Sample

Biotechnological Production of Plant
Secondary Metabolites
Edited by
Ilkay Erdogan Orhan

Faculty of Pharmacy
Eastern Mediterranean University
Gazimagosa (Famagusta)
The Northern Cyprus
&
Faculty of Pharmacy
Gazi University
Ankara
Turkey
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DEDICATION
This eBook is dedicated to Prof. Dr. Bilge Şener who is one of the most eminent
woman scientists in Turkey and has always been a great role model in my
academic life.
CONTENTS
Foreword i
Preface iii
List of Contributors iv
CHAPTERS
1. Plant Cell and Tissue Culture as a Source of Secondary Metabolites 3

Rodríguez-Sahagún A., Del Toro-Sánchez C.L., Gutierrez-Lomelí M. and Castellanos-
Hernández O.A.
2. Natural Product Extracts: Terpenes and Phenolics 21

Gutiérrez-Lomelí M., Del Toro-Sánchez C.L., Rodríguez-Sahagún A. and Castellanos-
Hernández O.A.
3. Biotechnological Production of Coumarins 36

Alev Tosun
4. Novel Biomedical Agents from Plants 53

Athar Ata
5. Production of Anthocyanins by Plant Cell and Tissue Culture Strategies 67

Claudia Simões, Norma Albarello, Tatiana C. de Castro and Elisabeth Mansur
6. In Vitro Organ Cultures of the Cancer Herb Castilleja tenuiflora Benth. as Potential
Sources of Iridoids and Antioxidant Compounds 87
Gabriela T.-Tapia, Gabriel R.-Romero, Alma R. L.-Laredo, Kalina B.-Torres and
Alejandro Zamilpa
7. Plant Cell Tissue and Organ Cultures in Terpenoids 107

Irem Tatli I.
8. Bioactive Chemical Constituents and Biotecnological Production of Secondary

Metabolites in Amaranthaceae Plants, Gomphreneae Tribe 124
Marcos J. Salvador, Nathalia L. Andreazza, Aislan C.R.F. Pascoal, Paulo S. Pereira,
Suzelei C. França, Orghêda L.A.D. Zucchi and Diones A. Dias
9. Biotechnology Approaches and Economic Analysis of Jojoba Natural Products 159

Mohammed A.M. Aly and Aydin Basarir
10. The Effects of Pesticides on Plant Secondary Metabolites 176

Monica Hancianu and Ana C. Aprotosoaie
11. Cardenolide Production as an Important Drug Agent 187
Sebnem Harput U.
12. Progress in Biotechnological Applications of Diverse Species in Boraginaceae Juss. 200
Ufuk Koca, Hatice Çölgeçen and Nueraniye Reheman
13. Production of Anticancer Secondary Metabolites: Impacts of Bioprocess Engineering 215
Sajjad Khani, Jaleh Barar, Ali Movafeghi and Yadollah Omidi
Subject Index 241
Plant Index 244

i
FOREWORD
Modern life is complex and evolution went along way starting from very simple organic molecules to larger
biomolecules and, in addition, they interact with other classes of molecules in the environment. It is the
essence of scientific research to be in constant evolution. Plant cell science, plant genetics, and plant
biotechnology of today bear only faint resemblance to what they used to be only twenty years ago. Plant
cell reports keep pace with this evolution.
Plants have evolved an amazing array of metabolic pathways leading to molecules capable of responding
promptly and effectively to stress situations imposed by biotic and abiotic factors, some of which supply
the ever-growing needs of humankind for natural chemicals, such as pharmaceuticals, nutraceuticals,
agrochemicals, food and chemical additives, biofuels, and biomass. Robotics and combinatorial techniques
allow chemists to synthesize single libraries that contain more compounds than ever before. Especially,
medicinal chemists but also chemists active in the catalysis area have embraced this efficient new synthesis
tool. Moreover, advances in molecular biology and genomics continue to improve our understanding of
biological processes and to suggest new approaches to deal with inadequately or untreated diseases that
afflict mankind. Despite of all the progress in both molecular biology/genomics and combinatorial
chemistry methods, it is generally recognized that the number of pharmaceutically relevant hits is not
directly proportional to the number of compounds screened. Both structural diversity and complexity in a
collection of molecules are essential to address.
13 Chapters in this eBook on medicinal plant biotechnology covers recent developments in this field. It
includes a comprehensive up-to-date survey on established medicinal plants and on molecules which gained
importance in recent years. The chapters published in this eBook today address highly relevant issues in
modern plant cell science and plant molecular biology.
In “biotechnological production of plant secondary metabolites”, expert researchers provide detailed

practical information on some of the most important methods employed in the engineering of plant
secondary metabolism pathways and in the acquisition of essential knowledge in performing this activity,
including the significant advances and emerging strategies. The chapters include introductions to their
respective topics, lists of the necessary materials and methods, findings along with discussions step-by-step.
Among secondary metabolites, the biosynthesis of phenolic compounds which have a potential use as an
antioxidant and terpenes extensively used as flavors and fragrances in perfumery and medicines are
described in detail. Recent advances in plant biotechnology have been explained by showing the potential
of plant cell and tissue cultures for the large-scale production of valuable secondary metabolites. One of the
most important secondary metabolite of the plants known as coumarins has been examined regarding to the
biotechnological view.
Natural products with potential biomedical applications along with the production of bioactive compounds
by using biotechnological methods have been described. The production of anthocyanins under in vitro
conditions has been given in detail for researchers in plant biotechnology. From Scrophulariaceae family,
Castilleja tenuiflora Benth. is one of the medicinal plants used in Mexican folk medicine in the treatment of
cancer. Root and shoot cultures of this plant species have been investigated for the production of flavonoids
and iridoids which are responsible for antioxidant and cytotoxic activities. Strategies to increase secondary
metabolite production in plant cell cultures have been explained by giving examples for terpenoids.
Biotechnological investigations on Amaranthaceae plant species have been summarized. Biotechnology

approaches have been shown for the utilization of Jojoba which is an economical important plant by giving
propagation and cloning of genes coding for economically important traits. The production of cardenolides
in Digitalis cultures was extensively described for the industrial production. Depending on their medicinal
and economical importance, the production of secondary metabolites of the plant species from
Boraginaceae family using biotechnological methods have been outlined. The impacts of cell and tissue
ii
culture technologies for the large-scale production of anticancer secondary metabolites have also been
included.
İlkay Erdogan Orhan has produced a magnificent effort covering relevant aspects of the medicinal plants in
this eBook. Botanists, chemists, biochemists, pharmacognosists and molecular biologists having any
interest potentially bioactive compounds will be satisfied in the eBook content.
Ilkay Erdogan Orhan is an experienced young pharmacognosist for many years now, she has been a
scientist at the Department of Pharmacognosy, Faculty of Pharmacy Gazi University, involving with
Turkish medicinal plant and marine organisms screening program. Her work includes the biological
evaluation and phytochemical studies of secondary metabolites for drug discovery. She has published many
scientific articles devoted to the discovery of new bioactive natural compounds and author/co-authored
several chapters on the subject of medicinal plants. This wide experience has given her a broad perspective
on drug discovery from biological sources and has benefited this eBook enormously. This eBook will be
useful by academic and industrial scientists having any interest in the potential of plants as a source of
bioactive lead compounds and to all who are interested in medicinal plants.
Prof. Dr. Bilge Sener

Professor of Pharmacognosy
Faculty of Pharmacy
Gazi University
Ankara-Turkey
iii
PREFACE
Biotechnology can be considered to be originated from prehistoric times, when microorganisms were
already used for processes like fermentation. In fact, the earliest use of biotechnology might have been
started with lactic acid fermentation by Louis Pasteur in 1857. Then, discovery of penicillin from
Penicillium sp. in 1929 by Alexander Fleming led to large scale production of this antibiotic during World
War II using cultures of this microfungus, which refers to another early application of biotechnological
methods. Later on, biotechnology has become a very important tool in every aspect of plant research and is
now extensively applied to production of secondary metabolites from many plant species. The field of plant
biotechnology has gained a lot of attraction from scientists due to its importance in pharmaceutical,
agricultural, forestry, food, and some other sectors. Especially, plant cell and tissue culture techniques are
important in vitro precise methods using various plant parts applicable in large-scale. Plant biotechnology is
such an immense scope varying from traditional plant breeding to genetically-engineered plants, which is
out of the borders of the current eBook. In this eBook, the chapters will cover biotechnological studies
performed on different groups of plant secondary metabolites such as terpenes, phenolics, coumarins,
anthocyanins, iridoids, and cardiac glycosides. Some of the chapters will also mention about
biotransformation aspects on several bioactive compound classes.
In this regard, I would like to thank to Bentham Publishers for offering me the kind invitation to edit this
valuable eBook. I would also like to extend my greatest thanks for the supports of the authors of the eBook,
who contributed qualified chapters by giving their precious times into this project. I am sure that the eBook
will provide an open platform to read and learn the latest knowledge on biotechnological production of
plant secondary metabolites and will give new perspectives on the scope.
Prof. Dr. Ilkay Erdogan Orhan

Faculty of Pharmacy
Eastern Mediterranean University
Gazimagosa (Famagusta)
The Northern Cyprus
&
Faculty of Pharmacy
Gazi University
Ankara
Turkey
iv
List of Contributors
N. Albarello
Núcleo de Biotecnologia Vegetal, Universidade do Estado do Rio de Janeiro, Brazil
M.A.M. Aly
Department of Arid Land Agriculture, and Department of Agribusiness, Faculty of Food and Agriculture,
United Arab Emirates University, P.O. Box 17555, Al Ain, United Arab Emirates
N.L. Andreazza
Curso de Farmácia, Departamento de Biologia Vegetal, Instituto de Biologia, Universidade Estadual de
Campinas (UNICAMP), C.P. 6109, 13083-970, Campinas, SP, Brazil
A.C. Aprotosoaie
Gr. T. Popa” University of Medicine and Pharmacy, Faculty of Pharmacy, Iasi, Romania
A. Ata
Department of Chemistry, The University of Winnipeg, 515 Portage Avenue, Winnipeg, MB, Canada R3B
2E9
J. Barar
Research Centre for Pharmaceutical Nanotechnology and School of Advanced Biomedical Sciences, Tabriz
University of Medical Sciences, Tabriz, Iran
A. Basarir
Department of Arid land Agriculture, and Department of Agribusiness, Faculty of Food and Agriculture,
K. Bermúdez-Torres
Departamento de Biotecnología, Centro de Desarrollo de Productos Bióticos, Instituto Politécnico
Nacional, P. O. Box 24, 62730, Yautepec, Morelos, México
T. Carvalho de Castro
O.A. Castellanos-Hernández
Centro Universitario de la Ciénega, Universidad de Guadalajara. Av. Universidad 1115, Col. Lindavista,
CP 47810, Ocotlán, Jalisco, México
H. Cölgecen
Zonguldak Karaelmas University, Faculty of Arts and Science, Department of Biology, 67100 İncivez,
Zonguldak, Turkey
C.L. Del Toro-Sánchez

D.A. Dias
Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo (USP), Via do Café,
s/n, 14040-903, Ribeirão Preto, SP, Brazil
v
S.C. França
Unidade de Biotecnologia, Universidade de Ribeirão Preto (UNAERP), 14096-900, Ribeirão Preto, SP,
Brazil
M. Gutiérrez-Lomelí
M. Hancianu
Gr. T. Popa” University of Medicine and Pharmacy, Faculty of Pharmacy, Iasi, Romania
S. Harput
Hacettepe University, Faculty of Pharmacy, Department of Pharmacognosy, 06100, Ankara, Turkey
S. Khani
Research Centre for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, Tabriz, Iran
U. Koca
Department of Pharmacognosy, Faculty of Pharmacy, Gazi University, 06330, Ankara, Turkey
A.R. López-Laredo
E. Mansur
A. Movafeghi
Research Centre for Pharmaceutical Nanotechnology and Plant Biology Department, Faculty of Natural
Sciences, University of Tabriz, Tabriz, Iran
Y. Omidi
Research Centre for Pharmaceutical Nanotechnology and School of Advanced Biomedical Sciences, Tabriz
University of Medical Sciences, Tabriz, Iran
A.C.R.F. Pascoal
P.S. Pereira
Unidade de Biotecnologia, Universidade de Ribeirão Preto (UNAERP), 14096-900, Ribeirão Preto, SP,
Brazil
N. Reheman
Department of Pharmacognosy, Faculty of Pharmacy, Gazi University, 06330, Ankara, Turkey
A. Rodríguez-Sahagún
vi
G. Rosas-Romero
M.J. Salvador
C. Simões
I.I. Tatli
Department of Pharmaceutical Botany, Faculty of Pharmacy, Hacettepe University, 06100 Ankara, Turkey
A. Tosun
Department of Pharmacognosy, Faculty of Pharmacy, Ankara University, 06100 Ankara, Turkey
G. Trejo-Tapia
A. Zamilpa
Centro de Investigación Biomédica del Sur, Instituto Mexicano del Seguro Social, Argentina No. 1, 62790,
Xochitepec, Morelos, México
O.L.A.D. Zucchi
Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo (USP), Via do Café,
s/n, 14040-903, Ribeirão Preto, SP, Brazil
Biotechnological Production of Plant Secondary Metabolites, 2012, 3-20 3
CHAPTER 1
Plant Cell and Tissue Culture as a Source of Secondary Metabolites
Rodríguez-Sahagún A., Del Toro-Sánchez C.L., Gutierrez-Lomelí M. and
Castellanos-Hernández O.A.*
Abstract: Plants are an important source of secondary metabolites that have been used throughout
history as drugs, pesticides, pigments, flavors and fragrances. However, one of the main constraints to
the use of cultivated plants as a source of these metabolites is the ability to ensure the constant and
efficient supply of the compounds, since the yields are usually affected by the genetic background, as
well as by the geographic location, edaphic and climatic conditions at the site of cultivation, combined
with the potential effect of harvest and transport methods. The use of plant tissue culture has been
proposed as an alternative to conventional agriculture for the production of secondary metabolites due
to the possibility of controlling the quality and quantity of the compound of interest by controlling the
factors affecting its synthesis and/or accumulation. Recent advances in the field of plant biotechnology
show the potential of using plant cell and tissue cultures as a source for the large-scale production of
valuable secondary metabolites instead of using whole plants and subsequent extensive land
exploitation. Moreover, the employment of molecular biology techniques has allowed for obtaining
novel products from genetically engineered plants.
Keywords: Secondary metabolites, biological activity, plant cell culture, tissue culture, biotechnology,
mass production, culture medium, organ culture, cell suspension, explants, callus, Agave tequilana, elicitor.
1. INTRODUCTION
Plant cell and tissues culture is based on the principle of cellular totipotency, which states that any cell from
a plant can regenerate a complete individual. With this tool, one may obtain cultures of undifferentiated
cells such as calli and cell suspensions, or organ culture as shoots and roots.
There are essentially three ways for in vitro culture: cells in suspension, immobilized cells or tissues or organs.
The kind of culture affects, among other things, cell growth, product formation, its purification and the type
of bioreactor that can be used. A commercial level has been used mainly by cell suspension cultures [1],
transformed root culture [2].
Plants produce a wide variety of chemical molecules that play important roles in its development and its
adaptation to the environment. Many of these compounds are used in the manufacture of drugs, flavors,
fragrances and pesticides (Table 1). These molecules may represent primary or secondary metabolites,
depending on whether they occur constitutively or in response to environmental aggression either by drastic
changes in biotic or abiotic factors [3].
Plant cell culture represents an alternative biotech for the production of secondary metabolites identified
and that are of interest to humans. There exist two important factors in a mass production system based on
the cultivation of cells in suspension, the prior establishment of callus culture in semisolid media and the
demonstration of in vitro fertilization to retain the ability to produce substances of interest or produce other
novel substances of interest [4], as reported by Konczak et al. [5] which identified that the culture
*Address correspondence to Castellanos-Hernández O.A.: Departamento de Ciencias Básicas, Centro Universitario de la Ciénega,
Universidad de Guadalajara. Av. Universidad 1115, Col. Lindavista, CP 47810, Ocotlán, Jalisco, México; E-mails:
ocnoscr@cuci.udg.mx and ocnoscr@gmail.com
Ilkay Erdogan Orhan (Ed)

All rights reserved-© 2012 Bentham Science Publishers
4 Biotechnological Production of Plant Secondary Metabolites Rodríguez-Sahagún et al.
temperature and ammonia levels in the culture medium influenced the composition of anthocyanins produced
by roots in vitro. This is evidence that plants can develop different compounds when grown in vitro as those
produced under natural conditions. The tissue culture technology can be used to produce plants regardless of
geographical location and poor weather conditions that may limit production in the field [6].
Table 1: Plant Species and secondary metabolites obtained from them using tissue culture techniques.
Plant Species Product Activity

Atropa belladanna Atropine Blocking of cholinergic
Catharanthus roseus Vincristine Antileukaemic
Ajmalthine Antiarrhythmic
Ajmalicine Tranquilizer
Serpentine
Campatotheca accminata Camptothecin Anticancer
Cephalotaxus Cephalotaxine Antitumour
harringtonia
Cinchona officinalis Quinine Antimalarial
Coffea arabica Cafeine Central nervous system stimulant
Coleus blumei Rosamarinic acid Spice, antioxidant
Conium spp. Coniine Hallucinogenic properties, First alkaloid to be synthesized; extremely toxic,
causes paralysis of motor nerve endings, used in homeopathy in small doses
Coptis japonica Berberine Antibacterial
Chondrodendrom (+)-Tubocurarine Nondepolarizing muscle relaxant producing paralysis, used as an adjuvant to
tomentosa anaesthesia
Chrysanthemum Pyrethrin Insecticide (for grain storage)
cinerariaefolium
Datura stramonium Scopolamine Antihypertension
Digitalis lanata Digoxill Cardiac tonic
Reserpine Hypotensive
Dioscorea deltoidea Diosgenin Antifertile
Erythroxylon coca Cocaine Topical anaesthetic, potent central nervous system stimulant, and adrenergic
blocking agent; drug of abuse
Eschscholzia californica Sanguinarine Antibacterial showing antiplaque activity, used in toothpastes and oral rinses
Eurycoma longifolia Jack 9-methoxycanthin- Aphrodisiac
6-one
Hyoscyamus niger Atropine Anticholinergic
Jasminum spp. Jasmine Perfume
Lithospermum Shikonines Dyes, pharmaceutical
erythrorhizon
Lycopersicum esculentum Tomatine Fungicidal properties
Morinda citrifolia (also Anthraquinones Laxatives, dyes
Cassia tora)
Nicotiana tabacum Nicotine Ganglion blocker
Glutathione cardiovascular agent
Ubiquinone-10
Papaver somniferum Morphine and Tranquilizers, muscle relaxers, pain killers, hallucinogens
(Opium) codeine
Papaver somniferum Morphine and Analgesic, sedative and antitussive
codeine
Peyote cactus Mescaline Hallucinogenic properties
Pilocarpus jaborandi Pilocarpine Peripheral stimulant of the parasympathetic system, used to treat glaucoma
P. bracteatum Codeine Analgesic
Rauwolfia serpentina Ajmaline Antiarrhythmic
Plant Cell and Tissue Culture Biotechnological Production of Plant Secondary Metabolites 5
Table 1 cont….
Solanum tuberosum Solanine Pesticidal and fungicidal properties
Stevia rebaudiana Stevioside Sweetener
Strychnos nux-vomica Strychnine Violent 5itanic poison, rat poison, used in homeopathy
Thaumatococcus danielli Thaumatin Sweetener
Uragoga ipecacuanha Emetine Orally active emetic, amoebicide
For producers, plant cell tissue and organ culture are of great interest as an alternative for obtaining
chemicals from plant species by reducing the time interval to harvest. To meet this objective one has used
different strategies, such as the optimization of culture medium [7], genetic modification [8], selection of
high producing cell lines, presence or absence of environmental factors [9] and chemical or biological
aspects [10], or the explant source, as reported for Impatiens balsamina whose results suggest that the
tissue cultures initiated from the high-yielding donor plants should be capable of producing higher content
of secondary compounds than those initiated from low-yielding donor plants [11].
2. PLANT CELL, TISSUE AND ORGAN CULTURES
The in vitro culture can be initiated from almost any part of the plant (stem, leaf, seed, fruit, embryo, cotyledon,
root, etc.). Commonly, selected parts of the plant that are actively dividing, such as meristematic regions. Although
found in the same plant are both juvenile and adult growth, the first is characterized by increased activity and by
the absence of reproductive structures, while the adult is slower growing and has sex structures for the
reproduction of the plant [12, 13]. For the generation of secondary metabolites by in vitro techniques, tissue culture
and cell suspension culture is mainly used. In the first, using small pieces of tissue inoculated in semisolid media,
in which, depending on the growth regulators used, is quite easily possible to induce callus formation.
Haberlandt in 1902 (cited by Gautheret [14]), involved in the most important experiments on tissue culture,
was also the first to employ the concept of callus to define an amorphous mass of cells. In the case of
suspension cultures, the calli were kept in liquid medium and under continuous stirring. The suspension
cultures are attractive because of their rapid growth, easy handling and simplicity, because of the
uniformity and limited number of cell types, and for the production of secondary metabolites. Most work
on drug production or secondary metabolites use cell suspension [15]. These rapidly differentiate in
response to organogenetic stimuli thereby varying the type and concentration of substances of plant growth
regulators. Meristematic cells are distinguished from other cells by their relatively small size, dense
cytoplasm, isodiametric shape, thin cell wall, minimal vacuolation and a large nucleus [16]. In vitro
cultures, such cells are found in the periphery of the callus or suspensions such as nodules pre-embryo
tissue, resulting in great variability in gene expression between the cell populations of these crops. It also
requires the presence of such cells to regenerate a plant from an in vitro culture.
Cell Culture
To initiate cell reproduction, called callogenesis, the culture must be established from a differentiated
explant, to which growth regulators must be added that activate dedifferentiation and cell division. The
facility may be divided into three main phases: induction, in whose cell metabolism is stimulated before
mitosis, cell division, in which the explant cells multiply, and finally cell differentiation and expression of
some metabolic pathways that lead to the formation of secondary metabolites [17].
The culture was incubated using ambient light, temperature and humidity, which together with
physicochemical and nutritional development of the explant lead to the formation of an amorphous cell
mass called callus, or to the differentiation in an organized tissue or organs that produced embryos. The
calli obtained from this procedure may be subcultured for maintenance and propagation or induce
differentiation to form organs (organogenesis), embryo (embryogenesis) or switch to a liquid culture
medium for cells and small aggregates in suspension.
The genetic variability between and within cultures is reflected first in the morphology of the callus and
subsequently in suspension cultures or culture systems with which they work. Some authors [18, 19]
CHAPTER 2
Natural Products Extracts: Terpenes and Phenolics
Gutiérrez-Lomelí M., Del Toro-Sánchez C.L., Rodríguez-Sahagún A. and
Castellanos-Hernández O.A.*
Abstract: The dependence of mankind upon the plant kingdom goes far beyond the production of food
crops. A great number of plant species produce secondary metabolites that possess valuable properties,
many of which have been studied and applied mainly to the pharmaceutical and food industries. Secondary
metabolites such as terpenes and phenolic acid compounds have become very important due to their
chemical and biological properties and play a major role in plant and human health. Terpenes are the
primary constituents of the essential oils of many types of plants and flowers, and have been extensively
used as natural flavor additives for food, as fragrances in perfumery, and in traditional and alternative
medicines. On the other hand, phenolic acid compounds are plant metabolites widely distributed throughout
the plant kingdom, which have a potential use as natural antioxidants in processed foods. The aim of this
chapter is to provide an overview of the current knowledge on these metabolites regarding their
biosynthesis, main sources and methods for their obtaining and use.
Keywords: Natural products, terpene, phenolic, coumarin, flavonoid, lignan, stilbene, secondary
metabolites, biological activity, biotechnological production, biosynthesis.
1. INTRODUCTION
In nature there are hundreds, even thousands of species of plants capable of synthesizing a large number of
compounds needed both for their own growth, as well as in response to the environment. Such compounds
are called natural products.
Natural products are organic compounds that are formed by living systems, these compounds may be
divided into three categories:
a) Primary metabolites, compounds which occur in all cells and play a central role in the
metabolism and reproduction of those cells (nucleic acids and the common amino acids and
sugars);
b) High-molecular-weight polymeric materials, as cellulose, the lignins and the proteins, which
form the cellular structures; and
c) Secondary metabolites, which do not appear to participate directly in growth and

development [1].
Plants form an important part of our everyday diet, and plant constituents and their nutritional value have
been intensively studied for decades. In addition to essential primary metabolites (e.g. carbohydrates, lipids
and amino acids), higher plants are also able to synthesize a wide variety of low molecular weight
compounds, which are called secondary metabolites. Plant secondary metabolites can be defined as
compounds that have no recognized role in the maintenance of fundamental life processes in the plants that
synthesize them, but they do have an important role in the interaction of the plant with its environment [2].
*
Address correspondence to Castellanos-Hernández O.A.: Departamento de Ciencias Básicas, Centro Universitario de la Ciénega,
Universidad de Guadalajara. Av. Universidad 1115, Col. Lindavista, CP 47810, Ocotlán, Jalisco, México; E-mails:
ocnoscr@cuci.udg.mx, ocnoscr@gmail.com
22 Biotechnological Production of Plant Secondary Metabolites Gutiérrez-Lomelí et al.
Many secondary metabolites have a complex and unique structure and their production is often enhanced
by both biotic and abiotic stress conditions [3]. Secondary metabolites are characterized by enormous
chemical diversity and every plant has its own characteristic set of secondary metabolites. Plant secondary
metabolites can be structurally divided into six major groups: polyketides and fatty acids, isoprenoids and
steroids, phenylpropanoids (phenolic acid compounds), alkaloids, specialized amino acids and peptides, and
specialized carbohydrates [1].
The terms “isoprenoid”, “terpenoid” and “terpene” are frequently used interchangeably [4]. Terpenoids are
perhaps the most diverse family of natural products synthesized from plants, serving a range of important
physiological and societal functions. Over 40,000 different terpenoids have been isolated from plant,
animal and microbial species [4, 5]. The terpenoid are often commercially attractive because of their uses
as flavor and color enhancers, agricultural chemicals, and medicinals [6], and are classified by the number
of carbons in the skeletal structure, typically in units of five carbons. The 5, 10, 15, 20, 25, and 30-carbon
terpenoids are referred to as hemi-, mono-, sesqui-, di-, sester-, triterpenes, respectively, and finally,
carotenoids, 40-carbon terpenoids [1, 4].
Phenolic acid compounds seem to be universally distributed in plants. They have been the subject of a great
number of chemical, biological, agricultural, and medical studies [7]. Plants, fruits and vegetables contain
numerous bioactive components and are especially rich in this kind of compounds such as flavonoids,
phenolic acids, stilbenes, and procyanidins [8]. In plants, these compounds have diverse functions such as
stabilization and protection. Furthermore, phenols are believed to work synergistically to promote human
health through a variety of different mechanisms, such as enhancing antioxidant activity, impacting cellular
processes associated with apoptosis, activities associated with inhibition of microorganisms,
antiinflamatory and antiviral [9-11]. The increased interest in polyphenols in the past decade has been
brought about by results from epidemiological studies linking the consumption of diets rich in plant foods
with decreased risk of these diseases [7].
2. IMPORTANCE AND ECOLOGICAL ROLE OF TERPENOIDS
Monoterpenes, are a large family of natural products that are best known as constituents of the essential oils
and defensive oleoresins of aromatic plants. In addition to ecological roles in pollinator attraction,
allelopathy and plant defense, monoterpenes are used extensively in the food, cosmetic and pharmaceutical
industries, with a range of medicinal proprieties, among which, anticarcinogenic, antioxidants, antifungal,
antimicrobial, and others [12].
Monoterpenes and sesquiterpenes are the majority of volatile compounds released from plants after
herbivore damage, attracting arthropods that prey on or parasitize herbivores, then avoiding further damage
[13]. In addition to volatile terpenoids, certain diterpenes and sesquiterpenes are phytoalexins involved in
the direct defense of plants against herbivores, and microbial pathogens [14].
Many of the terpenoids are commercially interesting because of their use as flavors and fragrances in foods and
cosmetics (e.g. menthol, nootkatone and sclareol) or because they are important for the quality of agricultural
products, such as the flavor of fruits and the fragrance of flowers, e.g. linalool [15, 16]. In addition, terpenoids
can have medicinal properties such as anticarcinogenic, antioxidant and antifungal, e.g. geraniol [17-19],
anticarcinogenic, e.g. linalool and limonene [20-23], antimalarial (e.g. artemisinin), anti-ulcer, hepaticidal,
antimicrobial or diuretic (e.g. glycyrrhizin) activity [24-28]. Other terpenoids play an important role in plant-
insect, plant-pathogen, and plant-plant interactions [13, 29].
3. BIOSYNTHESIS OF TERPENOIDS IN PLANTS
Terpenoids are derived from the mevalonate (MVA) pathway, which is active in the cytosol, or from the
plastidial 2-Cmethyl-D-erythritol-4-phosphate (MEP) pathway (Fig. 1). Both pathways lead to the
formation of the C5 units isopentenyl diphosphate (IPP) and its allylic isomer dimethylallyl diphosphate
(DMAPP), the basic terpenoid biosynthesis building blocks. In second phase of terpene biosynthesis, IPP
Natural Products Extracts Biotechnological Production of Plant Secondary Metabolites 23
and DMAPP are used by prenyltransferases in head-to-tail condensation reactions of these two C5-units to
produce geranyl diphosphate (GPP), the subsequent 1´4-additions or isopentenyl diphosphate to generate
farnesyl (FPP) and geranylgeranyl diphosphate (GGPP), the immediate precursors of monoterpenes (Fig.
2), sesquiterpenes (Fig. 3) and diterpenes (Fig. 4), respectively. These reactions are catalyzed by short-
chain prenyltransferases, including the GPP synthase, FPP synthase and GGPP synthase. GPP synthase
catalyzes the condensation reaction of IPP and DMAPP to form GPP. FPP synthase sequentially adds two
molecules of IPP to DMAPP to form the C15 diphosphate precursor of sesquiterpenes and triterpenes, and
GGPP synthase adds three molecules of IPP to DMAPP to form the C20 diphosphate precursor of
diterpenes and tetraterpenes [4, 12, 14, 30-32].
b) MEP pathway
a) MVA pathway Pyruvate D-Glyceraldehyde

3-phosphate
DXPS
Acetyl-CoA
AACT
1-Deoxy-D-xylulose
5-phosphate
Acetoacetyl-CoA
DXR
HMGS
2-C-Methyl-D-erythritol
4-phosphate
HMG-CoA
MCT
HMGR
4-(Citidine 5´diphospho)-
Mevalonate 2-C-methyl-D-erythritol
CMK
MK
2-Phospho-4-(citidine 5´diphospho)-
2-C-methyl-D-erythritol
Mevalonate-5-phosphate
MECPS
PMK
Mevalonate-5-diphosphate 2-C-methyl-D-erithritol-
2,4-cyclodiphosphate
MDC
IPPI
Isopentenyl Dimethyallyl
diphosphate diphosphate
GPP FPP GGPP
Monoterpenes - Sesquiterpenes - Diterpenes

- Triterpenes - Tetraterpenes
Figure 1: Biosynthetic pathways for the production of isopentenyl diphosphate (IPP) and dimethyallyl diphosphate
(DMAPP). The mevalonate pathway (a) and 2-C-methyl-D-erythritol-4-phosphate pathway (b). Abbreviations: AACT,
acetyl-CoA:acetyl-CoA C-acetyltransferase; CMK, 4-(cytidine-5′-diphospho)-2-C-methylerythritol kinase; DXPS, 1-
deoxyxylulose-5-phosphate synthase; DXR, 1-deoxyxylulose-5-phosphate reductoisomerase; FPP, farnesyl
diphosphate; GGPP, geranylgeranyl diphosphate; GPP, geranyl diphosphate; HMGR, 3-hydroxy-3-methylglutaryl-CoA
reductase; HMGS, 3-hydroxy-3-methylglutaryl-CoA synthase; IPPI, isopentenyl diphosphate isomerase; MCT, 2-C-
methylerythritol-4-phosphate cytidyltransferase; MDC, mevalonate-5-diphosphate decarboxylase; MECPS, 2-C-
methylerythritol-2,4- cyclodiphosphate synthase; MK, mevalonate kinase; PMK, phosphomevalonate kinase.
According to [4, 12, 30, 31].
36 Biotechnological Production of Plant Secondary Metabolites, 2012, 36-52
CHAPTER 3
Biotechnological Production of Coumarins
Alev Tosun*
Department of Pharmacognosy, Faculty of Pharmacy, Ankara University, 06100, Tandoğan, Ankara, Turkey
Abstract: Plants are useful sources of molecules for the development of new pharmaceutical products.
Coumarins are one of the most important secondary metabolites of plants and known as naturally occurring
benzo-α-pyrone derivatives from the metabolism of phenylalanine. Many kinds of coumarins such as
furocoumarins and pyranocoumarins arise from the biosynthetic pathway as being the substitution of the
coumarin ring following by some steps such as prenylation, cyclization or glycosylation. The coumarins
particularly exist in Apiaceae, Rutaceae, Fabaceae, Asteraceae and Rosaceae families, which have
considerable pharmacological properties and usages. To date, more than 1000 different types of coumarins
have been isolated from natural sources. Biotechnology is the most recent application in developing useful
products used in medicine or industry. The coumarin production was determined in the presence of some
precursors in suspensions. Thus, the biosynthesis of some coumarins has been carried out in cell cultures by
different types of applications. Moreover, dipyranocoumarins as cancer chemoprevention agents and
valuable dihydrocoumarin in flavor industry have been produced in callus cultures. Although the major
problem of these productions is very low efficiency; most of the coumarins especially important ones in
treatment are promising candidates for the production in the biotechnological process. In this chapter, these
procedures will be elucidated, and the coumarin production will be debated in the optimized systems in
relation to biotechnology under the light of recent articles. In addition, some beneficial information
concerning coumarins will be presented.
Keywords: Secondary metabolites, coumarins, furocoumarins, pyranocoumarins, biosynthetic pathway,

biological activity, phytochemistry, biotechnology, cell culture system, elicitor, suspension cultures.
1. INTRODUCTION
Natural biologically active pharmacopores have been produced by plants, fungi, bacteria and animals.
Natural compounds from plants are used as the source of medicine in all civilizations. Currently, many
natural products are explored to be new drugs depending on their ethnopharmacological background. Most
of them are already present in the pharmaceutical industry because of the generous structural diversity with
the interesting biological activities. Thus, natural compounds appear as novel therapeutic agents for the
future on behalf of mankind [1-4].
Coumarins are a group of natural compounds found in diverse plant sources. This group of secondary
metabolites of the plants is considered phytoalexins because of the defense substances. It has been reported
that especially 6-methoxy-8 - hydroxyl - 3 - methyl- 3, 4 dihydroisocoumarin belong to a different class of
the coumarin group known as isocoumarins, it is a phytoalexin that forms in carrot roots by fungal infection
and possesses notable antibiotic activity. Another example for the phytoalexins is for the
hydroxycoumarins. Hydroxycoumarins occur as secondary metabolites mostly in Rutaceae, Solanaceae,
and Apiaceae family. On the other hand, some of the hydroxycoumarins are constituent in some species
like scopoletin in sunflower and other plants that accumulate with mechanical wounding, insect feeding
damages, and fungal or bacterial infections. Thus, the group is also known as phytoalexins in these plants.
Hydroxycoumarins have various bioactivities and contribute essentially to the persistence of plants as
defence against phytopathogens, response to abiotic stresses, regulation of oxidative stress, and possibly
hormonal regulation [5]. Thereby, these groups of coumarins have remarkable therapeutic value following
commercial interest. So far, thousands of coumarins have been isolated from natural sources that comprise
*Address correspondence to Alev Tosun: Department of Pharmacognosy, Faculty of Pharmacy, Ankara University, 06100,
Tandoğan, Ankara, Turkey; E-mail: alevtosun@yahoo.com

Biotechnological Production of Coumarins Biotechnological Production of Plant Secondary Metabolites 37
of very large phenolic compounds [6, 7] with the interesting pharmaceutical utilizations. Thus, in this text,
some information in regard to biotechnological process will be given.
Coumarin was first isolated from tonka-fava bean (Dipteryx odorata or Coumarouna odorata) in 1820 by
Vogel. Thereby, the name of coumarin was given for this group of compounds. Coumarins are known in
a class of 2H-1-benzopyran-2-on derivatives, so called as benzo--pyrones which consists of the benzene
ring joined to a pyrone ring, and the oxygen atom present in the pyrone ring at -position. Coumarins are
also found as glycosides forms in the plants. Coumarin itself (Fig. 1) has a pleasant odor, like the the other
simple coumarins which give characteristic odor to grass. Many coumarins have been identified according
to their structure properties. Therefore, they are classified into the various groups [2, 4, 6, 8, 9] shown as
below, considering their structure diversity:
1. Simple coumarins
Hydroxylated, alkoxylated and alkylated derivatives with their glycosides (the substituent can
appear not only in benzene but also in the pyrone ring, or both)
2. Furocoumarins (Dihydrofurocoumarins)
1.1 Linear type (Psoralens)
1.2 Angular type (Angelicins)
3. Pyranocoumarins (Dhydropyranocoumarins)
1.3 Linear type (Xanthyletin type)
1.4 Angular type (Seselin type)
4. Coumarins substituted in the pyrone ring, for example;
1.5 4-hydroxycoumarins
1.6 3-phenylcoumarins
5. Benzocoumarins
6. Coumestans (e.g. Coumestrol)
7. Some complex structures involved coumarin system (Novobiocin, aflatoxin)
8. Isocoumarins (e.g. Cytogenin)
The biosynthetic pathway of coumarins has also been examined in plant physiology. In a biosynthetic
manner, the coumarins arise from phenylalanine metabolism, followed by photocatalyzed isomerization of
the double bond and eventuate with spontaneous lactonization [6, 8, 9].
5 4
4a
6 3
7
8 8a O 2 O
1
Figure 1: Coumarin (benzo--pyrone).

38 Biotechnological Production of Plant Secondary Metabolites Alev Tosun
Hydroxylation of umbelliferone brings about the formation of di- and tri- hydroxycoumarins. The
lactonization of corresponding cinnamic acid is a rare occasion, when compare to the hydroxylation
process. The prenylation of the benzene ring by dimethylallylpyrophosphate (DMAPP) in the structure of
hydroxycoumarins (6- and 7- position) constitutes furano- and pyranocoumarins, if it is 8-hydroxy-
coumarin, in this case angular homologs are formed [6, 8, 9].
Coumarins are mostly found free or in the glucosides type in Dicotyledonae families such as Apiaceae,
Rutaceae, Fabaceae, Hyppocastanaceae and Asteracea. In addition, the coumarins exist in some
Monocotyledons abundantly; on the contrary, the coumarins were not traced in lower plants except for a
few examples. They are mainly present in the leaves, fruits, roots and stems of the plants [6, 8, 9].
The physiological role of the coumarins in the plants is not utterly clear. However, it was identified that the
coumarins involved in growth regulation, absorb UV radiation, and protect plants against viral disorders [6, 8, 9].
Recently, the remarkable developments are present in plant tissue and cell culture technologies. The
biotechnological studies have been characterized with many applications. The applications have also
involved in obtaining secondary metabolites in plant cell cultures. Because of the preference to use natural
products, the sources have been widely investigated. The problem is the extinction of natural sources
excessively used for industrial purposes. Therefore, developing some secondary metabolites or
investigating under laboratory conditions for the biotechnological process would save the nature and its
resources. Consequently, biologically originated compounds would be acquired in the increasing demand
by humans.
Many interesting plants that contain coumarins are present in our diets, along with their economic and
therapeutic importance. Thus, in this section, along with useful information of the coumarins, the main
topic will be “biotechnological production of the coumarins” in the light of recently released articles.
2. BIOLOGICAL EFFECTS
Plenty of compounds isolated from plants have been investigated by many researchers for their biological
activities. However, many of them were not found to be suitable for therapeutic usages because of the side
effects which are toxic, mutagenic and carcinogenic. Nowadays, modifications and preparation of synthetic
derivatives of biologically active substances in order to reduce side effects and improve desirable outcomes
[9-11] are possible. A lot of coumarins have been identified in natural sources as well as their considerable
biological activities to promote human health and help reduce the risk of diseases such as the anti-
inflammatory, analgesic, antioxidant, antiallergic, hepatoprotective, anti-thrombotic, antiviral and
anticarcinogenic activities. In addition, coumarins act as anticoagulant, estrogenic, dermal photosensitizing,
antimicrobial, coronary-dilatator, mollucidal, antihelmintic, sedative, hypnotic and hypothermic agents.
Moreover, inhibition of 5-lipoxygenase, enzyme activity of the liver, monoamine oxidase and protein
kinases activity are observed on coumarins or coumarin consisting plant extracts [7, 9, 10, 12, 13].
Coumarins belong to a group of phenolic compounds exhibit wide diversity in their structures due to
substitutions of basic coumarin structure as much as various side chains in furo- and pyranocoumarins [10].
The structural diversity brings variety in biological effects. The coumarins have Isopentenyloxy-,
geranyloxy- and prenyloxy groups are very important side chains in coumarin structure. Oxyprenylated
natural compounds (isopentenyloxy-, geranyloxy- and the less spread farnesyloxy- compounds and their
biosynthetic derivatives) are also interesting groups of secondary metabolites. These compounds have
exhibited in vitro and in vivo interesting biological effects such as remarkable anti-cancer, anti-
inflammatory, anti-microbial and anti-fungal. Thus, these derivatives of coumarins have also important
biological effects probably attributed for their oxyprenyl side chains [14].
The history of original anti-thrombotic drugs has been reviewed in detail by Mueller (2004). In this
research, the coumarins are known as the classical anti-thrombotic agents [15]. Hovewer, currently some
coumarins have been already used for this purpose clinically.
CHAPTER 4
Novel Biomedical Agents from Plants
Athar Ata*
Department of Chemistry, The University of Winnipeg, 515 Portage Avenue, Winnipeg, MB, Canada R3B 2E9
Abstract: Natural product chemistry is playing a key role in providing structural diversity and this feature
makes them an important source of lead drug candidates to the drug discovery program. Nearly 50% of the
prescribed drugs available on the market are of natural product origin and 25% of these commercially
available drugs are of plant origin. Enzymes are responsible for performing several biochemical processes
including metabolisms, catabolism, signal transductions, cell development and their growth. Over
expression and hyper-activation of enzymes cause several human diseases. With the development of modern
techniques in the field of molecular biology and enzymology, over expression and hyper-activation of
enzymes in the body can easily be diagnosed that led to understand human diseases at the molecular level.
An understanding of diseases at the molecular level resulted in the successful applications of enzyme
inhibitors in clinics to treat these diseases. This chapter describes the discovery of novel glutathione S-
transferase, acetylcholinesterase and -glucosidase inhibitors from medicinally important plants and their
biomedical applications. Additionally, biotechnological method to produce potent bioactive compounds on a
large scale using biosynthetic information has also been proposed.
Keywords: Natural products, enzyme inhibition, glutathione S-transferase, acetylcholinesterase, -

glucosidase, phytochemistry, biosynthesis, isolation, pharmacophore, bioactivity.
1. INTRODUCTION
The importance of natural product chemistry is enormous in drug discovery program as it is one of the
major contributors to provide lead drug molecule. Natural product research has provided novel chemical
entities with desired bioactivities and potencies. Nearly 50% of the prescribed drugs to cure various
diseases are of natural product origin and 25% of these pharmaceuticals are of plant origin [1]. During the
last two decades, academic institutions and pharmaceutical industries considered the development of
combinatorial chemistry as a milestone in the drug discovery program. Undoubtedly, it is exceptionally cost
effective method in offering wide variety of chemical entities that are derivatives of naturally occurring,
pre-existing, working molecules. Unfortunately, it is impossible to emulate what the Mother Nature does.
The structural diversity with potent bioactivities against various biological targets that obtained from
different natural sources including plants and marine organisms is incredible in its range of multiplicity.
The molecules found in nature have resisted challenges of natural selections and overcame them during the
molecular evolution. An extensive research in the area of combinatorial chemistry has only provided one
drug, namely, sorafeinb (trade name, nexavar, manufactured by Bayer), and is used to treat kidney and liver
cancer. This molecule is in phase III clinical trail for thyroid cancer [2]. The complexity and diversity of
natural products is such that even through the combinatorial chemistry, their synthesis cannot be considered
by the existing methods. By studying the existing biological processes, we can find new naturally occurring
lead molecules potently effective against various diseases. Nearly 49% of 877 small molecules introduced
as pharmaceuticals between 1981-2002 are of natural product origin [2, 3]. Currently, natural product
chemists in collaboration with biochemists and cell biologist are actively involved in developing new in-
house bench-top bioassays that are compatible with in vivo-bioassays to perform rapid bioassay-guided
fractionations on crude extracts to isolate bioactive natural products [4].
Modern natural product research has provided a detailed understanding of the interaction of many
therapeutic agents with biological systems at a biochemical level. This information can be used as a tool to
*Address correspondence to Athar Ata: Department of Chemistry, The University of Winnipeg, 515 Portage Avenue, Winnipeg,
MB, Canada R3B 2E9: Tel: (204) 786-9389: E-mail a.ata@uwinnipeg.ca

54 Biotechnological Production of Plant Secondary Metabolites Athar Ata
discover new drugs against various ailments [5]. One of these aspects is to discover new natural products
inhibiting the activity of enzymes, which are over expressed or hyper-activated to cause human health
problems. These enzyme inhibitors can be used as pharmaceuticals to use them either as adjuvant to
improve chemotherapy or to cure diseases. In this chapter, novel glutathione S-transferase,
acetylcholinesterase and -glucosidase inhibitors, discovered in our lab, from plants and their biomedical
applications are described. A proposed biotechnological method to produce bioactive natural products,
using their biosynthetic information has also been discussed.
2. GLUTATHIONE S-TRANSFERASE INHIBITORS
Glutathione S-transferase (GST) (EC 2.3.1.18) is a multifunctional enzyme that protects cells from
cytotoxic and genotoxic stresses. GST catalyses the reaction between cytotoxic agents containing
electrophilic centers and glutathione to produce a chemically less reactive adduct [6]. This adduct is soluble
in water and can be excreted from the body. This enzyme has been suggested to play an active role in the
acquired drug resistance in the treatment of cancer and parasitic diseases. Anti-cancer and anti-parasitic
drugs contain electrophilic centers and can easily form adduct with glutathione, and will be excreted from
the body. This would lower the efficiency of anti-cancer and anti-parasitic chemotherapeutic agents [7].
Over expression of GST in various human cancers is discovered compared to the normal tissues [8, 9]. It
has also been documented in the literature that a 2-fold increase in GST activity was observed in
lymphocytes obtained from chronic lymphocytic leukemia (CLL) patients, resistant to chlorambucil when
compared with untreated CLL patients [10. 11]. Keeping these facts in view, it would be worthwhile to use
GST inhibitors as adjuvant during chemotherapy to overcome these acquired drug resistance problems.
There is an urgent need to discover new naturally occurring GST inhibitors as currently used GST
inhibitors exhibit either severe in vivo toxicity or are inactive in vivo. Toward this end, several medicinally
important plants were screened in GST inhibition assay in our research group and it was discovered that the
crude methanolic extracts of Caesalpinia bonduc, Artocarpus nobilis, Nauclea latifolia and Barleria
prionitis exhibited anti-GST activity with IC50 values of 83.0, 125, 10.5 and 160 g/ml, respectively. GST-
directed fractionations on these medicinally important plants were performed in order to isolate GST
inhibitors, present in them.
H3C
H 3C H3C
H 3C H3C
CH3
CH3
CH3 CH3 CH3
OH CH3 CH3
CH3 H3C
CH3 CH3 H 3C
H3C
HO (2) HO (3)
O (1)
O O O
O
O HO O
H3C
H
H3C H3C
H H CH3
H
CH3 CH3
H H
O O OAc
OH H AcO OH
MeO2C CH3 CH3
H3CO HO
(4) (5) (6) (7)
O
OH
OH CH3 O
H OH O
CH3 HO O
H
RO OH
H3C OAc H
O OH HO OH
OH
CH3 OAc
(9) R = H OH
(8) (10) R = CH3 (11)
Novel Biomedical Agents from Plants Biotechnological Production of Plant Secondary Metabolites 55
From the bioactive fractions of C. bonduc, 17-hydroxycompesta-4,6-dien-3-one (1), 13,14-seco-stigmasta-

5,14-dien-3-ol (2), 13,14-seco-9(11),14-dien-3a-ol (3), caesaldekarin J (4), neocaesalpin P (5),
neocaesalpin H (6), cordylane A (7), caesalpinin B (8), caesalpinianone (9), 6-O-methylcaesalpinianone
(10), and hematoxylol (11) were isolated [12-14]. Compounds 1-11 exhibited anti-GST activity with IC50
values of 380, 230, 248, 259, 200, 218, 250, 350, 16.5, 17.1 and 23.6 M, respectively. The bioactivity data
of all of these compounds indicated that homoisflavonoids, 10 and 11 have shown their potential as GST
inhibitors. The bioactivity data of these two compounds were more or less close to ethacrynic acid (IC50 =
16 M), a standard GST inhibitor [14].
CH3
H 3C CH3 H 3C H3C CH3 H3C
CH3 H
H H H3 C CH3 H CH3 H 3C CH3 H COO

H 3C CH3 H CH3 H
CH3 H CH3 H CH3
H CH3
AcO AcO HO
AcO H3C CH3
H 3C CH3 H3 C CH3
H 3C CH3
(12) (13) (14) (15)

H 3C CH3
H 3C HO OH H3C HO OH CH3 HO OH
H 3C H3C
O O H 3C
O O OH O O
O OH
CH3 H 3C CH3
CH3
OH O H3C
OH O CH3 OH O CH3
(16) (17) (18)
OH OH
HO O HO O
O OAc
CH3 O
OH O OH O O
CH3 HO
(19) OH
(20) OH
OHOH
GST inhibition-directed fractionations on the ethanolic extract of A. nobilis of Sri Lankan origin yielded
four known triterpenoids, cyclolaudenyl acetate (12), lupeol acetate (13), β-amyrine acetate (14), and
zizphursolic acid (15) along with five known flavonoids, artonins E (16), artobiloxanthone (17)
artoindonesianin U (18), cyclocommunol (19) and multiflorins A (20). Compounds 12-20 showed anti-GST
properties with IC50 values of 195.1, 146.1, 251.0, 68.5, 2.0, 1.0, 6.0, 3.0 and 14.0 M, respectively. The
higher potency of compounds 16-19 might be due to the presence of prenyl group in these compounds [15].
Phytochemical studies on the crude ethanolic extract of Nauclea latifolia resulted in the isolation of five
known compounds, strictosamide (21), naucleamides A (22), naucleamide F (23), quinovic acid-3-O--
rhamnosylpyranoside (24), and quinovic acid 3-O--fucosylpyranoside (25) from the bioactive fraction of
this plant [16]. Compounds (21-25) exhibited GST inhibitory activity with IC50 values of 20.3, 27.2, 23.6,
143.8, and 53.5 M, respectively. Compound 21 showed significant anti-GST properties and it was isolated
in a large quantity. It was, therefore, decided to carry out microbial reactions on this compound to prepare
its different analogues and to evaluate them for GST inhibition activity in order to study their structure-
activity relationships. To achieve this goal, we screened five different fungi, namely Mucor plumbeus
(ATCC 4740), Cunninghamella blakesleeana (ATCC 9245), C. echinulata (ATCC 9244), Curvularia
lunata (ATCC 12017), Rhizopus circinans (ATCC 1225) and Aspergillus niger (ATCC 1004), for their
capability to metabolize compound 21. During these biotransformation experiments, we discovered that R.
circinans metabolized compound 21 into 10-hydroxy-strictosamide (26), 10--glucosyloxyvincoside lactam
(27) and 16,17-dihydro-10--glucosyloxyvincoside lactam (28). A time-dependent biotransformation
experiment was also performed in order to determine the sequence for the formation of metabolites 26-28.
This experiment was carried out by incubating compound 21 in the liquid culture of R. circinans; this
afforded compounds 26 and 27. We incubated compound 27 in the liquid culture of this fungus to get
compound 28. These results indicated that R. circinans initially performed microbial hydroxylation at C-10
CHAPTER 5
Production of Anthocyanins by Plant Cell and Tissue Culture Strategies
Claudia Simões*, Norma Albarello, Tatiana C. de Castro and Elisabeth Mansur
Abstract: Plant cell and tissue culture strategies provide a valuable tool for the production of plant chemicals
and have been extended to commercial use for biosynthesis of various high-value metabolites of importance
to pharmaceutical, food and chemical industries. In this chapter, different systems for the production of
anthocyanins under in vitro conditions are discussed. Anthocyanins are used to color food as a substitute of
synthetic red dyes and recently, great attention has been focused on their multifaceted pharmacological
potential. In vitro production of these pigments has been obtained from several plant species. Most systems
are based on the use of callus and cell suspension cultures, although organ cultures have also been studied.
Several studies on the regulation of anthocyanins biosynthesis under in vitro conditions have been reported,
although additional research is still necessary in order to allow commercial production. In general, these
studies have shown that anthocyanins biosynthesis is strongly influenced not only by physical conditions as
light and temperature, but also by other parameters such as osmotic pressure, hormones, basal medium
composition and nutrient stress. Strategies such as elicitation and use of conditioned medium have also been
reported. In addition, the use of in vitro technologies has allowed the production of anthocyanins that usually
are not found in field-grown plants. Large scale production of these pigments in standardized conditions
remains as one of the great challenges for researchers in plant biotechnology.
Keywords: Anthocyanins, pigment, plant tissue culture, plant cell culture, secondary metabolites, large-
scale production, callus culture, biomass accumulation, biosynthesis, physical factors, chemical factors,
plant growth regulators, elicitation, biotechnology.
1. GENERAL ASPECTS
Anthocyanins (Greek anthos, flower and Greek kyanose, blue) are pigments widely distributed in the plant
kingdom that are responsible for most of the scarlet to blue colors in plants (reviewed by Strack and Wray [1];
Tanaka et al. [2]. They are members of the flavonoid class and derived from the basic structure of the flavylium
cation, which is composed of two aromatic rings connected by a three-carbon unit and condensed by an oxygen
atom. These compounds consist of an aglycone (anthocyanidin) esterified with one or more sugars that stabilize
the molecule. The glycosylation occurs more frequently in position 3, followed by position 5. Glucose,
rhamnose, xylose, galactose, arabinose and fructose are sugars commonly found in anthocyanins, although
other molecules might also be present, such as rufinose, sophorose, sambubiose and gentiobiose. Sugars
residues are often acylated with aromatic acids that include -coumaric, ferulic, caffeic, sinapic, gallic or -
hydroxybenzoic acid, and/or aliphatic acids such as acetic, succinic, oxalic or malic acid [3]. Anthocyanin
modifications contribute to the colour shade, as well as to stability and solubility of the pigment. About eighteen
aglycones occur naturally, specially cyanidin, delphinidin, malvidin, pelargonidin, cyanidin and petunidin.
Cyanidin is the most frequent (50%), followed by delphinidin, malvidin and pelargonidin (12% each), while
peonidin and petunidin represent each about 7% of the aglycones present in nature [4].
Anthocyanins are derived from the amino acid phenylalanine following a common pathway with most other
flavonoids. This is one of the most studied biosynthetic pathways involved in the production of secondary
metabolites in plants, at both biochemical and genetic levels, with most of the enzymes and genes already
characterized [5-7], demonstrating the interest that these substances have awakened among researchers
(reviewed by Schwinn and Davies [7]).
*
Address correspondence to Claudia Simões: Núcleo de Biotecnologia Vegetal, Universidade do Estado do Rio de Janeiro, Brazil;
Email: labplan_uerj@yahoo.com.br

68 Biotechnological Production of Plant Secondary Metabolites Simões et al.
The color of the same anthocyanin can vary depending on the pH. An intense red color is exhibited under
pH < 3, which tends to became non-colored when the pH value is increased to 4 - 5, and blue at pH values
higher than 7 [8]. Anthocyanin color is also influenced by copigmentation, resulting from interactions with
substances like alkaloids, amino acids, nucleosides and different flavonoids, including different
anthocyanins [9].
Due to the variety of colors provided by anthocyanins, the most striking function of the presence of these
pigments in plants is the attraction of pollinators. However, the fact that some species with anemophily
pollination present anthocyanins, and that their presence is also found in parts of plants that are not
involved in signaling to pollinators, suggest that these flavonoids have other functions in plants [10]. For
example, anthocyanins act as filters of ultraviolet radiation on leaves and protect the photosynthetic
apparatus from photoinhibition [11]. In some species, anthocyanins are associated with resistance to
pathogens [12, 13] and herbivores [14]. Several studies have also shown that anthocyanins accumulate as a
result of mechanical injury [15] and nutritional deficiencies [16]. Generally, these pigments increase the
antioxidant response in tissues affected by biotic or abiotic stress factors in order to maintain their
physiological stability [17].
Anthocyanins have been isolated from plants and characterized from several points of view. Most studies
on the identification, purification, separation and quantification of these pigments were performed using
different methods that include paper chromatography, thin-layer chromatography, column chromatography,
solid phase extraction, counter current chromatography, UV–visible absorption spectroscopy, high
performance liquid chromatography (HPLC), mass spectrometry (MS) and nuclear magnetic resonance
spectroscopy [18-21]. Recent advances in anthocyanins chemical investigation were reviewed by
Castañeda-Ovando et al. [22].
2. COMMERCIAL AND MEDICINAL INTEREST
Anthocyanins occur in relatively high concentrations in the human diet and have an especial nutraceutical
interest [4]. These pigments have also been used industrially as natural colorants for food and beverages
[23], since the use of synthetic dyes has been reduced due to adverse effects to human health [24]. It is also
important to highlight the great potential of using these natural pigments in the textile and cosmetics
industries [25, 26]. Although anthocyanins are widely spread in nature, there are few commercially usable
sources of these pigments. They are mainly extracted from the residual grape skins and seeds obtained as
by-products of wine production, but seasonal variations make the reproducibility and uniformity of the
color difficult to maintain [3].
Medicinal activities of anthocyanins have also been intensively studied [27, 28]. The best documented
activity is their antioxidant potential [29-31]. The role of anthocyanins as cancer chemo-preventive agents
has also been demonstrated [4, 32, 33]. In addition, studies linking chromosomal alterations and DNA
cleavage with the incidence of cancer [34] have increased the interest in antimutagenic and antigenotoxic
activities of anthocyanins [35-37]. Positive effects in preventing cardiovascular diseases [38-40] and
inflammation [41-43] have also been reported. Additionaly, beneficial anthocyanins effect have observed
on test involving diabetes and obesity prevention [44, 45], improvement of visual acuity [46] and the
prevention of the decline in neural function caused by aging, besides improving memory capacity [47, 48].
Table 1 contains additional information on anthocyanins medicinal properties.
Table 1: Medicinal properties investigated in anthocyanins.
Biological Activity Plant Source Anthocyanin/Extract Refs

Anti-angiogenic Wild blueberry, bilberry, Twenty different combinations of six berry extracts [32]
cranberry, elderberry, raspberry
seeds, and strawberry
Anti-angiogenic Eggplant peels (Solanum Nasunin (delphinidin-3-(p-coumaroylrutinoside)-5-glucoside) [49]
melongena, L. var. esculentum,
Ness.)
Production of Anthocyanins Biotechnological Production of Plant Secondary Metabolites 69
Table 1: cont…
Anti-carcinogenic Berry Berry extracts [50]
Anti-carcinogenic Vitis coignetiae Anthocyanin pigments: delphinidin-3, 5-diglucoside; cyanidin-3, 5-diglucoside; [51]
petunidin-3, 5-diglucoside; delphinidin-3-glucoside; malvidin-3, 5-diglucoside;
peonidin-3, 5-diglucoside; cyanidin-3-glucoside:petunidin-3-glucoside; peonidin-3-
glucoside; malvidin-3-glucoside
Anti-carcinogenic Sweet potato (Ipomoea batatas Anthocyanin-rich aqueous extracts from in vitro produced tissue [52]
L.)
Anti-carcinogenic Grape rinds and red glutinous Anthocyanidins [53]
rice
Anti-carcinogenic Red Soybeans and Red Beans Anthocyanin fraction [54]
Anti-carcinogenic Red and white wines Methanol extracts [55]
Anti-carcinogenic Roth (Karlsruhe, Germany) Aglycons: Cyanidin and delphinidin [56]
Anti-carcinogenic Fruits and berries Anthocyanidins, anthocyanins or anthocyan-containing fruit/vegetable extracts [4]
Antigenotoxic Eggplant (Solanum Anthocyanin: delphinidin [37]
melanogena)
Anti-inflammatory Tart cherries Aglycone: cyanidin [41]
Anti-inflammatory Saskatoon berries (Amelanchier Anthocyanins/anthocyanidins: pelargonidin, cyanidin, delphinidin, peonidin, malvidin, [43]
alnifolia Nutt.), blueberries, malvidin, 3-glucoside, and malvidin 3, 5-diglucosides and anthocyanin-rich crude
blackberries, and black currants extracts and concentrates of selected berries
Antimutagenic Sweet potato Ayamurasaki Anthocyanin derivatives deacylated: 3-sophoroside-5-glucoside of cyanidin and 3- [57]
variety sophoroside-5-glucoside of peonidin
Antimutagenic Sweet potato Ayamurasaki Anthocyanins: 3-(6, 6’-caffeylferulylsophoroside)-5-glucoside of cyanidin and 3-(6, 6’- [35]
variety caffeylferulylsophoroside)-5-glucoside of peonidin
Antioxidant Italian red wine Anthocyanin fraction [58]
Antioxidant Wild blueberry, bilberry, Twenty different combinations of six berry extracts [32]
cranberry, elderberry, raspberry
seeds, and strawberry
Antioxidant Flowers of Hibiscus sabdariffa Hibiscus pigments [59]
L.
Antioxidant Litchi (Litchi chinenesis Sonn.) Cyanidin-3-rutinside [60]
Antioxidant - Anthocyanin/anthocyanidin: cyanidin 3-O--D-glucoside and cyanidin [61]
Antioxidant Phaseolus vulgaris L. Anthocyanin pigments: pelargonidin 3-O--D-glucoside, cyanidin 3-O--D-glucoside [62]
and delphinidin 3-O--D-glucoside; and their aglycons: pelargonidin chloride,
cyaniding chloride, and delphinidin chloride
Antioxidant Petals of roselle (Hibiscus Delphinidin-3-sambubiose [63]
Sabdariffa L.)
Antioxidant Sweet potato (Ipomoea batatas Anthocyanin-rich aqueous extracts from in vitro produced tissue [52]
L.)
Antioxidant Red wine type Cabernet Anthocyanin dye [44]
Antiulcer Vaccinium myrtillus Anthocyanosides [64]
Bacteriostatic Malva sylvestris Anthocyanin fraction [65]
Cardiovascular Cranberry Cranberry extracts [66]
protection
Decrease lipid Phaseolus vulgaris L. Anthocyanin pigments: pelargonidin 3-O--D-glucoside, cyanidin 3-O--D-glucoside [62]
peroxidation and delphinidin 3-O--D-glucoside; and their aglycons: pelargonidin chloride,
cyaniding chloride, and delphinidin chloride
DNA triplex Grape (Vitis vinifera), Rose Anthocyanins: 3-O-β-D -glucopyranoside of malvidin, peonidin, delphinidin, petunidin [67]
stabilization (Rosa gallica), Malva (Malva and cyanidin
property sylvestris)
DNA-damaging - Delphinidin, delphinidin-3-glycoside, delphinidin-3-rutinoside, cyanidin, cyanidin-3- [36]
protection glycoside, cyanidin-3-rutinoside
Hypoglycemic Red wine type Cabernet Anthocyanin dye [44]
Protective against Cranberry Cranberry extracts [66]
atherosclerosis
Hepatoprotector Petals of H. rosasinensis Cyanididin derivative [68]
Hepatoprotector Purple-colored sweetpotato Crude anthocyanin extracts [69
]
Protective effect Aronia melanocarpa Anthocyanin dyes [70]
against pancreatitis
Antioxidant Litchi (Litchi chinenesis Sonn.) Cyanidin-3-rutinside [60]
3. ANTHOCYANIN PRODUCTION UNDER IN VITRO CONDITIONS
The stability of anthocyanins is influenced by several factors, particularly their chemical structure, pH,
temperature, light, presence of oxygen and interactions with other molecules [71]. The large number of
CHAPTER 6
In Vitro Organ Cultures of the Cancer Herb Castilleja tenuiflora Benth. as
Potential Sources of Iridoids and Antioxidant Compounds
Gabriela T.-Tapia1,*, Gabriel R.-Romero1, Alma R. L.-Laredo1, Kalina B.-Torres1
and Alejandro Zamilpa2
1
Nacional, P. O. Box 24, 62730, Yautepec, Morelos, México; 2Centro de Investigación Biomédica del Sur,
Instituto Mexicano del Seguro Social, Argentina No. 1, 62790, Xochitepec, Morelos, México
Abstract: Castilleja tenuiflora Benth. (Scrophulariaceae, “cancer herb”) is a wild plant widely
recommended in Mexican folk medicine to treat tumors. Root and shoot cultures of this species were
established for the production of secondary metabolites with cytotoxic and antioxidant activities. Root
cultures were initiated from root tips induced in leaf explants from wild-grown plants using MS medium and
10 µM -naphthalene acetic acid. Shoots spontaneously formed in 30-35 days and were excised. They
presented continuous multiplication and elongation during subsequent subculture in free-hormone liquid
medium. Antioxidant activity was measured using three in vitro models [scavenging of free radicals with
1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid)
diammonium salt (ABTS), and the transition metal reduction in the phosphomolybdenum assay], and the
strongest activity (p<0.05) was found in methanol extracts from shoot cultures. The highest contents of the
phenolic compounds, flavonoid and iridoids, were also found in shoot cultures (p<0.05). Total phenolic
compound levels correlated significantly with the antioxidant activity (p<0.05). Bioautography within TLC
using DPPH as a detection reagent indicated that quercetin 3--D-glucoside is one of the predominant
contributors to the free radical scavenging of C. tenuiflora organ cultures. Aucubin, an iridoid with cytotoxic
activity, was also found in the in vitro cultures. C. tenuiflora organ cultures are alternative sources for
iridoids and natural antioxidants such as flavonoids and other phenolic compounds.
Keywords: Castilleja tenuiflora, Scrophulariaceae, oxidative stress, secondary metabolites, iridoid,

biotechnology, micropropagation, cell suspension culture, callus, explant, organogenesis, root culture, shoot
culture, chemical composition.
1. INTRODUCTION
Oxidative stress is the result of the overproduction of oxygen free-radicals in cells and tissues. At high
concentrations, free radicals are hazardous for living organisms and damage all major cellular constituents.
Free-radicals increase the susceptibility of animal cells to irreversible injury by oxidative intoxication through
processes such as lipid peroxidation, protein oxidation, protein inactivation or disturbance in calcium
homeostasis [1, 2]. Because oxidative stress has been implicated in the pathology of many diseases,
inflammatory conditions, cancer and aging, there is a growing interest in plants with antioxidant and free-
radical-scavenging properties [3, 4]. However, in some cases, the availability of the bioactive compounds from
these natural sources is either limited or their abundance in plants is insufficient to meet demand. Plant cell
culture represents a biotechnological alternative to extraction from field-grown plants for the production of
secondary metabolites with pharmacological value. Biotechnology provides the possibility to produce
standardized material independent of environmental conditions [4]. The possibility of alterations in the
metabolic profiles of in vitro cultures that might affect the biological activity of the plant should be considered.
It is, thus, important to validate that the biological activity of the in vitro cultures [4-6].
Biosynthesis and accumulation of secondary metabolites in plants is generally related to cellular and tissue
differentiation [7]. In an attempt to obtain high-producing cultures, organized cultures have been proposed
*Address correspondence to Gabriela T.-Tapia: Departamento de Biotecnología, Centro de Desarrollo de Productos Bióticos,
Instituto Politécnico Nacional, P. O. Box 24, 62730, Yautepec, Morelos, México; Tel.: +52 735 39 4 20 20; Fax: +52 735 39 4 18 96;
E-mail: gttapia@ipn.mx
88 Biotechnological Production of Plant Secondary Metabolites T.-Tapia et al.
instead of undifferentiated cultures as an alternative to agricultural processes for producing valuable

phytochemicals [8, 9]. Roots, shoots and embryos have been cultured in vitro. Organ cultures provide
similar patterns and levels of secondary metabolites as the whole plant [10]. Moreover, organ cultures are
also appropriate for germplasm conservation and propagation.
Mexico is one of the most biodiverse countries of the world (mega diverse) and is well-known for its
ancient traditional knowledge in the use of plants to treat ailments. More than 10% of the 30, 000 species of
the Mexican flora are used in traditional medicine [11, 12]. However, scientific research on Mexican
medicinal plants is still scarce. Some plant species, known as “cancer herbs”, are recommended by
“chamanes” for the treatment of tumors and related diseases. At least 12 species belonging to several
families (Amaranthaceae, Euphorbiaceae, Sterculiaceae, Lythraceae and Scrophulariaceae) are known
under this name [13]. One of the most popular cancer herbs is Castilleja tenuiflora Benth.
(Scrophulariaceae) [14].
Castilleja tenuiflora has been used in folk medicine since the 16th century to heal tumors and other diseases
[15, 16]. Its propagation by seeds is extremely low, and, to meet demand, it is gathered from the wild.
Moreover, its habitat is exposed to falling trees and fires. Although it is largely used in traditional medicine,
little scientific research has so far been conducted on it [17].
In this work, we review what is known about Mexican medicinal plant C. tenuiflora and show results
pertaining to the establishment of organ cultures as sources for iridoids and other antioxidant compounds
such as phenolic compounds and flavonoids. Also, we present a brief review of the current status of
biotechnological research on other medicinal plants from related species of Scrophulariaceae.
2. CURRENT BIOTECHNOLOGICAL RESEARCH ON MEDICINAL PLANTS FROM

RELATED SPECIES (SCROPHULARIACEAE)
The family Scrophulariaceae belongs to the order Lamiales and contains approximately 200 genera
distributed worldwide. Most research has focused on Scrophulariaceae members as parasitic species because
of their suitability for investigations of molecular evolutionary processes, gene functions, and the evolution
of parasitism [18]. Recently, some authors proposed the reclassification of the parasitic and hemiparasitic
species into another family, the Orobanchaceae [19, 20]. However, the old classification as Scrophulariaceae
allows us to understand the chemical nature of the members of this family. Many members of the
Scrophulariaceae are well-known because of their use in many traditional medicines (Table 1). In general,
Scrophulariaceae species have been reported to be rich in bioactive compounds such as saponins, diterpenes,
triterpenes, iridoids, flavonoids and phenolic glycosides.
Table 1: Examples of Scrophulariaceae plants used in traditional medicines.
Species Traditional Medicine Refs

Agalinis spp. Andinean [21]
Anthirrhinum linarium Mexico [22]
Bacopa monnieri India [23]
Calceolaria spp. Andinean [24]
Castilleja arvensis Mexico (Nahua) [25]
Castilleja lithospermoides Mexico (Yecora) [25]
Castilleja tenuiflora Benth. Mexico (Nahua) [16]
Digitalis purpurea Mexico [22]
Halleria lucida Zulus (Africa) [26]
Neopicrorhiza scrophulariflora Himalaya [27]
Paulownia tomentosa China [28]
In Vitro Organ Cultures of the Cancer Herb Biotechnological Production of Plant Secondary Metabolites 89
Table 1: cont….
Pedicularis resupinata South Korea [16]

Penstemon barbatus
Mexico [15, 29]
P. rosseus
Picria fel-terrae China [16]
Picrorhiza kurroa Ayurvedic [30]
Rehmannia glutinosa China [31]
Scoparia ducis India; Bolivia (Kallawaya) [32]
Scrophularia yoshimurae China [33]
Scrophularia ningpoensis Vietnam [34]
Scrophularia nodosa Mexico [22]
Sibthorpia peregrine Madeira [16]
Veronica persica Peru [16]
Biotechnological research on medicinal plants from Scrophulariaceae has focused on two main aspects. The
first main area of research includes the development of propagation systems, which would help in
commercial cultivation and conservation of the germplasm of medicinally important species, especially
those considered “rare” or endangered. Propagation systems may be also useful for the transformation of
plants and in basic research (Table 2). The second main area of research consists of the evaluation of the
potential of in vitro cultures to produce bioactive compounds (Table 3).
Of the species listed in Table 1, Rehmannia glutinosa and Bacopa monnieri are the best studied from a
biotechnological point of view. Rehmannia glutinosa (“Chinese foxgloves”) has a very high value in
traditional Chinese medicine and displays a variety of beneficial effects and pharmacological actions that
are attributed mainly to the presence of catalpol, which is the major iridoid constituent from rhizomes [31].
This plant is propagated conventionally through roots because seeds have poor viability and low
propagation rates, which delays root harvests [35, 36]. Recently, Park et al. [31] reviewed the current
information regarding the application of plant biotechnology for this species.
Bacopa monnieri (“brahmi”) is an important medicinal plant mainly used for the treatment of neurological
disorders and depression. For this species, propagation systems through organogenesis and somatic
embryogenesis have been developed. In almost all cases, the resulting plantlets were successfully
transplanted to soil. By random amplified polymorphic DNA analysis, no polymorphisms were detected,
which reveals that the genetic integrity of micropropagated plants was high [39]. Morphogenetic potential
and shoot growth were found to depend on the type of explant [42-44], gelling agents [43] and plant growth
regulators used [39]. However, the type and concentration of cytokinins were found to be the main factors
influencing shoot multiplication [23, 41]. The usefulness of additives such as the fungicide bavistin for
mass propagation [42], and the phenolic compounds such as phloroglucinol and chlorogenic acid for root
induction has also been tested [39].
Table 2: Examples of medicinal plants from Scrophulariaceae established for micropropagation programs.
Species In Vitro Culture Refs

Antirrhinum majus Hairy roots; shoots [37, 38]
Bacopa monnieri Adventitious shoots [23, 39-44]
Paulownia tomentosa Shoots [45, 46]
Rehmannia glutinosa Plantlets [35, 36]
Scrophularia yoshimurae Shoots [47, 48]
Verbascum thapsus Adventitious shoots [49]
CHAPTER 7
Plant Cell Tissue and Organ Cultures in Terpenoids
Irem Tatli I.
Department of Pharmaceutical Botany, Faculty of Pharmacy, Hacettepe University, Sihhiye, 06100,

Ankara, Turkey
Abstract: This chapter deals with the production of terpenoids, including pharmaceuticals and food
additives through plant cell cultures, shoot cultures and root cultures obtained through biotechnological
means. Plant cell and hairy root cultures are promising potential alternative sources for the production
of terpenoids of industrial importance. Several strategies have been adopted for the enhancement of
metabolites. There has been tremendous success in the production of terpenoids such as capsaicin from
cell cultures of Salvia officinalis and Capsicum annuum. Procedures for the commercial production of
paclitaxel (Taxol®) by this technique are in advanced stages of development and may soon be
employed for the manufacture of these important drugs. The levels of sugar, nitrate, phosphate and
growth regulators have been shown to affect the productivity of secondary metabolite-accumulating
cultures. Precursor feeding has also been an obvious and popular approach to increase metabolite
production in plant cell cultures. Culture environmental conditions such as light, temperature, medium
pH and oxygen have been examined for their effect upon terpenoids accumulation in many types of
cultures. Organ cultures are relatively more stable. There are a number of medicinal plants whose shoot
cultures have been studied for terpenoids. Similarly, root cultures are valuable sources of medicinal
compounds. Until now, there is no commercial process as an alternative for root-derived compounds,
except in case of utilizing hairy root culture systems. The ability of Agrobacterium rhizogenes to induce
hairy roots in a range of host plants has lead to studies on it as a source of root-derived pharmaceuticals
and several hairy roots have been put to scale–up studies in bioreactors. Most remarkable developments
of scale-up in large vessels have been in the cultivation of Panax ginseng hairy root biomass.
Keywords: Terpenoids, secondary metabolites, biotechnology, iridoid, saponoside, micropropagation,

plant cell culture, organ culture, biotransformation, plant growth regulators, precursor, elicitation.
1. INTRODUCTION
Plants are a valuable source of a wide range of secondary metabolites, which are used as pharmaceuticals,
agrochemicals, flavours, fragrances, colours, biopesticides and food additives [1]. In 1985, of the 3500 new
chemical structures identified, 2600 came from the higher plants. Worldwide, 121 clinically useful
prescription drugs are derived from plants. Today, 75% of the world’s population relies on plants for
traditional medicine. In the US, where chemical synthesis dominates the pharmaceutical industry, 25% of
the pharmaceuticals are based on plant-derived chemicals [2]. Plants will continue to provide novel
products as well as chemical models for new drugs in the coming centuries, because the chemistry of the
majority of plant species is yet to be characterized [3]. Despite advancements in synthetic chemistry, we
still depend upon biological sources for a number of secondary metabolites including pharmaceuticals [4].
Elaborative pathways from basic primary metabolites, which are synthesized immediately as a result of
photosynthetic activity, produce secondary metabolites. Many of them are unique to the plant kingdom and
are not produced by microbes or animals. However, with the advancement of transgenic research, it is
possible to produce compounds and molecules, which were also not originally synthesized in plants.
Plant secondary compounds are usually classified according to their biosynthetic pathways. Four large
molecule families are generally considered: polyketides, shikimates, terpenes and steroids, and alkaloids. A
good example of a widespread metabolite family is given by terpenoids: because they are widely distributed
*Address correspondence to Irem Tatli I.: Department Of Pharmaceutical Botany, Faculty of Pharmacy, Hacettepe University,
Sihhiye, 06100, Ankara, Turkey; E-mail: itatli@hacettepe.edu.tr

108 Biotechnological Production of Plant Secondary Metabolites Irem Tatli I.
in the plant kingdom. Due to their large biological activities, plant secondary metabolites have been used
for centuries in traditional medicine. Nowadays, they correspond to valuable compounds such as
pharmaceutics, cosmetics, fine chemicals, or more recently nutraceutics. Recent surveys have established
that in western countries, where chemistry is the backbone of the pharmaceutical industry, 25% of the
molecules used are of natural plant origin. A good example could be aspirin (acetylsalicylate) which
derives from salicylate. The genuine molecule can be isolated in large quantities from many plants (Spiraea
ulmaria, Betula lenta…), but the chemical is synthesized as an acetyl-derivative in order to lower
secondary effects (stomachaches). Chemical synthesis apart, the production of plant secondary metabolites
has, for a long time, been achieved through the field cultivation of medicinal plants. However, plants
originating from particular biotopes can be hard to grow outside their local ecosystems. This has led
scientists and biotechnologists to consider plant cell, tissue and organ cultures as an alternative way to
produce the corresponding secondary metabolites [5].
Many constituents of volatile oils are unsaturated hydrocarbons (terpenes), with the molecular formula
C10H16, or alcohols and ketones, with similar skeletons. They could conceivably arise by a combination of
two branched C5 units, such as isoprene. Other compounds of this type are larger molecules containing 15,
20, 25, 30 or 40 carbon atoms. All these can be regarded as products resulting from the condensation and
hydrogenation of isoprene units. The compounds are called monoterpenes (C10), sesquiterpenes (C15),
diterpenes (C20), sesterterpenes (C25), triterpenes (C30) and tetraterpenes (C40). Also the steroids can be
fitted into this scheme if they are regarded as modifications of triterpenes. All these substances, the basic
skeleton of which may be conceived as derived from isoprene, are called isoprenoids. The hypothesis of
their biogenesis was developed especially by Ruzicka L., during the 1920s and 1930s and was named the
isoprene rule. In order to prove this rule, there was a long search to find the so-called ‘active isoprene’, i.e.
the C5 unit that could be incorporated into the isoprenoids. The solution came in 1956, when mevalonic
acid was isolated and found to be very readily built into isoprenoids. It is true that mevalonic acid is a C6
compound, but, before it takes part in biosynthetic reactions it is converted into isopentenyl diphosphate,
which thus represents the long sought ‘active isoprene’. It will be shown below that mevalonic acid is
formed from acetate and is thus a key substance in the second main biosynthetic pathway from acetate
which thus has been called the mevalonic acid pathway. However, since it has now been shown that
mevalonic acid is not the only precursor for isopentenyl diphosphate, it seems reasonable to change the
name of this pathway to the isopentenyl diphosphate pathway [6].
The iridoid and saponin glycosides are widely distributed in the genus Verbascum. A number of iridoid and
saponin glycosides isolated from Verbascum species growing in Turkey by our laboratory group. Although
the taxonomic and morphological aspects of the genus Verbascum appear more or less complex, the
frequent occurrence of the iridoid and saponin glycosides in the Scrophulariaceae has been used in
chemotaxonomic studies. Iridoids and saponins display an interesting spectrum of biological activity [7-
14].
Biothechnology offers an opportunity to exploit the cell, tissue, organ or entire organism by growing them
in vitro and to genetically manipulate them to get desired compounds. Many facets of biotechnological
approaches can be envisaged.
Aspects of biotechnological approaches to plant-derived secondary metabolites:
1. Plant cell tissue and organ cultures.
-Cell culture
-Shoot culture
-Root culture
-Scale-up of cultures
Plant Cell Tissue and Organ Cultures in Terpenoids Biotechnological Production of Plant Secondary Metabolites 109
2. Transgenic plants/organisms.
-Metabolic engineering
-Heterologous expression
-Molecular forming
3. Micropropagation of medicinal plants.
-Endangered plants
-High-yielding varieties
-Metabolically engineered plants
4. Newer sources.
-Algae
-Other photosynthetic marine forms
5. Safety considerations.
Since the world population is increasing rapidly, there is extreme pressure on the avaliable cultivable land
to produce food and fulfill the needs. Therefore, for other uses such as production of pharmaceuticals and
chemicals from plants, the avaliable land should be used effectively. Hence, it is appropriate to develop
modern technologies leading to plant improvement for better utilization of the land to meet the
requirements [15].
Studies on plant secondary metabolites have been increasing over the last 50 years. These molecules are
known to play a major role in the adaptation of plants to their environment, but also represent an important
source of active pharmaceuticals. Plant cell culture technologies were introduced at the end of the 1960s as
a possible tool for both studying and producing plant secondary metabolites. Different strategies, using in
vitro systems, have been extensively studied with the objective of improving the production of secondary
plant compounds. Undifferentiated cell cultures have been mainly studied, but a large interest has also been
shown in hairy roots and other organ cultures. Specific processes have been designed to meet the
requirements of plant cell and organ cultures in bioreactors [5].
The development of micropropagation methods for a number of medicinal plant species has been already
reported and needs to be adopted [16]. Cryopreservation of cells is an area of importance in the
conservation of medicinal plants. It has already been used in many plant species. The development and
adoption of plant cell culture and organ culture methods have lead to the production of plant products on a
large scale. The improvements in molecular biological research have given a new dimension to in vitro
culture as well as for plant improvement, enhancing the yields of the product and resulting in multiple
products or producing novel products from genetically engineered plants. These factors have laid emphasis
on the use of biotechnological methods to enhance the production of pharmaceuticals and food additives,
both in quality and quantity [15].
2. PLANT CELL CULTURE AS A SOURCE OF TERPENOIDS

Plant cell cultures are an attractive alternative source to whole plant for the production of high-value
secondary metabolites (For the groups of terpenoids: Carotenes, Monoterpenes, Sesquiterpenes, Diterpenes,
Triterpenes that were so far isolated from tissue and suspension cultures of higher plants) (Table 1). Plant
CHAPTER 8
Bioactive Chemical Constituents and Biotechnological Production of
Secondary Metabolites in Amaranthaceae Plants, Gomphreneae Tribe
Marcos J. Salvador1,*, Nathalia L. Andreazza1, Aislan C.R.F. Pascoal1, Paulo S.
Pereira2, Suzelei C. França2, Orghêda L.A.D. Zucchi3 and Diones A. Dias3
a
Campinas (UNICAMP), C.P. 6109, 13083-970, Campinas, SP, Brazil; bUnidade de Biotecnologia,
Universidade de Ribeirão Preto (UNAERP), 14096-900, Ribeirão Preto, SP, Brazil and cFaculdade de
Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo (USP), Via do Café, s/n, 14040-
903, Ribeirão Preto, SP, Brazil
Abstract: The Amaranthaceae family comprises of many species, which are used in nutrition and in
traditional folk medicine for the treatment of several diseases such as infections, inflammation and
fever. Gomphrena, Pfaffia and Alternanthera species are used in the extraction of natural pigments such
as betaxanthin and betalains for application as food colorants and antioxidants. Pfaffia paniculata
(Brazilian ginseng or Suma) has been indicated as a tonic, as well as having aphrodisiac, analgesic, and
antidiabetic properties and may act against cancer. Gomphreneae is the major tribe of Amaranthaceae
and previous chemical analyses have demonstrated the occurrence of anthraquinones, aurone,
betacyanins, betaxanthins, betalains, chromoalkaloids, ecdysteroids, flavonoids, protoalkaloids,
saponins, steroids and triterpenes. Biotechnological investigation with Amaranthaceae plants from the
Gomphreneae tribe, demonstrated their potential for bioprospection of bioactive natural compounds
such as flavonoids, steroids, terpenoides and saponins. Plant cell cultures, nowadays, are an important
strategy for bioprospection of natural products. The in vitro large-scale production of bioactive
compounds or extracts used as phytotherapics, pharmaceutical products, food additives and cosmetics
should be encouraged because of their scientific, economical or ecological importance. Therefore, the
present chapter reviews the literature data of the bioactive chemical constituents and biotechnological
production of secondary metabolites in Amaranthaceae plants (Gomphreneae tribe), species that have
many pharmacological properties and other applications.
Keywords: Traditional folk medicine, Amaranthaceae, Gomphrena, Pfaffia, Alternanthera, secondary

metabolites, biological activity, biotechnological production, chemical diversity, chemical composition,
saponin, triterpen, flavonoid.
1. INTRODUCTION
The Amaranthaceae family comprises of many species, which are used in nutrition and in traditional folk
medicine for the treatment of several diseases such as infections, inflammation and fever [1-6]. Members of this
plant family are used in the extraction of natural pigments such as betaxanthin, and betalains for application as
food colorants and antioxidants [4]. The Amaranthaceae family includes approximately 65 genera and 1000
species of annual and perennial herbaceous plants, shrubs and some trees occurring in tropical, subtropical and
temperate zone of Africa, South America and South East Asia [7, 8]. Previous chemical analyses have
demonstrated the occurrence of anthraquinones, aurone, betacyanins, betalains, betaxanthins, chromoalkaloids,
ecdysteroids, flavonoids, protoalkaloids, saponins, steroids and triterpenes [9-12].
Plant cell cultures, nowadays, are an important strategy for bioprospection of natural products. The in vitro
large-scale production of bioactive compounds or extracts used as phytotherapics, pharmaceutical products,
food additives and cosmetics should be encouraged because of their scientific, economical or ecological
importance. Some species of Amaranthaceae in Biotechnological study demonstrated their potential for
*Address correspondence to Marcos J. Salvador: Curso de Farmácia, Departamento de Biologia Vegetal, Instituto de Biologia,
Universidade Estadual de Campinas (UNICAMP), C.P. 6109, 13083-970, Campinas, SP, Brazil; E-mail: marcosjs@unicamp.br
Bioactive Chemical Constituents Biotechnological Production of Plant Secondary Metabolites 125
bioprospection of bioactive natural compounds. Thus, the present chapter reviews the literature data of the
bioactive chemical constituents and biotechnological production of secondary metabolites in
Amaranthaceae plants (Gomphreneae tribe).
2. BIOACTIVE SECONDARY METABOLITES FROM AMARANTHACEAE PLANTS,

GOMPHRENEAE TRIBE
The Amaranthaceae family, classe Magnoliopsida and ordem Caryophyllales, was established by A. L.
JUSSIEU in 1789 [7] and includes approximately 65 genera and 1000 species, with the occurrence of 14
genera in Brazil. The members of Amaranthaceae family are divided in four tribes: Celosieae,
Achyrantheae, Braylineae and Gomphreneae [7]. The species that happen in Brazil are distributed in
Celosieae, Achyrantheae and Gomphreneae tribes (Table 1). Gomphreneae is the major tribe and previous
chemical analyses have demonstrated the occurrence of chemical diversity with relate of the presence of
anthraquinones, aurone, betacyanins, betalains, betaxanthins, chromoalkaloids, ecdysteroids, flavonoids,
protoalkaloids, saponins, steroids and triterpenes. Several of these secondary metabolites show biological
activity. For the genus Alternanthera, Gomphrena and Pfaffia is reported the presence of species that have
many pharmacological properties and other applications.
Table 1: Distribution of the genus of the amaranthaceae family that happen in brazil in the respective tribes and
literature data of the genus of the gomphreneae tribe*.
Celosieae Tribe Achyrantheae Tribe Gomphreneae Tribe, , #

Celosia L. Achyranthes L. Alternanthera Forsskal.
(50, 3) (6, 2) (80, 30, 20, 30)
Amaranthus L. Blutaparon Raf.
(60, 12) (4, 2, 1, 2)
Cyathula Lour. Froelichia Moench.
(20, 2) (12, 3, 2, 3)
Chamissoa H. B. K. Gomphrena L.
(2, 2) (100, 46, 15, 33)
Herbstia Sohmer. Iresine P. Br.
(monotipic/monospecific) (80, 5, 2, 5)
Pfaffia Mart.
(50, 20, 12, 20)
Pseudoplantago Susseng
(monotipic/monospecific, - )
Quaternella Pedersen
(monotipic/monospecific, - )
*The American Chemical Society, 1907 to 2010;  The first number refers to the number of species that happen in the genus and the second
number indicate the species that happen in Brazil, respectively;  The third number refers to the number of studied species (biological,
chemical or other studies); # The fourth number refers to the number of pytochemistry studies; -: it indicates absence of studies in the
literature.
2.1. Alternanthera spp.

The genus Alternanthera Forkssal includes 80 species [7] and some are exclusive species of the sandbank
ecosystem, appearing so much in the sandy and rocky strings as well as in the dunes being, therefore,
indicative pioneers species of the before-beach [13]. On the other hand, some species present cosmopolitan
character, being found in different atmospheres as savannah, cerrado (an extensive tract of land composed
of stunted twisted trees), rocky fields, river edges and urban centers [7, 14]. Many species of Alternanthera
traditionally are used in the treatment of infections, as analgesic, anti-nociceptive, antiviral, antibacterial,
antifungal and diuretic agents [6, 14]. Some species have previously been studied regarding their chemical
composition (Table 2). For example, in Alternanthera Forkssal, the occurrence of flavonoids,
isoflavonoids, and flavone C-glycosides has been documented previously [10-16] as well as the occurrence
of betacyanins, saponins, steroids, and triterpenes [10, 15].
126 Biotechnological Production of Plant Secondary Metabolites Salvador et al.
Table 2: Main classes of constituents identified in species from the genus Alternanthera.
Compounds Species References

Steroids a
Alternanthera brasiliana
Alternanthera canescens a
Alternanthera flavescens a
Alternanthera halimifolia a [1, 10, 17]
H H Alternanthera maritima a
HO
H Alternanthera paronychioides a
Alternanthera tenella a
Campesterol
A. brasiliana a
A. canescens a
A. flavescens a
[1, 10, 17]
H H A. halimifolia a
HO A. maritima a
H
A. tenella a
Stigmasterol
A. brasiliana a, f
A. canescens a
A. caracasana a
A. flavecens a
A. halimifolia a [1, 10, 12,
H H A. maritima a 17-22]
HO A. philoxeroides a
H
A. pungens e
-sitosterol A. sessilis a
A. tenella a
H A. brasiliana a
22
A. canescens a
A. flavescens a
A. halimifolia a [1, 10, 17]
A. maritima a
3
7 A. paronychioides a
HO
A. tenella a
 -stigmastenol
7
A. brasiliana a
H A. canescens a
A. flavescens a
A. halimifolia a
[1, 10, 17,
A. maritima a
23]
A. paronychioides a
3
7 A. pungens a
HO
A. sessilis a
Spinasterol (alpha and beta)
A. tenella a
Triterpenes
29
30 20
28
12
13
25
1
26
A. sessilis d [24]
O
3 H 27
HOO
24 H
23
Lupeol
CHAPTER 9
Biotechnology Approaches and Economic Analysis of Jojoba Natural
Products
Mohammed A.M. Aly* and Aydin Basarir
Department of Arid land Agriculture, and Department of Agribusiness, Faculty of Food and Agriculture,
Abstract: Jojoba (Simmondsia chinensis (Link) Schneider) is a desert shrub which tolerates saline and
alkaline soils and minimal water requirements. Jojoba seed storage lipids are liquid waxes which are
esters of long chain monounsaturated fatty acids and alcohols. The oil content of the seed constitutes
approximately 40-50% of the seed weight. The liquid wax is of economic importance in industry
(machine lubricant) and medicine (e.g. cosmetics and anticancer compounds). Biotechnology
approaches can be utilized for jojoba propagation and cloning of genes coding for economically
important traits. Micropropagation of jojoba by in vitro seedling culture was achieved. This chapter will
discuss the following aspects: 1) Jojoba production and economics, 2) The medicinal metabolites and
their health related values, 3) Conventional and non-conventional, biotechnological, approaches to
improve and utilize jojoba.
Keywords: Jojoba, Simmondsia chinensis, biotechnology, micropropagation, gene cloning, in vitro

seedling culture, fatty acids, somatic embryogenesis, explants, plant growth regulators, precursor,
elicitation, molecular markers.
1. INTRODUCTION
Jojoba [Simmondsia chinensis (Link) Schneider], pronounced "hō-hō'-bə", is a dioeciously wind pollinated
desert shrub (2n = 26, 52) which can reach up to 4.5 meters high and typically lives more than 150 years (Fig.
1). It is the sole species of the family Simmondsiaceae, placed in the order Caryophyllales. It is also known as
goat nut, deer nut, pignut, wild hazel, quinine nut, coffee berry, and gray box bush. It is different than the
similar-sounding Jujube (Ziziphus zizyphus), an unrelated plant. Its origin is in north-west Mexico and south-
west the United States of America at 101-117 latitudes and 1,500 meters height. It tolerates saline and alkyl
soils as well as drought. The maximum amount of yield is reached after 10 years of planting.
It is an environment friendly crop that needs minimal agricultural practices, especially pesticides
treatments. Because of the little water requirement, it can be easily adapted to many different types of arid
regions in the world
Jojoba propagates by seeds as well as cuttings, and flowers after 3 to 7 years from germination [1]. Fruit set and
maturation is usually in a season characterized by temperature of 9.5 oC night/ 46 oC days, under 46 cubic cm
annual rainfall [1]. The fruit contains one seed (1-2 cm diameter). Modise [2] reported a full account of jojoba
cultivation and management. The seeds (Figs. 2 and 3) contain a liquid wax which reaches 50 percent of seed’s
dry weight and used in production of jojoba oil [3]. Jojoba oil is chemically different from other seed oils.
Instead of being a triglyceride, it is made up of liquid wax esters [4]. This characteristic liquid wax is of
economic importance in industry (machine lubricant), as a replacement for whale's sperm oil in manufacturing
of inks, varnishes and waxes and medicine, e.g. cosmetics and anticancer compounds [5]. The wax is beneficial
in alleviating minor skin irritations, stimulating hair growth and rejuvenation, in diets to decrease cholesterol
level and as antioxidant due to α-tocopherol found in the leaves. It is used as a folk medicine by the natives in
*Address correspondence to Mohammed A.M. Aly: Department of Arid land Agriculture, Faculty of Food and Agriculture, United
Arab Emirates University, P.O. Box 17555, Al Ain, United Arab Emirates, Tel: (+971) 693-8505, E-mail: mohammed.aly@uaeu.ac.ae

160 Biotechnological Production of Plant Secondary Metabolites Aly and Basarir
its center of origin and other locations. The pulp contains about 30% proteins. Furthermore, jojoba leaves, seeds
and cake are of high value in animal feed due to the high protein content (30-40-%) and the high metabolic
conversion rates, representing invaluable replacement to the expensive soybean component in animal feed. The
oil and cakes are organic agricultural products.
Figure 1: The jojoba shrub.
Figure 2: Jojoba immature.
.
Figure 3: Mature seeds.
2. ECONOMIC ANALYSIS OF JOJOBA PRODUCTION
Jojoba has started to be commercially produced since 1976. The product is produced in Argentina,
Australia, Chile, Egypt, Israel, Mexico, Peru, and USA. Compared to some other industrial agricultural
products, jojoba does not require intensive labor. Optimum number of plants per acre is 2000. The average
annual yield is 0.2-2.2 kg per plant, with an average of 2400 kg seeds per acre.
The major jojoba seed production area is in Argentina (Table 1). As a result of lower yields per hectare
even though the USA has less cultivation area, it has the highest jojoba seed production. The highest
yield/hectare is in Israel. This is mainly because of having superior varieties. Annual world production of
jojoba oil in 2000 was about 1500 tons.
Biotechnology Approaches and Economic Analysis Biotechnological Production of Plant Secondary Metabolites 161
Table 1: Jojoba seed and oil production (metric tons).
Cultivated Area Seed Production Oil Production

Country
Hectares (Metric Tons) (Metric Tons)
Argentina 4800 950 475
Australia 400 8 4
Egypt 140 15 7.5
Israel 675 1000 500
Mexico 470 90 45
Peru 300 75 37.5
USA 2000 1455 727.5
Sources: http://www.cicsoils.com/indexeng.htm.
http://www.agcensus.usda.gov/Publications/2007/Full_Report/Volume_1,_Chapter_2_US_State_Level/st99_2_028_028.pdf.
It was reported that a well-managed plantation with properly selected varieties could yield about 1 and 2 ton per
hectare with and without irrigation in Australia, respectively [6]. The calculated cost is mainly coming from
purchasing and rearing of the seedlings ($3750/ha), land preparation and planting ($1000/ha), irrigation
management (if implemented) and harvest ($0.2 to $1/kg). The 1996 price of seed in Australia was around
$4500/ton, with the oil extracted entering the high-priced cosmetics market. The jojoba cultivation expanded
ever since [6]. In North America, the jojoba production is mainly in USA, Arizona desert, and Mexico. The
total U.S. exports of jojoba were $10 million in 2000. Ninety percent of jojoba oil produced in USA is exported
mainly to cosmetic companies in France, Switzerland and Japan. Depending on the world demand and the
quality of product the cost of production was high and there were only about 40 jojoba growers in the United
States. Yield variability of jojoba plants causes export volatility [7]. The plant has also been used to combat and
prevent desertification in the Thar Desert and coastal sand dunes in India [8]. Recently, Saudi Arabia has started
to launch a giant jojoba project in collaboration with Japan to cultivate 10 acres-farms along its coasts on the
Red Sea and the Gulf. These farms are planned to be irrigated with desalinized sea water (personal
communication). Namibia (in Africa), is also planning a 75 hectares plantation of jojoba in Khan River below
Usakos in the Namib Desert. Production is expected to reach 300 tons of nuts yielding 65 tons of crude oil [9].
The economy of jojoba depends on three factors [10]:
1. Plantations which produce high yield which will overcome the unit cost of production.
2. The market size, which will match the total production of the product.
3. A price which will be high enough to cover expenses of producers, leave some profit for
them and stimulate new technology within the industry.
Several studies have been conducted to examine the profitability of jojoba cultivation in Australia [6, 11] and
Chile [12]. According to Brown and Hall [11], there was a 65 percent probability that jojoba farms will be more
profitable than the average of all farms and just over 50 percent probability that they will be more profitable
than wheat farms, the most profitable group. The economic analysis reported by Botti et al. [12] indicates that
commercial plantations using the best clones could be a profitable alternative for the arid and semi-arid regions
of Chile. According to Denham and Rowe [13] a cooperative marketing system is required in industry to
maximize the returns to growers. In addition, this system will prevent the large fluctuations in the supply and
demand of jojoba. On the other hand, a research conducted by United Nations [14] in sub Saharan Africa shows
that unless produced in large areas, it is not economical to use jojoba in biofuel production.
Another example of how profitable is jojoba cultivation, an analysis of the gain obtained from simmondsin
sales, a secondary metabolite included in the jojoba cake, is illustrated in Table 2. The cost of production
for 1 kg refined simmondsin is roughly €583. The retail price of the product is about €10000/kg. The profit
is 16 times higher than the cost of production.
176 Biotechnological Production of Plant Secondary Metabolite, 2012, 176-186
CHAPTER 10
The Effects of Pesticides on Plant Secondary Metabolites
Monica Hancianu* and Ana C. Aprotosoaie
Department of Pharmacognosy, Faculty of Pharmacy, Gr. T. Popa University of Medicine and Pharmacy
Iasi, University Street, No. 16, Iasi, Romania
Abstract: The controlled and uncontrolled use of pesticides along with unquestionable benefits can also
affect other living organisms, including human beings and plant metabolism by causing abiotic stress in
plants. Because possible effects on plants secondary metabolites are largely unknown, most researches focus
on the influence of pesticides, their metabolism products, and residues on human health and environment.
Other currently available data is related to effects of herbicides, fungicides, and plant growth regulators on
phenylpropanoid and derived flavonoid metabolites as well as terpenoid metabolism.
Pesticides may influence levels of secondary metabolites like flavonoids, hydroxycinnamic acids,
anthocyanins, tropane alkaloids, and volatile terpenoids by non-specific mechanisms or interfering the key
biosynthesis steps. The quality of volatile oils can be altered by changing their chemical composition,
especially the toxic or useful constituents. Moreover, pesticide residues can be solubilised in volatile oils.
Also, pesticides are able to modulate plant metabolism affecting assimilation rate of micronutrients.
The complete assesment of plants exposed to pesticides requires knowledge of the biochemical and
physiological responses of vegetal organisms to these substances in order to understand the magnitude
of the agrochemicals action, but also for the rational engineering of plant secondary metabolites.
Keywords: Secondary metabolites, phytochemistry, pesticides, chemical composition, human health,

environment, toxicity, herbicides, biosynthesis, fungicides, insecticides, plant growth regulators.
1. INTRODUCTION
Plants are able to synthesize a huge array of secondary metabolites, which are the structurally diverse
substances that are not part of such major organic categories as carbohydrates, fats and proteins. Flavonoids
and phenol-related compounds, tannins, lignins, isoprenoids (terpenoids and steroids) and alkaloids are just
a few examples of the secondary metabolites in higher plants. These compounds have many different
endogenous and exogenous functions such as serving as cell walls components, antioxidants, antimicrobial
agents, antifeedants, insect attractants, and chemical signal producers. Most of the secondary metabolites
are also of interest with therapeutic and economic importance due to their use as drugs, pharmaceutical and
industrial precursors, flavorings, cosmetic ingredients, dyes, adhesives. Recently, the role of some these
phytochemicals has been established as health protective dietary constituents.
2. FACTORS AFFECTING SECONDARY METABOLITES PRODUCTION IN PLANTS
The metabolism pathways of secondary compounds are under complex regulation mediated by genotype,
plant development, carbon-nutrition balance, and environmental conditions including biotic and abiotic
stimuli. Biotic factors can be counted as microbial infections, herbivores, and insects; whereas abiotic
stimuli include extremes in temperatures - cold and heat stress -, salinity, drought, heavy metals, oxidative
stress, radiation, and pesticides.
The factors such as temperature, light intensity, photoperiod, soil fertility, and water supply determine suitable
growing area and life forms of medicinal plants. Generally, production of non-nitrogenous shikimic acid-
derived metabolites increases when plants are grown under nutrient deficiency conditions. A raise in light
intensity tends to produce accumulation of phenolic compounds and terpenoids. Water stress has been reported
to lead to increase in cyanogenic glycosides, glucosinolates, terpenoids, alkaloids, and condensed tannins.
*Address correspondence to Monica Hancianu: Department of Pharmacognosy, Faculty of Pharmacy, Gr. T. Popa University of
Medicine and Pharmacy Iasi, University Street, No. 16, Iasi, Romania: Tel (40) 0232-301600: E-mail: mhancianu@yahoo.com
The Effects of Pesticides on Plant Secondary Metabolites Biotechnological Production of Plant Secondary Metabolite 177
Agronomic practices such as plant propagation, sowing dates, irrigation, fertilization, improving drainage,
pesticides, and post-harvest treatments also have a great impact on the final biomass and phytochemical
yields of medicinal plants.
3. ROLE OF PESTICIDES AND ITS IMPLICATIONS ON PLANT METABOLISM
Despite unquestionable benefits of pesticides, their uncontrolled and controlled use can affects other living
organisms including human being and plant metabolism by causing abiotic stress in plants.
Pesticide residues in plants are regulated in order to protect human health. However, this procedure does
not consider that pesticides modulate secondary metabolism in plants applied. The question that arises is
whether it is conceivable that pesticide-induced changes in the chemical composition of plants influence
human health. It might be possible that the modulation of plant phenols at the time of application and not
the usually low level of pesticide residues at the time of consumption is critical for human health. Another
complication in the interpretation of the role of pesticides originates from the additional effects of natural
factors such as nutrient availability, pathogens, herbivore pressure as well as UV radiation.
Exposure of plants to pesticides takes place mainly through leaf surface, fruits, and roots. Within the plant,
pesticides can be distributed via the plant vascular system or from cell to cell, dependent on their physical and
chemical properties. In general, the plants have mechanisms for the degradation or sequestration for most of the
pesticides. The initial metabolic biotransformation of pesticides includes oxidation, hydroxylation, epoxidation,
hydrolysis, reduction, N-dealkylation, O-dealkylation, desulfuration, dehalogenation, dehydrogenation, and
dehydrohalogenation reactions (Phase I). Some of these chemical changes are enzymatically mediated; while
some result from non-enzymatic mechanisms and others arise from photochemical reactions. Phase II and III
reactions involve the conjugation of the pesticide molecule or its metabolites with natural constituents (glucose,
glutathione, malonic acid, and amino acids). The plants decrease water solubility of toxic pesticides and reduce
the mobility of their compounds in the plant symplast by three major pathways; export into cell vacuoles,
export into the extracellular space, and deposition into lignin or other cell walls components. Another
mechanism may be the conjugation to insoluble structures in the plant such as co-polymerization with lignin
resulting in the formation of bound residues of pesticides [1].
Pesticide metabolism generally results in detoxification, but sometimes the metabolites are equal if not
greater toxicity than the respective parent molecules.
Most researches were focused on the influence of pesticides, their metabolism products, and residues on
human health and environment, while possible effects on plants secondary metabolites (targeted and non-
targeted organisms) are largely unknown [2].
More available data are related to the effects of herbicides and fungicides on phenylpropanoid and derived
flavonoid metabolites as well as terpenoid metabolism in plants.
Phenolic compounds such as flavonoids, isoflavones, coumarins, anthocyanins, xanthones, stilbenes,

phenolic esters, and benzoic derivatives represent a spectacular example of metabolic plasticity enabling
plants to adapt to changing abiotic and biotic environments and provide to the plant the product color, taste,
technological properties, and health-promoting benefits in cancer, cardiovascular, and neurodegenerative
diseases or for use in anti-aging and cosmetic products. The diverse modulating effects of pesticides on
phenolic compounds in plants may affect functioning of ecosystems. Direct effects involve the colonization
of plants with pathogenic microorganisms or the alteration of the attractiveness of plant to herbivores.
Pesticides can also affect the secondary metabolism indirectly by eliminating non-target plants that compete
for light and nutrients or serve as habitats for pathogens and herbivores. Plant phenols affect not only the
aboveground organisms and ecologic functions, but also they might be also important belowground if they
are applied to the soil or if the plants are exposed to pesticides in the field. During decomposition, these
plant tissues may either decay to carbon dioxide or become part of the soil organic matter; another part may
178 Biotechnological Production of Plant Secondary Metabolite Hancianu and Aprotosoaie
persist for centuries because of its chemical properties or physical protection. Plant phenols may persist for
weeks or months after plant death and affect suitability as a food resource for soil organisms and
decomposition rates of the soil organic matter.
Herbicides are able to modulate concentrations of secondary compounds at application regimes that are not
lethal for plants. These pesticides act by different mechanisms as follows:
 They reduce the carbon fixation by plants, which may decrease the proportion of carbon
allocated to the synthesis of secondary compounds;
 They block the shikimate pathway. The shikimate pathway is of particular importance in
plants since it provides the precursors for lignin and some secondary metabolites such as
flavonoids. A substantial proportion of carbon flux in plants is through the shikimate
pathway. Glyphosate (N-phosphomethylglycine) is the most widely used herbicide
worldwide. Its mode of action is the inhibition of the shikimic acid pathway which produces
aromatic amino acids, as well as the secondary plant products involved in the resistance of
plants to pathogens. This herbicide attacks 5-enolpyruvylshikimate-3-phosphate (EPSP)
synthase, an enzyme of the shikimate pathway. It is an enzyme common in the synthesis of
the aromatic amino acids tyrosine, phenylalanine, and tryptophan. In plants, these amino
acids are essential for critical processes other than protein synthesis, including cell wall
formation, defense against pathogens and insects, production of hormones as well as
compounds required in energy transduction such as plastoquinone. Some of the phytotoxicity
caused by glyphosate is due to deregulation of the shikimate pathway due to reduced
feedback inhibition, causing greatly increased amounts of carbon to flow into this pathway.
This fact results in two possible detrimental effects as debilitation of other pathways due to
loss of carbon skeletons to the shikimate pathway and accumulation of massive, possibly
phytotoxic levels of shikimate and benzoic acids. Application of the glyphosate in a sublethal
dose (50 M) to Cassia obtusifolia L. suppressed the biosynthesis of a phytoalexin derived
from the shikimate pathway. The contents of gallic acid and components of hydrolyzable
tannins are increased after application of glyphosate on soybean leaves and beans [Glycine
max (L.) Merr.]. Therefore, glyphosate (0.5 mM) decreases the accumulation of anthocyanin
and hydroxyphenolics in soybean [3, 4] in such ways;
 They increase PAL (phenylalanine ammonia-lyase) synthesis. This enzyme plays a key role
in phenylpropanoid and alkaloid biosynthesis. Most of the phenolic compounds derive from
phenylalanine via the core phenylpropanoid pathway (Fig. 1). The activities of PAL and
chalcone isomerase were greatly enhanced in maize (Zea mays L.) and soybean by the
herbicides; namely alachlor and rimsulfuron. Consequently, hydroxyphenolic compounds and
anthocyanin contents were shown to increase in both species [4, 5].
Legend: PAL= L-phenylalanine ammonia-lyase; C4H= cinnamate hydroxylase; 4CL= 4-

hydroxycinnamoyl-CoA; CAD= cinnamoyl-alcohol dehydrogenase; CCR= cinnamoyl-CoA reductase;
C3H= p-coumarate 3-hydroxylase; COMT= caffeoyl-CoA O-methyltransferase; F5H= ferulate 5-
hydroxylase; HCT= hydroxycinnamoyltransferase; SAD= sinapyl-alcohol dehydrogenase.
Dichlobenil, amitrol, acifluorfen, and paraquat can augment the PAL synthesis which determines an
increase of phenol derivatives. Treatment with acifluorfen of spinach leaves (Spinacia oleracea L.)
determines alterations in aromatic amino acid metabolism and phenylpropanoid biosynthesis involving
secondary phenolic compounds. Thus, acifluorfen (< 0.2 ppm) increased 25-fold the normal level of the
phenolic amide derived from 3-methoxytyramine and ferulic acid. Elevation of this compound’s
concentration is followed by the appearance of necrotic lesions in spinach leaves but is preceded by an
increase in phenylalanine ammonia-lyase activity that reaches 13-fold the normal values 20 h after
treatment (Table 1).
CHAPTER 11
Cardenolide Production as an Important Drug Agent
Sebnem Harput U.*
Hacettepe University, Faculty of Pharmacy, Department of Pharmacognosy, 06100- Sihhiye, Ankara,

Turkiye
Abstract: Leaves of Digitalis plants are still the major source for the isolation of cardenolides,
especially digitoxin and digoxin that are used to treat cardiac insufficiency in humans. Cardenolides are
characterized by a steroid nucleus with its four rings connected cis– trans–cis, having a 14-hydroxy
group and an unsaturated five-membered lactone ring at C-17. Typically, sugar side chains of variable
length are attached at position C-3 of the cardenolide genins. The use of tissue cultures for the
production of cardenolides was examined quite extensively, using suspension cultures; morphogenic or
embryogenic cell cultures as well as shoot or root cultures. It is important to found that cell cultures
without clear tissue or organ differentiation (undifferentiated cultures) were found to be unable to
produce considerable amounts of cardenolides, whereas cardenolide biosynthesis is restored as soon as
morphogenesis is induced. In addition, most workers have reported that undifferentiated cultured cells
either did not produce cardenolides or contained only trace amounts of it. However organ-
redifferentiating cultures have reported to accumulate considerable amounts of cardenolides. The
influence of light, phytohormones and nutrients on the production of cardenolides in Digitalis cultures
was examined extensively in different studies. Expression of the cardenolide-biosynthesis system
connected with differentiation of cells, different enzymes involved in biosynthesis, condition of plant
tissue cultures and effect of several plant growth substances on cardenolide formation will be discussed
together with the current expression systems used for the industrial production.
Keywords: Digitalis, cardiotonic glycosides, cardenolides, plant cell culture, biotransformation, digitoxin,
digoxin, biosynthesis, molecular data, genomic cloning, bioseparation, CD polymer.
1. INTRODUCTION
Cardiac or cardiotonic glycosides have been long used and continue to be used in the treatment of congestive
heart failure as positive inotropic agents [1, 2]. Chemically, cardioactive glycosides are compounds presenting a
steroid nucleus with lactone moiety at position 17 and sugar moiety at position 3. Their therapeutic action
depends on the structure of the aglycone and on the type and the number of sugar units attached. Two types of
cardiac glycosides are defined according to the structure of the aglycone: the cardenolides (with an unsaturated
butyrolactone ring) and the bufadienolides (with an -pyrone ring). Stereochemistry of the steroid nucleus is
very important for the activity: rings A/B and C/D are cis fused while B/C are trans fused, 3 and 14-hydroxyl
groups with the glycoside function at C-3, and an ,-lactone ring at C-17 [3]. The unsaturated lactone ring is
essential for the activity, if the ring is saturated, the activity is substantially decreased and opening the ring
renders the glycoside inactive. The fundamental pharmacological activity is derived from the aglycone portion,
but is considerably modified by the nature of the sugar at C-3. This increases water solubility and binding to
heart muscle. Up to 4 sugar molecules may be present in cardiac glycosides; attached in 3-OH group. Mainly
D-digitoxose and D-digitalose are unique to this group of compounds [1-3]. Cardiac glycosides occur in a
limited number of plant families. They are more common in the plant families Asclepiadaceae (e.g.
Convallaria), Plantaginaceae (e.g. Digitalis), Apocynacea (e.g. Strophanthus) and Ranunculaceae (e.g.
Helleborus). Some animal species and mainly toads are also recognized to contain cardiotonic glycosides. The
most well-known plants containing cardiac glycosides is the Digitalis species (D. lanata and D. purpurea, etc.).
It was first reported in 1542 by German physician Leonard Fuchs. He gave the plant its name because its
flowers were similar to a thimble. Today the word “digitalis” or “digitalis glycoside” is often used as a generic
term for all cardiotonic glycosides [2].
*Address correspondence to Sebnem Harput U.: Hacettepe University, Faculty of Pharmacy, Department of Pharmacognosy,
06100- Sihhiye, Ankara, Turkiye, E-mail: sharput@hacettepe.edu.tr
188 Biotechnological Production of Plant Secondary Metabolites Sebnem Harput U.
2. PLANT TISSUE AND CELL CULTURES FOR PRODUCTION OF CARDIAC GLYCOSIDES
Plant cell culture has the potential importance for the production of useful metabolites for pharmaceuticals and
various other uses. It can be used for the de novo production of phytochemicals and recombinant proteins. In
addition, plant cells in culture can be used as an enzyme source in the biotransformation of organic chemicals
into different valuable structures. It is especially valuable in the case of chemicals obtained from the plant with
stereospecificity and complex structure that affect chemical synthesis. Reactions which are restricted only to
plant cells and which produce economically valuable products can be commercially interesting with
biotechnological researches. Biotransformation of digitoxin into digoxin by Digitalis lanata cultures have been
studied for many years. Digitoxin and its 12-hydroxylated derivative, digoxin, are the most important cardiac
glycosides and in which digoxin has better pharmaceutical properties. That is the reason why plant cell culture
has been used for the biotransformation of digitoxin (Fig. 1) [4-7].
O
O
R
H OH
O Digoxin: R = OH
H Digitoxin: R = H
D
d
i
g
i
t
o
x
o
s
e

D
d
i
g
i
t
o
x
o
s
e

D
d
i
g
i
t
o
x
o
s
e
Figure 1: Chemical formula of digoxin and digitoxin.
3. BIOSYNTHESIS OF DIGITOXIN
Plants steroids are derived from cycloartenol, which in turn, originates from squalene. Demethylation of
cycloartenol results in cholesterol, which is oxidized to 20, 22- dihydroxycolesterol. The side chain of
this compound is cleaved to isocaproic aldehyde and pregnenolone. These two reactions are catalysed by an
enzyme called the cholesterol side chain-cleaving enzyme (SCCE) which has been detected in protein
extracts from leaves of Digitalis purpurea. The next reaction, isomerization and reduction to progesterone,
is catalysed by ∆5-3-hydroxysteroid dehydrogenase/∆5-∆4-ketosteroid isomerase (3-HSD). It has been
detected in extracts from suspension-cultured cells and intact leaves of Digitalis lanata. Reduction of the
double bound in ring A of progesterone is catalyesed by progesterone 5-oxidoreductase which transforms
5-pregnane-3,20-dione to 5-pregnane-3-ol-20-one. Formation of the butenolide ring involves transfer of
a malonyl moiety to the 21-hydroxy group to form a malonyl hemi-ester. This reaction is catalysed by
malonyl coenzyme A: 21-hydroxypregnane 21-hydroxy-malonyltransferase. A subsequent Claisen-type
condensation of the malonyl moiety with the C-20 ketone, accompanied by decarboxilation yields
digitoxigenin, to which three digitoxose residues are added, forming digitoxin (Fig. 2) [2, 8].
Taking cholesterol as the starting point, about 20 enzymes which probably affect the formation of
cardenolides have been identified and characterized in Digitalis. But only some of them have been purified,
including progesterone 5-reductase (5-POR), a key enzyme of cardenolide biosynthesis catalyzing the
conversion of progesterone to 5-pregnane-3,20-dione. This enzyme has been partially sequenced [9-11].
To find a possible route for manipulating cardenolide biosynthesis in plants, a more detailed knowledge of
the enzymes and genes involved in cardenolide formation is necessary for studying the regulation and
engineering of cardenolide pathway.
Cardenolide Production as an Important Drug Agent Biotechnological Production of Plant Secondary Metabolites 189
Figure 2: Biosynthesis of digitoxin [2].
3.1. Biosynthesis of Pregnane Derivatives in Somatic Embryos of Digitalis Lanata

There are a lot of studies on biosynthesis of cardenolides using different plant cell culture system.
Lindermann and Luckner was studied biosynthesis of pregnanes in somatic embryos of D. lanata [9]. They
determined different enzymes involved in biosynthesis of pregnane derivatives in different developmental
stages of somatic embryos of Digitalis lanata. Cardenolides are derived from sterols via pregnane
derivatives as it was mentioned above (Fig. 2). Schematic diagram of cardenolide biosynthesis is
summarized in Fig. 3. The first intermediate of the biosynthesis is pregnenolone (5). In mammals its
formation is catalyzed by cholesterol monooxygenase (SCCE; Fig 3 :enzyme a). It uses cholesterol (2) as
substrate and catalyses its subsequent hydroxylation at the positions 20 and 22R followed by the final
scission of isocapronal. Cholesterol (2), 20-hydroxycholesterol and sitosterol (3) are precursors of
pregnanes and cardenolides in D. lanata plants. Radioactive labeled cholesterol was incorporated into
pregnenolone in D. purpurea plants.
In cell cultures and plants of different Digitalis sp. pregnenolone (5) was transformed to progesterone (6)
and to cardenolides. Also progesterone (6) was incorporated into cardenolides. The synthesis of
CHAPTER 12
Progress in Biotechnological Applications of Diverse Species in
Boraginaceae Juss.
Ufuk Koca1,*, Hatice Çölgeçen2 and Nueraniye Reheman1
1
Department of Pharmacognosy, Faculty of Pharmacy, Gazi University, 06330, Ankara, Turkey;
2
Zonguldak Karaelmas University, Faculty of Arts and Science, Department of Biology, 67100 İncivez,
Zonguldak, Turkey
Abstract: During the past four decades plant cell biotechnology has evolved as a promising new area
within the field of biotechnology, focusing on production of secondary metabolites and in vitro
propagation of plants. Boraginaceae is one of the family that biotechnological tools were applied
extensively because of their economically, ornamentally and medicinally valuable seconder metabolites
as well as their endangered species. The Boraginaceae family is known as Borage or Forget-me-not,
contains more than 156 genera and about 2000 species including annual, perrenial herbs, shrubs and
trees. Members of the family were distributed mostly in sandy -drier regions of the world. The most
well-known members of the family are Forget me not (Myosotis sp.), Borage (Borago sp.), Comfreys
(Symphytum sp.), and Heliotrope (Heliotropium sp.). A good number of the family members are
used as a source of dye in cosmetics, food, textile and also in medical field. Selected species of the
family utilized for obtaining secondary metabolites including naphtaquinone derivatives, rosmarinic
acid and pyrrolizidine alkaloids. Due to their medicinal, economical and ecological importance,
biotechnological tools such as plant tissue culture, metabolic engineering and in vitro
micropropagation have been applied to produce biologically active compounds, pigments and to
increase the population of the endangered species.
The purpose of this chapter to review studies performed utilizing biotechnological methods in diverse
members of this family. Botanical aspects, traditional usage, chemical constituents and production of
secondary metabolites in cultures and via metabolic engineering in some of the family members were
also reviewed in brief.
Keywords: Boraginaceae, biotechnology, in vitro propagation, naphtaquinones, Arnebia, plant cell culture,
callus culture, plant tissue culture, elicitor, hairy root culture, Onosma, pyrrolizidine alkaloids.
1. INTRODUCTION
Boraginaceae was first described by Antoine Laurent de Jussieu [1] in his published Genera plantarum, as
'Borragineae', which was established based on genus Borago Linnaeus used the Latin name ‘burra’
meaning ‘hairy outfit’ when he established the genus Borago. The family is native to the Old World. Its
main centre of diversity in the Mediterranean and near Western Asia, with only a few species of the family
was found in Africa. The family known as Borage or Forget-me-not (Myosotis) includes a variety of
shrubs, trees, and herbs, totaling about 2,000 species in 156 genera found worldwide [2, 3]. Members of the
family are generally herbaceous most of which are rough or hispid, usually with rounded stems, and
alternate, exstipulate, entire leaves. The flowers are skorpioid, symoz or hirsus. The calyx is 5-toothed, the
corolla 5-lobed, saucer, funnel, or bell shaped, and is often yellow, pink in bud, changing later to blue.
There are 5 stamens, alternating with the corolla-lobes. The fruit is normally composed of 4 nutlets rarely
drupa [2]. Well-known members include Alkanet (Alkanna tinctoria), Borage (Borago officinalis),
Comfrey (Symphytum), Fiddleneck (Amsinckia spp.), Forget-me-not (Myosotis spp.), Geiger tree (Cordia
sebestena), Green alkanet (Pentaglottis sempervirens), Heliotrope (Heliotropium spp.), Hound's Tongue
(Cynoglossum), Lungwort (Pulmonaria), Oyster Plant (Mertensia maritima), Patterson's Curse (Echium
plantaginuem), Siberian Bugloss (Brunnera macrophylla), Viper's Bugloss (Echium vulgare).
*Address correspondence to Ufuk Koca: Department of Pharmacognosy, Faculty of Pharmacy, Gazi University, 06330 Ankara,
Turkey. Tel: (90) 312 2023197; E-mail: ukoca@gazi.edu.tr
Progress in Biotechnological Applications of Diverse Species Biotechnological Production of Plant Secondary Metabolites 201
Traditional usage of plants from the family goes back to 3rd Century BC that was reviewed by
Papageorgiou et al. [4] Medicinal usage of those dark pinkish red pigmented extracts was recorded by
Theophratus and Hippocrates in 3-5th centuries BC. Dioscorides, also described the properties of Alkanna
sp. in De Materia Medica at 70s. Root extracts of Alkanna species were used for treatment of ulcers and
burns in European folk medicine. Some Arnebia species, specifically endemic A. densiflora from the same
family were also applied for wound healing purposes in Anatolia [5]. Whereas, Lithospermum species were
used in fareast for the similar aims, both for dying of garments and healing of variety of symptoms. In vitro
and in vivo studies on the extracts of the plants and isolated compounds revealed majorly wound healing,
anti-inflammatory, antiallergic, antitumor, antimicrobial, antiplatelet aggregation, and anti-HIV1-RT
inhibitor activities [6-19]. The proliferation of granulation tissue induced by the root extracts correlated
well with the total naphthoquinone pigment content [6, 7]. Studies demonstrated the effects of root extracts
of the Boraginacea plants, in addition to shikonin and several derivatives as pure compounds on various
aspects of wound healing and anti-inflammatory effects [8]. Commercial preparation contains
alkannin/shikonin derivatives are used for healing puposes especially in Greece and Japan. Shikonin
derivatives significantly inhibit TNF-α promoter activation in a dose-dependent manner that indicates these
compounds can be a good candidate for antiinflammatory drugs. Morever, the anti-inflammatory and anti-
allergic effects of the essential oil of Cordia verbenacea were detremined and activity attributed to
sesquiterpenic compounds obtained from the essential oil revealing that Boraginaceae plants would be a
rich source for the antiinflammatory drug search [10]. L. erythrorhizon root extracts have been used as a
cancer treatment in Chinese traditional medicine for many years. Alkannin and its derivatives exhibited in
vitro cytotoxicity against KB cells [11]. Crude plant extract of L. erythrorhizon, as well as isolated
compounds, showed a high antitumor activity against the ascites cells of sarcoma 180 [12]. Early studies
reported that Boraginaceae plant extracts, alkannin, shikonin, and their derivatives exhibited antimicrobial
and antioxidant effects including bacteriostatic and bactericidal activities [13, 14]. The n-hexane-
dichloromethane (1:1) extract of Onosma argentatum and the methanol extract of Rubia peregrina was
effective on Staphyloccoccus aureus, Bacillus subtilis and Escherichia coli [14].
Shikonin, acetylshikonin, β,β -dimethylacrylshikonin and teracrylshikonin were isolated from Arnebia
euchroma shown to inhibit platelet aggregation induced by collagen [16]. Whereas, the methanol extract of
Trichodesma indicum R. Br. significantly inhibited the frequency of sulphur dioxide (SO2) induced cough
reflex in mice and confirmed the traditional use of this plant in the treatment of cough [17]. Decoction of
Rotula aquatica lour displayed inhibitory effect on establishment of stone in kidneys [18]. Lobostemon
trigonus aqueous leaf extracts reported as a potent HIV-1 RT inhibitor, thus showing a potential
mechanistic action of these plants in aiding HIV-positive patients [19].
2. CHEMICAL CONSTITUENTS OF BORAGINACEAE FAMILY
Boraginaceae family is well-known for its red pigments, Isohexenylnaphthazarins (IHN), commonly known as
naphtaquinones mainly alkannins and/or shikonins. These are mostly found in the outer surface of the roots of
species in genus Alkanna, Arnebia, Lithospermum, Echium, Onosma, Anchusa and Cynoglossum. Shikonin,
refering the Japanese name for Lithospermum erytrorhizon, was isolated first of all from the roots of Onosma
visianii Clem. [20], later from the air-dried rhizomes and roots of Onosma polyphyllum Led [21] and Echium
lycopsis L. [22]. In early studies mostly Alkannin derivatives were determined in Alkanna, Arnebia, Echium,
and Onosma sp. [23, 24]. Other naphthaquinones: β,β-dimethylacryl alkannin, β,β-dimethylacrylshikonin,
acetylalkannin, alkannin, shikonin, deoxyshikonin, β,β-dimethylacrylhydroxyalkannin, hydroxyalkannan and
acetyl hydroxy alkannin were isolated from the root of Arnebia euchroma [25], in further, besides
deoxyshikonin, acetyl shikonin, 3-hydroxy-isovaleryl shikonin and 5,8-O-dimethyl acetyl shikonin were
isolated from roots of Onosma argentatum [26]. Ten species of the genus Alkanna (A. calliensis, A.
corcyrensis, A. graeca, A. methanaea, A. orientalis, A. pindicola, A. primuliflora, A. sieberi, A. stribrnyi and A.
tinctoria) were analyzed for their constituents, main hydroxynaphthoquinones were determined to be similar to
the previous compounds such as β,β-dimethylacrylalkannin, isovalerylalkannin + α-methyl-n-butylalkannin and
acetylalkannin [27]. The hydroxynaphthoquinone constituents and their proportions were found to vary among
202 Biotechnological Production of Plant Secondary Metabolites Koca et al.
Alkanna species. Four hydroxynaphthoquinone derivatives from the roots of L. erythrorhizon Sieb. et Zucc.,
three naphthoquinone derivatives from the roots of Macrotomia euchroma (Royle) Pauls. were isolated [28].
Moreover, compounds derived from naphthalene-1,4-naphthoquinones and rarely also 1,2-naphthoquinones
were revealed in species of Boraginaceae [29] as well.
A phenolic compound Lithospermic acid, as the major compound, together with other constituents of
rosmarinic acid were isolated from Lithospermum ruderale and leaves of Cordia spinescens [30], whereas
cafeic acid salts and their isomers were isolated from roots of Arnebia euchroma. Rosmarinic acid was
reported also in L. erythrorhizon [31] and Arnebia densiflora [32]. The same compound was identified as
the major compound with an amount of 8.44% in the ethanolic extract of the leaves of Cordia americana
[33]. Although rosmarinic acid is, an ester of caffeic acid and 3,4-dihydroxyphenyllactic acid, widespread
in the species of Boraginaceae an Lamiaceae, there are limited number of reports on the identification and
isolation procedure of this compound from Boraginaceous plants.
The presence of rosmarinic acid with shown antiviral, antibacterial, anti-inflammatory and antioxidant
activities brought the attention on search of this metabolite in Boraginaceous plants [34].
Pyrolizidine alkaloids are also a major class of compounds in the family. Well-known medicinal plants in
Boraginaceae containing pyrrolizidine alkaloids are Alkanna tinctoria, Arnebia euchroma, Anchusa
officinalis, Borago officinalis, Cynoglossum sp. Cordia myxa, Echium plantagineum, Heliotropium sp.
Lithospermum officinale, Myosotis scorpioides, Symphytum sp. Forty pyrrolizidine alkaloids were only
detected in Anchusa milleri, Gastrocotyle hispida (syn. Anchusa hispida), Anchusa arveniis, , Alkanna
orientalis, and Alkanna tuberculata Lappula spinocarpos, Paracaryum rugulosum, P. intermedium,
Trichodesma africanum [35], whereas they have been detected and isolated from H. Curassavicum and
Heliotropium curassavicum var. argentinum. [36]. Fourteen pyrolizidine alkaloids were isolated from
Cynoglossum officinale and five pyrrolizidine alkaloids supinine, amabiline, rinderine, echinatine, and 3′-
O-acetylechinatine were recorded in C. amabile [37]. Uplandicine, was isolated as a major component from
Onosma arenaria [38] and (7S, 8R)-petranine, (7S, 8S)-petranine, (7R, 8R)-petranine, 7-
angeloylretronecine, 9-angeloylretronecine were isolated from Echium glomeratum Poir [39]. Moreover,
the novel pyrrolizidine alkaloids tessellatine and 3′,7-diacetylintermedine were isolated from genus
Amsinckia [40] and novel 7-acetyleuropine with known ones were isolated from Heliotropium bovei [41].
Flavonoids, although in low levels, mostly in flowers of the family species were found. European Nonea
species and in two Heliotropium species from Chile were reported to have flavonoids [42]. Hispidone, (2S)-
5,2'-dihydroxy-7,5'-dimethoxyflavanone, benzoic acid and 4-hydroxy benzoic acid has been isolated from
Onosma hispida [43]. Methyl rosmarinate, caffeic acid, quercetin, kaempferol, kaempferol 3-O-alpha-D-
arabinoside, quercetin 3-O-alpha-D-arabinoside, and p-hydroxy benzoic acid were isolated from Ehretia
thyrsiflora [44]. Whereas, isoquercetrin, hyperoside, trifolin, astragalin, kaempferol 3-O-
arabinosylgalactoside, quercetin 3-O-arabinosylgalactoside, with other phenolic acids were first isolated
from leaves of same species by different researchers [45].
Terpenoids were also reported in some species of Boraginaceae family plants. Sixteen compounds mostly
oleanane and ursane type triterpenes were isolated from Cordia multispicata [46]. Additionally, a
monodesmoside triterpenoid saponin (3β-O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl pomolic
acid) was isolated from Cordia piauhiensis Fresen [47]. The family plant seeds are one of the best known
sources of γ-linolenic acid (GLA; 18:3 n−6). Novel sources of GLA, belongs to omega-6 (w-6) family, are
under the scope of search due to its claimed beneficial effect on the treatment and control of cardiovascular
disease, diabetes, atopic dermatitis, premenstrual syndrome, hypertension, variety of tumors and cholesterol
levels as well as its importance in dietary and a cosmetic preparations [48]. The fatty acid γ-linolenic acid
(GLA, 18:3ω6) and α-linolenic acid (ALA, 18:3ω3) were recorded in the seeds of some Boraginaceae
species. GLA is also found in the seed oil of all Boraginaceae species and its content on total saponifiable
oil has been used as a taxonomic marker [49, 50].
Biotechnological Production of Plant Secondary Metabolite, 2012, 215-240 215
CHAPTER 13
Production of Anticancer Secondary Metabolites: Impacts of Bioprocess
Engineering
Sajjad Khani1, Jaleh Barar1,2, Ali Movafeghi1,3 and Yadollah Omidi1,2,*
1
Research Centre for Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tabriz University of Medical
Sciences, Tabriz, Iran; 2Ovarian Cancer Research Center, Perelman School of Medicine, University of
Pennsylvania, Philadelphia, PA 19104, USA and 3Plant Biology Department, Faculty of Natural Sciences,
University of Tabriz, Tabriz, Iran
Abstract: Higher plants produce a wide spectrum of secondary metabolites that have been used as
sources of a large number of industrial products (e.g. agricultural chemicals and pharmaceuticals).
Although some of the natural products have been replaced by synthetic substitutes because of cost
considerations, a number of medicinally important high value chemicals are still being extracted from
plants. These products are used as intermediate/model compounds for chemical synthesis of many
potent analogues/pharmaceuticals. Various natural products are used as antitumour agents or for the
synthesis of antitumour agents. Of note, the readily available baccatin III have been exploited for the
synthesis of paclitaxel by coupling baccatin III and the N-benzoyl-b-phenylisoserine side chain.
However, novel biotechnological approaches (e.g. cell based bioprocess engineering) appears to be the
best strategy since the extraction of natural products from plant sources may result in extinction of
medicinal plant species (e.g. Taxus species). In fact, this approach confers cost-effective technology for
the large-scale production of clinically/commercially important secondary metabolites such as
paclitaxel. Since the emergence of the tissue culture technology in early 1950s, it has been increasingly
advanced towards industrial production of secondary metabolites to overcome many problems
associated with such approach. Given the fact that the plant cells/tissue culture systems not only provide
means for biosynthesis of natural products but also serve as 'factories' for bioconversion of low value
compounds into high value products, in the current chapter, we will focus on impacts of this robust
technology regarding production of anticancer secondary metabolites.
Keywords: Secondary metabolites, natural products, cancer, antitumor, bioprocess engineering, large-
scale production, biosynthesis, semi-synthetic derivatives, biotechnology, precursor, cell culture, elicitation,
immobilized biocatalysts.
1. INTRODUCTION
To date, cancer has been considered as a growing health problem around the world and according to the
World Health Organization (WHO) report in 2005 cancer accounted for 13% of a total of 58 million deaths
worldwide. Unfortunately, there are more than 10 million cases of various cancers per year worldwide [1].
There exist no extremely effective chemotherapy modalities as complete treatment for most of the cancers
even though recent advancements in cancer immunotherapy and gene therapy appears to be very promising
[2]. There is a general solemn covenant for developments of new medicaments with maximal effectiveness
and minimal side effects. Among many compounds tested for cancer therapy, natural products offer great
opportunities to progress the drug development processes since it has been proved for many years that they
play a major role in prevention and treatment of various diseases. Accordingly, a considerable number of
anticancer agents currently used in the clinic are of natural origin. For instance, over half of all anticancer
prescription drugs approved internationally in past decades were natural products or their derivatives [3].
Plant based pharmaceuticals have been considered as basis of various compounds used against a number of
diseases. In this regard, for example, the National Cancer Institute (NCI) launched a large screening
approach to examine antitumor potentials of many plant extracts during the 1960s [1].
*Address correspondence to Yadollah Omidi: Ovarian Cancer Research Center, School of Medicine, University of Pennsylvania,
Philadelphia, USA; Tel: +1 (215) 573-5016, Fax: +1 (215) 573-7627, E-mail: yomidi@mail.med.upenn.edu

216 Biotechnological Production of Plant Secondary Metabolite Khani et al.
Plant based bioactive secondary metabolites play a significant role in the treatment of cancer at least for the
last four decades. Of note, the discovery and introduction of paclitaxel, vinca alkaloids, etoposide and
comptothein to market support drug discovery programs based on natural products [4]. In the late 1960s,
vinblastine and vincristine were introduced as the first plant based potent anticancer agents, as a result of
which long-term remissions and cures became practical in childhood leukaemia, testicular teratoma,
Hodgkin’s disease and many other cancers. Perhaps the undoubted cornerstone pharmaceutical for cancer
therapy is paclitaxel, which confers great efficacy against refractory breast and ovarian cancers. It is, at
present, one of the most effective anticancer drugs [5].
The market price for the plant-derived anticancer drugs appears to be very high – 1 kg of vincristine costs
about 20,000 USD and the annual world market is about 5 million USD. For paclitaxel, the cost is even
higher (~5 million USD/kg), while camptothecin, podophyllotoxin and demecolcine seem to be in the range
of vincristine [6].
Given the fact that the natural supply of anticancer bioactive compound is limited, several research groups
have led to employ plant cell/tissue cultures for the biotechnological mass production of these compounds
[6]. In fact, for more than 45 years, plant cell and tissue cultures have been used to produce secondary
metabolites such as anticancer bioactive compound [7]. Since the world demand for natural anticancer
compound is increasingly growing and the plant sources are limited, the application of bioprocess
engineering of plant cells/tissues is going to be one of the most promising methods for industrial production
of natural product such as paclitaxel. This can be more highlighted if the futuristic transgenic plants that are
commonly being exploited for sustainable production of in house compounds, recombinant proteins and
plantibodies. Our main focus, in this current chapter, is to provide the importance of bioactive natural
products for cancer therapy, the technical framework for improved production of secondary metabolites, the
bioreactor based scale up process and the successful industrial approaches.
2. SCREENING OF NATURAL PRODUCTS TOWARDS ANTICANCER DRUG DISCOVERY
Based on the number of known species (300,000- 500,000), plants represent the second largest source of
biodiversity (15%). Despite their large impacts on the health care systems (in particular in developing
countries), surprisingly only 15 – 20 % of terrestrial plants have been evaluated for their therapeutic
potential. Recent advancements in sample selection and collection methodologies, chemical separation
technologies and the bioprocess engineering as well as transgenic plants have favored the means for plant
based drug developments [8]. In 1955, the largest effort searching for new anticancer drugs was launched
by the NCI, upon which during 1960-1982 about 114,000 extracts from an estimated 35,000 plant samples
from different regions were screened for their antitumor potentials against primarily L1210 and P388
mouse leukemias [9]. Among a wide variety of compound classes isolated and characterized, some
clinically important chemotherapeutic agents were introduced to health market including paclitaxel (Taxus
brevifolia and other Taxus species), hycamptamine (topotecan), CPT-11, 9-aminocamptothecin, and
semisynthetic derivatives of camptothecin (Camptotheca acuminata) as shown in Table 1 [8].
3. NATURAL PRODUCTS VERSUS CHEMICALLY SYNTHESIZED COMPOUNDS
Synthetic methods for production of new drugs sometimes endure various pitfalls, while there exist several
theoretical/practical advantages for natural products as follow: A) Natural products offer unmatched
chemical diversity with structural complexity and biological potency [10] and the plant resources are
largely unexplored. Thus screening of plant resources can lead us towards discovery of novel bioactive
compounds. B) Natural products occupy a complementary region of chemical space compared with
synthetic compounds [11]. In fact, the natural product databases contain many more scaffolds with
important proportion of ring systems that are not found in other drug databases. C) The libraries of natural
product analogs can be generated using natural products as templates that are exploited in combinatorial
chemistry for generation of novel drugs that is also enhanced by combining the techniques of
biotransformation and combinatorial biosynthesis. D) The regulation of natural product biosynthesis can be
optimized [12] thus their biosynthesis can also be manipulated to yield new derivatives with possibly
Production of Anticancer Secondary Metabolites Biotechnological Production of Plant Secondary Metabolite 217
superior qualities and quantities. E) Natural product compounds may lead to the discovery and better
understanding of targets and pathways involved in the disease process as reported for the protein–protein
complexes “B-catenin” in the “Wnt pathway” and “HIF-1/p300” [13]. F) The natural product based
compounds can go straight from ‘hit’ to drug, where the synthetic drugs are typically the result of numerous
structural modifications over the course of an extensive drug discovery program [14].
Table 1: Some selected drugs derived from plant sources.
Drug Generic Name Source Plant

(year introduced)
Arglabin (1999) Artemisia glabella
Masoprocol (1992) Larrea tridentata
Paclitaxel (1992) Taxus brevifolia . and Taxus spp.
Solamargine (1987) Solanum incanum
Vinblastine (1965) Catharanthus roseus
Vincristine (1963) Catharanthus roseus
Docetaxel (1995) Paclitaxel-derived, Taxus brevifolia
Elliptinium acetate (1983) Synthetic modification from ellipticine, Bleekeria vitiensis
Etoposide phosphate (1996) Podophyllotoxin derived, Podophyllum peltatum
Irinotecan hydrochloride (1994) Camptothecin derived, Camptotheca acuminata Decne
Teniposide (1992) Podophyllotoxin derived, Podophyllum peltatum
Topotecan hydrochloride (1996) Camptothecin derived Camptotheca acuminata Decne
Vindesine (1980 Europe; 1985 Japan) Vinca alkaloid derived Catharanthus roseus
Vinorelbine (1989) Vinca alkaloid derived Catharanthus roseus
4. ANTITUMOR COMPOUNDS AND THEIR SOURCES
Several potent and active substances, which are now employed in clinics, are isolated compounds from
plant sources. Some of these compounds (e.g. alkaloids, such as vinblastine, vincristine, paclitaxel,
docetaxel, camptothecin, colchicine, demecolcine, or the lignan podophyllotoxin) stop cell division or act
as cytotoxic agents that employed as chemotherapy modalities. Figs. 1-5 represent the natural sources and
the chemical structures of some plant based natural products. Of these, vinblastine, demecolcine and
podophyllotoxin inhibit the polymerization of tubulin to microtubules. But, paclitaxel stabilizes the
microtubule complexes. Camptothecin mainly acts as an inhibitor of DNA topoisomerase I. The
podophyllotoxin derivatives (e.g. Etopophos) inhibit topoisomerase II. It is clear that the inhibition of
polymerization or dissociation of microtubules and DNA topoisomerase stop the multiplication of healthy
and tumour cells [6].
Apocynaceae. Catharanthus roseus (Apocynaceae) contains more than 95 alkaloids such as vinblastine and
vincristine. It is a short-lived perennial that is of up to 0.4m in height, with dark green, glossy leaves and
attractive pink/white flowers (Fig. 1A). Roots or leaves are used in traditional medicine as remedies,
whereas pure alkaloids are extracted from aerial parts which are considered as important medicines for
treatment of different malignancies (e.g. breast, uterine cancers; Hodgkin’s and non-Hodgkin’s lymphoma).
The two dimeric indole alkaloids, vincristine and vinblastine (Fig. 1B), are produced at very low levels (i.e.
< 3 g/metric ton) in the related plants [6].
Taxaceae. Yew of the genus Taxus (Taxaceae), as the main source for paclitaxel, is a slow-growing, long-
lived, evergreen tree, with a thick trunk, reddish brown bark and narrow, flat, dark green leaves arranged in
two ranks (Fig. 2A). Yew bark or leaves are the sources of paclitaxel and similar compounds which are
used to treat the advanced ovarian and breast cancers and appear to be highly effective in the treatment of
some other cancers [15]. Paclitaxel (Fig. 2B) was originally obtained from the bark of North American
Taxus brevifolia. Notwithstanding its first extraction from the leaves of Taxus baccata which is native in
Biotechnological Production of Plant Secondary Metabolite, 2012, 241-243 241
Subject Index
Abiotic 3, 8, 11, 22, 36, 68, 74, 90, 176, 177, 223, 235
Alkaloid 6, 10, 13, 14, 22, 48, 59, 60, 64, 68, 107, 110, 176, 178, 180, 200, 202,
203, 206, 216, 217, 220, 221, 222, 223, 225, 228, 235
Anthraquinone 11, 124
Anticancer 38, 41, 54, 110, 215, 216, 220, 221, 226, 227, 228, 230, 231, 233, 234,
236
Anthocyanin 4, 13, 26, 27, 28, 29, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 176,
178, 179, 180, 181
Antioxidant 8, 21, 22, 30, 31, 38, 41, 42, 68, 87, 96, 97, 98, 99, 101, 110, 111, 115,
117, 124, 147, 181, 202, 222
Antitumor 91, 215, 216, 217, 235
Auxin 90, 92, 94, 100, 112, 116, 149, 150, 167, 168, 221, 226
Benzylaminepurine (BAP) 73, 93, 94, 100, 149, 150, 167, 168, 183, 205, 208
Biocatalyst 11, 48, 118, 215, 224
Biomass 8, 9, 10, 67, 70, 73, 74, 119, 145, 150, 151, 177, 181, 205, 206, 224,
227, 231, 232, 233
Bioprocess 215, 216, 222, 223, 232, 233
Bioreactor 3, 8, 14, 76, 107, 109, 111, 117, 118, 119, 167, 168, 204, 206, 216, 224,
225, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236
Biosynthesis 6, 9, 14, 21, 22, 23, 25, 26, 27, 29, 36, 37, 46, 47, 53, 63, 64, 67, 70, 71,
73, 74, 75, 76, 87, 100, 108, 112, 113, 114, 146, 152, 165, 170, 171,
176, 178, 179, 180, 181, 183, 184, 187, 188, 189, 190, 191, 194, 195,
196, 197, 198, 203, 204, 205, 206, 207, 208, 215, 216, 220, 221, 222,
223, 224, 225, 229, 230, 233, 234, 235, 236
Biotransformation 8, 45, 48, 49, 55, 11, 107, 110, 111, 177, 188, 192, 193, 194, 198, 216,
227, 228
Callugenesis 5, 95
Callus culture 3, 36, 45, 46, 47, 67, 70, 71, 72, 73, 114, 116, 149, 150, 198, 200, 203,
204, 205, 222, 235
Callus induction 47, 112, 149, 221, 233
Carotenoid 22, 110, 112, 183
Culture medium 3, 4, 5, 8, 9, 10, 71, 72, 74, 75, 112, 115, 149, 150, 151, 168, 208, 221,
222, 224, 225, 232, 236
Coumarin 26, 27, 28, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48
Cytokinin 9, 89, 90, 92, 93, 94, 112, 149, 150, 167, 182, 183, 184, 221, 226
Cytotoxicity 41, 54, 61, 91, 92, 152, 217
De novo 11, 49, 90, 188, 195, 198, 236
Dichlorophenoxyacetic acid 73, 112, 149, 150, 167, 168, 206, 208, 221
Differentiation 5, 6, 7, 9, 41, 46, 112, 115, 116, 118, 187, 194, 195, 196, 197, 198, 204,
235
DNA 6, 13, 41, 42, 68, 75, 89, 168, 169, 170, 171, 191, 219, 226
Elicitor 3, 10, 11, 36, 45, 46, 47, 67, 73, 74, 114, 200, 203, 204, 205, 206, 207,
215, 221, 222, 223, 224, 225, 228, 234, 235, 236
Enzyme 9, 11, 14, 31, 38, 43, 48, 53, 54, 60, 62, 63, 64, 71, 75, 76, 117, 118,
163, 170, 177, 178, 181, 183, 187, 188, 190, 192, 196, 197, 206, 207,
208, 224, 227, 233
Essential oil 21, 22, 25, 110, 111, 117, 182, 183
Fermentor 114, 236
Fermentation 26, 49, 230
Field-grown 67, 87, 89, 90, 220

242 Biotechnological Production of Plant Secondary Metabolite Ilkay Erdogan Orhan
Flavonoid 22, 26, 27, 28, 29, 30, 67, 68, 75, 87, 88, 95, 96, 99, 101, 124, 125, 149,
176, 177, 178, 179, 180, 181
Food additive 43, 108, 109, 110, 124
GC–MS 112, 117
Gene cloning 170, 171, 187, 191
Gene expression 5, 165
Genetic engineering 6, 25
Genotype 92, 166, 167, 169, 176, 182
Germplasm 88, 89, 92, 166
Gibberellin (GA) 113,194, 208
Growth regulator 5, 8, 9, 38, 45, 47, 67, 72, 73, 89, 94, 100, 107, 112, 149, 150, 159, 166,
167, 168, 176, 179, 181, 183, 203, 205, 206, 208
Hairy root 9, 46, 47, 48, 49, 107, 109, 118, 119, 200, 204, 205, 206, 207, 226, 228,
231, 233, 235
HPLC 68, 112, 117, 192, 195
Immobilization 3, 8, 11, 118
Indole acetic acid (IAA) 72, 94, 149, 194, 203, 204, 205
Iridoid 87, 89, 90, 91, 96, 97, 100, 101, 108
Isoprene 6, 22, 25, 108, 176, 207
Jojoba 159, 160, 161, 162, 163, 167, 168, 169, 170, 171
Large-scale 3, 14, 15, 53, 63, 67, 70, 114, 124, 170, 215, 224, 225, 232, 235
Metabolic engineering 14, 25, 76, 109, 170, 171, 200, 203, 207, 228, 235
Micropropagation 90, 107, 109, 149, 150, 159, 166, 167, 171, 200
Naphthaleneacetic acid (NAA) 72, 73, 90, 92, 94, 95, 100, 113, 150, 167, 168, 206, 208, 236
Nitrogen 9, 72, 112, 150, 183, 184, 203
Nutrient 6, 8, 9, 29, 31, 67, 72, 75, 93, 112, 117, 176, 177, 181, 182, 224, 228,
231, 232, 233
Organ culture 3, 4, 7, 46, 48, 67, 94, 95, 97, 107, 108, 109, 115
Organogenesis 5, 89, 90, 92, 94, 95, 100, 112, 166, 195
Oxidative stress 36, 87, 176, 181, 224
Pesticide 3, 164, 171, 176, 177, 179, 181, 182, 184, 185
Pharmaceutical 14, 21, 28, 36, 37, 43, 45, 46, 49, 53, 54, 60, 67, 107, 109, 119, 124,
149, 163, 164, 165, 176, 188, 215, 216, 221, 226, 228
Phenolic 14, 21, 22, 26, 27, 28, 29, 31, 38, 88, 95, 98, 99, 100, 112, 176, 177,
178, 179, 180, 181, 202, 205
Pigment 3, 67, 68, 70, 72, 73, 74, 76, 124, 145, 150, 184, 200, 203, 204, 205,
207, 208
Plant cell culture 90, 107, 108, 109, 110, 111, 112, 113, 114, 124, 149, 200
Precursor 7, 14, 23, 25, 36, 45, 63, 75, 107, 113, 118, 183, 189, 196, 203, 205,
215, 220, 222, 225, 227, 234
Propagation 89, 92, 165, 166, 169, 171
Recombinant 13, 170, 188, 191, 216
Rhizogenesis 94, 95, 100
Rhizogenic callus (calli) 92, 93, 100
Root culture 3, 46, 93, 95, 96, 97, 98, 99, 100, 108, 115, 116, 117, 187, 194, 195,
198, 204, 226, 233, 235, 236
Salinity 90, 145, 171, 176, 181
Saponin 108, 114, 124, 125, 145, 149, 203
Scale-up 108, 111, 118, 221, 228, 231, 232, 233, 236
Shoot culture 7, 46, 95, 96, 97, 98, 100, 108, 115, 170, 187, 194, 195, 196, 206, 235
Shoot regeneration 90, 149, 166
Somatic embryo(genesis) 89, 159, 167, 189, 190, 191, 195, 208
Steroid 13, 48, 107, 108, 124, 125, 149, 187, 192
Sucrose 9, 72, 73, 90, 112, 221, 222, 223, 236
Subject Index Biotechnological Production of Plant Secondary Metabolite 243
Suspension culture 3, 5, 7, 14, 36, 46, 47, 67, 71, 73, 74, 75, 110, 113, 114, 117, 167, 193,
194, 195, 196, 197, 203, 204, 205, 206, 207, 208, 221, 222, 223, 224,
225, 228, 231, 232, 233, 234, 235
Terpenoid 13, 21, 22, 48, 107, 108, 109, 119, 149, 176, 177, 181, 183, 184, 203,
221, 228
Traditional medicine 21, 88, 107, 108, 124, 217
Tissue culture 3, 4, 5, 13, 38, 45, 67, 73, 74, 76, 111, 112, 150, 166, 167, 171, 187,
197, 198, 200, 204, 215, 216, 220, 221, 222
Transgenic 6, 14, 25, 75, 76, 107, 109, 170, 208, 216, 227, 236
Volatile oil 112, 113, 176, 181
Yeast 114, 145, 149, 206, 208
244 Biotechnological Production of Plant Secondary Metabolite, 2012, 244-252
Plant Index
Achillea millefolium 12, 117

Achyranthes 125
Achyranthes bidentata 145
Adenosma caeruleum 96
Aegilops biuncialis 184
Aegilops cylindrica 184
Aegle marmelos 61
Agalinis 88
Agave tequilana 3, 6
Ajuga reptans 73
Alkanna 201, 202, 203
Alkanna calliensis 202
Alkanna corcyrensis 202
Alkanna graeca 202
Alkanna methanaea 202
Alkanna orientalis 202
Alkanna pindicola 202
Alkanna primuliflora 202
Alkanna sieberi 202
Alkanna stribrnyi 202
Alkanna tinctoria 201, 202
Alkanna tuberculata 202
Alternanthera 124, 125, 147, 151, 152
Alternanthera bettzichiana 147
Alternanthera brasiliana 126, 131, 132, 146, 147, 149, 150
Alternanthera canescens 126
Alternanthera flavescens 126
Alternanthera halimifolia 126
Alternanthera lanceolata 147
Alternanthera maritima 126, 127, 128, 129, 130, 132, 133, 146, 147, 149
Alternanthera nodiflora 147
Alternanthera paronychioides 126, 147
Alternanthera philoxeroides 126, 131, 132, 146, 147, 151
Alternanthera polygonoides 132
Alternanthera pungens 126, 127, 132, 147
Alternanthera repens 128, 147
Alternanthera sessilis 126, 127, 131, 147, 149
Alternanthera tenella 126, 127, 128, 129, 130, 146, 147, 149
Alternanthera versicolor 132
Amaranthus 125
Amaranthus dubius 146
Amelanchier alnifolia 69
Ammi majus 39, 47
Amsinckia 201, 202
Anchusa arveniis 202
Anchusa 112
Anchusa hispida 202
Anchusa milleri 202
Anchusa officinalis 202
Anethum 113
Anethum graveolens 113
Plant Index Biotechnological Production of Plant Secondary Metabolite 245
Anethum sowa 113

Angelica 40
Angelica gigas 41, 47
Angelica koreana 41
Angelica officinalis 42
Antirrhinum majus 89
Anthirrhinum linarium 88
Apium graveolens 180
Arabidopsis 170
Arabidopsis thaliana 25, 26, 191, 227
Aralia cordata 72, 73, 77
Arnebia 201, 202
Arnebia densiflora 201, 202, 205
Arnebia euchroma 201, 202, 204, 205
Aronia melanocarpa 69
Artemisia absinthium 8, 13, 115, 117
Artemisia annua 7, 115
Artemisia glabella 217
Artocarpus nobilis 54, 55
Atropa belladona 7
Avena sativa 180
Azadirachta indica 9
Bacopa monnieri 88, 89, 90, 93
Barleria prionitis 55
Begonia 7
Beta vulgaris 8, 10
Betula lenta 108
Bidens 13
Bidens alba 8
Bleekeria vitiensis 217
Blutaparon 125
Blutaparon portulacoides 145, 146
Borago 200, 201
Borago officinalis 201, 202
Brassica 184
Brassica napus 167, 183
Brassica oleracea 183
Brosimum gaudichaudii 39
Brunnera macrophylla 201
Buxus 59
Buxus hyrcana 59
Caesalpinia bonduc 54, 55
Calceolaria 88
Calophyllum 41
Calophyllum brasiliense 41
Calophyllum dispar 41
Calophyllum inophyllum 47
Calystegia sepium 8
Camelia 77
Camptotheca acuminata 4, 72, 73, 110, 216, 217, 220, 221, 235
Campylotropis hirtella 41
Capsicum 11, 118
Capsicum annuum 107, 110, 112
Capsicum frutescens 11, 12, 110, 112, 113, 118
Carpentaria acuminata 228

Cassia 13
Cassia obtusifolia 178
Castilleja 87, 88, 91, 97, 98, 99
Catalpa ovata 97
Catharanthus roseus 4, 7, 10, 11, 12, 15, 75, 181, 217, 218, 221, 224,
225, 228, 230, 235
Centaurium erythraea 183
Chamissoa 125
Chamomila recutita 182
Celeus blumei 7
Celosia 125
Centaurium erythraea 183
Centella asiatica 6
Cephalotaxus harringtonia 4
Chenopodium ambrosioides 113
Chenopodium rubrum 10, 226
Chondrodendron tomentosa 4
Chrysanthemum cinerariaefolium 4
Chrysanthemum coronarium 77
Cichorium intybus 13, 46
Cinchona 7
Cinchona ledgeriana 13
Cinchona officinalis 4
Citrus 113
Citrus limon 12, 114
Citrus paradisi 12
Cleome rosea 70, 71, 73, 75, 76
Cnidium 40
Coffea arabica 4, 10, 11, 12
Colchicum autumnale 218, 219
Coleus blumei 4, 15
Coleus forskohlii 206
Conium 4
Convallaria 187
Coptis japonica 4, 221
Cordia americana 202
Cordia multispicata 203
Cordia myxa 202
Cordia sebestena 201
Cordia spinescens 202
Cordia verbenacea 201, 208
Coreopsis tinctoria 8
Coriandrum sativum 113, 167
Coumarouna odorata 37
Cupressus lusitanica 111
Curcuma zedoaria 12
Cyathula 125
Cymbidium 12
Cynara cardunculus 14
Cynoglossum 201, 202
Cynoglossum amabile 202
Cynoglossum officinale 202, 207
Daphne 40
Daphne gnidium 40
Datura 13
Datura stramonium 4, 180
Daucus 77
Daucus carota 11, 47, 71, 72, 73, 75, 76, 77, 112, 183
Decatropis bicolor 39
Decentra pergrina 7
Dianthus caryophyllus 25
Digitalis 187, 188, 191, 194, 198
Digitalis lanata 4, 7, 11, 12, 187, 188, 190, 191, 192, 194, 195,
197, 198
Digitalis obscura 191
Digitalis purpurea 7, 12, 88, 187, 188, 190, 191, 194, 195, 198
Dioscoria deltoidea 4, 7, 11, 110, 114
Dipteryx odorata 37
Dolichandrone falcate 63
Duboisia leichhardtii 13
Echinacea purpurea 8, 13, 15
Echium 202
Echium amoenum 206
Echium glomeratum 202
Echium italicum 204, 222, 225
Echium lycopsis 202
Echium plantaginuem 201, 202
Echium vulgare 201
Ehretia thyrsiflora 202
Eritrichium sericeum 206
Erythroxylon coca 4
Eschscholzia californica 4
Eucalyptus 113
Eucalyptus camaldulensis 112
Eucalyptus perriniana 12
Euphorbia milli 72, 77
Euphorbia pulcherrima 91
Eurycoma longifolia 4, 8
Ficus hispada 39
Foeniculum 113
Foeniculum vulgare 113
Froelichia 125
Galipea panamensis 27
Galphimia glauca 11
Garcinia brevipedicellata 62
Gardenia jasminoides 12
Gastrocotyle hispida 202
Ginkgo biloba 117, 227
Glehnia littoralis 72
Glycine max 11, 180
Glycyrrhiza glabra 12, 13, 110
Glycyrrhiza uralensis 13, 117
Gomphrena 124, 125, 133, 147, 151, 152
Gomphrena affinis 133, 134
Gomphrena boliviana 134, 135, 137, 140, 148
Gomphrena canescens 133, 134
Gomphrena celosioides 133, 148
Gomphrena claussenii 135, 136, 138

Gomphrena cunninghamii 133, 134
Gomphrena decumbens 140
Gomphrena dispersa 133,134
Gomphrena glabrata 146
Gomphrena globosa 137, 138, 139, 140, 146, 148, 149, 150
Gomphrena haageana 133, 134, 140, 148
Gomphrena holosericea 135, 140
Gomphrena macrocephala 136, 148, 150
Gomphrena martiana 134, 135, 136, 137, 138, 140, 148
Gomphrena meyeniana 133, 134, 135, 136, 140, 148
Gomphrena officinalis 133, 150
Gomphrena perennis 133, 140, 148
Gomphrena pulchella 148
Gossypium arboreum 114
Gossypium hirsutum 181
Fragaria ananassa 73
Hackelia venusta 208
Halleria lucida 88, 96, 98
Haplophyllum patavinum 46
Harpagophytum procumbens 117
Harpagophythum 97
Hedychium spicatum 62
Heliotropium 200, 201, 202
Heliotropium bovei 202
Heliotropium curassavicum 202
Heliotropium curassavicum var. argentinum 202
Heliotropium indicum 207, 208
Helleborus 187
Hemidesmus indicus 8
Herbstia 125
Hibiscus rosasinensis 69
Hibiscus sabdariffa 69, 72, 77
Hyoscamus albus 8, 13
Hyoscamus muticus 8, 13
Hyoscyamus niger 4, 13
Impatiens balsamina 5
Ipomoea batatas 69, 72, 73, 74, 77
Iresine 125
Jasminum 4
Jasmine officinale 110
Jatropha curcas 170
Lagerstroemia speciosa 62
Lappula spinocarpos 202
Larrea tridentata 217
Lavandula angustifolia 12
Lavandula vera 11
Leucaena 181
Linum 236
Linum album 236
Linum flavum 236
Linum nodiflorum 236
Litchi chinenesis 69
Lithospermum 201, 202, 203, 204, 207
Lithospermum canescens 205

Lithospermum erythrorhizon 4, 8, 10, 13, 15, 73, 201, 202, 203, 204, 205, 206,
207, 208, 221
Lithospermum officinale 202
Lithospermum ruderale 202
Lobostemon trigonus 201
Lunaria annua 170
Lycopersicon esculentum 4, 25, 68, 110
Macrotomia euchroma 202
Mallotus japonicus 77
Malus 76, 77
Malva sylvestris 69
Manihot esculenta 183
Matricaria chamomilla 110, 114
Mecardonia tenella 93
Melilotus alba 43
Melilotus officinalis 43, 49
Melissa officinalis 181
Mentha 12
Mentha arvensis 182, 183
Mentha longifolia 181
Mentha piperita 110, 113, 183
Mentha spicata 183
Mentha citrata 7, 183
Mertensia maritima 201
Morinda citrifolia 4, 7, 11
Myosotis 200, 201
Myosotis scorpioides 202
Mucuna pruriens 11, 12
Nauclea latifolia 54, 55
Neopicrorhiza scrophulariflora 88
Nicotiana tabacum 4, 7, 11, 12, 13, 15, 26, 151, 181
Nothapodytes foetida 220, 235
Ocimum basilicum 183
Ocimum sanctum 181
Olea europaea 31
Onosma 202
Onosma arenaria 202
Onosma argentatum 202
Onosma hispida 202
Onosma paniculatum 203, 204, 205, 207
Onosma polyphyllum 202
Onosma visianii 202
Ophiorrhiza mungos 235
Ophiorrhiza pumila 220, 228, 235
Orthosiphon aristatus 114
Oryza sativa 180, 191
Oxalis linearis 72
Papaver bracteatum 12
Papaver somniferum 4, 11, 12
Panax ginseng 9, 13, 15, 107,114, 117, 118, 227
Panax sikkimensis 76
Paracaryum intermedium 202
Paracaryum rugulosum 202
Paulownia elongate 10
Paulownia tomentosa 88, 89
Pedicularis resupinata 89
Pelargonium graveolens 183
Pelargonium peltatum 219, 236
Pelargonium tomentosum 7, 115
Penstemon barbatus 89
Penstemon gentianoides 98
Penstemon rosseus 89
Penstemon serrulatus 90, 97
Pentaglottis sempervirens 201
Perilla frutescens 77, 113
Petunia hybrida 25, 77
Pfaffia 124, 125, 140, 141, 147, 148, 151, 152
Pfaffia elata 145
Pfaffia grandiflora 145
Pfaffia glomerata 143, 148, 151
Pfaffia hookeriana 145
Pfaffia iresinoides 142, 143, 148
Pfaffia jubata 148
Pfaffia paniculata 124, 141, 142, 146, 148
Pfaffia pulverulenta 144
Pfaffia stenophylla 145, 148
Pfaffia tuberosa 148
Pharbitis nil 48
Phaseolus 180
Phaseolus vulgaris 69, 91
Phellolophium madagascariense 41
Picria felterrae 89
Picrorhiza kurroa 7, 89, 90, 115
Picrorhiza scrophulariiflora 96
Pilocarpus bracteatum 4
Pilocarpus jaborandi 4
Pimpinella anisum 110, 115
Pisum sativum 167, 180
Plantago lanceolata 91
Plumbago rosea 11
Podophyllum hexandrum 12, 219, 231, 236
Podophyllum peltatum 217, 235
Polygonum multiflorum 47
Polygonum tinctorium 7, 8
Populus 14
Populus tremuloides 191
Psoralea caryolia 39
Pyrenacantha klaineana 220
Pseudoplantago 125
Pulmonaria 201
Quaternella 125
Raphanus sativus 73, 77
Rauwolfia serpentina 4, 13, 15
Rehmannia glutinosa 89, 90, 96, 97
Rhodiola sachalinensis 13
Rosa gallica 69
Rosa hybrida 74
Rosmarinus officinalis 77, 111

Rotula aquatica 201, 208
Rubia akane 73
Rubia cordifolia 13
Rubia peregrina 201
Rubus idaeus 180
Ruta graveolens 7, 45, 46
Saccharum officinalis 184
Salacia reticulate 61
Salvia 110
Salvia miltiorrhiza 13, 117
Salvia officinalis 7, 107, 115, 183, 222
Salvia sclarea 111, 117
Scoparia ducis 89
Scrophularia ningpoensis 89
Scrophularia nodosa 89, 90, 97
Scrophularia striata 96
Scrophularia umbosa 90
Scrophularia yoshimurae 89,90
Seseli 40
Seseli gummiferum ssp. corymbosum 40
Seseli indicum 40
Sibthorpia peregrine 89
Simmondsia chinensis 159, 167
Solanum incanum 217
Solanum melongena 68, 69
Solanum paludosum 7
Solanum tuberosum 4, 74, 180, 184
Sorghum bicolor 184
Spinacia oleracea 178
Spiraea ulmaria 108
Spirulina platensis 12
Stevia rebaudiana 4, 7, 12, 110, 115
Strophanthus 187
Symphytum 200, 201, 202
Symphytum officinale 207
Strychnos nux-vomica 4
Tagetes patula 11
Tanacetum parthenium 46, 91
Taraxacum officinale 115
Taxus 74, 113, 216, 217, 221, 223, 228,233, 234, 235
Taxus baccata 11, 217, 218, 222, 224, 225, 228, 231, 233, 234
Taxus brevifolia 216, 217, 221, 222, 224
Taxus chinensis 222, 224, 225, 231, 233
Taxus chinensis var. mairei 113
Taxus cuspidata 113, 222, 224, 225, 232, 233
Taxus media 225
Taxus wallichiana 231, 233
Terminalia chebula 61
Terminalia superba 61
Thalictrum glaucum 187
Thalictrum rugosum 10
Thaumatococcus danielli 4, 110
Thymus minus 11
Torenia fournieri 96
Trichodesma africanum 202
Trichodesma indicum 201, 208
Trigonella foenum-graecum 117
Triticum aestivum 184
Uragoga ipecacuanha 4
Vaccinium myrtillus 69
Vaccinium pahalae 77
Valeriana officinalis 117
Valeriana wallichii 114
Vanilla planifolia 12
Verbascum 108
Verbascum thapsus 89
Veronica persica 89
Vicia faba 180
Vigna unguiculata 183
Vitis 73, 77
Vitis coignetiae 69
Vitis vinifera 10, 69, 72, 74, 75, 184
Withania somniferum 7
Zea mays 178, 180
Ziziphus zizyphus 159

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Biotechnological Production of Plant

Ilkay Erdogan Orhan

Permission for Use of Material and Reproduction

1. Plant Cell and Tissue Culture as a Source of Secondary Metabolites 3

2. Natural Product Extracts: Terpenes and Phenolics 21

3. Biotechnological Production of Coumarins 36

4. Novel Biomedical Agents from Plants 53

5. Production of Anthocyanins by Plant Cell and Tissue Culture Strategies 67

7. Plant Cell Tissue and Organ Cultures in Terpenoids 107

8. Bioactive Chemical Constituents and Biotecnological Production of Secondary

9. Biotechnology Approaches and Economic Analysis of Jojoba Natural Products 159

10. The Effects of Pesticides on Plant Secondary Metabolites 176

Plant Index 244

In “biotechnological production of plant secondary metabolites”, expert researchers provide detailed

Biotechnological investigations on Amaranthaceae plant species have been summarized. Biotechnology

Prof. Dr. Bilge Sener

Prof. Dr. Ilkay Erdogan Orhan

C.L. Del Toro-Sánchez

Ilkay Erdogan Orhan (Ed)

Plant Species Product Activity

2. PLANT CELL, TISSUE AND ORGAN CULTURES

c) Secondary metabolites, which do not appear to participate directly in growth and

2. IMPORTANCE AND ECOLOGICAL ROLE OF TERPENOIDS

3. BIOSYNTHESIS OF TERPENOIDS IN PLANTS

a) MVA pathway Pyruvate D-Glyceraldehyde

GPP FPP GGPP

Monoterpenes - Sesquiterpenes - Diterpenes

Keywords: Secondary metabolites, coumarins, furocoumarins, pyranocoumarins, biosynthetic pathway,

Ilkay Erdogan Orhan (Ed)

1.1 Linear type (Psoralens)

1.2 Angular type (Angelicins)

1.3 Linear type (Xanthyletin type)

1.4 Angular type (Seselin type)

4. Coumarins substituted in the pyrone ring, for example;

6. Coumestans (e.g. Coumestrol)

7. Some complex structures involved coumarin system (Novobiocin, aflatoxin)

8. Isocoumarins (e.g. Cytogenin)

Figure 1: Coumarin (benzo--pyrone).

Keywords: Natural products, enzyme inhibition, glutathione S-transferase, acetylcholinesterase, -

Ilkay Erdogan Orhan (Ed)

2. GLUTATHIONE S-TRANSFERASE INHIBITORS

From the bioactive fractions of C. bonduc, 17-hydroxycompesta-4,6-dien-3-one (1), 13,14-seco-stigmasta-

H H H3 C CH3 H CH3 H 3C CH3 H COO

(12) (13) (14) (15)

Núcleo de Biotecnologia Vegetal, Universidade do Estado do Rio de Janeiro, Brazil

Ilkay Erdogan Orhan (Ed)

2. COMMERCIAL AND MEDICINAL INTEREST

Table 1: Medicinal properties investigated in anthocyanins.

Biological Activity Plant Source Anthocyanin/Extract Refs

3. ANTHOCYANIN PRODUCTION UNDER IN VITRO CONDITIONS

Keywords: Castilleja tenuiflora, Scrophulariaceae, oxidative stress, secondary metabolites, iridoid,

instead of undifferentiated cultures as an alternative to agricultural processes for producing valuable

2. CURRENT BIOTECHNOLOGICAL RESEARCH ON MEDICINAL PLANTS FROM

Table 1: Examples of Scrophulariaceae plants used in traditional medicines.

Species Traditional Medicine Refs

Pedicularis resupinata South Korea [16]

Species In Vitro Culture Refs

Department of Pharmaceutical Botany, Faculty of Pharmacy, Hacettepe University, Sihhiye, 06100,

Keywords: Terpenoids, secondary metabolites, biotechnology, iridoid, saponoside, micropropagation,

Ilkay Erdogan Orhan (Ed)

Aspects of biotechnological approaches to plant-derived secondary metabolites:

1. Plant cell tissue and organ cultures.

3. Micropropagation of medicinal plants.