The drug discovery from plants involves a multidisciplinary approach combining botanical/ethnobotanical, phytochemical and biological techniques. Plants continue to provide us new chemical entitities (lead molecules) for the development of drugs against various pharmacological targets including cancer, HIV/AIDS, malaria, Alzheimer's and pain. Several natural product drugs of plant origin are in clinical use, which include paclitaxel, camptothecin-derived analogues, arteether, galanthamine, tiotropium to name a few or some are undergoing phase II and III clinical trials. Although plant-based drug discovery programmes continue to provide an important source of new drug leads, numerous challenges are encountered including procurement and authentication of plant materials, implementation of high-throughput screening bioassays and scale-up of bioactive lead compounds.

It is estimated that around 250,000 flowering plant species are reported to occur globally. Approximately half (1,25000) of the flowering plant species found in the tropical forests. They continue to provide natural product chemists with invaluable compounds of starting point for development of new drugs. The potential for finding new compounds is enormous as till date only about 1% of tropical species have been studied for their pharmaceutical potential. The success of drug discovery from plants resulted principally in the development of anti-cancer and anti-bacterial agents. The success of anti-cancer drug development can be illustrated from the efforts of National Cancer Institute (NCI), USA. In this effort, field explorations are largely guided by the so-called biodiversity or "random" collection approach, with ethnobotanical or ethnopharmacological information playing a minimal or no role. NCI launched its effort in 1955, and for the period of 1960-1982, about 114,000 extracts from an estimated 35,000 plant samples (representing 12,000-13,000 species) collected mostly from temperate regions of the world had been screened against a number of tumor systems. A wide variety of compound classes were isolated and characterized. Clinically significant cancer chemotherapeutic agents that emerged from this programme included paclitaxel (Taxus brevifolia Nutt. and other Taxus species, Taxaceae), hycamptamine (topotecan), CPT- 11 and 9-aminocamptothecin. The latter three compounds are semi-synthetic derivatives of camptothecin (Camptotheca acuminata Decne, Nyssaceae). The programme was extended from 1986 to 2004, with an emphasis on global plant collections and screening against tumor cell cultures.

Drug discovery from plants has evolved to include numerous interdisciplinary fields and various methods of analysis. The process typically begins with a botanist, ethnobotanist, ethnopharmacologist, or plant ecologist who collects and identifies the plants of interest. Collection may involve species with known biological activity for which active compound(s) have not been isolated or may involve taxa collected randomly for a large screening programme. It is necessary to respect the intellectual property rights of a given country where plants of interest are collected. Phytochemists (natural product chemists) prepare extracts from the plant materials, subject these extracts to biological screening in pharmacologically relevant assays, and commence the process of isolation and characterization of the active compound(s) through bioassay-directed fractionations. Molecular biology has become essential to medicinal plant drug discovery through the determination and implementation of appropriate screening assays directed towards physiologically relevant molecular targets.

IMPORTANCE OF MEDICINAL PLANTS

Numerous methods have been utilized to acquire compounds for drug discovery including isolation from plants and other natural sources, synthetic chemistry, combinatorial chemistry and molecular modelling. Despite the recent interest in molecular modelling, combinatorial chemistry, and other synthetic chemistry techniques by pharmaceutical companies and funding organizations, natural products and particularly medicinal plants, remains an important source of new drugs, new drug leads and new chemical entities (NCEs). According to a survey by D. J. Newman et al. of NCI, USA, 61% of the 877 small-molecule new chemical entities introduced as drugs worldwide during 1981-2002 were inspired by natural products. These include: natural products (6%), natural product derivatives (27%), synthetic compounds with natural product-derived pharmacophore (5%) and synthetic compounds designed from a natural product (natural product mimic, 23%).

It is evident that modifications of existing natural products can lead to NCEs and as possible drug leads, from medicinal plants. Calanolide A, a dipyranocoumarin compound, isolated from Calophyllum lanigerum var. austrocoriaceum (Whitmore) P.F. Stevens (Clusiaceae), a Malaysian rainforest tree. Calanolide A is an anti-HIV drug with specific mechanism of action as a non-nucleoside reverse transcriptase inhibitor of type-1 HIV and is effective against AZT-resistant strains of HIV and is currently undergoing phase II clinical trials. Recently, calanolde A is reported as anti-tubercular agent.

The current emphasis of new drug discovery processes from plants is the development of products with new pharmacological modes of actions apart from ages known advantage of structural novelty. From India, 3 drugs qualify, Flavopiridol, Forskolin, and Guggulsterone, on mode of action account. Flavopiridol is totally synthetic, but the basis for its novel flavonoid structure is a natural product, rohitukine, isolated as the constituent responsible for anti-inflammatory and immunomodulatory activity from Dysoxylum binectariferum Hook. f. (Meliaceae), which is phylogenetically related to the Ayurvedic plant, Dysoxylum malabaricum Bedd., used for rheumatoid arthritis. Flavopiridol was one of the over 100 analogues synthesized during structure-activity studies, and was found to possess tyrosine kinase activity and potent growth inhibitory activity against a series of breast and lung carcinoma cell lines.

It also showed broad spectrum in vivo activity against human tumour xenografts in mice, which led to its selection for preclinical and clinical studies by the NCI in collaboration with the company, Hoechst. It is currently in 18 phase I and phase II clinical trials, either alone or in combination with other anticancer agents, against a broad range of tumours, including leukaemia, lymphomas and solid tumours. Forskolin, a labdane diterpenoid isolated from the Indian herb, Coleus forskohlii Briq., is a unique, potent adenylate cyclase activator. In view of the cyclic AMP-dependent effects produced by forskolin, it was considered for development as an agent for the treatment of congestive cardiomyopathy, glaucoma and asthma. Later, several analogues were synthesized and structure-activity relationships are developed. The semi-synthetic derivatives approved for clinical use, mainly in the treatment of glaucoma. The gum resin of Commiphora mukul (Stocks) Engl. commonly referred to as the Guggul tree, has been used in traditional Ayurvedic medicine for nearly 3000 years. It was reported to be effective in the treatment of several conditions, including obesity and disorders of lipid metabolism. An organic extract of this gum resin, referred to as gugulipid, has been approved for use in India since 1987 as a treatment for hyperlipidemia. Studies of patients receiving this therapy and experiments with rodent models have demonstrated that gugulipid effectively lowers serum low-density lipoprotein and triglyceride levels. Guggulsterone, pregnadiene-3, 16-dione], the active component of gugulipid largely responsible for the anti-hyperlipidemic effects of this extract.

The hepatic conversion of cholesterol to bile acids is an important mechanism for the elimination of excess dietary cholesterol. Bile acid biosynthesis and transport are regulated by the farnesoid X receptor (FXR), a member of the of nuclear hormone receptor gene superfamily. Thus, therapeutic strategies that target FXR represent a promising new approach for the treatment of hypercholesterolemia. It has been reported that guggulsterone is a highly efficacious antagonist of the FXR. Guggulsterone treatment decreases hepatic cholesterol in wild-type mice fed a high-cholesterol diet but is not effective in FXR-null mice. Thus, it was proposed that inhibition of FXR activation is the basis for the cholesterol-lowering activity of guggulsterone.

CHALLENGES IN DRUG DISCOVERY FROM MEDICINAL PLANTS

In spite of the success of drug discovery programme from plants in the past 2-3 decades, future endeavours face many challenges. Natural product scientists and pharmaceutical industries will need to continuously improve the quality and quantity of compounds that enter the drug development phase to keep pace with other drug discovery efforts. The process of drug discovery has been estimated to take an average period of 10 years and cost more than 800 million dollars. Much of this time and money is spent on the numerous leads that are discarded during the drug discovery process. It is estimated that only one in 5000 lead compounds will successfully advance through clinical trials and be approved for use. In the drug discovery process, lead identification is the first step. Lead optimization (involving medicinal and combinatorial chemistry), lead development (including pharmacology, toxicology, pharmacokinetics, ADME and drug delivery), and clinical trials all take a considerable time. Drug discovery from plants has traditionally been lengthier and more complicated than other drug discovery methods. Many pharmaceutical companies have eliminated or scaled down their natural product research.

In addition, there is a decline in interest in natural product (plant)-based drug discovery and research globally. Natural product chemists and pharmacognosists can look for suitable employ-ment in other academic departments such as biology, chemistry, eco-logy and nutrition to continue research investigations on plants.

DRUG DISCOVERY PROCESS FROM PLANTS

As the drug discovery from plants has traditionally been time-consuming, the faster and better methodologies for plant collection, bioassay screening, compound isolation, and compound development must be employed. Innovative strategies to improve the process of plant collection are needed, especially with the legal and political issues surrounding benefit-sharing agreements. The design, determination, and implementation of appropriate, clinically relevant, high-throughput bioassays are difficult processes for all drug discovery programmes. Although the design of high-throughput screening assays can be challenging, but once a screening assay is in place, compound and extract libraries can be tested for biological activity. The common problem faced during screening of extracts is solubility and the screening of extract libraries is many times problematic, but new techniques including pre-fractionation of extracts can alleviate some of these issues. Challenges in bioassay screening remain an important issue in the future of drug discovery from medicinal plants. The speed of active compound isolation can be increased by using hyphenated techniques like LC-NMR and LC-MS. The development of drugs from the lead compounds isolated from plants, face unique challenges. Natural products in general are typically isolated in small quantities that are insufficient for lead optimization, lead development and clinical trials. Thus, there is a need to develop collaborations with synthetic and medicinal chemists, to explore the possibilities of its semi-synthesis or total synthesis. One can also improve the natural product compound development by creating natural product libraries that combines the features of natural products with combinatorial chemistry. After considering all these issues, we would like to discuss the Indian scenario in context to challenges in drug discovery from plants.

INDIAN SCENARIO

India represented by rich culture, traditions, and natural biodiversity, offer unique opportunity for the drug discovery researchers. This knowledge-based country is well recognized for its heritage of world's most ancient traditional system of medicine, Ayurveda. Even, Dioscorides (who influenced Hippocrates) is thought to have taken many of his ideas from India. In India we have 2 (Eastern Himalaya and Western Ghats) of the 18 worlds' hotspots of plant biodiversity and interestingly, we are 7th among the 16 Megadiverse countries where 70 % of the world's species occur collectively. We are rich in our own flora i.e. endemic plant species (5725 angiosperms, 10 gymnosperms, 193 pteridophytes, 678 bryophytes, 260 liverworts, 466 lichens, 3500 fungi, and 1924 algae). Unfortunately, due to inaccessibility of some areas and various other regions only 65% flora of the country could have been surveyed so far.

With the dwindling population of taxonomists and rare introduction of the youngsters in this field it might take another 20-30 years with the current pace to bring out complete Flora of the country. Now the question before us is, whether we could assess the pharmaceutical potential of all the floristic components that we know? Answer is - no. Realizing that we have approximately 17500 species of higher plants, 64 gymnosperms, 1200 pteridophytes, 2850 bryophytes, 2021 lichens, 15500 fungi, 6500 algae at our disposal, surprisingly, hardly a few concerns like Central Drug Research Institute (CDRI), Lucknow with its concerted efforts could test a few plants and have published results on 3488 species of plants for a limited indications in almost 28 years between 1968 - 1996. This resulted into some promising leads that were later developed as drugs viz., Gugulipid, the hypolipidaemic from Commiphora mukul (Stocks) Engl.; bacoside, the memory enhancer from Bacopa monnieri (L.) Penn.; Picroliv, the hepatoprotective from Picrorhiza kurroa Benth.; Curcumin, the anti-inflammatory from Curcuma domestica Valeton; Consap, the contraceptive cream from Sapindus mukorossi Gaertn., etc. Other CSIR laboratories and some private pharmaceutical companies have also made some efforts in this direction. However, assessing the pharmaceutical potential of our whole flora even for the important disease indications may take several decades. The reason could be availability of source plant material, expertise to authenticate the taxa, developing enough suitable in-vitro screens for all indications, reproducibility of results and so on. Whatever the case may be, but, can we afford to wait for any longer to evaluate our flora for its medicinal efficacy?

The procedure for access to biological resources now is very tedious. According to "The Biological Diversity Rules, 2003" of Govt. of India (Notified on 24 March, 2004), any person seeking approval of the Authority (National Biodiversity Authority) for access to biological resources and associated knowledge for research or for commercial utilization shall make an application in Form I as given in schedule. Every application shall be accompanied by a fee of Rs 10,000. The Authority on being satisfied with the merit of the application, may grant the approval as far as possible within a period of six months of receipt of the same. One has to specify each time the quantity to be collected of exact species, quantum of monetary and other incidental benefits and also guarantee to deposit a reference sample of the biological material sought to be accessed with the repositories identified and submitting to the Authority a regular status report of research and other developments. As the process of plant-based drug discovery involves continuous collection of plant material from different places at various point of times it is rather impractical to wait for obtaining permission each time.

At the same time the authorities can also not give blanket permission for any collector. We have to find a way out. A lot of field experience and wide florisitic knowledge is required if one wants to go for random collection programme required for preliminary screening. Once found active, target plant collection in bulk quantity may be a problem due to its threatened status in some cases or biomass and scattered distribution in others. Authentication of plant material is an important and most crucial factor in the plant-based drug discovery. This needs to be supported by a set of suitable voucher specimens of the target species authenticated by a botanist and then deposited with a recognized herbarium. In absence of vouchers it is next to impossible to remember the location/phytogeographical conditions and time/ season of collection of the exact plant material for repeat studies. The reproducibility of the results depends on various other factors too. The proper collection procedures need to be laid down and documented. Collection practices should ensure the long term survival of wild populations and their associated habitats. Management plans for collection should provide a framework for setting sustainable harvest levels and describe appropriate collection practices that are suitable for each medicinal plant species and plant part used. Another important issue here is the pharmaceutical evaluation of rare or endangered species. These species, including other redlisted threatened species, following the current IUCN norms, can not be collected from wild and in turn remain dead for science as far as their pharmaceutical potential is concerned. Interestingly, many of these species do find mention in our traditional Indian systems / tribal systems of medicines.

After collection, the drying procedures that vary for different plant materials, may lead to alter in the chemical properties of the material. The commonly employed drying procedures are sun and/or shade drying. Right kind of packaging procedures adopted in order to avoid the fungal infection also needs to be carefully worked out before transportation of material to the laboratory. The processing of plant materials mainly includes pulverization and then preparation of extracts. Various extracts such as hexane, chloroform, ethyl acetate, n-butanol and ethanol or 70% ethanol are generally prepared for chemoprofilings as well as for biological screening.

In order to screen thousands of plant species at one go for as many bio-assays as possible we must have a collection of large number of extracts. Globally it has been felt to build natural product extract libraries. The extract libraries offer various advantages such as reduction in cost and time for repeat collection of plants and availability of properly encoded and preserved extracts in large numbers for biological screening in the term of high throughput screenings and obtaining hits within a short period. In India though some institutions have small plant extract libraries but they are not in public domain. The only information is available from Nicholas Piramal India Ltd. (NPIL), one of the major pharma players India. NPIL has built up a plant extract library having 6000 extracts prepared from around 2300 plant species collected from all over India. Such libraries could serve as a powerful tool and source of extracts to be screened for biological activities using high throughput assays.

As evident, the nature is the best combinatorial chemist and possibly has answers to all mankind diseases. Till today, natural product compounds discovered from medicinal plants (and their analogues thereof) have provided numerous clinically used drugs. In spite of the various challenges encountered in the medicinal plant-based drug discovery, natural products isolated from plants will still remain an essential component in the search for new medicines. The fact that only about one tenth of the flowering species occurring globally, are investigated for their pharmaceutical potential, can be the obvious advantage to begin with plant/medicinal plant-based drug discovery programme. Thus, there is still a great hope that one may discover novel lead molecules from plants by employing modern drug discovery techniques and the coordinated efforts of various disciplines.

- (The authors are with Department of Natural Products, National Institute of Pharmaceutical Education and Research (NIPER), Punjab.)