Studies on the production of some important secondary

VBaont.isBreuell.eAt caal.d—. SiSn.tu(2d0ie0s4)on45t:h1e-p2r2oduction of some important secondary metabolites

1

(Review paper)

Studies on the production of some important secondary metabolites from medicinal plants by plant tissue cultures
Mulabagal Vanisree1, Chen-Yue Lee2, Shu-Fung Lo2,3, Satish Manohar Nalawade1, Chien Yih Lin3, and Hsin-Sheng Tsay*,1
1Institute of Biotechnology, Chaoyang University of Technology, 168, Gifeng E. Road, Wufeng, Taichung, Taiwan 413 2National Chung Hsing University, Taichung, Taiwan 402 3Taiwan Agricultural Research Institute, Wufeng, Taiwan 413
(Received January 8, 2003; Accepted April 22, 2003)
Abstract. Plants are a tremendous source for the discovery of new products of medicinal value for drug development. Today several distinct chemicals derived from plants are important drugs currently used in one or more countries in the world. Many of the drugs sold today are simple synthetic modifications or copies of the naturally obtained substances. The evolving commercial importance of secondary metabolites has in recent years resulted in a great interest in secondary metabolism, particularly in the possibility of altering the production of bioactive plant metabolites by means of tissue culture technology. Plant cell culture technologies were introduced at the end of the 1960’s as a possible tool for both studying and producing plant secondary metabolites. Different strategies, using an in vitro system, have been extensively studied to improve the production of plant chemicals. The focus of the present review is the application of tissue culture technology for the production of some important plant pharmaceuticals. Also, we describe the results of in vitro cultures and production of some important secondary metabolites obtained in our laboratory.
Keywords: Biotransformations; Cell suspension cultures; Hairy root cultures; Pharmaceuticals; Secondary metabolites.

Contents
Introduction .......................................................................................................................................................................... 2 Tissue Cultures Producing Pharmaceutical Products of Interest ......................................................................................... 6
Taxol ................................................................................................................................................................................. 6 Morphine and Codeine ................................................................................................................................................... 6 Ginsenosides .................................................................................................................................................................... 7 L-DOPA ............................................................................................................................................................................ 8 Berberine ......................................................................................................................................................................... 8 Diosgenin ........................................................................................................................................................................ 9 Capsaicin ......................................................................................................................................................................... 9 Camptothecin .................................................................................................................................................................. 9 Vinblastine and Vincristine ............................................................................................................................................. 9 Tanshinones ................................................................................................................................................................... 10 Podophyllotoxin ............................................................................................................................................................ 11 Studies on In Vitro Cultures and Production of Important Secondary Metabolites in the Author’S Laboratory ............... 11 Production of Taxol from Taxus mairei by Cell Suspension Cultures ......................................................................... 11 Formation of Imperatorin from Angelica dahurica var. formosana by cell Suspension Cultures .............................. 12 Production of Diosgenin from Dioscorea doryophora by Cell Suspension Culture .................................................. 12 Formation and Analysis of Corydaline and Tetrahydropalmatine from Tubers of Somatic Embryo Derived
Plants of Corydalis yanhusuo ................................................................................................................................... 12
*Corresponding author. Tel: +886-4-23323000 ext. 7578; Fax : +886-4-23742371; E-mail: [email protected]

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Botanical Bulletin of Academia Sinica, Vol. 45, 2004

In Vitro Synthesis of Harpagoside, an Anti -Inflammatory Irridoid Glycoside from Scrophularia yoshimurae Yamazaki.................................................................................................................................................................... 13
Gentipicroside and Swertiamarin from In Vitro Propagated Plants of Gentiana davidii var. formosana (Gentianaceae) .......................................................................................................................................................... 14
Conclusions and Future Perspectives ................................................................................................................................ 14 Literature Cited ................................................................................................................................................................... 14

Introduction
Many higher plants are major sources of natural products used as pharmaceuticals, agrochemicals, flavor and fragrance ingredients, food additives, and pesticides (Balandrin and Klocke, 1988). The search for new plantderived chemicals should thus be a priority in current and future efforts toward sustainable conservation and rational utilization of biodiversity (Phillipson, 1990). In the search for alternatives to production of desirable medicinal compounds from plants, biotechnological approaches, specifically, plant tissue cultures, are found to have potential as a supplement to traditional agriculture in the industrial production of bioactive plant metabolites (Ramachandra Rao and Ravishankar, 2002). Cell suspension culture systems could be used for large scale culturing of plant cells from which secondary metabolites could be extracted. The advantage of this method is that it can ultimately provide a continuous, reliable source of natural products.
Discoveries of cell cultures capable of producing specific medicinal compounds (Table 1) at a rate similar or superior to that of intact plants have accelerated in the last few years. New physiologically active substances of medicinal interest have been found by bioassay. It has been demonstrated that the biosynthetic activity of cultured cells can be enhanced by regulating environmental factors, as well as by artificial selection or the induction of variant clones. Some of the medicinal compounds localized in morphologically specialized tissues or organs of native plants have been produced in culture systems not only by inducing specific organized cultures, but also by undifferentiated cell cultures. The possible use of plant cell cultures for the specific biotransformations of natural compounds has been demonstrated (Cheetham, 1995; Scragg, 1997; Krings and Berger, 1998; Ravishankar and Ramachandra Rao, 2000). Due to these advances, research in the area of tissue culture technology for production of plant chemicals has bloomed beyond expectations.
The major advantages of a cell culture system over the conventional cultivation of whole plants are: (1) Useful compounds can be produced under controlled conditions independent of climatic changes or soil conditions; (2) Cultured cells would be free of microbes and insects; (3) The cells of any plants, tropical or alpine, could easily be multiplied to yield their specific metabolites; (4) Automated control of cell growth and rational regulation of metabolite processes would reduce of labor costs and improve productivity; (5) Organic substances are extractable from callus cultures.

In order to obtain high yields suitable for commercial exploitation, efforts have focused on isolating the biosynthetic activities of cultured cells, achieved by optimizing the cultural conditions, selecting high-producing strains, and employing precursor feeding, transformation methods, and immobilization techniques (Dicosmo and Misawa, 1995). Transgenic hairy root cultures have revolutionized the role of plant tissue culture in secondary metabolite production. They are unique in their genetic and biosynthetic stability, faster in growth, and more easily maintained. Using this methodology a wide range of chemical compounds have been synthesized (Shanks and Morgan, 1999; Giri and Narasu, 2000). Advances in tissue culture, combined with improvement in genetic engineering, specifically transformation technology, has opened new avenues for high volume production of pharmaceuticals, nutraceuticals, and other beneficial substances (Hansen and Wright, 1999). Recent advances in the molecular biology, enzymology, and fermentation technology of plant cell cultures suggest that these systems will become a viable source of important secondary metabolites. Genome manipulation is resulting in relatively large amounts of desired compounds produced by plants infected with an engineered virus, whereas transgenic plants can maintain constant levels of production of proteins without additional intervention (Sajc et al., 2000). Large-scale plant tissue culture is found to be an attractive alternative approach to traditional methods of plantation as it offers a controlled supply of biochemicals independent of plant availability (Sajc et al., 2000). Kieran et al. (1997) detailed the impact of specific engineering-related factors on cell suspension cultures. Current developments in tissue culture technology indicate that transcription factors are efficient new molecular tools for plant metabolic engineering to increase the production of valuable compounds (Gantet and Memelink, 2002). In vitro cell culture offers an intrinsic advantage for foreign protein synthesis in certain situations since they can be designed to produce therapeutic proteins, including monoclonal antibodies, antigenic proteins that act as immunogenes, human serum albumin, interferon, immuno-contraceptive protein, ribosome unactivator trichosantin, antihypersensitive drug angiotensin, leu-enkephalin neuropeptide, and human hemoglobin (Hiatt et al., 1989; Manson and Arntzen, 1995; Wahl et al., 1995; Arntzen, 1997; Hahn et al., 1997; La Count et al., 1997; Marden et al., 1997; Wongsamuth and Doran, 1997; Doran, 2000). The appeal of using natural products for medicinal purposes is increasing, and metabolic engineering can alter the production of pharmaceuticals and help to design new therapies. At present, researchers aim

Table 1. Bioactive secondary metabolites from plant tissue cultures.

Plant name Agave amaniensis
Ailanthus altissima Ailanthus altissima Allium sativum L.
Aloe saponaria Ambrosia tenuifolia
Anchusa officinalis Brucea javanica (L.) Merr. Bupleurum falcatum Bupleurum falcatum L. Camellia sinensis
Canavalia ensiformis
Capsicum annuum L. Cassia acutifolia
Catharanthus roseus Catharanthus roseus
Cephaelis ipecacuanha A. Richard Chrysanthemum cinerariaefolium Chrysanthemum cinerariaefolium
Cinchona L.
Cinchona robusta
Cinchona spec. Cinchona succirubra
Citrus sp.
Coffea arabica L.
Corydalis ophiocarpa Croton sublyratus Kurz

Active ingredient

Culture medium

Saponins
Alkaloids Canthinone alkaloids Alliin
Tetrahydroanthracene glucosides Altamisine
Rosmarinic acid Canthinone alkaloids Saikosaponins Saikosaponins Theamine, γ-glutamyl derivatives
L-Canavanine
Capsaicin Anthraquinones
Indole alkaloids Catharanthine
Emetic alkaloids Pyrethrins Chrysanthemic acid and
pyrethrins Alkaloids
Robustaquinones
Anthraquinones Anthraquinones
Naringin, Limonin
Caffeine
Isoquinoline alkaloids Plaunotol

MS + Kinetin (23.2 µM), 2,4-D (2.26 µM), KH2PO4 (2.50 µM), Sucrose (87.64 mM)
MS + 2,4-D (1 mg/l), Kinetin (0.1 mg/l), Sucrose (5%) MS + 2,4-D (1 mg/l), Kinetin (0.1 mg/l), Sucrose (5%) MS + IAA (11.4 µM), NAA (10.8 µM), Kinetin (9.3 µM),
Coconut water (15%) MS + 2,4-D (1 ppm), Kinetin (2 ppm) MS + Kinetin (10 µM), 2,4-D (1 µM), Ascorbic acid
and Cystine (10 µM) B5 + 2,4-D (1.0 mg/l), Kinetin (0.1 mg/l) MS + 2,4-D (1 mg/l), Kinetin (0.1 mg/l), Sucrose (5%) LS + 2,4-D (2 mg/l) B5 + IBA (8 mg/l), Sucrose (1-8%) MS + IBA (2 mg/l), Kinetin (0.1 mg/l), Sucrose (3%),
Agar (9 g/l) LS + NAA (1.8 mg/l), 2,4-D (0.05 mg/l), BA (4.5 mg/l),
Picloram (0.05 mg/l) MS + 2,4-D (2 mg/l), Kinetin (0.5 mg/l), Sucrose (3%) MS + 2,4-D (1.0 mg/l), Kinetin (0.1 mg/l), Sucrose (3%),
Myo-inositol (100 mg/l) MS + Sucrose (3%) MS + NAA (2 mg/l), IAA (2 mg/l), Kinetin (0.1 mg/l),
Sucrose (3%) MS + NAA (1 mg/l) or IAA (3 mg/l) MS + 2,4-D (2.0 mg/l), Kinetin (5.0 mg/l), Sucrose (3%) MS + Casein hydrolysate (1 g/l), 2,4-D (0.5 mg/l),
Kinetin (0.75 mg/l) MS + Koblitz and Hagen vitamins and amino
acids, 2,4-D (4.52 µmol/l), Kinetin (1 µmol/l), GA3 (0.3 µmol/l), Sucrose (0.09 mol/l) B5 + 2,4-D (2 mg/l), Kinetin (0.2 mg/l), Cystine (50 mg/l), Sucrose (2%) B5 + 2,4-D (1.0 mg/l), Kinetin ( 0.2 mg/l) MS + 2,4-D (1 ppm), Kinetin (0.1 ppm), Myoinositol (100 ppm), Coconut milk (5%), Sucrose (2%) MS + 2,4-D (0.66 mg/l), Kinetin (1.32 mg/l), Coconut milk (100 ml) MS + Thiamine. HCl (0.9×103), Cysteine. Hcl (10.0×102), Kinetin (0.1×103), 2,4-D (0.1×103), Sucrose (30×103) MS + 2,4-D (1 mg/l), Kinetin (0.1 mg/l) MS + NAA (2 mg/l), BA (0.2 mg/l), Sucrose (2%)

Culture type Callus
Suspension Suspension Callus
Suspension Callus
Suspension Suspension Callus Root Suspension
Callus
Suspension Suspension
Suspension Suspension
Root Callus Suspension
Suspension
Suspension
Suspension Suspension
Callus
Callus
Callus Callus

Reference Andrijany et al., 1999
Anderson et al., 1987 Anderson et al., 1986 Malpathak and David, 1986
Yagi et al., 1983 Goleniowski and Trippi, 1999
De-Eknamkul and Ellis, 1985 Liu et al., 1990 Wang and Huang, 1982 Kusakari et al., 2000 Orihara and Furuya, 1990
Ramirez et al., 1992
Johnson et al., 1990 Nazif et al., 2000
Moreno et al., 1993 Zhao et al., 2001b
Teshima et al., 1988 Rajasekaran et al., 1991 Kueh et al., 1985
Koblitz et al., 1983
Schripsema et al., 1999
Wijnsma et al., 1985 Khouri et al., 1986
Barthe et al., 1987
Waller et al., 1983
Iwasa and Takao, 1982 Morimoto and Murai, 1989

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Vanisree et al. — Studies on the production of some important secondary metabolites

Table 1. (Continued) Plant name Cruciata glabra

Active ingredient Anthraquinones

Cryptolepis buchanani Roem. & Schult
Digitalis purpurea L. Dioscorea deltoidea Dioscorea doryophora Hance Duboisia leichhardtii Ephedra spp.
Eriobotrya japonica Eucalyptus tereticornis SM. Fumaria capreolata Gentiana sp. Ginkgo biloba Glehnia littoralis Glycyrrhiza echinata Glycyrrhiza glabra var. glandulifera Hyoscyamus niger Isoplexis isabellina Linum flavum L.

Cryptosin
Cardenolides Diosgenin Diosgenin Tropane alkaloids L- Ephedrine D-pseudoephedrine Triterpenes Sterols and Phenolic compounds Isoquinoline alkaloids Secoiridoid glucosides Ginkgolide A Furanocoumarin Flavanoids Triterpenes Tropane alkaloids Anthraquinones 5-Methoxypodophyllotoxin

Lithospermum erythrorhizon Lithospermum erythrorhizon Lycium chinense Mentha arvensis Morinda citrifolia Morinda citrifolia

Shikonin derivatives Shikonin derivatives Cerebroside Terpenoid Anthraquinones Anthraquinones

Mucuna pruriens Mucuna pruriens Nandina domestica Nicotiana rustica Nicotiana tabacum L. Ophiorrhiza pumila Panax ginseng Panax notoginseng Papaver bracteatum

L-DOPA L-DOPA Alkaloids Alkaloids Nicotine Camptothecin related alkaloids Saponins and Sapogenins Ginsenosides Thebaine

Papaver somniferum L. Papaver somniferum

Alkaloids Morphine, Codeine

Culture medium
LS + NAA (2 mg/l) , Kinetin (0.2 mg/l), Casein hydrolysate (1 g/l)
B5 + 2,4-D (2 mg/l), Kinetin (0.5 mg/l)
MS + BA (1 mg/l), IAA (1 mg/l), Thiamine. HCl (1 mg/l) MS + 2,4-D (0.1 ppm) MS + 2,4-D (2 mg/l), BA (0.2 mg/l) LS or B5 or White + NAA (5×10-5 M), BA (5×10-6 M) MS + Kinetin (0.25 µM), 2,4-D or NAA (5.0 µM),
Sucrose (3%) LS + NAA (10 µM), BA (10 µM) MS + 2,4-D (2 mg/l) LS medium B5 + Kinetin (1 mg/l), 2,4-D (0.5 mg/l) MS + NAA (1 mg/l), Kinetin (0.1 mg/l), Sucrose (3%) LS + 2,4-D (1 µM), Kinetin (1 µM) MS + IAA (1 mg/l), Kinetin (0.1 mg/l) MS + IAA (5 ppm), or 2,4-D (1 ppm), Kinetin (0.1 ppm) LS + NAA (10-5 M), BA (5×10-6 M) MS + 2,4-D (5 µM), Kinetin (10 µM) MS salts+ B5 vitamins, Folic acid (0.88 mg/l), Glycine
(2 mg/l), Sucrose (2%) LS + IAA (10-6 M), Kinetin (10-5 M) LS + IAA (10-6 M), Kinetin (10-5 M) MS + 2,4-D (1.0 ppm), Kinetin (0.1 ppm) MS + BA (5 mg/l), NAA (0.5 mg/l) B5 + NAA (10-5 M), N-Z-amine 0.2%, Sucrose (2%) B5 + NAA (10-5M), Kinetin (0.2 mg/l), Sucrose (4%),
Pluronic acid F-68 (2% w/v) MS + IAA (1 mg/l), BA (1 mg/l), Sucrose (4%) MS + 2,4-D (2.5 mg/l), Coconut water (10%) MS + 2,4-D (1.0 mg/l), Kinetin (0.1 mg/l) LS + 2,4-D (1 µM), Kinetin (1 µM) MS + NAA (2.0 mg/l), Kinetin (0.2 mg/l) LS + 2,4-D (0.22 mg/l), NAA (0,186 mg/l), Sucrose (3%) MS (without glycine) + 2,4-D (1 mg/l) MS + 2,4-D (2 mg/l), Kinetin (0.7 mg/l), Sucrose (3%) MS + Kinetin (0.47 µM), 2,4-D (4.52 or 0.45 µM),
Sucrose (3%) MS (without Glycine) + Kinetin (0.1 mg/l) MS + 2,4-D (0.1 mg/l), Cystine. HCl (2.5 mg/l), Kinetin
(2 mg/l), Sucrose (3%)

Culture type
Suspension
Callus
Suspension Suspension Suspension Callus Suspension
Callus Callus Suspension Callus Suspension Suspension Callus Callus Callus Suspension Suspension
Suspension Suspension Suspension Shoot Suspension Suspension
Suspension Callus Callus Callus Suspension Callus Callus Suspension Callus
Callus Suspension

Reference
Dornenburg and Knorr, 1996
Venkateswara et al., 1987
Hagimori et al., 1982 Heble and Staba, 1980 Huang et al., 1993 Yamada and Endo, 1984 O’Dowd et al., 1993
Taniguchi et al., 2002 Venkateswara et al., 1986 Tanahashi and Zenk, 1985 Skrzypczak et al., 1993 Carrier et al., 1991 Kitamura et al., 1998 Ayabe et al., 1986 Ayabe et al., 1990 Yamada and Hashimoto, 1982 Arrebola et al., 1999 Uden et al., 1990
Fujita et al., 1981 Fukui et al., 1990 Jang et al., 1998 Phatak and Heble, 2002 Zenk et al., 1975 Bassetti et al., 1995
Wichers et al., 1993 Brain, 1976 Ikuta and Itokawa, 1988 Tabata and Hiraoka, 1976 Mantell et al., 1983 Kitajima et al., 1998 Furuya et al., 1973 Zhong and Zhu, 1995 Day et al., 1986
Furuya et al., 1972 Siah and Doran, 1991

Botanical Bulletin of Academia Sinica, Vol. 45, 2004

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Vanisree et al. — Studies on the production of some important secondary metabolites

Table 1. (Continued)

Plant name

Active ingredient

Culture medium

Culture type

Reference

Peganum harmala L. Phytolacca americana Picrasma quassioides Bennett

β-Carboline alkaloids Betacyanin Quassin

Podophyllum hexandrum royle Polygala amarella Polygonum hydropiper

Podophyllotoxin Saponins Flavanoids

Portulaca grandiflora Ptelea trifoliata L.
Rauwolfia sellowii Rauwolfia serpentina Benth. Rauvolfia serpentina x Rhazya stricta
Hybrid plant Rhus javanica Ruta sp.
Salvia miltiorrhiza
Salvia miltiorrhiza Scopolia parviflora Scutellaria columnae Solanum chrysotrichum (Schldl.) Solanum laciniatum Ait Silybum marianum Solanum paludosum

Betacyanin Dihydrofuro [2,3-b] quinolinium
alkaloids Alkaloids Reserpine 3-Oxo-rhazinilam
Gallotannins Acridone and Furoquinoline
alkaloids and coumarins Lithospermic acid B and
Rosmarinic acid Cryptotanshinone Alkaloids Phenolics Spirostanol saponin Solasodine Flavonolignan Solamargine

Tabernaemontana divaricata Taxus spp.

Alkaloids Taxol

Taxus baccata

Taxol baccatin III

Thalictrum minus Thalictrum minus Torreya nucifera var. radicans

Berberin Berberin Diterpenoids

Trigonella foenumgraecum Withaina somnifera

Saponins Withaferin A

MS + 2,4-D (2 µM) MS + 2,4-D (5 µM), Sucrose (3%) B5 medium + 2,4-D (1.0 mg/l), Kinetin (0.5 mg/l),
Glucose (2%) B5 + NAA (4 mg/l), Coconut water (5%), Sucrose (4%) MS + 1 mg/l IAA MS + 2,4-D (10-6 M), Kinetin (10-6 M), Casamino acid
(0.1%), Sucrose (3%) MS (without Glycine)+2, 4-D (5 mg/l), Kinetin (0.2 mg/l) MS + 2,4-D (1 mg/l), Kinetin (0.1 mg/l), Coconut water
(5%) B5 + 2,4-D (1 mg/l), Kinetin (0.2 mg/l), Sucrose (3%) LS + NAA (10 µM), BA (1 µM) LS medium
LS + IAA (10-6 M), Kinetin (10-5 M) MS + 2,4-D (1 mg/l), Kinetin (1 mg/l)
MS + 2,4-D (0.5 mg/l), BA (0.5 mg/l)
MS + 2,4-D (1 mg/l), Kinetin (0.1 mg/l) LS + 2,4-D (10-6 M), IAA (10-5 M) MS + 2,4-D (0.3 mg/l), Kinetin (1 mg/l) MS + 2,4-D (2 mg/l), Kinetin (0.5 mg/l), Scrose (3-4%) MS + 2,4-D (1 mg/l), Kinetin (1 mg/l), Sucrose (3%) Hormone free LS medium MS + BA (10-6 M), NAA (10-6 M) or MS + Kinetin
(10-6 M) + 2,4-D (10-6 M) MS + NAA (2 mg/l), BA (0.2 mg/l) B5 medium + 2,4-D (0.2 mg/l), BA (0.5 mg/l),
Casein hydrolysate (200 mg/l), Sucrose (3%) B5 (salts) + 3× B5 vitamins, 2,4-D (2×10-2 mM)
Kinetin (4×10-3 mM) + GA3 (10-3 mM) LS + NAA (60 µM), 2,4-D (1 µM), BA (10 µM) LS + NAA (60 µM), BA (10 µM) MS + 2,4-D (10 mg/l), Casamino acid (1 g/l), Coconut mulk
(7%), and K+ instead of NH4+ MS + 2,4-D (0.25 or 0.5 mg/l), Kinetin (0.5 mg/l) MS + BA (1 mg/l), Sucrose (3%)

Suspension Suspension Suspension
Suspension Callus Suspension
Callus Callus
Suspension Suspension Callus
Root Callus
Callus
Suspension Callus Callus Suspension Suspension Root Suspension
Suspension Suspension
Suspension
Suspension Suspension Suspension
Suspension Shoot

Sasse et al., 1982 Sakuta et al., 1987 Scragg and Allan, 1986
Uden et al., 1989 Desbene et al., 1999 Nakao et al., 1999
Schroder and Bohm, 1984 Petit-Paly et al., 1987
Rech et al., 1998 Yamamoto and Yamada, 1986 Gerasimenko et al., 2001
Taniguchi et al., 2000 Baumert et al., 1992
Morimoto et al., 1994
Miyasaka et al., 1989 Tabata et al., 1972 Stojakowska and Kisiel, 1999 Villarreal et al., 1997 Chandler and Dodds, 1983a Alikaridis et al., 2000 Badaoui et al., 1996
Sierra et al., 1992 Wu et al., 2001
Cusido et al., 1999
Kobayashi et al., 1987 Nakagawa et al., 1986 Orihara et al., 2002
Brain and Williams, 1983 Ray and Jha, 2001

Abbreviations: B5 = Gamborg’s (1968) medium; BA = 6-Benzyladenine; 2,4-D = 2,4-dichlorophenoxyacetic acid; GA3 = Gibberellic acid; IAA = Indole-3-acetic acid; IBA = Indole-3butyric acid; 2iP = N6-[2-isopentenyl]-adenine; LS = Linsmaier and Skoogs (1965) medium; MS = Murashige and Skoog (1962) medium; NAA = Napthaleneacetic acid.

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to produce substances with antitumor, antiviral, hypoglycaemic, anti-inflammatory, antiparasite, antimicrobial, tranquilizer and immunomodulating activities through tissue culture technology.
Exploration of the biosynthetic capabilities of various cell cultures has been carried out by a group of plant scientists and microbiologists in several countries during the last decade. In the last few years promising findings have been reported for a variety of medicinally valuable substances, some of which may be produced on an industrial scale in the near future. The aim of the present review is to focus on the importance of tissue culture technology in production of some of the plant pharmaceuticals reported earlier. We will also describe the successful research on tissue cultures for production of bioactive metabolites performed at our own laboratory.
Tissue Cultures Producing Pharmaceutical Products of Interest
Research in the area of plant tissue culture technology has resulted in the production of many pharmaceutical substances for new therapeutics. Advances in the area of cell cultures for the production of medicinal compounds has made possible the production of a wide variety of pharmaceuticals like alkaloids, terpenoids, steroids, saponins, phenolics, flavanoids, and amino acids. Successful attempts to produce some of these valuable pharmaceuticals in relatively large quantities by cell cultures are illustrated.
Taxol
Taxol (plaxitaxol), a complex diterpene alkaloid found in the bark of the Taxus tree, is one of the most promising anticancer agents known due to its unique mode of action on the micro tubular cell system (Jordan and Wilson, 1995). At present, production of taxol by various Taxus species cells in cultures has been one of the most extensively explored areas of plant cell cultures in recent years owing to the enormous commercial value of taxol, the scarcity of the Taxus tree, and the costly synthetic process (Cragg et al., 1993; Suffness, 1995). In 1989, Christen et al. reported for the first time the production of taxol (placlitaxel) by

Botanical Bulletin of Academia Sinica, Vol. 45, 2004
Taxus cell cultures. Fett-Neto et al. (1995) have studied the effect of nutrients and other factors on paclitaxel production by T. cuspidata cell cultures (0.02% yield on dry weight basis). Srinivasan et al. (1995) have studied the kinetics of biomass accumulation and paclitaxel production by T. baccata cell suspension cultures. Paclitaxel was found to accumulate at high yields (1.5 mg/l) exclusively in the second phase of growth. Kim et al. (1995) established a similar level of paclitaxel from T. brevifolia cell suspension cultures following 10 days in culture with optimized medium containing 6% fructose. Ketchum and Gibson (1996) reported that addition of carbohydrate during the growth cycle increased the production rate of paclitaxel, which accumulated in the culture medium (14.78 mg/l). In addition to paclitaxel, several other taxoids have been identified in both cell and culture medium of Taxus cultures (Ma et al., 1994). Parc et al. (2002) reported production of taxoids by callus cultures from selected Taxus genotypes. In order to increase the taxoid production in these cultures, the addition of different amino acids to the culture medium were studied, and phenylalanine was found to assist in maximum taxol production in T. cuspidata cultures (FettNeto et al., 1994). The influence of biotic and abiotic elicitors was also studied to improve the production and accumulation of taxol through tissue cultures (Ciddi et al., 1995; Strobel et al., 1992; Yukimune et al., 1996). The production of taxol from nodule cultures containing cohesive multicultural units displaying a high degree of differentiation has been achieved from cultured needles of seven Taxus cultivars (Ellis et al., 1996). Factors influencing stability and recovery of paclitaxel from suspension cultures and the media have been studied in detail by Nguyen et al. (2001). The effects of rare earth elements and gas concentrations on taxol production have been reported (Wu et al., 2001 and Linden et al., 2001).
Morphine and Codeine
Latex from the opium poppy, Papaver somniferum, is a commercial source of the analgesics, morphine and codeine. Callus and suspension cultures of P. somniferum are being investigated as an alternative means for production of these compounds. Production of morphine and codeine in morphologically undifferentiated cultures has been re-

Vanisree et al. — Studies on the production of some important secondary metabolites

7

ported (Tam et al., 1980; Yoshikawa and Furuya, 1985). Removal of exogenous hormones from large-scale culture systems could be implemented using a two-stage process strategy by Siah and Doran (1991). Without exogenous hormones, maximum codeine and morphine concentrations were 3.0 mg/g dry weight and 2.5 mg/g dry weight, respectively, up to three times higher than in cultures supplied with hormones. Biotransformation of codeinone to codeine with immobilized cells of P. somniferum has been reported by Furuya et al. (1972). The conversion yield was 70.4%, and about 88% of the codeine converted was excreted into the medium.

Ginsenosides
The root of Panax ginseng C.A. Mayer, so-called ginseng, has been widely used as a tonic and highly prized medicine since ancient times (Tang and Eisenbrand, 1992a). Ginseng has been recognized as a miraculous promoter of health and longevity. The primary bioactive constituents of ginseng were identified as ginsenosides, a group of triterpenoid saponins (Huang, 1993a; Proctor, 1996; Sticher, 1998). Among them, ginsenoside Rg1 is one of the major active molecules from Panax ginseng (Lee et al., 1997). Chang and Hsing (1980a) obtained repeatable precocious flowering in the embryos derived from mature gin-

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seng root callus cultured on a chemically defined medium. Also, plant regeneration through somatic embryogenesis in root-derived callus of ginseng has been reported (Chang and Hsing, 1980b). In recent years ginseng cell culture has been explored as a potentially more efficient method of producing ginsenosides. The effect of medium components like carbon (Furuya et al., 1984; Choi et al., 1994), nitrogen (Franklin and Dixon, 1994), and phosphate (Zhang and Zhong, 1997) concentrations and plant growth hormones (Furuya, 1988) were thoroughly studied to increase the production of ginsenosides. Influence of potassium ion was also studied (Liu and Zhong, 1996). Large-scale suspension culture of ginseng cells was first reported by Yasuda et al. (1972). Later on an industrial-scale culture process was initiated by Nitto Denko Corporation (Ibaraki, Osaka, Japan) in the 1980s using 2000 and 20000-1 stirred tank fermentors to achieve productivities of 500-700 mg/l per day (Furuya, 1988; Ushiyama, 1991). This process is considered an important landmark in the commercialization of plant tissue and cell culture on a large scale. In addition to this, Agrobacterium tumefaciens infected root cultures were introduced, productivity of which was found to exceed the callus of normal roots threefold (Choi et al., 1989). Other types of tissue cultures, such as embryogenic tissues (Asaka et al., 1993) and hairy roots transformed by Agrobacteria (Yoshikawa and Furuya, 1987; Hwang et al., 1991; Ko et al., 1996) have been examined. Yu et al. (2000) reported ginsenoside production using elicitor treatment. These developments indicate that ginseng cell culture process is still an attractive area for commercial development around the world and it possesses great potential for mass industrialization. Concentration of plant growth regulators in the medium influences the cell growth and ginsenoside production in the suspension cultures (Zhong et al., 1996). Recent studies have shown that addition of methyl jasmonate or dihydro-methyl jasmonate to suspension cultures increases the production of ginsenosides (Wang and Zhong, 2002). Also, jasmonic acid improves the accumulation of gensinosides in the root cultures of ginseng (Yu et al., 2002).
L-DOPA
L-3,4-dihydroxyphenylalanine, is an important intermediate of secondary metabolism in higher plants and is known as a precursor of alkaloids, betalain, and melanine, isolated from Vinca faba (Guggenheim, 1913), Mucuna, Baptisia and Lupinus (Daxenbichler et al., 1971). It is also a precursor of catecholamines in animals and is being used as a potent drug for Parkinson’s disease, a progressive disabling disorder associated with a deficiency of dopamine in the brain. The widespread application of this therapy created a demand for large quantities of L-DOPA at an economical price level, and this led to the introduction of cell cultures as an alternative means for enriched production. Brain (1976) found that the callus tissue of Mucuna pruriense accumulated 25 mg/l DOPA in the medium containing relatively high concentrations of 2,4-D. Teramoto and Komamine (1988) induced callus tissues of Mucuna hassjoo, M. Pruriense, and M. deeringiana and optimized

Botanical Bulletin of Academia Sinica, Vol. 45, 2004
the culture conditions. The highest concentration of DOPA was obtained when M. hassjoo cells were cultivated in MS medium with 0.025 mg/l 2.4 -D and 10 mg/l kinetin. The level of DOPA in the cells was about 80 mmol/g-f.w.
Berberine Berberine is an isoquinoline alkaloid found in the roots
of Coptis japonica and cortex of Phellondendron amurense. This antibacterial alkaloid has been identified from a number of cell cultures, notably those of Coptis japonica (Sato and Yamada, 1984), Thalictrum spp. (Nakagawa et al., 1984; Suzuki et al., 1988), and Berberis spp. (Breuling et al., 1985). The productivity of berberine was increased in cell cultures by optimizing the nutrients in the growth medium and the levels of phytohormones (Sato and Yamada, 1984; Nakagawa et al., 1984, 1986; Morimoto et al., 1988). By selecting high yielding cell lines, Mitsui group produced berberine on a large scale with a productivity of 1.4 g/l over 2 weeks. Other methods for increasing yields include elicitation of cultures with a yeast polysaccharide elicitor, which has been successful with a relatively low producing T. rugosum culture (Funk et al., 1987). The influence of spermidine on berberine production in Thalictrum minus cell cultures has been reported by Hara et al. (1991).

Vanisree et al. — Studies on the production of some important secondary metabolites

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Diosgenin Diosgenin is a precursor for the chemical synthesis of
steroidal drugs and is tremendously important to the pharmaceutical industry (Zenk, 1978). In 1983, Tal et al. reported on the use of cell cultures of Dioscorea deltoidea for production of diosgenin. They found that carbon and nitrogen levels greatly influenced diosgenin accumulation in one cell line. Ishida (1988) established Dioscorea immobilized cell cultures, in which reticulated polyurethane foam was shown to stimulate diosgenin production, increasing the cellular concentration by 40% and total yield by 25%. Tal et al. (1983) have been able to obtain diosgenin levels as high as 8% in batch-grown D. deltoidea cell suspensions. However, the daily productivity was only 7.3 mg/l. Several other groups have also attempted cell cultures for diosgenin production (Heble et al., 1967; Brain and Lockwood, 1976; Jain and Sahoo, 1981; Jain et al., 1984; Emke and Eilert, 1986; Huang et al., 1993). Kaul et al. (1969) studied the influence of various factors on diosgenin production by Dioscorea deltoidea callus and suspension cultures. The search for high-producing cell lines coupled to recent developments in immobilized cultures and the use of extraction procedures, which convert furostanol saponins to spirostanes such as diosgenin, should prove useful in increasing productivity in the years to come.
Capsaicin Capsaicin, an alkaloid, is used mainly as a pungent food
additive in formulated foods. It is obtained from fruits of green pepper (Capsicum spp.). Capsaicin is also used in pharmaceutical preparations as a digestive stimulant and for rheumatic disorders (Sooch et al., 1977). Suspension cultures of Capsicum frutescens produce low levels of capsaicin, but immobilizing the cells in reticulated polyurethane foam can increase production approximately 100fold (Lindsey and Yeoman, 1984). Further improvements in productivity can be brought about by supplying precursors such as isocapric acid (Lindsey and Yeoman, 1984). Lindsey (1985) reported that treatments which suppress cell growth and primary metabolism seem to improve capsaicin synthesis. A biotechnological process has been de-

veloped for the production of capsaicin from C. frutescens cells (Lindsey et al., 1983). Holden et al. (1988) have reported elicitation of capsaicin in cell cultures of C. frutescens by spores of Gliccladium deliquescens. The effects of nutritional stress on capsaicin production in immobilized cell cultures of Capsicum annum were studied thoroughly by Ravishankar et al. (1988). Biotransformation of externally fed protocatechuic aldehyde and caffeic acid to capsaicin in freely suspended cells and immobilized cells cultures of Capsicum frutescens has also been reported (Ramachandra Rao and Ravishankar, 2000).
Camptothecin Camptothecin, a potent antitumor alkaloid was isolated
from Camptotheca acuminata. Sakato and Misawa (1974) induced C. acuminata callus on MS medium containing 0.2 mg/l 2,4-D and 1 mg/l kinetin and developed liquid cultures in the presence of gibberellin, L-tryptophan, and conditioned medium, which yielded camptothecin at about 0.0025% on a dry weight basis. When the cultures were grown on MS medium containing 4 mg/l NAA, accumulation of camptothecin reached 0.998 mg/l (Van Hengal et al., 1992). 10-Hydroxycamptothecin, a promising derivative of camptothecin is in clinical trials in the US.
Vinblastine and Vincristine The dimeric indole alkaloids vincristine and vinblastine
have become valuable drugs in cancer chemotherapy due to their potent antitumor activity against various leukemias and solid tumors. These compounds are extracted commercially from large quantities of Catharanthus roseus. Since the intact plant contains low concentrations (0.0005%), plant cell cultures have been employed as an alternative to produce large amounts of these alkaloids. Vinblastine is composed of catharanthine and vindoline. Since

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vindoline is more abundant than catharanthin in intact plants, it is less expensive. Misawa et al. (1988) established an economically feasible process consisting of production of catharanthine by plant cell fermentation and a simple chemical or an enzymatic coupling. The significant influence of various compounds, like vanadyl sulphate, abscisic acid, and sodium chloride on catharanthin production have been described by Smith et al. (1987). Endo et al. (1988) attempted synthesis of anhydrovinblastine (AVLB from catharanthine and vindoline through enzymic coupling followed by sodium borohydride reduction). A crude preparation of 70% ammonium sulphate precipitated protein from

Botanical Bulletin of Academia Sinica, Vol. 45, 2004
the cultured cells of C. roseus was used as an enzyme source. The reaction mixture contained catharanthine, vindoline, Tris buffer, Ph 7.0, and the crude enzyme; the mixture was incubated at 300°C and for 3 h. The products of the reaction were various dimeric alkaloids including vinamidine, 3(R)-hydroxyvinamidine, and 3, 4anhydrovinblastine. Dimerization using ferric ion catalyst in the absence of enzyme resulted in anhydrovinblastine and vinblastine in 52.8% and 12.3% yields, respectively. The yield of vinblastine via chemical coupling was improved in the presence of ferric chloride, oxalate, maleate, and sodium borohydride. Influence of various parameters like stress, addition of bioregulators, elicitors and synthetic precursors on indole alkaloids production were studied in detail by Zhao et al. (2001a and b). Also, metabolic ratelimitations through precursor feeding (Morgan and Shanks, 2000) and effect of elicitor dosage on biosynthesis of indole alkaloids (Rijhwani and Shanks, 1998) in Catharanthus roseus hairy root cultures have been reported.
Tanshinones
Tanshinones are a group of quinoid diterpenoids believed to be active principles of Danshen (Salvia miltiorrhiza), a well known traditional Chinese medicine. Tanshinone I and cryptotanshinone prevent complications of myocardial ischemia; tanshinone II A has undergone