nicotine is active against piercing and
sap-sucking insects such as aphids, whiteflies, thrips, leafhoppers, and mites.
Nicotine and nornicotine compounds are extremely lethal and fast-
acting nerve toxins agonistic to acetylcholine. The synthetic compound of
nicotine such as imidacloprid, thiocloprid, nitempiram, acetamiprid, and
thiamethoxam are less toxic. They are binding to acetylcholine receptors in
the nervous system causing synaptic blocking and resulting in the failure of
the central nervous (Ononuju et al., 2016).
16.3.3 ROTENONES: MODE OF ACTION
It is a naturally occurring isoflavonoid with insecticidal, acaricidal, and

piscicidal properties (Walia et al., 2014). It is obtained from the plant roots of
species of Derris (D. elliptica, D. involuta) and Lonchocarpus. Subsequently,
rotenone has been isolated from several members of the other plant family
such as Tephrosia virginiana (Hoary pea), Tephrosia vogelii, Pachyrhizus
erosus (Mexican yambean, Jicama), Sphenostylis stenocarpa (African yam
bean), Mundulea sericea (cork bush), Piscidia piscipula (Florida fish poison
tree) Dalbergia spp. (African blackwood), and Millettia laurenti. (Ononuju
et al., 2016). It is a selective, nonspecific and has been extensively used as
insecticide, for household, gardens, and for insect control such as lice and
tick. It is potent poison for fish and used for eradication as part of water
body management. Rotenone is slightly soluble in water and is used either
as a dust or in an oil/kerosene solution. It exerts its toxic action by acting as
a general inhibitor of cellular respiration. (Walia et al., 2014).
Rotenone is a lipophilic, isoflavonoid compound, and a strong toxic
to insects and aquatic lives such as fish but moderately toxic to birds and
mammals (Ononuju et al., 2016). This plant-derived chemical reportedly
acts by electron transport inhibition at cytochrome b, denying cells to utilize

tissue oxygen, with cell death eventually ensuing (Enyiukwu et al., 2014).
The mechanism of this action is linked to the inhibition of the transfer of
electrons from iron-sulfur centers in complex 1 to ubiquinone in the mito-
chondria. This spills into interference with nicotinamide adenine dinucleotide
during the creation of energy in the form of cellular adenosine triphosphate
(ATP). Therefore, build a backup of electrons within the mitochondrial
matrix which leads to a reduction in cellular oxygen to radical, and inducing
a reactive oxygen species which then damages deoxyribonucleic acid and
other components of the mitochondria (Ononuju et al., 2016).
16.3.4 SABADILLA: MODE OF ACTION
Sabadilla (Schoenocaulon officinale) is alkaloid producing plants belonging

to the family Liliaceae contain many seeds-derived alkaloids—cevadine and
its close relative veratridine. These alkaloids with alkaline yield cevacine
(cevine) and protocevacine (cevadillin) which have been reported as the
active principles of the extracts (Kupchan et al., 1953). The insecticidal
properties of sabadilla come from hydrolysis, the alkaloid fraction, which
constitutes 3–6% of the extract. Two most important compound such as
veratridine and cevadine have been extracted and identified, the former
being more potent insecticidal (Walia et al., 2014).
The hydrolytic isolate is more toxic then the former and both act by
disrupting neuron cell membrane of nerve activity, symptomized by paralysis
and death of susceptible organisms. The mechanism of the sabadilla poisoning
is binding to sodium channels, which regulate to nerve excitability resulting
in paralysis in insects. Its mode of action is likely to that of the pyrethroids
and acts through disruption of nerve cell membranes causing loss of nerve
function, an increase in the duration of the action potential, repetitive firing,
and a depolarization of the nerve membrane potential due to effects on the
sodium channel (Walia et al., 2014; Ononuju et al., 2016). Sabadilla alkaloids
are labile and break down rapidly in sunlight. These are less toxic to mammals
than other insecticides and are therefore safe to use (Walia et al., 2014).
16.3.5 PYRETHRUM: MODE OF ACTION
Pyrethrum, the most widely used traditional botanical insecticide on the

market (Veer and Gopalakrishnan, 2016; Walia et al., 2014). It is highly
insecticidal in nature and effective against flying insects such as houseflies

and mosquitoes. This crop was extensively grown in considerably large
commercial quantities in Kenya, Tanzania, Rwanda, Japan, and Ecuador
(Schleier and Peterson, 2011). It is obtained by crushing dried flowers of
African daisies belonging to the family of Asteraceae such as Chrysan-
themum. spp., Pyrethrum. spp., and Tanacetum. spp. (Walia et al., 2014).
This plant species produces an insecticidal compound named, oleoresin
that can be extracted with organic solvents and contains six major pyrethrin
compounds such as pyrethrin I and II, jasmolin I and II, and cinerin I and II.
The toxins, namely pyrethrins, cinerins, and jasmolins, have some unusual
insecticidal properties (Arnason and Scott, 2012).
After the exposure of this compound, it acts both on the central nervous
system and the peripheral nervous system. After exposure of pyrethrins in
affected organisms generally presents symptoms of hyperexcitability, pros-
tration, convulsions, and finally death. It disrupted the membrane pumps
that participate in calcium ion-dependent ATPase, and calcium/magnesium
ion-dependent ATPase in different biological contexts on different receptors
like GABA-gated, and voltage sensitive chloride and calcium channels,
and peripheral benzodiazepine receptors (Schleier and Peterson, 2011).
It acts on sodium ions and voltage-gated sodium channels, disallows the
closing of these channels which result in convulsions and paralysis. The
rapid action of pyrethrins is called knockdown effect. However, it has low
toxicity to vertebrates but has significant toxicity for no targeted species,
especially aquatic organism likely to fish and invertebrates. Like most other
natural pesticides, pyrethrins have limited stability to sunlight, air, and
atmospheric moisture and reduce stability under field conditions, consider
the risks related to its use (Ntalli and Menkissoglu-Spiroudi, 2011). Owing
to it instability, the market for natural pyrethrum declined while synthetic
pyrethroids have become major commercial product. Thus, successful use
of traditional natural pyrethrin is now showing revival due to new bioactive
phytochemicals and extractives as possible source of pest control methods
(Walia et al., 2014).
16.4 PHYTOCHEMICAL-BASED GREEN PESTICIDE AND

INTEGRATED PEST MANAGEMENT (IPM) STRATEGIES
Insect pest and diseases cause substantial agricultural losses through

direct and indirect damage (Shukla et al., 2016). We are experiencing a
rapid control over the last 50 years on single control agents, particularly
chemical pesticides. These synthetic pesticides have increasing difficulties

and complications in managing these pests resulting in control failures,
resistance in target species, environmental degradation, and contamination
of food commodities. It has been reported that assurance of single tactics seri-
ously detracts the sustainability and environment (Koul and Cuperus, 2007).
However, different control strategies based on physical barriers, pesticides,
biotic agents, and host–plant resistances have been used to control these
pests and disease (Shukla et al., 2016).
But none of these strategies can effectively control the pest and diseases
owing to their limitation which do not make them ideal controlling agent.
At present, we need to implement a management system to deal with these
pests. It is a well-established fact that IPM has its own potential, issues,
and challenges (Dhaliwal et al., 2004); however, IPM practices control the
damage to the environment which is an essential component of sustainable
agriculture and environment. Therefore, one of the goals would be the
deployment of green chemistry-based IPM practices (Kennedy and Sutton,
2000). Phytochemical-based green pesticides are desirable because the tech-
nology could control the pest without affecting the environment. This idea
was to support the IPM pattern from centralizing on insect pest management
approach depend on pesticide management to a system strategies primarily
on plant-driven chemicals.
Botanical insecticides have long been offered an attractive alternative
to synthetic pesticides for pest management (Isman, 2006; Khanan et al.,
2006). Hence, botanical pesticides are natural, eco-friendly, cost-effective,
target specific and less persistent and biodegradable. There are a number
of native plants species that have been evaluated against a range of insect
pests and diseases on various crops. Their efficacy is more efficient against a
number of different pests compared to chemicals that give consistent results
under practical conditions, under laboratory, greenhouse, semi-field, field
conditions, and in different environments. Botanical pesticides act as a
synergistic component in IPM strategies. The IPM is necessary for environ-
mental sustainability to control the insect pests and various diseases (Koul
and Cuperus, 2007). This suggests that IPM programmes should represent
“a sustainable approach to control pests combining chemical, physical,
biological, and cultural practice to ensure favorable economic, ecological,
and sociological consequences” (Kennedy and Sutton, 2000). Development
of a new strategy for managing insect pests is necessary for sustainable
productivity and profitability of agriculture owing to pose substantial threats
to the production, quality, and yields of agricultural commodities (Koul and
Cuperus, 2007).
16.4.1 MAIN ADVANTAGES OF USING PHYTOCHEMICAL IN

IPM
The application of plant-driven insecticide in IPM offers several advantages

over synthetic chemical pesticides. As many native plants have adapted the
phytochemical in response to the combined selection pressure of phytopatho-
gens, insects, nematodes, and effective against diseases (Dhaliwal and Koul,
2011). Botanical biopesticide is naturally occurring from indigenous plant
sources, easily available, cost-effective, and easily accessible. It is potent in
very small quantities; generally, affect the selective insect pest and closely
related organisms. It is often less persistent, often decompose quickly in the
environment, avoiding the pollution leading to the less hazardous environ-
ment as well as farmers and consumers. There are harmful effects on plant
growth, seed viability and other plant’s physiologically process.
16.5 CURRENT RESEARCH AND COMMERCIALIZATION

CHALLENGES
The uses of natural plant-based insecticidal products in sustainable

agriculture have been increasing globally. It has been a considerable practice
happening in the world for finding solutions for economically important pest
control through botanicals. Everybody wants to have quick results for saving
cost, time, and energy. However, in developing countries, the expenditure
on research and development, technological facilities, and expertise are
lacking. The potential insecticidal values of plant products are not being
properly harnessed and research and development in this area are lacking
behind. Thus, most of the research is incomplete and not significant because
of its design have limited goals. If plant-based products are to be successful
and competitive it must develop and adopt the four major strategies such
as organize the resource, develop quality control, adopt standardization
strategies, and modify regulatory constraints. This section tries to elucidations
of all the necessary steps needed to conduct research and development in this
area to impose statutory regulations on ensuring the quality, safety, efficacy,
and commercial distribution of such products.
There is a need for large-scale utilization and advances in research of
botanicals to maintain the standards of quality and safety of the products.
The government and non-governmental organizations are expected to initiate
political and financial support needed to establish the infrastructure and to
encourage the necessary research and development in this area and impose
regulations for commercial production. To enhance the commercialized

utilization and efficacy of botanicals pesticides, there should be a developed
synergist such as piperonyl butoxide for crop protection. Once a new bioac-
tivity is identified in a plant, simultaneously, the commercialization of that
plant-based product, raw material sourcing such as cultivation and propaga-
tion of any wild species remains to be a great challenge. The consumption of
plant-based insecticide is widespread and increasing. The chief source of raw
material is native plant from the wild which is causing reduction of genetic
diversity and damage to their habitat. Thus, local farming is a feasible substi-
tute that offers the enabling ground to overcome challenges of discrepancy in
plant materials. Conventional plant breeding and genetically modified crops
may improve both agronomic and insecticidal traits. There has been signifi-
cant progress in the research in the biotechnological field. Attention should
be paid to develop the awareness, offering training programme, promote
improved processing, formulation, and marketing of plant-based pesticides.
ACKNOWLEDGMENT
All the authors acknowledge the necessary infrastructure facilities provided

by the Central University of Punjab, Punjab, India.
KEYWORDS
•• pests
•• biopesticides
•• insecticides
•• plant-derived chemicals
•• sabadilla
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CHAPTER 17
ESSENTIAL OILS IN PEST CONTROL

AND DISEASE MANAGEMENT
ARVIND SAROJ1,*, ATUL KUMAR SRIVASTAVA1,
ASHISH KUMAR NAYAK2, C. S. CHANOTIYA3, and A. SAMAD1
Department of Plant Pathology, Central Institute of Medicinal and
1
Aromatic Plants, Lucknow, India

2
Department of Microbial Genomics and Diagnostic Laboratory,
Regional Plant Resource Centre, Bhubaneswar, India
3
Department of Analytical Chemistry, Central Institute of Medicinal
and Aromatic Plants, Lucknow, India
*
*
ABSTRACT
Plant pathogens damage agriculture products to a large extent resulting

in poor crop yield and quality. The well-followed practice to curb plant
diseases are mostly involved with the application of conventional fungi-
cides (synthetic chemical). However, their continuous and uncontrolled
use has emerged as a big challenge for humans due to its hazardous
effect on the environment. This can also lead to the development of
resistance to plant pathogens. Green chemistry-based approaches have
revolutionized the development of new strategies to curb plant diseases.
Natural products mainly essential oils (EOs) have the potential to replace
conventional fungicides to some extent. The current chapter aims to
discuss the role of EO as biopesticide against plant pathogens (pests and
fungal diseases).
17.1 INTRODUCTION: ESSENTIAL OILS (EOs) IN SUSTAINABLE

AGRICULTURE
Around the 19th century, Bordeaux mixture as a first chemical fungicide was
used to treat pathogen causing downy mildew of grapes. Since then, many
conventional fungicides were developed in order to minimize substantial
crops damage. As a result, excessive use of conventional fungicides has
completely changed the scenario by now. Moreover, uncontrolled use of
these chemicals for many decades resulted in several adverse effects to the
environment, soil fertility, and human health. This also leads to the develop-
ment of resistant and more destructive plant pathogens. Therefore, demand
and necessity of eco-friendly and biopesticides is the foremost need of
modern agriculture.
Biopesticides are derived from natural materials such as plants and
microorganisms. For example, L-carvone, Citronellol, p-menthane-3,
8-diol, verbenone (terpenoids class), and methyl eugenol (phenylpropanoid
class) are considered biopesticides. There are 299 registered biopesticides
active ingredients and 1401 active biopesticides product registrations
were registered in The United States Environmental Protection Agency
(https://www.epa.gov/ingredients-used-pesticide-products/biopesticide-
active-ingredients). Essential oils (Eos) are well documented as natural
biopesticides and also proven their effectiveness against various plant
pathogens (Pragadheesh et al., 2013a; Maia et al., 2014; Saroj et al., 2015;
Ma et al., 2016). Owing to the complexity of oil composition, pathogens
may develop resistance slowly.
EOs are produced by plants mainly using two biochemical pathways
isopentenyl diphosphate pathway and its isomer dimethylallyl diphosphate
pathway (Rehman et al., 2016). EO has been used not only in perfumery,
cosmetics, detergents, pharmacology, fine chemistry, and food production
industries but also contributing in ethnobotanical medicines since ancient
time (Bakkali et al., 2008; Regnault-Roger et al., 2012). Besides this, EO
is also well documented as multiple toxic, fumigant, repellent, pesticidal
properties, ovicidal, larvicidal (Freitas et al., 2010), and antifeedant activi-
ties (Pavela, 2011).
A mixture of compound produced by plants can be divided into
primary and secondary metabolites. Primary metabolites are important for
the survival of the plant but secondary metabolites do not have a direct
impact on the survivability of the plants. EOs are the type of secondary
metabolite extracted from an aromatic plant through different hydrodistilla-
tion methods. Valgimigli (2012) defined EOs as concentrated hydrophobic
Essential Oils in Pest Control and Disease Management 343
liquids having many types of volatile compounds which produce aroma.

Terpenes are the main component of the EO, a collection of ethereal lipo-
philic compounds in a liquid form, obtained from aromatic plants using
different hydro or steam distillation techniques (Shah et al., 2012; Garcia
et al., 2012 and Amorati et al., 2013). EOs are named after “the oil of the
plant they extracted from” but some people have objection or get confused
with the other nonvolatile edible vegetable oils such as corn oil, soya oil,
and so forth. EOs are produced from all plant organs of aromatic herbs such
as buds, leaves, flowers, stems, seeds, twigs, roots, wood, fruits (Andrade
et al., 2014). Plant store EOs mainly in glandular trichomes though other
oil-bearing glands are also present such as secretory cells, cavities, canals,
and epidermis cells (Valgimigli, 2012).
17.2 JOURNEY OF EO
Evidence of EO use was recorded from Lascaux in the form of cave paint-
ings, located in the Dordogne region of France. These cave paintings are first
clear evidence of human understanding regarding knowledge of EO-bearing
plants and its healing properties. In the 16th century, first time Paracelsus
von Hohenheim coined the term “Quinta essential” (EO) as an effective
component of drug (Guenther, 1950).
Since ancient time Egyptians use “Kyphi,” as herbal medicine, incense,
and perfume, made up of 16 different ingredients. They do not have any
cleaning agent for body and hairs so used EO as cleaning alternatives like
soaps and shampoo. The knowledge of EO recorded in Greece between
400 and 500 BCE which is adapted from the Egyptians. The Greek physi-
cian Hippocrates (460–377 B. C. E.) is known as “Father of Medicine,”
given perfume fumigation and recognized the effect of 300 plants, includes
thyme, marjoram, saffron, peppermint, and cumin. The EOs during the
ancient time was used in the production of wines, aromatic, breath-
refreshing gums, and in the food industry. French used aromatic plants
such as rosemary, chamomile, lemon, and thyme in the field of cosmetics
and in perfume formation. They also used EOs in the body creams also
(Valgimigli, 2012). In China during the period of Huang Ti, aromatic oil
uses come in human knowledge. Huang Ti wrote a medicinal-based book
“The Yellow Emperor’s Book of Internal Medicine” in which he explained
the use of EO. Even today, this book is used for the medical purposes in
eastern medicine. Romans use EO abundantly for applying essence in their
bodies, clothes, and bedding. Roman physicians’ spread the knowledge
of EOs through books written by Galen and Hypocrites, the texts of this
book were later translated into different languages such as Arabic, Persian,
and some other as well. The process of EOs extraction through distillation
was discovered by Ali-Ibn Sana. The term “Aromatherapie” was first time
used by French Chemist René-Maurice Gattefossé while exploring the
medicinal properties of EOs. His book “Aromatherapie” was published in
1928, discussed medicinal properties of EOs and their healing potential.
Likewise in India, Ayurveda is well known as the traditional therapy based
on plant parts and its extracts. Ayurveda used plants as medicine in the
treatment of many chronic and acute diseases since 3000 years and EOs
for its healing property.
It is assumed that the extraction of EOs using the distillation method
began in Egypt, Persia, and India during middle ages (Guenther, 1948).
However, later on, extraction of EOs through liquid carbon dioxide, low or
high-pressure distillation, steam distillations was discovered as the knowl-
edge of EOs increases. For perfumery, extraction of EOs using lipophilic
solvents and supercritical carbon dioxide are found to be the best extraction
processes. Although steam distillation is preferred when the EO is used for
antimicrobial, pesticidal activities, and pharmaceutical uses as well as for
the flavor and preservatives in the food. Recent understanding regarding
the composition of EOs and its components such as the detection of hydro-
carbons and terpenes lays the foundation of advanced current distillation
techniques (Başer and Buchbauer, 2010).
Presently in our daily life, EOs directly or indirectly gets benefited due
to its role in medicine, flavor, and cosmetic industries. Depending on the
chemical composition of EO resulted in many biological activities such
as bactericidal, fungicidal, and pesticidal. Recent research revealed that
terpenoids and phenolic compounds are highly toxic to pests and microor-
ganism makes EO more effective in repelling the harmful insects and micro-
organism (Valgimigli, 2012). Some of the EOs also possess antimicrobial
effect including bergamot (Citrus aurantium bergamia), black pepper (Piper
nigrum), cinnamon (Cinnamomum cassia), eucalyptus (Eucalyptus glob-
ulus), orange (Citrus aurantium dulcis), rosemary (Rosmarinus officinalis),
ginger (Zingiber officinale), lavender (Lavandula officinalis), and lemon-
grass (Cymbopogon schoenanthus) (Andrade et al., 2014). The EOs are well
documented for bactericidal, fungicidal, and insecticidal activities (Amorati
et al., 2013; Yanishlieva et al., 2006; Bakkali et al., 2008; Valgimigli, 2012;
Pragadheesh et al., 2013a, 2013b and Saroj et al., 2015). Some plants of EO
importance were presented in Table 17.1.
TABLE 17.1 Essential Oils (EOs) are Derived from Various Parts of Plants.
Plant parts Name of the plants
Leaves Basil, bay leaf, cinnamon, eucalyptus, lemon grass, melaleuca, oregano,
patchouli, peppermint, pine, rosemary, spearmint, tea tree, wintergreen
thyme, palmarosa, citronella, petitgrain
Flowers Chamomile, clary sage, clove, geranium, hyssop, jasmine, lavender,
manuka, marjoram, orange, rose, neroli
Peel Bergamot, grapefruit, lemon, lime, orange, tangerine
Seeds Almond, anise, celery, cumin, nutmeg
Wood Camphor, cedar, rosewood, sandalwood
Berries Allspice, juniper
Bark Cassia, cinnamon
Resins Frankincense, myrrh
Rhizome Ginger
Root Valerian, vetiver
17.3 COMPOSITION OF EOS
EOs are normally mixtures of more than 200 components mainly terpenes
or derivatives of phenolic compounds. The chemical and structural
differences between components of EOs are minimal. On the basis of
nature of the compound, it can be classified into volatile and nonvolatile
component. More than 90–95% of the EO comprises volatile compounds,
primarily monoterpene and sesquiterpene hydrocarbons as well as their
oxygenated derivatives along with alcohols, aliphatic aldehydes, and esters.
A nonvolatile component comprises about 1–10% of the EO, containing
hydrocarbons, fatty acids, carotenoids, sterols, flavonoids, and waxes. Most
of the hydrocarbons found in the EO are present as isoprene (Fig. 17.1),
which act as a unit of terpenes. Terpenes are the cyclic molecule having the
chemical formula C10H16.
Limonene, camphene, pinene, methyl chavicol, geraniol, and so forth are
the example of terpenes and used as anti-inflammatory, bactericidal, anti-
viral, antifungal, and antiseptic agent. Terpenes are classified into monoter-
penes, sesquiterpenes, and diterpenes. Monoterpenes are a class of terpenes
that consist of two isoprene units and present in the form of either acyclic
or contain rings. When two isoprene units join head to tail, the result is a
monoterpene, when three joins, it is sesquiterpenes, and four linked isoprene
units are diterpenes. Terpenes are described below for more information:
FIGURE 17.1 Structure of isoprene
17.3.1 MONOTERPENES
The molecular formula of monoterpenes is C10H16 and made up of two

isoprene units, widely distributed in nature with more than 400 natural
monoterpenes (Fig. 17.2). Oxidation or biochemical modification of mono-
terpenes produces the derivative of monoterpenes such as alcohols, ketones,
and carboxylic acids that are known as monoterpenoids. Monoterpenes are
known for its analgesic, bactericidal, expectorant, and stimulant properties.
FIGURE 17.2 Structure of limonene (monoterpene).
Moreover, besides being linear derivatives (geraniol, citronellol), the

monoterpenes can be cyclic molecules (menthol—monocyclic; camphor—
bicyclic; pinenes—α and β). Borneol and camphor are two general monoter-
penes used for various purposes. Borneol, isolated from pine oil, is used as
a deodorant and disinfectant. Camphor is used as a counterirritant, expecto-
rant, anesthetic, and antipruritic among many other uses. Thujone is a type
of monoterpene which act as a toxic agent found in Artemisia absinthium
(wormwood) from which the liqueur, absinthe, is made.
17.3.2 SESQUITERPENES
Sesquiterpenes are a class of terpenes made up of three isoprene units.

The molecular formula of sesquiterpene is C15H24. Sesquiterpenes are
biogenetically derived from farnesyl pyrophosphate and like monoterpenes,
sesquiterpenes may be acyclic or contain rings, including many unique

combinations. Biochemical modifications such as oxidation or rearrangement
produce the related sesquiterpenoids. They are anti-inflammatory, analgesic,
antiseptic, anti-allergic, and antimicrobial in nature. Those sesquiterpenes
which are having structural features such as α, β-unsaturated-γ-lactones,
showing better biological activities. Beta-caryophyllene in basil and black
pepper, Farnesene in chamomile and lavender are the common examples of
sesquiterpene (Fig. 17.3).
FIGURE 17.3 Structures of sesquiterpenes.
17.3.3 DITERPENES
Diterpenes are a class of terpene, made up of four isoprene unit with the
molecular formula of C20H32. About 2500 natural diterpenes are reported
so far. They are biosynthesized by plants, animals, and fungi through the
HMG-CoA reductase pathway, with geranylgeranyl pyrophosphate being a
primary intermediate. Diterpenes form the basis for biologically important
compounds such as retinol, phytol, and retinal. They are known to exhibit
antimicrobial and anti-inflammatory property with expectorant, hypotensive,
and hormonal balancers. The biosynthesis occurs in plastids and interest-
ingly mixtures of monoterpenes and diterpenes are the major constituents
of plant resins. In a similar manner to monoterpenes, diterpenes arise from
metabolism of geranyl pyrophosphate (GGPP).
Besides terpenes, alcohols, aldehydes, esters, ketones, and lactones are
also present in the EO.
17.3.4 ALCOHOLS
Alcohols are compound have a hydroxyl group, present naturally either as

a free compound or in combined with a terpenes/ester. When terpenes are
attached to an oxygen atom, and a hydrogen atom, the result is an alcohol.

When the terpene is monoterpene, the resulting alcohol is called a monoter-
penol. Alcohols have a very low or totally absent toxic reaction in the body
or on the skin. Therefore, they are considered safe to use. They mainly used
individually as antiseptic, bactericidal, antiviral, and germicidal agent. Gera-
niol in geranium, linalool is in Ocimum and Lippia is an example of terpineol.
17.3.5 ALDEHYDES
It functions as anti-inflammatory, antifungal, bactericidal, antiviral,

antiseptic, disinfectant, sedative and also reported as a good insecticidal.
Cinnamaldehyde in cinnamon, citronellal in lemongrass, lemon balm, and
citrus eucalyptus and citral in lemon is a good example of an aldehydic
present in EO.
17.3.6 ESTERS
Esters are formed through the reaction of alcohols with acids. Esters are the
main source of balancing, pleasant, and soothing effects in EOs. Because
of the presence of alcohol, they are effective antibacterial and antifungal
agents. Medicinally, esters are reported as a sedative, with a balancing act
on the nervous system. Geranyl formate in geranium and linalyl acetate in
lavender is the best example of ester present in EO.
17.3.7 KETONES
EOs containing ketones are useful in wound healing and encouraging the
formation of scar tissues. Ketones are generally toxic such as thujone found
in sage, tansy, and thuja. Aromatic nontoxic ketones are jasmone in jasmine
oil, carvone in spearmint, and menthone in peppermint oil. Ketones are also
having some medicinal properties like anti-catarrhal, expectorant, healing,
and cell proliferant.
17.3.8 LACTONES
Lactones particularly known for their anti-inflammatory action play a role

in the reduction of prostaglandin synthesis and expectorant actions. The
lactones are present in nature in the form of saturated or unsaturated gamma

and delta-lactones. A number of substrates have been utilized to demonstrate
the microbial formation of lactones. Oleic acid, ricinoleic acid, castor oil
gamma keto-acid are some example of lactones.
17.4 BIOACTIVITY OF EOs
The indiscriminate use of antimicrobial agents has resulted in the emergence

of a number of drug-resistant bacteria and fungi. To overcome the increasing
resistance of pathogenic microorganism and insects, more effective agents
with a novel mode of action must be developed. Recently, rigorous research
in the field of EOs and extracts of certain plant materials have the potential
to replace synthetic pesticides against certain plant pathogens including
pest, fungal, and bacterial diseases. EOs derived from several plant families
especially Lamiaceae (Ocimum spp.), Myrtaceae (Syzygium spp.), Laura-
ceae (Cinnamomum camphora), and Asteraceae (Tagetes patula) have been
reported to have antimicrobial and pesticidal properties (Table 17.2–17.4).
17.4.1 ANTIFUNGAL ACTIVITY
EOs are composed of a number of different components such as aromatic

phenols, terpenes, oxides, ethers, alcohols, esters, aldehydes, and ketones in
different composition or combinations (Bakkali et al., 2008). Some of the
components remain present in very high concentration while some in very
low concentration. The antifungal efficacy of EO is mainly either attributed
to the overall synergistic effects of all the major and minor compounds or
to the bioactivity of the major compounds (Mishra et al., 2013; Saroj et al.,
2015). The chemical composition may vary according to the method of EO
extraction, the age of the plant, time of harvest, ecological and geographical
variations. Hence, before large-scale application, the chemical standardiza-
tion of EO must be endorsed. As it is well known that ergosterol is specific to
the fungal cell membrane and is its major sterol component responsible for
maintaining the cell function and integrity. One of the important mechanisms
of EO is the reduction in ergosterol synthesis in fungal cells. Some studies
have shown that EOs have the potency to cause a considerable reduction in
the quantity of ergosterol in the fungal cell membrane (Pinto et al., 2009).
The primary action mechanism of azole antifungal drugs is the interruption
of sterol biosynthetic pathways resulting in reduced ergosterol biosynthesis
TABLE 17.2 EOs as Antifungal Agents Against a Wide Range of Post-Harvest Fungi. 350
S/ Plant for essential oil (EO) Against storage fungi Method Used observation References
No. isolation
1 Brassica nigra (S) Aspergillus niger Vapor phase and 100% growth Inhibition at Mejia-Garibay et al.
Brassicaceae Aspergillus ochraceous direct contact 4 μL/mL concentration in direct (2015)
contact method and at 47/μL air
Penicillium citrinum concentration in vapor phase
2 Mentha arvensis (AP) Aspergillus fumigatus In vivo volatile 100% protection at 600 ppm Varma and Dubey
Lamiaceae Aspergillus flavus assay wheat seeds except A.f. (2001)
A. niger
Penicillium oxalicum
Rhizopus spp.
Curvularia spp.
Mucor spp.
3 Chenopodium ambrosioides A. glucans Poisoned food 100% growth inhibition at 0.3% Jardim et al. (2008)
(L) Amaranthaceae A. niger technique concentration
A. flavus
A. ochraceous
4 Cicuta virosa (F) Apiaceae A. ochraceous Poisoned food 100% protection at 300 ppm Tian et al. (2011)
A. niger technique
A. flavus
Alternaria alternata
5 Cinnamomum jensenianum A. flavus Poisoned food MIC at 8 μL/mL concentration Tian et al. (2012)
(Ba) Lauraceae technique
No. isolation
6 Cuminum cyminum (S) A. alternata Poisoned food 100% growth inhibition at Kedia et al. (2014)
Apiaceae Penicillium citrinum technique 0.6 μL/mL concentration except
R.s.
Aspergillus unguis
A. flavus
A. niger
Curvularia lunata
Aspergillus nidulans
Mucor spp
7 Cympopogon citratus (AP) A. niger Dilution method 100% growth inhibition at 500 Tzortzakis and
Poaceae Botrytis cinerea Economakis (2007)
8 Cymbopogon martini (L) A. fumigates Dilution by broth 100% growth inhibition at Mishra et al. (2015)
Poaceae A. flavus method 0.5 μL/mL concentration
A. niger
Fusarium spp.
Penicillium spp.
9 Foeniculum vulgare (S) A. fumigates Dilution by broth MIC at 10 μg/L concentration Roby et al. (2013)
Apiaceae method
10 Laurus nobilis (L) Botrytis cinerea Poisoned food At 1000 μg/mL concentrated Corato et al. (2010)
Lauraceae Penicillium digitatum technique 100% growth inhibition of
B.C. and M.l. but 71% inhibi-
tion to P.d.
No. isolation
11 Lippia rugosa (L) A. flavus Agar medium MIC at 1000 ppm Tatsadjieu et al.
Lamiaceae assay (2009)
12 Mentha spicata (AP) A. alternata Poisoned food 100% growth inhibition at Kedia et al. (2014)
Lamiaceae A. terreus technique 1.0 μL/mL concentration except
A.lu. and A.t.
A.flavus
A. niger
C. lunata
A. glutans
A. nidulans
Mucor spp.
13 Ocimum sanctum (AP) Rhizoctonia solani and Poisoned food 1200 and 900 ppm respectively Kumar et al. (2010)
Lamiaceae Choanephora cucurbitarum technique
14 Ocimum sanctum (L) C. cucurbitarum and R. solani Poisoned food MIC at 730 and 1200 ppm Pragadheesh et al.
Lamiaceae technique (2013b)
15 Rosmarinus officinalis (L) A. fumigatus Contact assay 100% growth inhibition at Prakash et al. (2015)
Lamiaceae A. alternata 1.5 μL/mL concentration except
A.a and C.c.
A. flavus
A. niger
Mucor spp.
16 Satureja hortensis (AP) A. flavus Agar dilution MIC at 500 ppm Omidbeygi et al.
Lamiaceae method (2007)
No. isolation
17 Syzygium cumini (L) R. solani and C. cucurbitarum Poison food MIC at 1200 ppm Pinto et al. (2009)
method and
volatile phase
18 Syzygium aromaticum (B) A. flavus Broth dilution 100% inhibition at 0.64 μL/mL Pinto et al. (2009)
Myrtaceae A. fumigatus method concentration
A. niger
19 Tagetes patula (Fl) Asteraceae Penicillium digitatum Poisoned food MIC for B.c. at 10 μL/mL Romagnoli et al.
Botrytis cinerea method and for P.d. at 1.85 μL/mL (2005)
concentration
20 Thymus pulegioides (AP) A. flavus Broth 100% inhibition at 0.32 μL/mL Pinto et al. (2006)
Lamiaceae A. fumigatus dilution concentration
A. niger method
21 Trachyspermum ammi (F) Aspergillus glucans Poisoned 100% growth Kedia et al. (2015)
Apiaceae A. alternata food inhibition at 0.8 μL/mL
A. flavus method concentration
A. niger
A. terreus
C. lunata
Penicillium citrinum
A. unguis
A. nidulans
Mucor spp.
No. isolation
22 Zataria multiflora (AP) A. flavus Liquid agar MIC at 400 ppm Gandomi et al.
Lamiaceae dilution method (2009)
23 Cinnamomum camphora (L) C. cucurbitarum Poisoned food MIC at 1200 ppm Pragadheesh et al.
Lauraceae method (2013a)
24 Ocimum basilicum (L) R. solani and C. cucurbitarum Poisoned food MIC at 1200 ppm Padalia et al. (2014)
Lamiaceae method
Plant part: AP = aerial part; B = bud; Ba = Bark; F = Fruit; Fl = flower; L = leaf; S = seed. MIC = Minimum inhibitory concentration.
TABLE 17.3 Some Commercial Plant Health Products from plant Natural Products used as Fungicide.
Botanical source Product Main bioactive Mode of action Examples of trade References
components names
Azadirachta indica neem (neem Azadirachtin, Moulting inhibitors (ecdysone Ecozin, azatrol EC, Copping and
A. Juss oil, medium, dihydroazadirachtin, variety antagonists), antifeedant/ agroneem, trilogy Duke (2007)
polarity extracts) of triterpenoids (nimbin, repellent, physical smothering,
salannin, and others) and desiccation
Cassia tora L., Cinnamaldehyde Cinnamaldehyde Disruption of the fungal Vertigo, Cinnacure Dayan et al.
cassia obtusifolia membranes, repellent and (2009)
attractant
Reynoutria Extract of giant Physcion, emodin Induction of SAR (phenolic Milsana, Regalia Regnault-Roger
sachalinensis knotweed phytoalexines) (2012)
(Fr. Schm) Nakai
Macleaya cordata Pink plume Anguinarine chloride, Induction of SAR (phenolic Qwel Regnault-Roger
R. Br. poppy extract alkaloids, and chelerythrine phytoalexines) (2012)
chloride
Trigonella foenum- Stifénia Unknown Stimulation of plant defence Stifénia Regnault-Roger
graecum L. (2012)
Plant-derived acid Citric acida Citric acid Not identified with Certainty Sharp shooter, Copping and
Repellex Duke (2007)
Simmondsia califor- jojoba essential straight-chain wax esters suffocation (eggs and immature Detur, Erasem, Eco Dayan et al.
nica Nutt., Salvia oil (EO) life stages), repellent, blocking E-Rase, Permatrol, (2009)
chinensis Link access to oxygen ERase
Capsicum spp. Capsicum Capsaicin Neurotoxic, repellent Hot pepper wax insect Copping and
(Capsicum oleoresin repellent, hot pepper Duke (2007)
frutescens Mill) wax
Thymus vulgaris Thyme EO thymol, carvacrol Neurotoxic, interference with Proud 3, Organic Yard Fischer et al.
Thymus spp. GABA-gated chloride channels Insect Killer, Promax (2013)
Rosmarinus Rosemary EO 1,8-cineole (borneol, Octopamine antagonists; Ecotrol, Sporan Isman and
officinalis camphor, monoterpenoids) membrane disruptors, others Machial (2006)

TABLE 17.4 List of Active Substance from Different Plants and its Mode of Action Against Microorganisms, Weeds, and Insects. 356
Source Active substance MoA References
Target: bacteria and fungi
Cassia plants (Cassia tora L.) Cinnamaldehyde Unknown Brown (2013)
Allium crops (garlic and onion) Diallylsulphide Unknown Davis (2007)
Laminaria digitata Laminarin Host plant defense Induction Aziz et al. (2003)
Oudemansiella mucida Oudemansin Inhibitor of mitochondrial electron Akita (2009)
transport (complex III)
Target: Insect
Azadirachta indica Azadirachtin Unknown López and Pascual-Villalobos (2010)
Capsicum spp. Capsaicin Nervous system dysfunction USEPA (1992)
Mentha spicata var. crispa (Lamiaceae) Carvone Repellent López and Pascual-Villalobos (2010)
Carumcarvi L. (Apiaceae)
Allium crops (garlic and onion) Diallylsulphide Repellent USEPA (2010)
Estragon and conifer Trees Estragole Repellent USEPA (2001)
Corymbia citriodora p-Menthane-3,8-diol Repellent Elmhalli et al. (2009)
Nicotiana tabacum L. Nicotine Nicotinic acetylcholine receptor Shi et al. (2006)
(nAChR) Agonists
Chrysanthemum cinerariaefolium and Pyrethrin I and II Sodium channel modulators Casida (2011)
Chrysanthemum coccineum
Tropical plants in the genus Derris, Rotenone Mitochondrial complex I electron Chauvin et al. (2001)
Lonchocarpus or Tephrosia transport inhibitors
Saccharopolyspora spinosa and Spinosyns Nicotinic, acetylcholine receptor Kirst et al. (1991)
Saccharopolyspora Pogona (nAChR) allosteric activator
(Mishra et al., 2015). Further, the studies of Cuminum cyminum, Mentha

spicata, Trachyspermum ammi, and Cinnamomum jensenianum. EOs have
a clear evidence of reduced ergosterol biosynthesis due to EO treatment on
Aspergillus flavus cells (Kedia et al., 2014). Being lipophilic in nature, the
EOs targets the plasma membrane (Fig. 17.4) and the membranous organelles
of the fungal cell by either crossing or accumulating in the cell membrane.
This results in interaction with the enzymes and proteins therein, disturb cell
permeability by producing a flux of protons toward the cell exterior which
ultimately disrupts the fungal cell organization and causes cell death (Kelly
et al., 1995). The effects included membrane swelling, change in fluidity,
and increased passive flux of protons. The releases of ions are not based
on their size and/or formation of holes in the membrane. However, the EO
accumulates in the plasma membrane, causes increased membrane bilayer
disorder and ion leakage. These effects cause a disturbance in the osmotic
balance of the cell, making its membrane-associated proteins inefficient
leading to inhibition of cell growth. Some of the well-known EO compo-
nents namely thymol, carvacrol, eugenol, and other phenolic components
have been reported to disrupt cell membrane by dissipating H+ and K+ ion
gradients causing leakage of vital cellular constituents which results in water
imbalance, depletion of intracellular ATP concentration, and finally cell
death (Mejia-Garibay et al., 2015).
FIGURE 17.4 (See color insert.) Diagrammatic representation of the antifungal mode of
action of essential oil (EO).
17.4.2 PESTICIDAL ACTIVITY
The compound of plant EO has been known as a traditional source of

pesticide, which controls/reduce the number of harmful insects from the
environment. Industries develop a large number of EO-based commercial
products including pesticide for human being. Secondary metabolites play an
important role in plant-insect interaction, work as insecticidal, antifeedant,
and repellent activity (Bernays and Chapman 1994). The physiological action
of EO on insects is not much clearer in knowledge but the action of EO or
it’s compound cause symptoms that suggest a neurotoxic mode of action
(Coats et al., 1991; Kostyukovsky et al., 2002). Monoterpene (like linalool,
citronellal) effect on ion transport system of insect nervous system to release
acetylcholine esterase (Re et al., 2000). Octopamine-1 and octopamine-2 are
two class of receptor; behave as a neurotransmitter or neurohormone work on
a biological system of insect (Evans, 1980; Hollingworth et al., 1984; Evans,
1981). Interrupting the functioning of octopamine results in total crash of the
nervous system of insects (Fig. 17.5). Therefore, an octopaminergic system
of insects represents a biorational target for insect control.
FIGURE 17.5 (See color insert.) Target sites in insects as a possible neurotransmitter-
mediated toxic action of essential oils.
17.5 ROLE OF EOS IN DISEASE MANAGEMENT
EO as biological control has been advanced and now an alternative to

synthetic fungicides and considerable success in laboratory and pilot scale
tests have been realized utilizing antagonistic microorganisms to control
crops diseases. EOs are also considered as a promising alternative with many
having antifungal properties (Hammer et al., 2003). Several antagonistic
fungi and bacteria have been isolated and shown to have a broad spectrum
of activity against a number of pathogens on different varieties of fruits.
Recently, an interest has been shown in combining microbial biocontrol
agents with other chemical components to increase their activity against
phytopathogens (Droby et al., 1998). Application of EO is a very attractive
method for controlling plant diseases.
EOs and their different components are gaining increasing interest
because of their relatively safe status, their wide acceptance by consumers,
and their exploitation for potential multi-purpose functional use (Ormancey
et al., 2001). EOs have been used successfully in combination with a variety
of treatments, such as antimicrobial agents, mild heat, and salt compounds
(Karatzas et al., 2000).
Production of EOs by plants is believed to be predominantly a defense
mechanism against pathogens and pests. EOs are made up of many different
volatile compounds that contribute in its composition quite often varies
between species (Mishra and Dubey, 1994). It is difficult to associate the
antimicrobial activity with single compounds or classes of compounds. It
seems that the insecticidal and antimicrobial effects are the result of many
compounds acting synergistically (Bagamboula et al., 2004). After applica-
tion of EOs, the chances of development of resistant races of fungi/insects
get reduced. Currently, the management of necrotrophic fungi infecting
tomatoes is controlled by the use of protectant fungicides such as mancozeb
and chlorothalonil or systemic fungicides in the strobilurin class (Diver
et al., 1999). However, studies carried out by many research groups revealed
that EOs are other sustainable disease management option. EO of spearmint
is associated with a number of antifungal properties. Spearmint has been
studied and found to be potential against necrotrophic phytopathogens.
Consequently, EOs are found to be more effective against many gram-posi-
tive and gram-negative bacteria. Spearmint has an inhibitory effect against
Staphylococcus aureus, Klebsiella sp., Escherichia coli, and Pseudomonas
aeruginosa.
EOs from “alecrim-pimenta” (Lippia sidoides Cham.), wild basil
(Ocimum gratissimum L.), lemongrass (Cymbopogon citratus Stapf) and
“cidrao” (Lippia citriodora Kunth) inhibited the germination and mycelial

growth of Colletotrichum gloeosporioides conidia (Souza Júnior, 2009).
Thyme, eucalyptus citriodora, citronella, and neem oils showed a direct
effect on Phakopsora pachyrhizi, because they reduce Asian soybean rust
severity. Some of the EOs are commercialized with different names such as
orange EOs on wheat seeds (Stülp et al., 2011) and neem EOs on rice seeds.
17.5.1 CURRENT SCENARIO OF EO AS BIOPESTICIDE
Natural products such as repellent EOs are becoming increasingly popular

because of their low toxicity and customer approval. Therefore, the market
of EO will have the strongest growth of all the botanical pesticides markets
in coming years. Similarly, the antimicrobial potential of Ocimum spp.
against E. coli, Bacillus subtilis, Staphylococcus spp makes it potential to
be commercialized at large scale. However, studies conducted by many
research groups revealed that Ocimum canum and Ocimum sanctum have
been reported as the most effective bactericide among the studied Ocimum
EOs. Bozin et al. (2006) reported that the antibacterial activity of Ocimum
basilicum against 13 human pathogenic bacteria and find that the minimum
inhibitory concentration ranges 8–30 μL/mL. Atanda et al. (2007) inves-
tigated the ability of sweet basil (O. basilicum) in the growth control of
aflatoxigenic fungus Aspergillus parasiticus.
17.5.2 EMERGING PROSPECT OF EO
A long list of plant-derived products as potential pesticides exists and this list
will keep counting in the future. Consequently, resistance against EOs may
develop slowly because of its complex composition and more than single
target sites. Antifungal activity of the EO has been established due to induc-
tion of changes in cell wall composition, cell lysis, cytoplasm coagulation,
and plasma membrane disruption (Knobloch et al., 1989; Pragadheesh et al.,
2013a) in the phytopathogens. Though, in order to exploit these products to
their full extent safety issue must be addressed.
EO, which showed fungistatic activity served as a better commercial
option for the management of damping-off diseases as compared to fungi-
cidal oils. As soil harbors natural microflora including bacteria and fungi.
These microfloras are required for the plant growth promotion. If treated with
fungicidal oils, it can also disturb the natural microflora of that particular
area leading to poor plant growth. Research on action mechanism of EO may

help to select suitable EO or combination of EOs which will be used to iden-
tify resistance/susceptibility status of the target pest/pathogen. Such studies
allow a more targeted approach, further adapted for the selection of EO,
also providing more sites to be explored. Combination of piperonyl butoxide
with other EO components such as eugenol and thymol may increase the
activity of these compounds to different sorts of arthropods (Yadav et al.,
2009; Rossi and Palacios, 2013). Studies on detoxification pathways may
also lead to an advantageous way to analyze kinds of EOs that can be
used on pesticide-resistant insects, similarly in chemotherapy treatment of
cancers where over express drug metabolizing enzymes are targeted using
pro-drugs. Additionally, it may be possible to increase the toxicity of EOs,
such as that of Mintostachy verticillate, further by inducing the expression
of detoxification enzymes.
Futuristic approach to make EOs more effective against a range of pests
and plant pathogens lies in its synergistic combinations. Combination of EO
may lead to activating multiple mechanisms to inhibit pathogens, eventually
resolve the problem of pathogens resistance development nature. Synergism
between carvacrol and some hydrocarbons monoterpenes (such as α-pinene,
camphene, myrcene, α-terpinene, and p-cymene) that typically showed low
antimicrobial properties has been observed (Moreno et al., 2007). However,
the ability of hydrocarbons to interact with cell membrane facilitates the
penetration of carvacrol into the cell (Jaenson et al., 2006; Moreno et al.,
2007, McAllister and Adams, 2010).
17.6 FUTURE PERSPECTIVES AND CONCLUSIONS
Prolong uncontrolled use of chemical pesticides for many decades resulted

in several adverse effects to the environment, soil fertility, and human health.
Therefore, sustainable development in agriculture is the foremost need of
the current global scenario to sustain the growing population, especially in
developing countries. In the on-going search to find out alternatives, EOs
showing the promising potential to establish EO-based products against
specific pests in a number of ways. For many of such products still needed
to prove efficacy, safety, and modes of action to identify its limitations such
as minimal residual activity with advances in fields such as slow-release
technology (nanocoating of EO). Molecular-based studies may enhance the
understanding of the identification of targets and detoxification pathways,
which ultimately leads to ensuring product safety and effectiveness.
Allowing EOs, compounds/combinations to be used on the same specific

modes of action which are used by existing synthetics in general activity
that target similar pathways. EOs and their components applied singly or in
specific combinations, are effective against a range of important arthropod
pests and plant pathogens.
To commercialize EO-based pesticides generally face three obstacles to
be overcome: (i) availability of natural resources; (ii) the need for chemical
standardization, mode of action, toxicity and quality control; and (iii) diffi-
culties in the registration of such products. Although most of the EOs are
found to be nontoxic to mammals and not persist in the environment for too
long these properties may play an important role to ease its commercializa-
tion (Isman, 1999; Isman, 2000).
KEYWORDS
•• plant pathogens
•• essential oils
•• pest control
•• disease management
•• botanicals
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CHAPTER 18
INHIBITION OF MILD STEEL

CORROSION IN ACIDIC MEDIA BY
PHYTOCHEMICALS
BASU MAAN DAAS
Department of Chemistry, Netaji Subhash Mahavidyalaya,
Udaipur, Tripura, India, Tel.: +917005115828,
ORCID: https://or-cid.org/0000-0003-2861-1914
ABSTRACT
The chapter deals with the phenomenon of corrosion of mild steel, its causes,
consequences, and inhibition by means of phytochemicals. Corrosion is a
major hazard in the application of metals in various fields and its inhibition
is to be addressed for rapid and smooth progress in civil, industrial, transport,
and other sectors. In this chapter, the inhibition of corrosion of mild steel
by means of chemicals extracted from plants is elaborately discussed. This
chapter is going to be very useful for researchers who are working or planning
to work in the field of corrosion inhibition by phytochemicals because many
organic corrosion inhibitors are toxic in nature and there is a constant search
for non-toxic inhibitors. In this chapter, the major achievements in the inhibi-
tion of corrosion of mild steel till recent years are compiled and discussed. The
electrochemistry and mechanism of corrosion of mild steel in acidic media
and its inhibition by green phytochemicals are discussed in this chapter.
18.1 INTRODUCTION
Roman philosopher, Pliny (23–79 AD) wrote about corrosion of iron in

an essay named Ferrum Corrumpitar (Ahmad, 2016). Corrosion is to eat
away or be eaten away gradually, especially by chemical action (Webster’s

Dictionary). Corrosion is the process of corroding or being corroded (Oxford
and Cambridge Dictionaries). Corrosion is deterioration of a metal because
of a reaction with the environment (NACE Corrosion Basics). The word
corrosion comes from the Latin words—rodere means gnawing and corro-
dere means gnawing to pieces.
Corrosion has been a major hazard since ancient times when metals were
first used. Corrosion affects the microstructure, mechanical properties and
the physical appearance of the metal. Corrosion is a continuous hazard for
buildings, bridges, oil pipelines, water supply lines, toilets and bathrooms,
chemical and industrial plants, and so on. Corroded electrical contacts can
catch fire due to short circuit and corroded medical implants may lead to
blood poisoning and tetanus as well as other problems. Furthermore, corro-
sion continuously damages arts and sculptures around the world. Corrosion
also damages the storage containers used for safe disposal of radioactive
waste for tens of thousands of years which can be or is a severe concern (The
Electrochemical Society).
Steel is a major ingredient in the progress of modern civilization. It is
invariably used in the construction of instruments, machines, engines,
buildings, roadways, bridges, highways, factories, industries, and the list is
too long to be mentioned in details here. In short, to think of progress and
development without the varied applications of steel in its various forms
is impossible as far as our latest research, discoveries, and inventions are
concerned. So, if steel gets corroded due to atmospheric influences, which
is a severe concern, then the progress of mankind gets jerked and retarded.
This is the reason why inhibition of steel corrosion is of prime importance
for mankind and researchers are toiling hard in the laboratories to find viable
solutions to address this phenomenon.
18.1.1 CORROSION TYPES
Broadly, corrosion can be classified as the following depending on the

causes, that is, environment, material, or chemical reaction and effects
(Chigondo and Chigondo, 2016).
• Uniform corrosion damages and thins out the whole metal surface.
• Galvanic corrosion takes place with an electrolyte with metals having
different values of electrical potentials.
Inhibition of Mild Steel Corrosion in Acidic Media 371
• Pitting corrosion damages particular parts of the metal surface

forming holes or pits which act as the anode, while the undamaged
metal surface the cathode.
• Stress corrosion cracking takes place due to stress and corrosive
environment. Corrosion fatigue takes place due to the combination of
cyclic stress and corrosion.
• Intergranular corrosion takes place onto the granular boundaries of
a metal.
• Crevice corrosion takes place due to the trapping of corrosive liquid
between the gaps in the metal.
• Filiform corrosion takes place on a metal surface coated with a thin
organic film.
• Erosion corrosion takes place due to the movement of corrosive
liquids on a metal surface.
• Fretting corrosion takes place due to the combination of corrosion
and fretting of metal.
18.1.2 CORROSION ELECTROCHEMISTRY
Most metals generally occur in nature as compounds, such as oxides, sulfides,

silicates, or carbonates, barring aside very few metals that occur in native
form. Extraction of a metal from ore involves reduction, while the oxidation
of metal is commonly called corrosion where metals tend to lose electrons
to oxygen or other oxidizing agents in air or water. The most common kinds
of corrosion result from electrochemical reactions which occur when water
or moisture gets trapped between two electrical contacts (Fig. 18.1), having
a voltage between them, generate an accidental electrolytic cell (The Elec-
trochemical Society).
The process of corrosion of mild steel in the presence of moisture
illustrated the preceding diagram shows that when an oxide-free metal
surface gets exposed to moisture, iron is oxidized to positively charged
metal ions yielding electrons which can freely move through the mild
steel structure up to the surface of the metal. Two electrochemical regions
are generated. At the cathodic region, these free electrons cause reduc-
tion of atmospheric oxygen into water, while at the anodic region, the
oxidized iron reacts with oxygen and water to create rust, thus corroding
the surface (Toussaint, 2015). These residual electrons provide a nega-
tive charge on the metal surface, thus increasing the potential difference
between the metal and adjacent solution. Hike in this potential difference
facilitates deposition of dissolved metal ions from the solution back onto
the metal surface till a point of equilibrium when the opposing rates of
dissolution and deposition become equal (Chatterjee et al., 1991). Once
this steady state is achieved, the potential is called reversible potential
and by this time a very small amount of metal gets dissolved into the
adjacent moisture. Potential of a metal in the solution remains positive but
lower than the reversible potential.
FIGURE 18.1 Process of corrosion of mild steel in the presence of moisture

Source: Modified from Toussaint (2015).
In acid media, the corrosion is expedited; in the presence of acidic solu-

tion, hydrogen ions are adsorbed on the metal surface from the solution and
electrons react with them to produce hydrogen gas (Fig. 18.2). Sustained
passage of metal ions into solution phase leads to corrosion of the metal
surface.
The anodic dissolution of mild steel in HA occurs as,
Fe + A− → (FeA−)ads
(FeA−)ads → (FeA)ads + e−
(FeA−)ads → (FeA+)ads + e−
(FeA+)ads → Fe2+ + A−
FIGURE 18.2 Process of corrosion of mild steel in the presence of moisture and acid
The cathodic hydrogen evolution in HA occurs as,

Fe + H+ → (FeH+)ads
(FeH+)ads + e− → (FeH)ads
(FeH)ads + H + e → Fe + H2 (Shahid, 2011)
+ −
18.2 CORROSION INHIBITION
Corrosion inhibitors are used to combat the damage due to corrosion

(Corrosionpedia). Corrosion inhibition is more economical, practical, and
convenient than cathodic protection in controlling corrosion on a metal
surface in an aqueous environment (Solomon and Umoren, 2015).
In fact, corrosion is a natural phenomenon that is thermodynamically
favored. Metals, barring aside the noble metals, have a tendency to be in
compounds (Toussaint, 2015). The uncombined metal is thermodynamically
unstable and with some assistance transform into some or other form of
a compound. The corrosion thus is a very favorable phenomenon in case
of exposed metal surfaces. Therefore, the role of corrosion inhibitors is to
somehow resist this thermodynamically favorable process by increasing the
activation energy required to perform the transformation reaction (Fig. 18.3).
This is done by causing hindrance to the anodic and/or cathodic reactions
and slowing them so that corrosion is inhibited.
FIGURE 18.3 Energy states of metal in various forms.

Corrosion inhibitors perform by any of the following ways (Toussaint,

2015):
• Hindering the anodic and/or cathodic region reactions by raising

polarization behavior therein
• Hindering ionic diffusion to the metal surfaces
• Raising electrical resistance of the metal surfaces
• Blocking the metal surfaces and/or the adjacent corrosive environ-
ment from coming into direct contact with each other
• Getting adsorbed on to the metal surfaces to form a resistant layer
between the metal surfaces and the adjacent corrosive environment
The most common trend to inhibit corrosion of iron-containing materials

has been painting one or two coats of zinc compound containing material over
the metal surfaces. However, zinc compounds are by themselves hazardous
toward mankind and environment alike. Zinc compounds are harmful to
both mankind and environment if their percentage in the composition of the
corrosion prevention materials is more than mere 2.5% of the total amount.
Zinc can locally jeopardize aquatic environment as it gets accumulated in
aquatic fauna and subsequently poison them and also those who eat them
(Toussaint, 2015; Scottish Environment Protection Agency, 2008). Thus, the
use of such near primitive methods to inhibit has to be discarded for the
betterment of the society at large.
Corrosion inhibitors actually inhibit the metal dissolution and acid
consumption by getting adsorbed on the metal surface (Chigondo and
Chigondo, 2016). Organic compounds containing nitrogen can effectively
inhibit corrosion of steel in acidic media by adsorption of the organic inhibi-
tors on the metal surface (Chatterjee et al., 1991; Elachouri et al., 1995;
Mernari et al., 1998; Elkadi et al., 2000; Bentiss et al., 2000; Elkanouni
et al., 1996; Walker, 1975; Kertit and Hammouti, 1996; Bentiss et al., 1999).
Organic corrosion inhibitors are generally toxic in nature and this toxicity
is the main reason of an urgent need to identify nontoxic alternatives to
inhibit corrosion of mild steel in acidic media for smooth and rapid develop-
ment in diverse sectors.
18.2.1 ORGANICVERSUSINORGANICCORROSIONINHIBITORS
Generally, the functioning of the two chemically different types of inhibitors

is primarily diverse in approach.
The chemical characteristics of organic and inorganic corrosion inhibi-
tors are dissimilar and thus the way they act on the metal surfaces in a totally
dissimilar manner to inhibit the corrosion of the metal surfaces (Vishnudevan
and Thangavel, 2007).
The organic corrosion inhibitors have high molecular mass with hetero-
atoms, unsaturation, and aromaticity. They generally act on the metal surfaces
by prominent adsorption on to the surfaces, thus forming a corrosion-resistant
coating on them reducing the chances of corrosion. These normally prevent
the intensity of corrosion of the metal surfaces by a combination of the
following ways (Toussaint, 2015).
• Facilitating adhesion of the coating which is termed as an interfacial

activity
• Forming insoluble complex salts on to the anodic defect sites which
is termed as an anodic activity
• Assisting formation of precipitate by making the cathodic sites more
alkaline which is termed as cathodic activity
• Diminishing permeability of the metal surfaces to the adjacent corrosive
environment which is termed as cathodic barrier activity
• Growing a hydrophobic coating on the metal surfaces which is termed
as cathodic adsorption activity
The adsorption of corrosion inhibitors on the metal surfaces is substitu-

tive adsorption between the inhibitor in the aqueous solution, Orgaq and the
water molecules adsorbed on the metal surfaces, H2Oads.
Orgaq + nH2Oads ⇔ Orgads + nH2Oaq
Here n, the size ratio, is the number of water molecules replaced by

each inhibitor molecule (Bastidas, 2000). Organic corrosion inhibitors have
cathodic and anodic and adsorption actions and the total efficiency of corro-
sion inhibitor is equal to a summation of all the factors mentioned.
ℇ = ∑ [fia + faa + fca + fcba + fcaa]
However, on the other hand, inorganic corrosion inhibitors are salts of

some metals such as Ca, Ba, Sr, Mo, or Al and amphoteric elements that can
react both as an acid as well as a base; they generally have cathodic actions
or anodic actions only, although in some cases they can even react with the
metal surfaces to scavenge corrosive ions (Kumar, 2014).
Again, if toxicity is considered then both the organic and inorganic corro-
sion inhibitors are toxic in nature toward mankind and the environment.
Although the toxicity of inorganic corrosion inhibitors is appreciable, the
organic corrosion inhibitors are far more toxic.
Thus, the emphasis on the naturally occurring phytochemical approach
towards inhibition of corrosion of metal surfaces such as mild steel,
aluminum, and so forth, in recent years is predictable since to resist the
hazard of corrosion of metal surfaces, we are automatically getting exposed
to poisonous chemical substances.
18.2.2 ADVANTAGES AND DISADVANTAGES OF ORGANIC

AND INORGANIC CORROSION INHIBITORS
Organic corrosion inhibitors are normally more effective in inhibiting corro-

sion of metal surfaces than their inorganic competitors. Organic corrosion
inhibitors are found to act on the metal surfaces by means of adsorption on to
the surfaces. On the contrary, the inorganic corrosion inhibitors are generally
found to act through anodic passivation but this is highly debatable in case
of high concentration of chloride ions (Vishnudevan and Thangavel, 2007).
This is because in organic corrosion inhibitors, the presence of conjugated or
single double bonds owing to the presence of π-electrons facilitates chelating
with the metal ions, thus favoring pulling away of the metal component from
the metal surfaces. This naturally corrodes away the metal from the exposed
surface. Thus, bigger inhibitor molecules decorated with such functional
groups that can form chelate compounds in the organic corrosion inhibitors
are instrumental in pulling away metal from the metal surfaces.
Another advantage of organic corrosion inhibitors is that they do not react
with the metal surfaces as they form an adsorbed hydrophobic coating over
the surfaces, while their inorganic counterparts may react with the surfaces
and this may change the chemical and physical characteristics of the metals.
The disadvantage of organic corrosion inhibitors compared to inorganic
ones is that the organic inhibitors are generally more toxic to human beings
and environment. However, it is not that the inorganic corrosion inhibi-
tors are harmless and nontoxic, inorganic corrosion inhibitors containing
chromate (CrO42−) and/or nitrate (NO3−) and/or nitrite (NO2−) ions are
highly toxic toward both human beings and the environment, while those
containing zinc and/or barium salts are highly toxic toward the environment
(Toussaint, 2015).
18.3 MILD STEEL AND ITS CORROSION
Mild steel is the most common form of steel used owing to relatively low
cost and properties suitable for the varied form of applications (Singh
et al., 2016). Mild steel is tough, ductile, and malleable with superb tensile
strength. It is extensively used as an engineering material in this modern era.
In fact, the invention of steel is a huge factor in the development of mankind.
However, its low corrosion resistance, especially in the acidic environment,
is a hurdle to be addressed for smooth application of steel in progress and
development (Alaneme et al., 2016a).
18.3.1 STEEL CATEGORIES
Steel can be prepared in various forms. It is an alloy of iron and carbon

and accounts for 90% of total steel production. Steel is classified into three
groups, based on their carbon content.
1. Low-carbon or mild steels (carbon < 0.3%)

2. Medium-carbon steels (carbon = 0.3–0.6%)
3. High-carbon steels (carbon > 0.6%)
18.3.2 MILD STEEL CORROSION
Most organic corrosion inhibitors have at least one polar unit with atoms of
nitrogen (N), sulfur (S), oxygen (O), and in some cases phosphorous (P).
It has been reported that the inhibition efficiency decreases in the order to
O < N < S < P. The polar unit is regarded as the reaction center for the chemi-
sorption process. Furthermore, the size, orientation, shape, and electric
charge on the molecule determine the degree of adsorption and hence the
effectiveness of inhibitor. On the other hand, iron is well known for its coor-
dination affinity to heteroatom-bearing ligands (Singh and Quraishi, 2010).
18.4 MILD STEEL CORROSION INHIBITION BY PHYTOCHEMICALS
A comprehensive list of the recent research and findings in the application

of phytochemicals in inhibiting corrosion of mild steel are illustrated in
Table 18.1.
18.5 MECHANISM OF CORROSION INHIBITION BY

PHYTOCHEMICALS
Inhibition of corrosion is attributed to the adsorption of inhibitor phytochemi-

cals at the metal/solution interface, thus forming a protective film. The rate of
adsorption is usually rapid, and hence, the reactive metal surface is shielded
from the acid solutions (Chao et al., 1981). The adsorption of inhibitor phyto-
chemicals depends on the chemical structure, molecular size, nature of metal
surface, and charge distribution over the inhibitor. Adsorption of inhibitor
phytochemicals occurs through the replacement of solvent molecules from
the metal surface. It is seen that all the phytochemicals contain basic polar
functional groups that facilitate adsorption on the metal/solution interface.
Higher the adsorption, higher is the inhibition of corrosion of mild steel or any
other metal as the case may be. Phytochemicals bearing polar electronegative
functional groups as well as a planar structure can get adsorbed easily and
cover the metal surface effectively. Moreover, organic inhibitors contain lone
pair of electrons in functional groups and π-electrons in conjugated double or
triple bonds and have the ability to supply electrons through π-orbitals that
can facilitate adsorption of the inhibitor molecules on to the metal surfaces to
inhibit corrosion of the metal surface by forming a protective coating on them
(Vishnudevan and Thangavel, 2007; Yadav et al., 2016). In short, the size
TABLE 18.1 Recent Findings World Over.
Green inhibitor Scientific name Phytochemicals present Work cited
African oil bean roots Pentaclethra Tannins, alkaloids, nitrogen bases, carbohydrates, amino acids, Nnanna et al. (2016)
extract macrophylla and proteins
Aloe vera extract Aloe barbadensis Mannose-6-phosphate Shah and Agarwal (2014),
Montemor (2016)
Anise herb extract Pimpinella anisum Conjugated double bonds and benzene rings, and alkoxy, ether, Khamis and Alandis (2002)
and carbonic groups in anethol and chavicol
Arjuna bark extract Terminalia arjuna Hydroxyl group in b-sitosterol Singh et al. (2012)
Bamboo leaf extract Gigantochloa Flavonoids and phenolic acids, such as chlorogenic acid, caf- Khan et al. (2015), Quraishi
manggong feic acid, ferulic acid, p-coumaric acid, orientin, homoorientin, et al. (2010)
vitexin, and isovitexin
Banana peel extract Musa sapientum Oxo, amino, and hydroxyl groups in gallocatechin and Eddy and Ebenso (2008),
dopamine Singh et al. (2012), Gunavathy
and Murugavel (2013)
Barley extract Hordeum vulgare Benzene rings and hydroxyl and carbonic groups flavonoids Saadawy (2014)
such as isovitexin 7-O-β-[6-O-(E)-p-coumaroyl]glucoside
(6-coumaroylsaponarin), isovitexin 7-O-β-[6′-O-(E)-feruloyl]
glucoside, isoorientin 7-O-β-[6‘‘‘-O-(E)-feruloyl]glucoside, and
tricin 7-O-β-glucoside
Bitter orange extract Citrus aurantium Carbonyl and hydroxyl groups in threonine Singh et al. (2012)
Bitter kola seed extract Garcinia kola Amino and carboxylic groups in 15 fatty and 18 amino acids Eleyinmi et al. (2006), Okafor
et al. (2007)
Black cumin herb Nigella sativa Double bonds and hydroxyl and ether groups in saponin Khamis and Alandis (2002)
extract
Black pepper extract Piper nigrum fem. Carbonyl, tertiary amino, alkoxyl groups in piperine and its Gorgani et al. (2017), Quraishi
Piperaceae isomers et al. (2009)
Black plum extract Syzygium cumini Carbonyl, hydroxyl, and carbonic groups in ellagic acid, gallic Singh et al. (2012)
acid, quercetin, and cafeic acid

Blue-green algae Spirulina platensis Amino and carboxylic groups in acids and carbohydrates de Oliveira et al. (1999),
extract Babadzhanov et al. (2004),
Alvarenga et al. (2011)
Brahmi leaf extract Bacopa monnieri Hydroxyl and carbonic groups in bacoside a Singh et al. (2012)
Brazilian rosewood Aniba rosaeodora Anibine as the major alkaloid Chevaliera et al. (2014)
plant extract
Ceylon spinach extract Basella alba Alkaloids, pseudo tannins, chorogenic acid, anthocyanin, steroi- Vimala et al. (2016)
dal glycosides, flavonoids, and coumarin
Coffee plum bark Flacourtia jangomas Hydroxyl, phenolic, ketone, and amine in tannin and flavonoids Sisodia and Hasan (2013)
extract
Common wormwood Artemisia Carbonic, hydroxyl, and alkyl groups and bicyclic rings in Boumhara et al. (2015),
plant extract mesatlantica β-thujone, camphor, 1,8-cineole, and α-thujone Boumhara et al. (2014)
Coriander herb extract Coriandrum sativum Benzene rings, conjugated double bonds and hydroxyl, ether Khamis and Alandis (2002)
and carbonyl groups in linalool, terpinine, piene, coumarins,
phthalides, and phenolic acids.
Corn oil Carbonyl group Abbasov et al. (2013)
Cotula leaf extract Cotula coronopifolia Tertiary and secondary amino, oxo groups in anagyrine and Raja and Sethuraman (2008a),
cytosine Singh et al. (2012)
Curry leaf extract Murraya koenigii Carbonyl, secondary amino and carbonic groups in murrafoline- Singh et al. (2012)
i, pyrayafoline-d, and mahabinine-a
Curry leaf extract Murraya koenigii Monoterpene hydrocarbons (pinene, camphene, myrcene, Sharmila et al. (2010)
and limonene) and monoterpene-derived alcohols: linalool,
terpinene-4-ol, nerol, and geraniol, also their acetates with at
least one functional polar group
Custard apple extract Annona squamosa Liriodenine, oleuropein, and oxoanalobine Lebrini et al. (2010), Singh
et al. (2012)
Davana plant extract Asteraceae Monoterpenes, sesquiterpenes, furan rings, bicyclic compounds, Zhigzhitzhapova et al. (2016)
isoprenologs, longipinanes, cubebane-type sesquiterpenoids,
aristolanes, and bourbonane
Desert germander Teucrium Neo-clerodane diterpenoids and their derivatives Al-Yahya et al. (2002), Al-
extract oliverianum Otaibi et al. (2014)
Devil pepper extract Rauvolfia serpentina Reserpine, ajmalicine, ajmaline, isoajmaline, ajmalinine, Raja and Sethuraman (2008a),
chandrine Singh et al. (2012)
Drumstick leaf extract Moringa oleifera Amino and carboxylic groups in arginine Singh et al. (2012)
False daisy extract Eclipta alba Hydroxyl, alkoxyl, and ketoxy groups in wedelolactone Shyamala and Arulanantham
(2009), Singh et al. (2012)
Fenugreek seed extract Trigonella Carboxylic, amino, tertiary amino, and sulfide groups in me- Noor (2007), Singh et al.
foenum-graecum thionine and choline (2012)
Garden cress herb Lepidium sativum Benzene rings, double bonds, and secondary amino, hydroxyl, Khamis and Alandis (2002)
extract sulfonate, sulfide, and ether groups in benzyl glucosinolate
Golden rain tree leaf Cassia fistula A range of carboxylic, amino, carbonic, and hydroxyl groups Omotosho et al. (2017)
extract
Gum exudate Khaya senegalensis, Myrcene, camphene, alpha-thujene/origanene, nopinen/ Ameh (2015)
Albizia ferruginea pseudopinen, 4(10)-thujene (bicyclo[3.1.0]hexane), alpha-
campholena, 2,6-dimethyl hepta-1,5-diene, nerolidol
isobutyrate, limonene/cajepetene, verbenol, cis-verbenol,
myrtenol, carvacrol, diisopropenyl-1-methyl-1-vinyl
cyclohexane, o-menth-8-ene-4-methanol, 7-hexadecenal,
2-methylene cholestan-3-ol, and 5 alpha-pregnane-12,20-dione
Guvar bean extract Cyamopsis 3-epikatonic acid 7-o-beta-(2-rhamnosyl-glucosyl) myricetin, Subhashini et al. (2010),
tetragonoloba ash, astragalin, caffeic acid, and chlorogenic acid Singh et al. (2012)
Hairy fig leaf extract Ficus hispida Hydroxyl group and double bonds in stigmasterol Muthukrishnan et al. (2015)
Henna extract Anvillea garcinii Germacranolide Abdel-Sattar and McPhail
(2000), Al-Otaibi et al. (2014)
Henna leaf extract Lawsonia Oxo and hydroxyl groups in lawsone (2-hydroxy-1,4-naphtho El-etre et al. (2005), Singh
quinone) and tannin et al. (2012), Montemor
(2016)
Hibiscus herb extract Hibiscus sabdariffa Carboxylic, carbonic, and hydroxyl groups in hibiscus, malic Khamis and Alandis (2002),
acid, citric acid and tartaric acid Mak et al. (2013)
‎Hop bush extract Dodonaea viscosa Dihydroxy, dimethyl, and other substituted catechols, which Leelavathi and Rajalakshmi
are postulated as pyrolysate derivatives of the anti-spasmodic (2013)
flavonoids quercetin, rutin, kaempferol, and sakuranetin
Indian bael extract Aegle marmelos Carbonic and tertiary amino groups, skimmianine Singh et al. (2012)
Jatropha leaf extract Jatropha curcas Carbohydrates, cardiac glycosides, keller kilani, kedde, Mshelia et al. (2017)
borntragers, monosaccharide, reducing sugar, saponins, and
falvonoids
Kalmegh leaf extract Andrographis Carbonyl, hydroxyl, and carbonic groups in andrographolide Singh et al. (2012)
paniculata
Kuchla seed extract Strychnos Oxo and tertiary amino groups in brucine Singh et al. (2012)
nux-vomica
Leadwood tree extract Terminalia catappa Hydroxyl, carbonic, alkoxyl, and tertiary amino groups in Eddy et al. (2009)
saponin, tannin, phlobatanin, anthraquinone, cardiac glycosides,
flavanoid, terpene, and alkaloid
Long pepper extract Piper longum Tertiary amino, oxo, carbonic, and hydroxyl groups in piperine, Singh et al. (2012)
piplartine, and rutin.
Marine red alga extract Kappaphycus Phenolic, carboxylic, and amino groups and conjugated double Kamal and Sethuraman (2013)
alvarezii bonds in phycocolloids, fatty acids, carrageenan, lectins, hema-
glutannin, and carotenoids
Mayweed extract Tripleurospermum Unsaturated fatty acids and sterols Al-Otaibi et al. (2014)
auriculatum
Moraceae extract Ficus asperifolia Alkaloids, flavonoids, saponins, tannins anthraquinones, and Fadare et al. (2016)
reducing sugars
Morroccan medicinal Salvia aucheri Carbonic, hydroxyl, and alkyl groups and benzene rings in El ouadi et al. (2014)
plant extract mesatlantica α-thujone, β-thujone, α-pinene, β-pinene, p-cymene, and many
other components
Muell leaf extract Acalypha torta Fused benzene rings and O-heteroatoms in the rings in polyphe- Krishnegowda et al. (2013),
nols, terpenoids, and plant sterols Adesina et al. (2000), Monte-
mor (2016)
Napier grass extract Pennisetum Crude protein, organic matter, and acid detergent lignin Alaneme et al. (2016b)
purpureum
Neem seed extract Azadirachta indica Azadirachtin, monosaccharide, reducing sugar, tannins, sapo- Okafor et al. (2010), Mshelia
nins, kedde, keller kilan, cardiac glycosides, and carbohydrates et al. (2017), Manickam et al.
(2016), Sharma et al. (2010)
Ochrosia oppositifolia Ochrosia Carbonyl, secondary amino and alkyl groups in isoreserpiline Raja et al. (2013)
bark and leaf extract oppositifolia
Olive leaf extract Olea europaea Ester, hydroxyl, and carbonic groups in oleuropein and El-etre (2007), Singh et al.
hydroxytyrosol (2012), Montemor (2016)
Olive leaf extract Osmanthus fragrans Carbonic and alkoxyl groups in flavonoids, ascorbic acid, and Li et al. (2012), Muthukrish-
gallic acid nan et al. (2015)
Pongam tree extract Pongamia pinnata Carbonyl, hydroxyl, and carbonic groups in karanjin, pongap- Singh et al. (2012)
ine, kanjone, and pongaglabrone
Red tasselflower Emilia sonchifolia Carbonic, hydroxyl, and alkoxyl groups in amino acids, flavo- Onuegbu et al. (2013)
extract noids, quinones, carbohydrates, cardio glycosides, saponins,
tannins, fats, and oils

Red-leafed pulai leaf Alstonia angustifolia Tertiary amino groups in alstogustine and 19-epialstogustine Raja et al. (2013)
extract
Retama leaf extract Retama raetam Tertiary and secondary amino, oxo groups in anagyrine and Raja and Sethuraman (2008b),
Sacred fig extract Ficus religiosa Hydroxyl group in lanosterol Singh et al. (2012)
Safflower extract Carthemus tinctorius Unsaturated fatty acids, flavanoids and their glycosides, adenos- Zhou et al. (2008), Al-Otaibi
ine, adenine, uridine, thymine, uracil, roseoside, acetylenic and et al. (2014)
aromatic glycosides.
Sagebrush extract Artemisia sieberi Flavanoids, terpenoids, and their glycosides Marco et al. (1993), Al-Otaibi
et al. (2014)
Saudi henna extract Cassia italica Coumarins, carotenoids, flavonoids, anthraquinones, sterols, Kazmi et al. (1994), Al-Otaibi
and triterpenes et al. (2014)
Soursop leaves extract Annona muricata 6-hydroxyundulatine and other alkaloids Iroha and Chidiebere (2017),
Vimala et al. (2012)
Taily weed extract Ochradenus Flavanoids and their glycosides Barakat et al. (1991), Al-
baccatus Otaibi et al. (2014)
Thyme herb extract Thymus vulgaris Benzene rings, bicycle rings, double bonds, and hydroxyl and Khamis and Alandis (2002)
alkyl groups in thymol, p-cymene, borneol, nerol, and carvacrol.
Winged prickly ash leaf Zanthoxylum Carbonic, alkoxyl, hydroxyl, alkyl, ester, phenyl, and carboxylic Gunasekaran and Chauhan
extract armatum groups, double bonds and benzene rings in methoxyanthra- (2004), Akhtar et al. (2009)
quinone, hydroxyanthraquinone, diphenyl alatumoic dimethyl
ester, salicylic acid, methoxysalicylic acid, zanthoxylumflavone
xyloside, and b-sitosterol-b-d-glucoside
Wormwood leaf extract Artemisia herba-alba Tertiary and secondary amino, oxo groups in anagyrine and Raja and Sethuraman (2008b),
of corrosion inhibitor molecules, the substituent groups linked to them, the

functional hetero adsorption atoms present in them, the extent of conjugated
bonds, and any presence of aromaticity play a vital role in determining the
efficiency and effectiveness of the corrosion inhibitors (El-Naggar, 2007).
18.6 CONCLUSION
Phytochemicals are a good alternative as corrosion inhibitors because

these are naturally occurring and comparatively very less toxic barring a
few examples. These environment-friendly compounds can provide a better
option in tackling the major hazard of corrosion of mild steel in acidic
media. The R&D around the globe is constantly at work in identifying better
phytochemical inhibitors of corrosion of mild steel in acidic media.
KEYWORDS
•• mild steel corrosion

•• organic corrosion
•• inorganic corrosion
•• adsorption
•• corrosion inhibitors
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PART IV
Recent Advances
CHAPTER 19
NOVEL TERPENOIDS AS ANTICANCER

STEM CELL AGENTS
REBATI MALIK1, SANTOSH KUMAR MAURYA1,
PREM PRAKASH KUSHWAHA1, PUSHPENDRA SINGH2, and
SHASHANK KUMAR1,*
1
School of Basic and Applied Sciences, Department of Biochemistry
and Microbial Sciences, Central University of Punjab, Bathinda,
Punjab 151001, India
2
National Institute of Pathology, New Delhi, India
*
[email protected], Mob.: +91 9335647413
*
ABSTRACT
Tumor tissue has some small subpopulation of cells known as cancer stem
cells (CSCs). These populations are capable of self-renewal, differentiation,
and have the unique property to evade radiotherapy and chemotherapy. This
subpopulation of cells is the major cause of resistance to current cancer
treatments. It is also reported that CSCs are associated with relapse in
cancer patients. Compared to the differentiated tumor cells, CSCs have
some important distinguishing feature that confers chemoresistance in
these cells. Different proteins such as Bcl2, CXCR4, carbonic anhydrase 2,
MTH1, CHK1, and VEGFR2 have been reported to be involved in cancer
cell stemness. Nowadays, natural products are popular remedies against
various diseases including cancer. These products have been reported
for their low/nontoxicity and cost-effectiveness. In the present chapter,
we have discussed the phytochemistry, natural, synthetic pathways and
pharmacological activities of terpenoids with special references to cancer
cell stemness and anti-cancer property. Furthermore, we also performed in

silico studies to identify potent terpenoids active against a protein involved
in cancer cell stemness.
19.1 INTRODUCTION
Terpenoids are the biggest class of naturally occurring compounds derived

from five-carbon isoprene units. They are produced by plants and also
known as isoprenoids. Majority of the terpenoids protect plants from abiotic
and biotic stress. Plants commonly use terpenoids in their development and
growth. Terpenoids have a crucial role in plant physiology such as hormone
regulation and chlorophyll pigment development. Several terpenoids are
being used by pharmaceutical, chemical, and food industries. Systematic
and genomic biology is exploring the potential of terpenoids with their high
value in microbes and plants. Scientists are giving attention to developing
terpenoids as a pest control agent. Literature shows the various benefits of
a terpenoid. Thus, terpenoid biosynthesis pathways should be explored in
more depth (Tholl, 2015).
Biosynthesis of terpenoids depends on the flux of precursors. The first step
of terpenoid biosynthesis is formation of the five-carbon (C5) units which
undergoes through the mevalonate or methylerythritol phosphate (MEP)
pathway to generate terpenes. Terpenoids can be classified as hemiterpenes
(C5), monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), sesqui-
terpenes (C25), triterpenes (C30), tetraterpenes (C40) and polyterpenes
on the basis of C5 unit (Ashour et al., 2010; Martin et al., 2003). Terpene
synthase (dimethylallyl diphosphate diphosphate-lyase) is the key enzyme
for terpene synthesis. The enzyme requires Mg2+ or Mn2+ as a cofactor
(Silver and Fall, 1995; Wildermuth and Fall, 1996). It produces isoprene and
pyrophosphate from dimethylallyl pyrophosphate (DMAPP) (Fig. 19.1).
Plants and many of the prokaryotes are known to produce isoprenes (Kuzma
et al., 1995). Cytosolic mevalonic acid (MVA) and plastidial MEP pathway
generate the key precursor (isopentenyl pyrophosphate (IPP) and DMAPP)
of terpenoids (Fig. 19.2).
MVA pathway synthesizes cytosolic precursor sesquiterpenoids, poly-
prenols, phytosterols, brassinosteroids, and triterpenoids. Ubiquinones
and polyprenols are the two important precursors for the synthesis of
mitochondrial terpenoids (Tholl, 2006). The first step of the mevalonic
acid pathway starts with the condensation of the acetyl-CoA with another
Novel Terpenoids as Anticancer Stem Cell Agents 395
acetyl-CoA in the presence of acetoacetyl-CoA thiolase (Fig. 19.3).

Acetoacetyl-CoA condenses with another acetyl-CoA to form 3-hydroxy-
3-methylglutaryl-CoA (HMG-CoA) with the help of HMG-CoA synthase.
These reactions are followed by different steps involving HMG-CoA reduc-
tase, mevalonate-5-kinase, mevalonate pyrophosphate decarboxylase, and
isopentenyl pyrophosphate isomerase enzyme resulting in the formation
of DMAPP. On the other hand, the MEP pathway is initiated with the
reaction of pyruvate and glyceraldehyde 3-phosphate (Fig. 19.4). Different
enzymes of the pathway work in a sequential step to form (E)-4-Hydroxy-
3-methyl-but-2-enyl pyrophosphate (HMB-PP) as the end product of the
pathway. Another enzyme called IspH finally converts the HMBPP to the
IPP and DMAPP.
FIGURE 19.1 Isoprene synthesis from dimethylallyl pyrophosphate and isoprene synthase.
FIGURE 19.2 Common pathway involved in terpene biosynthesis.

FIGURE 19.3 Step involved in the mevalonic acid pathway.
FIGURE 19.4 Steps involved in methylerythritol phosphate pathway.

Hemiterpene (C5), the simplest class of terpenes is mostly found in oil.

There are around 100 hemiterpene aglycones known. Mostly, they are water-
insoluble, but some of them contain sugar group and are water-soluble.
Isoprene is a very common and extremely known hemiterpene. Chlorinated
hemiterpenes such as utililactone and epiutililactone have been reported in
Prinsepia utilis leaves. Monoterpene is a type of hydrocarbon comprised
of two condensed isoprene units (C5). It is an ingredient of fragrant oils
obtained from leaves of the different plant. It is a monocyclic monoterpene
such as limonene that has remarkable chemopreventive/chemotherapeutic
potential against multiple types of cancer (Sun, 2007). Limonene is
known to induce apoptosis and can modulate polyamine metabolism in
colon cancer. Limonene treatment showed increased Bax/Bcl2 ratio with
upregulation of cleaved caspase-3, caspase-9, poly ADP ribose polymerase,
and cytochrome C. It has been reported that patients fed with limonene
conferred a striking effect in tumor regression. Monoterpene limonene
has very low toxicity and does not report any mutagenic, carcinogenic or
nephrotoxic risk to humans (Vigushin et al., 1998). A hydroxylated form of
limonene, Perillyl alcohol showed inhibition of azoxymethane-induced rat
colon carcinogenesis (Reddy et al., 1997). Carvacrol downregulates cyclin
B1 expression, arrests G2/M phase of cell cycle and initiates apoptosis
in HCT116 and LoVo cancer cell lines (Fan et al., 2015). Carvacrol also
showed anti-invasive potential via decreasing matrix metalloprotease-2
and matrix metalloprotease-9 expression. Sesquiterpenes are a cluster of
15 carbon compounds resulting from the assembly of three isoprenoids
(C5) units. Sesquiterpene lactones are kept at the top position in pharma-
cological studies and have an unusual anticancer activity (Orofino et al.,
2012). Sesquiterpene lactones, namely isobutyroplenolin and arnicolide
D, zerumbone, isolated from Centipede minima and Zingiber zerumbet
respectively, showed proliferation inhibition and induction of apoptosis
(Huang et al., 2014; Murakami et al., 2004). Four isoprene units or two
terpene units form diterpenes. Andrographolide diterpene lactones isolated
from Andrographis paniculata showed cytotoxic properties in different
cancers such as breast, melanoma, lung, and leukemia cell lines (Nanduri
et al., 2004). Pseudolaric acid B is another diterpene isolated from the root
bark of Pseudolarix kaempferi that showed antiproliferative potential in
different cancers (Pan et al., 1990). Triterpenoids such as saponins, betu-
linic acid, and phytosterols are well known for their biological activities.
Betulinic acid showed antiproliferative potential and induced apoptosis in
colon cancer cell (Rzeski et al., 2006). Another triterpenoid oleanolic acid
comprises antiinflammatory and cytotoxicity efficacy against various types

of colon cancer cell lines (Li et al., 2002). Glycyrrhizic acid or glycyr-
rhizin from the licorice root extracts can inhibit N-acetyltransferase enzyme
(Chung et al., 2000). Carotenoids and lycopenes are some other terpenoids
having the anticancer potential (Scolastici et al., 2007).
19.2 CANCER STEM CELLS (CSCS)
In a malignant tumor, there is some subpopulation of CSCs. Many over-

acting and abnormal signaling pathways contribute to the survival of cancer
stem cells (CSCs). CSCs are capable of differentiation, self-renewal and
generation of heterogeneous tumors in a different type of cancer. There
are two models which explain heterogeneity within the tumor: the clonal
evolution model and cancer stem cell model. Clonal evolution model is
also known as a stochastic model. According to this model, every cell can
develop into cancer cell through successive mutations (Merlo et al., 2006).
CSC model is also known as hierarchical model. CSCs are always placed
at the top in this model (Reya et al., 2001). According to CSC model, only
CSCs have the property of tumor formation within the tumor (Sottoriva
et al., 2010). In 2014, a new model was purposed which creates a bridge
between these models and this model is now called plasticity model.
According to this model, CSC has property to change into non-CSC cell
and vice versa (Tetteh et al., 2015). Like normal stem cells, all the proper-
ties of CSCs are regulated by their specific niche. CSCs present different
types of markers on their surface like oct4, CD44, aldehyde dehydrogenase
1 (ALDH1), CD24, CD29, CD90, CD133, epithelial-specific antigen,
and so forth. Expression of this marker is tissue type-specific or tumor
subtype-specific (Ajani et al., 2015). Expression of these markers depends
on singling pathways. These pathways are interconnected to each other, and
these interwoven networks of signaling meditator feed into one another. In
breast CSC-like cells, JAK/STAT including IFNK, IFNGR, IL6, STAT1,
and the activated form of STAT3 are highly upregulated and promote
cancer stemness (Stine and Matunis, 2013). Aberrant Hedgehog signaling
(overexpression of Gli1, SHH, and PATCHED1) in CSCs is documented
into basal cell carcinoma, multiple myeloma, glioblastoma, chronic
myeloid leukemia and colon cancer (Peacock et al., 2007). Aberrant Wnt
signaling (upregulation of β-catenin and downregulation of APC, GSK-3β)
show stemness properties in different types of CSCs (breast, gastric and
colorectal cancer) (Lindvall et al., 2006). A higher level of Notch signaling

genes notch1, notch3, jag1, jag2, and Notch target gene hes1, are docu-
mented in several types of cancers stem cells (pancreatic, blood and breast)
(Ranganathan et al., 2011). Similarly, overexpression of NF-kβ signaling
pathway has been reported in chronic lymphocytic leukemia and multiple
myeloma (Zhao et al., 2012). Stemness of cancer is also regulated by
their microenvironment or niche (Junttila and de Sauvage, 2013). Chronic
inflammation is one the major factors influencing the metastatic property of
CSCs (Grivennikov et al., 2010). CSCs have multidrug resistance (MDR)
property, for example, exposure of temozolomide against glioblastoma
expand the stem cell pool and increase the overexpression of markers such
as Oct4 and Nestin (Abubaker et al., 2013). Hypoxia also induces stemness
in different type of CSCs (Axelson et al., 2005).
19.3 PHYTOCHEMICALS AND CSC
Among anticancer drugs, 50% are natural products or their derivatives

extracted from plants and seeds. Many phytoextracts are used as an
anticancer drug, for example, gamma-tocotrienol which is extracted
from palm oil, polysaccharide peptide obtained from mushroom, inhibits
tumor formation capacity of CSCs (Luk et al., 2011). Triterpenes present
in many fruits (stop the self-renewal power of CSC of liver cell (Lee
et al., 2011). Genistein which is an isoflavone, has antiproliferative
effect on different types of cancer (Hewitt and Singletary, 2003). Bras-
sicaceae (watercress and broccoli) extracts contain isothiocyanates such
as phenethyl isothiocyanates and sulforaphane that target the prolif-
eration, stemness, metastatic potential in colorectal cancer (Table 19.1).
Curcumin (dietary polyphenol) is a chemopreventive agent in a variety
of cancers (breast, liver, prostate, hematological, gastrointestinal and
colorectal cancer) inhibits metastasis (Kunnumakkara et al., 2008),
growth and self-renewable (Kakarala et al., 2010) properties of CSCs.
Epigallocatechin-3-gallate (EGCG), a type of catechin synthesized by
green tea, is a polyphenolic constituent of green tea having the potency
for apoptosis and inhibits growth of various cancers through different
mechanisms (Jung and Ellis, 2001). Retinoic acid derived from vitamin
A, differentiates CSCs or depletes their formation by regulating Notch
signaling (Moreb et al., 2005). Selenoproteins are biologically active
form of selenium and act as anticarcinogenic nutrients.
TABLE 19.1 Cancer Stem Cell Signaling Targets by Phytochemicals.

Phytochemicals Targeted signaling References
Cyclopamine, Sulforaphane Hedgehog signaling Oh et al., 2016
Epigallocatechin-3-gallate (EGCG), Vita- Wnt signaling
min D, Curcumin
Retinoic acid, Curcumin Notch signaling
Selenium, Sulforaphane RTKs signaling
Genistein, Quercetin mTOR signaling
19.4 TERPENOIDS AND CSCS
In China, a special type of timber tree named Albizia julibrissin is distributed

(Pharmacopoeia committee of China, 2015). The stem bark of this plant is
used as medicine (Ikeda et al., 1997). The oleanane-type triterpenoids are
main constituents (Zou et al., 2000). Oleanane-type triterpenoid is made up of
acetic acid glycan and monoterpenoid glycosyl. Some triterpenoid saponins
have cytotoxic and anti-angiogenic activity against cancer cell line (Liang
et al., 2005). In recent times, ten new oleanane-type triterpenoid saponins and
six triterpenoid saponins have been identified (Zheng et al., 2010).
Hinokitiol shows the anti-CSC effect on glioma cancer. It is a compound
of aromatic tropolone derived from Chamaecyparis taiwanensis (Shih et al.,
2013). Besides the anti-CSC effect, it has antimicrobial, anti-inflammatory
effects (Shih et al., 2014). It has been shown that hinokitiol increases anti-
oxidant enzymes in CSCs and exerts anticancer effect (Huang et al., 2015b).
Hinokitiol suppresses the phosphorylation of mitogen-activated protein
kinase signaling and inhibits migration of cancer melanoma cells (Huang
et al., 2015a). In breast cancer, hinokitiol reduces the vasculogenic mimicry
activity by stimulating epidermal growth factor receptor (Tu et al., 2016).
Retinoic acid derived from vitamin A, plays an important role in limiting
the synthesis of ALDH and performs negative feedback effect (Moreb et al.,
2005). It was reported that exogenous addition of retinoic acid suppresses
ALDH activity in the cancer cell. This negative feedback of ALDH
renders it sensitive to 4-hydroperoxycyclophosphamide (4-HC). 4-HC is a
derivative of cyclophosphamide and an aldehyde substrate of ALDH (Moreb
et al., 2007). All-trans-retinoic acid (ATRA) induces cell differentiation in
malignant tumor type, including breast cancer (Sutton et al., 1997). Reti-
noic acid (RA) targets the CSCs and is less toxic to normal cell, thus more
conventional in chemotherapy (Garattini et al., 2007). It has been shown that
retinoic acid receptors (RARs) bind to retinoid X receptors (RXRs) present
as a heterodimer (RAR/RXR) to the RAR response element at the promoter

of the target gene. Once the RAR interacts with the RA ligand and binds to
RXR, it takes conformational changes to recruit the coactivator and disso-
ciate the corepressor, leading to the start of transcription of the target gene.
There are three forms of RARs present in a cell, RARα, RARβ, and RARγ.
Among them, the loss of RARβ expression is significantly correlated with
the tumor-forming capacity and RA-resistant property (Tang and Gudas,
2011). ATRA directs the breast cancer cells though RARβ-TET2-miR-
200c-PKCζ signaling pathway (Shimono et al., 2009). Ailanthus triphysa, a
therapeutic agent locally named Yom-Pa, is a deciduous tree distributed over
Asia and Australia. The dichloromethane and dichloromethane-methanol
extracts of the stem and its bark, show a good inhibition of cancer cell line
(Kundu and Luskar, 2010). Amoora rohituka is a medicinal plant, a member
of Meliaceae family and grows in India, Sri Lanka, Malaysia, Indonesia,
China, and Bangladesh. Many plants extracts comprises triterpenoids,
diterpenoids, limonoid, glycosides and alkaloids and are used in the treat-
ment of tumor, liver disease, and abdominal complaints (Ghani, 2003).
Petroleum ether extracted from A. rohituka causes the death of pancreatic
cancer cells (PANC-1, Mia-Paca2, Capan1, and MFC-7) (Chan et al.,
2011). A synthetic triterpenoid, methyl 2-cyano-3,12-dioxooleana-1,9 (11)
dien-28-oate, induces apoptosis and inhibits telomerase activity in PANC-1
and Mica-Paca2 cells (Deeb et al., 2012). Cucurbitacin B, a tetracyclin
terpenoid extracted from Trichosanthes kirllowii, inhibits JAK2, STAT3,
and STAT5, increases p21, and induces apoptosis of pancreatic cancer.
Gemcitabine affects the K-Ras mutant pancreatic cancer cell and reduces
the size of pancreatic tumor xenografts (Abbassi et al., 2009). AMR-MeoAc
is a monoacetate of a triterpene amooranin extracted from A. rohituka stem
bark, inhibits oncogenic K-Ras through ERK, Akt, and survivin in HPAF-II
cells of pancreatic cancer (Rabi and Venkatanarasimhan, 2014). Aphanin, a
triterpene, was extracted through column chromatography for the first time
from the stem of A. rohituka, and inhibits cell proliferation, induces G0-G1
cell cycle arrest and promotes apoptosis with a dynamic change in Bax/
Bcl-2 retio in lung cancer A549 cells. Aphanin inhibits the proliferation with
G0-G1 cell cycle arrest, induces apoptosis by inhibition of K-Ras, STAT3,
Akt, cyclin D1, c-Myc, and activates the caspase cascade of pancreatic
cancer (Rabi et al., 2007).
Tingenone is cytotoxic against many cancer types including breast
cancer (Gomes et al., 2011). The tingenin b (22β-hydroxytingenone) has
antibacterial (Maregesi et al., 2010), antiparasitic (de Sousa et al., 2015) and
anticancer activity (Bavovada et al., 1990). Tingenin b induces a cytotoxic
effect and apoptosis in breast CSCs (MCF-7). Cytotoxic effect of tingenin

b against any type of cancer still remains unidentified. Tingenin b has
cytotoxic activity by inducing apoptosis which is caused by mitochondrial
injury. Even endoplasmic reticulum is also involved in inducing apoptosis in
breast CSCs (in vitro) (Karakas et al., 2015).
LANGDU derived from Euphorbia prolifera Buch.-Ham found in
southest China is a herbal medicine and used to treat cancer and inflammation
(Li et al., 2008). The myrsinol diterpene extracted from E. prolifera Buch.-
Ham inhibits the proliferation of tumor cells (Li et al., 2011). P-glycoprotein
exports a verity of the cytotoxic drug from the cytoplasm and cell membrane
to body fluids and provides MRD (Hennessy and Spiers, 2007). Overex-
pression of P-gp reduces intracellular concentration of drugs and protects
cancer cells from cytotoxicity (Eichhorn and Efferth, 2012). A variety of
diterpenes extracted from Euphorbia species, including LANGDU, inhibits
P-gp-dependent efflux and reverse MDR of the breast cancer cell line (Corea
et al., 2004).
A sesquiterpene lactone named parthenolide (PTL), extracted from
the shoots of Tanacetum parthenium (Stojakowska and Kisiel, 1997), has
shown anticancer and anti-inflammatory activities (Wiedhopf et al., 1973).
PTL is the first small molecule selected against CSCs. PTL targets specific
signaling pathways and kills cancer cells at their roots. In 1973, it was found
that PTL has antitumor and anti-inflammatory effect by targeting of NF-kB
signaling pathways (Bork et al., 1997). Thus, PTL is often purchased as a
pharmacological NF-kB inhibitor. In 2005, the molecular mechanism of
its anticancer properties through epigenetic regulations was found. Thus,
PTL is also used as an epigenetic-based chemoprevention technique (Gopal
et al., 2007).
We performed an in silico study to find out active terpenoids (Fig. 19.5)
against cancer stem cell signaling pathway proteins Bcl2, CXCR4, CHK1,
MTH1, VEGFR2, and Carbonic anhydrase 2. These proteins are mainly
involved in cancer cell stemness initiation and progression. Various offline
and online tools were used for docking study (protein, ligand, and struc-
ture preparation). The best dock score of various terpenoids is shown in
Figures 19.6–19.11. Schematic representation of the different type of
interactions (hydrogen bonds and hydrophobic interactions) among lead
compounds and targeted proteins was developed using LigPlot+v.1.4.5
and results are depicted in Figure 19.12. We also predicted ADME/T and
drug-likeness properties by using PreADMET server. The result of these
parameters is shown in Tables 19.2 and 19.3.
FIGURE 19.5 Structure and PubChem-IDs of the terpenoid used in the present study.
FIGURE 19.6 Dock score of terpenoids with Bcl2 protein and standard inhibitor.
FIGURE 19.7 Dock score of terpenoids with VEGFR2 protein and standard inhibitor.
FIGURE 19.8 Dock score of terpenoids with CXCR4 protein and standard inhibitor.
FIGURE 19.9 Dock score of terpenoids with MTH1 protein and standard inhibitor.
FIGURE 19.10 Dock score of terpenoids with CHK1 protein and standard inhibitor.
FIGURE 19.11 Dock score of terpenoids with Carbonic anhydrase 2 protein and standard
inhibitor.
Bcl2-119034 CXCR4-181183
CHK1-3034821 MTH1-21577087
VEGFR2-159593 Carbonic anhydrase2-3503
FIGURE 19.12 (See color insert.) LigPlot of lead terpenoids with Bcl2, VEGFR2,
CXCR4, MTH1, CHK1 and Carbonic anhydrase 2 proteins.
TABLE 19.2 ADME Properties of the Terpenoid used in the Present Study.
PubChem BBB Caco2 Pgp- HIA MDCK PPB
ID inhibition
3503 1.24369 20.7934 Inhibitor 85.10669 0.0434204* 100
6654 5.5333 23.6322 Inhibitor 100 304.815 100
10,114 2.21347 21.5166 Inhibitor 96.71404 0.0467336 98.14341
17,100 5.11925 50.8083 None 100 191.284 23.41631
72,421 0.0742244 19.4817 Inhibitor 76.45951 0.0507847 55.11564
73,170 20.9638 47.1744 Inhibitor 100 1.43045* 100
73,296 0.0911556 20.6726 Inhibitor 59.91227 0.0434156* 87.67771
91,458 0.0545602 9.93766 None 20.68248 0.532271 25.22242
119,034 0.628392 20.9771 Inhibitor 91.23933 0.0434811 96.45518
159,573 0.304079 36.7279 None 98.82558 4.73654 86.62276
181,183 0.0720654 19.9336 Inhibitor 84.01712 0.044098 59.79623
289,984 3.04333 23.6383 Inhibitor 97.99076 0.0435672* 100
442,360 14.5943 23.4036* Inhibitor 100 63.7385* 100
455,262 2.68366 21.5394 Inhibitor 92.96128 197.161 93.0993
457,901 0.0383007 21.1051 None 82.10811 0.934989 60.86894
469,744 5.05936 22.2889 Inhibitor 97.76701 0.110106* 100
470,259 13.7696 31.1248 Inhibitor 94.40467 0.204598* 100
472,768 8.48839 21.8826 Inhibitor 95.99632 0.0498201* 100
500,219 0.528504 22.827 None 91.83539 7.32962 55.9652
588,303 0.0398137 20.0244 Inhibitor 88.70174 0.0440033 58.48968
636,756 0.557916 21.4232 None 84.48824 2.73672 69.58589
3,034,821 0.0282505 21.9967 Inhibitor 95.66251 0.165431 86.8339
5,281,520 14.2219 23.633 Inhibitor 100 60.6852* 100
5,282,108 1.73523 53.0214 Inhibitor 100 305.112 99.46857
5,318,379 2.78849 21.2642 Inhibitor 94.27844 0.0435056 99.22454
5,319,791 0.821186 22.7052 Inhibitor 87.92062 0.043855 85.97652
6,444,377 0.0573358 20.8179 None 94.40623 0.22335 86.39517
6,479,753 7.62409 23.176 Inhibitor 96.72772 0.0434161* 100
6,506,231 0.2271 18.9858 Inhibitor 90.54322 1.83684 87.60205
9,950,773 2.68366 21.5394 Inhibitor 92.96128 197.161 93.0993
9,974,918 1.83781 21.2963 Inhibitor 96.14894 108.741 93.92846
9,977,821 0.0104038 13.4916 Inhibitor 92.77509 13.1789 72.44668
PubChem BBB Caco2 Pgp- HIA MDCK PPB

ID inhibition
10,014,355 6.81085 23.3109 Inhibitor 100 11.2712 100
10,243,131 2.7124 6.45265 None 95.30722 152.927 100
10,327,653 0.0426706 25.8336 None 92.10519 0.0435758* 46.44425
10,369,667 2.03623 21.7023 Inhibitor 92.28835 0.0515458 94.03352
10,445,633 0.626686 20.5558 None 89.9543 11.8002 66.75749
10,466,743 5.13469 22.7531 None 90.6887 82.1451 100
11,120,895 0.0699499 22.3454 Inhibitor 94.52302 34.0304 87.77617
11,474,040 0.0120793 17.7412 None 80.57817 0.798585 47.01546
11,954,143 17.4 53.2259 Inhibitor 97.8963 0.044665 100
12,305,935 11.5378 31.7521 Inhibitor 96.01623 0.0435947 100
14,165,733 0.0766897 19.9277 Inhibitor 85.70879 0.0440213 65.90702
15,432,541 0.373606 19.2105 None 84.75231 0.979548 43.39387
16,401,759 0.278196 21.1146* None 80.01777 1.15224 53.06102
21,597,452 0.139588 20.3242 Inhibitor 91.81901 0.0486388 80.47152
44,555,454 0.488299 22.3963 Inhibitor 85.86073 0.0434181* 92.61243
44,566,526 0.0162074 29.1051 Inhibitor 97.58165 0.0930119 87.8218
44,566,960 0.22473 14.5047 None 88.89729 3.00037 68.63116
44,607,275 1.49938 20.8858 Inhibitor 83.90136 0.0452909 100
44,607,276 4.42918 21.514 Inhibitor 89.39868 0.0453023 100
44,607,277 5.40051 24.188 Inhibitor 92.65427 0.0460208 100
46,186,370 1.87206 24.0539 Inhibitor 94.19949 7.27395 95.43168
46,186,371 5.71784 28.9288 Inhibitor 93.38052 50.5665 100
46,186,620 0.0513656 45.4987 Inhibitor 98.23901 12.0251 88.83777
46,186,621 0.383625 37.7897 Inhibitor 94.83327 0.0536684 83.19236
46,912,852 8.55914 27.1709 Inhibitor 94.61309 0.0484552 100
52,947,022 4.03427 22.1689 Inhibitor 94.51553 0.045275 96.43383
52,947,048 0.937266 21.3756 Inhibitor 93.89857 0.227083 93.25572
426,077,999 6.17967 17.5039 Inhibitor 93.74117 0.0434744* 98.37829
Human Intestinal Absorption (HIA), Carcino Rat (Caco-2), Plasma Binding Protein (PPB),
Madin-Darby Canine Kidney cells (MDCK), Pgp inhibition (Pgp-I) and Blood Brain Barrier
(BBB) penetration, BBB+: penetrable to Blood-Brain Barrier; BBB-: not penetrable to Blood
Brain Barrier).
TABLE 19.3 Drug Likeness and Toxicity Profile of the Terpenoid used in the Present Study.
PubChem Drug-likeness Toxicity
id CMC like Rule of Ames test hERG Carcino
Rule Five inhibition Rat
3503 Not qualified Violated Mutagen Medium risk Positive
6654 Not qualified Suitable Mutagen Medium risk Positive
10,114 Not qualified Suitable Non-mutagen Low risk Positive
17,100 Not qualified Suitable Mutagen Low risk Negative
72,421 Not qualified Suitable Non-mutagen Ambiguous Positive
73,296 Not qualified Violated Mutagen Ambiguous Negative
91,458 Not qualified Suitable Mutagen Low risk Negative
159,573 Qualified Suitable Mutagen Medium risk Positive
181,183 Not qualified Suitable Non-mutagen Ambiguous Positive
289,984 Not qualified Suitable Mutagen Low risk Positive
442,360 Qualified Suitable Mutagen Medium risk Positive
455,262 Qualified Suitable Non-mutagen Low risk Positive
457,901 Qualified Suitable Mutagen Ambiguous Positive
470,259 Not qualified Suitable Mutagen Low risk Positive
500,219 Qualified Suitable Mutagen Low risk Negative
588,303 Not qualified Suitable Mon-mutagen Ambiguous Positive
636,756 Qualified Suitable Mutagen Low risk Positive
3,034,821 Not qualified Suitable Non-mutagen Ambiguous Positive
5,281,520 Qualified Suitable Non-mutagen Medium risk Positive
5,282,108 Qualified Suitable Mutagen Low risk Negative
5,318,379 Not qualified Suitable Non-mutagen Low risk Positive
6,444,377 Qualified Suitable Non-mutagen Low risk Positive
6,479,753 Not qualified Violated Mutagen Medium risk Positive
PubChem Drug-likeness Toxicity

id CMC like Rule of Ames test hERG Carcino
Rule Five inhibition Rat
9,977,821 Qualified Suitable Mutagen Low risk Positive
10,014,355 Failed Suitable Mutagen Low risk Positive
10,327,653 Not qualified Violated Non-mutagen Low risk Positive
42,607,999 Not qualified Suitable Non-mutagen Medium risk Negative
44,555,454 Not qualified Suitable Non-mutagen Low risk Negative
44,566,526 Not qualified Suitable Mutagen Medium risk Positive
44,607,275 Not qualified Suitable Mutagen Ambiguous Negative
ACKNOWLEDGMENT
Shashank Kumar acknowledges Central University of Punjab, Bathinda

and University Grants Commission, India for providing the necessary infra-
structure facility and financial support in the form of UGC-BSR Research
Start-Up-Grant, GP: 87 [No. F.30–372/2017 (BSR)] respectively. PPK
acknowledges financial support from University Grants Commission, India
in the form of CSIR-UGC Junior Research fellowship. Rebati Malik and
Santosh Kumar Maurya acknowledge Central University of Punjab, Bath-
inda, India for providing necessary infrastructure facility.
KEYWORDS
•• terpenoids
•• anticancer
•• stem cell
•• cytotoxicity
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CHAPTER 20
EVALUATION OF THE
PHYTOHEMAGGLUTININS ACTIVITIES
OF ECHINACEA SPECIES IN
ONTOGENESIS
SERGEY V. POSPELOV
Department of Agriculture and Agrochemistry, Faculty
Agrotechnology and Ecology, Poltava State Agrarian Academy,
1/3 Skovorody St., Poltava 36003, Ukraine, Tel.: +380951213218
ABSTRACT
It is established that purple coneflower (Echinacea purpurea (L.) Moench)

and pale purple coneflower (Echinacea pallida [Nutt.] Nutt.) contains
specific protein compound—lectins, which have the ability to reversibly
interact with carbohydrates and exhibit high biological activity at the
cellular and organism levels. An original methodology of hemagglutination
activity of Echinacea extracts evaluation was developed. The study of the
dynamics of lectin activity showed that in the E. purpurea high level of
hemagglutinins was found in roots with rhizomes, stems, both forming an
open inflorescences. It has been established that during the vegetation of E.
pallida the highest level of phytolectins observed in leaves—twice as much
higher as in roots with rhizomes. In the genesis period, hemagglutination
activity of herb extracts was higher than roots with rhizomes extracts.
Echinacea overground mass considered as important raw materials for
preparative isolation of lectins.
20.1 INTRODUCTION
Species of the Echinacea (Echinacea Moench, family: Asteraceae) are

used in many countries of the world as raw materials in the preparation
of immunostimulating, anti-inflammatory, and antiseptic effects due to its
unique chemical composition (Bauer and Wagner, 1990; Miller, 2004). This
is typical for Ukraine and all CIS countries, where Echinacea is actively
studied and widely used, especially in connection with the creation of new
products and drugs for the elimination of the consequences of the chernobyl
accident (Dubinska, 1998; Dreval et al., 2003).
In the Poltava State Agrarian Academy (Ukraine), as a result of 25 years
of systematic researches, the genus Echinacea species were collected and
studied; a varieties of Echinacea purpurea (Zirka Mykoly Vavilova) and
Echinacea pallida (Krasunya prerii) were selected; the largest planta-
tion cultivation in the CIS was organized, which is capable of providing
all consumers with eco-friendly raw materials of the highest quality; the
new technologies for growing Echinacea as medical raw material, feed for
livestock, melliferous plant for beekeeping were developed and patented
(Pospelov and Samorodov, 2009).
20.2 ANALYSIS OF PREVIOUS STUDIES
In connection with an in-depth study of the chemical composition (Samo-

rodov et al., 1996) and clarification of the nature of the immunostimulating
properties of Echinacea lectins—biologically active substances of a protein
nature that have properties to selectively and reversibly interact with carbo-
hydrates and carbohydrate-containing polymers, deserve attention, which
gives them a number of unique properties (Lutsik et al., 1981).
There are data about the search for lectins in the seeds of Echinacea
augustifolia DC among other 167 plant species of the North American flora
(Hardman et al., 1983). The authors used human erythrocytes (processed and
non-processed with enzymes) for this purpose, but no agglutinating activity
of the extracts was found.
A systematic search for E. purpurea lectins was conducted in Ukraine
(Pospelov and Samorodov, 1996) and Lithuania (Kondrotas et al., 1996).
In the studies, the employees of the Kaunas Medical Academy, the hemag-
glutinating activity of the E. purpurea extracts (it was not specified which
part of the plant) with the human erythrocytes of all four groups, rabbit,
pig, mouse, and frog has been estimated. At the same time, weak activity
Evaluation of the Phytohemagglutinins Activities 423
was registered when interacting with the blood cells of human B(III) blood
group and mouse blood cells. Earlier, in our experiments (Pospelov and
Samorodov, 1996) the extraction of roots, leaves, stems, inflorescences, and
fruits with buffered saline in raw material ratios extragent 1:5 and 1:10, was
carried out. In all cases, negative results were obtained.
Several other data were received by the employees of the Taras
Shevchenko National University of Kyiv (Pohorila et al., 1997). Water-
salt extracts of roots and stems of 10-day-old seedlings, as well as stems,
leaves, non-blossoming inflorescences, and plant seeds of the second year
of vegetation, did not interact with human erythrocytes. At the same time,
extracts of roots and inflorescences showed specific activity. The authors
found that the albumin fraction showed no activity, while the globulin
and gliadin fractions of the proteins reacted with extracts of stems, non-
blossoming inflorescences, leaves, flowering inflorescences and roots (by
the degree of activity decrease). A positive reaction was obtained in almost
all variants with the use of rat erythrocytes, which indicates the specificity of
the Echinacea lectin receptors.
20.3 MATERIALS AND METHODS
As a plant raw material, the aboveground and underground parts of the E.

purpurea (L.) Moench (Figs. 20.1 and 20.2) and E. pallida (Nutt.) Nutt.
(Figs. 20.3 and 20.4) were used. Evaluation of lectin activity was carried out
by setting the hemagglutination reaction in immunological plates (Lutsik
et al., 1981). For this, 0.05 ml of physiological solution or buffer mixtures
were added to each well of the plate, then 0.05 ml of extract was added and
the series of successive two-fold dilutions were prepared. After that, 0.05 ml
of a 2% suspension of washed red blood cells was added to each well and
the plate was left at 25°C for 2 h. The evaluation was carried out visually on
a five-point scale (Golynskaya et al., 1992):
• 3 points—sharply expressed agglutination. Erythrocytes in the form

of a thin film more or less evenly distributed at the bottom of the
well;
• 2 points—moderate agglutination. Erythrocytes diverge on the bottom
of the well at a distance of more than 2 mm in diameter, forming a
ring with sharply expressed granularity at the edges;
• 1 point—weak agglutination. Erythrocytes diverge on the bottom of
the hole at a distance of less than 2 mm, forming a ring or disk;
• 0.5 point—minimal agglutination. A small clearance appears in the

center of the aggregate of erythrocytes, which have settled on the
bottom of the well;
• 0 point—no agglutination. Erythrocytes accumulate in the center of
the well.
After a visual assessment of the agglutination in each well of the dilution

series, the sum was counted in all wells where the reaction was determined.
Thus, the maximum activity in eight wells can be 8 × 3.0 = 24 points.
FIGURE 20.1 Echinacea purpurea of the first year of vegetation.
FIGURE 20.2 E. purpurea of the second year of vegetation.

FIGURE 20.3 Echinacea pallida of the first year of vegetation.
FIGURE 20.4 E. pallida of the second year of vegetation.
Carbohydrate specificity was determined according to the method

developed by us earlier (Golynskaya et al., 1992). The preparation of lectins
with a constant titer (1:4) was placed in immunological plates, mixed with
equal volumes of carbohydrates in the series of their successive two-fold
dilutions. The system had been incubated for 1 h at room temperature, after
which a volume, equal to the initial volume of lectins, of 2% suspension
of triply washed red blood cells was put into each well and left for 1–2 h
at a temperature of 25°C. Similarly, the hemagglutination reaction with
lectins was carried out without the addition of an inhibitory carbohydrate.

To estimate the specificity, the sum of the activity of the lectins without the
carbohydrate inhibitor was calculated, then with the carbohydrate, and the
affinity of lectins to carbohydrates was determined by the difference. In this
case, the larger the difference, the higher the specificity.
The statistical evaluation according to Student’s t-test was carried out
using the analysis package in the Excel program.
20.4 DEVELOPMENT OF METHODS FOR DETERMINING THE

ACTIVITY OF LECTINS
The ambiguity of the obtained data stimulated us to improve the technique for
determining the activity of Echinacea lectins. It is known that fruits and seeds
of plants contain the maximum amount of lectins (Lutsik et al., 1981). We
studied the activity of fruit extracts after their fractionation. It was determined
by us step by step in the process of the stepwise low-temperature ethanol frac-
tionation up to 20, 35, 50, and 76% the final concentration (Pospelov, 1998).
We found that the optimal conditions for fractionation were saturation
of the extract with ethanol to 20% final concentration, bringing the solution
to pH 8.0, cooling and centrifugation (Table 20.1). The resulting sediment
showed high activity in the reaction with erythrocytes of different human
blood groups in the ABO system: with the first O(I) group it was 16 points,
A(II)—15 points, and AB(IV)—11 points.
TABLE 20.1 Hemagglutinating Activity of the Fruit Extract of Echinacea purpurea at the
Different Stages of Ethanol Fractionation.
Stages of fractionation Activity, points
Extraction with physiological solution 0,0
Sediment after 20% saturation, pH = 3.0 14,0 ± 0,9
Sediment after 20% saturation, pH = 8.0 22,0 ± 1,2
Supernatant at 20% saturation 0,0
Sediment after 35% saturation 0,0
According to the described method by Golynskaya et al. (1992), we

evaluated the interaction of partially purified lectins of leaves, stems, and
rhizomes with roots of E. purpurea with carbohydrates arabinose, glucose,

galactite, xylose, galactose, and fructose. At the same time, inhibition of the
agglutination reaction was practically not observed.
Antonjuk and Rybak (2002) isolated and received lectins of E. purpurea
with a high degree of purification, studied their carbohydrate specificity. The
authors concluded that lectins of Echinacea can be attributed to the group
of mannose-specific ones, although they differ from lectins of monocotyle-
donous and dicotyledonous, in particular, legume family, which also belong
to this group. The yield of the purified product is 133 mg/kg of air dry raw
material.
In our experiments, we used physiological saline as an extractant, and
the further evaluation was carried out with using a phosphate-citrate buffer
mixture on the basis of physiological saline (Table 20.2). The results indicate
that Echinacea lectins show their activity in the acidic zone in our experi-
ments at pH = 4.0–4.4. In this case, certain regularities can be traced. When
extracting protein compounds from the stem, complete and partial lysis at
pH = 4.0–4.2 and a well-defined location of red blood cells in the wells of
the plate at pH = 4.4 are observed. When extracting protein compounds from
the rhizomes, the lysis was only at pH = 4.0. At the same time, the lectins of
the leaves were well defined in the acidic zone (pH = 4.0–4.4).
TABLE 20.2 Dependence of Hemagglutinating Activity of Lectins on pH of the Solution.

Terms of determination Activity, points
Leaves Stem Rhizome
Physiological saline + PCBM, pH = 4.0 9,0 ± 0,7 L.comp. Lysis
Physiological saline + PCBM, pH = 4.2 9,0 ± 0,6 L. part. 3,0 ± 0,3
Physiological saline + PCBM, pH = 4.4 5,5 ± 0,2 7,5 ± 0,3 1,5 ± 0,1
Physiological saline + PCBM, pH = 4.6 0,0 0,0 0,0
Physiological saline + PCBM, pH = 5,0 0,0 0,0 0,0
Note: L. comp.—lysis is complete; L. part.—lysis is partial.
Evaluation of the results visually in scores allows you to quickly, objec-

tively, and accurately determine the intensity of the hemagglutination reaction.
The well-known Ukrainian lectinologist, Yevgenia Golynskaya, widely applied
this method in her studies, popularized it in every possible way and considered
it more accurate than the titre estimation (Golynskaya et al., 1992). In addition,
the obtained data can be compared and statistically estimated, which is also
very important. Table 20.3 shows the data of the activity evaluation by the
agglutination titer and in scores. The agglutinating activity of the extracts of
the E. purpurea inflorescence varies from 4.5 to 6.0 in the experiment, while
at the same time it is 1:8–1:16 by the agglutination titer. E. pallida extracts
have a high activity—20.5–24.0 points and a titer—1:256. Evaluation of the
results of the experiment on the agglutination titer does not allow to carry out
statistical calculations, at the same time, as the estimation in points allows to
compare experiment variants and to make judgments about the reliability of
the obtained data.
TABLE 20.3 Echinacea Lectin Activity and Their Statistical Evaluation.

Variants Inflorescences of Inflorescences of
Echinacea purpurea Echinacea pallida
Titer Evaluation Titer Evaluation
evaluation in points evaluation in points
Repetitions of the experiment: 1 1:8 4,5 1:256 20,5
2 1:16 6,0 1:256 21,0
3 1:16 5,0 1:256 22,0
4 1:8 4,5 1:256 24,0
Average – 5,0 – 21,9
Dispersion – 0,5 – 2,39
t0.01 (theor.)= 3,18*
t0.01 (act.) = 17,47
*
The difference is reliable if the value of t0.01 (actual) is more than t0.01 (theoretical).
Thus, it can be concluded, that the use of a phosphate-citrate buffer

mixture with pH = 4.4, prepared on the basis of physiological saline should
be considered as the optimal conditions for assessing the lectin activity of
Echinacea extracts (Pospelov, 2012).
20.5 ACTIVITY OF LECTINS AND THEIR DYNAMICS IN

ECHINACEA PURPUREA
An analysis of the available literature showed us that, despite the established

fact of the presence of lectins in E. purpurea, many aspects remain poorly
understood. In Table 20.4 we summarize data on the detection of lectins in
different parts and organs of Echinacea and the method of their evaluation.
It can be concluded that the results of the research are ambiguous, and the
authors themselves find them difficult to interpret. So, the employees of the
Taras Shevchenko National University of Kyiv (Pohorila et al., 1997) found
that lectins in extracts of leaves, stems, and achenes are not determined with
the help of human erythrocytes but roots and inflorescences gave a positive
reaction. At the same time, according to (Antonjuk and Rybak, 2002), human
erythrocytes do not react to lectins contained in the root system of Echinacea.
The situation is similar for the extracts of achenes in our studies, lectin activity
was registered (Pospelov, 1998; Pospelov et al., 2001), but according to the
data of the Kiev authors (Pohorila et al., 1997), it was not noted.
At the same time, certain regularities can be traced. Due to the presence
of the receptors of a certain type, the erythrocytes of animals (rat, mouse,
and rabbit) are more suitable for determining the activity of Echinacea
lectins. Human erythrocytes are more unstable and do not always give a
positive reaction, which may be associated with the technique, the condi-
tions of their storage, the preparation, and quality of the raw materials,
and so forth. It should be noted that the authors (Kondrotas et al., 1999;
Antonjuk and Rybak, 2002) are unambiguous in their opinion that the
erythrocytes of the human blood group B(III) react more specifically to
lectins of Echinacea. However, a more detailed research of carbohydrate
specificity (Antonyuk and Rybak, 2002) does not give us the right to state
this definitively yet.
TABLE 20.4 Results of the Study of the Activity of E. purpurea Lectins.

Object of study Receptors (erythrocytes Positive (+) or negative References
of human or animal) (−) reaction
Roots Human О(І) + Pohorila et al.
Human А(II) + (1997) Antonjuk
Human В(III) + and Rybak (2002)
Mouse +
Human −
Rabbit +
Inflorescences Human О(І) + Pohorila et al.
Human А(II) + (1997)
Human В(III) +
Mouse +
Leaves Human О(І) − Pohorila et al.
Human А(II) − (1997) Antonjuk
Human В(III) − and Rybak (2002)
Mouse +
Human −
Rabbit +
Object of study Receptors (erythrocytes Positive (+) or negative References

of human or animal) (−) reaction
Stems Human О(І) − Pohorila et al.
Human А(II) − (1997)
Human В(III) −
Mouse +
Fruits Human О(І) − Pohorila et al.
Human А(II) − (1997) Pospelov
Human В(III) − (1998) Antonjuk
Mouse + and Rybak (2002)
Human О(І) +
Human А(II) +
Human АВ(IV) +
Human −
Rabbit +
Extract of Human О(І) − Kondrotas et al.
Echinacea* Human А(II) − (1999)
Human В(III) +
Human АВ(IV) −
Rabbit −
Guinea pig −
Mouse +
Frog −
*
—not specified from which part of the plant.
The absence of system data on the dynamics of lectin accumulation at

different stages of the ontogenesis of E. purpurea prompted us to study this
issue (Pospelov and Pospelova, 2012). Figure 20.5 shows the evaluation of
the activity of lectins in extracts from different parts and organs of the E.
purpurea of the first year of vegetation. There is a general tendency of a high
level of hemagglutinating activity of extracts of blossoming inflorescences,
as well as rhizomes with roots and stems (from 4 to 8 units). The average
level was characteristic of the leafstalks of the leaves and of the developing
inflorescences (from 2 to 6 units) and in the leaf blades, the activity level of
the lectins did not exceed 0.5 units. At the end of the first year of vegetation,
there is a slight decrease in the activity of phytolectins.
A more detailed analysis of the lectin activity in the leaves made it
possible to reveal certain regularities (Fig. 20.6). If in the leaf blades of all
the studied leaves the activity of lectins was minimal during the vegetative
period (0.5 points) then in the leafstalks their level increased several times,
especially in the rosette leaves (3.0–4.5 points). This leads to a thought that
the leaf blade is the main place of the biosynthesis of lectins, which are
then transported through the vascular system of the leafstalks. Considering
the fact that E. purpurea contains a significant amount of polysaccharides
(Samorodov et al., 1996) as well as the ability of lectins to reversibly bind
to carbohydrates, the transport of lectin-polysaccharide complex is quite
possible.
FIGURE 20.5 (See color insert.) Dynamics of activity of lectins of E. purpurea of the
first year of vegetation. I—root system; II—leaf blade; III—leafstalk; IV—stems; V—not
blossoming inflorescences; VI—blossoming inflorescences. Sampling time: 1 June; 2 July; 3
August; 4 September; 5 October.
The study of the dynamics of the activity of lectins in parts and organs
of the E. purpurea of the second year of vegetation makes it possible to
draw the following conclusions (Fig. 20.7). Hemagglutinating activity of
extracts of leaf blades and leafstalks was low, in the range of 0.5–3.5 points.
In a wider range, it changed in developing inflorescences (0.5–5.0 points).
Stably high activity of lectins was in the roots with rhizomes, stems, and
blossoming inflorescences (5.5–9.0 points).
FIGURE 20.6 (See color insert.) Dynamics of activity of lectins in leaves of E. purpurea
of the first year of vegetation. Rosette leaves: I-blade; II-leafstalk; Stem leaves: III- blade;
IV-leafstalk. Sampling time: 1-June; 2-July; 3-August; 4-September; 5-October.
FIGURE 20.7 Dynamics of activity of lectins of E. purpurea of the second year of

vegetation. I—rhizome with roots; II—leaf blade; III—leafstalk; IV—stems; V—not
blossoming inflorescences; VI—blossoming inflorescences. Sampling time: 1—renewal of
vegetation; 2—regrowth; 3—formation of inflorescences; 4—flowering; 5—fruit formation;
6—ripening of fruits.
Figure 20.8 presents data on the evaluation of hemagglutinating activity

of Echinacea leaves. It can be concluded that in Echinacea of the second
year of vegetation, rosette leaves are the main place of the synthesis of
lectins—their activity in plates increased from April to June (0.5–1.5 points)
and decreased to zero at the end of vegetation (Fig. 20.8, I). In the leafstalks,
hemagglutinating activity was the highest at the beginning of the growing

season—3.5 points and in the subsequent samples, it was 1.5–2.0 points
(Fig. 20.8, II). In the stem leaves, lectins were not detected (Fig. 20.8, III
and IV). Only in August, there was little activity in leaf blades. However, it
would be a mistake to talk about their absence. More likely, our methods are
not sensitive enough to detect the activity of phytolectins.
FIGURE 20.8 (See color insert.) Dynamics of activity of lectins in leaves of E. purpurea
of generative period of ontogenesis. Rosette leaves: I—blades; II—stems; Stem leaves: III—
blade; IV—leafstalk; Sampling time: 1—renewal of vegetation; 2—regrowth; 3—formation
of inflorescences; 4—flowering; 5—fruit formation; 6—ripening of fruits.
20.6 ACTIVITY OF LECTINS AND THEIR DYNAMICS IN

ECHINACEA PALLIDA
The protein complex of E. pallida was not studied enough, which served us
as a basis for studying lectins in its samples. If there is information about the
presence of lectins in E. purpurea, then we have not found such information
for E. pallida. In this connection, the purpose of the present studies was to
study the activity of lectins in different parts and organs of E. pallida during
ontogenesis (Pospelov, 2013).
For 3 years we had been conducting systematic take samples of E. pallida
of the first year of vegetation. The results of their analysis are shown in
Figure 20.9. The evaluation of the activity of lectins in leaf blades (Fig. 20.9,
II) shows a sufficiently high level during the entire growing season. In young
blades, activity averaged 12.0 points, with time it increased and in October
reached 16.0 points. An analogous regularity was also characteristic of
leafstalks (Fig. 20.9, III). In July, the agglutination activity was minimal—9.0

points and at the end of the vegetation, it increased to 17.0 points. Determi-
nation of activity in the underground part showed that in young rhizomes
with roots lectins were not detected, and only in October their content was
estimated at 2.0 points.
FIGURE 20.9 (See color insert.) Dynamics of activity of lectins of E. pallida of the
first year of vegetation. I—root system; II—leaf blade; III—leafstalk; IV—stems; V—not
blossoming inflorescences; VI—blossoming inflorescences. Sampling time: 1 July; 2 August;
3 September; 4 October.
For E. pallida transition to the generative period of ontogenesis, as a

rule, occurs in the second year of vegetation (Samorodov and Pospelov,
1999). At the same time, in the conditions of Poltava region, a small number
of plants blossomed at the end of the first year, in October. We used them
for our research. It should be noted that the activity of lectins in stems and
developing inflorescences was relatively high—12.5 points. An even higher
level was noted in the blossoming baskets—14.0 points (Fig. 20.9).
The obtained data allow to draw a conclusion that in plants of the first
year of vegetation, the leaves act as the main place of synthesis and local-
ization of lectins in the plant. Young rhizomes with roots only accumulate
phytolectins to a small extent at the end of vegetation, they apparently enter
the stems and inflorescences together with other necessary substances and
subsequently localize there. Thus, the overground mass at the end of the
growing season accumulates a significant amount of lectins and is of interest
as a source of raw materials of these specific protein compounds.
We also determined the presence of lectins in the samples of E. pallida

of the second year of vegetation. The study of the dynamics of their activity
has been carried out by us for 3 years, indicates a greater accumulation in
the overground part in comparison with the rhizomes and roots. Figure 20.10
shows the change in the activity of lectins in the rosette leaves of E. pallida.
During the period of regrowth—the beginning of flowering (May–June) the
agglutinating activity of extracts of leaf blades was 5.5–6.0 points. Later
it increased to 21.0–24.0 points. Similarly, the activity of lectins in the
leafstalks also changed. If in the May samples it was 7.0 points, then in
June to September, at the level of 18.0–24.0 points. This leads to a thought
that in May to June lectins are actively synthesized in leaves and through
the leafstalks are transported to other parts of the plant. In the subsequent
(July to August) simultaneously with the synthesis, processes of agglutinin
accumulation in the leaves takes place.
FIGURE 20.10 (See color insert.) Dynamics of activity of lectins in rosette leaves of
E. pallida of the second year of vegetation. Sampling time: I—regrowth; II—formation of
inflorescences; III—flowering; IV—fruit formation; V—ripening of fruits.
Similar regularities are also characteristic for stem leaves (Fig. 20.11). In
spring, during the growth, the activity of lectins was 9.5 points in leaf blades
and 10.0 in leafstalks. From the beginning of flowering to the end of vegeta-
tion, the agglutinating activity of the extracts increased from 18.0–19.5 points
to 21.0–24 points. It is known that a specific feature of lectins is the ability
of reversibly and specifically bind to carbohydrate ligands, which makes it
possible to move agglutinins through the plant (Lutsik et al., 1981; Ignatov,
1997). It is highly possible that the polysaccharide complex of Echinacea
can not only perform a transport function in relation to lectins but also bind
to them, accumulating in parts and organs of the plant.
FIGURE 20.11 (See color insert.) Dynamics of activity of lectins in stem leaves of
E. pallida of the second year of vegetation. Sampling time: I–regrowth; II–formation of
inflorescences; III–flowering; IV–fruit formation; V–ripening of fruits.
For Echinacea species, the polysaccharide complex is an important link

in the complex of biologically active substances. It is believed that heter-
oxylan, arabinogalactan, and arabinogalactan, fructose-containing polysac-
charides (inulin) and others are responsible for the immunomodulating and
anti-inflammatory properties of Echinacea. Deserves the attention works of
Vasfilova and Bagautdinova (2011) who conducted original studies of E.
pallida for the presence of fructans in it. The authors found that the under-
ground part of E. pallida contains a significant amount of oligo- and poly-
fructans. In connection with the recently isolated arabinogalactan-proteins
(AGPs) compounds and their properties (Showalter, 2001) the study of
lectin-polysaccharide complexes of Echinacea can be developed.
The agglutinating activity of the extracts of the stems was detected from
the moment of their formation (Fig. 20.12). In May, activity was minimal
(12.0 points), later it increased and reached its maximum at the end of
vegetation (20.0–21.0 points).
Interesting regularities were revealed when studying the dynamics of
lectin activity of inflorescences (Fig. 20.13). In Echinacea, inflorescences,
located on the main shoot, begin to blossom first, and then on lateral shoots.
In the conditions of Ukraine, E. pallida massively blooms in June, but sepa-
rate inflorescences can be formed until August. It was found that the activity
of lectins in the forming baskets in June is maximal and is 19.5 points, in
inflorescences, which were formed in July—14.0 points, and in August— it
was not detected. The opposite relationship was noted by us when assessing
the level of activity of phytolectins in flowering inflorescences. In the
baskets selected in June, lectins were not detected and the activity of lectins
in the blossoming inflorescences (July) was 21.5 points. During the period
of fruit formation (August) agglutination activity reached a maximum of 24

points. It should be noted that when determining the activity of phytolectins
in fruits, extracted from baskets in August and September, it did not exceed
4.5 points. Thus, the physiological role of lectins in stems and inflorescences
is still unclear, when the maximum number of lectins accumulates at the end
of vegetation, while the aboveground mass actually dies.
FIGURE 20.12 (See color insert.) Dynamics of activity of lectins in stems and rhizomes
of E. pallida of the second year of vegetation. Sampling time: I—regrowth; II—formation of
inflorescences; III—flowering; IV—fruit formation; V—ripening of fruits.
FIGURE 20.13 (See color insert.) Dynamics of activity of lectins in inflorescences of E.

pallida of the second year of vegetation. I—date of inflorescences forming; II—blossoming
inflorescences Sampling time: 1 June; 2 July: 3 August.
The study of rhizomes with roots of E. pallida testifies to the absence

of lectin activity in samples taken in May, June, and July (Fig. 20.13). The
maximum activity was registered by us during in August (21.0 points) the
smaller in September (20.0 points).
20.7 CONCLUSION
As a result of the studies, a new technique for conducting mass analysis for
determining hemagglutination activity of Echinacea extracts is proposed.
The raw material should be extracted with physiological solution and
further evaluation should be carried out with using a phosphate-citrate buffer
mixture (pH = 4.4) prepared on the basis of physiological solution with the
help of human red blood cells, and the activity should be determined visually
in points.
As a result of the studies, the activity of E. purpurea lectins during
different periods of ontogenesis was studied. The results of many years
of the research allow us to conclude that they accumulate most of all in
roots with rhizomes (6.0–9.0 units), stems (5.5–7.0 units), inflorescences
(2.0–6.0 units).
It is established that the basal leaves blade a major role in the formation
of the lectin pool of the plant. The peculiarities of accumulation of lectins in
stems and root system lead to a thought that there is a lectin-polysaccharide
complex, which ensures the necessary functioning of hemagglutinins in
the plant.
Certain regularities of changes in the activity of lectins in the ontogenesis
of E. pallida have been established. In the first year of vegetation in young
plants, activity was low, as they grow and develop it increased in leaves
to 16.0 points, in generative stems to 14.0 points. In rhizomes with roots,
phytolectins are found at the end of the growing season.
In the second year of vegetation, beginning with flowering, high activity
of lectins is characteristic for rosette (21.0–24.0 points) and stem leaves
(18.0–24.0 points). The peak of hemagglutinating activity of extracts of
stems and inflorescences occurred at the end of vegetation (20.0–21.0 and
24.0 points, respectively), and rhizomes with roots in August to September
(20.0–21.0 points).
The aboveground part of the E. pallida contains a considerable amount
of lectins and can be a source of raw materials of these unique protein
compounds, which opens the possibility of bioconversion of wastes that are
formed on seed crops and after the plantation is eliminated.
KEYWORDS
•• lectins
•• hemagglutination
•• purple coneflower
•• pale purple coneflower
•• Echinacea purpurea (L.) Moench
•• Echinacea pallida (Nutt.) Nutt.
REFERENCES
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INDEX
1 A
1,3,5-trihydroxybenzene, 27 Aaptamine, 52, 53
10-hydroxycamptothecin, 50 Aaptossu beritoids, 53
14-bromoreticulatate, 49 Abiotic, 72, 132, 134, 175, 280, 394
1-naphthaleneacetic acid, 150 Absorbance, 74–84
Absorption, 73, 74, 80, 153, 205, 206, 235,
2 249, 283
2,2-diphenyl-1-picrylhydrazyl, 72 Acanthella carteri, 57
2,3,6-tribromo-1H-indole, 11 Acanthicifolin, 11
2,4-dinitrophenylhydrazine, 77 Acanthus illicifolius,, 11
2,5-dibromo-1-methyl-1H-indole, 11 Acaudina molpadioides, 19
Accelerated solvent extraction (ASE), 95
3 Acetic acid, 139, 170, 400
Acetylcholine-esterase, 21, 52
3,5,6-tribromo-1H-indole, 11 Achenes, 429
3,5-dibromo-1-methyl-1H-indole, 11 Acridine
3-alkyl piperidine, 51 alkaloids, 54
alkaloidal, 55 moiety, 55
3-bromofascaplysin, 49 ring system, 54
Acrolein, 55
4 Actinobacteria, 7
4-hydroperoxycyclophosphamide (4-HC), Activator protein 1 (AP-1), 214
400 Activity testing protocol, 25
Adaptation, 13, 141, 268, 293, 296
α Adenocarcinoma pancreatic cells, 48
α-carotene, 69 Adenosine
α-tocopherol, 13, 69, 77, 78 receptors, 22
α-tocopheroxyl radical, 13 triphosphate (ATP), 17, 58, 298, 299,
332, 357
β Adherence, 25, 118
Adipocytes, 26
β-carotene, 68–70, 261 Adsorption, 172, 375, 376, 378, 385
β-catenin, 398 Aegiceras corniculatum, 11
Affinity, 90, 299, 378, 426
γ Agar, 15, 168, 173, 354
γ-EC synthetase, 295 Agelas
dispar, 57
δ longissima, 56
Agglutinating activity, 422, 428, 435, 436
δ-lactone, 51
Agglutination, 423, 424, 427, 428, 434, 437
Agglutinin, 435
442 Index
Aging, 12, 23, 27, 70, 72, 253, 260, 261 Anemones, 11
Agitation, 115, 142, 146, 156, 158, 159, 164 Angina pectoris, 205
Aglycones, 171, 198, 397 Angiogenic phenotype, 17
Agrobacterium, 155, 188, 190, 199 Angioneurotic edema, 19
rhizogenes, 155, 199 Angiosperms, 5, 295
tumefaciens, 155, 199 Angiotensin converting enzyme (ACE), 19
Agrochemicals, 6, 131, 132, 319 Annonaceous acetogenins (ACGs), 312
Ailanthus triphysa, 401 Antheraxanthin, 69
Ailments, 8, 14, 72, 209, 254 Anthocyanidins, 13, 171
Ajmaline, 10, 381 Anthocyanins, 12, 68, 254
Albizia julibrissin, 400 Anthranilate, 11
Albumin, 423 Anthraquinones, 6, 134, 135, 147, 153, 192,
fraction, 423 382–384
Alcohol, 9, 21, 78, 189, 348, 397 Antiaging, 27
Aldehyde Antiallergic, 13, 14, 27, 68, 347
dehydrogenase, 398 activity, 27
substrate, 400 drug, 14
Algae, 4–6, 9, 10, 12–16, 19, 21, 22, 27, Antiangiogenesis, 58
65–70, 72, 94–96, 119, 380 Anti-angiogenic activity, 400
Algal meroterpenoids, 27 Antibacterial, 8, 21, 22, 28, 36, 56, 233,
Algicolous fungus, 11 330, 348, 360, 401
Alginate, 8, 15, 173, 200, 213, 246, 250 Antibiotic, 10, 191, 205, 209, 212, 235
Alginic acid, 8 activities, 27, 57
Alkaloids, 6, 9–11, 35, 36, 47–58, 134, 156, berberine, 10
192, 193, 199, 200, 268, 272, 288, 303, Anticancer, 8–10, 13, 15, 27, 35, 36, 47, 48,
312, 313, 327, 328, 331, 332, 355, 379, 51, 57, 58, 67, 198, 205, 210, 215, 235,
380, 382, 384, 401 288, 397–402, 415
Alkyl pyridine alkaloids, 50 activities, 10
Allergen, 208 activity, 11
Allergic dermatitis, 12 drug, 399
Allosecurinine, 145, 154 potentials, 47
All-trans-retinoic acid (ATRA), 400 Anticoagulant, 8, 16, 17, 211
Amines, 9, 18 Anticomplementary activity, 16
Amino Anticytotoxic, 13
acids, 9, 10, 18, 28, 36, 108, 230, 231, Antidiabetic, 20, 21, 25, 27
288, 297, 379, 383 activity, 20
group, 52 Antifeedants, 303, 313, 327, 328, 330
sugars, 15 Antifungal, 8, 13, 21, 36, 53, 55, 212, 233,
Ammonia, 9, 12, 55, 286 249, 330, 345, 348, 349, 357, 359
Ammonium nitrogen, 153 Antigen, 16, 398
Amoora rohituka, 401 Antihyperglycemic effect, 25
Amphetamines, 9, 10 Antihypertension actions, 28
Amphimedine, 51, 54 Antiinflammatory, 15, 21, 22, 26, 27, 36,
Amphimedon sp., 50, 51, 54 48, 206, 209, 211, 215, 235, 330, 345,
Ananas comosus, 205, 216, 232 347, 348, 398, 400, 402, 422, 436
Andrographis paniculata, 151, 397 modules, 22
Andrographolide diterpene, 397 Antileishmanial, 13
Anemia, 27 Antimalarial, 36, 49
Index 443
Antimicrobial, 10, 13, 15, 25, 27, 28, 48, Araguspongine, 55

49, 90, 97, 175, 215, 249, 268, 288, 344, Arena, 18
347, 349, 359–361, 400 Arenosclera brasiliensis, 51
activity, 25 Arnicolide, 397
peptides (AMPs), 19 Aroma, 20, 343
Antimitotic, 11 Aromatic
Antimutagenic, 8 compounds, 73
Antineoplastic, 10, 51 tropolone, 400
activity, 51 Aronia melanocarpa, 143, 149
Antioxidant, 8–10, 13, 14, 18, 19, 21, 23, 24, Arsenic hyperaccumulators, 300
27, 28, 65, 68, 70, 72–74, 81, 82, 94, 96, Artemisia annua, 136, 153, 173
97, 248, 255, 256, 259, 261, 288, 295, 400 Artemisinin, 153
activity, 13, 14, 21, 73 Arteriosclerosis, 22
Antiparasite bioassay, 55 Arthritis, 3, 23, 70, 210, 211, 234, 235, 260
Antiparasitic, 27, 401 Ascidians, 6, 11, 54
Antiplasmin inhibitor, 14 Ascophyllum nodosum, 68
Antiproliferative Ascorbate, 77, 84
effect, 399 Ascorbic acid, 24, 77, 383
potential, 397 Asetoposide, 51
Antithrombotic, 16, 28, 215 Aspergillus
Antitumor, 8, 16, 20, 22, 35, 51, 52, 57, flavus, 249, 350, 357
209, 214, 402 fumigatus, 350
indole alkaloids, 48 niger, 213, 350
quinolines, 52 ochraceous, 350
Anti-ulcer, 25 Astaxanthin, 68–71
Antiviral, 8, 21, 22, 27, 36, 54, 55, 57, 67, Asteraceae, 303, 315, 329, 333, 349, 353,
68, 233, 330, 345, 348 381, 422
activity, 8, 22, 54, 55 Asthma, 22, 23
drug, 67 Atherosclerosis, 16, 23, 72, 253, 260
Antivirus, 16 Athymic mice, 51
Apertures, 71 Atropa belladonna, 149
Aphanin inhibits, 401 Atropine, 9
Apiaceae, 20, 350, 351, 353, 356 Aurora globostellata, 73
Aplicyanins, 11 Auxin, 123, 139–141, 150, 153, 156, 173,
Apoptosis, 16, 24, 48, 49, 53, 55, 57, 209, 191, 196
213, 214, 231, 235, 397, 399, 401, 402 Avicennia officinalis, 15
Appetite, 20, 27 Avicides, 305
Aquatic Ayurveda, 14, 280, 344
environs, 7 Azadirachtin, 136, 143, 154, 157, 310, 311,
faunal, 7 329, 330, 355, 356, 383
life, 3
organisms, 7, 8, 10, 97 B
plants, 4 Bacillariophyta, 67
region, 3 diatoms, 5
Arabinogalactan-proteins (AGPs), 436 Bacteria, 5, 7, 13, 22, 25, 57, 66, 73, 121,
Arabinose, 427 175, 199, 223, 231, 330, 349, 356, 359,
Araceae family, 303 360
Arachidonic acids, 22 Bactericides, 305
444 Index
Bacteriostatic activity, 25 Biopesticides, 303, 305, 308, 317–319,

Baking industry, 207 325–328, 330, 336, 342
Balanced ecosystem, 4 Bioreactor, 108, 116, 127, 133, 134, 146,
Balms, 18 149, 150, 155, 156, 158–160
B-cell lymphoma 2 (Bcl-2), 24, 209, 213, Biosynthesis, 7, 10, 11, 72, 140, 141, 160,
401 163, 172, 173, 191, 193, 199, 293–296,
Benzoic acid, 66 298, 347, 349, 357, 394, 395, 431
Benzopyrene, 24 Biotic stress, 148, 394
Benzoquinones, 11 Biotransformation (BT), 128, 134, 153, 158,
Benzyl groups, 47 172, 174, 198, 199, 201
Betulinic acid, 397 Bisquinolinylpyrrole, 52
Biemna fortis, 51 Blood
Biemnadin, 51 cells, 8, 423, 425, 427, 438
Bilayer disorder, 357 groups, 232, 426
Bioactive, 72 lipids, 16
compounds, 6, 26, 35, 70–72, 89–91, 93, Blooms, 4, 436
94, 97–100, 109, 132, 147, 153, 169, Boiling, 80, 93, 95, 96, 165
254, 256, 258, 259, 303, 306 Bornyland isobornyl acetate, 199
ingredients, 70 Botanicals, 306–310, 315–319, 328, 335,
materials, 8, 94 336, 362
nitrogenous complexes, 28 Brassicaceae, 299, 350, 399
peptides, 18, 28 Brassinosteroids, 394
sterols, 22 Bromelain, 91, 205–216, 221, 223, 232–236
Bioactivities, 8, 13, 14, 20, 133 Bromopyrrole longamide, 56
Bioassay, 35, 54, 160, 172, 308, 309 Bromotopsentin, 49
Bioavailability, 18, 249, 250, 254, 261, 262, Bromotyrosine units, 56
296, 300 Bronchitis, 205, 212, 235
Biochemical, 6, 57, 58, 70, 71, 112, 147, Brown
158, 199, 300, 301, 342, 346 algae, 5, 14, 27, 67
Biochemistry, 6, 8, 110, 160, 194 seaweeds, 8
Biocontrol agents, 359 Brugine, 11
Biodiversity, 7, 65, 67, 89, 280, 309, 318, Bruguiera sexangula, 11
325 Bryozoans, 6, 7
Bioflavonoids, 9 Buffer mixtures, 423
Biogas, 21 Butanol, 74
Biological
active compounds, 65, 327 C
activities, 9–11, 13–16, 21, 36, 49, 90, Caffeic acid (CA), 223, 227, 228, 230, 249
268, 274, 288, 344, 347, 397 Caffeine, 10, 50, 54
amines, 10 Calcarea, 72
Biologically active compounds, 35, 66, 71, Calcium, 8, 20, 27, 72, 78, 121, 173, 206,
93, 256, 258, 286 333
Biomarkers, 215, 300 binding, 20, 27
Biomass, 7, 66, 71, 73, 94, 131, 133, 135, chloride, 200
140, 141, 146–150, 153, 155, 160–162, Calendula officinalis, 157
166, 173, 175, 176, 193, 298, 299 Callus, 115–117, 126, 135, 139–142,
Biomedical agents, 73 147–150, 153, 156, 162, 163, 167–169,
Biomolecules, 23, 316 190–196, 198, 200
Index 445
cultures, 143, 161 Catechin, 12–14, 68, 80, 171, 399

Callyspongia Catechol
clavata, 72 oxidase, 76
siphonella, 72 solution, 76, 79
Camptotheca acuminata, 10 Catharanthine, 146, 200
Camptothecin, 10, 50, 154 Cattle poisoning, 15
Cancer, 3, 7, 12–14, 18, 22–24, 26, 35, 36, Cavitation, 255
49–51, 54–56, 70, 72, 95, 96, 109, 125, Cell
128, 210, 211, 213–215, 254, 260, 261, adhesion, 16, 26, 214
393, 394, 397–402 cellular antioxidant, 18
apoptosis, 24 concentration, 120, 164, 167
cell, 24, 26, 56, 210, 214, 215, 393, 394, counting chamber, 163, 164
397, 398, 401, 402 culture, 18, 110, 117–121, 128, 135, 142,
stem cells (CSCs), 192, 199, 200, 393, 162, 166, 174, 175
398–400, 402 cycle, 24, 26, 49, 52, 213, 231, 397, 401
therapy, 22 death, 55, 163, 165, 176, 213, 214, 332,
Candida 357
albicans, 53, 212 division, 50, 123, 140, 148, 163, 164,
utilis, 8 166, 331
Capillary electrophoresis coupled with mass growth, 26, 112, 118, 121, 122, 134, 148,
spectrometry (CE-MS), 97 149, 167, 172, 175, 191, 214, 357
Capsaicin, 355, 356 measurements, 165
Captopril, 19 line, 49, 51, 53, 56–58, 119, 127, 128,
Carbohydrate, 94, 243, 422, 425–427, 429, 160, 172, 400–402
435 membrane, 23, 25, 298, 299, 332, 349,
Carbon, 9, 53, 67, 121, 132, 146, 158, 173, 357, 361, 402
229, 284, 286, 298, 344, 377, 394, 397 mobilization, 17
Carbonic anhydrase 2, 393 reinforcement, 16, 18
Carcinogenesis, 23, 397 signaling pathways, 36
Carcinogens, 24 stemness, 394
Carcinoma, 13, 16, 26, 51, 52, 55–58, surface, 17
213–215, 234, 398 suspension culture (CSC), 142, 148, 164,
Cardenolide, 200 188, 191, 193, 398–400
Cardiac ischemia, 23 toxicity, 21
Cardiovascular viability, 161, 163–165
illness, 19 volume, 165
maladies, 19 after sedimentation (CVS), 165
mortality, 68 wall, 8, 12, 15, 16, 67, 92, 94, 175, 360
protection, 20 Cellular
Caribbean marine sponge, 57 destruction, 23
Carnosic acid, 154 functions, 16
Carnosol, 138, 145, 154 protein content, 163
Carotenoid, 24, 68, 70, 171, 398 Cellulase, 147, 164
Carrageenan, 8, 15, 382 Cellulosic plant, 15
Carvacrol, 397 Cenchritis muricatus, 20
Caspase-3 stimulation, 16 Centipede minima, 397
Catalase, 23, 73, 74, 298 Central nervous system, 9, 333
Catalytic metal sites, 73 Centrifugal impeller, 159
446 Index
Centrifugation, 75, 77, 81, 118, 245, 426 Clathrina, 47

Cereals, 12, 190, 208 Climate change, 270, 280, 281, 285, 287,
Cerebral, 23 289
Chamaecyparis taiwanensis, 400 Clonal
Chemical evolution, 398
composition, 68, 97, 286, 344, 349, 422 model, 398
compounds, 6, 67, 268 propagation (CP), 193, 194, 196, 221,
diversity, 65, 66 223
ecology, 72 Coagulation proteases, 16
reaction, 198, 199, 370 Cocaine, 10
Chemoprevention, 22, 402 Codeine, 10
Chemopreventive, 22, 23, 397, 399 Codium iyengarii, 22
Chemoresistance, 393 Coffea arabica, 200
Chemoresistant, 50 Colon cancer, 24, 397, 398
Chemotaxonomy, 71 cell, 397
Chemotherapeutic mediators, 22 Colorectal cancer, 399
Chemotherapy, 36, 209, 213, 361, 393, 400 Commercialization, 24, 312, 336, 362
Chenopodium ambrosioides, 350 Conjugation, 75, 171
Chitosan (CS), 249 Conventional extraction, 89, 90, 92, 100
Chlorella Coronary heart disease, 13, 70
protothecoides, 70 Corrosion inhibitors, 373, 375–377, 385
pyrenoidosa, 69 Corymbia citriodora, 356
vulgaris, 69, 70 Cosmetic industry, 66
Chlorogenic acid, 12, 379, 381 Coumaric acid, 154
Chlorophyll, 5, 17, 18, 67, 77, 80, 81, 298, Crassostrea gigas, 19
394 Crown gall, 156
Chlorophyta, 5, 9, 67 cultures, 156
Choanephora cucurbitarum, 352 Crude phlorotannins, 14
Cholesterol, 8, 25, 244 Cryptophyta, 67
Chondroitin sulfate, 17 Crystallization, 246, 255
Chorismate, 11 Cultivation period, 162
Chromium, 164, 296 Culture period, 141, 146, 150, 164
trioxide, 164 Cuminum cyminum, 249, 351, 357
Chromogen, 74 Curcumin, 399, 400
Chronic Cuvette, 75, 76, 78
diseases, 23, 254 Cyanobacteria, 5, 7, 96, 97, 295
lymphocytic leukemia, 399 Cyanophyta, 67
myeloid leukemia, 398 Cyclic
ulcer, 18 nitrogen, 10
Chrysanthemum cinerariaefolium, 135, 356 peptides, 36
Chrysophyta, 67 tetrapyrroles, 17
Cicuta virosa, 350 Cyclin, 57, 397, 401
Cinnamaldehyde, 25, 26 Cyclodercitin, 54
Cinnamic acid, 66 Cyclomixer, 77
Cinnamomum jensenianum, 350, 357 Cyclopamine, 400
Circulatory Cyclophosphamide, 400
blood pressure, 19 Cymbopogon martini, 351
systems, 71 Cystic fibrosis, 23
Index 447
Cytokine, 16, 17, 194, 209 Digitalis

Cytotoxic, 10, 11, 15, 16, 21, 22, 35, 47–52, lanata, 117, 149, 198
54–58, 209, 210, 397, 400–402 purpurea, 160
discorhabdins, 53 Digitoxin, 117, 160, 198, 200
guanidines alkaloids, 55 Dihydrodeoxybromo topsentin, 49
imidazoles alkaloids, 47 Dilution, 351
penta, 22 series, 424
pyridines alkaloids, 50 Dimers, 200
pyrimidine alkaloids, 57 Dimethylallyl pyrophosphate (DMAPP),
pyrroles alkaloids, 52 394
Cytotoxicity, 18, 22, 26, 48–53, 55–58, 398, Dimethylketal, 52
402, 415 Dinoflagellates, 67
Dinophyta, 67
D Diode array detector, 6
Daidzein, 14 Dioscorea, 15
D-D-gluconolactone, 24 Diosgenin, 15
Defoliants, 305 Dioxinodehydroeckol, 8
Dehydroascorbate, 77 Diphlorethohydroxycarmalol, 27
Dehydroxylation, 56 Discorhabdin alkaloids, 53
Demethyl(oxy)aaptamine, 52 Disease management, 359, 362
Demethylation, 199 Disodium cromoglycate, 14
Demospongiae, 72 Distillation, 20, 90, 91, 170, 343, 344
Deoxyamphimedine, 51 Distilled water, 76, 80, 162
Deoxytopsentin, 49 Diterpenes, 175, 345, 347, 394, 397, 402
Dercitamide, 54 Diterpenoids, 381, 401
Dercitamine, 54 D-mannose, 15
Dercitin, 54 Docosahexaenoic acid, 12
Desiccants, 305 Dosidicus gigas, 19
Desiccator, 161, 162 Downregulation, 16, 398
Destructive methods, 160 Dragmacidin, 49
Desulfated fucoidan, 17 Drug discovery, 6, 65, 67, 99, 109
D-fructose, 15 Dry weight, 70, 117, 132, 134, 135, 141,
D-galactosamine, 15 153, 155, 161, 192, 300
D-galactose, 15 Drying, 161, 244, 245, 248, 249, 254, 257,
D-glucosamine, 15 258, 261, 262
D-glucose, 15 Dunaliella
Diabetes, 12, 23, 125, 235, 248, 253, 260 salina, 69, 70, 95
Diabetic tertiolecta, 68
pregnancy, 23 D-xylose, 15
rats, 25 Dynamic, 17, 93, 96, 401
Dianisidine, 75 receptor collaboration, 17
Diarrhea, 14, 212, 234
Dicotyledonous, 155, 227, 427 E
Dieckol, 8, 14, 27 Echinacea, 151, 421–430, 432, 435, 436,
Dietary nitrites, 24 438, 439
Differentiation, 16, 120, 123, 135, 140, 141, pallida, 421–423, 425, 428, 433–439
148, 188, 190, 194, 200, 231, 393, 398, purpurea, 421, 422, 424, 426, 428, 439
400 Echinoderms, 6, 27
448 Index
Ecklonia cava, 14 Erythrocytes, 422–424, 426, 429, 430

Eckol, 8, 14, 27 Escherichia coli, 125, 212, 359
Eclonia kurome, 14 Essential oils (EOs), 20, 23, 26, 213,
Ecosystem, 66, 280, 307 247–249, 271, 315, 327, 328, 341, 342,
Eelgrass, 4 345, 358, 362
Eicosapentaenoic acid, 12 Estrogen, 24
Eisenia bicyclis, 8, 95 Ethanol, 75, 78, 80, 82, 95, 96, 165, 170, 426
Elastase, 27 fractionation, 426
Electron, 73, 100, 298, 332, 356 Ethnopharmacological drugs, 66
acceptor, 73 Eunicella granulate, 11
Electrophilic metabolites, 24 Euphorbia prolifera, 402
Elements, 5, 7, 10, 121, 122, 214, 224, 376 Evaporation, 20, 161, 166, 245
Eleutherococcus koreanum, 153 Evaporative light scattering detector, 6
Elicitors, 153, 175, 231, 312 Exogenous, 135, 153, 155, 158, 400
Embryogenic, 149, 193 Explant, 115, 126, 135, 140–142, 147, 149,
Emmerie–engel reaction, 77 150, 176, 189–191, 195, 196, 198
Enalapril, 19 Exploration, 5, 7, 58, 90, 242
Encapsulation, 241–245, 247–250, 306 Extracellular, 16, 163, 172, 173, 214
Endogenous phytohormones, 156 Extrusion, 246, 250, 261
Endoplasmic reticulum, 402
Endothelial F
cell, 17 Fabaceae, 294, 303, 329
progenitor cell (EPC), 17 Fascaplysin, 49, 57, 58
Entrapped cells, 200 Fascaplysinopsis
Environmental bergquist, 49
factors, 108, 120, 249, 267–269, 275, reticulate, 49, 72
276, 279–282, 286–288 Fatty acids, 9, 12, 36, 68, 70, 97, 345,
Protection Agency (EPA), 318, 342 382–384
Enzymatic Fauna and flora, 289
antioxidants, 74 Fermentation, 8, 233
hydrolysis, 20, 100 Ferrous ions, 77
inactivation rates, 256, 258 Fertilizer, 21
Enzyme, 14, 24, 74–76, 92, 120, 122, 147, Fibrin, 206, 211
156, 161, 164, 198, 205–208, 213, 215, Fibrinolytic action, 206
216, 224, 225, 229, 230, 232, 260, 286, Flagella, 67
298, 331, 394, 395, 398 Flavanols, 12
activity, 74 Flavanones, 12
assay, 75 Flavanonols, 12
Ephedrine, 10 Flavones, 12, 171
Epidermoid, 51, 56, 213 Flavonoids, 12–14, 23, 66, 68, 77, 96, 133,
Epifaunal diversity, 72 141, 151, 153, 170, 171, 174, 175, 242,
Epigallocatechin, 13, 14 248, 254, 258, 259, 268–270, 272, 274,
3-gallate (EGCG), 399, 400 286, 288, 303, 312, 313, 345, 379, 380,
Epi-manzamine D, 49 382–384
Epitope structure, 208 Flavonols, 12, 24
Epiutililactone, 397 Flora, 7, 14, 279, 318, 422
Epoxidations, 199 Florentine flask, 20
Ergotamine, 10 Fluorescein diacetate, 164
Index 449
Foeniculum vulgare, 20, 317, 351 regions, 25

Foodborne, 8 Ginseng roots, 150
Forage, 208, 282 Girolline, 48
Formazon, 74, 83 Giwieng, 15
Fractionation, 54, 426 Glacial acetic acid, 74
Fragments, 28, 210 Glandular hairs, 20
Frangula alnus, 153 Gliadin, 207, 423
Freeze drying (FD), 245, 258 Glioblastoma, 398, 399
Fresh weight (FW), 81, 161 Glioma cancer, 400
Fructose, 173, 427, 436 Globulin, 423
Fruits and vegetables, 253–257, 259–262 Glossy infinitesimal cells, 5
Fucoidan, 16, 17, 27 Glucose, 26, 122, 173, 427
mobilization, 17 transporter, 26
starch backbone, 17 Glucosinolates, 24, 260
Fucoxanthin, 5, 68, 95 Glutathione (GSH), 23, 24, 68, 73, 75, 77,
Fucus 79, 293, 295, 296
serratus, 69 S-transferases (GSTs), 24, 74, 75
vesiculosus, 69 Glycans, 15
Fungi, 7, 25, 223, 231, 232, 330, 347, Glyceraldehyde 3-phosphate, 395
349–354, 356, 359, 360 Glycitein, 14
Fungicides, 196, 303, 305, 328, 341, 342, Glycosaminoglycans (GAGs), 16
359 Glycosides, 20, 22, 170, 171, 198, 274, 314,
380, 382–384, 401
G Glycosylation, 198, 199
Gadus morhua, 20 Gotten, 15
Galactan, 15 Gracilaria, 11, 15, 21, 68
Galactite, 427 verrucosa, 11
Galactose, 15, 427 Gracilariopsis, 21
Gallic acid, 13, 66, 383 Growth
Gallotannins, 14 index, 162
Gamma-tocotrienol, 399 kinetics, 150
Gas chromatography (GC), 97, 100, 247 parameters, 148, 160
Gastrointestinal, 23, 232, 249, 399 rate, 119, 133, 140, 141, 149, 160, 162,
ulcerogenesis, 23 167, 199, 283, 303, 327
Gel filtration chromatography (GFC), 98, regulators, 15, 117, 123, 135, 138, 142,
99 145, 147, 152–154, 157, 158, 305, 330
Gelidium, 15, 21
Gemcitabine, 401 H
Gene, 22, 125, 156, 161, 188, 293, 295, Haematococcus, 69, 70, 97
300, 399, 401 pluvialis, 69, 70, 97
Genera, 15, 47, 72, 299 Hairy root
Genetic stability, 127, 153 culture, 155
Genistein, 14, 144, 154, 399, 400 phenotype, 155
Genotype, 13, 160, 192 Halichondriidae, 49
Genus, 21, 27, 48, 50, 52, 55, 311, 312, 356, Haliclona tulearensis, 52
422 Halitoxin, 50
Geographical Halitulin, 52
origin, 68 Halogenated, 10, 22
450 Index
Halomon, 22 hepatocellular liver carcinoma cell line,

Halophytes, 7 26
Hampers, 26, 214 immunodeficiency virus (HIV), 12
Haplosclerida, 53, 55 leukemic cells, 16
Heavy metal tolerance, 295, 296, 301 ovarian carcinoma, 57
Hecogenin, 15 papillomavirus, 24
Hedgehog signaling, 398, 400 pathogenic permanency, 12
Helical ribbon impeller, 158 platelet aggregation, 13
Helicobacter pylori, 25 umbilical vein endothelial cell (HUVEC),
Helper T-cells, 16 17
Hemagglutinating activity, 422, 426, 427, Hydantoin, 47, 48
430–433, 438 Hydrocarbon, 397
Hemagglutination, 421, 423, 425, 427, 438, Hydrocortisone, 15
439 Hydrogen, 9, 58, 73, 75, 81, 117, 121, 228,
activity, 421 229, 306, 348, 372, 373, 402
Hemagglutinins, 421, 438 peroxide, 74
Hemiterpenes, 394, 397 Hydrolysis, 14, 20, 90–92, 94, 170, 231,
Hemocytometer slide, 164 245, 332
Hemolysis, 15 Hydrolyzable tannins, 14
Hemorrhagic shock, 23 Hydrolyzing peptide bonds, 208
Heparan sulfate, 17 Hydroperoxides, 73
Hepatopathy, 16 Hydroxybenzoic acid, 12, 154
Hepatoprotective, 27, 248 Hydroxycinnamic acid, 12
Herbicides, 303, 305 derivatives, 68
Herbivores, 9, 12, 231 Hydroxyl, 12, 18, 23, 72, 81, 84, 347,
Herbivorous, 21, 268, 304, 306, 312 379–384
Hesperidin, 12 groups, 12, 379, 381, 382
Heterodimer, 401 radicals, 23
Heterogeneity, 133, 398 Hydroxylation, 198, 199
Heterogeneous, 67, 139, 140, 172, 398 Hyoscyamus muticus, 200
Heteroxylan, 436 Hyperaccumulation, 293, 300, 301
Hexactinellida, 72 Hyperaccumulators, 299, 300
High Hypericin, 144, 151, 154
density lipoproteins (HDL), 25 Hypericum perforatum, 144, 149
performance liquid chromatography Hypertension, 8, 19, 27, 234
(HPLC), 6, 170, 269 Hypnea valitiae, 22
diode-array detection (HPLC-DAD), Hypoglycemic effects, 26
95 Hypoxia, 399
pressure homogenization, 261, 262
Hinokitiol, 400 I
Homaxinella sp., 56 Iduronic acids, 15
Homeostasis, 22, 297 Imidazole, 36, 47, 48, 228, 229
Homogenate, 75, 76, 79, 80, 244, 246 alkaloids, 48
Homophytochelatins, 294 ring, 47, 228
Hordenine, 10 Immobilization, 156, 172, 242, 293
Hormonal imbalance, 156 Immobilized plant cells (IPC), 200
Human Immune system, 8, 209, 210, 215, 234, 288
colon, 49, 52, 58 Immunodeficiency syndrome, 3, 23
Index 451
Immunological plates, 423, 425 Iyengadione, 22

Immunomodulatory, 16, 28, 209, 215 Iyengarosides, 22
Indole, 10, 11, 36, 139, 228
3-acetic acid (IAA), 136, 139 J
3-butyric acid (IBA), 143, 150 Jasmonic acid, 201
3-carbinol, 24 Jelly capsules, 8
3-glycerol-phosphate, 11 Jumbo squid, 19
Infertility, 23, 124
Inflammation, 12, 13, 25, 26, 36, 70, 206, K
211, 214, 235, 268, 399, 402
Inflorescences, 421, 423, 429–438 Kandelia kandel, 11
Inhibitor, 22, 24, 54, 98, 116, 205, 313, 314, Kinase, 16, 22, 49, 56–58, 395, 400
331, 376–385, 402, 407–409, 426 Krasunya prerii, 422
Inhibitory activity, 56, 58
Inoculum L
size, 142, 146, 150, 153 Labuanine, 51
weight, 162 Laccase, 76
Inorganic corrosion, 375–377, 385 Lactones, 347, 349, 397
Insecticides, 5, 135, 303, 310, 313, Lamiaceae, 20, 315, 349, 350, 352–354, 356
315–318, 326–328, 330, 332, 334, 336 Laminaria
Insulin, 8, 26, 125 digitata, 356
Intensity, 26, 71, 74, 79, 93, 174, 231, 279, ochroleuca, 69
280, 283, 375, 427 saccharina, 69
Interleukin-1 beta, 16 Laminarin, 8
Intertidal zones, 21 L-arabinose, 15
Intracellular, 17, 93, 116, 163, 210, 227, Lateral branching, 155
357, 402 Latrunculia brevis, 53
Inulin, 436 Laurencia
Invertebrates, 5, 7, 66, 71, 333 brongniartii, 11
Ionic trace minerals, 9 decumbens, 11
Iotrochota purpurea, 56 similis, 11
Ischemic coronary illness, 19 Laurus nobilis, 351
Isoaaptamine, 52 Lavandula angustifolia, 20
Isobatzellines, 53 Leaf blades, 430, 431, 433, 435
Isoflavones, 12, 96, 141, 171, 329, 399 Leafstalks, 430–432, 434, 435
Isoflavonoids, 12, 142 Lectin, 268, 382, 421, 422, 425–439
Isolated activity, 421, 423, 428–430, 436, 438
isomers, 49 polysaccharide complexes, 436
topsentins, 49 Leguminous plants, 12
Isomeric manzamines, 49 Lethal diseases, 3
Isomerization, 174, 199 Leucetta, 47, 48
Isomers, 95, 379 chagosensis, 48
Isopentenyl pyrophosphate (IPP), 394, 395 Leucettamine C, 48
Isoprene, 345–347, 394, 395, 397 Leucorrhoea, 14
Isoprenoids, 394, 397 Leucosolenia, 47
Isoquinolines, 36 Leukemia
alkaloids, 52 cell lines, 397
Isothiocyanates, 24, 399 THP-1, 55
452 Index
L-galactose, 15 biotechnologists, 66
Ligand, 48, 293, 294, 378, 401, 402, 435 dispensation, 27
Lignans, 198, 199, 288 ecosystems, 4
Lignin, 12, 13, 383 environment, 4–6, 35, 58, 65, 66
Limanda aspera, 19 food chain, 5, 68
Limonene, 345, 346, 380, 381, 397 life, 18–20, 26, 66
Limonoid, 312, 401 metabolites, 36
Lipid, 12, 18, 66, 73, 210, 243, 244, 249, nitrogenous composites, 10
260, 298 organisms, 4–6, 9, 10, 20, 22, 27, 28, 35,
oxidation, 12 51, 65–67, 73, 74, 89, 94, 98
Lipopolysaccharide, 26 phytochemicals, 6, 13, 26, 89, 100
Liposomes, 244, 250 phytochemistry, 3
Lippia sidoides, 359 plants, 3–5, 10, 12, 13
Liquid chromatography (LC), 95, 170 red alga, 11
Lisinopril, 19 sediments, 4
Littoral
species, 4
habitats, 67
sponge, 35, 36, 47, 48, 50, 52, 56, 58
zone, 67
alkaloids, 47
Livestock, 284, 422
vegetation, 7
Longamide B, 57
Mass spectrometry (MS), 97, 99, 100,
Long-chain dialdehyde, 55
136–138, 142–145, 150–154, 157, 173,
Lozenge, 8
Lutein, 69 190, 191, 196, 247
Lycopene, 77–79, 260, 261, 398 detector, 6
Lymphocyte, 50, 209 Matrix, 27, 72, 94, 99, 156, 170, 173, 190,
Lymphocytic leukemia, 56 214, 243, 245, 246, 261, 332, 397
Lymphoma, 16, 24, 209 assisted laser desorption ionization
Lysine, 10 (MALDI), 99
metalloproteases, 27
M Maximum diameter (MD), 49, 169
Mechanically-agitated bioreactors, 158
Macroalgae, 7, 67, 68, 95–97
Medications, 7, 16, 19, 132, 234
Macrophages, 16, 26, 56
Medicinal plants, 21, 109, 132, 149, 150,
Malignant tumor, 36, 398, 400
Mammalian 153, 155, 188, 247, 267–270, 272,
cell cycle, 57 274–276, 279–282, 284, 285, 288, 289
systems, 22 Melanin, 27, 76
Mangrove, 7, 9, 11, 13 Melanoma, 16, 52, 55, 213, 397, 400
plant, 9 Meliaceae, 303, 329, 330, 401
Manzamine, 48, 58 Melliferous plant, 422
Marine Membrane filtration, 97, 98, 100
algae, 10, 67 Mentha
alkaloid, 10 arvensis, 350
animals, 4, 5 citrata, 158
antioxidants, 84 spicata, 352, 356, 357
bioactive Mesohyl, 71, 72
compounds, 26 Metabolism, 22, 24, 71, 99, 122, 132, 134,
peptides, 18 140, 283, 288, 293, 296, 299, 308, 347,
biodiversity, 4 397
Index 453
Metabolite, 22, 49, 65, 66, 72, 112, 117, Molecular

122, 133, 140, 146–148, 150, 153, 155, modeling, 57
165, 172, 173, 176, 187, 192, 199, 285, weight, 13, 14, 16, 97–99, 212, 299
286, 342 Molecule, 9, 26, 97, 212, 223, 229, 345,
Metal 376, 378, 402
chelation, 73 Molluscicidal, 15, 314
chelators, 13 Molluscicides, 303, 328
hyperaccumulation, 300 Mollusks, 6
ions, 294, 296, 371, 372, 377 Momentous, 70
Metalloproteinase (MMP), 214 Monanchocidin, 55, 56
Metallothioneins (MT), 294 Monanhora pulchra, 55
Metastasis, 16, 26, 96, 214, 215, 399 Monoacetate, 401
Methicillin, 8 Monocotyledonous, 427
Methyl Monocyclic, 346, 397
ester, 56, 201 Monomeric, 258
eugenol, 154, 342 Monomers, 53, 226
putrescin, 201 Monosaccharide, 15, 382, 383
Methylation, 174, 199 Monostroma undulatum, 68
Methyldorimidazole, 48 Monoterpene, 22, 345, 346, 348, 380, 397
Methylerythritol phosphate (MEP), 394 limonene, 397
pathway, 396 Monoterpenoid glycosyl, 400
Mevalonic acid (MVA), 192, 394, 396 Morphine, 7, 10
Microalgae, 7, 67, 70, 71, 95, 97 Morphology, 67, 71, 72, 139, 156, 167, 194,
Microbial, 36, 120, 128, 134, 233, 244, 253, 243, 298
254, 256, 258, 259, 296, 327, 349, 359 Motuporamines, 54, 55
Microflora, 7, 360 Mucous membrane irritation, 15
lingers, 7 Multidrug resistance (MDR), 399
Micromixing, 254 Murine, 16, 49, 51, 52, 56–58, 210
Microorganisms, 4, 5, 11, 20, 22, 25, 70–72,
leukemia, 51, 52, 56, 57
93, 99, 116, 132, 174, 255, 257, 259, 280,
melanoma cells apoptosis, 16
297, 317, 342, 359
solid tumor, 49
Micropropagation (MP), 114, 153, 187, 188,
tumor cells, 49
201
Mutagen, 413, 414
Microscopic, 3–5, 17, 244
Mycotoxin, 256, 257
algae, 3, 4
Myeloma, 398, 399
Microwave, 89, 92, 94, 254
Myristate, 199
assisted extraction (MAE), 92
Myristica fragrans, 154
Migration, 16, 209, 214, 281, 285, 400
Myristin, 154
Mild steel corrosion, 385
Mytilus edulis, 19
Miticides, 305
Mitochondrial injury, 402
Mitogen, 24
N
activated protein kinase (MAPK), 24 Naamidines, 47, 48
Mitotic N-acetylneuraminic corrosive, 15
index, 164 Nanobiopesticides, 306
spindle, 22 Nanomoles, 76, 79
Mobility, 306 Nanoparticles (NPs), 306
Moisture diffusion, 255 Naringin, 12
454 Index
National Agency for Food and Drug Admin- Normonanchocidin D, 56

istration and Control (NAFDAC), 319 Nuclear magnetic resonance (NMR), 99,
Natural antioxidants, 12, 13, 24, 72 171
Necrotrophic fungi, 359 Nucleic acid, 54
Neem seed kernel extract (NSKE), 311 Nucleosides, 36
Nematicides, 303, 305 Nudibranchs, 7
Neoamphimedine, 51 Nutraceuticals, 12, 26, 66, 71, 94, 109, 131
Neochlorogenic acid, 154 Nutrient
Neoplasm, 24, 25 medium, 115, 147, 190, 191, 200
Neoplastic transformation, 56 mist bioreactors, 159
Nephrotoxic risk, 397 Nutrition security, 256
Neubauer chamber, 164 Nutritional compounds, 65, 66
Neurite outgrowth, 52 Nylon, 162
Neuritogenic activity, 52
Neurodegenerative O
diseases, 23 Ocimum sanctum, 352, 360
disorders, 23 Octaphlorethol, 27
Neurohormone work, 358 Octopaminergic system, 358
Neuroprotection, 20 Odoriferous, 20
Neuroprotective activities, 27 Ohmic heating, 256, 259, 261
Neurotoxic mode, 358 Okinawan, 50, 51, 53, 56
Neurotoxin, 7 Oligoand polyfructans, 436
Neutraceuticals, 66 Ontogenesis, 430, 433, 434, 438
Nicotiana tabacum, 317, 328, 356 Ophiorrhiza rugosa, 154
Nicotinamide adenine dinucleotide phos- Opulent cradles, 27, 28
phate (NADPH), 17 Oral
Nicotine, 10, 135, 146, 158, 328, 331 agent, 26
Nicotinia tabacum, 199 sepsis, 18
Niphates furcate, 72 Organ culture, 131, 134, 142, 148–150, 155,
Nitrate, 24, 146, 153, 165, 167, 189, 298, 377 188, 200, 201
Nitric oxide (NO), 26, 56 Organic
Nitroblue tetrazolium (NBT), 74 corrosion, 369, 375–378, 385
Nitrogen, 5, 9, 10, 28, 121, 126, 132, 153, farming system, 307
173, 208, 228, 229, 284, 375, 378, 379 Organogenic redifferentiation, 149
atoms, 9 Orientalidine, 200
Nitrosamines, 24 Ornithine, 10
Non-agitated bioreactors, 158 Osazone crystals, 77
Nonblossoming, 423 Oscules, 71
Nondestructive methods, 160, 168 Osmundea pinnatifida, 13
Nonenzymatic Osteogenic sarcoma, 58
antioxidants, 77 Osteoporosis, 27
methods, 74 Ostia, 71
Non-flavonoid, 12 Oudemansiella mucida, 356
Nonionizing radiation, 259 Overground mass, 421, 434
Non-mutagen, 413, 414 Oxidation, 12, 13, 24, 54, 66, 76, 174, 199,
Non-photic regions, 4 257, 260, 297, 311, 347, 371
Nontoxicity, 393 Oxidative
Nordercitin, 54 initiation, 73
Index 455
stress, 23, 210, 231, 253, 261, 297, 299 Pesticidal activity, 358
Oxide, 26, 56, 81, 83, 371 Pesticide, 256, 257, 305, 307, 318, 319,
Oxygen, 9, 17, 18, 23, 49, 73, 83, 99, 121, 326, 327, 334, 342, 358, 361
158, 159, 173, 229, 242, 283, 284, 294, Phaeophyceae, 27
332, 348, 355, 371, 378 Phaeophyta, 5, 9
Oysters, 19, 94 Phagocytosis, 71, 212
Ozone, 256, 257 Pharmaceutical
industry, 15, 66, 124, 241, 247
P products, 10, 234
Pachastrellidae, 54 Pharmacognosy, 35, 133
Pale purple coneflower, 421, 439 Pharmacological
Palmaria palmata, 14, 68 activities, 3, 36, 207, 393
Palmitate, 199 properties, 15, 314
Panacea, 15 Phenazine methosulphate (PMS), 74
Panax ginseng, 135, 141, 151, 193 Phenethyl isothiocyanates, 399
Pancreatic Phenol
cancer cell, 401 compounds, 14
islet carcinoma apoptosis, 16 oxidases, 76
Papain, 91, 209, 221–230, 232–234, 236 Phenolic
Papaver bracteatum, 137, 200 acids, 12, 13, 24, 25, 68, 143, 149, 379,
Parkinsonism, 23 380
Parthenolide (PTL), 402 compounds, 12, 13, 96, 147, 244, 253,
Pathogenic 258, 274, 286, 287, 344, 345
bacteria, 8, 25 content, 8, 13, 269, 270
fungus, 22 polymers, 12
Pathogens, 8, 9, 12, 115, 121, 126, 212, 231, rings, 13
268, 307, 315, 341, 342, 359, 361
Phenols, 11, 12, 23, 77, 80, 96, 97, 272,
Pectinase, 147, 164
274, 303, 349
Pelteobagrus fulvidraco, 20
Phenylalanine, 12, 286
Pelvetia canaliculata, 69
ammonia lyase (PAL), 12
Penicillium
Phenylethylamine
citrinum, 350, 351, 353
alkaloids, 10
digitatum, 351, 353
oxalicum, 350 groups, 10
Pentamer, 14 Pheophytin, 69
Pentosan sulfate, 17 Phlorofucofuroeckol, 8, 14
Peptides, 18–20, 27, 28, 94, 210, 224, 227, Phloroglucinol, 14, 27
234, 294, 295, 300 polymers, 14, 27
Peritassa campestris, 151 tetramer, 14
Permeability, 23, 206, 249, 357, 375 Phlorotannins, 14, 27, 68
Peroxidant effects, 73 extracts, 14
Peroxidase, 73–75, 206 group, 12
Peroxidation, 18, 66, 260, 298 Phorbol esters (PEs), 314
Peroxides, 36 Phosphate buffer, 74–76, 79, 83
Peroxyl radicals, 23 Phospholipids, 23, 244
Peroxynite, 23 Phosphorylation, 214, 400
Pest control, 306, 314, 316, 318, 326, 333, Photoautotrophic roots, 156
335, 362, 394 Photomixotrophic cell, 159
456 Index
Photosynthesis, 4, 17, 132, 280, 282–284, protoplasm, 20

286, 298 species, 5, 9, 20, 108, 109, 112, 115, 124,
Photosynthetic organisms, 68 126, 133–135, 156, 175, 242, 268–270,
Phycobilins, 68 275, 281, 283–285, 287, 299, 300, 307,
Phycocyanins, 68 309, 312, 333, 422
Physiological tissue, 14, 92, 110, 111, 113–115, 118,
saline, 427, 428 125, 126, 129, 133, 172, 201, 259, 260
solution, 423, 426, 438 culture (PTC), 110, 111, 113–115,
variations, 68 124–127, 129, 133, 172, 187, 201
Phytochelatin, 293–296, 301 Plasma, 8, 211, 357, 360
synthase (PCS), 293–296, 299–301 Plasticity model, 398
Phytochemical Platelet, 13, 205, 211, 214, 215, 288
accumulation, 134, 275, 276 aggregation, 205, 214
biopesticides, 303, 318 Plumbago
pesticides, 306 indica, 151
Phytochemistry, 109, 314, 393 rosea, 152
Phytoconstituent, 10, 90, 268 Pneumatically-driven bioreactors, 158
Phytoestrogens, 26 Podophyllum, 199
Phytoextracts, 399 hexandrum, 160
Phytohormone, 155, 177, 191 Poltava region, 434
Phytolectins, 421, 430, 433, 434, 436–438 Polycyclic aromatic alkaloids, 54
Phytopathogens, 335, 359, 360 Polygonum multiflorum, 152, 153
Phytoplankton, 5, 9, 67 Polyketide precursors, 55
Phytoremediation, 21, 126, 293, 300, 301 Polymerization, 22, 243, 246
Phytostabilization, 300 Polymers, 14, 26, 245, 306, 422
Phytosterols, 25, 268, 394, 397 Polyphenol, 11, 13, 270, 298, 399
Phytotherapeutic agent, 205 oxidase (PPO), 74, 76
Phytotherapy, 241, 242, 247, 250 Polyphenolic, 24, 190, 399
Phytovolatilization, 300 Polyphenols, 9, 11–14, 24, 26, 27, 68, 134,
Pigment, 5, 71, 394 242, 253, 259, 268, 270
Pimpinella anisum, 20, 379 Polysaccharide, 8, 15, 16, 27, 68, 94, 175,
Piper nigrum, 20, 344, 379 399, 431, 435, 436, 438
Piperaceae, 20, 379 Polyterpenes, 394
Piscicides, 305 Polyunsaturated fatty acids (PUFAs), 12
Pityrogramma calomelanos, 300 Poriferan, 72
Plakinidines, 55 Porphyra
Plant tenera, 69, 95
age, 25 umbilicalis, 68
biotechnology, 126, 129 Porphyridium cruentum, 68
cell culture (PCC), 108–110, 112, 117, Portieria hornemannii, 22
119, 121, 124, 125, 127–129, 135, 155, Potassium, 8, 74, 78, 121
177, 188 Potency, 7, 49, 54, 349, 399
cysteine proteases, 231, 236 Potent
derived chemicals, 132, 336 antiulcer agents, 25
growth regulators (PGRs), 135–138, 141, carcinogen inhibitors, 24
143–146, 150–152, 154, 157 cytotoxicites, 53
pathogens, 307, 341, 342, 349, 361, 362 Potentiators, 308
protectants, 303 Power ultrasound technique, 255
Index 457
Prasinophyta, 67 Pseudaxinyssa cantharella, 48

Predators, 4, 116, 119, 132, 231, 306, 307, 317 Pseudolaric acid, 397
Pressurized Pseudolarix kaempferi, 397
hot water extraction (PHWE), 96 Pseudomonas aeruginosa, 359
liquid extraction (PLE), 92, 95 Pseudosaberities clavatus, 72
Prianos melanos, 53 Psoriasis, 22
Prianosinsalkaloids, 53 Pteridaceae, 300
Prinsepia utilis, 397 Pteris
Proand antiapoptotic proteins, 26 cretica, 300
Proanthocyanidins, 12 longifolia, 300
Procarcinogen, 24 umbrosa, 300
Proctology, 18 vittata, 300
Procyanidin, 26 Ptilocaulis
Production of phytochemicals, 107, 115, spiculifer, 55
134, 156, 173, 201 walpersi, 52
Progesterone, 15 Pulsed-electric field extraction (PEF), 93
Programmed cell death (PCD), 231 Pupil dilator atropine, 10
Pro-inflammatory prostaglandin, 206 Purpurea glycoside, 117, 200
Prokaryotes, 394 Purpurogalli, 75
Proliferation, 16, 118, 135, 140, 146, 194, Pyelonephritis, 205, 212, 235
195, 209, 210, 214, 259, 397, 399, 401, Pyrazinone moiety, 56
402 Pyricularia oryzae, 22
Proliferative response, 16 Pyridines, 36
Proline, 208 Pyridoacridine, 10, 11, 51
Prooxidant activity, 255 alkaloids, 51
Propagation, 14, 24, 108, 114, 118, 126, Pyrogallic acid, 14
134, 187–189, 191, 193–195, 197, 280, Pyrogallol, 75
336 Pyronaamide, 48
Prosobranch mollusk, 11 Pyrophosphate, 74, 346, 347, 394, 395
Pyrrolesquinolones, 36
Prostaglandin E2 (PGE2), 210
Pyrrolophenanthroline, 53
Prostate, 24, 25, 56, 399
Pyruvate, 395
Protamine, 17
Protease, 27, 206–208, 213, 225, 226, 232,
233, 235
Q
Protein Quillaja saponaria, 14
compounds, 427, 434, 438 Quinone, 24, 76, 382
content, 165, 298
degradability, 208 R
hydrolysates, 27 Racemate mixture, 199
kinases, 26, 57 Racemic form, 56, 57
Proteinaceous, 28 Radical, 18, 23, 24, 68, 72, 73, 81, 82, 84, 332
Proteinases, 205 scavenging, 68, 72, 73, 81, 82, 84
Proteolytic fraction, 206 Radiotherapy, 393
Protocatechuic aldehyde, 153 Raphanus sativus, 152
Protoplasmic streaming, 163 Rauwolfi serpentina, 200
Protozoa, 25, 223 Raw material, 254, 258, 286, 336, 422, 423,
Prunella vulgaris, 152 427, 438
Prymnesiophyta, 67 Reactive oxygen, 23, 332
458 Index
species (ROS), 23, 294 Sanguisorba officinalis, 153

Reagents, 74–84 Sapogenins, 15
Receptor, 10, 17, 19, 24, 48, 212, 213, 233, Saponaria vaccaria, 14
316, 331, 333, 356, 358, 400, 423, 429 Saponification, 78, 199
Red algae, 5, 12, 67 Saponins, 14, 15, 134, 149, 193, 242, 248,
Redox signaling, 26 268, 272, 288, 314, 379, 382, 383, 397, 400
Reductase, 24, 73, 164, 298, 347, 395 Sarcina maxima, 70
Refractive index detector, 6 Sarcoplasmic reticulum, 50, 54
Regrowth, 432, 433, 435–437 Sargassum, 21, 27, 96
Rehmannia glutinosa, 154 carpophyllum, 22
Reproduction, 6, 12, 120, 194, 299, 315, 327 polycystum, 22
Retinoic acid (RA), 399, 400 Saturation, 426
receptors (RARs), 400 Satureja hortensis, 352
Retinoid X receptors (RXRs), 400 Scavenging activities, 13, 294
Reversed-phase high-performance liquid Schizogenous, 20
chromatography (RP-HPLC), 97 Scylla paramamosain, 20
Revolutions per minute (rpm), 76, 77, Seafood, 12, 13
79–81, 146, 163, 164 Seagrass, 3, 4, 13
Rhabdastrella globostella, 72 beds, 3, 4
Rhaphidiophyta, 67 Seasonal variation, 267–269, 272, 275, 276
Rheumatic disorder, 23 Seaweed, 4, 7, 8, 13–16, 21, 28, 67, 68
Rhinorrhea, 14 Secondary
Rhizofiltration, 300 ecological functions, 12
Rhizomes, 20, 421, 427, 430, 431, 434, 435, metabolism, 13, 115, 175
437, 438 metabolite, 155, 176
Rhizophora Securinine, 145, 154
mucronata, 11 Sedative scopolamine, 10
stylosa, 11 Selenium, 399
Rhizophrine, 11 Selenoproteins, 399
Rhodophyta, 5, 9, 11 Senescence cardiovascular disorders, 23
Rhodophytes, 16 Sesquiterpene, 117, 345, 347, 381, 394
Rhus javanica, 152 lactone, 397, 402
Rodenticides, 305 Sesquiterpenoids, 347, 381, 394
Root Shoot culture, 153, 169
culture, 150, 153, 155, 160, 174 Shooty teratoma, 155, 156, 158
growth ratio, 162 cultures, 156, 158
Rosmarinic acid, 137, 138, 145, 154, 157, Silurian period, 4
173 Simmondsia californica, 355
Rummaging, 18 Singlet oxygen quenching, 73
Rutin, 12, 210, 212, 269, 270, 382 Sinusitis, 205, 212, 235
Sodium pyrophosphate, 74
S Solanaceae, 193, 200, 303, 329, 331
Sabadilla, 332, 336 Somaclonal variations, 155
Saccharopolyspora spinosa, 356 Somatic embryogenesis (SEg), 193
Salicylic acid, 154 Sorbitol, 76, 122
Salinity, 7, 72 Spectrophotometer, 74–84
Salvia miltiorrhiza, 150, 157 Spermidine, 55
Sanguinarine, 10, 200 Sphaerococcus coronopifolius, 22
Index 459
Sphingosine, 22 pesticides, 303, 305, 308, 317–319, 326,

Spirastrella inconstans, 72 328, 334, 349
Spongiochloris spongiosa, 69, 96 Syzygium aromaticum, 353
Spongsorites ruetzleri, 48
Spray drying (SD), 256, 258 T
Standardization, 208, 326, 335, 349, 362 Talinum paniculatum, 152
Staphylococcus aureus, 8, 359 Tanacetum parthenium, 402
Stearate, 199 Tannins, 12–14, 270, 272, 274, 286, 287,
Sterilization, 133, 169, 188–190, 254 380, 383
Steroidal Tanshinone, 153
hormones, 15 Target gene, 401
saponins, 15 Taxol, 10, 125
Steroids, 198, 286, 288, 303, 310, 327, 328 Taxus brevifolia, 10
Sterols, 8, 36, 68, 345, 383, 384 T-cell, 16, 209
Stigmast sterol, 22 Teichaxinella morchella, 52
Stilbenes, 12 Teratomas, 156, 158
Stochastic model, 398 Terpenes, 15, 36, 134, 268, 288, 314,
Stramonium, 157, 174, 199, 317 344–347, 349, 394, 397
Streptozotocin (STZ), 25 synthesis, 394
Strobilurin class, 359 units, 397
Stypoldione, 22 Terpenoid, 21, 394, 401, 406
Stypopodium zonale, 22 Terrestrial
Suberea sp., 56 bioactive peptides, 18
Substrates, 19, 76, 134, 198, 222, 223, 226, organisms, 4
260, 349 plants, 10
Sucrose, 121, 147, 150, 153, 173, 174, 190, Tetracyclic
moiety, 54
246
pyrroloiminoquinone, 53
Sugar acids, 15
Tetraterpenes, 394
Sulfated
Tetrazolium test, 164
oligosaccharide, 16
Thalassiosira pseudonana, 68
polysaccharides, 16
Thebaine, 200
Sulfation, 16, 17
T-helper cells, 16
Sulforaphane, 24, 399, 400 Theonella swinhoe, 50
Supercritical fluid Theonelladins, 50
chromatography (SFC), 100 Therapeutic
extraction (SFE), 92 agents, 21
Superhelical density, 54 determinations, 9
Supernatant, 74–77, 79–81, 147, 426 mechanism, 6
Superoxide, 18, 23, 73, 81, 83 properties, 12
dismutase (SOD), 73, 74 Thermosonication, 256, 258
Surgical traumas, 205, 235 Thiourea solution, 77
Suspension culture, 133, 140, 142, 146–149, Thorectandra, 55
159, 163–166, 177 Thorectandramine, 55
Swinholide macrolides, 50 Threshold, 28, 297
Synthetic Thrombophlebitis, 205, 212, 235
compounds, 24 Thrombotic thrombocytopenic purpura, 26
pathways, 393 Thymus vulgaris, 154, 355, 384
460 Index
T-lymphocytes, 16 Tumorigenesis, 22
Tocopherol, 68, 69, 77, 78 Turbid, 21
Topographical factors, 25 Tyrosinase inhibitors, 27
Topotecan, 50
Topsentia genitrix, 49 U
Topsentin, 49 Ubiquinones, 394
Torment, 18 Ulcers, 18, 22, 235, 330
Toxic Ultrasound, 89, 92, 93, 254–257
compounds, 71, 312 assisted extraction (UAE), 92, 93, 96
effects, 52, 55, 58, 314 rays, 256
metal, 13, 21, 293, 294, 299 Ultraviolet (UV), 12, 70, 73, 74, 133, 171,
substances, 163 175, 259, 268, 287, 308
Toxicity, 13, 22, 49, 90, 99, 213, 275, 293, Ulva
297, 298, 306, 310–312, 314, 315, 317, fasciata, 22
327, 333, 360–362, 375, 376, 397 lactuca, 22
Toxins, 9, 118, 119, 175, 317, 331, 333 Unicellular, 4, 67, 70
Trachycladindoles, 50 Urease activity, 25
Trachycladus laevispirulifer, 50 Uropathy, 16
Trachyspermum ammi, 353, 357 Uterine cancer, 24
Transfer DNA (T-DNA), 156 Utililactone, 397
Transgene, 199
Transgenic organs, 155 V
Transition metal ions, 73
Valeriana officinalis, 157
Trichosanthes kirllowii, 401
Vascular system, 431
Triglyceride, 8, 25
Vasculogenic mimicry activity, 400
Triple quadrupole-mass spectrometers
Vegetation, 14, 421, 423–425, 430–438
(QqQ-MS), 97
Vermifuge, 8
Trisulfated disaccharide, 17
Versatile, 16, 195
Triterpene, 14, 173, 384, 394, 401
Vinblastine, 10
amooranin, 401
Vincristine, 158
saponins, 14
Vindoline, 200
Triterpenoid, 355, 394, 397, 400, 401
Vitamin, 9, 23, 24, 68, 70, 71, 108, 122,
oleanolic acid, 397
173, 190, 288
saponins, 400
A, 399, 400
Tropane alkaloid, 201
C, 68, 253
Tropical
E, 68
coasts, 5
Volatile oils, 6, 20, 21, 242
oceans, 21
Tryptophan, 11, 193 W
Tumor
cell, 11, 16, 22, 55, 58, 120, 213, 214, Wettability, 208
234, 393, 402 Withania somnifera, 135, 152
inducing, 156 World Intellectual Property Organization
inflammation, 23 (WIPO), 233
necrosis factor-related apoptosis-inducing
ligand (TRAIL), 48 X
tissues, 16 Xanthones, 12
xenografts, 401 Xenobiotics, 155
Index 461
Xestospongia, 48, 51, 52 Z

caycedoia, 53
Zeaxanthin, 69, 70
exigua, 54
Zingiber zerumbet, 397
Xestospongin, 55
Xylose, 427

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PHYTOCHEMISTRY

© 2019 by Apple Academic Press, Inc.

Library and Archives Canada Cataloguing in Publication

Phytochemistry / edited by Chukwuebuka Egbuna, Jonathan Chinenye Ifemeje, PhD,

QK861.P65 2019 572’.2 C2018-904824-7 C2018-904875-1

CIP data on file with US Library of C

Jonathan Chinenye Ifemeje, PhD

(ICCON), the Nigerian Society of Biochemistry and Molecular Biology

Shashank Kumar, PhD

Nadia Sharif, PhD

University, Multan, Pakistan, and did her PhD in Biotechnology, Department

PART I: Marine Sources of Secondary Metabolites............................... 1

2. Marine Sponge Alkaloids: A Source of Novel Anticancer Agents..............35

3. Marine Antioxidants and Assay Methods....................................................65

4. Extraction of Marine Phytochemicals: Methods and Techniques.............89

PART II: Industrial and Medicinal Applications of

7. Practical Processes Involved in the Production of Phytochemicals by

8. Medicinal and Industrial Applications of Bromelain...............................205

9. Cysteine Proteases from Plants and Their Applications..........................221

11. Effective Processing Methods for Fruits and Vegetables..........................253

PART III: Environmental Concerns and Eco-Friendly

13. Effects of Environment on the Chemical Constituents and

14. Phytochelatins and Heavy Metal Tolerance in Plants..............................293

15. Phytochemical Biopesticides.......................................................................303

16. A Sustainable Approach in Integrated Pest Management:

17. Essential Oils in Pest Control and Disease Management.........................341

18. Inhibition of Mild Steel Corrosion in Acidic Media by

PART IV: Recent Advances................................................................... 391

20. Evaluation of the Phytohemagglutinins Activities of Echinacea

Syeda Nazish Arshad

Basu Maan Daas

Azza Migdam Elsheikh

Sechene Stanley Gololo

Rakesh Kumar Gupta

Prem Prakash Kushwaha

Santosh Kumar Maurya

Ashish Kumar Nayak

Onyeka Kingsley Nwosu

Walter David Obregón

David Okechukwu Okeke

Maria Catherine B. Otero

Juan Abreu Payrol

Atul Kumar Srivastava

Inidia Rubio Vargas

PAL Phenylalanine ammonia lyase

I am pleased to write the foreword for this book, Phytochemistry, Volume 3:

This book, Phytochemistry, Volume 3: Marine Sources, Industrial Applica-

et al. discusses the sustainable approach to integrated pest management.

The aquatic region has a wide range of temperature underneath freezing

and far-reaching photic and non-photic regions. From microorganisms to

As sunlight is required for the manufacturing of food in all marine

a unique source of chemical compounds that may have potential industrial

1.2 DISCOVERY OF MARINE PHYTOCHEMICALS

A lot of drugs are derived from aquatic organisms. One example is a

1.3 UNIQUENESS OF MARINE PHYTOCHEMICALS

The marine vegetation comprises of microflora (cyanobacteria, bacteria,

and floral species is essentially unheeded. Various complexes derivatives

1.4 CLASSES OF PHYTOCHEMICALS

Marine organisms are a rich source of biologically active and therapeuti-

The term alkaloid was formally suggested by Meissner in 1819 to describe