South African Journal of Botany 2004, 70(1): 89–96 Copyright © NISC Pty Ltd Printed in — All rights reserved SOUTH AFRICAN JOURNAL OF BOTANY EISSN 1727–9321

Medicinal at the ethnobotany–biotechnology interface in Africa

SA Nigro1, NP Makunga1* and OM Grace2

1 Research Centre for Growth and Development, School of Botany and Zoology, University of KwaZulu-Natal Pietermaritzburg, Private Bag X01, Scottsville 3209, South Africa 2 Ethnobotany Unit, National Botanical Institute, PO Box 52099, Berea Road, Durban 4007, South Africa * Corresponding author, e-mail: [email protected]

Received 5 September 2003, accepted 15 October 2003

Africa bears a long history of people–plant interaction useful plants. African ethnomedicinal plants with valu- that has been significantly enhanced by biotechnology. able secondary metabolites and established in vitro Ethnomedicinal plants have been targeted in the search propagation protocols may be likely candidates for for new natural products but their exploitation has led to genetic transformation when biotechnology becomes concern for their conservation. Biotechnology has more accessible on the continent. The ethnic and floral applications that extend beyond conservation to opti- wealth of southern Africa lends a prime example of how mising natural product research and adding value to biotechnology may enrich people–plant interactions.

Introduction

Africa, the cradle of mankind, bears a long history of people- commercial value (Coombes 1986), biotechnology is an plant interaction. It is the second largest of the earth’s seven important instrument for the manipulation of plants to better continents, and is characterised by rich ethnic and biotic suit the needs of Man. In adding value to useful plants, it mosaics that represent 13% of the earth’s human population plays a developmental role at the interface of ethno- and and the largest of continental floras, estimates of which economic botany. The term was first applied by the range between 50 000 and 70 000 plant taxa (Klopper et al. Hungarian engineer Karl Ereky in 1917 to ‘all lines of work 2002, Smith and Van Wyk 2002). The African flora is remark- by which products are produced from raw materials with the able not only for its diversity but its distinctiveness: as many aid of living things’ (Glick and Pasternak 1994). Although as 88% of its species are endemic (Davis et al. 1994). High biotechnology referred originally to a system for the large- levels of endemism indicate that many of the continent’s scale production of pigs, the term was used loosely until plant resources are uniquely African. the establishment of the journal Biotechnology and The terms ‘ethno-’ and ‘economic’ botany to describe the Bioengineering in 1961. Biotechnology was redefined as the study of people–plant interaction have been used inter- ‘industrial production of goods and services using biological changeably and deserve clarification. We refer to Wickens organisms, systems, and processes’ within the disciplines of (1990) who defined ethnobotany as ‘the study of useful microbiology, biochemistry and chemical engineering (Glick plants prior to their commercial exploitation and eventual and Pasternak 1994). The discovery of recombinant DNA domestication’, and economic botany as an umbrella term technology in 1973 (Cohen et al. 1973) introduced biotech- applied to ‘the study of plants utilised either directly or indi- nology at the molecular level, where an organism’s machin- rectly for the benefit of Man’. Economic plants therefore ery could be modified to manufacture desired products. include those used in agriculture, horticulture and forestry The ethnobotanical approach to natural products research (Wickens 1990) and in turn provide for the many industries highlights that those plant taxa used traditionally are most that make use of plant products. In Africa, where approxi- likely to yield useful products, and, consequently, are most mately 43% of the population are impoverished (Africa South likely to be threatened by over-exploitation (Grace et al. of the Sahara 2001), the same plant species may be used at 2002). It is not surprising, therefore, that useful plant species all levels between subsistence and commercial agriculture, have been targeted in the search for and optimisation of new thus blurring the definition of ‘ethno-’ and ‘economic’ plants. natural products, or that the value added to them using The transitory process of a useful plant species assuming biotechnology has strengthened the call for their conserva- commercial importance has been enhanced significantly in tion. Of the multitude of purposes for which Man exploits modern times by biotechnology. Described as the use of bio- plants, their use in ethnomedicine is among the most rele- logical entities to create products with novel functions or vant to modern society. Ethnomedicinal plants have con- 90 Nigro, Makunga and Grace tributed many important phytochemicals to allopathic medi- ing the same period (Bajaj 1988–1997). Progress was made cine. African examples include molluscicides from in particular on in vitro techniques such as micropropaga- Phytolacca octandra L. (Phytolaccaceae) used to control the tion, induction of high yielding somaclones, protoplast fusion schistosomiasis vector (Lemma 1991 cited in Horeau and for the induction of novel somatic hybrids, cyropreservation DaSilva 1999), and several compounds from Catharanthus of germplasm, and Agrobacterium rhizogenes-mediated roseus G.Don (Apocynaceae) used in therapy of diabetes transformation for hairy roots (Bajaj 1998). and tumours (George et al. 2001). Familiar plant products The main objectives of such research are to enhance the with African origins include Old World cotton (Gossypium production of secondary metabolites, by manipulating plant herbaceum L., Malvaceae), coffee (Coffea spp., cells to increase metabolic flux into specific pathways (Dixon Rubiaceae), and frankincense (Boswellia papyrifera (Del.) and Bolwell 1986). Since many widely used natural products Hocsht, Burseraceae) (Geldenhuys and Van Wyk 2002). are remarkably complex in structure, and despite advances In this paper we consider the role of biotechnology at the in synthetic chemistry, synthesis is frequently difficult and interface of ethno- and economic botany in Africa. We review economically non-viable (Hamill and Lidgett 1997). Indeed, progress made in the disciplines over recent decades, and plants remain the major sources of a variety of indispensa- speculate on the way forward, with particular reference to ble industrial compounds, examples of which include phar- the search for natural products from ethnomedicinal plants in maceuticals (e.g. steroids, alkaloids), food additives (e.g. the southern African flora. emulsifiers, natural flavourants and colourants) (Balandrin and Klocke 1988, Bajaj and Ishimaru 1999) and, more Understanding Medicinal Plants recently, nutraceutics (Bourgaud et al. 2001).

The documentation and study of people-plant interaction in Conserving the African Flora Africa began relatively recently with the arrival of European colonists (Van Wyk 2002). Ethnobotanical accounts of the Taxa used for their secondary metabolites on a commercial east African flora include those of Williamson (1955) and or industrial level may be considered ‘economic plants’, but Lindsay (1978). Burkhill’s (1985) volumes for West Tropical the material from which useful compounds are extracted is Africa are keystones for that region, as is Dounias’ review not always cultivated, or wild-harvested on a sustainable (2000) for Central and West Africa. Plant use throughout the basis. A case in point is Prunus africana (Hook.f.) Kalkm. continent was considered recently by Iwu (1993) and (Rosaceae) bark, from which several secondary metabolites Neuwinger (2000), and continues to be documented by pro- are extracted for use in European pharmaceuticals. The grammes such as the Survey of Economic Plants for Arid principal sources of the bark are natural forest populations in and Semi-Arid Lands (SEPASAL) (Davis et al. 1998, SEPA- Africa and Madagascar and, despite legislation to ensure SAL 2003). otherwise, harvesting is non-sustainable (Cunningham and The history of ethnobotanical research in southern Africa Mbenkum 1993, Prunus Net 2003). was thoroughly reviewed by Van Wyk (2002), but a literature A similar situation affects ethnomedicinal plant taxa in search of English sources will yield several key publications southern Africa; they are almost exclusively wild-harvested (Grace et al. 2002). Ethnomedicinal plants in southern Africa to meet the demands of a booming informal trade and con- were accounted for in the famous tome of Watt and Breyer- sumer industry (see, for example, Cunningham 1988, Brandwijk (1962), and proceeded by those of, for example, Mander 1998, Williams et al. 2000). Germplasm conserva- Mabogo (1990), Hutchings et al. (1996), Van Wyk et al. tion, including propagation and breeding to improve various (1997) and Arnold et al. (2002). Concern for the conserva- traits (Nessler 1994), and cryopreservation, is focussed on tion of medicinal plants in recent decades stimulated crop plants but is equally relevant to wild-harvested medici- research that gave rise to a collection of cornerstone publi- nal taxa. This branch of conservation seeks to arrest genet- cations on their trade. Those dealing with southern Africa ic erosion in indigenous taxa (Bukenya-Ziraba 1998) that include Cunningham (1988), Scott-Shaw (1990), Mander et may occur as a consequence of localised extinction and dis- al. (1997), Mander (1998), Botha et al. (2001) and Williams placement. To this effect, germplasm collections have been et al. (2000, 2001). established in several African countries, including Kenya Although the focus of biomedical research shifted early (Isahakia et al. 1998), Nigeria (Ng 1998), South Africa (Van last century to pure chemistry for pharmaceuticals, natural Zijl and Botha 1998), Tanzania (Marandu and Nkya 1998), product research has continued to yield therapeutic com- Uganda (Bukenya-Ziraba 1998) and Madagascar pounds from secondary plant metabolites (Cragg et al. 1997, (Zoeliarisoa 1998). Seedbanks fulfil a similarly critical role in McChesney 2001). Phytochemical and pharmacological providing seed for living collections of rare and threatened investigations have concentrated on American and Asian taxa, habitat restoration, species reintroduction and sustain- healthcare and relatively limited work has been conducted able utilisation programmes (Smith et al. 2002). on South African medicinal plants (Iwu 1993, George et al. Biotechnology contributes to these processes via seed 2001). Nonetheless, bioactivity of some popular ethnomedi- research that may include dormancy studies, genetic finger- cinal taxa used in southern Africa has been confirmed in printing and propagation studies (Smith et al. 2002). Given recent decades by in vitro tests and the elucidation of active that 86 southern African ethnomedicinals are Red Data constituents (for further discussions, see George and Van Listed and 4% of the nearly 4 000 ethnomedicinals Staden 2000 and George et al. 2001). International biotech- Threatened (Arnold et al. 2002), the reasons for germplasm nology research on medicinal plants gained momentum dur- and seed conservation are clear. South African Journal of Botany 2004, 70: 89–96 91

Advances in DNA-based tools have extended the applica- researchers focus on herbaceous taxa (see Fennell and Van tions of molecular biotechnology to complement conserva- Staden, this issue, for a review of bulbous taxa), advances tion. Restriction fragment length polymorphism (RFLP) and have been reported on the micropropagation of less compli- techniques based on the polymerase chain reaction (PCR) ant woody subjects, including the economically important are most widely applied; they include amplified fragment yellowwoods, Podocarpus elongatus (Aiton) L’Hér. ex Pers. length polymorphism (AFLP), random amplified polymorphic and P. henkelii Stapf. ex Dallim. & Jacks. (Podocarpaceae) DNA (RAPD), simple sequence repeat (SSR) and single (Kowalski and Van Staden 1998, 2001a), and Cussonia pan- strand conformation polymorphism (SSCP) (Guarino and iculata Eckl. & Zeyh. (Araliaceae) (Tetyana and Van Staden Dawson 1998). PCR-based techniques, isozyme analyses, 2001). Southern Africa’s two most threatened ethnomedici- DNA sequencing and RFLP are widely used by conservators nal trees, Ocotea bullata (Burch.) Baill. (Lauraceae) and for genetic fingerprinting, establishing phylogenetic relation- Warburgia salutaris Bertol.f. Chiov. (Canellaceae), have also ships and analysing genetic linkages that help to answer been successfully propagated in vitro (Kowalski and Van questions of plant relationships and distributions. Staden 2001b). In vitro-propagated ethnomedicinal taxa of Techniques such as nucleic acid hybridisation, polyacry- commercial interest to the horticulture and floriculture indus- lamide gel electrophoresis (PAGE) tests and DNA hybridisa- tries include numerous liliaceous (s.l.) taxa; examples from tion are used routinely to detect pathogens by plant quaran- the Hyacinthaceae are Boweia volubilis Harv. ex Hook.f. tine controllers governing international germplasm (Hannweg et al. 1996), spp. (McCartan and Van exchange (Abdallah and Black 1998). Staden 1995, Ault 1995, McCartan et al. 1999, Taylor and Van Staden 2001b) and Scilla spp. (Chakravaty and Sen Mass Producing Medicinal Plants 1989, McCartan and Van Staden 2002). Successful micropropagation protocols for southern The need to propagate economically important plants aris- African ethnomedicinal taxa known to contain commercially es not only from the obvious implications of harvesting important secondary metabolites indicate likely candidates pressure on natural populations, but also for the practical for future genetic transformation studies. Crouch et al. benefits of optimising secondary metabolite production. (1990) reported an in vitro system for the insectivorous Indeed, an efficient and reproducible system of regenerat- Drosera spp. (Droseraceae) that are known to contain ing plant material in vitro is preferable, but not a pre-requi- plumbagin and related compounds. Micropropagation proto- site for all genetic transformation techniques (Birch 1997). cols have similarly been reported for the colchicine-contain- Micropropagation is perhaps the largest component of plant ing Gloriosa superba L. (Colchicaceae) (Finnie and Van biotechnology exploited on a commercial scale world wide, Staden 1989) and Salvia chamelaeagnea P.J.Bergius and micropropagation of medicinal taxa has assumed its (Lamiaceae) for rosmarinic acid, a compound with anti- own economic importance. oxidative properties (Hung and Van Staden 2002). The scope of this paper does not extend to provide a com- Hypoxoside, a valuable medicinal compound that has prehensive list of all African taxa for which micropropagation proved difficult to obtain synthetically, can be produced and protocols have been, or are being, developed. Examples concentrations manipulated using an in vitro culture system from the southern African flora suffice to demonstrate the for Hypoxis rooperi T.Moore (syn. H. hemerocallidea Fisch. twofold benefits of ex situ conservation and commercial & Avé-Lall., Hypoxidaceae) (Page and Van Staden 1984, value that micropropagation protocols offer to ethnomedici- 1987, Appleton and Van Staden 1995). Taylor and Van nal taxa. Among the earliest reports of in vitro propagation of Staden (2001a, 2001c) showed that in vitro bioactivity of southern African ethnomedicinal taxa was one for Aloe pre- Eucomis autumnalis (Mill.) Chitt. against the inflammatory toriensis Pole-Evans (Groenewald et al. 1975). It was fol- response may be upregulated by manipulating the micro- lowed by those of Bayley and Van Staden (1987), Meyer and propagation conditions. Van Staden (1991), Richwine (1995) and Abrie and Van Staden (2001) for other members of the Asphodelaceae Optimising secondary metabolite production (Aloe, Gasteria, Haworthia). The research history of the aloes alone serves to indicate the volume and possible time- The use of cultivated rather than wild-harvested medicinal frame of work required in establishing viable micropropaga- plants not only alleviates pressure on natural populations, tion systems. but facilitates standardisation and increased safety, as Micropropagation plays an important role in modern con- inconsistencies in the quality and composition (due to geno- servation and is used not only to mass-propagate threat- typic and phenotypic variation) are reduced, risks of adulter- ened taxa, but also for seed germination, long-term ation lowered, and yields raised by management practice germplasm storage and establishing pathogen-free material (Grace et al. 2002). The controlled environments of tissue (Fay 1996). Valuable contributions made to southern African culture systems and cell suspension cultures offer the bene- conservation by micropropagation have included the first fits of optimised plant uniformity, continuous and consistent successful protocol for in vitro morphogenesis in the critical- natural product extraction (Dixon and Bolwell 1986). In vitro ly endangered cycads Stangeria eriopus (Kunze) Baill. plant cell and callus cultures, first described by Nickell (Stangeriaceae) (Osborne and Van Staden 1987) and (1962), are a firmly established means to produce second- Encephalartos cycadifolius (Jacq.) Lehm. (Jäger and Van ary metabolites from rare or threatened plants (Bajaj and Staden 1996), and the rare Gerbera aurentiaca Sch. Bip. Ishimaru 1999) and a range of products have been pro- () (Meyer and Van Staden 1988). Although duced and patented (Dodds and Roberts 1985). For high 92 Nigro, Makunga and Grace volume secondary metabolite production, cell suspension vitro. Somaclonal variation, although frequently considered cultures are favoured for their rapid growth cycles and the an undesirable phenomenon in cell cultures, has been totipotency of plant cells (Fu 1998). Whole organs may be recognised as a ‘source of exploitable variation’ (Larkin and cultured in vitro for the production of secondary metabolites Scowcroft 1981 cited in Van Den Bulk 1991). Somaclonal requiring a differentiated system. The profile of metabolites variations have given rise to cell lines exhibiting novel or generated by such specialised cultures resembles those of escalated levels of useful secondary metabolites due to whole plants (Fu 1998, Luczkiewicz et al. 2002) and surpass genetic instabilities that have attained significant commercial cell and callus cultures in their genetic stability and metabol- value (Bajaj 1998). Similarly, protoplast fusion has been suc- ic consistency. cessfully used to establish somatic medicinal hybrids with In vitro cultures tend to accumulate secondary metabolites increased alkaloid levels and pathogen resistance. Gene when subjected to chemical stress or stimuli such as fungal transfer studies have been conducted on some 70 medicinal elicitors, and varied environmental conditions (Balandrin and plant taxa worldwide (for an overview, see Bajaj and Klocke 1988, Toivonen and Rosenqvist 1995, Dixon and Ishimaru 1999) with the intent of optimising secondary Steele 1999, Kovalenko et al. 2002). Indeed, these factors metabolite production and harvesting. Both direct and indi- may even elicit de novo synthesis of novel compounds not rect methods of gene transfer have been used in medicinal usually present in the plant taxon (Bajaj and Ishimaru 1999). plants, of which Agrobacterium-mediated transfer has The growth, production and excretion of secondary metabo- proven the most popular. Early transformation studies used lites from cell suspension cultures in a continuous produc- direct DNA transfer methods such as the polyethylene glycol tion process have been aided by numerous emerging tech- (PEG) technique (Krens et al. 1982), electroporation niques. These include immobilisation of plant cells (see Fu (Neumann et al. 1982), particle bombardment (Klein et al. 1998 for a review), permeabilisation, two-phase technology, 1987), and to a lesser extent, microinjection (Crossway et al. the use of bioreactors and elicitors (reviewed by Bourgaud 1986). More recently, the use of Agrobacterium rhizogenes et al. 2001). has been preferred for its root-inducing (Ri) plasmid that Shikonin, the first phytochemical product to be derived induces hairy roots (Chilton et al. 1982) resulting from rol T- from in vitro cell cultures of Lithospermum erythrorhizon DNA expression and upon integration into the plant genome. Sieb. & Zucc. (Boraginaceae) (Tabata and Fujita 1985) was The transgenic hairy root organ cultures typically contain succeeded by only a limited number of similar products, higher levels of extractable secondary metabolites (Hamill et despite intensive research efforts (Buitelaar and Tramper al. 1987) or novel compounds (Bajaj and Ishimaru 1999) 1992, Hamill and Lidgett 1997). Natural products from south- than non-transgenic whole plants or cell suspension cultures ern African taxa that are manufactured and marketed on a (Rhodes et al. 1990). A wealth of literature exists on the commercial basis include immunostimulatory phytosterols genetic transformation of medicinal plants; for reviews see from Hypoxis hemerocallidea L. (Hypoxidaceae) in Bajaj (1988–1997). Moducare®, as well as over-the-counter herbal formulations The commercial importance of plants used in holistic ther- of Aloe spp. (Asphodelaceae), Harpagophytum procumbens apies such as homeopathy and aromatherapy, or as sources DC. ex Meisn. (Pedaliaceae), Senna italica Mill. (Fabaceae) of phytochemicals used in allopathic drugs, has made them and Sutherlandia frutescens R.Br. ex W.T.Aiton (Fabaceae). attractive candidates for genetic transformation. Few African Other southern African ethnomedicinal taxa of commercial ethnomedicinals have benefited from similar research atten- interest include afra L. (Asteraceae), Aspalathus tion, but an increase is likely in the current climate of active linearis (Burm.f.) R.Dahlgren (Fabaceae), Combretum caf- bioprospecting on the continent. African ethnomedicinal taxa frum (Eckl. & Zeyh.) Kuntze, Combretum kraussii Hochst. to have been genetically transformed are the north African (Combretaceae), Hoodia pilifera (L.f.) Plowes Pimpinella anisum L. (Umbelliferae) and Thapsia garganica (Apocynaceae), Siphonochilus aethiopicus Wood & Franks L. (Apiaceae). Cell culture, somatic embryogenesis and the (Zingiberaceae), some of which are the subject of patent production of anise oil from P. anisum were reviewed by applications (George et al. 2001, Geldenhuys and Van Wyk Ernst (1989). A co-cultivation technique using 2002, Van Wyk 2002). Agrobacterium tumefaciens was initially employed for DNA Limitations to the success of in vitro phytochemical pro- transfer (Komari 1989) but the establishment of plants duction have been ascribed to a lack of basic knowledge of regenerated from transformed callus was not reported. biosynthetic pathways and mechanisms involved in second- Later, the transformation and biochemical studies of Salem ary metabolic processes (Buitelaar and Tramper 1992), and, (1994) and Salem and Charlwood (1995) produced stable concomitantly, inadequate metabolite accumulation (Hamill transformed callus tissue and shoot cultures using direct et al. 1987). Progress in the fields of cell, developmental and Agrobacterium infection at different sites of P. anisum molecular biology of secondary metabolism have led to a seedlings (for a summary, see Charlwood and Salem 1999). renewed appreciation of the complexity of biosynthetic path- For T. garganica, a two-step protocol combining particle ways, their regulation and exploitation for the production of transfer and Agrobacterium-mediated transformation was valuable phytochemicals (Facchini 2001). recently published (Makunga et al. 2003). Transient expres- sion was shown concomitantly with stable integration of Transgenic medicinal plants reporter and rol genes, although the regeneration of hairy roots true to phenotype was not indicated. The commercial value of secondary metabolites has driven Catharanthus roseus, indigenous to Madagascar, has much research into ways of optimising their production in received much attention due to its high alkaloid content. South African Journal of Botany 2004, 70: 89–96 93

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