If cloning is the norm among animals and there is no chance for mating, then the earth would be full of the organisms as ‘Dollies’. Similarly in a situation where all plants multiply by simple detachment of parts and growth of each part into a faithful photocopy of the mother plant, then there would be very little room for variation and evolution without which living organisms cannot adapt to unique and changing environments, pests and diseases. Fortunately it is not the case to be. Variety is the spice of life and basic characteristic of life is its unlimited diversity. Nature has myriads of life forms on this planet among which variations are of ubiquitous occurrence. It is particularly so in the megacentres of diversity in the tropics which harbour approximately two-thirds of the biota and where many species of economic importance presumably had their origin. As a matter of fact, variations between individuals of the species were observed over the millennia and were considered as real ‘hot spots’ of evolution.
The notion that no two individuals of a sexually reproducing population are 100% identical prevailed even when methods of scientific scrutiny were not available. In 17th century when the concept of a species was poorly understood, the English naturalist John Ray (1628-1705) showed that within a species there might occur individuals different from the normal in one or more characters. Later Charles Darwin (1809-1882) developed this concept into a supremo and effectively linked the variations with natural selection, survival of the fittest and origin of new species. Down the years, variations among plants and animals had always fascinated an inquisitive mind and helped an evolutionary biologist or a breeder to select a desirable variant or breed a new form of greater agronomic value. Traditional physicians and village doctors of yester years were no different. They collected herbs of certain morphological attributes (of flowers, fruits, leaves etc.) and preferred root drugs of specific colour, smell, size, fibrous content, itching quality etc. obviously from locations known only to them. Even within a medicinal plant species, sometimes one variety was preferred over others. It is also not surprising that curative properties of a plant species change according to seasons or developmental stages and hence vaidyas prefer to collect required plants or their parts during certain periods only (Bharat, 1997).
In the wilderness of the tropics, plants grow in extreme situations along longitudinal, latitudinal and temperature gradients and therefore variations within and between populations of a species are not uncommon. Although plants in general show habitat and distribution preferences, there are many a species which are neutral and adapted to other environmental regimes. Much of the variations in phenotype observed in natural populations of a species were earlier attributed to environmental influences (Briggs and Walters, 1984). Thus individuals of a species adapted to a particular soil type and climatic zone were designated as edaphic ecotypes and climatic ecotypes respectively. Many botanists reasoned that distinct intraspecific variations of plants were merely due to habitat modifications and adaptation to environment was by phenotypic plastic response. Phenotype was accepted as a resultant product of interaction between genotype and environment and different phenotypes of a given genotype could occur in different environments. Since a plant remains static and possesses persistent meristems and succession of organs of limited growth, it was thought to show greater phenotypic variability than a higher animal.
In certain plants developmental variations were observed as evident from differential morphological characteristics of the juvenile and adult forms. In early 1920s, the European botanist Turesson added yet another dimension to the problem of variations by demonstrating the persistence of morphological variations in the same species under standard conditions of cultivation (Turesson, 1922 a, b). The results of his ingenious experiments proved beyond doubt the widespread occurrence of intraspecific, habitat correlated genetic variations. It was soon realized that adaptation to environment was sometimes by plastic responses but more frequently had a genetic basis. Since selection operated in natural populations, well-adapted genotypes were thought to be selected preferentially over others in each habitat.
The problem of variations is further compounded in medicinal plants which apart from displaying visible variations, synthesize and accumulate an array of plant-specific chemicals. These compounds together called secondary metabolites are mostly high-value, low-volume compounds biosynthetically derived from primary metabolism and accumulated by certain plants or groups of plants in trace quantities. In plants 'defense chemicals' form a therapeutic arsenal to fight a variety of biotic and abiotic stresses. A study of variation in the active principles is often an important element in the investigation of variation in such plants. Although only limited number individuals in a plant population were usually subjected to chemical scrutiny to represent the population of a taxon in earlier studies, subsequent investigations brought out significant variations within as well as between populations. Thus it became reasonable to assume that chemotypes or chemical variants occurred in wild populations of medicinal plant species.
A wide spectrum of simple and overlapping variations is now documented in plants (Sen and Sharma, 1990; Connoly et al, 1994; Stewart and Porter, 1995; Demeke et al, 1996; Sonnante et al, 1997; James and Ashburner, 1997). In general, all observed variations are broadly grouped into two categories: epigenetic and genetic. Genetic variations in plants are strictly heritable i.e. truthfully passed on from one generation to another through seeds and do not change under conditions of cultivation. They occur invariably due to alterations in the genetic material and may affect both phenotypic and chemical characteristics of a medicinal plant. Epigenetic variations on the other hand, are mostly induced by the environment in which the plants grow and are also partially affected by developmental events. Epigenetic changes in medicinal plants in general include morphological and chemical as well as physiological variations. Therefore a great deal of information- morphological, biochemical, physiological and genetic is necessary before the observed pattern of variation may be interpreted. It is also true that beneath these intraspecific variations, there exists a fixed unchangeable genetic spectrum of characteristics that makes up the species.
Definitive evidence for genetic variations became available only in late 1960s (Ayala and Kiger, 1980). This was provided by artificial selection experiments in cultivated crops such as rice, wheat etc. and laboratory model organisms like Drosophila. This kind of variation may be ultimately related to variation in the sequence of DNA base pairs, that constitutes the genetic code. Genetic variation may arise in individuals by gene and chromosome mutations which can be spread through the population by recombination. The occurrence of two or more indigenous forms in the same population/area and adaptability of these forms to different environment being maintained by natural selection thus became clear. In few such species showing balanced polymorphisms, the heterozygote is favoured compared to either of the homozygotes, and this maintains a high degree of genetic diversity in the species. In fact plants show a rather high degree of heterozygocity. Although reliable estimates of average heterozygocity are now available for more than 100 species of plants and animals, they do not include medicinal plants (Narain, 2000).
References for genetic variation among medicinal plants are scanty although analysis of such variations holds great promise owing to the location specific attributes of the herbs and the attendant diversity of plant-specific compounds of therapeutic and industrial value. It is also now understood that the loss of genetic variation within a given species (genetic depletion) is usually much more serious and occurs much earlier than the total extinction of the species itself. In the case of Dioscorea zingiberensis, plants collected during 1950s accounted for a maximum of 17% diosgenin whereas during 1980s the content was reduced to such an extent that even 4% was considered as high (He and Sheng, 1997). The only solution proposed to this rapid exhaustion of diversity is selection and cultivation of promising chemotypes. Standing examples in this line are Coleus forskohlli (Hegde and Gangadharappa, 1997), Chamomilla recutita (Bettray and Vomel, 1989), Mentha arvensis (Tandon et al, 1998), Aconitum napellus ssp. Tauricum (Colombo et al, 1989), Valeriana officinalis (Bos et al, 1998), Mentha spicata (Misra et al, 1989), Eucommia ulmoides, Coptis chinensis, Magnolia officinalis, Louicera japonica and Tripterygium wilfordii (Anonymous, 1989; Peng and Xiao, 1993).
India is a known mega-diversity centre harbouring a multitude of medicinal plant species each presumably studded with as yet unknown genetic and chemical variations of economic importance. Out of an estimated 17,000 higher plant species occurring in India, more than 1000 species are used over several centuries in the traditional systems of medicine viz. Ayurveda, Siddha, Unani and Amchi. The villagers and tribal folks spread across the length and breadth of the country make use of more than 7000 plant species through oral traditions (Pushpangadan et al., 1997). Nearly 3/4 of the herbal drugs and perfumery products used in the world are available in natural state in India. Therefore, the rich and varied plant diversity, especially the genetic diversity of medicinal and aromatic plants, is one of India's important strengths and is the bedrock for all future bioindustrial developments. Unfortunately, the renowned medicinal plant wealth of India has seldom been subjected to genetic scrutiny keeping in mind the latent and patentable properties and economic utility of the selected plant types. As severe habitat losses and consequent endangerment and extinction of known and hitherto lesser known species of economic value are not uncommon in the Indian subcontinent, it is imperative that heritable variations within the otherwise unimproved natural populations of prospective taxa are studied for selection, improvement and development of suitable cultivars. Otherwise called bioprospecting, this line of research is essential to fish out useful genes and gene products for commercialisation in the now unfolded patent regime. Knowledge of the genetic diversity is also a prerequisite for any in situ and ex situ conservation schemes (Hamrick et al., 1991) as it is not practical to conserve all genotypes of a given species against the mass extinction spasm projected for the 21st century (Raven, 1999).
Andrographis paniculata Nees (Acanthaceae), the Kalmegh of Ayurveda selected for the present investigation is an erect annual herb (Fig. 1.1) extremely bitter in taste in each and every part of the plant body. The plant is known in north-eastern India as ‘Maha-tita’, literally ‘king of bitters’ and known by various vernacular names (Table 1.1). It is also known as ‘Bhui-neem’, since the plant, though much smaller in size, shows similar appearance and has bitter taste as that of Neem (Azadirachta indica). Incidentally, the genus Andrographis consists of 28 species of small annual shrubs essentially distributed in tropical Asia (Fig 1.2). Only a few species are medicinal, of which A. paniculata is the most popular.
It grows erect to a height of 30-110 cm in moist shady places with glabrous leaves and white flowers with rose-purple spots on the petals. Since ancient times, A. paniculata is used as a wonder drug in traditional Siddha and Ayurvedic systems of medicine as well as in tribal medicine in India and some other countries for multiple clinical applications. The therapeutic value of Kalmegh is due to its mechanism of action which is perhaps by enzyme induction. The plant extract exhibits antityphoid and antifungal activities (Anonymous, 1985). Kalmegh is also reported to possess antihepatotoxic, antibiotic, antimalarial, antihepatitic, antithrombogenic, antiinflammatory, antisnakevenom, and antipyretic properties to mention a few, besides its general use as an immunostimulant agent (Table 1.2). A recent study conducted at Bastyr University, USA confirms anti-HIV activity of andrographolide (Calabrese et al., 2000).
A. paniculata is distributed in tropical Asian countries often in isolated patches. It can be found in a variety of habitats viz. plains, hill slopes, waste lands, farms, dry or wet lands, sea shore and even road sides. Native populations of A. paniculata are spread throughout south India and Sri Lanka which perhaps represent the centre of origin and diversity of the species (Hooker, 1885; Bhat and Nanavati, 1978). The herb is also available in northern stations of India, Java, Malaysia, Indonesia, West Indies and elsewhere in Americas where it is probably introduced (Hooker, 1885; Ridley, 1967; Backer and Brink Jr., 1967; Correll and Correll, 1982).
As per personal communication from Krishna Murti (Royal Botanic Gardens, Kew), the species is also available in Hong Kong, Penang, Malacca, Pangkor Island (south of Penang), Malaya, Thailand, West Java, Borneo, Celebes, Brunei, West Indies, Jamaica, Barbados, Bahamas etc. However, precise data are lacking on the introduction and naturalization of the species in these countries.
Unlike other species of the genus, A. paniculata is of common occurrence in most of the places in our country including the plains and hilly areas up to 500 m, which accounts for its wide use. Since time immemorial, village and ethnic communities in India have been using this herb for treating a variety of ailments (Table 1.3).
It is one of the important ingredients in Ayurvedic preparations recommended for fever and liver diseases. These include 'Liv 52' for treating hepatotoxicity (Dwivedi et al., 1987); 'TBR-002' for fever (Subramaniam et al., 1995), 'Kan Jang' for cold, flu and sinusitis (Hancke et al., 1995; Panossian et al., 2000), ‘Tephroli’ for treating viral hepatitis (Dutta and Sukul, 1982); ‘Ayush-57’ effective in treating vitiligo (Rao et al., 1980) and ‘Hepatogard’– an indigenous formulation exhibiting hepatoprotective activity (Saraf et al., 1991). Some industrial units like Natural Remedies India, Bangalore prepare semi-purified leaf extracts containing high concentrations (15-60%) of andrographolide and supply to other drug and fine compound manufacturing companies as the case may be for further value addition. Besides, Natural Remedies India Pvt Ltd (Bangalore), Alpha Omega Labs (USA), Sabinsa Corporation (USA) etc are also preparing extracts and drugs out of this important medicinal plant. Several Web sites deal with ethnobotanical, chemical, pharmaceutical and other aspects of A. paniculata (Table 1.4).
Andrographolide, chief constituent extracted from the leaves of the plant, is a bitter water-soluble lactone exhibiting protective effects in carbon tetrachloride induced hepatopathy in rats. Its LD50 in male mice was 11.46gm/kg, ip (Handa and Sharma, 1990). This bitter principle was isolated in pure form by Gorter (1911). Andrographolide is also attributed with such other activities like liver protection under various experimental conditions of treatment with galactosamine (Saraswat et al, 1995), paracetamol (Visen et al, 1993) etc. The hepatoprotective action of andrographolide is related to activity of certain metabolic enzymes (Choudhury and Poddar, 1984, 1985; Choudhury et al, 1987).
Despite its enormous medicinal and economic importance, attempts to cultivate A. paniculata have seldom been undertaken in any part of the country; hence local vaidyas as well as drug companies depend on wild sources for the supply of raw material. Gupta and Srivastava (1995) have reported a systematic cultivation experiment using different accessions of A. paniculata at NBPGR, New Delhi. Cultivation experiments are also reported by various authors from different parts of SE Asia (Zhou, 1987; Ramesh et al, 1997; Alagesaboopathi and Balu, 1997; Nandi, 1992; Muniramappa et al, 1997). Production of a variant line of A. paniculata by plant tissue culture has been standardised by Roy and Dutta in 1998.
Consequent upon the enforcement of the Convention of Biological Diversity and other patent/IPR regimes, it is imperative that the gene rich third world countries including India work out suitable strategies and mechanisms to evaluate their resources and develop new plant types and plant-specific value added products and processes. Unless these countries develop necessary competence and know-how to prospect their resources, they will be low paid for the unimproved genetic resources they export and may get trampled by the technologically as well as economically rich powerful countries. Against this growing realism and necessity in the developing world, it is of considerable significance to use modern tools and techniques of biotechnology and identify components of biodiversity for conservation and sustainable utilization. Taking A. paniculata as the experimental material, the author has attempted to study the biology of the species to understand its genetic fidelity and the pattern of gene flow and to evaluate the germplasm collected from different phytogeographical regions to identify genotype(s) of greater economic potential. In this process, variability existing in the wild populations of the species purported to have ecological and more importantly genetic basis, has been addressed at morphological, cytological, phytochemical, biochemical and molecular levels. It should be noted that even basic biological data are lacking in A. paniculata as also the case with many other medicinal plants. It is needless to say that in the wilderness of nature, the populations of a sexually reproduced species like A. paniculata are presumably so varied, that selection of high biomass and product yielding genotypes is distinctly possible and holds much promise for upgradation into cultivars.
