Biotechnological Production of Plant Secondary ...

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 Rodríguez-Sahagún A., Del Toro-Sánchez C.L., Gutierrez-Lomelí M. and CastellanosHernández O.A.

3

2.

Natural Product Extracts: Terpenes and Phenolics Gutiérrez-Lomelí M., Del Toro-Sánchez C.L., Rodríguez-Sahagún A. and CastellanosHernández O.A.

21

3.

Biotechnological Production of Coumarins Alev Tosun

36

4.

Novel Biomedical Agents from Plants Athar Ata

53

5.

Production of Anthocyanins by Plant Cell and Tissue Culture Strategies Claudia Simões, Norma Albarello, Tatiana C. de Castro and Elisabeth Mansur

67

6.

In Vitro Organ Cultures of the Cancer Herb Castilleja tenuiflora Benth. as Potential Sources of Iridoids and Antioxidant Compounds 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 Irem Tatli I.

8.

Bioactive Chemical Constituents and Biotecnological Production of Secondary Metabolites in Amaranthaceae Plants, Gomphreneae Tribe 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 Mohammed A.M. Aly and Aydin Basarir

87

107

124

159

10. The Effects of Pesticides on Plant Secondary Metabolites Monica Hancianu and Ana C. Aprotosoaie

176

11. Cardenolide Production as an Important Drug Agent Sebnem Harput U.

187

12. Progress in Biotechnological Applications of Diverse Species in Boraginaceae Juss. Ufuk Koca, Hatice Çölgeçen and Nueraniye Reheman

200

13. Production of Anticancer Secondary Metabolites: Impacts of Bioprocess Engineering Sajjad Khani, Jaleh Barar, Ali Movafeghi and Yadollah Omidi

215

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, United Arab Emirates University, P.O. Box 17555, Al Ain, United Arab Emirates 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 Núcleo de Biotecnologia Vegetal, Universidade do Estado do Rio de Janeiro, Brazil 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 Centro Universitario de la Ciénega, Universidad de Guadalajara. Av. Universidad 1115, Col. Lindavista, CP 47810, Ocotlán, Jalisco, México 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 Centro Universitario de la Ciénega, Universidad de Guadalajara. Av. Universidad 1115, Col. Lindavista, CP 47810, Ocotlán, Jalisco, México 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 Departamento de Biotecnología, Centro de Desarrollo de Productos Bióticos, Instituto Politécnico Nacional, P. O. Box 24, 62730, Yautepec, Morelos, México E. Mansur Núcleo de Biotecnologia Vegetal, Universidade do Estado do Rio de Janeiro, Brazil 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 Curso de Farmácia, Departamento de Biologia Vegetal, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), C.P. 6109, 13083-970, Campinas, SP, Brazil 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 Centro Universitario de la Ciénega, Universidad de Guadalajara. Av. Universidad 1115, Col. Lindavista, CP 47810, Ocotlán, Jalisco, México

vi

G. Rosas-Romero Departamento de Biotecnología, Centro de Desarrollo de Productos Bióticos, Instituto Politécnico Nacional, P. O. Box 24, 62730, Yautepec, Morelos, México M.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 C. Simões Núcleo de Biotecnologia Vegetal, Universidade do Estado do Rio de Janeiro, Brazil 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 Departamento de Biotecnología, Centro de Desarrollo de Productos Bióticos, Instituto Politécnico Nacional, P. O. Box 24, 62730, Yautepec, Morelos, México 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.* Centro Universitario de la Ciénega, Universidad de Guadalajara. Av. Universidad 1115, Col. Lindavista, CP 47810, Ocotlán, Jalisco, México 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: [email protected] and [email protected] 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

Atropa belladanna

Atropine

Activity Blocking of cholinergic

Catharanthus roseus

Vincristine Ajmalthine Ajmalicine Serpentine

Antileukaemic Antiarrhythmic Tranquilizer

Campatotheca accminata

Camptothecin

Anticancer

Cephalotaxus harringtonia

Cephalotaxine

Antitumour

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 tomentosa

(+)-Tubocurarine

Nondepolarizing muscle relaxant producing paralysis, used as an adjuvant to anaesthesia

Chrysanthemum cinerariaefolium

Pyrethrin

Insecticide (for grain storage)

Datura stramonium

Scopolamine

Antihypertension

Digitalis lanata

Digoxill Reserpine

Cardiac tonic 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-methoxycanthin6-one

Aphrodisiac

Hyoscyamus niger

Atropine

Anticholinergic

Jasminum spp.

Jasmine

Perfume

Lithospermum erythrorhizon

Shikonines

Dyes, pharmaceutical

Lycopersicum esculentum

Tomatine

Fungicidal properties

Morinda citrifolia (also Cassia tora)

Anthraquinones

Laxatives, dyes

Nicotiana tabacum

Nicotine Glutathione Ubiquinone-10

Ganglion blocker cardiovascular agent

Papaver somniferum (Opium)

Morphine and codeine

Tranquilizers, muscle relaxers, pain killers, hallucinogens

Papaver somniferum

Morphine and codeine

Analgesic, sedative and antitussive

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]



suggest that this phenomenon may be due to the high frequency of cell division that takes place in cultures in vitro. Others indicate that it may be caused by alterations or changes caused by epigenetic factors in establishing culture. Thus, there are studies showing callus established from different organs of the same plant but differing in appearance and unique characteristics (Fig. 1) [19, 20]. But even more, calli from the same explant also differ in their morphology and intrinsic characteristics such as color, friability, hardness, size, degree of differentiation, and production of metabolites [21, 22]. Regarding the latter, upon observing cultures started naturally, tissues have the highest production of certain metabolites, cultures are obtained that give better returns compared to other organs or tissues from which they do not produce, or in very small quantities [21]. The production and accumulation of secondary metabolites is an expression of a particular state of cellular differentiation, which is influenced by a number of factors, different classes of secondary metabolites requiring different degrees of cell differentiation or tissue. The formation of gradients due to physical or biochemical cellular organization is also an important factor. For example, the accumulation of isoprenoids depends on the differentiation of plastids, because most of the enzymes in the biosynthetic pathway are located in these organelles. In other cases it is necessary to develop storage organs, such as glandular hairs and cells, laticifers in the case of alkaloids [23]. In the biosynthesis of tropane, alkaloids occurs mainly in the roots of producing species [24] or synthesis of essential oils of lavender is done in the glands of aerial parts [25-28] Centella asiatica in the asiaticoside accumulation is more in the aerial parts of the plant [29], the triterpenoids from leaf tissue were more easily quantifiable in each phenotype than in the case of the undifferentiated cells (callus and cell suspensions), which had lower, but still quantifiable, levels of these targeted secondary metabolites. Leaves contained the highest triterpenoid levels (ranging from 1.8 to 5% dry weight for the triterpenoid acids and their glycosides, respectively), with the free acids occurring in a ratio of approximately 1:2.5 in relation to the glycoside content [30]. If the cultures are incubated in vitro or subjected to conditions of physiological stress, characteristics of adaptation can be expressed, and resistance under natural conditions are never expressed, selectively growing only those cells capable of adapting to new conditions. This genetic variation can also be induced by mutation techniques, genetic engineering, protoplast fusion and genetic transformation by inclusion of foreign DNA in a manner similar to those used commonly in microorganisms [15, 31, 32]. In the latter case transgenic crops or plants are obtained where the foreign DNA must be integrated into plant genome to ensure stable expression in their progeny.

Figure 1: Calli production in solid medium MS. Calli from the same explant Agave tequilana Weber that produce calli with different phenotypic characteristics.

Cell Suspension In suspensions, individual cells are distributed evenly across the medium, because it facilitates the transfer of nutrients and oxygen into the individual cells. This type of culture has the advantage of actively monitoring variables such as pH, temperature and dissolved oxygen, but some characteristics of plant cells may change in their differentiation and intercellular communication [33], which implies in many cases the decrease in the production of secondary metabolites and/or increase or initiation of another type of


Biotechnological Production of Plant Secondary Metabolites 7

compound (Table 2) [34], as has been reported in some species the synthesis of certain metabolites that require the coexistence of different cell types or intracellular compartmentalization [35]. The difference generated in the accumulation of secondary metabolites depending on the quality of cell aggregation has been reported. Also reported is the accumulation of secondary metabolites in cell culture increased when working with friable callus than when generating compact callus. The accumulation of ursolic acid in suspension culture of Salvia officinalis was constant and increase in time when derived from the friable callus but it declined, when derived from the compact callus [36]. Table 2: Comparison of the performance of natural products derived from cell suspension culture and plant. Product

Plant Species

In Vitro Culture %DW

Plant %DW

Performance Ratio

Glutathione

Nicotiana tabacum

1.0

0.1

10.0

Anthraquinones

Morinda citrifolia

18.0

2.2

8.0

Rosmarinic acid

Celeus blumei

15.0

3.0

5.0

Ajmalincine

Catharanthus roseus

1.8

0.3

6.0

Serpentine

Catharanthus roseus

1.3

0.5

2.6

Diosgenine

Dioscorea deltoide

2.0

2.0

1.0

DW = Dry Weight [37].

Plant Organ Culture Some compounds of primary and secondary metabolism are highly dependent on the degree of cell differentiation, leading to the use of tissue culture as a source of secondary metabolites. There are a number of medicinal plants whose shoot cultures have been studied for metabolites (Table 3). Similarly, root cultures are valuable sources of medicinal compounds (Table 4). Table 3: Shoot cultures of medicinal plants. Plant Species

Product

References

Mentha citrata

Terpenes

[38]

Ruta graveolens

Furanocoumarins and their biogenetic precursor umbelliferone

[39]

Furanocoumarins

[40]

Origanum vulgare

Rosmarinic acid

[41]

Solanum paludosum

Solamargine

[42]

Catharanthus roseus

Catharanthine and vindoline

[43]

Vindoline

[44]

Artemisia annua

Artemesinin

[45]

Atropa belladona

Atropine

[46]

Begonia spp.

-

[47]

Cinchona spp.

Vinblastine

[48]

Quinine

[49]

Digitalis lanata

Cardenolides

[50]

Digitalis purpurea

Cardenolides

[51, 52]

Pelargonium tomentosum

Essential oils

[53]

Picroprrhiza kurroa

Kutkin

[54]

Stevia rebaudiana

Steviosides

[55]

Withania somniferum

Withanolides

[56]

Polygonum tinctorium

Indirubin

[57]

Decentra pergrina

Alkaloids

[58]

Adapted from Ramachandra Rao and Ravishankar [59].



Korean ginseng root contains many desirable compounds, including the glycoside panaquilon (a saponin panaxin), oils, vitamins B1 and B2, alkaloids, and polysaccharides. These ingredients have various pharmacological effects on the human body, as well as antioxidant and effective drugs. Table 4: Root cultures of medicinal plants. Plant Species

Product

Beta vulgaris

Betalaines

Reference [60]

Bidens alba

Polyacetylenes

[61]

Calystegia sepium

Tropane alkaloids

[62]

Coreopsis tinctoria

Phenylpropanoids

[63]

Hyoscamus albus

Hyoscyamine

[64]

Hyoscamus muticus

Hyoscyamine

[65]

Hemidesmus indicus

2-hydroxy-4-methoxybenzaldehyde

[66]

Artemisia absynthium

Volatile oils

[67]

Polygonum tinctorium

Indigo and Indirubin

[57]


Shikonin derivatives, red naphthoquinone pigments

[68]

Echinacea purpurea

Caffeic acid derivatives

[69]

Eurycoma longifolia

9-methoxycanthin-6-one

[70]

Adapter from Payne et al. [3].

3. STRATEGIES FOR IMPROVING PRODUCTION OF SECONDARY METABOLITES IN PLANT CELL CULTURES In recent years, researches in cell culture have focused on encouraging the formation and accumulation of commercially important secondary metabolites such as drugs, fungicides, fragrances, flavorings and colorings [71, 72]. An alternative to increasing the production of active biomolecules is to use biotechnological procedures for cell cultures, tissues and organs that can be high producing, in addition to present considerable advantages over conventional phytochemical research at the time of isolating the active principle of plants. The different ways to improve production are: (1) search for cell lines with high production of the metabolite of interest, (2) optimize culture media and physicochemical parameters, cell permeability, eliciting systems, cell immobilization, cell biotransformation, genetic transformation, and (3) scale cell cultures to bioreactors. All these procedures were developed as a viable option to increase the synthesis of secondary metabolites. Search for Cell Lines with High Production of the Metabolite of Interest The selection of explant type is one of the strategies to achieve a highly productive cell line of a metabolite of importance. Recommended is the use of part of the plant where it is usually observed the accumulation of the metabolite, as has been shown in lines obtained from different parts of the plant having different productivity of secondary metabolites [73, 74]. When the in vitro culture is established, a cell line must be selected based on its growth rate, performance and stability, maintaining cell culture lines that have the characteristics of interest [75]. This selection is achieved by exposing them to toxic inhibitors or some abiotic stress, because they develop under these extreme conditions. Optimization of Culture Media and Physicochemical Parameters This includes during cell growth the production of secondary metabolites that involved various variable and its effects such as the concentration of nutrients, growth regulators, light, temperature and agitation, among others. The production of metabolites is reflected in the composition and concentration of nutrients involved in the culture medium to induce optimal cell growth as the first stage and second by the production of metabolite [76]. Next, different components are described in the culture mediums that affect the production of biomass and the production of the active expected.



Carbon Source In regard to carbon sources such as sucrose, glucose, maltose and other carbohydrates [77, 78], their nature and concentration are crucial for growth, nutrient consumption, and production of secondary metabolites [79]. It has been observed that high concentrations of sucrose in the adaptation process to increase biomass slows, moreover, they increase the intracellular content or the final concentration of the metabolite, this is due to osmotic stress, as the normal MS medium is approximately 140 mmol/kg and 170 mmol/kg are considered high; while low initial concentrations increase the rate of growth [80, 81]. Source Phosphate This is directly related to growth and energy metabolism, because it is involved in the biosynthesis of nucleotides, phospholipids and other biomolecules [82]. Low levels of phosphate can stimulate secondary metabolism by either decreasing the rate of growth that encourages the flow of nutrients to the production of secondary metabolites, or activation of enzymes involved in the biosynthesis of these metabolites [76, 59]. Source of Nitrogen Like phosphorus, nitrogen is an essential component for cell growth because it is used in the synthesis of proteins and nucleic acids. It can be added in the form of inorganic or organic sources. While the culture is added to the medium in the form of nitrate or ammonium salts, their assimilation is mainly as a nitrate ion at concentrations of approximately 1-5 mM per cell, by entering the proton pump mechanism (Na+, H+ ó K+). Inside the cell, nitrate reduction to incorporated nitrite suffers as glutamine is provided when available glutamate acts as the carbon skeleton. The assimilation of nitrate is regulated by various factors such as lighting, CO2 concentration, cytokinin present, type of carbon source, presence of oxidizing compounds and nitrogen compounds such as ammonium. A study of the relationship of nitrate/ammonia, found that both affect both growth and production of secondary metabolites [59]. In Azadirachta indica, strong relations of nitrate/ammonium (above the relation 1-1) have been shown to foster both growth and metabolite production, while finding the best results when nitrate is the only source of nitrogen, as ammonia is an inhibitor of growth and production [78]. Nitrogen can act as a limiting nutrient to a critical growth concentration in the culture medium (about 10 mM), however, it has also been observed that high concentrations (approximately 80 mM) may be inhibitory for growth and/or the production of secondary metabolites [83]. Growth Regulators They are molecular structures with a specific configuration in order to join specific receptors, thus transmitted to the cell patterns of development and differentiation that must be followed. The modification of the culture medium in concentration and type, permitted a wide variety of species a significant increase in both biomass production and on the accumulation of secondary metabolites [84, 85]. Sometimes the extreme difference from the control and other species in the induction and expression of metabolites is due to hormonal type and concentration, another factor is the type of media and the injunction of different hormones, the most significant factor is finding the right mix in order to achieve success in obtaining the metabolite. Light and Temperature In nature, light is one of most crucial environmental signals for developmental and physiological processes in various organisms [86]. Wavelength, intensity and photoperiod are light irradiation characteristics that influence the growth and production of secondary metabolites in heterotrophic cell cultures, depending on the species [87, 88]. Plant development is influenced by light quality through photoreceptors known as phytocrome (which detects red and far reed light), cryptochrome (blue and UV-B detector) and phototropin (blue and UV-A detector) [89]. All of them are involved in one or more of the processes of photomorphogenesis, photoperiodism, circadian rhythm and phototropism, working independently or together to enable the plant to adapt as efficiently as possible to its environment [90]. The characteristics described above influence the growth of "hairy roots" and activate specific signaling pathways in P.



ginseng [91], increased biomass production in suspension cultures of Vitis vinifera and C. arabica [88], morphologic changes of the cells to the formation of chloroplasts [88] also, [92] reported the in vitro induction of roots in Paulownia elongate (Fig. 2). Temperature is an important factor in the cultivation of plant cells (between 15-35 ºC), which affects different variables related to cell growth, rate of biomass generation, accumulation and/or segregation of compounds, and oxygen consumption rate. But most importantly, the heat stress stimulates the production of metabolites [93]. Some studies report an increase in the accumulation of secondary metabolites, as is the case for F. ananasa [94] to select the optimal process temperature was reached in a 300% improvement in production of anthocyanins and C. Roseus [95] a 100% increase in production of ajmalicine. Cell Permeability The release of intracellular secondary metabolites in the culture medium during the process, may facilitate the recovery and purification of the product. Therefore, physicochemical procedures have been developed including electropermeation, ultrasonication and the permeability of cell membranes with chemicals [84, 96, 97]. Electropermeation involves electrical currents low (1-5 mA) applied for several hours [98], this technique has been applied to the production of alkaloids from Thalictrum rugosum and Chenopodium rubrum (5kV cm-1) [99]. The ultrasonication is based on the full implementation of electromagnetic waves of the type of microwave, producing a series of cavitation between the medium and the gas dissolved in it, forming a temporary pore membranes of the cells. This technique varies depending on the frequency, power and application time [100]. Wu and Lin [101] used this technic for the recovery of saponins from cell cultures of Panax ginseng, and shikonina of Lithospermum erythrorhizon. Permeabilizant chemical agents often used are dimethylsulfoxide, glycerol, triton, polyethylene glycol, have been applied in dedifferentiated cultures, organs and complete plants which promote the formation of micropores, where intracellular secondary metabolites are excreted into the culture medium, posiblementes by a diffuse process [99]. Thimmaraju et al. [102] used Tween 80 (0.15% v/v) to release betalaines of hairy roots of Beta vulgaris without affecting the viability of cell culture, TX-100 was applied to the same species to permeate betacyanines [103].

Figure 2: Root culture of Paulownia elongata.

Eliciting System Elicitors are molecules that stimulate secondary metabolite product formation in plant cell cultures, thereby reducing the process time to attain high product concentrations and increased culture volumes. Depending



on their origin they are classified as either biotic or abiotic. Abiotic elicitors are environmental stress factors such as osmotic shock, presence of heavy metal ions or other chemicals and UV radiation. Polysaccharides, glycoproteins and low molecular weight organic acids are biotic elicitors [84, 104]. Cell Immobilization Immobilization is defined as the confinement of a biocatalyst, enzyme or cell, in or on a solid matrix to facilitate the release of the product and reuse of biocatalyst itself. The technique is to generate clusters of cells that allow some contact between cells and thus greater degree of organization. Generally, the cells are added with different types of gels such as agar, agarose, gelatin, polyacrylamide or calcium alginate (Table 5) [105]. A disadvantage of this technique is the high cost that can be long term because when you are not excreted metabolites is necessary to use other delivery systems such as sonication, solvents or other, therefore the system is costly. Table 5: Immobilized plant cell systems used for production of secondary metabolites. Plant Species

Substrate/Precursor

Product

Immobilization Method

Catharanthus roseus

Cathenamine

Ajmalicine

Agarose

Digitalis lanata

Digitoxin

Digoxin

Alginate

Daucus carota

Digitoxigenin

Periplogenin

Alginate

Nicotiana tabacum

Keto esters

Hydroxy esters

Alginate

Bioconversions

Papaver somniferum

Codeinone

Codeine

Polyurethane foam

Mucuna pruriens

L-Tyrosine

L-DOPA

Alginate

Capsicum spp.

Ferulic acid, vanillylamine

Vanillin, capsaicin

Alginate

Catharanthus roseus

Tryptamine, secologanin

Ajmalicine

Alginate, agarose

Capsicum frutescens

Apric acid, vanillylamine, valine and ferulic acid

Capsaicin

Polyurethane foam

Coffea arabica

Theobromine

Synthesis from Precursors

Galphimia glauca [106]

Caffeine

Membrane

Galphimine-B

Alginate

Nicotiana tabacum

Phenylalanine

Caffeoyl putrescine

Alginate

Plumbago rosea [106]

Chitosan

Plumbagin

Alginate

Capsicum futescens

Capsaicin

Polyurethane foam

Catharanthus roseus

Ajmalicine

Alginate, agarose

Glycine max

Phenolics

Hollow fibres

Lavandula vera

Pigments

Polyurethane foam

De Novo Synthesis

Dioscorides deltoidea

Diosgenin

Polyurethane foam

Thymus minus

Berberine

Alginate

Tagetes patula

Thiophenes

Alginate

Taxus baccata [107]

Paclitaxel and baccatin III

Alginate

Morinda citrifolia

Anthraquinones

Alginate

Adapted from Ramachandra Rao and Ravishankar [59].

Cell Biotransformation Biotransformations are those reactions catalyzed by enzymes in whole or partially purified, in the form free, immobilized cells or complete (Table 6) [108].



Table 6: Biotransformation of flavour compounds and pharmaceuticals. Plant Species

Substrate

Product

Flavour Compounds

Citrus limon

Valencene

Nootkatone



Citrus paradisi

Valencene

Nootkatone



Nicotiana tabacum

Linalool

8-hydroxylinalool



Pulegone

Isomenthone



Menthol

Neomenthol



Stevia rebaudiana

Steviol

Stevioside



Lavandula angustifolia

Geranial

Geraniol



Neral

Nerol



Citronellal

Citronellol



Mentha spp.

Pharmaceuticals

Vanilla planifolia

Ferulic acid

Vanillin



Capsicum frutescens

Ferulic acid

Vanillin, capsaicin





Vanillylamine

Vanillin, capsaicin



 

Protocatechuic aldehyde



Caffeic acid Digitoxin

Digoxin, purpureaglycoside A



Isoeugenol

Morphine Ramachandra Ca. frutescens Isoeugenol Vanillin, capsaicin



Isoeugenol and eugenol

Isoeugenyl b-rutinoside and eugenyl b-glucoside



Menthol

Menthol 3-O-b-gentiobiosides



Camphor

Camphor glucosides



Borneol

(-) Borneol b-gentiobioside



Taxol

Taxol derivatives

Gardenia jasminoides

Acetophenone

(S)-aromatic alcohol



Curcuma zedoaria

Germacrone

Guaiane-type sesquiterpenes



Glycyrrhiza glabra

Glycyrrhitinic acid

Hydroxyglycoside esters



Coffea arabica

Vanillin

Vanillin glucosides



Capsaicin

Capsaicin glucoside



Papaver bracteatum

Linalyl acetate

Linalool, -terpineol Geraniol



Achillea millefolium

Borneol, mentol thymol and farnesol

Many products



Nicotiana tabacum

Carvone

Dihydrocarvone neodihydrocareol



Pulegone

Menthone 4-hydroxymenthone



Piperitone

Hydroxypiperitone



Geraniol

10-hydroxygeraniol



Glycyrrhizin

Glycyrrhetic acid



Cymbidium spp.

Menthyl acetate

Menthol



Papaver somniferum

Thebaine

Codeine



Digitalis purpurea

Digitoxin

Digoxin



Digitalis lanata

Digitoxin

Digoxin



Podophyllum hexandrum

Coniferyl alcohol

Podophyllotoxin



Mucuna pruriens

Tyrosine

DOPA



Spirulina platensis

Codeine

Morphine



Eucalyptus perriniana

Catharenthus roseus

Adapted from Rao and Ravishankar [59].





Genetic Transformation Genetic transformation of medicinal plants has been possible due to two basic facts: recombinant DNA technology and systems for plant tissue culture. The transformation process consists of introducing foreign DNA to the plant cell, breaking new ground in knowledge and production of chemical structures as phenylpropanoids, alkaloids, terpenoids, quinones and steroids, as well as some new ones that are not found in wild plants, or in non-transformated vitro cultures. Table 7: Secondary metabolites produced by transformed roots. Plant Species

Product

Bidens spp.

Polyacetylenes

Cinchon. ledgeriana

Quinolene alkaloids

Cichorium intybus

Esculetin

Datura spp.

Tropane

Cassia spp.

Anthroquinones

Duboisia leichhardtii

Tropane alkaloids

Echinacea purpurea

Alkaloids

Glycyrrhiza uralensis

Glycyrrhizin

Hyacyamus albus

Alkaloids

Hyoscyamus niger [109]

Scopolamine

Nicotiana tabacum [110]

Tropane alkaloids

Panax ginseng

Saponin

Salvia miltorrhiza

Diterpenes

Artemi. Absynthium

Volatile oil


Shikonin


Ajmaline, serpentine

Rhodiola sachalinensis [111]

Salidroside

Rubia cordifolia

Anthroquinones

Glycyrrhiza glabra

Isoprenylated flavonoids

Panax ginseng

Ginsenoside

Hyascyamus muticus

Hyoscyamine

Adapted from Rao and Ravishankar [59].

There have been genetic transformations using two main methods. The first is using physical agents, by microinjection, microwave, sonication and biolistics. The second method is by biological agents such as viruses or bacteria such as Agrobacterium tumefaciens and Agrobacterium rhizogenes (Table 7), these microorganisms naturally introduce their genes in the plant genome, benefiting the production of secondary metabolites in the laboratory, such as polyphenols, alkaloids, terpenes and anthocyanins (Fig. 3) [112-116]. To clone a large number of genes for secondary metabolism and to map the metabolic pathways of useful compounds in this species, scientists at Eugentech Inc., a Korean venture capital company, have launched a project for functional genomics of secondary metabolism. Hairy root cultures of ginseng are used for transgenic mutant analysis because most of the useful secondary metabolites are produced in the root. Numerous hairy root cultures are easily generated from leaf tissues through inoculation with Agrobacterium rhizogenes, which harbors the Ti-plasmid with an activation-tagging sequence, while UniGen a Korean venture capital company collected data and built a library of 40,000 natural compounds from medicinal plants worldwide by 2005. Since a holistic approach to plant secondary metabolism was introduced, based on genomics, highthroughput biology, and bioinformatics, the paradigm for such research has been drastically changed. New



functional compounds can now be discovered via (high-throughput screening) HTS. Likewise, the pathways for secondary metabolites and the genes involved in those pathways can be determined through functional genomic approaches in conjunction with data-mining tools. In addition, plants can be metabolically engineered with three to five, or more, heterologous genes for large-scale production of useful secondary metabolites. For example, a few heterologous genes under the control of a promoter as an operon can be introduced into a plastid. The gene products are then be safely compartmentalized from the degrading enzymes found in the cytosol [117].

Figure 3: Using antisense/cosuppression technologies or overexpression, medicinal plants can be tailored to produce pharmaceutically important alkaloids by eliminating interfering metabolic steps or by introducing desired metabolic steps. Expressing an entire alkaloid biosynthesis pathway of 20 to 30 enzymes in a single microorganism is currently beyond our technical capability. However, altering the pathway in a plant and producing the desired alkaloid either in culture or in the field may now be possible. For example, to accumulate more of the end product alkaloid, a side pathway that also uses the same precursor may have to be blocked (A). To accumulate an alkaloid not normally produced in a particular plant species, a transgene (from another plant or a microorganism) may be introduced (B). If the end product alkaloid would be more useful as a particular derivative, for example, as a more soluble glycoside, a gene that encodes a glycosyl transferase could be introduced (C). Source: Buchanan 2000 [118].

Through transgenic modification, Busov et al. [34] found that DELLA-less versions of GAI (gai) and RGL1 (rgl1) in a Populus tree have profound, dominant effects on phenotype, producing pleiotropic changes in morphology and metabolic profiles. The transgenic modifications elicited significant metabolic changes. In roots, metabolic profiling suggested increased respiration as a possible mechanism of the increased root growth. In leaves, metabolite changes suggesting reduced carbon flux through the lignin biosynthetic pathway and a shift towards allocation of secondary storage and defense metabolites, including various phenols, phenolic glucosides, and phenolic acid conjugates. In other case, Cynara cardunculus suspension cells were transformed by particle bombardment to overexpress the cypro11 gene coding for cyprosin B. The results encourage the overexpression of cypro11 gene in transformed C. cardunculus cells leading to high yields of cyprosin B production in bioreactor, which can be considered adequate for industrial production (Table 8) [119].



Table 8: Large-scale suspension cultures reactors for plant cell cultures. Plant Species

Product

Bioreactor Capacity

Catharanthus roseus

Serpentine

100 l airlift

Coleus blumei

Rosmarinic acid

300 l airlift

Lithospermum erythrorhizon Nicotiana tabacum

Shikonin

750 l agitated

Biomass Biomass

20,000 l agitated 1500 l bubble column

Panax ginseng

Saponins

20,000 l agitated

Echinacea purpurea

Biomass

750-75,000 l agitated


Biomass


Panax ginseng

Biomass


Adapted from Rao and Ravishankar (2002).

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[119] De Sousa SP, Neto H, Poejo P, Serrazina SM, Soares PM. Overexpression and characterization of cyprosin B in transformed suspensión cells of Cynara cardunculus. Plant Cell Tiss Org Cult. DOI 10.1007/s11240-010-9690-z. 2010.


21

CHAPTER 2 Natural Products Extracts: Terpenes and Phenolics Gutiérrez-Lomelí M., Del Toro-Sánchez C.L., Rodríguez-Sahagún A. and Castellanos-Hernández O.A.* Centro Universitario de la Ciénega, Universidad de Guadalajara. Av. Universidad 1115, Col. Lindavista, CP 47810, Ocotlán, Jalisco, México 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: [email protected], [email protected] Ilkay Erdogan Orhan (Ed) All rights reserved-© 2012 Bentham Science Publishers


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 plantinsect, 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


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 shortchain 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

D-Glyceraldehyde

Pyruvate

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)2-C-methyl-D-erythritol

Mevalonate

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-erithritol2,4-cyclodiphosphate

MDC IPPI Isopentenyl diphosphate

Dimethyallyl diphosphate

GPP

FPP

GGPP

Monoterpenes

- Sesquiterpenes - Triterpenes

- Diterpenes - 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, 1deoxyxylulose-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-Cmethylerythritol-4-phosphate cytidyltransferase; MDC, mevalonate-5-diphosphate decarboxylase; MECPS, 2-Cmethylerythritol-2,4- cyclodiphosphate synthase; MK, mevalonate kinase; PMK, phosphomevalonate kinase. According to [4, 12, 30, 31].



GPP

Acyclics

Geraniol

cis-Ocimene

Linalool

Boranes, camphanes and fenchanes

Caranes

p-Menthanes

Camphor

3-Carene

Limonene

Isoborneol

4-Caranol

Carvone

Fenchone

5-Carenol

Menthol

Pinanes

α-Pirene

Thujanes

α-Thujene

β-Pirene

Sabinene

Verbenone

Sabinene hydrate

Figure 2: Representative members of monoterpenes subfamilies. Abbreviations: GPP, geranyl diphosphate. According to [12].

α-Bisabolene

Germacrene C

FPP γ-Humulene

5-epi-Aristolochene

Figure 3: Representative sesquiterpenes. Abbreviations: FPP, farnesyl diphosphate. According to [31].



(-)-Abietadiene

Casbene

GGPP Taxadiene

(-)-Kaurene

Figure 4: Representative diterpenes. Abbreviations: GGPP, geranylgeranyl diphosphate. According to [31].

4. METABOLIC ENGINEERING AND MANIPULATION OF MONOTERPENE BIOSYNTHESIS In the current, metabolic engineering is considered as one of the main areas of support of biotechnology. At present, genetic engineering allows the transfer of a biosynthetic pathway to any selected host. In this field, has been demonstrated that some relevant genes involved in the biosynthesis of isoprenoid can be reassembled from different biological sources (e.g. plants, bacteria and fungi) in a heterologous microorganism [30, 33]. Although the feasibility of increasing monoterpene yield and altering metabolite composition in essential oil plants by overexpression the enzymes that produce terpene precursors or modify parent terpenene structures has been demonstrated [12, 34, 35], currently, most research has focused on the improvement in the production of terpenes by terpenes syntanses overexpression in transgenic plants under the control of constitutive promoters [36]. The application of modern biotechnology techniques to isoprenoid production can be divided into the engineering of native or heterologous hosts. Native host engineering has focused on elevating levels of terpenoids normally found in a species, typically plants [4, 37]. The heterologous hosts more used are those with the most advanced genetic tools: Escherichia coli, Saccharomyces cerevisiae and Arabidopsis thaliana [4]. Some of the research related to the production of monoterpenes in native or heterologous hosts are show in Table 1. Table 1: Reports of metabolic engineering of monoterpenes in different hosts. Engineered species (host)

Target

Result

References

Petunia hybrida

S-Linalool synthase

S-linalyl-beta-D-glucopyranoside ↑

[38]

Tomato (Lycopersicon esculentum)

S-Linalool synthase

S-linalool ↑, 8-hydroxylinalool ↑

[39]

Carnation(Dianthus caryophyllus)

S-Linalool synthase

S-linalool ↑, cis- and trans-linalool oxides ↑

[40]



Table 1: cont…. Arabidopsis thaliana and potato

S-linalool/(3S)-E-nerolidol synthase

S-linalool ↑, hydroxylated and glycosylated linalool ↑

[41]

Tobacco (Nicotiana tabacum)

γ-Terpinene, β-pirene and limonene synthases

β-pinene ↑, limonene ↑, and γ-terpinene ↑, and other products

[42, 43]

Tobacco (Nicotiana tabacum)

Limonene synthase

Limonene ↑

[44]

Escherichia coli

(E)-beta-ocimene synthase

(E)-beta-ocimene ↑

[45]

Escherichia coli

1-Deoxyxylulose-5-phosphate synthase, Isopentenyl diphosphate isomerase, farnesyl diphosphate synthase

α-pinene ↑, myrcene ↑, sabinene ↑, 3-carene ↑, α-terpinene ↑, limonene ↑, β-phellandrene ↑,α –terpinene ↑, terpinolene ↑

[46]

Saccharomyces cerevisiae

S-linalool synthase

S-linalool ↑

[47]

Saccharomyces cerevisiae

Geraniol synthase

Geraniol ↑, linalool ↑

[48]

Lactococcus lactis

Linalool/nerolidol synthase

S-linalool ↑

[49]

5. SYNTHESIS OF PHENOL COMPOUNDS IN PLANTS Phenolics are very stable products in plant organisms and are one of the major groups of secondary metabolites. External stimuli such as microbial infections, ultraviolet radiation, and chemical stressors induce the synthesis of these compounds [8, 50]. Their common feature is the presence of at least one hydroxyl-substituted aromatic ring system. Phenylalanine, synthesized in the shikimate pathway, is a starting point of the synthesis of plant phenols. As the first step, phenylalanine is deaminated to yield cinnamic acid (cinnamate) by the action of phenylalanine ammonia lyase (PAL). Cinnamic acid is hydroxylated by cinnamate-4-hydroxylase (C4H) to 4-coumaric acid, which is then activated to 4coumaroyl-coenzyme A (CoA) by the action of 4-coumarate-CoA ligase (4CL). Then it is divided into two major pathways—the flavonoid biosynthesis pathway and the lignin biosynthetic pathway (Fig. 5). Lignin, a complex polymer of phenylpropane units is, quantitatively, the most important phenolic compound in plants constituting from a quarter to a third of the dry mass of wood. It is a valuable phenolic polymer that gives wood its characteristic brown color, density and mass [51]. Other abundant compound classes are the stilbenes, coumarins, and polyflavonoids (condensed tannins) (Fig. 6). However, the major or common phenolic constituents of plants can be divided broadly into two main groups: phenolic acids and coumarins, and flavonoids, including anthocyanidins. 6. PHENOLIC ACIDS These compounds could be divided in two classes: derivatives of benzoic acid and derivatives of cinnamic acid (Fig. 6). The hydroxybenzoic acids, such as gallic acid, is found in very few plants eaten by humans; this is the reason why these compounds are not currently considered to be of great nutritional interest [52]. Their content of edible plants is generally very low, except for certain red fruits, i.e. blackberries which contain up to 270 mg/kg fresh wt [53]. Tea is an important source of gallic acid: tea leaves may contain up to 4.5 g/kg fresh wt of gallic acid [54, 55]. The hydroxycinnamic acids are more common than are the hydroxybenzoic acids and consist chiefly of pcoumaric, caffeic and ferulic acids (Fig. 6). These acids are rarely found in the free form, except in processed food that has undergone freezing, sterilization, or fermentation. The bound forms are glycosylated derivatives or esters of quinic acid, shikimic acid, and tartaric acid [7]. Caffeic acid, both free and esterified, is generally the most abundant phenolic acid and represents between 75% and 100% of the total hydroxycinnamic acid content of most fruit. Kiwis contain up to 1 g caffeic acid/kg fresh wt [56].



Ferulic acid is found chiefly in the trans form, which is esterified to arabinoxylans and hemicelluloses in the aleurone and pericarp of grains. Only 10% of ferulic acid is found in soluble free form in wheat bran [57]. Several dimers of ferulic acid are also found in cereals and form bridge structures between chains of hemicelluloses [58]. Additionally, ferulic acid and 4-coumaric acid esters are widely distributed in vascular plants.

Figure 5: Phenolic compounds biosynthetic pathway. Abbreviations: PAL= phenylalanine ammonium lyase; 4CL= 4coumarate-CoA ligase; C4H= cinnamate-4-hydroxylase; CHS= chalcone synthase; CHI= chalcone isomerase; F3H= flavonone 3-hydroxylase; FLS= flavonol synthase; DFR= dihydroflavonol reductase; ANS= anthocyanidin synthase; LDOX= leucoanthocyanidin dioxygenase; C3H= coumaroyl-quinate/shikimate 3-hydroxylase; COMT= caffeic acid:5hydroxyferulic acid O-methyltransferase; CCR= cinnamoyl-CoA reductase; CAD= cinnamyl alcohol dehydrogenase; F5H= ferulate 5-hydroxylase; UFGT= UDP-glucose:flavonoid 3-O-glucosyltransferase . According to [8].

7. COUMARINS The double-ring phenolic compound called coumarin are lactones of cis-o-hydroxy cinnamic acid derivatives (Fig. 6). These molecules impart the distinctive sweet smell to newly-mown hay [59]. Coumarins are less widely distributed in seeds and occur primarily as glucosides. They exhibit limited chemical reactivity and have little effect on the organoleptic or nutritive value of food. New coumarins have been extracted from some plants, i.e. pyrone-coumarin, 7,8-dihydroxy-3-(3-hydroxy-4-oxo-4H-pyran-2-yl)-2H-chromen-2-one was isolated from the leaves of bamboo [60]. Two new coumarins (7-{[(2R*)-3,3-dimethyloxiran-2-yl]methoxy}-8[(2R*,3R*)-3-isopropenyloxiran-2-yl]-2H-chromen-2-one and 7-methoxy-8-(4-methyl-3-furyl)-2H-chromen-2one ) from the leaves of Galipea panamensis were also isolated [61]. 8. FLAVONOIDS Flavonoids are themselves divided into 6 subclasses, depending on the oxidation state of the central pyran ring: flavonols, flavones, flavanones, isoflavones, anthocyanidins and flavanols (catechins and proanthocyanidins) (Fig. 6). More than 4000 flavonoids have been identified in plants, and the list is constantly growing [52, 62].



Phenolic acids Hydroxybenzoic acid O

O O

HO

HO

HO

OH

OH

OH

OH

HO

O

HO

OH

O

O

O

O

O

OH

HO

Hydroxycinnamic acid O

HO

HO

OH

Syringic acid

Vannilic acid

Gallic acid

Ferulic acid

Caffeic acid

Coumaric acid

Flavonoids Flavanols

Flavones

HO

O

OH

OH

OH

HO

HO

O

O OH

OH OH

O

OH

Kaempferol

Quercetin

O

OH

Apigenin

O

Luteolin

Flavanols OH

HO

O

OH

HO

O

OH

OH

OH

OH

OH

OH

Catechin

Epicatechin

Epigallocatechin

Epigallocatechin gallate

Anthocyanidins

Flavanones

O

OH

O

HO

O+

HO

O

OH

OH

OH

O+

HO

O

OH

OH

Narigen

O

Hesperitin

OH OH

Cyanidin

Stilbenes

Isoflavones HO

OH

Malvidin

Coumarin

O O

OH

O OH

Genistein

Figure 6: Classification of phenolic compounds.

Resveratrol

Coumarin

O



These compounds are present in glycosylated forms. The associated sugar moiety is very often glucose or rhamnose, but other sugars may also be involved (e.g. galactose, arabinose, xylose, glucuronic acid). Fruit often contains between 5 and 10 different flavonol glycosides [55]. These flavonols accumulate in the outer and aerial tissues (skin and leaves) because their biosynthesis is stimulated by light. Marked differences in concentration exist between pieces of fruit on the same tree and even between different sides of a single piece of fruit, depending on exposure to sunlight [63]. Similarly, in leafy vegetables such as lettuce and cabbage, the glycoside concentration is high in the green outer leaves as in the inner light-colored leaves [64]. Flavanones are generally glycosylated by a disaccharide at position 7: either a neohesperidose, which imparts a bitter taste (such as to naringin in grapefruit), or a rutinose, which is flavorless [65]. Isoflavones are flavonoids with structural similarities to estrogens. Although they are not steroids, they have hydroxyl groups in positions 7 and 4 in a configuration analogous to that of the hydroxyls in the estradiol molecule. This confers pseudohormonal properties on them, including the ability to bind to estrogen receptors, and they are consequently classified as phytoestrogens. Isoflavones are found almost exclusively in leguminous plants [66]. Flavanols exist in both the monomer form (catechins) and the polymer form (proanthocyanidins). Black tea contains fewer monomer flavanols, which are oxidized during "fermentation" (heating) of tea leaves to more complex condensed polyphenols known as theaflavins (dimers) and thearubigins (polymers). Catechin and epicatechin are the main flavanols in fruit, whereas gallocatechin, epigallocatechin, and epigallocatechin gallate are found in certain seeds of leguminous plants, in grapes, and more importantly in tea [67, 68]. In contrast to other classes of flavonoids, flavanols are not glycosylated in foods. Proanthocyanidins, which are also known as condensed tannins, are dimers, oligomers, and polymers of catechins that are bound together by links between C4 and C8 (or C6). Anthocyanins are pigments dissolved in the vacuolar sap of the epidermal tissues of flowers and fruit, to which they impart a pink, red, blue, or purple color [69]. They exist in different chemical forms, both colored and uncolored, according to pH. Although they are highly unstable in the aglycone form (anthocyanidins), while they are in plants, they are resistant to light, pH, and oxidation conditions that are likely to degrade them. anthocyanins are stabilized by the formation of complexes with other flavonoids (copigmentation). 9. LIGNANS Lignans are formed of 2 phenylpropane units (Fig. 1). They are mostly present in nature in the free form, while their glycoside derivatives are only a minor form [52]. Linseed represents the main dietary source, containing up to 3.7 g/kg dry wt of secoisolariciresinol [70]. Intestinal microflora metabolizes lignans to enterodiol and enterolactone. The interest in lignans and their synthetic derivatives is growing because of potential applications in cancer chemotherapy and various other pharmacological effects [71-73]. 10. STILBENES Stilbenes are found in only low quantities in the human diet. One of these, resveratrol, for which anticarcinogenic effects have been shown during screening of medicinal plants and which has been extensively studied [74], is found in low quantities in wine (0.3–7 mg aglycones/L and 15 mg glycosides/L in red wine) [75]. However, because resveratrol is found in such small quantities in the diet, any protective effect of this molecule is unlikely at normal nutritional intakes [7]. 11. PHENOLS FUNCTION IN PLANTS Phenols in plants have diverse functions such as stabilization of the structure, protection from herbivory, protection from ultraviolet (UV) light, exchange of information with symbionts, coloration of blossoms, and biocidal effects against bacteria and fungi [76, 77]. After the death of plants, phenolics may persist for weeks or months and affect decomposer organisms and decomposition processes in soils [78]. Therefore, their effects are not restricted to single plants but may extend to the functioning of whole ecosystems. Phenolics, such as chlorogenic acid and caffeic acid have been shown to exhibit fungicidal properties [79]. Phenolics also contribute to colour, astringency, bitterness, and flavour in fruits. The synthesis of isoflavones and some other flavonoids is induced when plants are infected or injured or under low temperatures and low nutrient conditions. Flavonoids in plants fulfill a variety of functions.



Certain isoflavonoids attract insects, promote growth of several microorganisms, and induce the nodulation gene in the nitrogen-fixing bacteria Rhizobium sp. and Bradyrhizobium sp. [80]. Other flavonoids help to repel herbivorous insects. In woody plants these are mainly the condensed tannins, whereas in herbs the flavonoles (quercetin, rutin) predominate [81]. In recent years there has been a growing interest in antioxidant properties of phenolic compounds. Many reports of induced accumulation of these molecules and peroxidase activity in plants treated with high concentrations of metals have been reported. Their antioxidant action is due to their high tendency to chelate metals. Phenolics possess hydroxyl and carboxyl groups, able to bind particularly iron and copper [82]. The roots of many plants exposed to heavy metals exude high levels of phenolics [83]. They may inactivate iron ions by chelating and additionally suppressing the superoxide-driven Fenton reaction (Scheme 2), which is believed to be the most important source of ROS (Reactive Oxigen Species) [84]. Generally a plant’s cells try to keep the concentration of ROS at the possible low level because they are more reactive than molecular oxygen (O2) [85], and they react with almost every organic constituent of the living cell. ROSs are known to damage cellular membranes by inducing lipid peroxidation [86]. When the fatty acid chains in lipids become damaged by free radicals, they become cross-linked and damaged. They also can damage DNA, proteins, lipids and chlorophyll. The most popular ROS are . O2 -superoxide radical, H2O2 –hydrogen peroxide, and .OH -hydroxyl radical originating from one, two or three electron transfers to dioxygen (O2) (Scheme 1). The half-life (t1/2) is variable for each metabolite [87]:

Scheme 1

H2O2 in the presence of .O2- can generate highly reactive .OH hydroxyl radicals via the metal-catalyzed Haber-Wiess reaction (Scheme 2), thus the scavenging of H2O2 in cells is critical to avoid oxidative damage [88, 89]. In the presence of redox active transition metals such as Cu+ and Fe2+, H2O2 can be converted to . OH molecule in a metal-catalyzed reaction via the Fenton reaction [90]:

Scheme 2



The antioxidant properties of phenolic compounds in plants of our diet, impact directly to human health in the prevention of some diseases. 12. PHENOLS IN HUMAN HEALTH Phenol compounds are constituents of higher plants found in a wide range of commonly consumed plant foods such as fruits, vegetables, cereals and legumes, and in beverages of plant origin, such as wine, tea and coffee [91]. Polyphenols are abundant micronutrients in our diet, and evidence for their role in the prevention of degenerative diseases such as cancer and cardiovascular diseases is emerging. The health effects of polyphenols depend on the amount consumed and on their bioavailability. Polyphenols, which constitute the active substances found in many medicinal plants, modulate the activity of a wide range of enzymes and cell receptors. In this way, in addition to having antioxidant properties, polyphenols have several other specific biological actions that are as yet poorly understood [7]. The human body is incapable of producing the natural oxidants that it needs. That is the reason why we are solely dependant on our diet so that our body gets the necessary amount of antioxidants. Several studies have been realized about the antioxidant activity from extracts of plants, especially medicinal plants. However, is important to think that phenol compounds are no the only molecules with this property. In this context, different extracts from the leaves of Urticaceae Family showed that the antioxidant activity was not correlated with the phenolics content suggested that non-phenolic compounds might play major free radicals scavenging activity in studied plant materials [92]. On the other hand, some extracts coincided in the relation from antioxidant and antiinflamatory activities. Salvia and red and white wine extracts demonstrated these effect [9, 93]. Researches in cancer studies from extract of plants are actually an important part of human health. Bisphenol A and estradiol are equipotent in antagonizing cisplatin-induced cytotoxicity in breast cancer cells [10]. On the other hand, 8-hydroxyquinoline is a crucial scaffold for anticancerigen activity tested uterine cancer [94]. Furthermore, poliphenols from sea buckthorn berries extracts showed antiproliferative effect on human colon and liver cancer cell lines [95]. Individual and combined phenolics from Olea europaea leaf extract were showed that oleuropein and caffeic acid presented inhibition against microorganisms. Furthermore, the antimicrobial effect of the combined phenolics was significantly higher than those of the individual phenolics [97]. Liver protector and antiviral are other properties that have been attributed to phenols [11, 96]. The value of phenols from plants in the human health is increasing by their properties, having an opportunity to increase the option to choice always natural products. REFERENCES [1] [2] [3] [4] [5] [6] [7]

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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: [email protected] Ilkay Erdogan Orhan (Ed) All rights reserved-© 2012 Bentham Science Publishers

Biotechnological Production of Coumarins


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

4a

4

6

3

7 8

Figure 1: Coumarin (benzo--pyrone).

8a

O 1

2

O


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-hydroxycoumarin, 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 antiinflammatory, 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, antiinflammatory, 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.



Some pharmacological studies on coumarin have been reviewed by Egan et al. (1990), whilst the comprehensive information had already been given by Murray et al. (1982) regarding occurrence, chemistry and biochemical properties of natural coumarins. The other elaborate information on biology, application and mode of action of coumarin and its derivatives was released by O’Kennedy and Thornes in 1997 [6, 8, 13]. 2.1. Photosensitizing Properties The most famous and popular effect of coumarins is the photosensitive activity, particularly the psoralens named as furocoumarins that form a class of heterocyclic aromatic compounds which are famous for this effect. In fact, it is the undesirable and ominous effect in some cases due to the formation of dark color spots on the skin if the plants including furocoumarins are rubbed on the skin while exposed to sunlight. Furocomarins are well-known heterocyclic photosensitizers already used for the treatment of some skin problems in ancient India and Egypt. They are still utilized as remedy for various skin diseases (psoriasis, vitiligo, cutaneous T-cell lymphoma, mycosis fungoides, polymorphous light eruption and more), because of the phototherapeutic activity due to photoadducts with DNA, the mechanisms of photoadduct have been studied by some researchers entirely [16-20]. Psoriasis is defined as a common skin disease characterized by epidermal keratinocyte hyperproliferation, abnormal keratinocyte differentiation and immune-cell infiltration. Psoralens are mainly used in the treatment of psoriasis. Administration of oral and topical psoralens with UV radiation in 320-400 nm range (UVA) is widely used in systemic treatment of both psoriasis and vitiligo named as PUVA therapy. Various derivatives of psoralen, such as 8-methoxysoralen (8-MOP), 5-methoxypsoralen (5-MOP), trimethylpsoralen (TMP), 7-methyl pyridopsoralen and khellin, have been used in conjunction with UVA irradiation (PUVA therapy) for the control psoriasis and repigmentation of the skin in vitiligo [16, 21]. In the past, several plants were used for this purpose due to the unavailability of psoralens in purified forms. Egyptians used an extract obtained from Ammi majus L., whereas Indians used Ficus hispada and Psoralea caryolia for thier photosensitizing effects. Even now, these plants are very popular in the treatment of vitiligo in phytomedicines and phytocosmetics because of their furocoumarin content such as psoralen, bergapten, pimpinellin, and isopimpinellin in the concentrations between 0.001 and 1% [22]. Vitiligo is also a common skin disease characterized by the destruction of melanocytes caused of depigmented maculae [22-24]. Xanthotoxin is an effective linear furocoumarin in vitiligo. However, linear furocoumarins and UVA are used for not only in psoriasis and vitiligo, but also used in cutaneous T-cell lymphoma, atopic dermatitis, alopecia areata, urticaria pigmentosa and lichen planus [6, 8, 10]. A Moraceae plant, Brosimum gaudichaudii Trecul, is used as medicinal plant in Brazil, especially in vitiligo treatment. After that, psoralen and bergapten were isolated from the roots which are well known for their photosensitize activities [23]. The mode of action of PUVA is stated as suppression of accelerated proliferation of the keratinocytes, while another direction of the action is through toxic effect on lymphocytes in the treatment of such skin diseases [6, 8, 10]. 2.2. Anti-Inflammatory Properties Many coumarins have been concerned in the anti-inflammatory activities of carrageenan-induced inflammation test and paw edema test induced by dextran or TPA (12-O-tetradecanoylphorbol-13-acetate) in rats. In addition, the coumarins have exhibited analgesic effects. Underline mechanism of this process, showed that the carrageenan stimulated the release of several inflammatory mediators such as histamine, serotonin, bradykinin and prostaglandins. Non-steroidal anti-inflammatory drugs (NSAID) have blocked the synthesis of prostaglandins by inhibiting cyclo-oxygenase (COX). COX and 5-LO (5-lipoxygenase) catalyze the peroxidation of arachidonic acid. In this manner, it is thought that the polyphenols like coumarins and flavonoids might be expected to intervene with this process [10, 13]. Based on these activity tests, the coumarins are not potent, but the effect exists in coumarins moderately. Seselin isolated from aerial parts of Decatropis bicolor (Zucc.) Radlk. (Rutaceae) has efficacy in the carrageenan-induced


Alev Tosun

inflammation test. Furthermore, seselin isolated from Seseli indicum (Apiaceae) was reported with a significant and dose-dependent anti-inflammatory activity in carrageenan-induced acute inflammations in rats and analgesic effect assessed by acetic acid induced writhing and hot plate tests in mice. We reported that the extracts from the different kind of species of Seseli genus growing in Turkey, including different types of coumarins were screened for their anti-inflammatory and antinociceptive activities, and then noted some considerable effects on some species for further examinations [6, 8, 10, 13, 25, 26]. In earlier studies, the coumarins had been reported for their anti-nociceptive effects in the acetic acid-induced writhing and hot plate thermal stimulation tests [10, 13]. A simple coumarin known as osthol possesses diverse pharmacological and biochemical properties. Osthol with a methoxyl group at 7th position and an isopentenyl group at 8th position has been isolated from many medicinal Umbelliferous (Apiaceae) plants such as Cnidium, Angelica and Seseli species. Osthol has not only calcium antagonistic effect but also has the anti-inflammatory effect as well as many other important activities mentioned in literature published previously. However, the compound also showed a selective inhibitor activity on 5-LO in vitro studies which is important point for many activities. Since 5-LO is activated by the calcium influx, this effect is involved in calcium antagonistic properties. Thereby, osthol is an important coumarin known as the calcium channel blocker. Similarly, several coumarins such as visnadin, columbianadin, ostruthol have also calcium blocker effects. In fact, the activity was mostly observed on dihydropyrano-coumarins. Seseli gummiferum ssp. corymbosum belongs to Apiaceae family is a rich source for osthol content beside the other khellactone derivatives. Thus, the species can be a promising source for the agents of anti-inflammatory and calcium antagonistic effect [10, 13, 25-27]. 2.3. Antimicrobial Properties The determination of the antibacterial and antifungal activity on the crude extracts as much as on the pure compounds, have been increasing interest in recent studies to find new active molecules. The exploring of antimicrobial drugs is important for the control of bacterial infections for some pathogens, which rapidly become resistant to many of the established antibiotics. A lot of plants are constantly being screened for their antimicrobial effects. There are also many studies which are performed on the antimicrobial activity of the coumarins. The antibacterial properties of coumarins were first recognised in 1945 when dicoumarol was found to inhibit the growth of several strains of bacteria in a study [4]. The antimicrobial activity of the methanol extract obtained from the stems of Daphne gnidium L. growing in Italy was evaluated against 6 strains of standard and clinically isolated Gram (±) bacteria as well as two strains of fungi. Whilst the extract showed antibacterial activity against Bacillus lentus and Escherichia coli, but was inactive against fungi; daphnetin was the most active coumarin among the other coumarins isolated from the extract [28]. Several reports showed that free hydroxyl group at 6th position of the coumarin nucleus plays an important role for antifungal activity, and at 7th position for antibacterial activity. In addition, daphnetin isolated from Daphne species also displays antimalarial activity [6, 8].

Figure 2: Structure of Novobiocin.

A few antibiotics including the coumarin skeleton have also been isolated from natural sources. The most active one is novobiocin, isolated from Streptomyces niveus, mainly active against Gram (+) bacteria. Novobiocin (Fig. 2) is an interesting example of the naturally-occurring coumarins being as amino coumarin in complex



structure which target DNA gyrase (the bacterial type II topoisomerase) as potent inhibitor of DNA replication. In fact, the coumarins are much more potent inhibitors of DNA gyrase in vitro than clinically utilized quinolones [1, 6, 13]. However, there is no more coumarins using for their antimicrobial effects. The antimicrobial activity of 43 natural and synthetic coumarins was studied by a research group. In this study, the coumarin derivatives exhibiting good bioactivity against two S. aureus strains were then assessed for their antimicrobial activity against a range of eight clinically isolated MRSA strains. The study indicated that most of the tested coumarinic compounds displayed antimicrobial activity [4]. 2.4. Other Biological Effects As we mentioned previously, a wide variety of biological activities has been observed on coumarins. A number of plant extracts and natural compounds can provide the protection of cancer by stimulating the immune system. Recent studies are related to the estimation of cytotoxicity against human leukemia cell 60, KB cells and melanoma cells, and the possible induction of apoptosis (against HL-60 cells). The genus Calophyllum (Clusiaceae) which comprises 200 species is widely distributed in the tropical rain forest where several species are used in folk medicine. This genus is a rich source of biologically active compounds such as coumarins, xanthones and terpenoids. The 4-phenylcoumarins from C. dispar P. F. Stevens exhibited cytotoxic activities as well as the other minor coumarins showed a significant cytotoxic activity against KB cells [29]. In addition, the coumarins of C. brasiliense were also cytotoxic against human tumor cell lines [30]. The coumarins from Campylotropis hirtella (Franch) Schindl. showed inhibitory activities against the prostate cancer cells (LNCaP) and decreased the prostate specific antigen (PSA) with IC50 values from 24 to -1 102 μg·L . DU145, PC-3 and LNCaP are human prostate cells. Oxypeucedanin, a well-known furocoumarin could inhibit the growth of DU145 cell. Isoimperatorin isolated from Angelica koreana could cause the growth inhibition and apoptosis of DU145 cells. Decursin from the roots of Angelica gigas, could strongly inhibit the cell growth and induce cell death on human prostate DU145, PC-3 and LNCaP cell lines. These effects were associated with cell cycle arrest and apoptosis. The coumarins from Phellolophium madagascariense Baker showed inhibitory activities against the prostate cancer cells LNCaP, PC3 and DU145 [31]. Esculetin and daphnetin (7, 8-dihydroxycoumarin) have notable antiproliferative activity in several tumour cell lines, and have been proposed as potential anticancer agents. Despite the fact that the antiproliferative activity of esculetin, the molecule produced biphasic effects, similar to those observed with other pythoestrogens in breast carcinoma MCF-7 cells. The inhibitory activity of esculetin occurred only at μM concentrations. Meanwhile, lower concentrations produced estrogenic effects in vitro and in vivo that would limit its use as an anticancer agent in estrogen-dependent tumors. The results indicated that daphnetin possesses more selective type of antiproliferative activity. This makes it a promising antitumoral agent for several types of cancer, including hormonal-dependent tumours. The introduction of an additional hydroxyl group (6-hydroxy or 8-hydroxy) to the 7-hydroxycoumarin (umbelliferone) caused differential effects on proliferation of MCF-7 cells [32]. Moreover, 7-hydroxycoumarin inhibits the proliferation of several human malignant cell lines in vitro, and some of the animal tumors in vivo. Generally, ortho-dihydroxy substituted coumarins exhibits cytotoxic activity due to structure-activity relationships. Therefore, the presence and position of hydroxyl groups are important for the different potency of cytotoxicity. In addition, coumarin and its derivatives cause important changes in the regulation of immune responses as well as cell growth and differentiation [7, 27]. Antioxidant activity studies and inhibition of NO production using RAW 264.7 cells have a high attraction for many research groups. The natural antioxidants possess multiple pharmacological activities such as neuroprotective, anticancer, antimutagenic and anti-inflammatory activities, and these activities may be related to their antioxidant properties. Reactive oxygen species (ROS), major free radicals generated in many redox processes, often induce oxidative damage to biomolecules (carbohydrates, proteins, lipids and DNA). Biomolecule degeneration causes accelerated aging and many chronic diseases, including


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neurodegenerative diseases, cancer, cardiovascular diseases and inflammation. Some natural coumarins also affect the formation and scavenging of ROS and influence free radical-mediated oxidative damage. In a study, the antioxidant capacity of the Cortex Fraxini and its coumarins was evaluated. Cortex Fraxini coumarins; esculetin and fraxetin had good radical-scavenging capacity. Moreover, esculetin (6,7dihydroxy-coumarin) and fraxetin had selective scavenging activity on hydroxyl radicals and hydrogen peroxide. This potency was better than known antioxidants and equal to quercetin in scavenging hydrogen peroxide [33]. In addition to that, cholinesterase inhibitory and antioxidant activities of the methanol extract, furanocoumarin fraction, and major coumarins (imperatorin, xanthotoxin, and bergapten) of the fruits of Angelica officinalis L. growing in Poland were investigated. The major coumarins of the extracts such as imperatorin, isoimperatorin, xanthotoxin, and bergapten displayed strong inhibition towards BchE which is used in the cholinesterase activity test [34]. Moreover, praeruptorin A, xanthotoxin, psoralen and bergapten, inhibit monoaminoxidase (MAO) found in mouse brain [10]. In a research, the osthol, a component in a lot of medicinal plants, prevented anti-Fas antibody-induced diseases in mice. Fas (Apo-1/CD95) ligand is a major inducer of apoptosis, and fas mediated apoptosis is involved in the development of variety of diseases such as viral hepatitis, autoimmune hepatitis, alcoholinduced hepatitis, cholestatic hepatitis, and hepatitis in metabolic disorders. Therefore, osthol could be evaluated for the treatment of a wide variety of liver diseases [35], as well as calcium antagonistic and antiinflammatory effect mentioned previously in the text. Coumestrol and related constituents similar to estrogen structure in some points, have estrogenic effects [6, 13]. Unfortunately, the coumarins fail in clinical applications because of the unsatisfied cell penetration, low solubility and toxicity. Thereby, these compounds generally are unsuccessful in drug developments, even if they have impressive activities. However, the coumarins have many significant effects which are developed for the promising agents using in diseases. 3. TOXICOLOGY OF COUMARINS Until 1954, when the first toxicological concerns about coumarin were raised, synthetic coumarin was widely used as flavour. After that, the use of coumarin as a food flavouring was abondened based on reports of hepatotoxicity. Coumarin was first suspected to have genotoxic and carcinogenic effects in the 1980s. For this reason, the Codex alimentarius commission determined maximum levels of coumarin as natural flavourings in the final product ready for consumption. European Union Scientific Committee concluded the maximum level of coumarin as 2 mg/kg for foods and beverages in general; with the exception of special caramels and alcoholic beverages as 10 mg/kg. The Codex alimentarius maximum levels were subsequently introduced into European law in 1988. Eventually, coumarin is not a genotoxic agent, and exposure to coumarin (in foods and cosmetics) has not possessed a health risk in humans according to more recent evidence [36, 37]. Coumarin is also found in the essential oils of a number of plants. Coumarin’s aroma has been described as sweet-herbaceous, cherry flower-like odour, aromatic, a creamy vanilla bean odour with nut-like tones that are olid, but not sharp or pleasant [36]. Meanwhile, coumarin derivatives have undesirable side effects in high dosages such as headache, nausea, vomiting, sleepiness, and even in some cases, liver damage. It is known that furocoumarin derivatives show phototoxic properties. Moreover, according to some hypothesis cutaneous melanoma, it shows very high risks on photochemotherapy with 8-methoxy psoralen (8-MOP) in combination with ultraviolet-A radiation (PUVA) as well as genetic predisposition with exposure to ultraviolet radiation. It is known that psoralens and other furocoumarins, are phototoxic and photocarcinogenic, because of the intercalate DNA and photochemically induce mutations. Thus, according to some ideas, the increases in cutaneous melanoma incidence may be related to increases in dietary photocarcinogenic furocoumarins [38].



In addition, umbelliferous vegetables such as parsnips, celery and parsley contain significant amounts of furanocoumarins. A number of furanocoumarins act as inhibitors of drug metabolizing enzymes. As an example, 6,7-dihydroxybergamottin and related linear furanocoumarins derivatives such as xanthotoxin and bergapten found in grapefruit juice act as highly potent inhibitors of cytochrome P450 and other CYP isozymes affecting drug metabolism [10, 39]. Aflatoxins, naturally occurring mycotoxins, are hepatotoxic and carcinogenic agents produced by Aspergillus species. Furthermore, dicoumarol derivatives are known as anticoagulant effects that cause bleeding in cattle [3, 6, 8, 9]. 4. IMPORTANCE OF COUMARINS The coumarins have many applications in pharmaceutical and chemical industry. Psoralens such as xanthotoxin and bergapten, and dicoumarol derivatives are used in antipsoriatic and anticoagulant agents, respectively. While the xanthotoxin is used often for photochemotherapy of psoriasis, bergapten is a valuable alternative for the same purpose in clinical treatments [9]. Warfarin which is a vitamin K antagonist is a parent molecule of coumarin. It is known clinically as a useful anticoagulant agent, and widely used as rodenticide. Warfarin sodium (coumadin) is a synthetic derivative of dicoumarol, which is metabolized from coumarin in the sweet clover (Melilotus alba and M. officinalis) under the influence of molds, and causes bleeding in farm animals. It has also been used for the treatment of various cancers types [9]. Beside all these usages mentioned above, fluorescent properties of many coumarins are also very useful for a variety of different applications. Furthermore, coumarin is an important factor for the industry. Its strong, pleasant odor evokes the usage as sweeteners and fixers in perfumes as well as enhancers of natural oils and as a food additive together with the combination of vanilla and as a flavor or odor stabilizer in tobaccos, odor maskers in paints and rubbers. Pharmaceutically, coumarin was reported to play a significant role in the inhibition of lymphoedema, renal carcinoma and melanoma [6, 8]. Finally, it is obvious that the coumarin and coumarin derivatives are quite beneficial in many fields, and very important groups in the nature for biological activity. 5. PHYTOCHEMICAL PROPERTIES Coumarins are mostly to be colorless or sometimes yellow crystalline substances, well soluble in organic solvents, fats and fatty oils. Coumarins have been isolated successfully from plants by using simple extraction methods with the help of solvents such as n-hexane, petroleum ether, diethyl ether, ethyl acetate and ethanol. In further applications, the extracts are purified in different isolation methods explained in many articles. The purification of the coumarins can be performed by the ability of the lactone ring (pyrone) to open under alkaline conditions resulted with the formation of coumarinates (o-coumaric acid salts) and to close again under acidification. However, this method has disadvantages in practice and may not reach starting compounds, so it is not suggested to be used in the applications. In addition to this, the most important problem in the purification of coumarins is to possess close solubility in organic solvents due to enantiomeric structures in many cases; even to apply subsequently re-crystallization could not give successful results. For this reason, it needs various chromatographic applications and professional knowledge to obtain pure coumarins [6, 9]. The presence of coumarins can be detected by the characteristic fluorescence under UV irradiation, or positive response in chemical reactions such as treatment with alkaline solutions (potassium hydroxide or ammonia solutions) or Emerson reagent. UV irradiation is also useful to detect the coumarin spots on TLC (Thin Layer Chromatography) plates at specific wavelength. The fluorescent color may give a prediction on the identification of the structure, thus can indicate about the functional groups or class of the coumarins [6, 9].


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TLC has many advantages in chromatographic separations such as rapid separation of components in mixtures or extracts, and identification of the components using reference samples. TLC is used in coumarin separation by itself or sometimes with joint techniques. The separation of coumarins can be performed by classical chromatographic methods with self modifications on silica gel, polyamide or dextran sorbents as well as TLC applications on aluminum oxide and silica gel to check the fractions or extracts. The coumarin compounds can also be successfully separated using preparative TLC techniques [6, 9]. Conventional spectroscopic methods such as UV (Ultraviolet), IR (Infrared), NMR (Nuclear Magnetic Resonance) and MS (Mass) as well as some physical methods such as melting points and specific optical rotation are generally used for the identification of coumarins’ structure to reach the absolute configurations. The structures of coumarins can be confirmed by some methods such as Perkin condensation, Pechmann reaction, Knoevenagel reaction and other techniques [6, 9]. There have been various techniques for the analysis of coumarins such as titrimetric, colorimetric and polarographic methods. Nevertheless, these methods are used rarely in the quantitative determination of coumarins. On the other hand, the UV spectroscopy is based on the ability of coumarins to absorb the light in the UV spectral range. The presence of several characteristic high-intensity bands (220-350 nm) helps for the quantitative determination of coumarins. The UV spectrometry is the most promising method for analysis of coumarins as well in the other compounds, due to it being a simple analytical method. Determination of the components can be carried out without pre-separation with their different absorption values. However, this method needs standard samples in order to provide exact results. The UV spectrophotometric measurements can aklso help the determination of coumarins molecular weight [6, 9]. The IR spectra of coumarins shows characteristic absorption bands at 1750-1700 cm-1 (-C=O groups) and 1620-1470 cm-1 (-C=C- groups of aromatic rings). If the coumarins are obtained in crystalline forms, the structure can be easily determined by X-ray analysis [6, 9]. Nuclear magnetic resonance (NMR) and mass spectrometry (MS) are very useful methods for the elucidation and confirmation of the expected coumarin structure. In the 1H-NMR spectrum (in CHCl3), observation of a pair of doublets located at  6.1-6.4 ppm and 7.5-8.3 ppm (J 9.5 Hz) belong to H-3 and H-4, respectively strongly indicates coumarin structure. However, the majority of natural coumarins have an oxygen atom at C-7 position. The position of aromatic proton in 5and 8-alkoxypsoralens (in furocoumarins) can be easily defined by the 1H-NMR spectrum. In addition, the presence of an unsubstituted furan ring is easily recognizable by the pair of doublets. Natural pyranocoumarins are characterized by the geminal methyl groups which resonate as six-proton singlet(s) around  1.45 ppm. The identity of coumarin esters such as acetic acid, angelic acid, senecioic acid, 2methylbutanoic acid, isovaleric acid and tiglic acid esters can be determined by 1H NMR spectra by the help of specific signals [6]. Measurement of nuclear overhauser effects (NOE) gives useful data to determination of the geometry on the coumarin structure having a substituent in the benzene ring [6]. In addition, 13C-NMR spectroscopy is a quite convenient method in the structural elucidation on natural product chemistry. The coumarins have been determined by the assignment of 13C chemical shifts and carbon-proton couplings. In most of the coumarins; the chemical shift of the carbonyl-carbon atom has been detected as to be almost160 ppm [6]. The MS spectrum of coumarins is very useful in determining the structure as being all natural compounds. The basic structure of coumarin gives a strong molecular ion (M+, m/z: 146, 76%) ionizated by EI/MS and a



base peak (m/z 118,100 %). In addition, the mass fragmentation is a very important point for the determination of ester groups in coumarins [6]. Recently, GC (Gas Chromatography) and HPLC (High-Performance Liquid Chromatography) are used dominantly for the isolation, purification and identification of the coumarins with hyphenated techniques. In addition, these methods have been advantageous in the quantitative and qualitative analysis of coumarins for many applications. HPLC is widely and effectively used in the analysis of coumarins not only by itself but also with incorporated techniques. Combination of HPLC with mass spectrometry (HPLC-MS) is a promising method in coumarin analysis for various purposes [6, 9, 40]. Some information about methods of identification, analysis, preparation of the compounds and their properties are found in the pharmacopoeias to help in our studies. Nonetheless, it was mention that limited information has been given about coumarins in some pharmacopoeias. 6. BIOTECHNOLOGICAL APPROACH ON COUMARINS It is reported that many new drugs have been introduced to the market originating from natural sources in the past couple of decades. Plant metabolites are successful sources for drugs that are used in pharmaceutical industry as well as some simple preparations used for human health problems. Various multidisciplinary approaches have many notable applications to reach the plant derived pharmaceuticals. In recent years, the cell culture system has been used for the production of compounds that have medicinal importance. Thus, many reports on the plant cell cultures for the commercial use of phytochemical production exist. Nowadays, there are remarkable progresses in technologies of plant tissue and cell culture. The biotechnological studies have been characterized with many processes such as growing plant tissue on gel-solidified nutrition media, regeneration of plants from cultured cells, production of virus free crop plants, culture of anthers and microspores to produce haploid and homozygous plants, production of new plant hybrids, and integration of tissue culture with molecular genetics as well as many biotransformation studies described in resources. However, there are many disadvantages of these techniques due to high cost of the process, fewer amounts of the final compounds and of course time consumption [41-44]. Biosynthetic pathway of the secondary metabolites is not so clear. Therefore, the effect of the elicitors cannot easily be predictable. The results generally stay empirical. Nowadays, biotransformation is considered to be an economically competitive technology by synthetic organic chemists in search of new production routes for chemicals, pharmaceuticals, and agrochemicals. Biotechnological methods involved in obtaining coumarins are main subject in this report. There are several studies on these topics outlined below, and these studies will be debated regarding production of coumarins: Rutaceae is a famous family with coumarin content. Most of the coumarins in this family occur as to be cinnamic acid-derivatives. Several studies have dealt with in vitro production of coumarins by tissue culture. Some studies concerned with the effect of the precursors on the coumarin contents of Ruta graveolens from Rutaceae family. R. graveolens has attracted commercial interest because of its interesting secondary metabolites. Some important coumarins were determined from aerial parts of this species such as umbelliferone, scopoletin, isopimpinellin, xanthotoxin, isoimperatorin, psoralen, bergapten etc. Thus, it is an interesting biotechnological source of biologically active phytoalexins. The callus culture of R. graveolens has been investigated for the effect of plant growth regulators. It was found that the callus culture produced coumarins more than that of the aerial parts of the plant. Moreover, the effects of cinnamic acid presence in different concentrations (as a precursor) to the coumarin contents, has also been investigated, and it was found that cinnamic acid concentration increased the amount of coumarins. Pursuant to this study, some non-identified coumarins have been detected and isolated in callus cultures of stems and roots of R. graveolens. It was mentioned that a culture of R. graveolens in liquid media produced relatively high amounts of coumarins. In this experiment, precursors of some coumarins were added to the culture to increase the concentration. Octanol led to the release of coumarins into the medium. In another research, growth of R. graveolens shoots induced by Bacillus sp. cell lysates in the culture medium was investigated. Elicitation of coumarins by the lysate was effected and increased the coumarin content,


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including isopimpinellin, xanthotoxin and bergapten as well as psoralen and rutamarin. Moreover, these conditions induced the biosynthesis of chalepin which is not detectable in the shoot sample. Elicitation is used extensively for enhancing secondary metabolites in biotechnological applications. In a study, the effects of butylated hydroxy toluene (BTH) and saccharin on the biosynthesis of coumarins in shoots of R. graveolens cultivated in vitro was also examined. At the end of this study, biosynthesized metabolites were analyzed and identified by GC/MS; bergapten, chalepin, isopimpinelin, pinnarin, psoralen, rutacultin, rutamarin, and xanthotoxin were detected. Thus, the use of BTH and saccharin had increased the production of coumarins. In a biotransformation study, hairy root culture of R. graveolens obtained after genetic transformation of plant tissue with Agrobacterium rhizogenes led to the production of coumarins. The analysis of these coumarins was performed by GC and GC/MS, and the content of hairy root tissue was compared with in vitro that of shoot culture in this study. The research indicated that hairy root culture of R. graveolens produced a high level of bergapten and isopimpinellin [45-48]. Haplophyllum patavinum (L.) G. Don fil. is also a species that belongs to Rutaceae family. There are no usages in folk medicine regarding to this plant and scarce phytochemical studies available. However, the species is important for its notable biological effects which are caused by chemical constitutents. Callus and suspension cultures of H. patavinum were found to produce several coumarin compounds such as umbelliferone, scopoletin, 7-isoprenyloxy-coumarin, umbelliprenin, osthenol, columbianetin, angelicin and psoralen [49]. It is obvious that the plant cell and organ culture systems are alternative production methods for the secondary metabolites which have commercial importance in pharmaceutical and in food industries. Hairy root cultures possess biosynthetic capabilities of differentiated tissues, and have a high growth ratio comparable with cell cultures. Usages of elicitors lead to new approaches for the production of secondary metabolites, because of their enhancement role in the production. There are also several examples on the study of hairy root culture as stated below: Cichorium intybus L., belonging to Asteraceae (Composiate) family, is also known as common chicory, witloof and blue-sailor’s succor. The plant is native to the temperate parts of the Old World such as India where it grows wild. Phytochemical and pharmacological properties of this species were clarified with all details because of its economic importance. It has been also reported that the chicory can be found easily in the markets as healthcare products; dry roots and leaves are used as a vegetable in salads, and extracts in beverages, and coffee blends. Biotechnological developments and evaluation of genetically modified chicory crops have been expressed by Bais and Ravishankar (2001). The plant contains coumarins such as esculin, esculetin, cichoriin, umbelliferone, scopoletin beside some sesequiterpenes and a polyholoside as inulin. C. intybus is able to form inflorescence in vitro. Thus, root and leaf segment cultures of this plant for the flowering process were investigated in vitro cultures. Possible coumarin production in hairy root cultures of C. intybus, were reported under the influence of microbial agents used as elicitors. In the study, some pathogens such as Pythium aphanidermatum and Phytophora parasitica var. nicotianae have been used as fungal elicitors in coumarin production. The results showed that the products were found as esculin (6, 7-dihydroxycoumarin-6-glucoside), and esculetin (6,7-dihydroxycoumarin), which are markers in microbiological media and UV filter in cosmetics, respectively. In hairy roots, the cultures have been strongly correlated with growth and elicitors. It was also noted that exogenous feeding of polyamines (Pas) induced the root culture, and thereby coumarin production. Some earlier studies mentioned that putrescine or other polyamines would be substrates for coumarin synthesis in chicory. The study has found that putrescine influences development and differentiation of the plant root. Therefore, it was deduced that there should be a relationship of polyamine and coumarin biosynthesis [50-52]. Another Asteraceae species, Tanacetum parthenium produced isofraxidin and isofraxidin drimenyl ether in transformed roots. The transformed roots with Agrobacterium rhizogenes LBA 9402 of T. parthenium were extracted with hexane and hexane:acetone (1:1) mixture. Isofraxidin and an isofraxidin drimenyl ether were separated from the latter extract [53].



The coumarins are o-hydroxycinnamic acid lactones that constitute a large class of allelochemicals mostly found in higher plants. Coumarin (1, 2 benzopyrone) is a strong inhibitor of seed germination and root growth. Moreover, the coumarin decreases respiration and photosynthesis in the plants by inhibiting electron transport. The coumarin does not only inhibit plant growth but also has the ablilty to develop it. Abenavoli et al. (2003) investigated the effects of the allelochemical coumarin on cell growth and utilisation of exogenous nitrate, ammonium and carbohydrates in cell suspension culture of Daucus carota L. from Apiaceae family (Umbelliferae). As a result, they found that exposure to micromolar levels of coumarin caused serious inhibition of cell growth and the presence of 50 μmol/L coumarin that caused accumulation of free amino acids and of ammonium in the cultured cells. As a result, the hypothesise indicate that such allelochemical compounds may act as an inhibitor of the cell cycle and/or as a senescence-promoting substance in nature [54]. Chemistry of Calophyllum inophyllum L. (Clusiaceae) has been studied, and many compounds have been identified. Dipyranocoumarins are the most important group of bioactive components isolated from this species due to cancer chemopreventive properties. Quantitative analysis of dipyranocoumarins in callus culture has been performed by the optimized conditions of extraction and HPLC methods. The effects of different hormone combinations and concentrations on callus induction from various explants and the pattern of expression of dipyranocoumarins in the callus cultures have been investigated by Pawar et al. [55]. Angelica gigas Nakai from the Apiaceae family is an endemic species growing in the Korean peninsula. The plant contains many coumarins including decursin and decursinol which are important in health-food supplements in Korea. These compounds have been isolated from the roots of this species. Decursinol, an isopentenyl conjugate of coumarin, is considered to be a symptom-alleviating agent in Alzheimer disease. Because of the important effects and nutraceutical value of this compound, its chemistry and biology were investigated in the biosynthetic pathway. Biosynthetic pathway of decursin has been determined by deuterium-labeled intermediates to the hairy root culture of A. gigas [56]. Because of the therapeutic uses of coumarins, it evoked the requirement to work on the production of coumarins in vitro cultures from different plants such as bishop weed, parsley, carrot, cucumber, celery and parsnip. The results showed that accumulation of secondary metabolites can be sufficiently induced by elicitors. Using to this concept, there are other examples on cell suspension and hairy roots of Ammi majus L. to obtain secondary metabolites by abiotic elicitors. Ammi majus L. (Bishopsweed), also belonging to the Apiaceae family, is a rich source for coumarin content, particulary linear furocoumarins such as psoralen, imperatorin, bergapten, marmesin used as photosensitizes effects in skin diseases (especially in vitiligo and psoriasis). Thus, the induction of secondary metabolites production in elicited callus, cell suspension and hairy root cultures of A. majus have also been investigated to obtain possible novel compounds. The presence of umbelliferone and other coumarins like substances in methanolic extract of callus, cell suspension and hairy root cultures, and the presence of furocoumarins in the fruit extracts were determined. However, it was not possible to observe any differences in the content of coumarins after changing the level of growth regulators and trophic compounds. On the other hand, in earlier studies, induction of coumarins content had been reported after some changes in this conditions. In further studies, the growth rate of in vitro cultures by applying elicitors did not change notably except for an addition of jasmonic acid to the cell suspension. The umbelliferone level increased doubled by a Gram () microorganisms called as Erwinia chrysantemi and jasmonic acid. Even though the highest level has been observed by the treatment of callus cultures with SiO2, the production of linear furocoumarins such as marmesinin increased by the treatment of the cell suspension with scleroglucan and lysats of culture from E. chrysantemi. In a different study, ADR-4® plates have been used as an elicitor in A. majus callus culture to obtain the pharmacologically important secondary metabolites. The biosynthesis of bergapten and umbelliferone in A. majus tissue has been induced by ADR-4® plates. In this study, three different types of extraction techniques have been used for the extraction of coumarins from transformed callus of A. majus elicited by ADR-4® [57-59]. The suspension cultures of hairy roots have also been used for the glycosylation of hydroxycoumarins. The study was performed on suspension cultures of hairy root of Polygonum multiflorum induced by Ri plasmid Agrobacterium rhizogenes, and eventually two coumarin glycosides were biosynthesized. Another


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glycosylation study was carried out on the hairy roots of Pharbitis nil (L.) Choisy (Morning glory). The plant is known as “asago” in Japanese and belongs to the Convolvulaceae family. The hairy roots of this plant exhibited potent glycosylation activity against umbelliferone and esculetin. In this research, the possible glycosylation capability of hairy roots against some phenolic compounds such as coumarins or flavonols was clarified [60, 61]. Aspergillus strains such as Aspergillus ochraceus, Aspergillus niger and Aspergillus flavus have been used often in coumarin metabolism. Aspergillus ochraceus and Aspergillus niger carried out the reduction of the C3-C4 double bond of the coumarin and gave dihydrocoumarin in 24 h. A. niger demonstrated to have two divergent catabolic pathways; one of them is opening of the lactone moiety and further reduction of the carboxylic acid furnishing the primary alcohol 2-(3-hydroxypropyl) phenol, and the other being the hydroxylation of the aromatic ring of dihydrocoumarin at a specific position to give 6-hydroxy-3,4dihydrochromen-2-one. Aspergillus flavus did not perform double bond reductions, and only produced oxygenated metabolites, mainly 5-hydroxycoumarin [62] The biotransformation of coumarin skeleton was carried out by A. niger more than 40 years ago by means of basic chromatographic techniques. In Aguirre-Pranzoni study (2011), the metabolism of coumarin and derivatives were reported by several Aspergillus spp., especially A. niger. Final products and intermediates were isolated and characterized by GC/MS NMR. As a conclusion, the ability of some members of the Aspergillus species to metabolize coumarins was understood by methabolic pathways. This knowledge will be very useful for the biocatalytic preparation of interesting coumarin derivatives [62]. Plants are valuable natural sources for the wide variety of secondary metabolites, drugs, flavor and fragrance agents, and pigments. Most of the biochemical reactions occurring in plant cells cannot be carried out by synthetic reactions, for this reason, the natural components could be obtained only from natural sources. The plant cell and organ cultures, and plant enzymes as biocatalysts play a significant role in reaching complex reactions. Thus, aromatics, steroids, alkaloids, coumarins and terpenoids can be applied for biotransformation using plant cells, organ cultures, and enzymes. It is reported that the biocatalystmediated reactions can be regiospecific and stereospecific, and the reaction types are found to be oxidation, reduction, hydroxylation, methylation, acetylation, isomerization, glycosylation and esterification. Natural flavors and fragrance have been produced by the new biotechnological processes. Biotrans-formation methods using microorganisms as efficient and selective catalyst have obtained natural flavors and fragrances used for industrial purposes [39, 63-65]. The microbial metabolism of coumarins may be able to produce metabolites that are difficult to obtain from chemical synthesis or animal systems. Moreover, this may be promising for some novel metabolites that can serve as starting compounds for semisynthesis. Marumoto and Miyazawa have studied the microbial transformation of furanocoumarins by a fungus called Glomerella cingulata and evaluated the transformation products Coumarin, psoralen, and xanthyletin have been used for the transformation process. The main reaction pathways was the reduction at α-β-unsaturated δ-lactone ring on coumarin derivatives. Coumarin was metabolized to give the hydrocoumaric acid. In the biotransformation of psoralen, two reduced metabolites, 6,7-furano-hydrocoumaric acid, and 6,7-furano-o-hydrocoumaryl alcohol were isolated from the incubation of psoralen. Xanthyletin was converted to reduced products 9,9dimethyl-6,7-pyrano-hydrocoumaric acid and 9,9-dimethyl-6,7-pyrano-o-hydro-coumaryl alcohol by G. cingulata. The structures of the new compounds were identified by spectroscopic techniques. In addition, all of compounds including methyl ester derivatives of the metabolites were tested for the β-secretase (BACE1) inhibitory activity in vitro. The coumarin derivative, 6,7-furano-hydrocoumaric acid methyl ester, possesses BACE1 inhibitory activity, and IC50 value was 0.84±0.06 mM. It is obvious that biotransformation is a powerful method for the structural modification of natural products which has been performed on coumarins [39]. It has been indicated that the coumarins have created great interest in the flavor industry because of their pleasant odor. Natural dihydrocoumarin which also has a high attraction in the flavor industry has been produced from coumarin or tonka bean, with Saccharomyces cerevisiae. Dihydro-coumarin (DHC) (Fig. 3)



is found in Melilotus officinalis (Sweet clover) and Dipteryx odorata Willd. (Tonka beans), and it is used as a flavoring agent in foods, soft drinks, yogurt and muffins as well as a common fragrance in cosmetics, lotions and soaps. DHC has a sweet herbaceous aroma that is released and smelt when grass has been freshly mown. DHC is produced by synthetic methods, generally hydrogenation of coumarin to be used in the market. However, chemical compounds or synthetically obtained compounds are not desired in general because of the possible poisonous effects during the chemical process. Thus, natural or biologically originated food or cosmetic additives are preferred in daily life usage. However, natural ones are always high in cost; in this case de novo microbial process (fermentation) or bio conversions using natural precursors with microorganisms or isolated enzymes (bio catalysis) are very useful processes for these reasons [65, 66].

O

O

Figure 3: Dihydrocoumarin (DHC).

There are many examples of applications using biotransformation procedures on natural sources. In addition, the biotransformation by cell cultures and hairy root cultures can be applied on the structural modification of compounds possessing for useful and important therapeutic activity [39, 63-65]. 7. CONCLUSION Natural products have been a great interest as a good source of novel drug molecules and have attracted high attention in the pharmaceutical industry as well as in human health problems. The discovery of drugs originated from natural sources and is still being used for the treatment of illnesses. However, new drug development or production should be performed with multidisciplinary approaches to avoid loss in their popularity around the World [41]. Natural coumarins are present in our daily life as spice, fruits and vegetables and are used in herbal remedies. These principles have triggered to investigate the production of the coumarins and coumarin consisting plants for their promising and interesting effects and usages. It is obvious that the coumarins like other natural unsaturated lactones, exhibits various important biologic effects. Coumarins can be recommended with beneficial usages and effects in many applications. Moreover, their mild antimicrobial and anti-inflammatory effects make them possible to be used as dietary supplements. Furthermore, the most important and common usage of these compounds are in medicinal agents for skin diseases. The pharmacological and biochemical properties of these compounds may possibly have pharmaceutical interest. Nonetheless, specific actions are rather limited. The compounds show multi-actions, which means that these compounds will not be able to cause specific potential. However, low toxicity, relatively low cost, presence in diet and occurrence of various herbal remedies indicate their importance for investigation and evaluation of their properties, applications and further isolations of novel coumarins. There are still important studies on coumarins and their derivatives. Limited studies exist on biotechnological applications and biotransformation researches on plant cells, organs and enzymes in vitro. However, it helps many applications and is useful for many biochemical reactions and structure modifications. There are only several biotechnological applications of coumarin. It is hoped that many researches will be performed on the group of natural compounds. In future, the biotechnological approach will continue to develop to reveal new potential drugs and to discover economic and advantageous crops. Coumarins promise impressive biologic effects and beneficial utilization. Therefore, the production of promising coumarin molecules is quite necessary to use in the therapeutic, cosmetic and dietetic fields by developing biotechnological techniques.


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53

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 Stransferase, 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 inhouse 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 [email protected] Ilkay Erdogan Orhan (Ed) All rights reserved-© 2012 Bentham Science Publishers


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. GSTdirected fractionations on these medicinally important plants were performed in order to isolate GST inhibitors, present in them.

H 3C

CH3

CH3

OH

CH3

CH3 CH3

CH3

(2)

O

H3C

O

H3C

H

H3CO

(4)

O H3C

H

CH3

O MeO2C

(3)

HO O

H

H3C

HO

O O

O OH CH3

H CH3

HO

(5)

H H

CH3

H

OAc OH

AcO

(7)

(6)

O OH CH3

OH H

H H3C CH3

OH

(8)

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CH3 OAc

OAc

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OH RO

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H

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OH OH

(9) R = H (10) R = CH3

CH3

CH3

H 3C

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(1)

H3C

H3C

CH3 CH3

H3C

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H 3C

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(11)

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CH3

Novel Biomedical Agents from Plants


From the bioactive fractions of C. bonduc, 17-hydroxycompesta-4,6-dien-3-one (1), 13,14-seco-stigmasta5,14-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

H 3C

CH3

CH3 H

H3 C

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(19)

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(17)

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(18)

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(16)

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(15)

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OHOH

OH

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 structureactivity 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


Athar Ata

of compound 21 to yield compound 26. This metabolite further underwent glycosylation, followed by reduction of the 16-17 double bond to give compounds 27 and 28, respectively [16]. Compounds 26-28 were also found to be active in GST inhibition assay with IC50 values of 18.6, 12.3, and 16.6 M, respectively. R H N

O

N

H

H

H

N H

OH

N

O

H

H

N

O

N

H

OH

O

OH

O

HO HO

OH

OH

O

O

(22)

(21) R = H (26) R = OH (27) R = -D-glucose

OH

O H

O

H

(23)

OH

OH O

HO HO

O OH

H

COOH

N

H

H

COOH

O

N

OH

RO

(24) R = -Rhamnose

O O

HO HO

(25) R = -Fucose

O OH

(28)

'

From the GST inhibiting fraction of Barleria prionitis, barlerinoside (29), shanzhiside methyl ester (30), 6O-trans-p-coumaroyl-8-O-acetylshanzhiside methyl ester (31), barlerin (32), acetylbarlerin (33), 7methoxydiderroside (34), and lupulinoside (35) were isolated. Compounds 29-35 showed GST inhibitory activity with IC50 values of 12.4, 49.2, 22.9, 38.9, 40.6, 122.0 and 134.0 M, respectively [17]. These studies suggested that phenylethanoids have potential to inhibit the activity of GST. OH OH

O

HO HO

OH

O

HO

O

O O

R 2O OH

H

C

(34)

O

(30) R1 = H, R2 = OH (32) R1 = H, R2 = Ac (33) R1, R2 = Ac O

CH3

H

O

C O H3CO AcO H CH 3 O OH O HO HO OH

H

C

AcO CH3H OH O O

HO HO OH HO HO

O OH

O

O

O

(35)

CH3

H

HO

CH3

O

O OH

(31) R =

O

H

OH CH3 HO HO

C

O

AcO

OH

(29) O

O RO

CH3

O

HO HO

O

O

O

O

O OH

OH

H

C

O

O

H 3C HO

HO

R 1O

OH

O

H 3C HO

O

OH

OH

O C



3. ACETYLCHOLINESTERASE INHIBITORS Alzheimer’s disease (AD) is a neurodegenerative disorder that causes severe health problems in aged population [18]. It is believed that the major cause of memory impairments in AD patients was due to the deficiency of acetylcholine [19, 20]. Restoring acetylcholine in the brain using potent acetylcholinesterase (AChE) inhibitors is one of the effective methods to treat AD [20]. AChE inhibitors also prevent the deposition of -amyloid, as deposition of -amyloid in the brain cells also causes the death of neuronal cells [21]. Four AChE inhibitors, tacrine (36), donepezil (37), galanthamine (38) and rivastigmine (39), approved by FDA, are in clinical practices [22-25]. All of these inhibitors have limited effectiveness and a number of side effects [26-28]. For instance, tacrine (36) exhibits hepatotoxic liability and rivastigmine (39) has a short half-life [27]. Two types of AChE inhibition assays (microplate reader and TLC based assays) are used to screen plant extracts and pure natural products [29]. Microplate reader bioassay is more accurate and reliable in order to determine the accurate potency of pure natural products while TLC based bioassay can be used to guide the bioactivity of fractions or crude extracts. O

NH2

H3CO N H3CO

N

(36)

(37) OH CH3

O

H3C N

H3CO

O

CH3

(38)

CH3 N CH3

O

N CH3

(39)

In this area, during the last decade, a few natural products or their analogues have been reported as potent AChE inhibitors. Tan et al. report the isolation of hopeahainol (40) as a potent AChE inhibitor with an IC50 value of 4.33 M [30]. This bioactivity is more closely related to huperzine A (41), a prescribed drug used to treat AD and is active in this bioassay with an IC50 value of 1.6 M. Bastida et al. discovers N-alkylated galanthamine derivatives, N-allylnorgalanthamine (42), and N(14-methylallyl)norgalanthamine (43) exhibiting anti-AChE activity with IC50 values of 0.18 and 0.17 M, respectively [31]. Compounds 42 and 43 are found to be more potent than galanthamine (39) that exhibit AChE inhibition activity with an IC50 value of 1.82 M. HO

HO

R

OH OH O

O

CH3 H3CO

HO

N

H3C O OH

OCH3 O

(40)

NH2

(41)

N H

O

CH3

(42) R = H3C

(43) R =

Helicid (44) is one of the major active constituent of a Chinese herb, used as an antalgic and hypnotic in China [32]. The aglycone exhibits potent GABA transaminase activity with an IC50 values of 4.1 g/ml. Studies on the structure-activity relationships indicate that this bioactivity was due to aldehyde and hydroxyl groups on benzene ring [32]. Song et al. report the weak AChE inhibition activity (IC50 = 37.8 mM) of this aglycone. These authors synthesize compounds 45, 46 and 47 using compound 44 as a


Athar Ata

template. Compounds 45, 46 and 47 exhibit anti-AChE activity with IC50 values of 0.45, 0.49 and 0.20 M, respectively [32]. OH

OH O

HO

O

CHO

OH

HO

O

HO HO

OH

(44)

CHO

(45) OH

OH O HO HO

O

OH

O

N-OCH3

O

O

HO OH

HO

(46)

CHO

(47)

Olafsdottir et al. report the isolation of ten lycopodane-type alkaloids from the Icelandic Lycopodium annotinum [33]. These compounds include annotinine (48), annotine (49), lycodoline (50), lycoposerramine M (51), anhydrolycodoline (52), gnidioidine (53), lycofoline (54), lannotinidine D (55), acrifoline (56) and a previously unknown N-oxide of annotine (57). Compounds 48-57 are moderately active in AChE inhibition assay as compared to huperzine A (41). The weak inhibitory activity might be due to incompatibility of their functional groups with the active site gorge of the enzyme. The structural modifications of these alkaloids (48-57) may improve their AChE inhibitory potentials. Interestingly, acrifoline (56) occurs as an equilibrium mixture of the ketone (56a) and the hemiketal form (56b). Both of these compounds appear to be artifacts. Compounds 52-54 are isolated for the first time from this plant.

O

H3C

O

CH3

N

R2

H3C

H

N

H

H

(48) H3C

H

O

H

N

(50) R1 = H, R2 = OH (51) R = OH, R2 = H

(49) OH H

H3C

H3C R1

O

O N

O

OH

OH H

OH

O

H N

H

N

O

H

OH N

(54) R = H (55) R = feruloyl

(53)

H

N

OH

O

(52) H3C

O H

H3C

H

H

(56b)

(56a)

Li et al. synthesize several analogues of berberine (58) and discover one of the synthetic analogues (59) exhibit significant AChE inhibitory activity with an IC50 value of 0.097 M. This compound is more potent than galanthamine (38) [34]. In this compound, berberine (58) is linked with phenol by 4-carbon spacers. Initial structure-activity relationship studies indicate that AChE inhibition is mainly due to the functional group present at the end of the chain and the length of the connecting tether. H3C OH

O

O + ClN

O O O

+ - N HO

+ BrN

O

O

O

H

(58) (57)

O

(59)

O

)CH2)4O Ar



H3C H3C

N

H

CH3

H3C N O

H3C

CH3

N

H CH3

O

H

N

CH3

H3C H3C

CH3

H

H

O

OH CH3 N H HH3C CH 3

O

H O Ph

N

O Ph

H

CH3

H

N H H H3C CH -OAc 2

CH3

H3C H3C

H

OH CH3 N H HH3C CH3

(65)

CH3 CH3 R3

OH

H

OH H

(71)

Ph

H

H3C H3C H O

N H

H

HO O

CH3 H3C N CH3

H3C H3C

O

(72)

Ph

H

CH3 CH3

(67) R1 = C=O, R2 = CH3, R3 = OH 1,2 (68) R1 = NH-tigloyl, R2= CH2-OH, R3 OAc (69) R1 = NH-benzoyl, R = CH2-OH, R3 = OH, 6,7 (70) R1 = NH-benzoyl, R = CH2-OH, R3 = OAc 6,7

N

CH3

N

N

CH3

R1 H H3C R2

(66)

H

H3C H3C

H

H3C H3C

H O

(64)

CH3

N H HH3C CH 3

H3C H3C

N H HH3C CH3

CH3

H

CH3

H

CH3

CH3

(62)

CH3

OH CH3

(63)

H3C H3C

N

CH3

H CH3

O

H3C

(61)

H3C H3C

N

CH3

H3C N

H CH3

(60)

O

H

H

H CH3

CH2

H3C

CH3

H3C H3C

CH3

N H

CH3 CH3 OAc

CH3

N H H3C

H

R O

(73) R = OH (74) R = H

CH3 OH

CH3 N H H H3C CH 3

(75)

Buxus alkaloids have shown potential as anti-AChE inhibitors. Most of the steroidal bases isolated from B. hyrcana have shown activity in AChE inhibition assay. These steroidal bases include moenjodarmine (60), hyrcanine (61), homomoenjodaramine (62), N-benzoylbuxahyrcanine (63), N-tigloylbuxahyrcanine (64) Nisobutyroylbuxahyrcanine (65), hyrcanone (66), Nb-demethylcyclobuxoviricine (67) hyrcamine (68), buxidine, (69), buxandrine (70), buxabenzacinine (71), buxippine-K (72) O6-buxafurandiene (73), 7-deoxyO6-buxafurandiene (74) benzoylbuxidienine (75), buxapapillinine (76), buxaquamarine (77), and irehine (78) Compounds 60-78 exhibit this bioactivity with IC50 values of 50.8, 312.0, 19.2, >1000, 443.6, >1000, 145.0, 310.0, 83.0, 210.6, 175.4, 468.0, >1500, 17.0, 13.0, 35.0, 76.0, 80.0, and 100.0, respectively [35-38]. Among all of these isolates, compounds 73 and 74 are found to be significantly active against AChE. AntiAChE properties of these compounds might be due to the presence of dimethylamino moieties at C-20, or


Athar Ata

the presence of the ether linkage between and C-31 and C-6 in these alkaloids. However, it is difficult to comment on the structure-activity relationships of these bioactive compounds from their data. These moderately active alkaloids can be used as a template to synthesize potent AChE inhibitor. For instance, using Buxus alkaloid, N-3-isobutyrylcyclobuxidine-F (79), alkaloids (80-84) are synthesized by Guillou and collaborators [39]. These synthetic alkaloids (80-84) exhibit potent AChE inhibitory activity with IC50 values of 31, 13, 27, 18 and 14 nM, respectively [39]. These compounds represent the first example of triterpenoidal alkaloids exhibiting potent anti-AChE activity. This indicates that studies on structureactivity relationships of moderately bioactive compounds is worthwhile in the discovery of new pharmaceuticals.

H3C H3C H

O

H3C H3C

H

O

O

CH3 N CH3

H

OH H

H N

CH3

O

N H H H3C CH -OH 2

CH3 N CH3

O

OH

R

O

H

CH3

H N

H CH3

R

O

CH3 N CH3 OH H

CH3

H CH3

(81) R = CH3 (82) R = CH3CH-CH2-CH3

H3C H3C H

O C

CH3

H

H3C H3C

H

(80)

(79)

CH3

(78)

H3C H3C

O

H

N

HO

(77)

H O C

H3C

H OAc CH3

(76)

H3C H3C

H3C H3C

CH3

CH3

N H H3C

Ph

H CH3

CH3

OH H

AcO O

CH3 H3C N

N

N

CH3 CH3 OH

H CH3

N H H H3C H2N

(83) R = iPr (84) R = (S)-(CH3)CH(C2H5)2

4. -GLUCOSIDASE INHIBITORS -Glucosidase, a membrane bound enzyme of intestinal cells, is involved in catalyzing the final step of carbohydrates digestion. During this process, glycosidic bonds in carbohydrates are cleaved to liberate free glucose causing postprandial hyperglycemia. This results in type 2 diabetes mellitus that affects approximately 311 million people worldwide. Its treatment mainly relies on suppressing hyperglycemia that includes reduction of glucose absorption in gut. This task can be accomplished by using potent glucosidase inhibitors making them method of choice to cure this ailment [40]. These inhibitors can also be used to overcome obesity problems [41]. Recent studies indicate that natural products adopt a novel mode of action in inhibiting the activity of -glucosidase. For instance, aegeline (85), a hydroxyl amide alkaloid,



is reported to suppress both blood glucose and plasma triglycerides levels [42]. Kokpol et al. discover phenyl-ethylcinnamides: anhydromarmeline (86), anhydroaegeline (87), tembamide (88), dehydromarmeline (89), aegeline (85), O-methyl ether aegeline (90), aegelinosides A and B (91-92) from Aegle marmelos leaves as -glucosidase inhibitors. Among all the isolates, compound (87) is reported to exhibit significant -glucosidase inhibition activity with an IC50 value of 35.4 M [43]. H3CO

O OR

R

N H

O

O N H

(85) R = H (90) R = CH3 (91) R = Glucose

H3CO

O

(86) R =

OH

(87) R = CH3 RO

N H

(88)

O N H

H3CO

O N H

(89) R = OR

(92) R = Glucose

Tabopda et al. report the isolation of two new ellagic acid derivatives, 3,4’-di-O-methylellagic acid 3’-O-D-xylopyranoside (93) and 4’-O-galloy-3,3’-methylellagic acid 4-O--D-xylopyranoside (94) from Terminalia superba, exhibiting -glucosidase inhibition activity with IC50 values of 7.95 and 21.21 M, respectively [44]. Additionally, both of these compounds also show significant immunoinhibitory activities with no cytotoxic effects. Chebulagic acid (95), purified from T. chebula, exhibits anti--glucosidase activity with an IC value of 6.6 M. Kinetic studies on this inhibition activity suggest that this compound is a reversible and non-competitive inhibitor of maltase [45]. HO

OH HO

OH

HO OR1

OH O

OR2 O

O

O H2C

O

O

O O

(93) R1 = CH3, R2 = Xylose, R3 = H (94) R1 = xylose, R2 = CH3, R 3 = galloyl

H

OH

H O O

OH

O

H

HO

HO

O

H3CO OR3

OH

O

O O

O O

OH

(95)

Salacia reticulate (Kothala-himbutu) is used to treat diabetes mellitus in folk medicines in Sri Lanka [46]. Investigation on this plant afforded salacinal (96), kotalanol (97) and a polydroxylated cyclic 13-membred sulfoxide (98) as active principles [47-49]. All of these compounds exhibit potent -glucosidase inhibitory activity: 96 IC50: maltase 9.58 M, sucrase 2.51 M isomaltase 1.77 M; 97 1C50: maltase 6.60 M, sucrase 1.37 M, isomaltase 4.48 M; 98 1C50: maltase 0.227 M, sucrase 0.186 M, isomaltase 0.099 M, respectively. The bioactivity data of 98 indicate that it is 42, 14 and 18 fold more potent against maltase, sucrase and isomaltase compared to compounds (96 and 97) [47-49]. It is also reported in the literature that high potency of compounds 98 is due to the orientation of hydroxyl, ring structure and sulfoxide groups [49].


Athar Ata OH

OH

OH

OH

OH

HO

OH OH

OSO3

S+

HO HO

OH

HO

(96)

OH

O S

S + OSO - OH 3

HO

OH

OH

HO

OH

OH

HO

(97)

(98)

Chen et al. identify triterpene acids, oleanolic acid (99), arjunolic acid (100), asiatic acid (101), maslinic acid (102), corosolic acid (103) and 23-hydroxyursolic acid (104) from Lagerstroemia speciosa as glucosidase inhibitors. Compounds 99-104 display this bioactivity with IC50 values of 6.29, 18.63, 30.03, 5.52, 3.53 and 8.14 M, respectively [50]. Among all of these compounds, oleanolic acid, maslinic acid, corosalic acid and 23-hydroxyursolic acid display a significant enzyme inhibition activity and these compounds need to be tested in vivo for this bioactivity and structure-activity relationship studies on these compounds are warranted to further improve their enzyme inhibition activity. H3C CH3

CH3 H3C

R1

H3C

CH3 H H

HO

COOH

CH3

R1

H3C

CH3 H H

HO

H R2 CH3

(99) R 1 = H, R2 CH3 (100) R1 = OH, R2 = CH2OH (101) R1 OH, R2 CH3

COOH

CH3

H R2 CH3

(102) R1 =OH, R2 CH2OH (103) R1 = OH, R2 = CH3 (104) R1 H, R2 CH2OH

Phytochemical studies on Garcinia brevipedicellata afford four new depsidones, namely, brevipsidones AD (105-108). All of these compound exhibit -glucosidase inhibition activity with IC50 values of 21.22, 27.80, 59.64 and 7.04 M, respectively [51]. Compound 108 is more potent in this bioassay and its high potency is due to the presence of two prenyl groups [51]. O HO

O

R2

O OCH3

O

R1

O

(105) R1= OH, R2 = prenyl group (106) R1 = H, R2 = prenyl group (107) R1 = OH, R = H

HO

O

R1

O OCH3

OH

R2

OH

(108) R1 and R2 = prenyl group

Rao et al. isolated labdane diterpenes, 6-oxo-7,11,13-labdatrien-16,15-olide (109), spicatanol (110), spicatanol methyl ether (111), hedychenone (112), 7-hydroxyhedychenone (113), yunnacoronarin D (114), 7-acetoxyhedychenone (115) and hedychia lactone B (116) from anti-hyperglycemic extract of Hedychium spicatum rhizomes. Compounds 109-116 inhibit 21.2, 35.1, 89.5, 54.1, 55.6, 13.5, 15.1, and 25.7 % activity of -glucosidase at the concentration of 100 g/ml, in the initial screenings of these compounds against the prescribed bioactivity [52]. Compound 110 is significantly active in this bioassay and IC50 value of this compound is determined to be 34.1 M. These authors further report that presence of , -unsaturated lactone and furan ring in these diterpenes is required to inhibit the activity of -glucosidase [52]. The presence of hydroxyl group in lactone ring significantly increases this bioactivity as evident by compound 110.


R

H3C

O


O O

CH3

H3C CH3 O

H3C

O

H3C

R1 R2

H3C CH3 O

(109) R = H (110) R = OH (111) R = OCH3

O

OH H3C CH3

(112) R1 =CH3, R2 = H (113) R1 = CH3, R2 OH (114) R1 = CH2OH, R2 = H (115) R1 = CH3, R2 = OAc

(116)

Dolichandroside A (117), a new phenylpropanoid glycoside, is isolated from Dolichandrone falcate and exhibits -glucosidase inhibition activity with an IC50 value of 18.72 M [53]. OH

O C O

H3CO

O O

H3CO HO

O

OH

O HO

OH OH

OH

(117) 5. BIOSYNTHESIS OF BIOACTIVE NATURAL PRODUCTS Mostly, bioactive natural products are available in minor quantities and their supply for further in-vivo testing and clinical trails is a major problem. Most of these bioactive natural products contain chiral centers and their synthesis on large scales seems to be difficult and always end up in low yield. These bioactive compounds are mainly supplied for detailed in vivo and clinical trails by extracting plants and marine organisms. This method of supply will damage our rainforests or coral reefs. Damaging rainforests also creates environmental problems. To overcome all these problems, new biotechnological methods should be discovered. A lot of active research is going on in the area of tissue cultures, plant cell cultures and biosynthesis of natural products. Studies on biosynthesis of natural products provide information about enzymology of plants involved in synthesizing potent bioactive natural products. These biosynthesis experiments can be used as an assay to purify key enzymes involved in the synthesis of bioactive natural products. Cloning of these enzymes in a suitable vector, commonly used Escherichia coli, can over express them to use in the synthesis of bioactive natural products by incubating suitable precursors. These enzymatic reactions would help to overcome the synthesis of chiral centers as enzymes perform stereospecific reactions. These enzymatic reaction would also be environmentally safe. For instance, homoisoflavonoids, 9 and 10, exhibit potent anti-GST activity. The biosynthesis of 9 and 10 might involve tyrosine, acetate and methionine to give 2’methoxychalcone (118) that undergoes cyclization to afford 3benzylchroman-4-one (119) [54-56]. Hydroxylation of 119 might yield compounds 9 [56]. Methionine might have methylated the C-6 hydroxyl group to afford 10. Purification and cloning of enzymes involved in the biosynthesis of intermediate 119 in E. coli may help in devising biotech method to produce 9 and 10. Incubation of tyrosine, malonyl CoA, acetate and methionine with this particular E. coli strain would provide the homoisoflavonoids skeleton. This skeleton can then be subjected to microbial hydroxylation reaction to produce compounds 9 and 10 in the lab. A possible biosynthesis of compound 9 and 10 is shown in Fig. 1. Similarly, biosynthetic origin of other bioactive compounds needs to be elucidated to establish biotech methods to produce these bioactive natural products.


Athar Ata

O OH

S

HO Malonyl CoA

HOOC

NH2

CH3

OCH3

NH2 HO

Acetate

O

OH

(118)

OH O

OH

Hydroxylation

HO O (9)

O

HO

OH O

O S

HO

OH

(119)

CH3

NH2 OH O

OH

H3CO O

(10)

OH

Fig. 1. Biosynthesis of homoisoflavonoids

Figure 1: Biosynthesis of homoisoflavoniods.

In summary, natural products have shown potential in the area of enzyme inhibitors and this area is still under-populated. In this area, sometime, moderately active enzyme inhibitors are also discovered. These moderately bioactive natural products can be used as a template to synthesize unnatural analogues with improved bioactivity. Another important aspect in the drug discovery area is to determine the active pharmacophore by studying the structure-activity relationships of bioactive natural products. This would provide a rational in designing new lead drug molecules against that particular enzyme. For instance, a very careful look at the structures of compounds 16-21, 26-27 and 29 indicate that , -unsaturated carbonyl functionality is common to all of these natural products. This means that -unsaturated carbonyl group is required to have GST inhibitory activity. This suggests that glutathione makes adduct with these natural products via Michael addition reaction in the presence of GST to lower the reactivity of glutathione. It provides a rational to screen all other natural products having , -unsaturated carbonyl group incorporated in their structures for their anti-GST potentials. Additionally, it is also observed that for AChE inhibition activity, alkaloids have shown more potential than non-nitrogenous natural products. This area of discovering new natural enzyme inhibitors remains unexplored and needs an active research in this important area of drug discovery program. 6. ACKNOWLEDGEMENTS I would like to thank all of my undergraduate and graduate students who were involved in discovering GST and AChE inhibitors and their names are cited in the references. Funding provided by the University of Winnipeg and Natural Sciences and Engineering Research Council of Canada is gratefully acknowledged. I am also grateful to the Department of Chemistry, The University of Manitoba for my appointment as adjunct professor. REFERENCES [1] [2] [3]

McChesney JD, Venkataraman SK, Henri JT. Plant natural products: Back to the future or into extinction? Phytochemistry 2007; 68: 2015-22. Newman DJ, Cragg GM. Natural products as source of new drugs over the last 25 years. J Nat Prod 2007; 70: 461-77. Newman DJ, Craig GM, Sanderb KM. The influence of natural products upon drug discovery. Nat Prod Rep 2000; 17: 215-34.


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CHAPTER 5 Production of Anthocyanins by Plant Cell and Tissue Culture Strategies Claudia Simões*, Norma Albarello, Tatiana C. de Castro and Elisabeth Mansur Núcleo de Biotecnologia Vegetal, Universidade do Estado do Rio de Janeiro, Brazil 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, largescale 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: [email protected] Ilkay Erdogan Orhan (Ed) All rights reserved-© 2012 Bentham Science Publishers


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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, cranberry, elderberry, raspberry seeds, and strawberry

Twenty different combinations of six berry extracts

[32]

Anti-angiogenic

Eggplant peels (Solanum melongena, L. var. esculentum, Ness.)

Nasunin (delphinidin-3-(p-coumaroylrutinoside)-5-glucoside)

[49]

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; petunidin-3, 5-diglucoside; delphinidin-3-glucoside; malvidin-3, 5-diglucoside; peonidin-3, 5-diglucoside; cyanidin-3-glucoside:petunidin-3-glucoside; peonidin-3glucoside; malvidin-3-glucoside

[51]

Anti-carcinogenic

Sweet potato (Ipomoea batatas L.)

Anthocyanin-rich aqueous extracts from in vitro produced tissue

[52]

Anti-carcinogenic

Grape rinds and red glutinous rice

Anthocyanidins

[53]

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 melanogena)

Anthocyanin: delphinidin

[37]

Anti-inflammatory

Tart cherries

Aglycone: cyanidin

[41]

Anti-inflammatory

Saskatoon berries (Amelanchier alnifolia Nutt.), blueberries, blackberries, and black currants

Anthocyanins/anthocyanidins: pelargonidin, cyanidin, delphinidin, peonidin, malvidin, malvidin, 3-glucoside, and malvidin 3, 5-diglucosides and anthocyanin-rich crude extracts and concentrates of selected berries

[43]

Antimutagenic

Sweet potato Ayamurasaki variety

Anthocyanin derivatives deacylated: 3-sophoroside-5-glucoside of cyanidin and 3sophoroside-5-glucoside of peonidin

[57]

Antimutagenic

Sweet potato Ayamurasaki variety

Anthocyanins: 3-(6, 6’-caffeylferulylsophoroside)-5-glucoside of cyanidin and 3-(6, 6’caffeylferulylsophoroside)-5-glucoside of peonidin

[35]

Antioxidant

Italian red wine


[58]

Antioxidant

Wild blueberry, bilberry, cranberry, elderberry, raspberry seeds, and strawberry

Twenty different combinations of six berry extracts

[32]

Antioxidant

Flowers of Hibiscus sabdariffa L.

Hibiscus pigments

[59]

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 and delphinidin 3-O--D-glucoside; and their aglycons: pelargonidin chloride, cyaniding chloride, and delphinidin chloride

[62]

Antioxidant

Petals of roselle (Hibiscus Sabdariffa L.)

Delphinidin-3-sambubiose

[63]

Antioxidant

Sweet potato (Ipomoea batatas L.)

Anthocyanin-rich aqueous extracts from in vitro produced tissue

[52]

Antioxidant

Red wine type Cabernet

Anthocyanin dye

[44]

Antiulcer

Vaccinium myrtillus

Anthocyanosides

[64]

Bacteriostatic

Malva sylvestris


[65]

Cardiovascular protection

Cranberry

Cranberry extracts

[66]

Decrease lipid peroxidation

Phaseolus vulgaris L.

Anthocyanin pigments: pelargonidin 3-O--D-glucoside, cyanidin 3-O--D-glucoside and delphinidin 3-O--D-glucoside; and their aglycons: pelargonidin chloride, cyaniding chloride, and delphinidin chloride

[62]

DNA triplex stabilization property

Grape (Vitis vinifera), Rose (Rosa gallica), Malva (Malva sylvestris)

Anthocyanins: 3-O-β-D -glucopyranoside of malvidin, peonidin, delphinidin, petunidin and cyanidin

[67]

DNA-damaging protection

-

Delphinidin, delphinidin-3-glycoside, delphinidin-3-rutinoside, cyanidin, cyanidin-3glycoside, cyanidin-3-rutinoside

[36]

Hypoglycemic

Red wine type Cabernet

Anthocyanin dye

[44]

Protective against atherosclerosis

Cranberry

Cranberry extracts

[66]

Hepatoprotector

Petals of H. rosasinensis

Cyanididin derivative

[68]

Hepatoprotector

Purple-colored sweetpotato

Crude anthocyanin extracts

[69 ]

Protective effect against pancreatitis

Aronia melanocarpa

Anthocyanin dyes

[70]

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


Simões et al.

factors affecting the stability of anthocyanins in plants, along with problems on the availability of raw material producing pigments at adequate quality and quantities, hinder the commercial use of these substances. Thus, studies concerning the development of new technologies for anthocyanin induction and/or optimization have attracted great attention. Plant cell and tissue culture strategies are an attractive alternative to the use of whole plants for the production of high-value secondary metabolites and have been the subject of extended research in the last decades. The ability control both physical and chemical conditions allows the establishment of methodologies that result in increased production of plant metabolites or even in the synthesis of new compounds [72, 73]. An important requirement for large-scale production of secondary metabolites under in vitro conditions is the understanding of their metabolic pathways. In this context, the anthocyanins are highlighted, since their biosynthetic pathway is one of the most studied. The first results on in vitro production of anthocyanins were published in the 1960’s [74-76] and were previously reviewed by Seitz and Hinderer [77] and Zhang and Furusaki [78]. Several studies reports that the anthocyanin production in callus cultures is first characterized by the appearance of reddish-pink spots in the callus surface. The mechanical isolation and repeated subcultures of the cells forming these pigmented spots can lead to a progressive increase in pigment induction, as observed in callus cultures of Cleome rosea (Fig. 1) [79].

Figure 1: Anthocyanin production in callus culture of Cleome rosea. A) Increased production of anthocyanins during six (a), seven (b), eight (c) and nine (d) months in culture; B) Anthocyanin-producer cell line already established, showing a homogeneous pigmentation. Bar: B = 0.80 cm.

In most in vitro culture systems, biomass accumulation and secondary metabolite production require different media conditions to induce a shift from the growth state to the metabolite production state, thereby limiting the efficiency of these systems for commercial used. Therefore, much effort has been done in



developing in vitro systems mainly based on callus and cell suspension cultures that provide high biomass accumulation and metabolite production in a single-stage culture regime. An efficient example of high production cell lines were achieved in the establishment of callus and cell suspension cultures of Cleome rosea [79]. In general, biotechnological studies have explored the influence of various factors on the anthocyanins production and many methodologies have been applied in order to increase their productivity. 3.1. Modulation of Anthocyanin Production 3.1.1. Physical Environment It is well known that the biosynthesis of anthocyanins in plant tissues either requires light or is enhanced by it. In general, anthocyanin accumulation occurs as a response from prolonged exposure to red and far-red light mediated by phytochrome, while responses to blue and UV-light are mediated by cryptochrome and/or a UV-B photoreceptors [71]. The light effect is expressed in the activation of different enzymes involved in anthocyanin biosynthesis, especially phenylalanine ammonia-lyase (PAL), a key enzyme in the pathway [80, 81] and chalcone synthase (CHS). Nakatsuka et al. [82] demonstrated that light-induced anthocyanin accumulation is regulated through activation of transcription factors. Several studies have demonstrated that light irradiation has also a significant influence in anthocyanin production in in vitro cultures [80, 81, 83-93]. The regulation of enzymes involved in anthocyanin biosynthesis in response to UV light was also analyzed [94]. Continuous UV irradiation leads to a transient induction of the catalytic activity of the enzymes and the induction of pigment formation in response to irradiation plays a role in protecting the cells from the adverse effects of UV light. Significant enhancement of anthocyanin accumulation was achieved in carrot cell cultures continuously treated with UV-A light [95]. Although light is considered an important controlling agent in anthocyanin biosynthesis, dark-acclimated anthocyanin producing cells have been established from some plant species [96-106]. The development of dark-producing cell lines is economically profitable, albeit cultures establish under dark condition may be very unstable and are prone to necrosis and death [107]. Although temperature is not frequently considered while developing protocols for callus cultures, this parameter has been proved to be an important environmental factor for in vitro anthocyanin production and seems to be species-dependent. In callus cultures of Cleome rosea, the highest rate of pigment production was obtained at 24±2ºC, while higher temperatures resulted in callus browning [79]. This was probably due to the hydrolysis of glycosidic bonds by glucosidases, resulting in anthocyanin degradation and formation of brown condensation products [108]. On the other hand, in callus cultures of Daucus carota the highest anthocyanin production was achieved at 30ºC when compared to lower temperatures [109]. Temperature also affected significantly the cyanidin:peonidin ratio in cell suspension cultures of Ipomoea batatas causing suppression of peonidin-based pigments accumulation at low temperatures [110]. Although lower temperatures seem to be more suitable to anthocyanin accumulation, they can reduce cell growth. To further improve both anthocyanin yield and biomass accumulation in suspension cultures of strawberry cells, Zhang and Furusaki [78] proposed a two-stage temperature-shift, where a first stage is established in higher temperature to improve cell growth and a second stage is performed in low temperature to induce anthocyanin production. The accumulation of anthocyanins in cell cultures is also influence by medium pH. Strawberry cultures submitted to a large range of pH (3.7 - 8.7) presented the highest anthocyanin accumulation (143 mg L-1) at pH 8.7 after nine days in culture [111]. The alkalinization of the culture medium can be a consequence of the cytoplasmic acidification through a mechanism of reduction in proton extrusion. High pH levels in the culture medium could increase phenylalanine ammonia-lyase (PAL) activity and consequent anthocyanin accumulation [77, 112].


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3.1.2. Chemical Factors In addition to physical factors, modifications on the nutrient composition of the plant cell culture medium are an efficient strategy to induce secondary metabolites production under in vitro conditions [72]. Hence, various chemical parameters have been studied in order to increase the production of anthocyanins in cell cultures. For example, nature and concentration of the sugar added to the culture medium have pronounced effects on pigment accumulation. Although sugars are mainly used as carbon source, they also act as osmotic agent when present at high concentrations. Furthermore, sugars represent not only energy sources and structural components, but also physiological signals regulating the expression of a variety of genes [113]. The mechanisms underlying the control of anthocyanin accumulation under sugar influence involves several components of general signal transduction pathways such as Ca2+, calmodulin and protein kinases/phosphatases [114]. Sucrose is the most frequently sugar used in in vitro cultures and its influence on anthocyanin production has been widely reported in the literature [79, 90, 96, 115-121]. However, the influence of other sugars has also been evaluated. Compared to other sugars, frutose-containing medium was the most efficient to anthocyanin accumulation in callus cultures of Aralia cordata [100], Hibiscus subdariffa [122] and strawberry cells [118]. A mutant cell line of carrot showed anthocyanin production in the presence of lactose [123], while the supplementation with manitol in association with sucrose resulted in a significant increase in anthocyanins in callus cultures of Daucus carota [124]. Nutritional restriction is an efficient strategy to induce anthocyanin synthesis in in vitro cultures. In general, the depletion of some nutrients leads to enhancement of secondary metabolites, but with growth limitations [108]. In plants, many nutrient deficiencies, especially nitrogen, phosphorus and sulfur, are accompanied by anthocyanin accumulation as a strategy used by plants to avoid the over-accumulation of carbohydrates in tissues in order to prevent physiological disorders [125]. Nitrogen source as well as the ratio of ammonium (NH4+) to nitrate (NO3-) has also been shown to markedly affect the production of anthocyanins by plant cell cultures. The basal culture medium MS [126] supplemented with a total nitrogen molar concentration at 70 mM sustained a maximum anthocyanin accumulation in callus cultures of Daucus carota [120]. On the other hand, best production was achieved with 60 mM in Vitis vinifera [96] and with 30 mM with Euphorbia millii [116], Aralia cordata [100] and strawberry cells [118]. High levels of anthocyanin were achieved in cell cultures of Aralia cordata maintained in modified MS medium with a 1:4 molar ratio of NH4+ to NO3-, instead of the standard ratio of 1:2 [100]. Similar results were achieved in callus cultures of strawberry [90], Daucus carota [120] and Cleome rosea [79]. However, the increase of ammonium concentration in cell suspension cultures led to a decrease on the accumulation of anthocyanin in Vitis vinifera [96, 127], Daucus carota [128] and Euphorbia millii [116]. Changes in anthocyanin composition influenced by ammonium level in culture medium were detected in cell suspension cultures of Ipomoea batatas, which presented an inhibition of acylation in the presence of high ammonium concentration [129]. According to Hirasuna et al. [117] the up-regulation of anthocyanin production may be due to the involvement of a nitrate sensitive tonoplast ATPase in the accumulation of these pigments in vacuoles. In addition to nitrogen, the manipulation of phosphate concentration has also been used to increase anthocyanin content. Callus cultures of Daucus carota maintained on MS medium under reduced levels of phosphate showed an increase in anthocyanin content up to 7.2% when compared to cultures maintained on full strength MS medium [124]. High anthocyanin accumulation under phosphate deprivation was also achieved in cell suspensions of Vitis vinifera [130, 131]. The effects of plant growth regulators on anthocyanin induction are apparently variable. Callus cultures of Glehnia littoralis maintained in the presence of NAA achieved almost a two-fold increase in anthocyanin content as compared to those obtained in the presence of 2, 4-D or IAA [101]. On the other hand, NAA repressed anthocyanin production in callus cultures of Oxalis linearis [132]. In cell cultures of Camptotheca acuminata, anthocyanin content was significantly greater in the presence of kinetin (KIN),



compared to benzyladenine (BAP) [121]. The maximum productivity of anthocyanin in callus of Daucus carota was observed in the presence of NAA and KIN [109]. Although some authors have suggested that 2, 4-D inhibits the production of a wide range of secondary metabolites, including anthocyanins [108, 133], culture medium supplementation with this growth regulator proved to be essential to support biomass increase as well as high anthocyanin production in callus of several species, such as Fragaria ananassa [134], Ipomoea batatas [106], Daucus carota [135] and Cleome rosea [79]. The regulation of anthocyanin synthesis by 2, 4-D was evaluated by Ozeki [136] in cell suspension cultures of carrot. This work indicated that there are two phenylalanine ammonia-lyase (PAL) genes. One of them is specifically induced in the presence of 2, 4-D, while the other is transient and rapidly activated by stress conditions. The increase on anthocyanin production by interaction between growth regulators and light has been well demonstrated in callus cultures of Aralia cordata. While cells cultured under light conditions achieved high pigment production on medium supplemented with 2, 4-D, the use of NAA was superior for cultures maintained in the dark [100]. Growth regulators supplementation has also been used to induce anthocyanins in organ cultures. Adventitious root cultures of Raphanus sativus cultivated in MS liquid medium produced anthocyanin in the presence of NAA or IBA and a significant increase on pigment accumulation was achieved when the cultures were maintained on medium supplemented with 0.5 mg L-1 IBA [137]. Although the manipulation of individual environmental stimuli has been effective for anthocyanins induction, one of the most efficient strategies for further increase in their biosynthesis level is to develop an integrated process that rationally combines the effects of various system and enhancement strategies. For example, in callus cultures of Cleome rosea an optimized medium formulation was established on halfstrength MS medium (MS1/2) containing 1:4 ratio of NH4+ to NO3-, 70 g L-1 sucrose and supplementation with 0.90 µM 2, 4-D, which were previously determined individually. Pigmented calluses transferred to this formulation maintained a high biomass accumulation and presented 150% increase in the anthocyanin content when compared to the original MS medium used to induce the pigment production [79]. The use of an optimized medium on Camptotheca acuminata cell cultures, that consists of Gamborg medium [138] added with 292 mM sucrose and supplemented with 2 M kinetin and 2 M 2, 4-D was also efficient to increase anthocyanin accumulation [121]. Another important aspect related to the use of in vitro technologies is the production of anthocyanins that usually are not found in field-grown plants. For example, callus cultures from Vitis sp. produced cyanidin and peonidin, whereas the intact pericarp contained malvidin and peonidin [139]. Differences in anthocyanin composition under in vivo and in vitro conditions were also reported by Mizukami et al. [140] in roselle and by Mori et al. [141] in strawberry. In callus cultures derived from stems tissues of Cleome rosea obtained in medium supplemented with 2, 4-D, two penidins were identified, while in stems only cyanidins were detected [79]. The presence of peonidin in extracts from callus of C. rosea, but not from field-grown plants, could be related to bioconversion of cyanidin to peonidin by methylation of the B-ring of the aglycon. In strawberry cell cultures, reduction in 2, 4-D concentration enhanced the methylation of anthocyanins and increased the level of peonidin-3-glucoside [134]. In addition to qualitative analysis, the stability of anthocyanin composition under in vitro condition was evaluated by Callebaut et al. [102] in Ajuga reptans callus and cell suspension cultures during a time span of 5 years. Although no new anthocyanin classes appeared, there were quantitative differences among the pigments produced, with a decrease in delphinidin-based anthocyanins with time in culture. 3.1.3. Elicitation Despite the undeniable value of plants as sources of substances of great commercial value, the production of plant secondary metabolites by in vitro cultures is still facing many limitations, mainly due to the low yield. Although considerable efforts have been made in order to increase the commercial application of cell and tissues cultures, only the production of the red naphthoquinone pigment shikonin by Lithospermum erythrorthizon cultures [142], the red anthraquinone pigment purpurina by Rubia akane [143] and the


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terpenoid taxol by Taxus cell cultures [144] were successfully industrialized for commercial application so far. A useful biotechnological tool applied to improve the production of secondary metabolites under in vitro condition is elicitation. Elicitors are chemicals or biofactors from various sources that can trigger a response in living organisms resulting in accumulation of secondary metabolites. They can be abiotic such as metal ions, inorganic compounds and UV irradiation, or biotic, obtained from fungi, bacteria or viruses [145, 146]. Elicitation has been efficiently applied to anthocyanin production. The elicitors more frequently used are culture filtrates and cell extracts of fungi or bacteria [86, 147, 148], methyl jasmonate [149-153], jasmonic acid [154-156], salicylic acid [152, 157] and inorganic ions [148, 150]. The enhancement in anthocyanin synthesis was also reported in the presence of phycocyanin [104], fluridone [158], ethylene [159], β-glucan [150], chitosan [150], riboflavin [160] and exposition to UV light [86]. The efficiency of the elicitation process depends on the plant material, contact time and elicitor concentration. The simultaneous use of elicitation by jasmonic acid and light irradiation in Vitis vinifera suspension cultures resulted in a significant synergistic enhancement of anthocyanin accumulation, showing the potential of integrated processes on anthocyanin accumulation under in vitro condition [154]. The influence of different elicitors on biomass production and anthocyanin biosynthesis on non-morfogenic calluses of Vitis vinifera was investigated by Mihai et al. [161]. In this work the supplementation with 10 M jasmonic acid positively influenced anthocyanin yield but not cell proliferation. On the other hand, salicylic acid (20 μM) had a positive effect on the biosynthetic capacity of the calluses. In cell suspension cultures of Ipomoea batatas the addition of methyl jasmonate induced significant changes in the composition of anthocyanin pigments, promoting the biosynthesis of anthocyanins with more complex molecular structures, including di-acylated compounds [153]. The elicitation of suspension cultures of Solanum tuberosum with cerium promoted culture growth, increased the accumulation of anthocyanins, and enhanced the expression of five anthocyanin biosynthetic genes: chalcone synthase (CHS), flavanone 3hydroxylase (F3H), flavonoid 3', 5'-hydroxylase (F3'5'H), dihydroflavonol 4-reductase (DFR) and flavonoid 3-0-glucosyltransferase (3GT) [162]. 3.1.4. Conditioned Medium Conditioned medium (CM), i.e. medium filtered from growing cultures, have been used to stimulate cell growth and accumulation of metabolites in plant cell and tissue cultures. The effect of CM is attributed to the production and release of growth factors into the culture medium, the so-called conditioning factors that are necessary for growth and cell division [163]. The first study related to anthocyanin accumulation using CM was performed by Mori and Sakurai [164] in strawberry cell suspension cultures. After evaluating the effect of different concentrations of CM on anthocyanin accumulation and composition, the authors observed a great enhance on pigment accumulation, with an 8-fold increase at 100% CM and also significant changes in the content of the two major anthocyanins (cyanidin-3-glucoside and peonidin-3glucoside) present in these cultures. The production of anthocyanins in these cultures was also strongly influenced by culture duration and concentrations of CM [165, 166]. Anthocyanin accumulation was investigated using CM obtained from different plant species. For example, a CM prepared from suspension cultures of strawberry induced anthocyanin production and accumulation in cell culture of rose (Rosa hybrida sp.), which did not produce anthocyanin [167]. On the other hand, CM prepared from anthocyanin producing cultures of rose as well as from grape cells were used to stimulate anthocyanin synthesis of strawberry cells [168]. Sakurai and Mori [169] tried to elucidate the mechanism involved in CM action by evaluating the changes of major mineral and sugar composition presented in the CM derived from strawberry cultures. Their findings suggested that the stimulation of anthocyanin accumulation was caused by some substance(s) released from cells during culture. The same authors using dialysis membranes to separate the CM



constituents demonstrated that a fraction of molecular weight smaller than 10, 000 Da induced a significant increase in anthocyanin synthesis, indicating that some substance(s) stimulating anthocyanin accumulation exist in CM [170]. In addition, Mori et al. [171] demonstrated that the phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) activities as well as CHS transcript levels were significantly increased in the CM-cultured cells when compared to transcript abundance in synthetic media-cultured cells. 3.1.5. Precursor Feeding Precursor feeding is also considered an efficient approach to increase secondary metabolite production in plant cell cultures, since supplementation of the culture medium with an intermediate, of a biosynthetic route, stands a good chance of increasing the yield of the final product [73]. This strategy has been applied for enhancement of anthocyanin production in cell cultures from different species [97, 172-177]. Among the most used precursors for anthocyanin synthesis are derivatives of cinnamic acid, such as the sinapic acid and L-phenylalanine (Phe). Compared to other precursors in the pathway, Phe is relatively inexpensive and more effective for anthocyanin accumulation [178]. Kakegawa et al. [179] suggested that the intracellular Phe is used as a biosynthetic precursor material for anthocyanin production and related flavonoids, and at the same time, has a function as a signal that promotes the transcription of the genes that code for enzymes involved in the anthocyanin biosynthetic pathway, such as the phenylalanine ammonialyase (PAL) and chalcone synthase (CHS). An increase of 81% in anthocyanin production was achieved in suspension cultures of strawberry cells by repetitive feeding with Phe [177]. In addition to increasing anthocyanin productivity, precursor feeding can also result in the induction of novel anthocyanins, as observed in carrot cell cultures supplemented with cinnamic and benzoic acid analogues, which showed the production of fourteen novel monoacylated anthocyanins [175]. 3.1.6. Growth Culture Phases and Cell Aggregation An important aspect to be considered when cell cultures are used as producers of anthocyanins is the influence of the growth culture phases as well as the degree of cell aggregation on pigment production. In many cell cultures, secondary metabolite production is enhanced by nutrient depletion and usually begins when the cultures moves from the exponential into the stationary phase, as observed in cell suspension cultures of Catharantus roseus [83, 180]. However, a decrease in anthocyanin production during the stationary phase was observed in Cleome rosea [181], strawberry [134] and Vitis vinifera [154] cell cultures, suggesting that the substrate supply is an important factor in the biosynthesis of these pigments in in vitro cultures. Moreover, the production of other secondary metabolites during the stationary phase could inhibit anthocyanin biosynthesis. Another important aspect to be considered is the cell aggregation, which is strongly influenced by several parameters and often determines the profile of metabolite production. It is well known that a degree of cell aggregation in cell suspension cultures influence secondary metabolite production [182, 183]. Anthocyanin content in suspension cultures of Daucus carota showed an increase in cell aggregation size up to 500-850 µm, but with a significantly decrease when aggregate reached higher diameters [184]. Increased aggregate sizes can cause lack of light at the aggregate core, restriction of oxygen supply, differential exposure to the microenvironmental factors and local concentration gradients that might alter the cell growth and pigment production [88, 185]. 3.1.7. Molecular Biology Molecular aspects of anthocyanin biosynthesis have been extensively studied, and genes encoding almost all the enzymes involved in anthocyanin pathway have been cloned. Characterization of anthocyanin mutants in a variety of plant species has led to the identification of genes that encode not only enzymes of the anthocyanin biosynthetic pathways, but also of regulatory elements that confer tissue-specific accumulation of anthocyanin [186-193]. Anthocyanin genes have been used extensively in transformation experiments in basic and applied studies. In addition, anthocyanin pigmentation could also be used as a marker in nondestructive cell-specific assays, both in transgenic and transient assay systems [194-199].


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Differential transcript abundance between unpigmented and pigmented cell lines of Daucus carota was investigated by Marshall et al. [200]. Nineteen partial cDNA clones were isolated and used in northern analysis, confirming that seven of the corresponding genes were preferentially expressed in a particular cell line. Anthocyanin biosynthesis was also evaluated through gene silencing techniques. In a transgenic red Malus hybrid, anthocyanin biosynthesis was almost completely blocked by silencing the enzyme anthocyanidin synthase, a key enzyme in the pathway that performs the penultimate step in anthocyanin biosynthesis [201]. A field of great interest is metabolic engineering, which consists of a series of strategies that can be used to enhance or modify the production of secondary metabolites. Metabolic engineering approaches can involve decrease in the catabolism of a desired compound, enhancement of the expression or activity of a ratelimiting enzyme, prevention of feedback inhibition of a key enzyme, decrease in the flux through competitive pathways, enhancement of expression or activity of all genes involved in the pathway or compartmentalization of the desired compound or the conversion of an existing product into a new product [202]. Despite the great potential use of metabolic engineering in the production of secondary metabolites, there have been few successes in modifying pathways to form compounds with commercial interest. The best results were achieved by modifying anthocyanin pathway leading to changes in flower colors [203206]. The accumulation of polyacylated anthocyanins in flowers of transgenic plants may serve as an efficient strategy for the development of blue flowers [207, 208]. 4. PERSPECTIVES OF COMMERCIAL EXPLOITATION The commercial interest on athocyanins produced through plant cell and tissue cultures strategies may be evaluated considering the number of patent applications related to this subject, as summarized in Table 2. However, despite the great number of studies and promising systems developed, the commercial exploitation of anthocyanins produced under in vitro conditions needs to overcome problems such as the maintenance of a high and constant production. The use of large-scale liquid cultures and automation has the potential to resolve the manual handling of the various stages of in vitro culture and decreases production cost significantly. A variety of bioreactor types providing growth and expression of bioactive substances is available today [209]. The in vitro cell production of anthocyanins using bioreactors is reported by some authors, although economic feasibility has not been achieved to date [185, 210-214]. Table 2: Patent applications related to anthocyanins production by plant cell and tissue cultures strategies. Patent Publication Year/Number

Plant Material

Assignee

2007/BR200703625

Cleome rosea

Rio de Janeiro State University

2006/ KR20060065221

Rice

Lee, SH

2006/JP2006067945

Maize

Saneigen FFI KK

2006/KR2006065221

Black rice

Kwank, SH; Jeon, Y; Lee, SH

2003/EP1329500

Panax sikkimensis

Council Scient. Ind. Res. (India)

2002/US6368860

Panax sikkimensis


2001/JP2001000177

Sweet potato

Kyushu Natl. Agricultural Exper.

2001/JP2001000062

Sweet potato

Kyushu Natl. Agricultural Exper.

2001/JP2001275694

Strawberry

Niigata Prefecture

2001/JP2001275660

Panax sikkimensis


1999/JP11018798

Strawberry

Ishikawajima Harima Heavy Ind.

1997/JP9308496

Strawberry


1996/JP8070883

Strawberry


1995/JP184679

Strawberry

Tokyo Gas Co. Ltd.

1995/JP72274992

Strawberry


1995/JP7313182

Strawberry



Biotechnological Production of Plant Secondary Metabolites 77 Table 2: cont….

1994/JP6133792

Statice


1994/JP06007185

Strawberry


1993/JP5184380

Strawberry


1993/JP5103685

Buckwheat

Japan Stell works Ltd.

1993/JP5153994

Strawberry


1993/JP5153995

Strawberry


1993/JP5153996

Strawberry


1993/JP05268979

Rosmarinus officinalis

Kondo, T

1992/JP4058894

Strawberry


1992/JP4365498

Aralia cordata

PCC Technology

1991/US5039536

Daucus carota

International Genetic Science Partnership

1991/KR9107823

ACG-1 KCTC 8473P

Korea Ginseng & Tob. Res. Institute

1991/JP03269060

Malus sp.

Takeda Chemical Ind.

1991/KR9100455

Vitis sp.

Lotte Confectionary

1991/JP3183493

Strawberry


1991/JP5007467

Western madder

Sanei Chemicals Ind. Ltd.

1990/JP02222691

Chrysanthemum coronarium

Nippon-Kayaku

1990/WO9000193

Petunia hybrida

Plantoma

1990/JP02265475

Raphanus sativus

Nippon Shokubai Kagaku Kogyo Co. Ltd.

1990/JP02276580

Safflower

Mitsui Eng. and Shipbuild Co. Ltd.

1990/JP02276581

Safflower


1990/JP02276582

Safflower


1989/JP64002593

Perilla frutescens

Pokka

1898/JP01030594

Hibiscus sabdariffa

Sekisui Plastics Co. Ltd.

1988/JP63199766

Safflower

Kibun K. K.

1988/JP63233993

Ipomoea batatas

Nitto Electric Ind. Co. Ltd.

1988/JP63240795

Euphorbia millii

Nippo Paint Co. Ltd.

1987/EP249772

Daucus carota

CPC International

1987/JP62181796

Brassicaceae

Sanei Chemicals Ind. Ltd.

1986/BE904804

Daucus sp.

International Genetic Sciences

1986/JP61019493

Camelia sp.

Pias Corporation

1986/GB2175913

Daucus carota

International Genetic Sciences

1979/JP54011281

Derris

Mitsui Petrochem. Ind. Co. Ltd.

1974/JP49094897

Mallotus japonicus

Nihon Shinyaku Co. Ltd.

Adapted and updated from Zhang and Furusaki [78].

Another important aspect regarding the commercial exploitation of in vitro systems is the maintenance of the productivity over time, which can be changed depending on the inherent genetic and epigenetic instability of plant cell cultures [215, 216]. Hence, the application of in vitro conservation strategies such as cryopreservation allows the maintenance of chosen cell lines protecting them against somaclonal variation during the storage period. This methodology was successfully applied to anthocyanin producing cells of Vaccinium pahalae that were criopreserved through the encapsulation-dehydration method, maintaining the capacity to produce anthocyanins [217]. The great number of biotechnological strategies that have been applied to in vitro production of anthocyanins, as described in this chapter, show the increased interest in the establishment of a scale-up production of these pigments. Considering the marketing selling price, the limited number of effective


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sources for anthocyanin extraction, and the economical interest as both, a natural dye and medicinal application, the development of new technologies to facilitate the success of commercial exploitation of anthocyanins using plant cell, tissue and organ cultures strategies is an attractive alternative to conventional extraction procedures, besides allowing the selection of anthocyanins with desirable application properties. REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11]

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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

Departamento de Biotecnología, Centro de Desarrollo de Productos Bióticos, Instituto Politécnico 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 98% accuracy. Most of the plant sexspecific molecular markers used rely on identifying male genotypes [83]. However, in some species, there exist female-sex specific genes. The cytokinin trans-zeatin (t-Z) was correlated with female sex in annual mercury [84]. The female-specific coding DNA fragment of a t-ZR β-ribosidase was cloned. Hirotoshi [85] utilized the isozyme technique to identify jojoba plants sex where peroxides and esterase proved to be sex markers. Agrawal et al. [86] tested 72 RAPD primers where only one was reported to amplify a 1, 400 base pair (bp) DNA fragment unique to male plants, which may be used as a sex identification RAPD marker for jojoba. Inter-simple sequence repeats (ISSR) marker-assisted selection for the early detection of male and female jojoba plants was employed by Sharma et al. [87]. Only one ISSR primer out of 42 examined amplified a 1200 bp in the males only. Agarwal et al. [88] reported two combinations of sex-linked amplified fragment polymorphism (AFLP) markers in jojoba male plants, a 525 bp fragment with the primer combination EcoRIGC/Msel-GCG and 325 bp with the primer combination EcoRI-TAC/Msel-GCG, respectively. They further identified a female-specific AFLP marker of 270 bp with the primer combination EcoRI-TAC/Msel-GCG. Gul [89] reported a male-specific marker using touch-down polymerase chain reaction. In our laboratory, the SCAR markers developed for papaya by Parasnis et al. [82] distinguished the male from the female jojoba plants, confirming the male-s pecific [90] markers reported by those authors [26]. We also examined several other SCAR and RAPD markers developed for other species where jojoba male-


Aly and Basarir

specific and female-specific markers were obtained. Furthermore, a marker was developed to distinguish male and female plants in one-step reaction. 7. GENE CLONING AND GENOMICS Clearly, jojoba has numerous important trades which can be further enhanced by jean cloning into selective jojoba lines or transformed into other plant specious. The obvious question would be the selection of the candidate gene (s) to clone. Wax esters are, as mentioned above, of considerable commercial importance and are produced on a scale of 3 million tons per year. The oil from the jojoba plant is the main biological source of wax esters. The fatty acid elongation (FAE) system in plants is thought to consist of four separable enzymes: β-ketoacyl-CoA syntheses (KCS), β-ketoacyl-CoA reeducates, β-hydroxyl-CoA dehydrate, and β-enoyl-CoA reductase. Of these four enzymatic steps, the first one, catalyzed by KCS, is thought to play a key role in the determination of the overall extent and rate of the elongation process [91, 92]. Finally, wax synthase (WS) transfers an acyl moiety from a second acyl-CoA to the fatty alcohol to form the wax molecule. In jojoba embryos, three key enzymatic activities are required for conversion of the CoA ester of oleic acid into wax. Fatty acyl-CoA reductase (FAR) carries out the reduction of acyl-CoA to yield fatty alcohols [93]. Cloning of a cDNA coding for KCS, from developing jojoba embryos, involved in microsomal fatty acid elongation was reported [91]. The gene was homologous to the cloned Arabidopsis fatty acid elongation1 (FAE1) gene that has been suggested to encode KCS. This gene was transformed into the low erucic acid rapeseed where the transgenic seed contained high levels of very long chain fatty acids. In another report, the introduction of KCS genes cloned from S. chinensis or B. napus into rapeseed mutants of low erucic acid showed complementation of the Canola fatty acid elongation mutation (fae) leading to the restoration of erucic acid synthesis in transgenic rapeseed [94]. Jojoba WS, cloned from developing jojoba embryos, in combination with jojoba fatty acyl-coA reductase (FAR) and a KCS from Lunaria annua (a plant that accumulates large amounts of very-long-chain fatty acids in its seed oil), was expressed with high levels of wax in transgenic Arabidopsis seeds [95]. Recently, cloning and functional characterization of an acyl-acyl carrier protein thioesterase (JcFATB1) from Jatropha curcas was achieved [96]. An extensive review of the different benefits of plant oils and enhancing plant seed oils for human nutrition utilizing gene cloning approaches was reported [97]. Heterologous wax ester biosynthesis was established in a recombinant Escherichia coli strain by coexpression of a fatty alcohol-producing bifunctional acyl-coenzyme A reductase from jojoba and a bacterial wax ester synthase from Acinetobacter baylyi strain ADP1, catalyzing the esterification of fatty alcohols and coenzyme A thioesters of fatty acids [98]. Jojoba oil-like wax esters such as palmityl oleate, palmityl palmitoleate, and oleyl oleate and fatty acid butyl esters were produced at up to ca. 1% of the cellular dry weight in the presence of oleate. This opens new perspectives for the biotechnological production of cheap jojoba oil equivalents from inexpensive resources employing recombinant microorganisms. Transfer of a wax synthesis pathway to a crop more amenable to large-scale agriculture may provide an economical source of a new feedstock for large-scale industrial applications such as biodegradable lubricants. Metabolic engineering of oilseed fatty acids has become possible and transgenic plant oils represent some of the first successes in design of modified plant products. Gene down-regulation strategies made possible the manipulation of specific fatty acids in several oilseed crops. Transferring novel fatty acid biosynthesis genes from noncommercial plants allows the production of novel oils in oilseed plants [99]. On the other hand jojoba can be a source of new genes which can be cloned, studies and utilized to improve other crops. Genes coding for salt tolerance can be discovered in jojoba which is a salinity tolerant plant. An 837 bp cDNA designated ScRab encoding a full length 200 amino acid long polypeptide was found to be homologous to the Rab subfamily of small GTP binding proteins. The fragment was isolated from shoot cultures of the salt tolerant jojoba, and cloned in E. coli, where the protein was expressed [100]. The same

Biotechnology Approaches and Economic Analysis


group isolated a cDNA fragment from salt stressed jojoba shoots chloroplasts post-transcription RNA [100]. They reported that salinity stress inhibited the post-transcriptional processing of chloroplast 16S rRNA. Jojoba cDNAs prepared from wounded parts of leaves under drying stress as a tester and cDNAs from unstressed parts of leaves as a driver, was used to identify genes regulated by wound–water stress. Suppression subtractive hybridization (SSH) was performed, resulting in 1344 clones as wound–water stress induced. Sequence analysis generated 385 unique ESTs (Expressed Sequence Tags), of which 139 ESTs in 13 main categories were annotated. Ninety-six genes were identified. These genes were found to be involved in 63 pathways. Some pathways, such as energy metabolism, lipid metabolism, amino acid metabolism, translation, and MAPK signaling pathway, are associated with wound–water stress. These results should prove of high value in understanding the genetic regulation process under wound–water stress in jojoba [101]. Jojoba proved to be susceptible to the soil born Agrobacterium rhizogenes which was utilized to induce root formation in jojoba [55]. This indicates that Agrobacterium can be utilized in gene transfer studies in jojoba cells and/or explants. This along with the fact that the particle accelerated bombardment technique can be utilized with numerous, almost all, plants and the availability of plant regeneration protocols from jojoba explants, emphasize that jojoba is amenable for gene transfer studies. 8. CONCLUSIONS Jojoba seed storage lipids are waxes rather than the triacylglycerols (TAG) found in other plants. The high price of jojoba oil limits its use to cosmetic applications. The above mentioned advances in jojoba breeding, propagation, tissue culture, gene cloning, metabolic engineering of oilseed fatty acids, genomics and proteomics should open great possibilities for jojoba cultivation and/or utilization. At the cellular level, micropropagation, cell selection, plant regeneration techniques are major areas that are still open for further research and development. At the molecular level, gene discovery and cloning in jojoba or other organisms, e.g. microorganisms, may provide significant advances. Obvious choices are genes coding for, or involved in the biosynthesis pathways of: 1) Fatty acylCoA: fatty alcohol acyltransferase (wax synthase, WS), 2) Specific fatty acids such as Omega-3, Omega-6 and others, according to the proposed industrial and pharmaceutical uses, 3) Simmondsin synthesis, 4) Biopesticides, 5) Bio-fuel, and 6) Sex determination genes. The cultivation of jojoba seems to be very profitable. However, the price of jojoba nuts fluctuates which may not be attractive for farmers to produce. The profit of the product is mainly coming from the after farm gate sales. The processing industry is taking a huge share of the profit. That is why; the industry of the product should be restructured to channel some of the profit to the farmers. Thus, as the profit of farmers increases the cultivation area of product and production amount will increase. A vertical integration might be a good alternative to handle the production of jojoba. Since the process will be managed by integrator from production to consumers, the distribution of profit to too many distribution channels will be prevented and more can be given to the producers. 9. ACKNOWLEDGEMENTS The authors are grateful to Mrs. Vidhya Anish for her assistance with the literature survey. REFERENCES [1] [2] [3]

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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 acidderived 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: [email protected] Ilkay Erdogan Orhan (Ed) All rights reserved-© 2012 Bentham Science Publishers

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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 nontargeted 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= 4hydroxycinnamoyl-CoA; CAD= cinnamoyl-alcohol dehydrogenase; CCR= cinnamoyl-CoA reductase; C3H= p-coumarate 3-hydroxylase; COMT= caffeoyl-CoA O-methyltransferase; F5H= ferulate 5hydroxylase; 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).


Phenylalanine


FLAVONOIDS ISOFLAVONOIDES ANTHOCYANINS STILBENES

PAL

p-coumaryl alcohol

Cinnamic acid CH4

CAD 4CL

p-coumaric acid

CCR p-coumaraldehyde

p-coumaroyl-COA HCT

p-coumaroyl shikimate

4CL

CH3

Caffeic acid

CH3

Caffeoyl shikimate

Caffeoyl-CoA

LIGNINS

COMT 4CL Ferulic acid

Feruloyl CoA

CCR Coniferyladehyde Coniferyl alcohol

F5H 5-hydroxy-ferulic acid

4CL

5-hydroxyferuloyl CoA

CCR

5-hydroxyconyferaladehyde

5-hydroxy-coniferyl alcohol

COMT

COMT Sinapic acid

CAD

Sinapaldehyde SAD/CAD SINAPOYL DERIVATIVES

COMT

Sinapyl alcohol

Figure 1: Biosynthetic pathway of phenol compounds from phenylalanine [5].

Most pesticides are applied together with adjuvants which enhance effects of pesticides. Triton X-100 used as adjuvant at 0.01% in 50 to 100 mL/L acifluorfen, increases the production of flavonoids in the soybean leaves [6]. Other pesticides, such as triazine, urea, amide, and carbamate classes are able to reduce the PAL synthesis in soybean seedlings (Table 2). Cultivation of soybean plants in a soil that was treated with higher concentration of pesticide initiates some kind of abiotic stress in plants triggering formation of phenolic compounds like isoflavones (genistein, daidzein), polyphenolic acids (elagic, tannic, and vanillic acids) and hydroxycinnamic acids (ferulic, phydroxybenzoic, and p-coumaric acids). For soybean, beyond a certain threshold concentration, the pesticide began to act as a growth inhibitor instead of growth promoter or regulator [11].



Table 1: Herbicides that increase the content of secondary metabolites in plants [2, 7-9]. Herbicide

Acifluorfen

Plant

Secondary Metabolites

Pisum sativum, pea

pisatin

Factor of Increase 19

Phaseolus sp.

phaseolin

> 47

Vicia faba, broad bean

medicarpina

> 45

Apium graveolens, celery

xanthotoxin

4

Glycine max, soybean

glyceollin I-III

>79

Chlomethoxyfen

Oryza sativa, rice

biphenyl-2-ol

3

Chlorsulfuron


anthocyans

1.77

Avena sativa, oat Glyphosate


phenols, total

1.43

gallate

9

glyceollin

2.22

protocatechuate

102

Table 2: Herbicides that diminish the content of secondary metabolites in plants [2, 7-10]. Herbicide

Plant

Secondary Metabolite

Factor of Reduction

Atrazine


anthocyanins

4

Buthidazol

Zea mays, maize

anthocyanins

4.3

Dinoterb

Pisum sativum, pea

flavonols

3.3

Glycine max, soybean;

anthocyanins

2

Phaseolus sp.

phaseolin

1.4-1.5

Glyphosate Metribuzin

Solanum tuberosum, potato

phenols, total

1.1-3.3

Sethoxydim

Rubus idaeus, raspberry

o-phenols

2

It is also known that the flavonoids accumulate when plants are exposed to diphenyl ether type of herbicides. Lactofen was found to cause isoflavone accumulation in the leaves of high-protein soybean cultivars through the generation of reactive oxygen species (ROS), thus the activity of this herbicide on plant polyphenols is a secondary effect resulting from photooxidative damage and ROS formation. The predominant isoflavones induced in cotyledon tissues are daidzein, formononetin, and glycitein aglycones, whereas daidzein, formononetin aglycones, and the malonyl-glucosyl conjugate of genistein exist in the leaves [12, 13]. Treatment of the wheat seedlings with the herbicide-safener cloquintocet mexyl causes a selective depletion in the content of flavone C-glycosides of luteolin, apigenin, and an accumulation of the methylated flavones; tricin along with ferulic acid. Changes in phenolic content were associated with an increase of Omethyltransferase and C-glucosyltransferase enzyme activity as well as the enhancement of detoxifying glutathione transferases. In addition to enhancing herbicide selectivity, safeners can also determine a shift in the metabolism of endogenous phenols [14]. Since phenylalanine is involved in tropane alkaloid biosynthesis, herbicidal treatment with glyphosate may have an inhibitory effect on the formation of the alkaloids such as hyoscyamine and scopolamine. Glyphosate and chlorsulfuron lead to a decrease of tropinone and tropine concentrations in overall tropane alkaloid biosynthesis in jimsonweed (Datura stramonium L.) treated with these herbicides. Certain wide-spectrum fungicides may be involved in the activation of some defensive responses of plants; such substances may influence the key steps of the phenolic and oxidative processes [15]. Production of phenols in the plants exposed to fungicidal spray is a response of those plants that both helps them to cope with the resulting chemical stress, but at the same time acts as protection against the growth of invaded pathogens by limiting it. It is presumed that the increase in total phenols may act as a prophylactic measure against pathogens before the invasion. For example; maneb, benomyl, and nabam induced the synthesis of hydroxyphaseollin in



soybean. Also, in maize, tetraconazole; a triazolic fungicide, affected the phenylpropanoid flavonoid biosynthesis by increasing the anthocyanin content [15, 16]. Application of the fungicide treatment with higher dosage of carbendazime (5.2 mM) was resulted in a decrease in dry weight and in all of the foliar pigments and nutrient levels in tobacco (Nicotiana tabacum L.). Therefore, the excessive application of carbendazim (5.2 mM) could be harmful for healthy plants, because of inhibiting the phenolic metabolism, such treatment would also sharply reduce the capacity of these plants to respond against pathogen attack [17]. The foliar application with carbendazim (2.6 mM) led to an increase in PAL activity and a foliar accumulation of phenols in tobacco (Nicotiana tabacum L. cv. Tennessee 86). The joint application of carbendazime (2.6 mM) and boron (8 mM) augmented biosynthesis and oxidation of the phenolic compounds, while carbendazim plus 32 mM or 64 mM boron reduced these processes as well as the foliar biomass [18]. Low concentrations of carbendazim and methylthiophanate foliar applied to artichoke (Cynara scolymus L.) increased polyphenol content in this plant, with flavonoid biosynthesis was exacerbated by PAL activity stimulation [19]. An increase in flavonoid amount was also observed after foliar treatment with low methylthiophanate concentrations to horsemint [Mentha longifolia (L.) Huds.] and lemon balm (Melissa officinalis L.) [20]. Triazoles were stated to raise anthocyanin content to a larger extent in the leaves and tubers of tapioca. On the other hand, triadimefon increased anthocyanin content in Catharanthus roseus L. and its effect can be compared to that produced by cytokinin. Treatment with abscisic acid was reported to enhance anthocyanin accumulation in holy basil (Ocimum sanctum L.) and in oilseed rape cultivars (Brassica napus var. oleifera). Benomyl, a benzimidazole fungicide, enhanced resorcinol biosynthesis in green rye seedlings (Secale cereale L.) grown at 15º, 22º, and 29ºC. In the plants kept in the dark, benomyl and carbendazim increased the content of alkylresorcinols at 29ºC and decreased it at 22º and 15ºC, respectively. The qualitative pattern of resorcinolic homologues was also importantly modified in the presence of these fungicides, while also depending on other physical stimuli. 4. PESTICIDES AND OXIDATIVE STRESS IN PLANT CELLS In addition to herbicides or other organic pollutants, insecticides may cause oxidative stress in plant cells affecting the different metabolic activities and growth components. Activation of metabolic process in plant cells in response to chemical stress initiated by pesticides is manifested in accumulation of proline (amino acid) and in the increase of various enzymatic and non-enzymatic antioxidants in different parts of the plant. Proline in plant cells acts as an osmoregulator, a signal of senescence, and an indicator of the resistance of plant to stress. Therefore, small metabolites like proline accumulate at high levels in plants when they are under stress conditions such as drought, extreme temperatures, and salinity. Several concentrations (0.05-0.20%) of delthamethrin, a synthetic pyrethroid insecticide, applied to soybean leaves, increased proline and total glutathione contents in a dose-dependent manner, whereas the total ascorbate content was observed to decrease. The application of insecticides as methylparation and 1,1dimethyl-piperidinium chloride to cotton (Gossypium hirsutum L.) suppressed foliar proanthocyanidins content. Insecticide foliar treatments with methidathion, dicofol, and dimethoate applied at recommended rates to Leucaena species reduced the proanthocyanidins concentrations by 59%, 40% and 23%, as they changed the level of biosynthesis and/or accumulation of these compounds in the plant tissues. Plant growth regulators have been shown to increase the biosynthesis of certain secondary constituents like flavonoids. Chlormequat chloride and V-2307 [(3-chlorobenzyl-3,6-dichloro-2-methoxy)-benzoate], two plant growth bioregulators, sprayed on cotton led a decline in the flavonoids by 19% each within the leaves and squares, while the leaf accumulation of anthocyanins decreased by 53% with V-2307. 5. PESTICIDES EFFECT ON VOLATILE OILS COMPOSITION It is well-known that the presence, yield, and spectrum of volatile terpenoids (essential oils) can be affected in a number of ways from their biosynthesis in the plant to their isolation. The factors that determine the chemical variability of terpenoids include:





Plant genotype;



Environmental conditions (soil, temperature, light, water and nutrient availability, precipitation, wind, radiation, pesticide treatment);



Geographic area;



Agronomic practices;



Harvest time;



Post-harvest conditions;



Extraction technology.

Among these factors, the environmental conditions affect the essential oil quantity directly through metabolic process and indirectly through the influence of plant growth. Pesticides interfere more with the quantitative composition of volatile constituents than total essential oil content. Another effect of pesticide concerns solubilization of these chemicals in essential oils. As a result, quality of essential oils can be altered by changing their chemical composition, especially regarding the toxic or useful constituents. For instance; the herbicides; mecoprop (MP 58), ethofumesate (tramat), carbethamide, trifluralin, and propyzamide applied in Chamomila recutita (L.) Rauschert cultures caused lower chamazulene content, where bisabolol content was found to be lower in the second harvest. Growth of the chamomille plants and the composition of their essential oils were strongly influenced by more substantial atrazin treatment. Other two herbicides; linuron (afalon) and 2,4-dichlorophenoxyacetic acid dimethylamine salt (monosan) decreased bisaboloxide A and chamazulene concentrations in the essential oil obtained from C. recutita. Fungicide treatment with methylthiophanate (topsin M) 0.1 and 0.4% applied to the leaves of horsemint affected chemical composition of the essential oil in the follows: 

Both fungicide concentrations increased the monoterpene content (from 33.38% for control at 36.29% and 39.82% respectively, in treated plants);



Low concentrations of methylthiophanate increased sesquiterpene content (from 2.15% for control at 5.01% for treatment), whereas high concentrations of pesticide decreased sesquiterpene level (from 14.15% at 8.70% in treated plants);



Methyltiophanate 0.1% and 0.4% increased terpenoid phenols (thymol and carvacrol) content in the essential oil by 1.61 and 1.18 times, respectively.

For lemon balm, high concentrations of methyltiophanate affected sesquiterpene spectrum of the essential oil from the leaves. In the treated plants, sesquiterpenes predominant were caryophyllene derivatives, while sesquiterpenes were muurolene and cadinene type in the control [20-24]. Application of purine and phenylurea-type cytokinins to foliar of some Lamiaceae species including Mentha piperita L. (peppermint), M. spicata L. (spearmint), M. suaveolens Ehrh. (apple mint), Lavandula vera DC. (lavender), and Salvia officinalis L. (common sage) was demonstrated to stimulate plant growth and essential oil content without alteration of the oil composition. For example; treatment with thidiazuron (100 mg/L, in the spring) of carvone type of spearmint cultivar increased the essential oil productivity (max. by 56% compared to the control) and produced changes in the proportions of carvone and limonene, while the level of carvone decreased to about 34.04% and limonene increased to 32.22%. After treatment with N,N′-2-phenylurea, thidiazuron, and N-(2-chlor-4-pyridyl)-N′-phenylurea of cineole type of spearmint cultivar established an increased yield of the essential oil up to 50 kg/ha. In field experiments with Japanese mint (Mentha arvensis L.), chlormequat chloride (2-chloroethyltrimethylammonium chloride) significantly



increased the oil content and inhibited growth only to some extent [25]. In a similar study, foliar treatment of daminozide, a plant growth regulator, at 1000 ppm reduced the growth of common sage and decreased essential oil yield, however it increased both growth and essential oil yield of Mentha piperita at the same concentration. Ethephon at 250 ppm reduced the growth and essential oil yield of peppermint and slightly increased growth and essential oil content in the common sage. Both growth regulators reduced noticeably the level of menthone and menthol in peppermint oil and increased the level of isomenthone and neoisomenthol. Consequently, they also decreased the level of camphor and increased the level of β-pinene in the common sage volatile oil [26]. Changes in the essential oil composition induced by these growth regulators are most readily explained by alterations in the levels or activities of the relevant biosynthetic enzymes. Phosphon D (tributyl-2,4-dichlorobenzylphosphonium chloride) at 50-100 ppm stimulates the growth of Salvia officinalis and moderately retarded the growth of M. piperita, while increasing the essential oil yield of both species by 50–70% phosphon D increased the proportions of (−)-3-isothujone and (+)-3-thujone in sage volatile oil and decreased the level of (−)-β-pinene and (+)-camphor. Foliar application of Cycocel (2-chloroethyltrimethyl ammonium chloride) at 250-500 ppm slightly stimulated growth and essential oil formation in peppermint with a tendency to increase the level of (−)-β-pinene and decreased the level of (−)-3-isothujone under severe stunting [27]. The growth regulators; AMO-1618 and DCPA applied to the common sage leaves increased the essential oil yield. In response to the various treatments, quantitative changes were also observed in the principle monoterpenes of the sage oil; namely β-pinene, 1,8-cineole, 3-isothujone, and camphor. Since monoterpene components examined arise by independent routes from a common precursor, the observed changes in the oil composition indicates that the applied bioregulators exert a direct effect on terpene metabolism by independent means of growth and development [28]. Foliar application of the cytokinins such as kinetin, diphenylurea, benzylaminopurine, and zeatin at the 110 ppm level increased the essential oil yield of M. piperita, M. spicata, and Salvia officinalis. In vitro assay of the enzymes catalyzing the rate limiting steps of camphor and menthone biosyntheses indicated to the fact that the increase in oil yield under the influence of cytokinin was a result of increased monoterpene biosynthesis [29]. Moreover, some studies showed that the nitrogen fertilization affects content and composition of essential oils in aromatic plants (geranium - Pelargonium graveolens L. Hér., bergamot mint - Mentha citrata Ehrhart, corn mint - Mentha arvensis L., basil - Ocimum basilicum L). 6. EFFECTS OF PESTICIDES ON OTHER SECONDARY METABOLITES In general, nitrogen treatments increase the yield of volatile oils by enhancing the amount of biomass per unit field and photosynthetic rate [30]. Therefore, the glucosinolates content may be significantly altered by both nitrogen and sulphur fertilization. Abundant nitrogen fertilization increases the progoitrin content in oilseed rape (Brassica napus L.), whereas its sinigrin concentration decreases. Sulphur fertilization may affect the glucosinolates level more than nitrogen application. A 40% glucosinolate reduction has been noticed in the plants cultivated at low sulphur fertilization condition. In broccoli sprouts (Brassica oleracea L. italian group cultivar), sulphur treatment has a detrimental effect on the content of aliphatic glucosinolates [31]. The sulfonyl urea and imidazolinone based herbicides acted by inhibiting acetolactate synthase, a key enzyme in branched chain aminoacid biosynthesis, and phosphinothricin acted by inhibiting glutamine synthetase. Simazine 50WP (1 kg/ha) applied in the second year of cultivation in common centaury (Centaurium erythraea Raf.) led to diminish in secoiridoid glucosid content [32]. Diclobutrazol fungicide inhibited sterol 14 - demethylation, causing an increase in the content of 4,14-dimethyl sterols, and it also increased proportion of campesterol of winter wheat seedlings [33]. Triadimefon (20 mg/L per plant) and hexaconazole (15 mg/L per plant) fungicides increased the carotenoid content in tapioca leaves (Manihot esculenta Crantz) [34].



The carrots (Daucus carota L.) grown in soil treated with organophosphate insecticides (chlorfenvinphosbirlane; bromofos-nexion; fonofos-dyfonate) and the herbicide; afalon S presented a higher content of total carotene than in control with the largest difference being about 21% [35]. Triadimefon treatment induced a higher level of carotenoid content in cowpea [Vigna unguiculata (L.) Walp.], whereas high doses (100 and 200 ppm) of dimethoate alone and together with UV-B (0.4 W/m2) determined a considerable reduction in carotenoid concentration. The last condition led to decrease in photosynthetic efficiency and the growth of the cowpea plants. Inhibitory effect of dimethoate on photosynthesis could be explained by direct action of insecticide on electron transport chain. Similar results were observed in Madagascar periwinkle (Catharanthus roseus) treated with the fungicides; ketoconazole and paclobutrazol [36]. Increased level of cytokinin particularly trans-zeatin and its riboside has been reported in sunflower cell suspension, rice, soybean and rape seedlings after uniconazole treatment; thus, it was concluded that the increased zeatin might be responsible for the increased synthesis of carotenoid in the plants. Metolachlor, a chloroacetanilide herbicide, applied premergence (0.125-1 ppm concentration) to sorghum (Sorghum bicolor L.) influenced terpenoid biosynthesis, whereas it decreased carotene content [37]. High concentrations of paraquat brought carotenoid and chlorophyll (a+b) loss in wild (Aegilops biuncialis and A. cylindrica) and cultivated wheats (Triticum aestivum L.); a higher photooxidation profile induced by paraquat was noticed in wild wheats than cultivated wheats [38-40]. Many triazine and urea herbicides, commonly used in potato (Solanum tuberosum L.) cultivation, increased nitrogen compound contents and also intensified enzymatic darkening of tubers, changing potato quality. In addition, herbicides belonging to the phenoxyacetic acids group develop off-flavor and off-odor potato tubers [41]. Besides, pesticides are able to modulate plant metabolism by interfering with assimilation rate of micronutrients (Cu, Zn, Mn, and Fe) by water distress and depolarization of plasma membrane of the root cells. Thus, low doses of glyphosate herbicide can prevent uptake and/or assimilation of iron and manganese micronutrients. The inability of plants to take up the essential micronutrients might be reflected in the irregularity in the diverse growth parameters. The biochemical changes brought about by pesticides may affect the plants growth by an osmotic shock effect which determines the release in structural protein and loss of carrying in the cells. Thus, pesticides applications retarded the protein and carbohydrate synthesis by inducing alteration in cytochrome oxidase activity, blocking alternative respiratory pathways and accumulation of succinate. Systemic fungicides have been found to increase NAD and NADP ratios interfering with electron transport system and increases ATP levels by inducing change in enzymes system which results in the conservation of leaf protein and chlorophyll in detached wheat leaves [42]. The herbicidal treatment with methabenzthiazuron [N-(benzothiazol-2-yl)-N,N'-dimethylurea] of the spring wheat (Triticum aestivum L.) led to a decreased concentration of soluble reducing sugars. Secondary effects following methabenzthiazuron treatment included a delayed chlorophyll breakdown, a decreased chlorophyll a/b ratio, enlarged chloroplasts, an increased concentration of soluble amino acids and of soluble protein, as well as an increase in in vitro nitrate reductase activity. These responses are taken to indicate an increased photosynthetic and metabolic capacity in methabenzthiazuron treated wheat plants [43, 44]. Fludioxonil and pyrimethanil, two fungicides commonly used in vineyards against Botrytis cinerea Pers., stimulated photosynthesis and increased pigment concentrations. They had a beneficial effect on grape (Vitis vinifera L.) physiology through the stimulation of leaf carbon nutrition [45]. On sugarcane (Saccharum officinalis L.), low dosage application of glyphosate modified carbohydrate metabolism by changing activities of enzymes involved in this process. The application of high concentrated chemical preparations led to stress on the plant and, therefore, decreased chlorophyll levels which affected the photosynthesis process negatively. The reduction of photosynthetic rate by some pesticides (paraquat, cuproxat, cyazofamid, chlorpyrifos, abamectin, imidacloprid) was accompanied by both stomatal and non-stomatal factors. The higher doses (1000 mg/L) of the herbicide propyl 4-[2-(4,6-dimethoxypyrimidin-2-yloxi)benzylamino] benzoate (ZJ0273) induced physiological disorderness leading to the decreased plant



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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 organredifferentiating 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: [email protected] Ilkay Erdogan Orhan (Ed) All rights reserved-© 2012 Bentham Science Publishers


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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 H O 

e s o x o t i g i d D



Digoxin: R = OH Digitoxin: R = H





H

OH

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


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


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progesterone (6) is catalyzed by the enzyme 5-3-hydoxysteroid dehydrogenase/5-4-ketosteroid isomerase (3-HSD). 3-HSD was shown to occur in cell cultures and plants of D. lanata. Progesterone (6) is reduced to 5-pregnane-3,20- dione (7) by progesterone 5-reductase. Occurrence of this enzyme was demonstrated in leaves of D. purpurea. Progesterone 5-reductase is of special importance in the biosynthesis of cardenolides, because the cardenolides occurring in Digitalis sp. are 5-pregnane derivatives. 5-Pregnane-3,20-dione (7) is converted in D. purpurea cell cultures to a series of different pregnane derivatives. 5-Pregnane-3-ol,20-one (8) was built by 3-hydroxysteroid 5-oxidoreductase (Fig. 3, enzyme d), an enzyme found in preparations of cell cultures of D. lanata. 5-Pregnane-3,20-dione (7) is transformed to 5-pregnane-3-ol,20-one (9) by 3-hydroxysteroid 5-oxidoreductase (Fig. 3, enzyme e). Progesterone 5-reductase (Fig. 3, enzyme f) forming 5-pregnane-3,20-dione (10) competes for progesterone (6) with progesterone 5-reductase. Progesterone 5-reductase was found in cell cultures of D. lanata. 5-pregnane-3,20-dione (10) is transformed to 5-pregnane-3 ol,20-one (11) by 3-hydroxysteroid 5-oxidoreductase in cell cultures of D. purpurea. It has been suggested that progesterone 5-reductase and 3-hydroxysteroid 5-oxidoreductase withdraw precursors from cardenolide biosynthesis [9].

Figure 3: Enzymes involved in pregnane biosynthesis and transformation in somatic embryos of D. Lanata.

1: Campesterol; 2: cholesterol; 3: sitosterol; 4: stigmasterol; 5: pregnenolone; 6: progesterone; 7: 5pregnane-3,20-dione; 8: 5-pregnane-3-ol,20-one; 9: 5-pregnane-3-ol,20-one; 10: 5-pregnane-3,20dione; 11: 5-pregnane-3-ol,20-one [9]. a)

Cholesterol monooxygenase (side chain cleaving) (= side chain cleaving enzyme, SCCE),

b)

5-3-hydoxysteroid dehydrogenase/5-4-ketosteroid isomerase (3-HSD),

c)

Progesterone 5-reductase,

d)

3-Hydroxysteroid 5-oxidoreductase,

e)

3-Hydroxysteroid 5-oxidoreductase,

f)

Progesterone 5-reductase,

g)

3-Hydroxysteroid 5-oxidoreductase.

Activities of the biosynthesis involved enzymes cholesterol monooxygenase (Side chain cleaving - SCCE), 5-3-hydoxysteroid dehydrogenase/5-4-ketosteroid isomerase, progesterone 5-reductase, 3hydroxysteroid 5-oxidoreductase, 3-Hydroxysteroid 5-oxidoreductase, Progesterone 5-reductase and 3-Hydroxysteroid 5-oxidoreductase were determined in different developmental stages of somatic embryos of Digitalis lanata. All enzymes were found to be present in proembryogenic masses (PEMs) as well as in globular and bipolar embryos. Most SCCE activity was found in the mitochondrial fraction. Cholesterol, sitosterol and stigmasterol, precursors of the pregnanes, occurred in somatic embryos in



amounts of about 1g.mg-1 protein. Pregnenolone was found in traces only (about 20 ng.mg-1 protein). Feeding of progesterone caused an increase of the contents of 5- and 5-pregnandione, progesterone, 5pregnane-3-ol,20-one and 5-pregnane-3-ol,-20-one. In contrast, administration of cholesterol caused a small increase of pregnenolone only. These results indicated that the rate limiting step in pregnane (and probably in cardenolide biosynthesis) was compartmentation of CSSE in the mitochondria of the somatic embryos of D. lanata [9]. 3.2. Molecular Studies on Different Enzymes Involved in Cardenolide Biosynthesis So far, molecular data from Digitalis are available only for a few housekeeping genes (tRNA-Leu, 18S ribosomal RNA) and several enzymes such as aldo-keto reductase, acyl-CoA-binding protein, cyclophilins, and so on [12]. Cardenolide specific genes are described for: cardenolide-16'-O-glucohydrolase, lanatoside15'-O-acetylesterase, and 5-3-hydroxysteroid dehydrogenase. The gene for progesterone 5-reductase of D. obscura (Dop5r; AJ555127) was reported by Roca-Perez et al. [13]. The progesterone 5-reductase (p5r) gene from D. purpurea was cloned and a partial genomic clone from D. obscura has been used to analyse the cardenolide production in 10 natural populations under the seasonal aspects. The cloning and heterologous functional expression of 5-POR from the leaves of Digitalis lanata Ehrh. and the biochemical characterization of the recombinant enzyme were reported by Herl et al. [11] PCR Amplification and Cloning of Progesterone 5-Reductase The early steps of cardenolide biosynthesis are summarized in Fig. 2. The crucial step leading to 5configured pregnanes supposed to be the direct precursors of Digitalis cardenolides were studied Initial experiments were carried out using degenerated oligo nucleotide primers derived from the peptide sequences of progesterone 5 -reductase from D. purpurea [10, 11] taking Kazusa’s codon usage system into account. The resulting fragments showed high sequence homology to the genomic clone of Dop5 r gene of D. obscura and to the progesterone 5reductase (p5r) from D. purpurea. After submission of the sequence for p5r gene, the results were confirmed by PCR amplification with distinct primers. The 5-POR was amplified by RT-PCR from cDNA prepared from D. lanata, D. purpurea and D. obscura. DNA fragments of nearly identical length were also obtained when genomic DNA of D. purpurea was used as template. A full-length cDNA clone that encodes a progesterone 5-POR from leaves of D. lanata was isolated. An identical match was observed between the deduced and directly determined amino acid sequence of the progesterone 5reductase peptides from D. purpurea. RT-PCR using RNA and/or mRNA from mature leaves resulted in one single DNA fragment of the appropriate size. The DNA fragments and the nucleotide sequences obtained from D. lanata, D. purpurea and D. obscura were found to be not different in size. PCR amplification with genomic DNA as a template resulted in a fragment of 1247 bp, slightly different in size from the cDNA fragments. The sequence of the genomic clone was found to containe a small intron as obtained after sequencing data analysis [10, 11, 13]. Alignments of Progesterone 5-Reductase Several authors proposed that 5-POR has a key function in cardenolide biosynthesis producing the required 5-configured pregnane intermediates leading to the various cardenolide genins [9, 10]. When the alignment of deduced 5-POR protein sequences for D. lanata (AY574950), D. purpurea (AY585868) and the Dop5r gene of D. obscura (AJ555127) were compared, the sequences of the deduced 5-POR gene products were found 95–99% identical. A high degree of homology was also seen when the nucleotide sequence of the cDNA was analysed in silico and compared with the reports for Dop5r of D. obscura [13] and Dop5r of D. purpurea (AJ310673). Hence, it seems as if the 5-POR genes are highly conserved within the genus Digitalis. Recently, Brauchler et al. proposed a phylogenetic cladogramme of the genus Digitalis on the basis of ITS- and trnL-F sequences and found D. lanata and D. obscura more closely related to each other than to D. purpurea supporting the Herl’s data [11, 14]. The deduced 5-POR protein sequences were found similar to those of Oryza sativa (about 58%), Populus tremuloides (about 64%) and to the Arabidopsis thaliana wound-induced gene AWI [15].


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Function of r5-POR The 5-POR gene over-expressed in E. coli yielded an enzymatically active protein. The Ni-NTA-purified r5-POR was checked for 5-POR. TLC analysis revealed the formation of one single product, namely 5pregnane-3,20-dione, when progesterone was used as the substrate. 5-Pregnane-3,20-dione, the 5-isomer of the pregnane-3,20-dione was not formed and not observed in TLC plate. The results have been also confirmed by GC analyses which proved that only the 5-isomer but not the 5-isomer of pregnane-3,20dione was produced. Interestingly, 5-pregnane-3,20-dione, the product of the enzyme reaction was not converted to other products as it was seen in partially purified enzyme preparations from D. lanata leaves, indicating that those extracts contained other pregnane-modifying enzymes and that r5-POR catalyses only the 5reduction of progesterone [11]. Substrate Preferences of Progesterone 5-Reductase The substrate preferences and kinetic properties of r5-POR were investigated using an HPLC method for product identification and quantification [11]. Besides the putative natural substrate progesterone, other steroid substrates were tested. The Km and max values for the putative natural substrate progesterone were calculated to be 0.120 mM and 45 nkat mg-1 protein, respectively. According to Km and max-values, the r5-POR did not only accept progesterone but also testosterone, 4-androstene-3,17-dione, cortisol and cortisone. Other substrates, such as pregnenolone, 21-OH-pregenenolone and isoprogesterone were not accepted by r5-POR. NADPH is the only co-substrate and cannot be replaced by NADH. From the data obtained in the experiments described above two important conclusions can be drawn. (1) Essential structural elements for substrates of r5-POR are the carbonyl group at C-3 and D4-double bound in conjugation to it. Less important are the side chain at C-17 and the substitution pattern of the steroid ring system in its periphery [11]. In a continuation of the studies on cardenolide pathway, the recombinant 5POR was also subjected to chrystallization to understand its mode of action and substrate discrimination. Sequence considerations of 5-POR suggested that 5-POR is a member of the short chain dehydrogenase/reductase (SDR) family of proteins but at the same time revealed that the sequence motifs that in standard SDRs contain the catalytically important residues are missing. This is in contrast to mammalian steroid 5-reductase, which is a member of the aldo-keto-reductase (AKR) family. The crystal structures of 5-POR from Digitalis lanata in complex with NADP+ at 2.3A˚ and without cofactor bound at 2.4A˚ resolution were determined together with a model of a ternary complex consisting of 5-POR, NADP+, and progesterone [16] (Thorn et al. 2008). 5-POR displays the fold of an extended SDR. The architecture of the active site is, however, unprecedented because none of the standard catalytic residues are structurally conserved. A tyrosine (Tyr-179) and a lysine residue (Lys-147) are present in the active site, but they are displayed from novel positions and are part of novel sequence motifs. Mutating Tyr-179 to either alanine or phenylalanine completely abolishes the enzymatic activity. It was proposed that the distinct topology reflects the fact that 5-POR reduces a conjugated double bound in a steroid substrate via a 1–4 addition mechanism and that this requires a repositioning of the catalytically important residues. Their observation that the sequence motifs that line the active site were conserved in a number of bacterial and plant enzymes of yet unknown function caused to the proposition that 5-POR defines a novel class of SDRs [16]. 4. BIOTRANSFORMATION STUDIES FOR THE PRODUCTION OF DIGOXIN The biotransformation reactions from digitoxin to digoxin using Digitalis lanata plant cell culture were investigated by several different authors. It is the most interesting approach in terms of commercial application since digoxin has a higher demand as a drug for heart diseases than digitoxin. One of the study on biotransformation from digitoxin is the usage of cylodextrin polymer for increasing the production of digoxin.



Schematic diagram of digitoxin biotransformation is shown in Fig. 4. Two enzymatic conversion steps are involved. One is 12-hydroxylation (step 1) and the other is 16'-O-glucosylation (step 2). In order to produce digoxin effectively, step 2 reaction should be minimized not to produce unwanted side products such as purpureaglycoside A and deacetyllanatoside C.

Figure 4: Digitalis lanata plant cell cultures: ─►, traffic; ----►% enzyme reaction; 1, 12-hydroxylase; 2, 16'-Oglucosyltransferase [4].

Interesting fact is that both glucosylated side products are accumulated in the vacuoles, although digitoxin and digoxin can permeate cell membrane. Therefore, if we can remove digoxin selectively from the medium as soon as it is produced, its productivity can be enhanced theoretically. Any forms of integrated bioprocessing (extractive bioconversion) composed of cell culture and bioseparation in situ can be applied for this process. Extractive bioconversion is the concept of increasing the productivity of performance of biotechnological processes by the continuous removal of a product from the site of its production. In situ adsorption with digoxin-selective resins was already found to be successful in enhancing digoxin production [17]. Cyclodextrins (CDs) are cyclic oligosaccharides composed of 6, 7, or 8  -l,4-linked D-glucose molecules. They are usually named as -, -, or  -CD, respectively [18]. CDs are water-soluble and able to form inclusion complexes by accommodating hydrophobic guest molecules within the cavity. Since they are biocompatible and enhance solubilization of organic compounds, it is expected that they can be used to increase the solubility of hydrophobic plant secondary metabolites produced by cell suspension cultures in aqueous media. Formation of inclusion complexes may reduce toxicity of the product which results in enhancing the productivity. Stimulatory effects of CD in biotransformation were reported using microorganisms [19]. In addition to the utilization of various CDs, CD polymers can also be used for different biotransformation reactions. CD polymers can be prepared with various cross-linking agents such as epichlorohydrin, diisocyanatohexane, phenyl isocyanate, and divinylbenzene. Polymeric forms of CD are insoluble in water so that they can be recycled and function as specific adsorbents. Therefore, it may be possible to use CD polymer as one of the adsorbents in situ with cell cultivation. Various kinds of CD polymers have been used in the removal of bitter components from fruit juices and decaffeination process [20, 21]. -CD polymer was used to enhance the production of digoxin from digitoxin biotransformation in Digitalis lanata cell suspension cultures. Addition of -CD polymer during the biotransformation of digitoxin into digoxin using cell suspension cultures of Digitalis lanata enhanced the conversion yield. Digitoxin showed better adsorption to CD


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polymer compared to digoxin, so that the optimization of addition time was found to be necessary. In the case of adding CD polymer 24 hours after the feeding of substrate digitoxin, the highest digoxin production could be achieved. At this period, digitoxin was almost consumed by cells and productivity was proportionally enhanced according as the amount of substrate was increased. Immobilization of CD polymer did not promote the biotransformation. When 1.67 g/L of CD polymers was added, 50% of both cardenolides were adsorbed very rapidly. However, when the amount of CD polymer was increased to 3.33 g/L, much more digitoxin was adsorbed compared to digoxin. Preferential adsorption of substrate digitoxin may inhibit the biotransformation apparently. At low concentration of CD polymer, considerable amount of digitoxin was not adsorbed and hydroxylated into digoxin. In conclusion, adequate use of CD polymer at proper concentration enhanced the production of digoxin via biotransformation by D. Ianata plant cell suspension cultures. By optimizing the mode of addition and their concentration, much more efficient biotransformation may be possible [4]. 5. EFFECT OF LIGHT AND PLANT GROWTH SUBSTANCES ON DIGITOXIN FORMATION BY DIFFERENT PLANT CELL/TISSUE CULTURE SYSTEM Comprehensive investigations were performed on cardenolide production of cultured cells of Digitalis species. Most workers have reported that undifferentiated cultured cells either did not produce cardenolides or contained only trace amounts of cardenolides. However, organ-redifferentiating cultures have been reported to accumulate considerable amounts of cardenolides. Undifferentiated cells of Digitalis have been reported to accumulate only extremely low amounts of cardenolide; there are no reports dealing with cardenolide-production by undifferentiated cells possessing chloroplasts. The main site of cardenolide storage and synthesis in Digitalis is the leaves, so that the development of the cardenolide biosynthesis system seems to be associated with their differentiation and is not prominent in undifferentiated cells as suspension cultures or in differentiated cells in roots. In the cells of D. purpurea leaf tissue, most of the cardenolide glycosides present were localized in the mitochondrial and chloroplast fractions. If the chloroplast is involved in cardenolide synthesis, then it can be speculated that undifferentiated cells with chloroplasts should synthesize cardenolides. Four liquid-cultured cell lines of Digitalis purpurea L., i.e. undifferentiated green cells; undifferentiated white cells; green, shoot-forming cultures; and white, shoot-forming cultures were compared for their cardenolide production. These cell lines were used to study the effect of light on the expression of cardenolide-production. In this study, it was established highly chlorophyllous, undifferentiated cells of D. purpurea L., the chlorophyll content and RuBPCase activity of which were about the same as those of the green, shoot-forming cultures. However, even the highest digitoxin content of the undifferentiated green cells (0.18 jug/g dry weight) was found to be much lower than that of the green, shoot-forming cultures (40,ug/g dry weight) and about the same as that of the dark-grown, undifferentiated white cells (0.21 ,ug/g dry weight); however, in the subculturing condition (IAA-indoleaceticacid, 1 mg/L), the green cells contained about 6 times more digitoxin than did the white cells. On the other hand, the dark-grown, white, shoot-forming cultures without chloroplasts accumulated about one-third as much digitoxin as did the lightgrown, green, shoot-forming cultures. These results suggest that the chloroplast is not essential for the synthesis of digitoxin. The stimulatory effect of light on digitoxin formation by shoot-forming cultures seems to allow the cells to be closer to the status of leaf cells. However, it is possible that the proplastids, which would develop into chloroplasts if illuminated, contain the cardenolide-biosynthesis system. Furthermore, it can be speculated that, when the cells differentiate to form shoots, the system in the proplastid is expressed regardless of light conditions [22, 23]. The effect of light and dark –grown to cardenolide biosynthesis was also studied on Digitalis lanata shoot culture [24]. The use of tissue cultures of D. lanata with a view towards producing cardenolides was examined quite extensively, using suspension cultures, morphogenic or embryogenic cell cultures, as well as shoot or root cultures. In summary, cell cultures without clear tissue - or organ differentiation (socalled 'undifferentiated' cultures) - were found to be unable to produce considerable amounts of cardenolides, whereas cardenolide biosynthesis is restored as soon as morphogenesis is induced. The influence of light,



phytohormones and nutrients on the production of cardenolides in Digitalis purpurea shoot cultures was examined. Dark-grown shoot cultures contained considerable amounts of cardiac glycosides, but green shoot-forming cultures had three times the concentration of cardenolides. Tissue differentiation could be stimulated by gibberellic acid in light- and dark-grown embryogenic cultures. Under these conditions, the cardenolide content of darkgrown cultures tripled, whereas that of light-grown cultures was considerably depressed. Similar effects were observed after the addition of Mn 2+, which is known to stimulate isoprenoid pathways in plants. White root cultures are almost cardenolide free, whereas low levels of cardenolides were detected in green, light-grown root cultures. In summary, it is not yet clear whether organogenesis, light or a combination of both are necessary for triggering cardenolide biosynthesis [23, 25]. About 80 different cardiac glycosides are known to occur in D. lanata. However, the cardenolide composition of the in vitro systems was not carefully evaluated in all cases. In several reports, radioimmunoassay techniques, low-resolution TLC or HPLC methods were used to demonstrate and quantify cardenolides in the tissues under investigation. From the information available, it can be tentatively deduced that the more differentiated the tissue cultures, the higher the cardenolide amount and the more complex the cardenolide spectrum. The main cardiac glycosides of embryogenic and morphogenic cultures have already been identified but, only recently, the cardenolide compositions of various D. lanata shoot cultures have been established using HPLC, TLC and chemical degradation [23, 24, 26, 27]. In Eisenbeig’s study, shoot cultures grown in liquid medium, which can still regenerate into whole plants when rooting was used for the effect of light. The potential of these cultures for active cardenolide biosynthesis was investigated through feeding studies with radioactive labelled pregnenolone and progesterone. These cultures were found to contain cardenolides of the 'early' type, e.g. disaccharides comprising fucose or digitalose, as well as of the 'late' type, e.g. tetrasaccharides comprising digitoxose. The cardenolide content of both light- and dark-grown shoot cultures was determined, and the changes in the cardenolide levels of cultures transferred from light into dark or vice versa were monitored. In addition, cardenolide-specific enzymes were measured [27]. Cardenolide production seems to be closely correlated with tissue differentiation in D. lanata. Cultivation under continuous dark reduces the leaf size of the shoot-cultures of D. lanata and their metabolic status changes in terms of cardenolide production. After 12 weeks in the dark, the cardenolide level decreased to non-detectable levels. Subsequent illumination with white light led to cardenolide levels and leaf size of light-grown controls after 4 weeks. Kuberski et al. detected no cardenolides in an embryogenic strain of D. lanata cultivated in the dark [27]. When transferred to continuous white light, the aggregates turned green and the cardenolide content reached about 130 mol.g-1 DW. Previously, it was reported that root cultures only accumulate negligible amounts of cardenolides when cultured in the dark, whereas light enhanced cardenolide accumulation reaches levels of up to 21 mol cardenolides.g -1 DW. Hagimori et al. found considerable amounts of cardenolides (up to 13 g.g -l DW) in dark-grown shoots of D. purpurea. In above researches, authors did not comment on the length of time the cultures had been subcultured in the dark [23, 24]. The length of time of the cultures is also important for the production, it was found that cardenolide production was decreased during cultivation in the dark depending on the time course [24]. The presence of chloroplasts itself did not lead to cardenolide production in heterotrophic or photoautotrophic green cell suspension cultures of Digitalis sp., which had lost the potential to produce cardenolides. Even inhibited chlorophyll synthesis in the light led to cardenolide production in somatic embryos of D. lanata. Therefore, chloroplasts do not seem to be essential for cardenolide biosynthesis [23, 25]. It was demonstrated by Eisenbeig et al. 1999 that cardenolides are still synthesised de novo in dark-grown shoots of D. lanata, as established by the incorporation of radiolabelled pregnenolone into cardenolide genins. The green shoots incorporated 0.74 % of the administered radiolabelled pregnenolone in


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digitoxigenin and 0.19 % in gitoxigenin. The white shoots showed a lower incorporation rate; only 0.15 % of the administered radiolabelled pregnenolone was found in digitoxigenin and 0.02 % in gitoxigenin. Even though this rate was about 5 times lower than in green shoots, the white shoots still showed the ability to synthesise cardenolides. After administering radiolabelled progesterone to D. purpurea shoot cultures, Hagimori et al. found an incorporation rate of 1% in digitoxigenin in green shoots and 0.3 % in digitoxigenin in white shoots. Their results were similar to Eisenbeig, as they found cardenolide biosynthesis to be independent of the chlorophyll content of D. purpurea [24]. The question of whether progesterone or pregnenolone would be the better precursor for cardenolide biosynthesis is not easy to answer. Both precursors entered the cardenolide pathway and resulted in similar amounts of aglycons. The different transformation rates of both substrates in dark- and light-grown shoot cultures seemed to be a matter of their changed physiological status and differentiation while growing in the dark. Dark-grown cultures may have different substrate specificities for the fed precursors than lightgrown cultures; this could be true for various strains of shoot cultures as well. The white cultures could obviously transiently produce cardenolides when precursors were fed, but they were not capable of accumulating cardenolides. The reason for this behavior is not yet clearly understood. The availability of enzymes may differ during adaptation and cultivation in the dark. Usually, the compartmentalization in the cell may serve more or less as a barrier between enzyme and substrate at a normal physiological status. An alteration of enzyme locality or solubility during dark cultivation might be feasible. It was found that the cell wall bound cardenolide-specific enzyme LAE could no longer be solubilized from dark-grown shoots using the standard extraction protocol. One also has to consider enzymes competing for the same substrates, namely progesterone or pregnenolone, which may result in the formation of 5-pregnane-3-ols or 5pregnane-3-ols. These products can not be used for cardenolide formation [2]. The enzyme activities might perhaps change under dark conditions and would be a reasonable explanation for the measured shifts in cardenolide contents. Actually, the progesterone 5-reductase, which catalyses the conversion of progesterone to 5-pregnane-3,20-dione in the putative cardenolide pathway, was shown to be very active in young leaves, fairly active in light-grown, green shoot cultures but was not seen in dark-grown, white shoots [28]. It should also be noted that the cardenolide concentrations measured may represent, to some extent, the dynamic value of the sum of anabolism and catabolism of this compounds. It may also be possible that dark cultures have a higher turnover of cardenolides. The white shoot cultures had reduced leaf size as well as a lower cardenolide content. Kuberski et al. showed that the main storage location for cardenolides are the mesophyll cells of the leaves. The reduction in mesophyll cells is paralleled by the reduction in cardenolide content. The cardenolide specific enzymes responsible for catabolic functions follow the same trend, as their activities dropped to non-detectable levels or were down-regulated in the dark. Cardenolide 16'-Oglucohydrolase (CGH I) reacted immediately to the dark conditions, with activity dropping drastically within 14 d. This cytosolic membrane-bound enzyme is known to occur only in tissues producing cardenolides. Although the activity of this enzyme correlates directly with the decrease in cardenolides, it is unlikely that this enzyme is substrate-regulated. Cardenolides were degraded in the dark; therefore, the flow of primary cardenolides from the vacuole towards the cytoplasm, the location of CGH I, was decreased. These observations indicate that CGH I is not involved in cardenolide degradation in situ, but may instead play a role in cardenolide remetabolisation from storage sites and activation after wounding or in developmental programs [27]. The other catabolic enzyme, lanatoside 15'-O-acetylesterase (LAE), is apparently down-regulated in the dark and quickly restored under light conditions. Since LAE was found to be present in various plant organs and cell suspension cultures of D. lanata, it was inferred that this enzyme is independent of tissue differentiation. Actually, the changes in LAE activity were found mainly due to the fact that the enzyme could not be solubilised completely from the cell walls of darkgrown shoots with the standard extraction protocol. This in turn indicates that structural modifications at the wall matrix have occurred, which resulted in an altered affinity of the matrix to LAE.



Both catabolic enzymes are separated from their substrates through compartmentalisation in the cell. Only remetabolisation processes will bring substrates and enzymes into contact, except for the newly synthesized cardenolides or those taken up by the cell. The two anabolic enzymes, DAT and DGT, are both enzymes with transferase abilities, the first transferring an acetyl-moiety, the latter a glucose-moiety to the sugar side-chain of the cardenolides. DAT is a soluble cytosolic enzyme with high activities in cardenolideproducing tissues and low activities in suspension cultures of D. lanata. It responded rapidly to dark conditions, as its activity dropped to nondetectable levels after 2 weeks in the dark. Two days after subsequent illumination enzyme activity could be measured again. The only enzyme which was not affected when transferring the shoot cultures from light to dark and vice versa was DGT, which is responsible for the transformation of a secondary glycoside to a primary one. Primary cardenolide glycosides were stored in the vacuole as a sink for secondary metabolism products. They are also known to be the only cardenolides capable of passing through the cell membrane against their concentration gradient. The slight accommodation to dark conditions was the response of the cell to reduced turnover and synthesis of cardenolides in dark-grown cultures. DGT has no regulatory role in cardenolide biosynthesis, as it reaches activities of 5-10 kat.kg-1 protein in both cardenolide-producing tissues as well as non producing tissues. It still seems to be active enough to enable upcoming cardenolides to enter the vacuole via an active transport mechanism [24]. Interestingly enough, the two enzymes CGH I and DAT, which were not active in the dark, showed high activities in cardenolide-producing tissues, such as leaves, whereas the others, LAE and DGT, were active in both producing and non-producing tissues. This also illustrates the close relationship between illumination, differentiation and cardenolide production. Cardenolide production in close correlation with morphological differentiation was discussed by Garve et al. and their views are consistent with those of Hagimori et al. and Eisenbeig et al. 1999., namely that the formation of chlorophyll or chloroplasts plays a subordinate role to differentiation [23, 24, 29]. Cardenolide accumulation could also be triggered independently of light through stimulation of differentiation by phytohormones. The cardenolide pathway is most probably not regulated by a single enzyme ('key enzyme'), but is influenced by the reduction of several enzyme activities, as was shown, for example, for Thalictrum glaucum suspension cultures where several enzymes of the berberine pathway are downregulated in protoberberinefree strains. Further studies with our D. lanata shoot culture system might provide information on the overall regulation of cardenolide biosynthesis [24]. 6. DISCUSSION Cardiac glycosides are a group of natural products characterized by their specific effect on myocardial contraction and atrioventricular conduction. They are important drugs in the treatment of congestive heart failure. They are currently produced by the direct extraction, mainly from Digitalis lanata plants for commercial production [30]. A digoxin product, Lanoxin, is a brand name of a Burroughs Wellcome product and has the largest market of the company's cardiovascular drugs. The major markets of Lanoxin are in the U.S.A. and Italy, and the total sales are approximately 6000 kg a year with 50 million U.S. dollars. Other companies such as Boehringer Mannheim, Merck Darmstadt and Beiersdorf AG in Germany also sell cardiac glycosides [31]. The direct production of cardenolides with Digitalis cell and tissue cultures has been studied for several years in Japan and in East Germany. The studies of plant tissue and cell cultures for production of cardiac glycosides were begun more than 30 years ago and the report presented by Hildebrandt and Riker in 1959 was probably the first one in this field. Staba investigated the nutritional requirements of tissue cultures of Digitalis lanata and D. purpurea in 1962. These two species are being commonly used by many scientists [25, 27, 31]. Although there are a number of papers describing production of cardiac glycosides in Digitalis tissue cultures, generally the yield was very low, the productivity has not yet reached the level necessary for


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economic application and moreover, during the successive transfers of the cultured cells the amount of cardenolides often decreased and disappeared completely [32]. On the other hand, many researchers indicated that morphological differentiation caused an increase in productivity. For example, organ cultures of D. lanata leaves and roots produced cardenolides and during the cultivation the level of digoxin in the tissues rose with increasing age. It was also found that renewed organ differentiation from callus tissues of D. purpurea led to a new formation of cardenolides [25, 27, 33]. Nutritional factors including growth regulators, sugars, nitrogen sources, vitamins and so on in the medium affect on differentiation of shoots and other organs and the effect of light and dark conditions on cardiac glycosides production are often studied. Hagimori et al. of Japan Tobacco Inc. cultivated shoot-forming tissues of D. purpurea in a 3 L jar fermentor and detected high concentrations of cardiac glycosides including digitoxin [23, 24]. The differentiated tissue culture is prerequisite for secondary metabolites production in some cases, however in general, the method requires much longer culture time than the suspension cell culture and consequently it is not efficient [31, 32]. In addition to the de novo synthesis of cardenolides, the biotransformation process with Digitalis plant cells seems to be more promising from a commercial point of view. It was reported that D. lanata and D. purpurea callus cultures rapidly transformed progesterone to pregnane. Leaf and root cultures of D. lanata and shoot-forming callus tissues of D. purpurea accumulated an increased level of digoxin and/or digitoxin when progesterone was added to the cultures [10, 17, 34]. For the biotransformation reaction, detailed biosynthesis of cardenolides was also studied by different authors. Molecular researches from Digitalis were performed a few housekeeping genes and several enzymes such as aldo-keto reductase, acyl-CoAbinding protein, cyclophilins, and so on. Cardenolide specific genes were also described for: cardenolide16'-O-glucohydrolase, lanatoside-15'-O-acetylesterase, and 5-3-hydroxysteroid dehydrogenase. The gene for progesterone 5-reductase of D. obscura, D. purpurea and D.lanata was reported and used to analyze the cardenolide production [9,11-13]. Molecular studies on different enzymes involved in cardenolide biosynthesis, different cell and tissue culture researches and biotransformation reactions for the production of digoxin are still very important research area for the developing and producing important drug in the treatment of congestive heart failure. REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10]

Mijatovic T, Van Quaquebeke E, Delest B, Debeir O, Darro F, Kiss R. Cardiotonic steroids on the road to anti-cancer therapy. Biochim Biophys Acta. 2007; 776:32-57. Samuelsson, G., 2004. Drugs of natural origin: A textbook of pharmacognosy. 5 ed. Stockholm: Swedish Pharmaceutical Press. Dewick PM, Medicinal a Natural Poducts, A biosynthetic approach, 2nd ed. UK, Wiley, 2004. Lee JE, Lee SY, Kim D, Increased production of digoxin by digitoxin biotransformation using cyclodextrin polymer in Digitalis lanata cell cultures. Biotechnol Bioprocess Eng. 1999; 4: 32-35. Bourgaud F, Gravot A, Milesi S, Gontier E, Production of plant secondary metabolites: a historical perspective. Plant Sci 2001; 161: 839–851. Ramachandra Raoa S and Ravishankarb GA, Plant cell cultures: Chemical factories of secondary metabolites. Biotechnol Adv 2002; 20: 101–153. Mulabagal V and Tsay HS , Plant Cell Cultures - An alternative and efficient source for the production of biologically important secondary metabolites. Int J Appl Sci Eng 2004; 2, 1: 29-48. R. Tschesche, Biosynthesis of cardenolides, bufadienolides and steroid sapogenins. Proc R Soc Lond B 1972; 180: 187-202. Lindemann, P and Luckner, M., Biosynthesis of pregnane derivatives in somatic embryos of Digitalis lanata. Phytochemistry 1997; 46: 507–513. Gartner DE, Keilholz W, Seitz HU,. Purification, characterization and partial peptide microsequencing of progesterone 5-reductase from shoot cultures of Digitalis purpurea. Eur J Biochem 1994; 225: 1125–1132.


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Herl V, Fischer G, Muller-Uri F, and Kreis W. Molecular cloning and heterologous expression of progesterone 5reductase from Digitalis lanata Ehrh. Phytochemistry 2006; 67: 225–231. Luckner, M., Wichtl, M., Digitalis. WVGmbH, Stuttgart, 2000. Roca-Perez L, Boluda R, Gavidia I, Perez-Bermudez P, Seasonal cardenolide production and Dop5 gene expression in natural populations of Digitalis obscura. Phytochemistry 2004; 65: 1869–1878. Brauchler C, Meinberg H, Heubl G, Molecular phylogeny of the genera Digitalis L. and Isoplexis (Lindley) Loudon (Veronicaceae) based on ITS- and trnL-F sequences. Plant Syst Evol 2004; 248: 111–128. Yang KY, Moon YH, Choi KH, Structure and expression of the AWI 31 gene specifically induced by wounding in Arabidopsis thaliana. Mol Cells 1997; 7: 131–135. Thorn A, Egerer-Sieber C, Jager CM et al. The crystal structure of progesterone 5reductase from Digitalis lanata defines a novel class of short chain dehydrogenases/ reductases. J Biol Chem 2008; 283: 17260–17269 Hong, H.-J., J.-E. Lee, J.-E. Ahn, and D.-I. Kim, Enhanced production of digoxin by digitoxin biotransformation using in situ adsorption in Digitalis lanata cell cultures. J Microbiol Biotechnol 1998; 8: 478-483. Szejtli, J, Downstream processing using cyclodextrins. Trends Biotechnol 1989; 7: 170-174. Bar, R. Cyclodextrin-aided bioconversion and fermentation. Trends Biotechnol 1989; 7: 2-4. Shaw PE and Buslig BS. Selective removal of bitter compounds from grapefruit juice and from aqueous solution with cyclodextrin polymers and with Amberlite XAD-4. J Agric Food Chem 1986; 34: 837-840. Yu EKC, Novel decaffeination process using cyclodextrins. Appl Microbiol Biotechnol 1988; 28: 546-552. Hagimori M., Matsumoto T., Mikami Y., Digitoxin biosynthesis in isolated mesophyll cells and cultured cells of Digitalis. Plant Cell Physiol 1984; 23: 947-953. Hagimori M., Matsumoto T., Obi Y., Studies on the production of Digitalis cardenolides by plant tissue culture. II. Effect of light and plant growth substances on digitoxin formation by undifferentiated cells and shoot-forming cultures of Digitalis purpurea L. Grown in liquid media. Plant Physiol 1982; 69: 653-656. Eisenbeig M, Kreis W, Reinhard E, Cardenolide biosynthesis in light- and dark-grown Digitalis lanata shoot cultures. Plant Physiol Biochem 1999; 37: 13-23. Hagimori M, Matsumoto T, Mikami Y, Photoautotrophic culture of undifferentiated cells and shootforming cultures of Digitalis purpurea L. Plant Cell Physiol. 1984; 25: 1099-1102. Seidel S., Reinhard E., Major cardenolide glycosides in embryogenic suspension cultures of Digitalis lanata. Planta Med 1987; 3: 308-309. Kuberski C, Scheibner H, Steup C, Diettrich B, Luckner M, Embryogenesis and cardenolide formation in tissue cultures of Digitalis lanata. Phytochemistry 1984;23: 1407-1412. Stuhlemmer U., Kreis W., Cardenolide formation and activity of pregnane-modifying enzymes in cell suspension cultures, shoot cultures and leaves of Digitalis lanata. Plant Physiol Biochem 1996; 34: 85-91. Garve R., Luckner M., Vogel E., Tewes A., Nover L.,Growth, morphogenesis and cardenolide formation in longterm cultures of Digitalis lanata. Planta Med 1980; 40: 92-103. Misava M, Plant tissue culture: an alternative for production of useful metabolite, FAO Agricultural Services Bulletin, 108, Chapter 7, 1994. Tuominen UL, Toivonen V, Kauppinen P, Markkanen and L. Bjork Studies on the growth and cardenolide production of Digitalis lanata tissue cultures. Biotechnol Bioeng 1989; 33: 558-562. Lui, J.H.C. and Staba, E.J., Effect of age and growth regulators serially propagated Digitalis lanata leaf and root culture. Planta Med 1981; 41: 90-95. Furuya T, Kawaguchi K, Hirotani M, Biotransformation of progesterone by suspension cultures of Digitalis purpurea cultured cells - Phytochemistry 1973; 1: 1621-1626.

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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; Zonguldak Karaelmas University, Faculty of Arts and Science, Department of Biology, 67100 İncivez, Zonguldak, Turkey 2

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: [email protected] Ilkay Erdogan Orhan (Ed) All rights reserved-© 2012 Bentham Science Publishers

Progress in Biotechnological Applications of Diverse Species


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 antiallergic 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-hexanedichloromethane (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


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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, 7angeloylretronecine, 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-Darabinoside, quercetin 3-O-alpha-D-arabinoside, and p-hydroxy benzoic acid were isolated from Ehretia thyrsiflora [44]. Whereas, isoquercetrin, hyperoside, trifolin, astragalin, kaempferol 3-Oarabinosylgalactoside, 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].



3. PRODUCTION OF SECONDARY METABOLITES Production and biosynthesis of shikonin/derivatives have been one of the first and most intensively studied topic in the area of secondary metabolites. The compounds were listed in ‘Color index’ as Tokyo violet (shikonin) (75520) and natural red 20 (75530). They are used as natural colorants. A commercial ointment prepared from Alkanna extract named as Helixderm®, Histoplastin Red® and Epouloderm® commercially used for wound healing purposes in Greece and a part of Europea. Due to medicinal and economical value of the compounds, as a dye in food and cosmetic industry, production and increase of major phytochemical groups, napthaquinones, rosmarinic acid, polyphenol compounds and pyrolizidine alkaloids were found worth to manipulate by utilizing several strategies such as optimization of growth and production media, induction by elicitors, culturing of differentiated cells and metabolic engineering. Naphthaquinones The value of pigments, as a mean of naphtaquinones, motivated biotechnologists for utilizing plant cell cultures in order to produce those compounds by using biotechnological tools. Production of naphtaquinones in callus and suspension cultures was initiated with L. erythrorhizon by Tabata’s group from Japan at the begining of 1970s. First of all, regulatory factors in the biosynthetic pathway of naphtaoquinones have been identified by using cell culture system. The strongest inhibitor of shikonin biosynthesis was determined as light. Therefore cultures were incubated in dark in order to produce pigmented cultures. Shikonin derivatives, in differentiated callus cultures of L. erythrorhizon increased in dark with the growth regulator Indole-3-acetic acid (IAA), however, decreased by adding 2,4Dichlorophenoxyacetic acid (2,4-D) radiated with blue light [51]. The cell culture of Onosma paniculatum in dark is capable of producing a large quantity of shikonin and its derivatives, which were completely inhibited when the cell cultures were irradiated with continuous red, blue and white light. The inducible expression of phenylalanine ammonia lyase (PAL1), 4-coumarate-CoA ligase (4CL1), and CYP98A6 as well as the inhibitory transcription of Lithospermum dark-inducible genes 2 (LDI2) mediated by continuous red light irradiation were likely accounted for the reduced accumulation of shikonin in O. paniculatum cell cultures [52]. Administration of various supposed precursors to the callus cultures of L. erythrorhizon grown on the Linsmaier-Skoog (LS) medium supplemented with growth regulators Indole-3-acetic acid (IAA) and kinetin, produced shikonin derivatives in various levels [53]. Moreover, addition of ascorbic acid, sucrose, nitrogen sources, L-phenylalanine and streptomycin sulphate in callus cultures of L. erythrorhizon had positive effect on the production of shikonin derivatives, while increase of Ca2+ and Fe2+ and, nitrogen sources inhibited the biosynthesis of them [54]. Addition of nitrate, as the nitrogen source, stimulates the production of shikonin derivatives while increase in ammonium concentration in the medium and treatment with ethylene, acounted for decreased production of shikonin derivatives in cell suspension cultures of L. erythrorhizon [55, 56]. Besides callus cultures, pigmented naphthoquinone derivatives of shikonin increased by adding of abiotic (CuSO4) or biotic (fungal elicitor) factors in cell suspension culture of L. erythrorhizon roots [57]. Moreover the gamma irradiation significantly stimulated the shikonin biosynthesis and increased the total shikonin yield in cell suspension culture of L. erythrorhizon [58]. Despite the significant success on producing pigments, among different callus and cell suspension cultures, levels of naphtaquinone derivatives varied. Soon after two high pigment-producing strains of L. erythrorhizon have been established whose content of shikonin derivatives are stable and similar to that of intact plant root [59]. Shikonin produced by cell cultures of L. erythrorhizon in medium containing L-phenylalanine, 2 mg/L IAA and 800 mg/L Ca(NO3)2 4H2O [60]. Red naphthoquinone pigments were also produced in callus of a different Lithospermum species, L. officinale, which is cultured in Linsmaier-Skoog (LS) medium containing IAA and kinetin [61]. Cultured cells of L. erythrorhizon capable of producing red naphthoquinone derivatives on LS agar medium stopped synthesizing these compounds when they were transfered to liquid medium without agar. When the liquid medium was supplemented with a small amount of activated carbon, the cells produced echinofuran B, a shikonin derivative in cultured cells of L. erythrorhizon with no difference present in intact plant (Ko-shikon) [62, 63]. Another study revealed that L. erythrorhizon cell suspension cultures in a growth medium produced no shikonin but an unusual


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metabolite, dihydroechinofuran, which is thought to be derived from geranylhydroquinone, a key intermediate in the biosynthesis of shikonin [64]. Different species from the same family were utilized as explant source to establish the plant tissue culture. Cell culture of Arnebia euchroma (Royle) Jonst., regularly synthesizes naphthoquinone pigments, produced naphtaquinones in higher levels with addition of methyl jasmonate [65]. Rapid induction and increased shikonin production was also obtained from suspension cultures of A. euchroma by combined fungal elicitation and in situ extraction [66]. A. euchroma cell lines able to accumulate shikonin up to 20% in dry biomass are presented [67]. The increase of the cell biomass of A. euchroma and shikonin derivatives may due to increase in the activities of peroxidase and PAL caused by the addition of the rare earth elements [68]. Studies on A. euchroma were quite promising for the production of red pigments. Therefore research focused on transfering A. euchroma culture to bioreactor for large scale production. When A. euchroma was grown in submerged/non-submerged airlift bioreactor periodically, 4.6%, w/w shikonin content and, 16.8 g/l cell dry mass were obtained in suspension culture [69]. The effects of elicitor extracted from Aspergillus oryzae (Ahlb.) Cobn mycelia and Ca2+ on the production of shikonin derivatives in O. paniculatum cell suspension cultures withn M 9 medium was analyzed. The rapid drop of cytoplasmic Ca2+ carries the elicitor signal and in turn regulates the biosynthesis of shikonin derivatives. Results suggested that Ca2+ plays a significant role in an early stage of the elicitation process of Onosma cells [70]. Shikonin derivatives were produced by a two-layer culture of L. erythrorhizon containing an organic solvent, which was made from paraffins and a fatty acid ester [71]. The same system also efficiently induced production of shikonin and alkannin derivatives in suspension cultured cells of Echium italicum [72]. Plant cell cultures of L. erythrorhizon in situ extraction by n-hexadecane and cell immobilization by calcium alginate gave higher specific shikonin productivities of 7.4 and 2.5 times respectively than those from the cultures of free cells without extraction [73]. Differentiated cells, mostly hairy root cultures were extensively used to produce secondary metabolites and specifically for naphtaquinones since they were mostly produced in roots of the plants. Effects of in situ extraction, fungal elicitation, a permeabilizing agent, and the oxygen transfer rate on shikonin production in transformed suspension and hairy root cultures of L. erythrorhizon were studied. Results showed that combined addition of n-hexadecane and extract of the fungus Penicillium as an elicitor seemed to result in a higher production of shikonin both in cell suspensions and transformed root cultures [74]. A. tumefaciens transformed L. erythrorhizon cell suspension cultures showed 2-3-fold increase at the accumulation of shikonin derivatives in sucrose-rich (C-rich) and nitrate-rich (N-rich) medium [75]. Hairy roots of L. erythrorhizon transformed with A. rhizogenes 15834 produced a large amount of red pigments culturing in root culture solid or liquid media with addition of adsorbents [76]. Hairy roots of L. erythrorhizon in Murashige-Skoog (MS) medium produced new brown benzoquinone derivatives instead of red naphthoquinone (shikonin) derivatives as the main secondary metabolites [77]. Shikonin pigments were yielded from hairy root cultures of L. canescens (Michx.) Lehm and A. euchroma using three strains of A. rhizogenes: ATCC 15834, LBA 9402, NCIB 8196 [78]. Established L. erythrorhizon hairy root system with A. rhizogenes is a suitable model system for molecular characterization of shikonin biosynthesis via reverse genetics [79]. The biosynthetic pathway to 4hydroxybenzoate (4HB), a precursor of the naphthoquinone pigment shikonin, was modified in L. erythrorhizon hairy root cultures by introduction of the bacterial gene ubiC (ubiquinone C) [80]. Shikonin production in shoot cultures of L. erythrorhizon was controlled by use of various culture media and light irradiation. L. erythrorhizon dark-inducible genes were isolated to investigate the regulatory mechanism of shikonin biosynthesis in cell suspension and hairy root cultures [81]. The dark-inducible gene LEDI-2 expressed in the root system of the intact plants when they were producing shikonin [82, 83]. The shikonin derivative content of p-fluorophenylalanine (PFP) resistant culture was two times higher than that of the control in BK-39 callus culture of L. erythrorhizon [84]. Besides adding different kinds of growth regulators and elicitors to the media, studies focused on using two different media, one is ‘growth media’ for increasing biomass and the second one is a production media for



producing pigments. Naphtaquinone production by Lithospermum cell cultures was induced by transferring the cells into the production medium. The study also displayed that addition of p-hydroxybenzoic acid increased pigment level in the production medium, phenolic compounds also increased when the cells produced shikonin [85]. Cell growth and formation of shikonin/derivatives in Onosma paniculatum cell culture was studied in B5 and M9, a production medium, addition with IAA and 6-benzylaminopurine (BAP). Only brassinolide (BR) addition (+BR/−IAA/−BAP) at 0.001–100 ppb in B5 medium significantly increased the cell fresh weight compared with a growth control while BR addition with IAA and BAP (+BR/+IAA/+BAP) in B5 medium slightly increased the cell growth at 0.01–0.1 ppb concentration [86]. Cell suspension cultures of L. erythrorhizon produced shikonin derivatives in significant levels when cultured in the production medium M9 in darkness, whereas light and ammonium ion inhibited shikonin production in LS medium. It has been determined that the production of naphtaquinone derivatives regulated by ubiA gene as well as enzymes in L. erythrorhizon [87]. Cell suspension cultures of L. erythrorhizon when cultured in shikonin production medium M9 produced a large amount of lithospermic acid B, a caffeic acid tetramer and shikonin derivatives, which were inhibited by 2,4-D or NH4+, whereas stimulated by Cu2+. In particular, blue light showed a stimulatory effect on lithospermic acid B production, while shikonin production was strongly inhibited [88]. The M9 medium showed beneficial effect on red pigments accumulation in comparison to LS medium in culturing hairy roots of L. canescens’ two cell lines. Biomass increase achieved by hairy roots of Lc-1A line was 10-fold and 27-fold, and for Lc-1D roots 2-fold and 8-fold in LS and M9 media, respectively [89]. Callus and suspension cultures of Arnebia densiflora were established after accomplishing the painstaking sterilization processes [90-92]. In order to increase the naphtaquinone derivatives, variety of elicitaion media were applied to the A. densiflora callus cultures. Aplication of M10 media displayed the best result followed by M9 and NH4+ free media for tthe production of the red pigments (Fig. 1) [93].

A

B

Figure 1: A. densiflora calli cultures: A) 15 day old culture in M9 and M10 media. B) 20 day old culture in M10 media.

Rosmarinic Acid Rosmarinic acid is an ester of caffeic acid and 3,4-dihydroxyphenyllactic acid. The biosynthesis of rosmarinic acid starts with the amino acids l-phenylalanine and l-tyrosine [94]. All eight enzymes involved in the biosynthesis were already characterized and cDNAs of several involved genes have been isolated. A cytochrome P450 cDNA was isolated by differential display from cultured cells of L. erythrorhizon, and the gene product was CYP98A6. The P450 was shown to catalyze the 3-hydroxylation of 4-coumaroyl-4Phydroxyphenyllactic acid in biosynthesis of rosmarinic acid [95]. Expression studies in yeast confirmed that the P450 clones encode Cinnamate-4-hydroxylase (C4H). The level of cinnamic acid 4-hydroxylase transcripts in the L. erythrorhizon callus culture was unaffected by environmental factors and not involved in the regulation of shikonin and rosmarinic acid biosynthesis [96]. A transient increase in phenylalanine ammonialyase and rosmarinic acid content in cultured cells of L. erythrorhizon was observed after addition of yeast extract to the suspension cultures [97]. Production of rosmarinic acid were studied in the past by biotechnological researches such as plant cell cultures, shoot culture, producing hairy root, using bioreactor and the treatment of elicitors [98]. It has been observed that plant cell cultures accumulate rosmarinic acid in amounts much higher than in the plant itself. L. erythrorhizon cells cultured in pigment production (M-9)


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medium, Lithospermic acid, a cafeic acid-rosmarinic acid conjugate, was isolated as a main constituent in Lithospermum hairy root cultures [99]. A cytochrome P450 cDNA, product of CYP98A6, was isolated from L. erythrorhizon cell suspension cultures. The data displayed that the expression level of CYP98A6 is dramatically increased by addition of yeast extract or methyl jasmonate to L. erythrorhizon cells, and its expression pattern reflected the elicitorinduced change in rosmarinic acid production [100]. Immobilized cells accumulated less than 15 μg rosmarinic acid g/1 [101]. Additionally, cytochrome P450-catalyzed hydroxylations of 4-coumaroyl-4hydroxyphenyllactic acid, in order to form rosmarinic acid, were dramatically up-regulated by the elicitor treatments in cultured cells of L. erythrorhizon [102]. Different methods and tools were applied to obtain naphtaquinone derivatives in significant amount. Differentiated cultures such as hairy root culture a promising field to produce and increase the seconder metabolites. The hairy root cultures of Coleus forskohlii was established in hormone-free MurashigeSkoog's (MS) medium to produce rosmarinic acid with methyl jasmonic acid as an elicitor [103]. Rosmarinic acid was produced in the callus culture of Echium amoenum, which was established from the seeds of the plant in MS media with three different ratios of plant growth regulatories: kinetin, 2,4-D and 1naphthylacetic acid (NAA) [104]. The rolC-transformed cell cultures of Eritrichium sericeum and L. erythrorhizon seedlings yielded two to three fold less levels of rabdosiin and rosmarinic acid than respective control cultures. That is irrespective of the methyl jasmonate-mediated and the Ca2+-dependent NADPH oxidase pathways [105]. Addition of yeast extract or methyl jasmonate to the cell suspension cultures of L. erythrorhizon drastically increased Isorinic acid 3-hydroxylase (IA3-H), which catalyzes a final step in the biosynthetic pathway leading to rosmarinic acid. The activity was in parallel with increased rosmarinic acid production, whereas addition of salicylic acid enhanced neither Isorinic acid 3-hydroxylase (IA3-H) activity nor rosmarinic acid production [106]. Polyphenol compounds consisting of caffeic acid oligomers, (-)-rabdosiin, (+)-rosmarinic acid and eritrichin, were detected in cell cultures of Eritrichium sericeum Lehm. The total content of polyphenol compounds as biotechnological raw material was 6.9% of dry tissue weight, which was 26.5 times greater than the content in the roots of the native plant [107]. Pyrrolizidine Alkaloids The N-oxides of pyrrolizidine alkaloids (senecionine or monocrotaline) are rapidly taken up and accumulated by cell suspension cultures of Symphytum officinale transporting N-oxides into the cells [108]. Hairy roots of L. canescens cultures were established using three strains of A. rhizogenes: ATCC 15834, LBA 9402 and NCIB 8196. Eight cell lines had biomass increase, moreover pyrrolizidine alkaloids (PA), canescine and canescenine, were found in all lines of transformed hairy roots [78]. Pyrrolizidine alkaloids were also synthesized by shoot cultures of Heliotropium indicum and A. rhizogenes transformed hairy roots cultures of Cynoglossum officinale as well as S. officinale [109]. 4. METABOLIC ENGINEERING Manipulation of shikonin biosynthetic pathway brought attention after defining and determination of the regulatory enzymes of the pathway. The enzymatic formation of p-hydroxybenzoate from p-coumarate in cell-free extracts of L. erythrorhizon cultures was investigated. P-coumaroyl-COA was detected as the activated intermediate in this biosynthetic reaction [110]. Northern analyses using total RNA of Lithospermum cell cultures demonstrated that the mRNA level of the L. erythrorhizon dark-inducible genes (LEDI-1) increased rapidly and kept high for a while when they were inoculated in M9 medium in order to initiate shikonin production, without any funga1 elicitation [111]. The ratio of the activities of p-hydroxybenzoic acid geranyltransferase and p-hydroxybenzoic acid glucosyltransferase regulates shikonin biosynthesis in L. erythrorhizon cell suspension cultures [112]. The activities of the biosynthetic enzymes PAL and 3-hydroxy-3-methylglutaryl-CoA-reductase (HMGR) were



measured in cell cultures of L. erythrorhizon in a two-stage batch culture. It has been determined that these enzymes transiently increase, thus production of shikonin/derivatives should have a positive relationship with level of PAL activity [113]. Endogenously occurring nitric oxide (NO) stimulates shikonin formation in Onosma paniculatum cells by up-regulating the expression of PAL, p-hydroxybenzoate metageranyltransferase gene (PGT) and HMGR, which encode key enzymes involved in shikonin biosynthesis [114]. Another research group was investigated the effect of enzymes under the influence of blue and white light. Regulatory roles of enzymes, 3-hydroxy-3-methylglutaryl-coenzyme A reductase, PAL, the effectors of methyl jasmonate and arsenate were analyzed in the biosynthesis of naphtaquinones and/or shikonin biosynthesis in cell suspension cultures of L. erythrorhizon. 2-Aminoindan-2-phosphonic acid, an inhibitor of PAL, completely suppressed the formation of acetylshikonin, p-hydroxybenzoic acid-O-glucoside and rosmarinic acid. Mevinolin, an inhibitor of HMG-CoA reductase specifically blocked the formation of acetylshikonin [115]. Endogenous oligogalacturonides capable of inducing the biosynthesis of naphthoquinone pigments, shikonin derivatives, in cell cultures of L. erythrorhizon [116]. Methyl jasmonate, in L. erythrorhizon cell suspension cultures, caused a rapid increase in the activities of enzymes involved in the biosynthesis of shikonin, which is similar to those elicited by oligogalacturonides [117]. Feeding of [l-13C] glucose to cell cultures of L. erythrorhizon revealed a labelling pattern in the isoprenoid part of shikonin consistent with a biosynthesis via the mevalonate pathway [118]. Research on localization and secretion of the naphtaquinone pigments in the cell cultures of L. erythrorhizon revealed that shikonin derivatives might accumulate in "secretion vesicles", which originate from the rough endoplasmic reticula [119]. The precursor p-hydroxybenzoic acid is stored in the form of a glucoside (p-O-β-D-glucosylbenzoic acid) when the cell of L. erythrorhizon are not synthesizing shikonin [120]. Intracellular localization of p-O-β-D-glucosylbenzoicacid and its aglycone, p-hydroxybenzoic acid was investigated in L. erythrorhizon cell cultures. p-hydroxybenzoic acid glucosylated in the cytosol was transported into the vacuole to be stored until utilization as a precursor upon induction of shikonin biosynthesis [121]. An NAD-dependent alcohol dehydrogenase has been purified to apparent homogeneity from cell suspension cultures of L. erythrorhizon using protamine sulphate and ammonium sulphate precipitation and chromatographical methods [122]. Two cDNA clones (L. erythrorhizon phenylalanine ammonia lyase 1 (LEPAL-1) and L. erythrorhizon phenylalanine ammonia lyase 2(LEPAL-2)) encoding PAL were isolated from cell suspension cultures of L. erythrorhizon, which are expressed mainly in the root of the intact plant [123]. A 4-coumaroyl-CoA 3-hydroxylase activity was purified from cell cultures of L. erythrorhizon, which are polyphenol oxidases rather than specific enzymes of secondary metabolism [124]. Two distinct enzymes geranyldiphosphate:4-hydroxybenztiate geranyltransferase and solanesyldiphosphate:4-hydroxybenztiate solanesyltransferase were measured in cell-free extracts from L. erythrurhizon cell cultures [125]. UDP-glucose: 4-hydroxybenzoate glucosyltransferase was purified to near homogeneity from cell suspension cultures of L. erythrorhizon, using ammonium sulphate precipitation, and column chromatography [126]. 3-hydroxy-3-methylglutaryl-coenzyme A reductase, a key enzyme of the mevalonate route to isoprenoids, plays a significant role in the regulation of shikonin biosynthesis in L. erythrorhizon cell-suspension cultures, partly derived from the isoprenoid biosynthetic pathway [127]. Geranyldiphosphate:4-hydroxybenzoate 3-geranyltransferase, is a regulatory enzyme in the biosynthesis of shikonin, produced cell cultures of L. erythrorhizon in LS medium. The activity of the enzyme enhanced after adding methyl jasmonat [128]. Geranylhydroquinone 3’-hydroxylase, which is likely to be involved in shikonin and dihydroechinofuran biosynthesis, was identified in cell suspension cultures of L. erythrorhizon [129]. Two cDNAs encoding geranyl diphosphate:4-hydroxybenzoate 3geranyltransferase were isolated from L. erythrorhizon by nested PCR using the conserved amino acid sequences among polyprenyltransferases. They were functionally expressed in yeast COQ2 disruptant and showed strict substrate specificity for geranyldiphosphate as the prenyl donor, suggesting that they are involved in the biosynthesis of shikonin [130]. In Vitro Regeneration Plant regeneration from in vitro cultures may be important as a source of secondary metabolite production. The establishment of a reliable procedure for regenerating plants is essential for the recovery of transgenic plants and the application of techniques of genetic engineering on compound production. Protoplasts


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isolated from cell cultures of L. erythrorhizon divided repeatedly and formed callus colonies in the protoplast-culture medium with glucose, and coconut milk rapidly [131]. The highest organogenic and embryogenic efficiency was obtained on MS medium supplemented with NAA and kinetin by cell lines (LE87) of L. erythrorhizon roots produced from shoots on plant growth regulator-free MS medium and developed into plantlets and regenerated plants being transferred to soil [132]. Shoots of Hackelia venusta were cultured on MS media supplemented with agar and benzyladenine (BA) produced axillary microshoots for reintroduction with minimal growth regulators [133]. Heliotropium indicum L. plantlets were regenerated in vitro from nodal and hypocotyl explants and also from hypocotyl callus, on (MS) medium supplemented with NAA, BAP, asparagine (Asp) and glutamine (Glu). The regenerated plantlets were rooted on MS supplemented with Glu or gibberellic acid [134]. The second most studied species of Boraginaceae A. euchroma coatless seeds achieved rapidly efficient callus by culturing in N6 solid medium containing kinetin (KT) and sucrose in darkness [135]. Leaf derived callus of A. euchroma showed simultaneous organogenesis in IAA combined with BA, whereas somatic embryogenesis occurred in indole-3-butyric acid (IBA) combined with BA [136]. Thidiazuron utilized for the induction of shoot organogenesis on cotyledon and hypocotyl explants of A. euchroma and maximal number of shoots was obtained on the modified LS medium [137]. Indirect somatic embryogenesis, encapsulation, and plant regeneration were achieved with Rotula aquatica Lour. Friable callus was developed from leaf and internode explants on MS medium with 2,4-D was most effective for the induction of somatic embryos [138]. Apical and nodal segments of Cordia verbenacea L. were cultured on MS medium supplemented with kinetin and NAA, showed useful to regenerate large populations of plants [139]. Callus and shoots were achieved through in vitro regeneration from Trichodesma indicum zygotic embryos on MS medium with kinetin, BA or NAA [140]. Boraginaceae is one of the largest plant family containing mostly endemic plants. Due to their endangered species, and valuable secondary metabolites mostly plant pigments in medicine, cosmetic and textile, biotechnological studies will continue with gradual increase. Those studies will probably bring valuable information for understanding the mechanism of secondary metabolite production in plants and utilizing them in health and industrial field. REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10]

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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, largescale 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: [email protected] Ilkay Erdogan Orhan (Ed) All rights reserved-© 2012 Bentham Science Publishers


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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


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 (year introduced)

Source Plant

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, longlived, 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


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Europe and the Mediterranean, varieties of different species in Asia have also been used as sources of taxanes. Nowadays, diterpenes (e.g. 10-deacetylbaccatin III) are extracted (from the leaves of Taxus baccata and other species) and converted by partial synthesis into structural analogues of paclitaxel, such as docetaxel (marketed as Taxotere™) [6]. (A) Catharanthus roseus

OH

(B)

CH2CH3

N

N H

N C

H3CO

O H H3CO

R: CH3 (Vinblastine) R: CHO (Vincristine)

CH2CH3

N

OCOCH3

H

OHCOOCH3

R

Figure 1: Catharanthus roseus (A) and chemical structure of its main compound, vinblastine/vincristine (B). (A) Taxus baccata

(B)

O

O

O

NH

O

OH

O H

O OH OH

O

O O O O

Paclitaxel

Figure 2: Taxus baccata (A) and chemical structure of its main compound, paclitaxel (B).

Colchicaceae. Colchicine and demecolcine are isolated from Colchicum autumnale (Colchicaceae) which is an attractive perennial with strap-shaped leaves and originates from Europe and North Africa (Fig. 3A). It emerges from a fleshy corm in spring, along with the fertilized young fruit produced in the former flowering stage. The main active compound “colchicine” (Fig. 3B) accounts for approximately 0.3–1.2% of dry weight of the corm, flower or seeds. It has formerly been used in cancer therapy, nevertheless today only its derivate “demecolcine” is employed as a cytostatic drug [6].



(A) Colchicum autumnale

OCH3

(B) H3CO

OCH3

H3CO

O NH O

Colchicine

H3C

Figure 3: Colchicum autumnale (A) and chemical structure of its main compound colchicines (B).

Berberidaceae. Podophyllotoxin is obtained from May apple, “Podophyllum hexandrum” and Pelargonium peltatum (Berberidaceae). May apple seems to be an unusual perennial plant with branched rhizomes spreading below the ground and a single pair of large, soft and deeply lobed umbrella-like leaves (Fig. 4A). The fleshy fruit is toxic when it is green, but edible when matures. The rhizome and roots contain up to 6% of a resinous substance known as podophyllum resin or podophyllin. This is a rich source (up to 50%) of podophyllotoxin, a lignan with antimitotic activity. Related compounds are - and -peltatins, deoxypodophyllotoxin and their glycosides. This traditionally used “podophyllotoxin” (Fig. 4B) as a strong purgative can be chemically converted into etoposide, teniposide and etoposide phosphate (Etopophos™), which are useful chemotherapeutics for the treatment of some malignancies such as testicular carcinomas and lymphomas. These semi-synthetic derivatives are believed to function as inhibitors of DNA topoisomerase II, that also act as mitotic spindle poisons in some cases [16]. (A) Podophyllum hexandrum

OH

(B)

H O O O H

H3CO

O

OCH3 OCH3

Podophyllotoxin

Figure 4: Podophyllum hexandrum (A) and chemical structure of its main compound, podophyllotoxin (B).


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Cornaceae. Camptothecin is primarily obtained from the Happy tree (Camptotheca acuminata (Fig. 3A) and related species “Cornaceae”), nonetheless it is also produced by some other plants such as Nothapodytes foetida, Pyrenacantha klaineana, Ophiorrhiza pumila [17]. For extraction of camptothecin, the stem wood, bark or seeds have been used, however now the young leaves are mainly utilized. Camptothecin (Fig. 3B), as a pentacyclic indole alkaloid, is the major compound of the extraction which is found about 0.1% in stem bark, 0.2% in root bark, 0.3% in seeds and 0.5% in young leaves. Notwithstanding the extraction of camptothecin from natural sources, the semi-synthetic analogues of this poorly water soluble compound have been also developed, including 9-amino-20S-camptothecine, irinotecan (also known as irinotecan hydrochloride trihydrate, CPT-11 or Camptosar) and topotecan (Hycamtin™). Extracted alkaloids or semi-synthetic derivatives (e.g. topotecan and irinotecan) are currently prescribed against some cancers [18]. (A) Camptotheca acuminata

O

(B) N N

O

Camptothecin

OH

O

Figure 5: Camptotheca acuminata (A) and chemical structure of its main compound, comptothecin (B).

5. PLANT CELL AND TISSUE CULTURE FOR PRODUCTION OF BIOACTIVE COMPOUNDS Currently, anticancer bioactive compounds and many other natural products are extracted in massive quantities from whole plant parts. The source plants are usually cultivated in tropical or subtropical, often geographically in a specific region. However, this approach is largely dependent upon many environmental factors including drought, disease and changing land use patterns. Furthermore, selection of high-yielding strains appears to be a difficult task and the long cultivation periods between planting and harvesting make the resultant bioactive compounds expensive [19]. Alternatively, anticancer compounds can be produced through a total chemical synthesis, semisynthesis using the isolated precursors or genetic engineering of plant pathways in microbial hosts, and plant cell culture. Each method offers distinct advantages and/or disadvantages depending on the specific system of interest. The total synthesis methodology is independent from the field-grown materials, however it demands use of hazardous solvents whose disposal appear to be very problematic. This approach typically results in low product yields in some cases such as production of paclitaxel. Further, transfer of biosynthetic genes into microbial hosts may favor the production of the anticancer compounds. It is considered to be environmentally friendly and inexpensive methodology that is also capable of genetic manipulation to increase yields. However, biosynthesis pathways of bioactive compounds are sometimes too complex for efficient transformation, and accordingly it is very difficult to reconstitute complete pathways within the microbial systems. Thus, the plant cell culture appears to be an efficient viable option, which provides an environmentally friendly alternative method for production of secondary metabolites. In preference to the extraction from natural sources, the culture condition can be



selectively adjusted towards demanded safety requirements as well as high production yields. This provides much better manipulation of the production process, by which the biosynthesis of key pathways can significantly and selectivity be controlled. The plant cell suspension culture has so far been successfully recruited for production of many anticancer bioactive compounds, most of which appear to be of note pharmaceuticals such as taxanes from Taxus sp., [20] terpenoid indole alkaloids (TIAs) from Catharanthus roseus [21]. Cell culture has also been investigated for production of other pharmaceutically active agents such as shikonin from Lithospermum erythrorhizon, berberine from Coptis japonica, and camptothecin from Camptotheca acuminate [22] . To date, various processes have been developed using plant suspension cultures as a result of the ease in scale-up and implementation towards mass production of herbal medicines both for known anticancer bioactive compounds as well as some unidentified anticancer bioactive compounds. This approach can be exploited for identification of important biosynthetic/metabolic pathway genes as reported for elucidation of the paclitaxel biosynthetic pathway [23]. Owing to the considerable pharmacoeconomic impacts of the plant based anticancer medicines in clinic, it is well clear that plant cell culture systems are important tools for development and mass production of these bioactive compounds. Accordingly, the plant cell and tissue culture systems are complementary approach that is believed to confer a robust platform for competitive metabolite production of the demanded pharmaceuticals in comparison with whole plant extraction or other chemically synthetic approaches [19]. 6. STRATEGIES AND TECHNICAL FRAMEWORKS FOR OPTIMIZED PRODUCTION OF SECONDARY METABOLITES For economic viability, the consistent generation of high yields of products from cultured cells seems to be an unquestionable fact, as a result of which it is important to develop reliable platforms for optimized productions of the bioactive anticancers. Such technology should ideally provide a robust framework for higher yields than that is normally found in intact plants. This clearly means that a careful selection of productive calli/cells and cultural conditions as well as some other factors should be considered during the setting up procedure. Nevertheless during cultivation, plant cell cultures usually lose their efficiency for generation of secondary metabolites, whose consistent productions are characteristic of the intact plant. Thus many researchers have focused on stimulation and/or elicitation of biosynthetic activities of cultured cells using various approaches to obtain efficient yields for commercial exploitation. Physical optimization. For optimized cultivation of plant cells/tissues, numerous chemical and physical parameters affecting culture system have been widely examined, including: media components, phytohormones, pH, temperature, pO2, pCO2, agitation and light among others. In 1962, Murashige and Skoog (MS) medium was recognized as superior for the production of secondary metabolites in the suspension culture of plant cells, although this is not always the case for other species in culture since some modifications should be applied for optimal yielding. Sucrose and glucose are the preferred carbon source for plant tissue cultures even though other carbohydrate sources are sometimes used. Likewise, we have used combination of several carbohydrates to control culture condition (our unpublished data produced by Khosroshahi and co-workers). In fact, the concentration of the carbon source may affect cell growth and yield of secondary metabolites (e.g. paclitaxel) in many cases. Hormones (e.g. auxins and cytokinins) levels have also been shown to impose the most remarkable effects on the growth as well as the productivity of plant metabolites. Accordingly, increased auxin (e.g. 2,4-dichlorophenoyacetic acid “2,4-D”) levels in culture medium promote dedifferentiation of cells and diminishes the accumulation of secondary metabolites. In fact, auxins added to solid media appear to stimulate callus induction, nevertheless low levels or removal of auxins from culture is recommended for high yield production. For example, cytokinins stimulated alkaloid synthesis when auxins were removed from the medium of a cell line of Catharanthus roseus [19]. Further, reduced phosphate levels often stimulate product accumulation. Nitrogen sources may also play a key role in product accumulation in plant cells. For example, ammonium ions are able to inhibit the synthesis of shikonin in Lithospermum erythorhizon, even though these ions promoted cell growth. It often seems necessary to change the nitrate ions concentration in media at the end of the growth phase [24]. So far the medium composition impacts on production of bioactive compounds


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have been well addressed, thus to characterize paclitaxel production we have studied the effects of some antioxidants (e.g. citric acid and ascorbic acid) and some carbohydrates (e.g., glucose, fructose and sucrose) on the tissue browning phenomenon and subsequent cultures for mass production of paclitaxel in Taxsus brevifolia subcultures. We found some interesting results upon impacts of these supplementary factors on growth, activities of polyphenol oxidase and peroxidase enzymes, browning and paclitaxel production [25, 26]. What is more, the gases (e.g. oxygen, carbon dioxide, and ethylene) are also important in plant cell culture systems. Oxygen is essential for cellular respiration and metabolism, while carbon dioxide is the main metabolic gas component in cell growth. Ethylene is believed to be a gas component produced by plants correspondence to protective response under provocation of environmental stress (e.g. wound, pathogen attack, or elicitation). The impacts of various concentrations and combinations of necessary gases have been investigated on cell growth and paclitaxel production in suspension cultures of Taxus cuspidata [27] with the results that highlighted a low headspace oxygen concentration (l0%, v/v) promoted early production of paclitaxel, while a high carbon dioxide concentration (l0%, v/v) inhibited its production. In fact, they found that the most effective gas mixture composition for paclitaxel production was 10% (v/v) oxygen, 0.5% (v/v) carbon dioxide, and 5 ppm ethylene. Such findings suggested that interaction between CO, and C, H, and between ethylene and methyl jasmonate (MJA) could significantly influence paclitaxel production. Given the fact that under osmotic stress cell physiology changes drastically, the effect of osmotic pressure on paclitaxel production was investigated in suspension cell cultures of Taxus chinensis [28] with the results showing production of the highest paclitaxel at an initial concentration of 60 g sucrose (300 mOsm/kg). High osmotic pressure conditions generated by non-metabolic sugars (e.g. mannitol or sorbitol) and the addition of a non-sugar osmotic agent (e.g. polyethyleneglycol) could enhance the production of paclitaxel in cell culture systems [29]. It looms that the manipulation of the media compositions and physical condition in a culture is the most fundamental approach for optimization of cell/tissue culture productivity. Indeed, various reports and patents have highlighted the importance of the optimization process of culture conditions, upon which improved growth rates of cells and/or higher yield of desirable products have been expected [30, 31]. For example, we have previously developed a 2-stage suspension cell culture of Taxus baccata using modified B5 medium to improve cell growth and productivity using various elicitors such as MJA, salicylic acid and fungal elicitor resulting in the highest amount of paclitaxel (40 mg/L). This led us to propose implementation of such improved process for mass production of paclitaxel [31]. We also have recently established an in vitro cell suspension culture of Echium italicum which was used to produce shikonin and alkannin derivatives. The callus tissues were induced using cotyledon explants incubated onto the solidified B5 medium and a two-liquid-phase system suspension culture was utilized to elicit pigments of shikonin and alkannin derivatives by means of liquid paraffin. We found that such bioprocess engineering approach can be used to induce shikonin and alkannin derivatives in cell suspension culture [30]. Addition of precursors. There exist great chances to boost the biosynthesis of specific secondary metabolites by addition of precursors to cell cultures. For example, addition of amino acids to cell suspension culture media was shown to exert productive effects on production of tropane alkaloids, indole alkaloids and other products. Such an impact could occur through direct or indirect (i.e. through degradative metabolism and entry into interrelated pathways) incorporation of the precursors towards production of the bioactive compounds. Likewise, addition of precursor of rosmarinic acid “Lphenylalanine” to the cell suspension cultures of Salvia officialis was reported to stimulate the production of rosmarinic acid, that was also able to decrease the time of production [19]. Interestingly, feeding callus and cell suspension cultures of Taxus cuspidata with L-phenylalanine and other potential paclitaxel sidechain precursors (e.g. benzoic acid and N-benzoylglycine) was shown to improve the production yield of paclitaxel. This may be due to the possible role of L-phenylalanine and other compounds in the biosynthesis of the N-benzoylphenylisoserine paclitaxel side chain. Besides, in suspension cultures of Taxus chinensis, a combination of an initial low sucrose concentration (20 g/L) and fed-batch mode improved both cell growth and taxane production [32]. Treatment of Taxus chinensis through intermittent feeding with sucrose (3%, l%, and 2% respectively on days 1, 7 and 21) resulted in paclitaxel production up



to 26 mg/L, while intermittent feeding with maltose (1% and 2% respectively on days 7 and 21) increased the paclitaxel production to 67 mg/L [33]. Thus, by controlling the combination of sucrose/maltose feeding and dissolved O2, paclitaxel production can be improved [34]. It should be also evoked that some evidences indicated critical importance of the precursor addition time for an optimum effect on production of bioactive compounds such as paclitaxel. Nevertheless, it seems that the effect of feedback inhibition must be considered when adding products of a metabolic pathway to cultured cells. Selection of high yielding cell lines. Basically, plant cells in culture represent a heterogeneous population wherein physiological characteristics of individual plant cells are different [19]. The selection process appears to be the foremost step of the bioprocess engineering since the strain improvement begins with the choice of a parent plant with high contents of the desired products. What is more, statistically, highproducing cell lines can be obtained from high-producing plants, nonetheless production levels in plant cells may vary. This means the isolation/selection method is often essential during cultivation since a major problem with plant cells is their inherent genetic and epigenetic instability. In addition, plant cells’ variability may lead to a diminished productivity in subcultures, which is believed to be attributed to genetic changes perhaps by mutation in the culture or epigenetic changes. Although the screening process itself is important for a desired cell selection from the heterogenous population typically present in plant cell cultures, the above mentioned issues can be resolved to some extent by some necessary alterations in the culture environment [24]. The techniques used in selection and screening plant cell for improved product synthesis have been previously well documented; readers are directed to see [35]. Cell selection and somatic cloning provide useful approaches for recovering cells that produce increased levels of secondary metabolites, nevertheless paucity of rapid screening methods often limits the application of screening processes. Further, a fluorescence assay was reported to be used for selection of high yielding cells in Taxus cell suspension cultures. Following cell sorting, single cell cultures can be used to establish new cell lines for further applications [36], that seems to be a better approach compare to the radioimmunoassay which is a time-consuming and costly method. In general, the cell selection approach through somatic cloning is useful when colored products can serve as morphologic visual selection cues [19]. Impacts of elicitors on yield improvement. Among all manipulation techniques for promotion of the productivity of secondary metabolites within plant cell cultures, implementation of elicitors is considered to be one of the most widely used modalities that has been shown to result in a drastic increase in product yield. The “elicitation” terminology refers to an improved production of bioactive secondary metabolites through enhanced biosynthesis paths induced by molecules called elicitors that can be originated endogenously or added to the culture system exogenously. Therefore, based upon their origin, they are classified as “biotic” and “abiotic” [37]. For a “biotic elicitor”, the primary reaction is deemed to be via recognition of the elicitor and its binding to a specific receptor protein on the plasma membrane followed by inhibition of plasma membrane ATPase resultant in reduced proton electrochemical gradient. The biotic elicitors could be the polysaccharides (e.g. pectin or cellulose) derived from plant cell walls; microorganisms based chitin, chitosan, or glucans, as well as glycoproteins, and low-molecular-weight organic acids. Abiotic elicitiors include a diverse range of physical elicitation such as ultraviolet irradiation, salts of heavy metals, and chemicals additives that disturb membrane integrity [38] For example, improved production of paclitaxel in plant cell cultures by some heavy metals (e.g. lanthanum, cerium, and silver) have been reported [29]. Jasmonic acid (JA) is an important plant stress signaling molecule. It induces the biosynthesis of defense proteins and protective secondary metabolites. These fatty acid based derivatives with a 12-carbon backbone are synthesized from 18-carbon intermediates via the so-called octadecanoid pathway. JA and its volatile derivative MJA collectively called jasmonates appear to be widely successfully utilized for production of secondary metabolites [39]. The effect of jasmonates on secondary metabolism have been well addressed for production of paclitaxel [31] and alkaloid biosynthesis, whose gene expression pattern is yet to be fully understood despite some recent evidences upon gene activation. Yukimune et al. (2000) reported that MJA treatment can provide great improvements in both cost and efficiency over the use of undefined fungal extracts, for instance 100 µM MJA increased the paclitaxel level from 0.4 to 48 mg/L in


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Taxus baccata [40]. Cell cultures of Taxus chinensis in bioreactor have resulted in significant enhancement of taxoid production via repeated elicitation using MJA [41]. The MJA-based elicitation may induce the activities of some major enzymes within pathway of the taxane biosynthesis, in which important enzymes such as geranylgeranyl diphosphate synthase (GGPP synthase) and taxadiene synthase have been identified [29]. In 1991, for the first time, christen and coworkers [42] reported the usefulness of fungal elicitors and other selected compounds for the production of paclitaxel in the cell suspension cultures of Taxus brevifolia (U.S. patent 5019504). Nonetheless, based on our data and those of others, it has been found that oxidative stress caused by treatment with a fungal elicitor can lead cells towards a programmed cell death, resulting in low production of paclitaxel [31, 43]. Further investigations of the underlying mechanisms seem to be necessary for understanding these phenomena as well as signal transduction processes in elicitor-induced synthesis of secondary metabolites in plant cell cultures [29]. Immobilization of cells. The plant cell immobilization (PCI) technique confers some benefits such as continuous process operation, reuse of biocatalysts, and separation of growth and production phases. Above all these, a simplified separation of biocatalysts from the culture medium provides not only a product orientated optimization of the medium but also a reduction in processing time of cultivation periods [24]. In comparison with plant cell suspension cultures, the PCI methodology held adherent plant cells (i.e. immobilized biocatalysts) which provide many advantages [44]. The PCI technology confers an extended viability to the cells in the stationary and producing stage, a better maintenance of biomass over a prolonged time period, a simplified downstream processing, a high cell density within relatively small bioreactors, a reduced shear stress, an increased product accumulation and a minimized fluid viscosity in cell suspension. All these advantages are believed to improve the yielding while granting simplicity and ease of handling processes. Such technology has been utilized for production of L-asparaginase which is deemed to be a promising chemotherapeutic agent [45]. It should be literally highlighted that the biosynthetic potential of normally slow growing plant cells appear to grant a better culture system for plant cells immobilization technique since such systems provide higher yields of bioactive compounds [19]. Gel entrapment methods. Among methods used for immobilization of cultured plant cells, gel entrapment looms to be one of the promising approaches, in which calcium alginate gel is prevalently used for the immobilization. The first report upon exploitation of this technique was immobilization of cultured plant cells using calcium alginate gel to cultivate Catharanthus roseus cells. Using such methodology, cell viability can be improved during the cultivation period, in which the calcium alginate are able to entrap with minimal biological impacts and cells are literally prepared by suspending in a solution of sodium alginate which is then subjected to extrusion through an appropriate small diameter opening. The resultant gel can be converted to the alginate beads through introduction of the gel to a solution of calcium chloride (50-100 µM) and the entrapped or immobilized cells can be recovered by chelation of the calcium ion using EDTA or polyphosphates [46]. It should be stated that, however, the cell viability appears to be a bit poor when some toxic materials (e.g. acrylamide and glutaraldehyde) are used and the continued growth of cells in the beads may also disrupt the integrity of the gel, at which the process can be interfered. The gel entrapment is conceivably considered as a useful technique when we deal with some non-dividing cells in a nutrient or hormone-limited liquid medium [47]. Nevertheless, this technique appears to be either expensive or laborious for large scale application which is associated with some disadvantages, in particular the growing cells tend to rupture the confining matrix and cells are not easily recovered during extended cultivation. Thus, the technique has not been applied commercially for the production of plant derived bioactive compounds. Surface immobilization. The cultured plant cells possess propensity to adhere to the inert surfaces, thus the “surface immobilization” technology takes advantage of such biological functionality which is a feature common to the most of cell suspension cultures although it may complicate process control. For example, the surface immobilization of Catharanthus roseus and Taxus cuspidata cultured cells has also been reported [48].



In practice, a porous ceramic matrix is used to effectively retain the cells. Accordingly, rigid but porous materials (e.g. acrylic or rayon fibers bound by phenolic resin, and a series of non-woven polyester or polypropylene textile) have been reported to be effectively used for immobilization process. Among various advantages of this methodology, some important issues should be emphasized including: a) the surface tension and electrostatic properties can be controlled; b) the downstream processing for excreted products (e.g. proteins and bioactive compounds) can be improved; c) the processing cost can be lowered via reuse/recycling process; d) rapid immobilization of high density and large-scale cultures can be achieved in bioreactors [49]. Such process seems to be relatively rapid and efficient under physiological conditions, that is also a firm and irreversible process which is able to be maintained for a designated longer period. The use of glass fiber mats for cell immobilization was tested in a bench scale, in which alkaloids such as berberine and columbamine were produced [50]. It was found that the accumulation of indole alkaloids and tryptamine, by glass-fiber immobilized cells of Catharanthus roseus was lower than that of freely suspended cells. Overall, the glass-fiber immobilized cells yield decreased production of bioactive compounds even though it is more applicable compare to the gel entrapment technique since it demonstrates industrial potential of the system [19]. Two-phase cultivation. Secondary metabolites produced by the plant cells are either intracellular or extracellular. Thus, some critical issues regarding culture condition should be taken into consideration. Of these, the two-phase plant cell cultures enable us to exploit a partitioning system for redirection of the extracellular products into a non-polar phase. Having utilized such methodology, low levels of secondary metabolites are accumulated in culture media since various factors (e.g. solubility, feedback inhibition, and degradation) can be easily manipulated towards higher yields. In fact, removing metabolites from an aqueous medium can alter the intracellular/extracellular equilibrium to favor the production yield. For example, it has been reported that application of amberlite XAD adsorbent resins to cell cultures of Taxus cuspidata on day 16 can significantly increase the biosynthesis of paclitaxel [51]. Given the difficulties encountered in adding and removing solid sorbents, organic solvents are seen as alternatives for the recovery of secondary metabolites. Recently, we have successfully employed parafin in a two-liquid-phase suspension culture of Echium italicum for improved production of shikonin and alkannin derivatives [30]. These kinds of liquid phases have included glycerol derivatives and compounded silicon oils. Similarly, Wang et al. [52] has reported that addition of dibutylphthaltate (10% v/v) during the late-log phase may favor paclitaxel production. Despites these remarkable findings, it is deemed that more detailed investigations have to be accomplished to address the possible interactions between sorbents/solvent and cells/ media. Two-stage cultivation. Two-stage suspension culture appears to be a similar methodology to the two-phase cultivation, but in fact, during this process the cultured cells itself undergo two different culture conditions and/or compositions. For example for production of paclitaxel, two-stage suspension culture of Taxus media cells was successfully implemented in a small bioreactor using a cell-growth medium for 12 days (the first stage) and a production medium supplemented with the MJA and two putative precursors, mevalonate and N-benzoylglycine (the second stage) resulting in approximately 22 and 56 mg/L of paclitaxel and baccatin III, respectively. To improve cell growth as well as productivity of Taxus baccata, we have also developed a two stage suspension cell culture using modified B5 medium as follow: at the first stage, the B5 medium was supplemented with vanadyl sulfate (0.1 mg/l), silver nitrate (0.3 mg/l) and cobalt chloride (0.25 mg/l) on day 1, with sucrose (1%) and ammonium citrate (50 mg/l) on day 10 and sucrose (1%) and phenylalanine (0.1 mM) on day 20; at the second stage, different concentrations of several elicitors such as MJA (10, 20 mg/l), salicylic acid (50, 100 mg/l) and fungal elicitor (25, 50 mg/l) were added to the medium. At stage II, treated cells with MJA (10 mg/l), salicylic acid (100 mg/l) and fungal elicitor (25 mg/l) produced the highest amount of paclitaxel (39.5 mg/l), which is 16-fold higher than that of untreated B5 control (2.45 mg/l). Thus, we suggested that the exploitation of this two stage methodology can be used for mass production of paclitaxel [31]. Further, to optimize a suspension culture of Taxus chinensis, Choi et al. studied the effects of temperature shift on the cell growth and paclitaxel production. They found that the cell growth was optimum at 24°C, while paclitaxel synthesis reached its


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maximum at 29°C. Thus, shifting the temperature to 29°C after maintaining the culture at 24°C for a certain time resulted in drastic increased in paclitaxel production, i.e. reaching the maximum of 137.5 mg/L (average productivity of 3.27 mg/L per day), reader is directed to see [53]. Permeabilization of plant cells. Permeabilization of plant cell membranes appear to be an essential step for the release of secondary metabolites, which is associated with the loss of viability of the plant cells because of application of permeabilizing agents. Such artificially induced release of intracellular retained products seems to be the palpable difficulty in this processing step, where various methods are basically applied to trigger the release of bioactive compounds from cultured plant cells. Of the physical and chemical methods used for permeabilization of plant membranes, the widely used strategies include: application of eletroporation, ultrasonic ultra-high-pressure, solution of high ionic-strength, change of external pH, transfer to media lacking phosphate, dimethylsulfoxide and chitosan. In fact, the intracellularly stored products are often excreted by the cells corresponding to immobilization per se. Cell permeabilization depends on the formation of pores in the membranes of the plant cells, enabling the passage of various molecules into and out of the cell. To avoid/minimize the cell damage and to release the metabolite in a short time period of cultivation, huge challenges have been directed towards transient permeabilization of the plant cells [54]. For example, the polycationic polysaccharides (e.g. chitosan) are able to induce pore formation only in the plasmalemma of the plant cell cultures such as Chenopodium rubrum, by which the leakage can performed from a relatively small compartment. However, its use for a longer period or with higher concentrations may cause an increase in pigment release leading to the cell death of plant cells. Further, since the highly charged chitosan polymers can induce a higher degree of pore formation in plasmalemma and causing faster release, it seems that there exists a significant correlation between the charge density and the loss of cell viability. The addition of pectin to chitosan-treated cell cultures had positive effects on cell growth and secondary metabolites accumulation in some plant cells such as Chenopodium rubrum. Although the additional interphasic membrane of pectin and chitosan may play as an additional barrier to mass transport of substances with larger molecular weights, its use have been shown to exert protective effects too [24]. Hairy roots’ culture. For decades, huge attentions have been devoted for the production of important secondary metabolites from hairy roots which develop as the consequence of the interaction between Agrobacterium rhizogenes (a gram-negative soil bacterium) and the host plant. In practice, for generation of hairy roots, wounded plant tissues are basically inoculated with Agrobacterium rhizogenes, which transmits the transferred-DNA (T-DNA) which harbor the loci between the left region (TL-DNA) about 1520 kb, and the right region (TR-DNA) about 8-20 kb of the root-inducing (Ri) plasmid into the plant genome. The rhizogenic strains contain a single copy of a large Ri plasmid [55]. The T-DNA carries the “rol” and the “aux” genes that are respectively responsible for the phenotype of hairy roots and the root induction [56]. High yield production of secondary metabolites appears to be due to the hairy roots’ uncontrolled division induced by rol genes and a rise in the number of root hairs with a higher ratio of metabolically active meristem tissues. The transformed roots are auxoautotrophic and vigorous which provide robust and stable cultures in hormone-free media, whereas the ordinary root cultures often require a balanced supplement of auxins and cytokinins to maintain growth and phenotype. Besides, such robust rapid growing hairy roots were shown to be exploited as a continuous source for the production of anticancer bioactive molecules [56]. For example, production of some plant derived anticancer agents such as comptothecin vinblastine, and ginsenosides that are literally root originated agents can be efficiently accomplished using such promising approach [17]. Altogether, it is now becoming more obvious that the hairy root technology is not only a strong option for the bench scale production of plant derived pharmaceuticals but also large scale industrial production is achievable by this technology. Adventitious roots’ culture. The adventitious root cultures represent good potential for production of various valuable biological materials, by which different bioactive compounds can be produced without any need to foreign gene(s). The adventitious root cultures display higher stability in the physicochemical conditions, as a result of which large quantities of secondary metabolites can be accumulated and easily isolated. Having expressed their in vitro photosynthetic capabilities, these roots can be simply cultivated in a medium with low inoculums resulting in high growth rate [56].



The adventitious roots cultures in bioreactors have so far been successfully used for production of anticancer secondary metabolites such as ginsenosides and antioxidant α-tocopherol. For example, enhanced production of the bioactive anti-cancer protopanaxatriol group of ginsenosides has been reported using cultured adventitious roots of Panax ginseng in bioreactor, in which addition of the biogenetic precursor “squalene” appeared to improve the production process. Using analytical methods, higher accumulation of the protopanaxatriol group of ginsenosides was found in cultures of 40 days old squalenefed adventitious roots compared to controls [57, 58]. Biotransformation. Cultivated plant cells possess great biochemical capability for biotransformation of the exogenously supplied compounds, for which the enzymatic potentials of cultured plant cells are exploited for bioconversion purposes. Such biological characteristics offer a broad potential for in vitro modification of natural and synthetic chemicals, where plant enzymes catalyze different reactions such as hydroxylation, oxidation of hydroxyl group, reduction of carbonyl group, hydrogenation of carbon–carbon double bond, glycosyl conjugation, and hydrolysis [59]. Using this approach, anticancer drug “vinblastine” has been produced from catharanthine and vindoline [60]. Further, bioconversion of taxadiene ring which is a precursor of paclitaxel has been shown using the cultured cells of Ginkgo biloba The product exhibited good result in co-administration with paclitaxel in treatment of paclitaxel-resistant human non-small cell lung cancer [59]. In fact, the biotransformation processes along with addition of substrates into cultures of appropriate cell lines confers a good in vitro platform which is perhaps one of the most commercially realistic approaches even though in some cases the costly precursors may limit the economic viability of the methodology to some extend [19]. Despite co-exploitation of the biotransformation and the immobilization of plant cells for improved production of bioactive compounds, a few samples have so far been solely reported base upon the biotransformation of foreign substrates using the immobilized plant cells [61]. Utilization of the immobilized plant cultured cells along with the biotransformation of exogenous substrates may provide some advantages. The immobilized cells can be protected from shear damage, used repeatedly over a prolonged period, while higher concentrations of biomass are feasible. Further, it facilitates recovery of the cell mass and products, while sequential chemical application is practical [61]. Biosynthetic pathway analysis and genetic manipulation of plant cells. For further improvement of high yielding cell line in plant cell culture, one needs to identify and efficiently manipulate the genes encoding the critical and rate limiting enzymes in the pathway. This process includes isolation, characterization, and reordering of genetic material as well as the transfer of the designated genes to the desired organisms. For instance, successful transformation of Arabidopsis thaliana was accomplished by some researchers with the aim of production of taxadeine synthase which is the first crucial enzyme in paclitaxel biosynthesis pathway [62, 63]. To date, there have been important advances based upon the identification of the biosynthetic pathways of plant based anticancers such as paclitaxel and comptothenin. However, by virtue of the regulatory modulation of such pathways, we need literally to perform new researches for enhanced accumulation of secondary metabolite compounds using engineered/transgenic plants. This will help us to clarify the undefined secondary metabolite biosynthetic pathways, which need implementation of some proof of concept steps. Nevertheless, we still need to resolve some pitfalls which are perhaps due to existence of multiple rate-influencing steps, unsubstantial genomic information of many plants, weakness of transformation techniques, and variability in secondary metabolite product. Since many secondary metabolite biosynthetic pathways remain partially undefined, a range of methods have been used for identification of the designated genes which encode the biosynthetic pathways, while pathway regulation, mutant selection, gene silencing, and gene overexpression via genetic engineering also are important issues to be considered.


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Now, the question is how efficiently can genetic engineering be used to improve the production of secondary compounds such as paclitaxel and camptothecin? Still surprisingly there is no reliable method for gene transfer. Genetic transformation of Taxus sp. cell suspensions with putative rate influencing genes has not been successfully accomplished, nonetheless Agrobacterium mediated transformation protocol has recently been established for Taxus sp. suspension cultures with stable transformation for up to 20 months [64]. Furthermore, given the complexity of paclitaxel biosynthesis pathway, the expression of the complete taxane biosynthetic pathway genes in alternate hosts is highly unlikely to be plausible. Nevertheless, direct genetic engineering in Taxus sp. seems to be the promising methodology to increase yielding [23]. The biosynthetic pathway of camptothecin is not fully understood, thus metabolic engineering in source plants appears to be an intricate mission. In fact, several genes of some part of camptothecin biosynthetic pathway have been identified in Carpentaria acuminata and Ophiorrhiza pumila [17]. The transformation and regeneration of transformed plants, via hairy root induction by Agrobacterium, suggest that genetically modified camptothecin producing plant is a possible approach regardless of the complexity of such methodology. The overexpression of camptothecin encoding genes in Catharanthus roseus seems to increase only intermediate products, but accumulation of the end product loom to be difficult to obtain. It is deemed that implementation of an inducible promoter may result in successful engineering of the indole pathway of Catharanthus roseus hairy roots [65]. Figs. 6 and 7 show biosynthetic pathways of paclitaxel and vinblastine, respectively. It has been reported that the fungal elicitor and jasmonates can enhance expression of several important key biosynthetic genes (e.g. strictosidine synthase (STR), tryptophan decarboxylase (TDC), cytochrome P450 reductase (CPR) in Catharanthus roseus resulting in enhancement of terpenoid indole alkaloids (TIAs) biosynthetic pathway [66]. In contrast to Catharanthus roseus, it has been shown that the expression of STR, TDC and CPR from O. pumila was not induced by fungal elicitor and jasmonate, suggesting the difference in regulation mechanism [67]. However, we have shown that these elicitors can be successfully applied for production of paclitaxel in Taxus baccata [31]. It should be evoked that the isolation of transcription factors that recognize promoter region of paclitaxel or camptothecin biosynthetic genes would be an interesting approach, in which manipulation of transcription factor expression might lead to improved production of related secondary metabolites in the plants [18]. 7. BIOREACTORS FOR SCALE UP PRODUCTION OF ANTICANCER BIOACTIVE COMPOUNDS Bioreactor technology. Taking advantages of low cost and high effective technology for production of natural products, the next main mission would be a translational approach to shuttle a bench scale product designs to an industrial large scale production. This is a keystone step, by which ideally high-tech biopharmaceuticals from plant organ should be reproducibly generated to provide a safe and economically competitive anticancer bioactive agents. To attain such translational modality, the bioreactor culture is deemed to be the key step that was shown to offer various advantages over conventional culture procedures. It can be automatically implemented to save labor and reduce production costs since it is easy to control the culture conditions (e.g. temperature, pH, and concentrations of oxygen, carbon dioxide and nutrients in medium) in a bioreactor. Above all these, we can also use an on-line medium circulation to maximize the uptake of necessary nutrients by the cultures, by which the cell proliferation and regeneration rates can also be increased. Thus, bioreactor is a useful technology for large scale cultivations of anticancer bioactive compounds because of its reduced cost and time as well as its potential for controlling the production consistency among various products batches without significant variations and geographical constraints [56] Routinely used bioreactor for production of anticancer bioactive compounds. For achievement of high yield production of bioactive compounds in bioreactor, one should optimize the production process by choosing appropriate bioreactor types, where some important key issues such as flow, shear pattern, mixing efficiency and oxygen transfer should be taken into consideration. For example, it is tremendously vital to monitor shear forces beforehand to predict how they will change vis-à-vis cultivation factors and bioreactor efficiency, and how the design of various parts (e.g. spargers, impellers and baffles) of bioreactors will



impact on these forces. In fact, when choosing the most suitable bioreactor type various issues (i.e. the morphology, rheology, shear tolerance, growth and production behavior of the culture) should be taken into account [7]. Following sections will provide some brief information upon different types of bioreactors.

OPP

OPP

Farnesyl diphosphate Isopentenyl diphosphate

GGPPS Geranylgeranyl diphosphate TS Taxa-4(5),11(12)-diene TYH5α Taxa-4(20), 11(12)-dien-5-α-ol TAT Taxa-4(20),11(12)-dien-5α-yl acetate TYH10b Taxadiene-5α,10ß-diol monoacetate 2-Debenzoyltaxane TBT 10-Deacetylbaccatin III DBAT

O

O

O

OH

H

HO OH

O

Baccatin III

O O O O

β-Phenylalanine baccatin III O

O

O

NH

O

OH

O H

O OH OH

O

O O O O

Taxol Figure 6: Paclitaxel biosynthesis pathway. GGPPS: Genranylgenranyl diphosphate synthase; TS: taxadiene synthase; TYHa: taxadiene 5α-hydroxylase; TAT: taxa-4(20), 11(12)-dien-5a-ol-O-acetyltransferase; TYHb: taxane 10βhydroxylase; TBT: taxane 2a-O-benzoyltransferase; DBAT: 10-deacetyl baccatin III-O-acetyltransferase. PAM: phenyalanine aminomutase.


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Glyceraldehyde 3-phosphate

Pyruvate

Shikimate pathway

DXS Geranyl diphosphate

Chorismate

Geraniol

AS

G10H CPR 10-Hydroxy-geraniol

Anthranilate

Loganin

Tryptophan TDC

SLS

Tryptamine

Secologanin

STR Strictosidine

SGD Cathenamine

Ajmalicine

Catharanthine

Tabersonine T16H 16-Hydroxy tabersonine D4H Deacetyl vindoline DAT

PRX1

Vindoline OH

CH2CH3

N

N H

N C

H3CO

O H H3CO

N H CH3

CH2CH3 OCOCH3

OHCOOCH3

Vinblastine Figure 7: Biosynthetic pathway of vinblastine in Catharanthus roseus. AS: anthranilate synthase, CPR: cytochrome P450 reductase, D4H: desacetoxyvindoline 4-hydroxylase, DAT: acetyl-CoA:4-O-deacetylvindoline 4-Oacetyltransferase, DXS:D-1-deoxyxylulose 5-phosphate synthase, G10H: geraniol 10-hydroxylase, PRX1: peroxidase 1, SGD: strictosidine b-D-glucosidase, SLS: secologanin synthase, STR: strictosidine synthase TDC: tryptophan decarboxylase, T16H: tabersonine 16-hydroxylase, adapted and modified from [68].

Stirred-tank bioreactor. One of the most important bioreactor types appears to be the stirred-tank bioreactor that is widely used for commercial production of anticancer bioactive compounds. The main advantages of these bioreactors are their potential to provide high volumetric mass transfer in a homogeneous environment. This confers an easy control on the production of anticancer bioactive compounds. The mixing “impeller” system is the most important part of the stirred-tank bioreactor. Of the mixing systems, the “Rushton turbine” is the most frequently employed for microbial fermentation because of its ability for



a complete dispersion of solids and gas as well as the sufficient oxygen mass transfer. Due to its radial flow pattern, it may result in high turbulence around the impeller region in comparison with the other impellers with axial flow patterns (e.g. helical ribbon impellers, paddle impellers, marine impeller, pitched-blade impellers), for more details reader is directed to see [69]. Thus, due to the resultant shear stress even at the minimum agitation speed, the plant cells may be damaged [69]. In general, the impeller systems exhibit slow-moving axial flow patterns with low tip speed (i.e. up to 2.5 m/s), upon which the longevity of the cultivated cells can be improved with less cellular damages. This is why they are considered as a suitable and conventional mixing system for plant cell cultures [70]. Having employed the stirred tank bioreactor, the cultivation of Taxus chinensis for 30 days resulted in 18.5 g/L of the dry cells weight and 20.2 mg/L of paclitaxel concentration [71]. What is more, under low shear conditions in batch and fed-batch modes of operation, a 3-1 stirred-tank bioreactor was reported to be successfully used for growth of the Podophyllum hexandrum cells to produce podophyllotoxin which is the precursor for etoposide, teniposide and etopophose. As a result, an improved production of podophyllotoxin (up to 48.8 mg) in a cell culture of Podophyllum hexandrum was reported when the reactor was operated in continuous cell-retention mode [72]. Pneumatic bioreactor. A pneumatic bioreactor (e.g. bubble column, air-lift and modified air-lift) is a type of gas–liquid dispersion bioreactor. They basically consist of a cylindrical vessel, in which compressed air/gas mixture is introduced at the bottom of the vessel through nozzles, perforated plates or a ring sparger. This process is deemed to facilitate the aeration, mixing and fluid circulation, without moving mechanical parts. The air-lift and modified air-lift bioreactors contain a draft tube as “internal loop” or “external loop” which confers the following advantages: a) prevention of bubble coalescence, b) improvement of oxygen mass transfer, c) uniformly dissemination of shear stress, and d) promotion of the cyclical movement of fluid which provides a shorter mixing time. However, these types of bioreactors have also some pitfalls when one deals with a high cell density culture of plant cells. Such problems include the inadequate oxygen mass transfer and poor fluid mixing, which may result in somewhat inhomogeneity in terms of various factors such as biomass, nutrient, oxygen and pH, and extensive foaming. This appears to negatively enforce the whole operation process of the pneumatic bioreactor. For operation of plant cell cultures at moderate levels of biomass concentrations, however, this technique is the one of the preferred systems because of low operational cost, efficient fluid circulation (using internal or external recirculation loops), simplicity of the scale up process, and adequate oxygen mass transfer by increasing the gas bubble–liquid interfacial area. Further, these relatively low shear stress resultant pneumatic bioreactors are deemed to particularly desirable for shear-sensitive plant cells [70]. Thus, it should be considered that this type of bioreactor is mainly used for production of anticancer bioactive compounds from hairy root rather than other kind of plant tissues or cells. Using this approach, suspension cultures of Taxus wallichiana were grown in a 20-L airlift bioreactor running for 28 days in a batch mode. The growth rate and capacity to accumulate paclitaxel and baccatin III were measured when cultures were in the highest productive state (i.e. from day 24 to day 28). Taxus wallichiana culture showed a great accumulation of paclitaxel (21.04 mg L−1) and baccatin III (25.67 mg L−1) [73]. Disposable bioreactor. Having aimed to minimize validation efforts and production costs, several disposable bioreactors have so far been developed for cultivation of plant cell cultures. Of these disposable bioreactors (e.g. life reactor, ebb-and flow-bioreactor, plasticlined bioreactor, wave reactors, Nestlé’s wave and undertow bioreactor, and slug bubble bioreactor), the wave reactors are suitable for cultivating highly shear-stress sensitive plant cell lines since there is no direct mechanical agitation [74]. Recently, high levels of paclitaxel productivity have been observed in Taxus baccata cells cultured in the wave bioreactor “BioWave” [75]. Another two new types of disposable bioreactors (i.e. the wave and undertow bioreactor; the slug bubble bioreactor) were reported by the Nestle R&D Centre in Tours, France. Their working volumes were at a range from 10 to 100 L, providing a wave/undertow motion that ensures mixing and bubble-free aeration of the culture [76]. The slug bubble reactor provides working volumes ranging from 10 to 70 L, in which bottom-to-top moving large cylindrical bubbles are generated for aeration. [77].


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Bioreactor design considerations. During processing of plant cell suspension cultures in a designated bioreactor, a number of cell culture operational variables such as bioactive compound productivity and quality, and cell growth need to be monitored, optimized and controlled. The distinctive biological and physiological characteristics in suspension cultures often limit their application in large-scale production; hence such biological, morphological and physiological properties of plant cells in suspension culture as well as the bioreactor engineering considerations should be taken into account. Of the most growth influencing factors, nutrient uptake and production kinetics, oxygen and heat transfer, and fluid hydrodynamics must be altogether adjusted in a well-designed and effective bioreactor to get the maximal output [70]. Here, in the following paragraphs, we provide some crucial information on these factors impacts on the whole bioprocessing matter. In addition to the nutrients dissolved in the culture medium, oxygen is the most important gaseous substrate required for in vitro cellular growth and aerobic metabolism of suspended plant cells cultures. In fact, the oxygen uptake rate can be used as an indicator for monitoring the physiology of plant cells during suspension culture in a bioreactor. Basically, a typical oxygen uptake rate value for plant cells is about 5–10 mmol/L per hour depending on the cell density and the cell type. Due to slow metabolism process, the oxygen demand of plant cells for cell growth is relatively low. However, the volumetric productivity within the high cell density plant cell culture has been shown to be dependent upon oxygen mass transfer in bioreactors. In fact, an inadequate oxygen mass transfer resulting from the high viscosity of the cell culture broth and dissolved oxygen concentration may negatively affect the outcome of the end-point bioactive compound production. However, it should be evoked that high aeration rates may result in inevitable extensive foaming problems and gas-stripping effects that are able to impose some conflict with plant cell growth and accordingly the production of desired secondary metabolites. This can be exacerbated in some bioreactors such as pneumatically agitated bioreactors [70]. For example, in a bench scale investigation, Mirjalili and Linden studied the effects of different concentrations and combinations of oxygen, carbon dioxide and ethylene on the cell growth and paclitaxel production in suspension cultures of Taxus cuspidate. They found that 10% (v/v) oxygen, 0.5% (v/v) carbon dioxide, and 5-ppm ethylene provided the most effective gas mixture composition regarding Paclitaxel production [78]. Besides, “cell aggregation” and “rheological properties” of a cell suspension culture may impose somewhat complications during the cell culture process. In general, plant cells in suspension culture tend to form aggregates and large clumps. The failure of the daughter cells to separate from the parent cells after division and the secretion of extracellular polysaccharides are deemed to be responsible for the cellular aggregation. Size distribution of aggregates is largely dependent on some key factors such as the type of plant cells, method of inoculums preparation, medium composition, culture conditions and bioreactor types. Under the formation of cell aggregates, the inner cells of the aggregates may become nutrient and oxygen deficient. This may have subsequential adverse effects on plant cell growth, at which the yield and quality of the final bioactive product can be affected. Having stated the downside of the cellular aggregation, it should be also highlighted that moderate cell aggregation (i.e. 200–500µm) is often beneficial for bioprocess engineering strategy. In fact, it enhances the sedimentation rates, facilitates the media exchange, and improves the in situ recovery of culture broth. But the generation of large cell aggregates (∼1–2mm) is not favorable with the bioprocessing approach as it may oppose with normal operation of bioreactor and makes cell aggregates more vulnerable to hydrodynamic stress and consequently cell damage can occur. Further, the rheological properties of plant cells suspension culture appear to be dependent on the cell aggregate size and morphology, biomass concentration, cell growth stage and culture conditions. Most importantly, it should be considered that scale-up of a plant cell suspension culture from a bench scale (shake flask) to an industrial scale (bioreactor system) is often associated with somewhat reduction in cellular productivity commonly attributed to the hydrodynamic stress [70]. Finally, foaming occurs during the exponential growth phase, perhaps due to the secreted or released extracellular proteinaceous compounds, polysaccharides and fatty acids. This phenomenon may become exacerbated by cell lysis during the stationary phase. Since the suspended cells tend to be entrapped in the foam layer, they may be subjected to somewhat deficiency of nutrients and oxygen, which may cause



marked reduction of suspended biomass and productivity. The entrapped cells in layers of foam may also release some harmful enzymes as well as undesired secondary metabolites. They also can generate a thick layer that adheres to the reactor wall, impeller shaft and the sensors, upon which the flow pattern of culture fluid may get inadvertent disruption [70]. Bioreactors and hairy root culture. In comparison with plant cell suspension cultures, hairy root cultures demand different bioprocess considerations (i.e. culture condition and bioreactor status) for mass production of secondary metabolite. Thus, we discuss the bioreactor parameters for the hairy root culture in the separate section even though there exist some commonalities between these two cell culture-based in vitro approaches. Hairy root cultures, as a stable source of biologically active chemicals, grant great potential for production of anticancer bioactive compounds, upon which vast attention has been devoted within the scientific community for its exploitation. In fact, by scaling up of hairy roots in designated bioreactors, one can set the best culture condition for optimal culture growth in order to yield high production of secondary metabolites similar to or even higher than that of the native roots. The growth of hairy root is often heterogeneous, upon which the performance of reactor mediated bioprocessing can be markedly impacted. This, together with dynamic morphology of the hairy root, makes optimization of the bioprocessing system an intricate process. Hence, we need to compromise the bioreactor design for root cultures based on the biological needs of such tissues and the final objectives. We know that the mechanical agitation may result in damage in cultures of hairy roots and thus leading to lower output perhaps due to revival/induction of calli. This, in return, can make an interlocked resilient matrix which may hinder the nutrient flow to some extent. In the hairy roots cultures, relatively small reduction in the dissolved oxygen in media is significantly able to decrease the growth rate, affecting the biosynthesis of desired secondary metabolites. The reason for such impact is the solid phase nature of the roots and the development of oxygen gradients within root tissues, so that the hairy roots can be oxygen limited in different types of bioreactors. This clearly indicates that exploitation of hairy root culture as a source of bioactive chemicals is greatly dependent upon various factors such as nutrient availability, nutrient uptake, oxygen, and hydrogen depletion in the medium, mixing and shear sensitivity. Also, factors such as the requirement for a support matrix and the possibility of flow restriction by the root mass in certain parts of the bioreactor seem to be very important for the design of bioreactors for hairy root cultures. Of the various bioreactors used for hairy root culture, liquid-phase, gas-phase, or hybrid reactors appear to be the most widely utilized systems [79]. Production of Paclitaxel using bioreactor. Reduced productivity in the scale-up of Taxus cell cultures has been often reported, perhaps based upon attribution of some influencing factors such as shear stress, oxygen supply, and gas composition. Given the fact that improvement of the cell cultures through rational modification of the reactor environment appears to be a key point, it has been shown that the cultivation of Taxus chinensis in a novel low-shear centrifugal impeller bioreactor (CIB) resulted in improved productivity and less cell adhesion to the reactor wall [29]. Altogether, it seems that the pneumatically agitated bubble columns and airlifts have been more widely used for taxoid production than stirred reactors, perhaps because of its low hydrodynamic stress and easy scale-up procedure with low operating cost. Suspension cultures of Taxus baccata and Taxus wallichiana were successfully grown in a small airlift bioreactor in batch mode, while a balloon-type bubble reactor (BTBR) was shown to be more efficient in promoting cell growth and cultures of Taxus cuspidate compared to the bubble-column reactor (BCR) with various internal cultivation loops. It was reported that the productivity of Taxus. chinensis cells was markedly reduced upon its transfer from shake flasks to bioreactors [78]. Such downside was resolved through utilization of ethylene in the inlet air of a BCR. This clearly highlighted ethylene as a key factor for the scale-up process during translational procedure of shake flask to a designated bioreactor. In such pragmatic industrial scale, to achieve an optimized culture system, fluid mixing process should be taken into account in order to attain maximum output of the suspension cultures of Taxus species such as Taxus chinensis. It has been shown that a low-shear CIB may provide such benefit upon more rapid mixing in Taxus chinensis with little biologic impacts [80].


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8. PARADIGMS OF SUSPENSION CULTURES It is now an accepted fact that the plant cell culture provides an alternative bioproduction platform for commercially valuable bioactive compound, however only few examples have been commercially developed. It should be mentioned that the lower product yields indicates needs to enhance productivity. This together with increasing demands for implementation of plant toward production of not only secondary metabolites but also animal based proteins such as antibodies emphasize the importance of the cell culture systems similar to the last decade impressive approaches that yielded improvements in mammalian cell culture. Thus, in the following sections, we will provide some pragmatic examples to show the potential of this modality for production of some important anticancer agents. Production of paclitaxel and related toxoids. Early works on biotechnological production of paclitaxel showed that calli and cell suspensions obtained from young stems of Taxus sp. were able to produce somewhat paclitaxel [81]. To increase the productivity of paclitaxel and related taxoids in cell and tissue cultures, many different strategies (e.g. optimization of culture conditions, selection of high-producing cell lines, use of elicitors, and addition of precursors) have been investigated; reader is directed to see the following works [20, 82, 83]. As mentioned previously, the main aim of all the studies was to obtain healthy calli and high productive cell line(s). Fig. 8 represents such viable calli and cells for paclitaxel production. (A)

(B)

(C)

(D)

(E)

(F)

Figure 8: Light and fluorescent microscopy of calli and cells of Taxus baccata. (A) and (B) Healthy and unhealthy calli; (C) and (D) Dark-field microscopic images of cultured cells; (E) and (F) Fluorescent images of the untreated and treated cells in stage II of suspension culture. , data were adopted from our previously published works [31].

For the industrial scale for production of paclitaxel, cultivation of Taxus cells has successfully been carried out in different types of bioreactors, including pneumatically mixed and stirred tank [84]; balloon-type bubble, bubble-column, with split-plate internal loop, with concentric draught-tube internal loop, with fluidized bed and stirred tank [85]; airlift [86]; turbine stirred tank, tower airlift and wave [87]. It has been reported that when cell suspensions of the Taxus baccata were immobilized on 2% alginate beads and cultured in a stirred bioreactor based on utilization of an optimum medium for paclitaxel biosynthesis with the addition of MJA, the paclitaxel production was significantly improved [88]. Such



production was shown to be drastically enhanced by more than 15 folds upon medium supplementation (e.g. with methyljasmonate and various other biotic and abiotic stress factors) up to 40 mg/L [31]. Interestingly, the yield of paclitaxel and taxanes can be up to 70 mg/L in a 500-litre bioreactor [85] and accordingly the production has been successfully scaled up in bioreactors of up to 70,000 litres as reported by Phyton Biotech (Germany, press release, 10 July 2002) to supply some part of the yearly paclitaxel demands of Bristol- Myers-Squibb company. Further, root and hairy root cultures have been also established from Taxus species to produce taxanes and paclitaxel [6]. Production of dimeric indole alkaloids. The main limitation for commercial production of indole alkaloids (e.g. vincristine and vinblastine) by Catharanthus roseus cell cultures appears to be the low level of alkaloid production of the cells in the bioreactor. To tackle this issue, biochemical engineering approaches have been recruited and resulted in optimized conditions for growth and production in bioreactors – we have discussed many of these issues previously in this current chapter. It is now clear that a close collaboration between the biochemical engineers, metabolic engineers and cell biologists is a key stone for resolving all the problems associated the cost-beneficial production of the desired bioactive compounds [89]. Unfortunately, up until now, the most efforts employing undifferentiated cell cultures have resulted in unacceptable yields. At the best condition, such cell culture in vitro systems were shown to produce extremely low traces (if any) of the dimeric alkaloids even though monomeric indole alkaloids (e.g. ajmalicine) have been detected [90]. Likewise, root and hairy root cultures failed to provide satisfactory yielding [91], but not organized shoot cultures. Since the yields of the dimeric alkaloids were shown to be low which is associated with difficulties in large-scale cultivation of the shoot cultures, the practice of growing Catharanthus roseus in the field seems to be much more economic [6]. However, we believe that still there exists plenty of room for some sophisticated investigations (e.g. implementation of various elicitors, pathway engineering, transformed organisms), at which high production of the related compounds of the Catharanthus roseus may become feasible in the future. Production of camptothecin. Various plants of different families were shown to produce camptothecin, whose in vitro cultures have so far been successfully established from Camptotheca acuminata, Nothapodytes foetida and Ophiorrhiza pumila. Intact plants of Camptotheca acuminata contain approximately 0.2–5 (mg/g dry wt) of camptothecin [92], while its levels in callus and suspension cultures were shown to be about 0.002–0.004 (mg/g dry wt) or even lower [93]. Besides, it was shown that the undifferentiated callus and suspension cultures may fail to produce significant amounts of camptothecin. This clearly indicates that the biosynthesis of camptothecin is strictly controlled through cellular differentiation and environmental factors. Cultures obtained from root and/or hairy root of Ophiorrhiza pumila have resulted in production of approximately 1 (mg/g dry wt) of camptothecin [94, 95]. To translate the bench scale production towards mass production of camptothecin, [96] a 3-litre bioreactor was scaled up, achieving a final concentration of 0.0085% camptothecin (fresh weight). Despite difficulties in establishment of hairy root cultures from Camptotheca acuminata, Lorence et al. (2004) reported that the hairy root cultures are able to produce and secrete camptothecin as well as 10-hydroxycamptothecin (HCPT) into the medium. It should be evoked that this nature camptothecin analogue, “HCPT” has been shown to have a strong antitumor activity against various cancers such as gastric carcinoma, hepatoma, leukemia, and tumor of head and neck. Compare to camptothecin, HCPT is more potent and less toxic in experimental animals and in human clinical evaluations as used and reported in China. The alkaloid level of root cultures were in the same range as those in roots of the intact plant. Similarly, the undifferentiated cultures of Ophiorrhiza mungos were shown to yield low levels of camptothecin, while its formation is substantially higher in root and hairy root cultures. Accordingly once cultured in bioreactors by ROOTec bioactives Ltd (a privately-held Swiss biotech company established in 2005), successful large-scale production of camptothecin in hairy root cultures were reported [6]. Production of podophyllotoxin and related lignans. The North America growing plant “Podophyllum peltatum” has been reported to contains the antitumor lignan “podophyllotoxin” that acts against KB cells (a cell line derived from a human carcinoma of the nasopharynx) and skin cancer. A semi-synthetic


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derivative of podophyllotoxin “etoposide” is a FDA approved active agent used against various malignancies such as brain tumor, lymphosarcoma and Hodgkins' disease [19]. For improved production of such lignan, various plant cultures of Linum album, Linum flavum, and Linum nodiflorum have been examined. The cultures of Linum album was shown to accumulate podophyllotoxin. The other two species appeared to accumulate a trace amount of podophyllotoxin, nonetheless they produced mainly the 6methoxypodophyllotoxin. In fermenter vessels, to improve the growing undifferentiated cells suspension potential, the culture media need to be supplemented with some phytohormones. For example, the optimal cultivation of Linum album cell suspensions can be achieved using Murashige & Skoog medium supplemented with 0.4 mg/L naphthalene acetic acid and 40 g/L sucrose. The amount of podophyllotoxin in cultured Linum album cells appears to be about 0.2–0.5% on a dry weight basis, which is markedly lower than that of Podophyllum hexandrum roots or rhizomes (i.e. 4–7%). However, in Linum album cell suspensions, the biomass production seems to be greater than that of Podophyllum hexandrum. In the Linum nodiflorum cultures, the amount of 6-methoxypodophyllotoxin was shown to be up to 1.5% comparable to podophyllotoxin amount found in Pelargonium peltatum roots and rhizomes [97]. Furthermore, great attention has been devoted for efficient production of the aryltetralin lignans in the Linum cell cultures through inducing of biosynthetic paths by different elicitors such as MJA and salicylic acid. Nevertheless, little success (if any) was observed upon such treatment. Although because of costprohibitive nature of the bioprocessing of plant cells in bioreactors, at least for some cases it appears that the agricultural approach provide a better option. It should be further highlighted that the production of podophyllotoxin by plant cell cultures will be economically attractive if such approach provides substantial yields, in which fast growth of cell cultures may compensate low product accumulation. Furthermore, accumulation (up to 5% dry weight) of 6- methoxypodophyllotoxin has been reported in root cultures of Linum flavum. This literally clarifies that the secondary metabolites can be accumulated in plant cells/tissue cultures comparable to that of the intact plants. Having emphasized that the cell suspensions of Linum album have been successfully cultivated in small bioreactors for a long period of time, still there are some unresolved issues associated with cost-effective yielding of the desired bioactive compound(s). In fact, scaling-up of the cultures towards a designated pilot-plant size can confer some of note data that are deemed to be necessary for translation of the methodology for large scale production by plant cell cultures as cell factories [97]. 9. CONCLUDING REMARKS In the third millennium, mankind will face with increasing demands for potent plant based anticancer agents, whose production need to employ de novo advanced cost-effective biotechniques to avoid the extinction of the natural product source plants. To attain a sustained platform for production of some key important anticancer agents, plant cell culture systems are believed to provide new means for the commercial production of plants derived anticancer bioactive compounds. In fact, such pragmatic approach can ultimately provide a continuous, reliable source for production of secondary metabolites with minimum environmental consequences; in particular some rare species can be saved from extermination. Nevertheless, the major advantage of the plant cell culture system includes synthesis of anticancer bioactive compound in a fully controlled environment, independently from climate and soil conditions. Furthermore, the increased use of genetic tools along with emergence of regulation of pathways for secondary metabolism can confer a robust platform for production of commercially adequate levels of the designated product. We are now expecting to get increased level of natural products for medicinal purposes using large-scale plant cell culture technology. Our knowledge about biosynthetic pathways of anticancer bioactive compounds in plant cells is still in its infancy, thus we need to perform necessary strategies to attain such information in a cellular and molecular level. Emergence of newer techniques of molecular biology as well as implementation of transgenic plants are deemed to provide a strong cell cultures tools to investigate the expression and regulation of biosynthetic pathways [37]. Use of appropriate bioreactor appears to grant a significant step towards making cell cultures more generally applicable to the commercial production of anticancer bioactive compound.



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241

Subject Index Abiotic Alkaloid Anthraquinone Anticancer Anthocyanin Antioxidant Antitumor Auxin Benzylaminepurine (BAP) Biocatalyst Biomass Bioprocess Bioreactor Biosynthesis

Biotransformation Callugenesis Callus culture Callus induction Carotenoid Culture medium Coumarin Cytokinin Cytotoxicity De novo Dichlorophenoxyacetic acid Differentiation DNA Elicitor Enzyme Essential oil Fermentor Fermentation Field-grown

3, 8, 11, 22, 36, 68, 74, 90, 176, 177, 223, 235 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 11, 124 38, 41, 54, 110, 215, 216, 220, 221, 226, 227, 228, 230, 231, 233, 234, 236 4, 13, 26, 27, 28, 29, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 176, 178, 179, 180, 181 8, 21, 22, 30, 31, 38, 41, 42, 68, 87, 96, 97, 98, 99, 101, 110, 111, 115, 117, 124, 147, 181, 202, 222 91, 215, 216, 217, 235 90, 92, 94, 100, 112, 116, 149, 150, 167, 168, 221, 226 73, 93, 94, 100, 149, 150, 167, 168, 183, 205, 208 11, 48, 118, 215, 224 8, 9, 10, 67, 70, 73, 74, 119, 145, 150, 151, 177, 181, 205, 206, 224, 227, 231, 232, 233 215, 216, 222, 223, 232, 233 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 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 8, 45, 48, 49, 55, 11, 107, 110, 111, 177, 188, 192, 193, 194, 198, 216, 227, 228 5, 95 3, 36, 45, 46, 47, 67, 70, 71, 72, 73, 114, 116, 149, 150, 198, 200, 203, 204, 205, 222, 235 47, 112, 149, 221, 233 22, 110, 112, 183 3, 4, 5, 8, 9, 10, 71, 72, 74, 75, 112, 115, 149, 150, 151, 168, 208, 221, 222, 224, 225, 232, 236 26, 27, 28, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 9, 89, 90, 92, 93, 94, 112, 149, 150, 167, 182, 183, 184, 221, 226 41, 54, 61, 91, 92, 152, 217 11, 49, 90, 188, 195, 198, 236 73, 112, 149, 150, 167, 168, 206, 208, 221 5, 6, 7, 9, 41, 46, 112, 115, 116, 118, 187, 194, 195, 196, 197, 198, 204, 235 6, 13, 41, 42, 68, 75, 89, 168, 169, 170, 171, 191, 219, 226 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 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 21, 22, 25, 110, 111, 117, 182, 183 114, 236 26, 49, 230 67, 87, 89, 90, 220 Ilkay Erdogan Orhan (Ed) All rights reserved-© 2012 Bentham Science Publishers


Flavonoid Food additive GC–MS Gene cloning Gene expression Genetic engineering Genotype Germplasm Gibberellin (GA) Growth regulator Hairy root HPLC Immobilization Indole acetic acid (IAA) Iridoid Isoprene Jojoba Large-scale Metabolic engineering Micropropagation Naphthaleneacetic acid (NAA) Nitrogen Nutrient Organ culture Organogenesis Oxidative stress Pesticide Pharmaceutical Phenolic Pigment Plant cell culture Precursor Propagation Recombinant Rhizogenesis Rhizogenic callus (calli) Root culture Salinity Saponin Scale-up Shoot culture Shoot regeneration Somatic embryo(genesis) Steroid Sucrose

Ilkay Erdogan Orhan

22, 26, 27, 28, 29, 30, 67, 68, 75, 87, 88, 95, 96, 99, 101, 124, 125, 149, 176, 177, 178, 179, 180, 181 43, 108, 109, 110, 124 112, 117 170, 171, 187, 191 5, 165 6, 25 92, 166, 167, 169, 176, 182 88, 89, 92, 166 113,194, 208 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 9, 46, 47, 48, 49, 107, 109, 118, 119, 200, 204, 205, 206, 207, 226, 228, 231, 233, 235 68, 112, 117, 192, 195 3, 8, 11, 118 72, 94, 149, 194, 203, 204, 205 87, 89, 90, 91, 96, 97, 100, 101, 108 6, 22, 25, 108, 176, 207 159, 160, 161, 162, 163, 167, 168, 169, 170, 171 3, 14, 15, 53, 63, 67, 70, 114, 124, 170, 215, 224, 225, 232, 235 14, 25, 76, 109, 170, 171, 200, 203, 207, 228, 235 90, 107, 109, 149, 150, 159, 166, 167, 171, 200 72, 73, 90, 92, 94, 95, 100, 113, 150, 167, 168, 206, 208, 236 9, 72, 112, 150, 183, 184, 203 6, 8, 9, 29, 31, 67, 72, 75, 93, 112, 117, 176, 177, 181, 182, 224, 228, 231, 232, 233 3, 4, 7, 46, 48, 67, 94, 95, 97, 107, 108, 109, 115 5, 89, 90, 92, 94, 95, 100, 112, 166, 195 36, 87, 176, 181, 224 3, 164, 171, 176, 177, 179, 181, 182, 184, 185 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 14, 21, 22, 26, 27, 28, 29, 31, 38, 88, 95, 98, 99, 100, 112, 176, 177, 178, 179, 180, 181, 202, 205 3, 67, 68, 70, 72, 73, 74, 76, 124, 145, 150, 184, 200, 203, 204, 205, 207, 208 90, 107, 108, 109, 110, 111, 112, 113, 114, 124, 149, 200 7, 14, 23, 25, 36, 45, 63, 75, 107, 113, 118, 183, 189, 196, 203, 205, 215, 220, 222, 225, 227, 234 89, 92, 165, 166, 169, 171 13, 170, 188, 191, 216 94, 95, 100 92, 93, 100 3, 46, 93, 95, 96, 97, 98, 99, 100, 108, 115, 116, 117, 187, 194, 195, 198, 204, 226, 233, 235, 236 90, 145, 171, 176, 181 108, 114, 124, 125, 145, 149, 203 108, 111, 118, 221, 228, 231, 232, 233, 236 7, 46, 95, 96, 97, 98, 100, 108, 115, 170, 187, 194, 195, 196, 206, 235 90, 149, 166 89, 159, 167, 189, 190, 191, 195, 208 13, 48, 107, 108, 124, 125, 149, 187, 192 9, 72, 73, 90, 112, 221, 222, 223, 236

Subject Index

Suspension culture Terpenoid Traditional medicine Tissue culture Transgenic Volatile oil Yeast


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 13, 21, 22, 48, 107, 108, 109, 119, 149, 176, 177, 181, 183, 184, 203, 221, 228 21, 88, 107, 108, 124, 217 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 6, 14, 25, 75, 76, 107, 109, 170, 208, 216, 227, 236 112, 113, 176, 181 114, 145, 149, 206, 208

244


Plant Index Achillea millefolium Achyranthes Achyranthes bidentata Adenosma caeruleum Aegilops biuncialis Aegilops cylindrica Aegle marmelos Agalinis Agave tequilana Ajuga reptans Alkanna Alkanna calliensis Alkanna corcyrensis Alkanna graeca Alkanna methanaea Alkanna orientalis Alkanna pindicola Alkanna primuliflora Alkanna sieberi Alkanna stribrnyi Alkanna tinctoria Alkanna tuberculata Alternanthera Alternanthera bettzichiana Alternanthera brasiliana Alternanthera canescens Alternanthera flavescens Alternanthera halimifolia Alternanthera lanceolata Alternanthera maritima Alternanthera nodiflora Alternanthera paronychioides Alternanthera philoxeroides Alternanthera polygonoides Alternanthera pungens Alternanthera repens Alternanthera sessilis Alternanthera tenella Alternanthera versicolor Amaranthus Amaranthus dubius Amelanchier alnifolia Ammi majus Amsinckia Anchusa arveniis Anchusa Anchusa hispida Anchusa milleri Anchusa officinalis Anethum Anethum graveolens

12, 117 125 145 96 184 184 61 88 3, 6 73 201, 202, 203 202 202 202 202 202 202 202 202 202 201, 202 202 124, 125, 147, 151, 152 147 126, 131, 132, 146, 147, 149, 150 126 126 126 147 126, 127, 128, 129, 130, 132, 133, 146, 147, 149 147 126, 147 126, 131, 132, 146, 147, 151 132 126, 127, 132, 147 128, 147 126, 127, 131, 147, 149 126, 127, 128, 129, 130, 146, 147, 149 132 125 146 69 39, 47 201, 202 202 112 202 202 202 113 113 Ilkay Erdogan Orhan (Ed) All rights reserved-© 2012 Bentham Science Publishers

Plant Index

Anethum sowa Angelica Angelica gigas Angelica koreana Angelica officinalis Antirrhinum majus Anthirrhinum linarium Apium graveolens Arabidopsis Arabidopsis thaliana Aralia cordata Arnebia Arnebia densiflora Arnebia euchroma Aronia melanocarpa Artemisia absinthium Artemisia annua Artemisia glabella Artocarpus nobilis Atropa belladona Avena sativa Azadirachta indica Bacopa monnieri Barleria prionitis Begonia Beta vulgaris Betula lenta Bidens Bidens alba Bleekeria vitiensis Blutaparon Blutaparon portulacoides Borago Borago officinalis Brassica Brassica napus Brassica oleracea Brosimum gaudichaudii Brunnera macrophylla Buxus Buxus hyrcana Caesalpinia bonduc Calceolaria Calophyllum Calophyllum brasiliense Calophyllum dispar Calophyllum inophyllum Calystegia sepium Camelia Camptotheca acuminata Campylotropis hirtella Capsicum Capsicum annuum Capsicum frutescens


113 40 41, 47 41 42 89 88 180 170 25, 26, 191, 227 72, 73, 77 201, 202 201, 202, 205 201, 202, 204, 205 69 8, 13, 115, 117 7, 115 217 54, 55 7 180 9 88, 89, 90, 93 55 7 8, 10 108 13 8 217 125 145, 146 200, 201 201, 202 184 167, 183 183 39 201 59 59 54, 55 88 41 41 41 47 8 77 4, 72, 73, 110, 216, 217, 220, 221, 235 41 11, 118 107, 110, 112 11, 12, 110, 112, 113, 118


Carpentaria acuminata Cassia Cassia obtusifolia Castilleja Catalpa ovata Catharanthus roseus Centaurium erythraea Chamissoa Chamomila recutita Celeus blumei Celosia Centaurium erythraea Centella asiatica Cephalotaxus harringtonia Chenopodium ambrosioides Chenopodium rubrum Chondrodendron tomentosa Chrysanthemum cinerariaefolium Chrysanthemum coronarium Cichorium intybus Cinchona Cinchona ledgeriana Cinchona officinalis Citrus Citrus limon Citrus paradisi Cleome rosea Cnidium Coffea arabica Colchicum autumnale Coleus blumei Coleus forskohlii Conium Convallaria Coptis japonica Cordia americana Cordia multispicata Cordia myxa Cordia sebestena Cordia spinescens Cordia verbenacea Coreopsis tinctoria Coriandrum sativum Coumarouna odorata Cupressus lusitanica Curcuma zedoaria Cyathula Cymbidium Cynara cardunculus Cynoglossum Cynoglossum amabile Cynoglossum officinale Daphne

Ilkay Erdogan Orhan

228 13 178 87, 88, 91, 97, 98, 99 97 4, 7, 10, 11, 12, 15, 75, 181, 217, 218, 221, 224, 225, 228, 230, 235 183 125 182 7 125 183 6 4 113 10, 226 4 4 77 13, 46 7 13 4 113 12, 114 12 70, 71, 73, 75, 76 40 4, 10, 11, 12 218, 219 4, 15 206 4 187 4, 221 202 203 202 201 202 201, 208 8 113, 167 37 111 12 125 12 14 201, 202 202 202, 207 40

Plant Index

Daphne gnidium Datura Datura stramonium Daucus Daucus carota Decatropis bicolor Decentra pergrina Dianthus caryophyllus Digitalis Digitalis lanata Digitalis obscura Digitalis purpurea Dioscoria deltoidea Dipteryx odorata Dolichandrone falcate Duboisia leichhardtii Echinacea purpurea Echium Echium amoenum Echium glomeratum Echium italicum Echium lycopsis Echium plantaginuem Echium vulgare Ehretia thyrsiflora Eritrichium sericeum Erythroxylon coca Eschscholzia californica Eucalyptus Eucalyptus camaldulensis Eucalyptus perriniana Euphorbia milli Euphorbia pulcherrima Eurycoma longifolia Ficus hispada Foeniculum Foeniculum vulgare Froelichia Galipea panamensis Galphimia glauca Garcinia brevipedicellata Gardenia jasminoides Gastrocotyle hispida Ginkgo biloba Glehnia littoralis Glycine max Glycyrrhiza glabra Glycyrrhiza uralensis Gomphrena Gomphrena affinis Gomphrena boliviana Gomphrena canescens Gomphrena celosioides


40 13 4, 180 77 11, 47, 71, 72, 73, 75, 76, 77, 112, 183 39 7 25 187, 188, 191, 194, 198 4, 7, 11, 12, 187, 188, 190, 191, 192, 194, 195, 197, 198 191 7, 12, 88, 187, 188, 190, 191, 194, 195, 198 4, 7, 11, 110, 114 37 63 13 8, 13, 15 202 206 202 204, 222, 225 202 201, 202 201 202 206 4 4 113 112 12 72, 77 91 4, 8 39 113 113 125 27 11 62 12 202 117, 227 72 11, 180 12, 13, 110 13, 117 124, 125, 133, 147, 151, 152 133, 134 134, 135, 137, 140, 148 133, 134 133, 148


Gomphrena claussenii Gomphrena cunninghamii Gomphrena decumbens Gomphrena dispersa Gomphrena glabrata Gomphrena globosa Gomphrena haageana Gomphrena holosericea Gomphrena macrocephala Gomphrena martiana Gomphrena meyeniana Gomphrena officinalis Gomphrena perennis Gomphrena pulchella Gossypium arboreum Gossypium hirsutum Fragaria ananassa Hackelia venusta Halleria lucida Haplophyllum patavinum Harpagophytum procumbens Harpagophythum Hedychium spicatum Heliotropium Heliotropium bovei Heliotropium curassavicum Heliotropium curassavicum var. argentinum Heliotropium indicum Helleborus Hemidesmus indicus Herbstia Hibiscus rosasinensis Hibiscus sabdariffa Hyoscamus albus Hyoscamus muticus Hyoscyamus niger Impatiens balsamina Ipomoea batatas Iresine Jasminum Jasmine officinale Jatropha curcas Lagerstroemia speciosa Lappula spinocarpos Larrea tridentata Lavandula angustifolia Lavandula vera Leucaena Linum Linum album Linum flavum Linum nodiflorum Litchi chinenesis Lithospermum

Ilkay Erdogan Orhan

135, 136, 138 133, 134 140 133,134 146 137, 138, 139, 140, 146, 148, 149, 150 133, 134, 140, 148 135, 140 136, 148, 150 134, 135, 136, 137, 138, 140, 148 133, 134, 135, 136, 140, 148 133, 150 133, 140, 148 148 114 181 73 208 88, 96, 98 46 117 97 62 200, 201, 202 202 202 202 207, 208 187 8 125 69 69, 72, 77 8, 13 8, 13 4, 13 5 69, 72, 73, 74, 77 125 4 110 170 62 202 217 12 11 181 236 236 236 236 69 201, 202, 203, 204, 207

Plant Index

Lithospermum canescens Lithospermum erythrorhizon Lithospermum officinale Lithospermum ruderale Lobostemon trigonus Lunaria annua Lycopersicon esculentum Macrotomia euchroma Mallotus japonicus Malus Malva sylvestris Manihot esculenta Matricaria chamomilla Mecardonia tenella Melilotus alba Melilotus officinalis Melissa officinalis Mentha Mentha arvensis Mentha longifolia Mentha piperita Mentha spicata Mentha citrata Mertensia maritima Morinda citrifolia Myosotis Myosotis scorpioides Mucuna pruriens Nauclea latifolia Neopicrorhiza scrophulariflora Nicotiana tabacum Nothapodytes foetida Ocimum basilicum Ocimum sanctum Olea europaea Onosma Onosma arenaria Onosma argentatum Onosma hispida Onosma paniculatum Onosma polyphyllum Onosma visianii Ophiorrhiza mungos Ophiorrhiza pumila Orthosiphon aristatus Oryza sativa Oxalis linearis Papaver bracteatum Papaver somniferum Panax ginseng Panax sikkimensis Paracaryum intermedium Paracaryum rugulosum


205 4, 8, 10, 13, 15, 73, 201, 202, 203, 204, 205, 206, 207, 208, 221 202 202 201 170 4, 25, 68, 110 202 77 76, 77 69 183 110, 114 93 43 43, 49 181 12 182, 183 181 110, 113, 183 183 7, 183 201 4, 7, 11 200, 201 202 11, 12 54, 55 88 4, 7, 11, 12, 13, 15, 26, 151, 181 220, 235 183 181 31 202 202 202 202 203, 204, 205, 207 202 202 235 220, 228, 235 114 180, 191 72 12 4, 11, 12 9, 13, 15, 107,114, 117, 118, 227 76 202 202


Paulownia elongate Paulownia tomentosa Pedicularis resupinata Pelargonium graveolens Pelargonium peltatum Pelargonium tomentosum Penstemon barbatus Penstemon gentianoides Penstemon rosseus Penstemon serrulatus Pentaglottis sempervirens Perilla frutescens Petunia hybrida Pfaffia Pfaffia elata Pfaffia grandiflora Pfaffia glomerata Pfaffia hookeriana Pfaffia iresinoides Pfaffia jubata Pfaffia paniculata Pfaffia pulverulenta Pfaffia stenophylla Pfaffia tuberosa Pharbitis nil Phaseolus Phaseolus vulgaris Phellolophium madagascariense Picria felterrae Picrorhiza kurroa Picrorhiza scrophulariiflora Pilocarpus bracteatum Pilocarpus jaborandi Pimpinella anisum Pisum sativum Plantago lanceolata Plumbago rosea Podophyllum hexandrum Podophyllum peltatum Polygonum multiflorum Polygonum tinctorium Populus Populus tremuloides Psoralea caryolia Pyrenacantha klaineana Pseudoplantago Pulmonaria Quaternella Raphanus sativus Rauwolfia serpentina Rehmannia glutinosa Rhodiola sachalinensis Rosa gallica Rosa hybrida

Ilkay Erdogan Orhan

10 88, 89 89 183 219, 236 7, 115 89 98 89 90, 97 201 77, 113 25, 77 124, 125, 140, 141, 147, 148, 151, 152 145 145 143, 148, 151 145 142, 143, 148 148 124, 141, 142, 146, 148 144 145, 148 148 48 180 69, 91 41 89 7, 89, 90, 115 96 4 4 110, 115 167, 180 91 11 12, 219, 231, 236 217, 235 47 7, 8 14 191 39 220 125 201 125 73, 77 4, 13, 15 89, 90, 96, 97 13 69 74

Plant Index

Rosmarinus officinalis Rotula aquatica Rubia akane Rubia cordifolia Rubia peregrina Rubus idaeus Ruta graveolens Saccharum officinalis Salacia reticulate Salvia Salvia miltiorrhiza Salvia officinalis Salvia sclarea Scoparia ducis Scrophularia ningpoensis Scrophularia nodosa Scrophularia striata Scrophularia umbosa Scrophularia yoshimurae Seseli Seseli gummiferum ssp. corymbosum Seseli indicum Sibthorpia peregrine Simmondsia chinensis Solanum incanum Solanum melongena Solanum paludosum Solanum tuberosum Sorghum bicolor Spinacia oleracea Spiraea ulmaria Spirulina platensis Stevia rebaudiana Strophanthus Symphytum Symphytum officinale Strychnos nux-vomica Tagetes patula Tanacetum parthenium Taraxacum officinale Taxus Taxus baccata Taxus brevifolia Taxus chinensis Taxus chinensis var. mairei Taxus cuspidata Taxus media Taxus wallichiana Terminalia chebula Terminalia superba Thalictrum glaucum Thalictrum rugosum Thaumatococcus danielli Thymus minus


77, 111 201, 208 73 13 201 180 7, 45, 46 184 61 110 13, 117 7, 107, 115, 183, 222 111, 117 89 89 89, 90, 97 96 90 89,90 40 40 40 89 159, 167 217 68, 69 7 4, 74, 180, 184 184 178 108 12 4, 7, 12, 110, 115 187 200, 201, 202 207 4 11 46, 91 115 74, 113, 216, 217, 221, 223, 228,233, 234, 235 11, 217, 218, 222, 224, 225, 228, 231, 233, 234 216, 217, 221, 222, 224 222, 224, 225, 231, 233 113 113, 222, 224, 225, 232, 233 225 231, 233 61 61 187 10 4, 110 11


Torenia fournieri Trichodesma africanum Trichodesma indicum Trigonella foenum-graecum Triticum aestivum Uragoga ipecacuanha Vaccinium myrtillus Vaccinium pahalae Valeriana officinalis Valeriana wallichii Vanilla planifolia Verbascum Verbascum thapsus Veronica persica Vicia faba Vigna unguiculata Vitis Vitis coignetiae Vitis vinifera Withania somniferum Zea mays Ziziphus zizyphus

Ilkay Erdogan Orhan

96 202 201, 208 117 184 4 69 77 117 114 12 108 89 89 180 183 73, 77 69 10, 69, 72, 74, 75, 184 7 178, 180 159