2. ethno medicine in complementary therapeutics - · pdf fileethno medicine in complementary...

Research Signpost 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Ethnomedicine: A Source of Complementary Therapeutics, 2010: 29-52 ISBN: 978-81-308-0390-6

Editor: Debprasad Chattopadhyay

2. Ethno medicine in complementary therapeutics

Pulok K. Mukherjee, S. Ponnusankar and M. Venkatesh

School of Natural Product Studies, Dept. of Pharmaceutical Technology, Jadavpur University Kolkata 700032, India

1. Introduction The relationship between man and plants has been very close throughout the development of human culture. The evidences of the use of various plants for various ailments by our ancestors, indicates that the plant derived medicines, are from rich traditions of ancient civilizations and scientific heritage. A great number of these natural products have come to us from the scientific study of remedies traditionally employed by various cultures. At no time in the development of man kind, there has been more rapid and more deeply meaningful progress was made in our understanding of plants and their chemical constituents. The gradual sophistication of phytochemistry, pharmacology etc and the hope for scientific remedies from plant sources setup a tendency concerning the potential value of natural products. The plants of various regions have been well documented for their medicinal values

Correspondence/Reprint request: Dr. Pulok K. Mukherjee, School of Natural Product Studies, Dept. of Pharmaceutical Technology, Jadavpur University, Kolkata 700032, India. E-mail: [email protected]

Pulok K. Mukherjee et al. 30

but there is still need for proper documentation of these plants because such type of study basically lies in finding out the usage of plants or its parts for betterment understanding from ethno-medical uses. Herbs have provided the basis for the great medical systems in human history of Hippocrates and Galen and the great Ayurveda of the Indian subcontinent, Traditional Chinese Medicine of Chinese medical system, Islamic medical system over two millennia and many other cultural traditions that were often hybrids of the various systems of medicine. All these systems were formed in large part by the peculiar characteristics of the respective materia medica, plants have clearly demanded and been granted their own therapeutic approach (1). In India for drug development from ethno medicine there is a strong historical base, where from the ancient literature on Indian system of medicine several documents on therapeutics can be derived. The classical Indian text like Rig-Veda, Atherveda, Charak Samhita and Sushruta Samhita are the evidences of the use of plants by our ancestors (Table 1). Table 1. The Historicity and the available ancient literature on Indian ancient system of medicine – Ayurveda.

Ayurveda is the most ancient system of medicine, its antiquity going back to the Vedas, surviving today through the classic texts Name of texts Author Historicity Subject Brihattrayi- The three major texts: 1. Charaka Samhita Charaka 1000 – 700 BC Philosophy

& Medicine 2. Sushruta Samhita Sushruta 1000 – 600 BC Practice

of Surgery 3. Vagbhatta Samhitas

Vagbhatta 300 – 600 AD Medicine and therapeutics

Laghuttrayi – The three major texts 1. Madhav Nidana Madhavkara 900 AD Diagnostics 2. Sarangadhar Samhita

Sarangdhara 1300 AD Medicine

3. Bhava Prakasa Bhava Misra 1600 AD Drugs and Herbs

Herbal medicine is a triumph of popular therapeutic diversity and used as complementary therapies in many developing countries. Until the discovery of modern medicines, any system of medicine that relieved the patient and their ailment(s) was considered to be a therapeutic system without further investigation. Whether the system was properly analyzed, researched or organized did not matter. But with the rapid development of conventional medicines, this ethno medicinal plants and its knowledge required to be

Ethno medicine in complementary therapeutics 31

proved scientifically with proper evidence to prove the therapeutic efficacy of the ethnic system of medicine to be used as complementary therapies. Medicinal uses of plant and animal species have been practiced for centuries in many parts of the world. Even today, hundreds of millions of people, mostly in developing countries, derive a significant part of their subsistence needs and income from gathered plant and animal products. Gathering of high value products such as mushrooms (morels, matsutake, truffles), medicinal plants (ginseng, black cohosh, goldenseal) also continues in developed countries for cultural and economic reasons (2). Ethno medicine has been utilized for a long time for various disorders, now the time has come to use these resources as complementary therapeutics based on scientific validations. Several approaches for the development of drugs from ethnic medicine are given in Figure 1. The era of grand systems has probably passed but it may be time to develop a new coherent approach to herb use for a scientific age. Apart from a general view that herbs are safer, there has been only a fragmentary rationale for using them as medicines in modern times.

Multiprone approach

Chemical biology approach System biology approach Combinatorial library Personalized medicine

Evidence based approach To Science based mechanism understanding

Drug discovery engineLead potentials identification Ethnopharmacology approach

Standardization of traditional formulations (used by various cultures, tribal and ethnic groups)

Figure 1. Ethno medicine – approaches in drug development.

2. Biodiversity of ethno medicinal plants Biodiversity has been touted as a mechanism for both discovering new pharmaceutical product and saving endangered ecosystem. Since time immemorial, people have gathered plant and animal resources for their needs. However, medicinal plants play a central role, not only as traditional medicines used in many cultures, but also trade commodities which meet the


demand of often distant markets (1). There has been considerable recent interest in the search of medicines from ethnic resources. In nature, these medicines are prepared by plants as metabolites. These chemical compounds might be of considerable commercial value if adapted to industrial, agricultural and particularly preparation of phytomedicines and pharmaceuticals. Bio-diversity responds to a number of new, emerging concerns including, the result of new developments in technology, in particular, biotechnology and information technology, and the ongoing degradation of the environment, inevitably accompanied by an erosion of biological diversity (3). Biodiversity encompasses all biological entities occurring as an interacting system in a habitat or ecosystem and plants constitute a very important segment of such biological systems. Demand for a wide variety of wild species is increasing with growth in human needs, numbers and commercial trade. With the increased realization that some wild species are being over-exploited, a number of agencies are recommending that wild species be brought into cultivation systems (4). Despite the increasing use of medicinal plants, their future is being threatened by complacency concerning their conservation. Reserves of herbs and stocks of medicinal plants in developing countries are diminishing, several important species are in danger of extinction as a result of growing trade demands for safer and cheaper healthcare products and new plant-based therapeutic markets in preference to more expensive target-specific drugs and biopharmaceuticals. The number of plant species which have at one time or another been used in some countries for medicinal purposes can only be estimated. Enumerations of the WHO from the late 1970’s listed 21,000 medicinal species are used in various parts of the globe (5). Developing countries like China and India have a vast biodiversity of medicinal and aromatic plants. In China alone 4,941 of 26, 092 native species are used as drugs in Chinese traditional medicine. If this proportion is calculated for other well-known medicinal floras and then applied to the global total of 4,22,000 flowering plant species are used for various purposes and it can be estimated that the number of plant species used for medicinal purposes is more than 50 000 (6, 7). In India, medicinal and aromatic plants have been in use in one form or another, under indigenous systems of medicine like Ayurvedha, Siddha and Unani etc. India is having a well-recorded and well practiced knowledge of traditional herbal medicine (8). India officially recognizes over 3000 plants for their medicinal value. It is generally estimated that over 6000 plants in India are in use in traditional, folk and herbal medicine, representing about 75% of the medicinal needs of the third world countries (9). India is one of the 12 mega biodiversity centers having 45, 000 plant species; its diversity is


unmatched due to the 16 different agroclimatic zones, 10 vegetative zones, and 15 biotic provinces. The country has a rich floral diversity and reported to have15,000-18,000 flowering plants, 23,000 fungi, 25,000 algae, 1,600 lichens, 1,800 bryophtes and 30 million micro-organisms (1). 3. Plant derived pharmaceuticals and its delivery Many plant based drugs were used as complementary therapies in industrial countries that were originally discovered by folk healers. Around 60% medicinally useful formulations and other health products, which are either derived or developed from plant origin dominate the global market of health care products. Quinine from cinchona tree had its origin in the royal households of the South American Incas. In the early 1500s, Indian fever bark was one of the first medicinal plants to find appreciative consumers in Europe. Taken from the cinchona tree (Cinchona officinalis), the bark was used as an infusion by native people of the Andes and Amazon highlands to treat fever (10). In Andean cultures, the leaves of the coca tree have been primarily chewed to obtain perceived benefits. From ancient times, indigenous people have added alkaline materials such as crushed seashells or burnt plant ashes to the leaves in order to accentuate the pharmacologically active moiety of coca. Pot curare arrowhead poison used in the East Amazon is predominately from the species Strychnos guianensis. Tube curare in the West Amazon is from Chrondrodendron tomentosum; curare in modern medicine is made from this and named as tubocurarine. The jaborandi tree (Pilocarpus jaborandi) secretes alkaloid- rich oil. Several substances are extracted from this aromatic oil, including the alkaloid pilocarpine, a weapon against the blinding disease, glaucoma(11). American Indians on the island of Guadeloupe used pineapple (Ananas comosos) poultices to reduce inflammation in wounds and other skin injuries, to aid digestion and to cure stomachache (11). In Ayurveda and other Indian systems of medicines, use of different plants for treating ailments was based on the fact that the additive or synergistic effects of the secondary metabolites present in those plants enhance therapeutic viability of the phytoconstituents. This knowledge and experiential database can provide new functional leads to reduce time, money and toxicity – the three main hurdles in drug development. These records are particularly valuable, since effectively these medicines have been tested for thousands of years on people. Efforts are underway to establish pharmaco-epidemiological evidence base regarding safety and practice of Ayurvedic medicines. Randomized controlled clinical trials for rheumatoid and


osteoarthritis, hepatoprotectives, hypolipedemic agents, asthma, Parkinson’s disease and many other disorders have reasonably established clinical efficacy. Many conventional drugs originate from plant sources: a century ago, most of the few effective drugs were plant based. Examples include artemisinin, atropine, digoxin, ephedrine, gallanthamine, morphine, physostigmine, quinine, reserpine, salicylic acid, sennoside, Taxol, vincristine, vinblastine, glycyrrhizin, and psoralen (12). Similarly a second antimalarial in addition to quinine is now available for cerebral malaria resistant to chloroquine. Other analogues of artemisinin are now being evaluated and arteether and artemether were found to be more effective. Flavopiridol is totally synthetic, but the basis of it is rohitukine which is isolated from Dysoxylum binectariferum Hook. f. (Meliaceae), which is phylogenetically related to the Ayurvedic plant D. malabaricum Bedd. used for rheumatoid arthritis. The successful introduction of these plants into modern therapeutics indicates that other discoveries are waiting to be made. The plants used in Indian Systems of Medicine showed the presence of a variety of chemical entities, belonging to different classes (13). Combining the strengths of the knowledge base of complementary alternative medicines like Ayurveda with the dramatic power of combinatorial sciences and High Throughput Screening (HTS) will help in the generation of structure–activity libraries. The development of drugs from ethnic plants continues, with drug companies engaged in large scale pharmacologic screening of herbs (14). There is a revival of interest in Ayurvedic herbal products at a global level; herbs such as turmeric, neem, ginger, holi basil and ashwagandha are a few examples of what is gaining popularity among modern physicians. 3.1. Novel delivery of phytoconstituents Every nation is seeking health care beyond the traditional boundaries of modern medicine; turning to self-medication in the form of herbal remedies (15,16). Now-a-days extensive research in novel drug delivery systems is going on to improve the therapeutic efficacy of the existing natural molecules. Toxicity and limited absorption of different phytoconstituents obtained from herbs are crucial problems in exploring their real potentials against different diseases. Value added formulation, as its name indicates is a formulation with added value, which gives better therapeutic efficacy of its main chemical constituents inside our body. The development of value added herbal formulations having better absorption and utilization profiles in our body is of paramount importance (17). To minimize drug degradation and loss during herbal drug consumption and to


increase herbal bioavailability, various drug delivery and drug targeting systems are currently under development. Among them, Phytosomes are advanced forms of herbal products that are better absorbed, utilized, and as a result produce better effects than conventional herbal extracts. Phytosomes are produced via a patented process whereby the individual components of an herbal extract are bound to phosphatidylcholine (18). The Phytosome process has been applied to many popular herbal extracts including Ginkgo biloba, grape seed, milk thistle, and green tea (19). Improved therapeutic efficacy of phytosomes can be best presented with the results obtained from the studies with Ginkgo biloba phytosomes. A representation of phytosomes as effective delivery systems for herbal constituents has been shown in Figure 2. It has been reported that Ginkgo phytosome produced better results compared to the conventional extracts (20, 21). There are also other phytosomal formulations like Grape seed phytosome, glycyrrhetinic acid phytosome, hawthorn phytosome, Panax ginseng phytosome etc. which have been proved to be a trust-worthy and useful pharmaceutical product keeping in view, their improved therapeutic activities in phytosome form. Phytosomes have also got tremendous impact in skin care products and cosmetology. Enhanced microcirculation provided by the phytosomes is very much useful for skin care and opened a new avenue in cosmetic science with significant upper hand over the crude extracts or uncomplexed phytoconstituents, used in this field (22-24). Our recent studies with phospholipids complex of different potent phytomolecules like Quercetin,

Figure 2. Drug activity enhancement through phytosomes drug development.


naringenin and curcumin showed that the therapeutic efficacy, in terms of free radical scavenging activity of the molecules increased upon complexation with phospholipids and the phospholipid complexes at different dose levels showed better effects than respective free molecules at same doses (25-27). 4. Bioactive phytoconstituents – lead for drug development Research into the isolated plant constituents is of great importance for the development of bioactive substances from ethnic medicine. With the emergence of latest technologies and enhanced knowledge about the isolated plant constituents, characterization and analytical tools, lot of compounds are efficiently isolated from potential plants and have been of great contribution to the drug discovery from ethnic plants. Separating a medicinal herb into its constituents cannot often explain exactly the way in which it works in the natural form. The whole herb is worth more than the sum of its components. A plant contains hundreds of chemical constituents that interact in a complex way to produce therapeutic effects of the remedy. We may not understand the detailed mechanism, in which a particular herb works – even though its medicinal benefits are well established. With the development of standardization tools like HPTLC, HPLC, LC MS/MS etc., several plant extracts as well as their formulations, will be standardized in a better way to enhance the bioactivity of the ethnic medicines (9). In the development of drugs and therapeutics, the use of plants as medicines has involved the isolation of active compounds, beginning with the isolation of morphine from opium in the early 19th century (28, 29). Drug discovery from medicinal plants led to the isolation of early drugs such as cocaine, codeine, digitoxin, and quinine, in addition to morphine, of which some are still in use (30,31). Plant based drugs provide outstanding contribution to modern therapeutics; for example: serpentine isolated from the root of Indian plant Rauwolfia serpentina in 1953, was a revolutionary event in the treatment of hypertension and lowering of blood pressure. During 1950-1970 approximately 100 plants based new drugs were introduced in the USA drug market including deserpidine, reseinnamine, reserpine, vinblastine and vincristine etc which are derived from Ethnomedicinal plants. From 1971 to 1990 new drugs such as etoposide, E-guggulsterone, teniposide, nabilone, plaunotol, Z-guggulsterone, lectinan, artemisinin and ginkgolides appeared all over the world. Drugs introduced from 1991 to 1995 include paciltaxel, toptecan, gomishin, irinotecan etc. Drugs isolated from ethnic medicines can serve not only as new drugs themselves but also as drug leads. In the recent past, many bioactive phytoconstituents were isolated from natural products or derived from natural


products. The following are some of the plant derived drugs available in market after the approval of USFDA and few in early stages of clinical trials. The bioactive phytoconstituents derived from ethnic medicine showed the presence of variety of chemical entities, belonging to different classes as represented in Figure 3 and were made available in the market.

OOO

OO

N

O

O

HO

Arteether (1) Galanthamine (2)

O

O O

F

FF

N+

O-O

N+ OO

O

S

S OH

Br- Nitisinone (3) Tiotropium (4)

N

HO

O

OO

O

HO

HO

OH

OH

N

O

O

O

OHON

N

NH

F

F

O

O

O

Morphine-6-glucuronide [M6G] (5) Vinflunine (6)

O

O

O

N

F

N

NH2

HO

O

OO

O

OH

Exatecan (7) Calanolide A (8)

OH

O

HO

N

OHO

O

O

OO

O

O

O

O

O

Betulinic acid (9) Pervilleine A (10)

Figure 3. Several potent bio-active phytoconstituents from ethnic medicine.


Arteether (1), [trade name Artemotil] is a potent antimalarial drug and is derived from artemisinin, a sesquiterpene lactone isolated from Artemisia annua L. (Asteraceae), a plant used in traditional Chinese medicine (TCM) (32). Galanthamine (2), [trade name Reminyl] is a natural product discovered through an ethnobotanical lead and first isolated from Galanthus woronowii Losinsk. (Amaryllidaceae) in Russia in the early 1950s (33). It is approved for the treatment of Alzheimer’s disease, slowing the process of neurological degeneration by inhibiting Acetylcholinesterase (AChE) as well as binding to and modulating the nicotinic acetylcholine receptor (nAChR)(34). Nitisinone (3), [trade name Orfadin] is a newly released medicinal plant-derived drug that works on the rare inherited disease, tyrosinaemia, demonstrating the usefulness of natural products as lead structures (35). Nitisinone is a modification of mesotrione, an herbicide based on the natural product leptospermone, a constituent of Callistemon citrinus Stapf. (Myrtaceae). All three of these triketones inhibit the same enzyme, 4-hydroxyphenylpyruvate dehydrogenase (HPPD), in both humans and maize. Tiotropium (4), [trade name Spiriva] has been introduced to the United States market for treatment of chronic obstructive pulmonary disease (COPD) (36). Tiotroprium is an inhaled anticholinergic bronchodilator, based on ipratropium, a derivative of atropine that has been isolated from Atropa belladonna L. (Solanaceae) and other members of the Solanaceae family. Tiotropium has shown increased efficacy and longer lasting effects when compared with other available COPD medications (37). Compounds M6G (5) is in later stages of Phase III clinical trials and its subtle modifications of drugs currently in clinical use (38). M6G or morphine-6-glucuronide (5) is a metabolite of morphine from Papaver somniferum L. (Papaveraceae) and will be used as an alternate pain medication with fewer side effects than morphine (39). Vinflunine (6) is a modification of vinblastine from Catharanthus roseus (L.) G. Don (Apocynaceae) for use as an anticancer agent with improved efficacy (40). Exatecan (7) is an analog of camptothecin from Camptotheca acuminata Decne. (Nyssaceae) and is being developed as an anticancer agent (41). Modifications of existing natural products exemplify the importance of drug discovery from medicinal plants as NCEs and as possible new drug leads. Calanolide A (8) is a dipyranocoumarin natural product isolated from Calophyllum lanigerum var. austrocoriaceum (Whitmore) P.F. Stevens (Clusiaceae), a Malaysian rainforest tree (42). Calanolide A is an anti-HIV drug with a unique and specific mechanism of action as a non-nucleoside reverse transcriptase inhibitor (NNRTI) of type-1 HIV and is effective against AZT-resistant strains of HIV (43). Calanolide A is currently undergoing Phase II clinical trials (44). Drug discovery from medicinal plants has played an important role in the treatment of various diseases including life threatening conditions such as


cancer. Most new clinically useful bioactive phytoconstituents are from plant secondary metabolites and their derivatives (38). Anticancer agents from plants currently in clinical use can be categorized into four main classes of compounds: vinca (or Catharanthus) alkaloids, epipodophyllotoxins, taxanes, and camptothecins. Vinblastine and vincristine were isolated from Catharanthus roseus (L.) G. Don (Apocynaceae) (formerly Vinca rosea L.) and have been used clinically for over 40 years. Vinblastine isolated from the Catharanthus rosesus is used for the treatment of Hodgkins, choriocarcinoma, non-hodgkins lymphomas, leukemia in children, testicular and neck cancer. Vincristine is recommended for acute lymphocytic leukemia in childhood advanced stages of hodgkins, lymophosarcoma, small cell lung, cervical and breast cancer(9). Podophyllotoxin was isolated from the resin of Podophyllum peltatum L. (Berberidaceae) but was found to be too toxic in mice so derivatives were made with the first clinically approved drug being etoposide. The epipodophyllotoxins bind tubulin, causing DNA strand breaks during the G2 phase of the cell cycle by irreversibly inhibiting DNA topoisomerase II (45). Podophyllotoxin is also a constituent of Phodophyllum emodi currently used against testicular, small cell lung cancer and lymphomas. Indian indigenous tree of Nothapodytes nimmoniana (Mappia foetida) are mostly used in Japan for the treatment of cervical cancer. Teniposide and etoposide isolated from Podophyllum species are used for testicular and lung cancer. Paclitaxel (Taxol) isolated from Taxus brevifolia Nutt. (Taxaceae) is used for the treatment of metastatic ovarian cancer and lung cancer. The taxanes, including paclitaxel and derivatives, act by binding tubulin without allowing depolymerization or interfering with tubulin assembly (46). The above drugs came into use through the screening study of medicinal plants because they showed less side effects, were cost effective and possessed better compatibility. Camptothecin was isolated from Camptotheca acuminata Decne. (Nyssaceae) but originally showed unacceptable myelosuppression. Interest in camptothecin was revived when it was found to act by selective inhibition of topoisomerase I, involved in cleavage and reassembly of DNA (41). Together, the taxanes and the camptothecins accounted for approximately one-third of the global anticancer market. Several of these plant derived compounds are currently undergoing further investigation including betulinic acid (9), pervilleine A(10), and silvestrol. Betulinic acid, a pentacyclic triterpene, is a common secondary metabolite of plants, primarily from Betula species (Betulaceae). Betulinic acid was isolated from Ziziphus mauritiana Lam. (Rhamnaceae) collected in Zimbabwe (47). The ethyl acetate-soluble extract displayed selective cytotoxicity against human melanoma cells (MEL-2). Betulinic acid was isolated using


bioassay-guided fractionation including silica gel chromatography and crystallization techniques. Pervilleine A, along with eight other tropane alkaloids, was isolated from the roots of Erythroxylum pervillei Baill. (Erythroxylaceae) collected in southern Madagascar(48). The chloroform-soluble extract was found to be selectively cytotoxic against a multi-drug resistant (MDR) oral epidermoid cancer cell line (KB-V1) in the presence of the anticancer agent vinblastine. The pervilleines were isolated using bioassay-guided fractionation including silica gel chromatography and aluminum oxide chromatography. Silvestrol was first isolated from the fruits of Aglaia sylvestris (M. Roemer) Merrill (Meliaceae) (later re-identified as Aglaia foveolata Pannell) collected in Indonesia(49). The chloroform-soluble extract was found to be cytotoxic to several human cancer cell lines and, more importantly, the extract was active in the P-388 in vivo test system. Bioassay-guided fractionation was performed using silica gel chromatography and reversed-phase high-pressure liquid chromatography (HPLC) leading to the isolation of silvestrol. 5. Drug development from ethnomedicine Drug development from ethnic medicinal plants has evolved to include various fields of inquiry and numerous methods of analysis. The process typically begins with a botanist, ethnobotanist, ethnopharmacologist, or plant ecologist who collects and identifies the plant(s) of interest. Collection may involve species with known biological activity for which active compound(s) have not been isolated (e.g., traditionally used herbal remedies) or may involve taxa collected randomly for a large screening program. 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-guided fractionation. 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 (9). Despite the recent interest in molecular modeling, combinatorial chemistry, and other techniques by pharmaceutical companies and funding organizations, ethnic medicines remain an important source of new drugs, new drug leads, and new chemical entities (NCEs) (30, 31). Numerous methods have been utilized to acquire compounds for drug discovery including isolation from plants, synthetic chemistry, combinatorial chemistry, and molecular modeling (50-52). Natural products provided a starting point for new synthetic compounds, with diverse structures and often with multiple stereocenters that can be challenging


synthetically(53-55). Many structural features common to natural products (e.g., chiral centers, aromatic rings, complex ring systems, degree of molecule saturation, and number and ratio of heteroatoms) have been shown to be highly relevant to drug discovery efforts (55-58). Furthermore, since the escalation of interest in combinatorial chemistry and the subsequent realization that these compound libraries may not always be very diverse, many synthetic and medicinal chemists are exploring the creation of natural product and natural-product like libraries that combine the structural features of natural products with the compound-generating potential of combinatorial chemistry (59-61). Several factors have contributed to the revival of interest in plant derived products which include, undisputed clinical efficacy of the product, compounds with less direct therapeutic potential may offer new molecular templates for the design of more effective drugs (1), as an alternative to established therapy and a valuable, inexpensive sources of “feed stock” molecules that can be really transformed into drugs. Biodiversity is a major source for drug development which fuels this reviving interest. For the exploration of this resource for new leads for drug development, high throughput screening for bioactivity has great potential. However, for an efficient exploration of this resource new methods are required that enable the rapid identification or false-positives and known-active compounds. This can help in developing several new chemical entities (NCE) from ethno medicine. 5.1. Combinatorial biosynthesis of medicinal plant secondary metabolites The approach to combine genes from different microorganisms for the production of new and interesting metabolites has become known as combinatorial biosynthesis. It is now possible to combine various genes and extend the realm of combinatorial biosynthesis far beyond the biosynthesis. The diversification of products will increase dramatically when genes of very different origins are used. However, there is no need to concentrate on new compounds only; there are many interesting natural products, of which the application (e.g. as a drug or fine chemical) is hampered by its availability. The biodiversity is endless and there are still possibilities to enlarge the diversity from a chemical point of view, by combining genes and products from different sources that in nature would never meet. This strategy will deliver compounds that are not influenced by selection pressures, by a habitat, or the biochemical limitations of an organism (such as compartmentalization or storage). These compounds can be selected for a specific pharmaceutical mode of action or an activity can be adjusted to a more specific pharmaceutical demand.


There are several pharmaceuticals in the market that are highly expensive, due to the fact that these compounds are only found in rare plants and often in extreme low concentrations. Podophyllotoxin and paclitaxel are clear examples of pharmaceuticals that can only be produced through the isolation from plants. To achieve a sustainable source of such compounds scientists all over the world have been experimenting with biotechnological approaches aiming at the development of an alternative production system. With this aim in mind, combinatorial biosynthetic strategies are expected to yield interesting alternatives in the near future. With regard to the production of podophyllotoxin it has been shown that plant cell cultures of Linum flavum L. can be used to convert deoxypodophyllotoxin, a major lignan of Anthriscus sylvestris L. into 6-methoxypodophyllotoxin. The combination of the product of one species and the enzymes of another species to yield a desired product is a good example of combinatorial biosynthesis (62). 5.2. Ethno pharmacology approach Ethnopharmacologic approach is based on botany, chemistry and pharmacology (observation, identification, description and experimental investigation) but other disciplines have made vital contributions. Based on these considerations, ethnopharmacology is defined as “the interdisciplinary scientific exploration of biologically active agents traditionally employed or observed by man”. This study of traditional drugs is not meant to advocate a return to the use of these remedies in their aboriginal form, or to exploit traditional medicine. The objectives of ethnopharmacology are to rescue and document an important cultural heritage before it is lost, and to investigate and evaluate the agents employed. Thus, it plays an immense role in evaluation of natural products and more particularly the herbal drugs from traditional and folklore resources. Field observations and descriptions of the use and effects of traditional remedies, botanical identification, phytochemical and pharmacological studies are all within the scope of ethnopharmacology. It is essential that anthropologists interested in ethnopharmacology seek contact and collaboration with experts in botany, chemistry and pharmacology. Such a multidisciplinary approach presents added advantages. Even in recent times an anthropologist can give a detailed composition of an African poison ordeal without bothering about the chemical composition of the poisonous drink used or even its plant origin. The identification of medicinal plants and other traditional drugs is of course a crucial point, and good ethnopharmacological research can only be based on properly prepared voucher specimens, carefully authenticated by experts. Wherever possible, phytochemical studies


on medicinal plants should be followed by a careful search for the biological activities of the compounds isolated. When biologically active principles have been found, the findings must be interpreted in the light of the traditional use. It is impossible to establish a dose-effect relationship unless the original drug preparations are analyzed and evaluated chemically and pharmacologically. In ethnopharmacological research, it is essential to have a proper sampling and analysis methods, and this necessity requires close cooperation by pharmacologists, with anthropologists and ethnobotanists on the one hand and specialists in chemical analysis on the another (1). Most traditional drugs are administered as mixtures of many components, and with today knowledge of the many possible interactions between drugs, and between food and drugs, ethnopharmacological research must deal with this aspect too. Additive, synergistic, or antagonistic effects are all possible. Various admixtures have also been shown to affect the bioavailability of pharmacologically active principles. Pharmacological studies of traditional medicinal agents should be therefore initiated prior to, or in parallel with, chemical research and should guide the isolation of active principles. Field observations of traditional therapies and the pharmacological effects in humans should be carried out by trained pharmacologists, and when interesting activity is found, controlled experiments should be initiated. Ethnopharmacology is not just a science of the past using an outmoded approach. It still constitutes a scientific backbone in the development of active therapeutics based upon traditional medicine of various ethnic groups with the ultimate aim of validating these traditional preparations, either through the isolation of active substances or through pharmacological findings. 5.3. Chemical biology approach Unlike modern drugs in form of single chemical ingredient, ethnic medicines are usually derived from aqueous extracts of a few herbs and contain hundreds of chemical compounds. Modern clinical trial proved that a complex formulation composed of up to 20 herbs had greater efficacy than single herb used (63). Obviously, there exists certain relation between biological activity and chemical composition of herbal medicine and it is called as quantitative composition- activity relationship (QCAR) (64). Experimental studies, such as random controlled trials (RCT), often provide the most trust-worthy methods for establishing causal relationships from data, in which one or more variables is changed (typically random) to measure its effect on other variables. In recent years, the relation between active ingredients of herb medicine and biological activity is one type of


causal relationship was attempted. When the amount of active components in certain formulation varied, its therapeutical effect was correspondingly changed. Thus, causal analysis method can be employed in studying relationship of chemical composition and bioactivity of herbs and help to discover active components. There are few methods available for discovering causal relationships, in which the actual process of controlled experiment could be stimulate through series of conditional dependence tests. A recent approach called “STEPCARD” {stepwise causal adjacent relationship discovery} method has been developed to overcome the disadvantage of existing causal discovery algorithm, as well as the unreliability of traditional statistical methods, e.g. stepwise regression. The main idea of STEPCARD is using conditional dependency test to determine causal adjacent relationships between explanatory variables and predictor. For a given data set containing chemical composition matrix (parameter X) and bioactivity information matrix (parameter y), STEPCARD algorithm can be applied to choose the components or components combinations most correlative to the biological activity of original formulation (comparison drug). The computational results of STEPCARD algorithm dealing with chemical and biological data, it represents the minimal significant level used in conditional independent test to pick out at least one variable. But there is lack of scientific approach to study correlations of their chemical constitution and pharmacological mechanism. However, this work affords a new strategy to identify active component or component combinations of ethnic medicine and will be helpful to accelerate the speed of new drug discovery. 5.4. Genomics approach Completion of human genome project and role of genomic and proteomics have revolutionized natural products based drug discovery. Several business collaborations are taking place around the world for genetic targets of small-molecule drugs for new discovery leads. This strategy is primarily coming from the pressing need to increase productivity and success rate of new drug discovery. Molecular markers identify the plant at genomic level and establish new standards in standardization and quality control of botanicals. In order to get better quality of herbal drugs generating breeds for disease resistance plant is inevitable, for which marker assisted selection (MAS) is employed. To authenticate the plant species and their adulterant technique like Sequence Characterized Amplified Region (SCAR) analysis was employed (65). Molecular techniques are more superior in sensitivity as well as specificity than conventional techniques (66). Molecular markers such as Random Amplified Polymorphic DNAs (RAPDs) also plays an


important role in assessment of genetic variability and in strain identification (67) and differentiation from its related species. Ultra-HTS (uHTS) assays require an accurate and reliable means of fluid handling in the submicroliter volume range. This relates to the design of instrumentation for dispensing fluids, as well as assay plates. In a chemical genomic research approach, ultra-high throughput screening of genomic targets takes place early in the drug discovery process, before target validation to generate drug leads (68). As more genomes have been sequenced and gene functions elucidated, time has come to bring the valuable ethnic medicinal knowledge to the rapidly expanding genomic landscapes. It is envisioned that systematic identification and characterization of gene targets could lead to deeper appreciation of the chemo-diversity in ethnic medicinal plants. 5.5. In-silico approach Bioinformatics has gained popularity during the last few years to describe tools and techniques for storing, handling, and communicating the massive and ever increasing amounts of scientific (primarily biological) data. It is made possible by dramatic improvements in computational power and computer accessibility. Bioinformatics has become a major scientific discipline and applications in other fields are currently underway. It is essential that informatics technology be devoted to increasing our understanding of earth’s biodiversity, and to develop new tools for archiving global diversity. The need for dynamically updated databases to support informed conservation decision-making is becoming increasingly recognized (69). Traditional approaches to gathering and disseminating ethnobotanical information are clearly inadequate to deal with the global increase of ethnobotanical research data. Consequently, numerous electronic databases have recently been developed to disseminate information on plant uses. However, because these databases were designed and developed independently, they are often oriented toward particular user groups (e.g. students, researchers, or the general public). As a consequence, the information they contain is very variable in its content and quality, often only having a regional or cultural focus. Although the current ethnobotanical databases are of great value in promoting the awareness of the need to record and conduct ethnobotanical research, they are insufficient, especially for collaborative and comparative analysis. A coordinated global approach is necessary to currently manage the increasing amount of bio-cultural knowledge being documented by ethnobiologists worldwide. The growing number of independent ethnobotanical databases is itself a compelling argument for a unified coordinated data management system.


The system’s primary goals would be to function as both a comprehensive digital library with a search engine for retrieving information on the present, past and future uses of plants, providing a “one-stop educational resource” for users. As new technologies for data retrieval should be developed, and the system could evolve into a meta-database – that is, a system that allows researchers to analyze comparative data pooled from multiple databases. The lack of a unified approach and standard data model for recording research data has led to a paucity of comparative ethnobotanical studies that not only examine different uses of the same type of plants in different cultures, but also compare the ways plants figure in different world views (70). Few papers published by leading scientific journals address comparative analysis of possible patterns of medicinal plant selection and use by humans across cultures, regions or hemispheres. Indeed the absence of readily accessible comparative sources of ethnobotanical data has been recognized as a serious hindrance (71). In fact, a dynamic resource would encourage a unified approach by facilitating a greater opportunity for comparative analysis of research data through direct contributions by members of the ethnobiological research community. 5.6. Reverse pharmacology approach The ethno-medicine is based on its use for many years and its clinical existence is presumed. For bringing more objectivity and to confirm ethnic claims, systematic clinical trials are necessary. In normal drug discovery course “laboratories to clinic” approach is followed, in herbal medicine research “clinics to laboratories” approach – a true reverse pharmacology approach is followed. In latter, clinical experiences, observations or available data becomes a starting point, where as with conventional drug research it comes at the end. Reverse pharmacology is the science of integrating documented clinical/experiential hits, into leads by transdisciplinary exploratory studies and further developing these into drug candidates by experimental and clinical research. In reverse pharmacology approach process safety remains the most important starting point and efficacy becomes a matter of validation. The scope of reverse pharmacology is to understand the mechanisms of action at multiple levels of biological organization and to optimize safety, efficacy and acceptability of the leads in natural products, based on relevant science. Thus the drug discovery based on Ayurveda or ethnic medicine should follow a ‘reverse pharmacology’ path (72).


5.7. Systems biology approach Systems biology aims at understanding biological complexity by unbiased measurements of as many as possible parameters, without having any hypothesis. Such measurement can be of very different kind, on the level of the genome, transcriptome, proteome, metabolome as well as physiological parameters, such as blood pressure, pulse, pain, fever, weight, length, gender and age. By the use of suitable biostatistical methods such as multivariate analysis and principle component analysis these data can be analyzed, and for example correlations between certain parameters can be made. One of the key technologies in the systems biology is metabolomics. Metabolomics aims at qualitatively and quantitatively determining as many compounds as possible in an organism. This can be in extracts of tissues, but also in body fluids such as serum or urine in case of humans. Chromatographic methods in combination with mass spectrometry (MS), mass spectrometry with electron spray ionization (MS-ESI), and nuclear magnetic resonance spectrometry (NMR) etc are used for such analyses. By combining the results of such analyses with other parameters novel correlations can be found, for example a relation between the occurrence of certain compounds in extracts and a biological activity. Analysis of metabolites in urine by means of 1H NMR is already extensively applied for studying toxicity of drugs. Also for the quality control of botanicals the metabolomics approach is very promising tool. The said technique was applied to metabolic profiling by means of 1H NMR in the quality control of Ginkgo biloba pharmaceutical preparations. Beside a recognizable pattern the quantitative analysis of ginkgolides and bilobalide could be done with a 5 min acquisition time of the spectrum, without the need of any elaborate sample preparation. Also for other preparations such as Strychnos, Ephedra and Cannabis this method found to be suitable. Such studies are the first step on a long way to a better understanding of the activity of medicinal plants. Because of the many years of documented use, one may start immediately from clinical trials, in that way also shortening the whole processes of developing a novel drug (73). 5.8. Personalized approach Despite all the improvement in technology the number of novel drugs coming to the market is decreasing every year, because almost for all known targets drugs are available, and to find a better one which is still affordable for the patients is getting increasingly difficult. The costs are now upto 1 billion Euros for developing a novel drugs and the total duration is 12 – 15


years. That means only for major diseases it is worth while to develop novel drugs. Diseases of the developing countries, such as malaria, TB are consequently not the targets for drug development by big pharmaceutical companies. In fact, the approach of “single target single compound” does not work anymore. Novel approaches should be thought of. Our ancestors did not discover active plants, but they also developed a holistic approach in their medical systems, for example, well-known Ayurvedic medicine, Traditional Chinese medicine, Siddha medicine etc each have principles in treating the disease. This is among others reflected in a tailor-made prescription that is made for each individual patient after an extensive diagnosis, a concept that now is also considered to be of the interest for the pharmaceutical industry. Pharmacogenomics and pharmacogenetics research, will in the coming years lead to a more individualized pharmacotherapy. The holistic traditional approach requires also a holistic way of studying it. The reductionist approach of the modern drug development will not be able to detect activity in case of the presence of several (may be weak) active compounds, synergy between compounds and pro-drugs. The reductionist approach is so far used to try to proof activity, i.e., studying the activity of known targets such as receptor binding assays. This approach failed for example in case of St John’s wort. Despite positive results in clinical trials, no single active compound has been found that can explain the proven clinical activity. That pro-drugs exist is best proven by one of the most successful drug salicylate. Based on the use of Salix bark as an analgesic preparation, this compound was developed at the end of the 19th century. However, in Salix bark there is no salicylate, instead there is a glucoside of salicylic alcohol that first needs to be hydrolyzed and then oxidized before the active compound is formed. This all happens in our bodies, a clear example of a pro-drug that never will be found be the reductionist high throughput screening methods. Only in vivo systems will be able to detect such activities. Until some 30 years ago in vivo pharmacology was the most important tool for drug development, and all compounds such as morphine, atropine, salicylate, and reserpine were found through such in vivo pharmacology. In fact our ancestors probably developed drugs only by testing plants on themselves; the reductionist trend in drug development is only of the past century. So, go back to a more holistic personalized approach in drug development, particularly to come to evidence-based medicinal plants (73). 6. Conclusion In many part of the developing countries, with ethnic groups and informal settlements, ethnic healers are the important source(s) for the treatment of


various diseases, such as cut wounds, skin infection, swelling, aging, mental illness, asthma, diabetes, jaundice, scabies, eczema, snake bite, gastric ulcer etc,. They provide instructions to local people on how to prepare medicine from herbal or they prepare and give it to the patients. There are no records and the information is mainly passed on verbally from generation to generation. The local knowledge on various medicinal plants, ethnomedicinal preparations are useful resources, which may be scientifically evaluated and disseminated for efficacious drug development and improved health status. Thus traditional knowledge from various part of the world provides a good source of drug discovery for the future. Future directions for traditional herbal medicines may have several paths including: • Development of human safety and activity data based on pharmaco-

epidemiology approach and its documentation. • Reverse pharmacology path for rapid R & D for natural drugs • Advanced multicentric and trans-disciplinary network of drug discovery

from ethnic medicine • Making standardized herbal extracts to therapeutic equivalence

acceptance • Develop active plant principles to form basis for combinatorial chemistry

and high throughput screening Evaluation of traditional medicines clinically is difficult, in addition if herbs are prepared traditionally, selecting placebo for comparison will be problematic, and herbal formulation usually take longer to work than conventional medicines or drugs. At the same time, a single medicinal plant can contain hundreds of natural constituents and they may have synergistic effect. Establishing which constituent is responsible for effect be time consuming and expensive. Although pre-clinical and clinical studies of botanicals are unique in many respects, they should be carried out and evaluated the same basic criteria applied to other types of investigations related to any drug development and should take into considerations of international initiatives and existing guidelines. Challenges in clinical research of botanicals are multiple and vary according to the type of the product (i.e single vs combination) and the history of use (i.e., new vs traditional). Yet given the worldwide popularity of herbal medicines, a widely applicable, appropriate and effective means of evaluating herbal medicines with limited resources is urgently needed. Considering the synergistic, antagonistic and interacting activities of natural health products particularly botanicals the need for adequate research and development of these products has a strong impact on the research community, the


pharmaceutical industry and the consumers. Reliable standardization and manufacturing processes, analytical methods, pharmacological test systems and well-designed clinical studies are essential for cost-effective development of botanicals and should follow international initiatives and existing guidelines. 7. Acknowledgement Authors wish to thank All India Council for Technical Education (AICTE), New Delhi for providing financial assistance through RPS scheme to School of Natural Product Studies, Jadavpur University, Kolkata and providing QIP - Fellowship to Sri S Ponnusankar 8. References 1. Mukherjee, P. K. 2002, Quality control of herbal drugs – an approach to

evaluation of botanicals, Business Horizons, New Delhi. 2. Jones, E. T., Mc Lain, R, J., and Weigand, J. 2002, Nontimber forest products in

the United States, University Press of Kansas, USA. 3. Romulo, R. N., and Ierece, M. L. 2007, J. Ethnobiol. Ethnomed., 3, 14. 4. Lambert, J., Srivastava, J., and Vietmeyer, N. 1997, Medicinal plants – rescuing a

global heritage, World Bank technical paper 355, Washington DC, USA. 5. Penso, G. 1980, WHO inventory of medicine plants used in different countries,

WHO, Geneva, Switzerland. 6. Sandhya Wakdikar, S. 2004, Electronic J. Biotechnology, 7, 217-223. 7. Kamboj, V. P. 2000, Curr. Sci., 78, 35. 8. Balunas, M.J., and Kinghom, A.D. 2005, Life. Sci., 78, 431. 9. King, S. 1992, Pac. Discovery., 45, 23. 10. Patwardhan, B., Ashok, D.B., and Chorghade, M. 2004, Curr. Sci., 86, 789. 11. Mukherjee, P. K., Rai, S., Mukherjee, K., Hylands, P. J., and Hider, R. C. 2007,

Expert. Opin. Drug. Discov., 2, 633. 12. Mukherjee, P. K., 2003, Clin. Res. Regul. Aff., 20, 249. 13. Mukherjee, P. K., Sahu, M., and Suresh, B., 1998, The Eastern Pharmacist,

42, 21. 14. Gold, J. L., Laxer, D. A., and Rochon, P. A. 2000, Ann. R. Coll. Physicians.

Surg. Can., 33, 497. 15. Mukherjee, P. K., 2001, Drug. Inf. J., 35, 623. 16. Mukherjee, P. K. 2005, Promotion and development of botanicals with

international coordination: exploring quality, safety, efficacy and regulations, Allied Book Agency., Kolkata, India.

17. Mukherjee, P. K., and Wahile, A. 2006, J. Ethnopharmacol., 103, 25. 18. Bombardelli, E., Curri, S, B., Della Loggia, R., et al. 1989, Fitoterapia., 60, 1. 19. Della Loggia, R., Sosa, S., Tubaro, A, Morazzoni, P., Bombardelli, E., and

Griffini, A. 1996, Fitoterapia., 67, 257.


20. Arpaia, G., Bombardelli, E, Curri, S. B., Della Loggia, R. 1989, Fitoterapia., 60 (Suppl 1), 11.

21. Bombardelli, E., and Spelta, M. 1991, Cosmet. Toiletries., 106, 69. 22. Bombardelli, E., and Spelta, M., Della Loggia, R., Sosa, S., Tubaro, A. 1991,

Fitoterapia., 62, 115. 23. Bombardelli, E., Cristoni, A., Morazzoni, P. 1994, Fitoterapia, 65, 387. 24. Maiti, K., Mukherjee, K., Gantait, A., Saha, B. P., Mukherjee, P. K. 2007, Int. J.

Pharm., 330, 155. 25. Maiti, K., Mukherjee, K., Gantait, A., Saha, B. P., Mukherjee, P. K. 2006, J.

Pharm. Pharmacol., 58, 1227. 26. Maiti, K., Mukherjee, K., Gantait, A., Ahamed, K. H. N., Saha, B. P., Mukherjee,

P. K. 2005, Iranian. J. Pharmacol. Ther., 4, 84. 27. Kinghorn, A. D. 2001, J. Pharm. Pharmacol., 53, 135. 28. Samuelsson, G. 2004, Drugs of Natural origin: a textbook of Pharmacognosy,

Swedish Pharmaceutical Press, Stockholm. 29. Newman, D, J., Cragg, G. M., Snader, K. M. 2000, Nat. Prod. Rep., 17, 215. 30. Butler, M. S. 2004, J. Nat. Prod., 67, 2121. 31. Graul, A. I. 2001, Drug News Perspect., 14, 12. 32. Heinrich, M., Teoh, H. L. 2004, J. Ethnopharmacol., 92, 147. 33. Pirttila, T., Wilcock, G., Truyen, L., Damaraju, C. V. 2004, Eur. J. Neurol.,

11, 734. 34. Frantz, S., and Smith, A. 2003, Nat. Rev. Drug. Discov., 2, 95. 35. Frantz, S. 2005, Nat. Rev. Drug. Discov., 4, 95. 36. Mundy, c., Kirkpatrick, P. 2004, Nat. Rev. Drug. Discov., 3, 643. 37. Butler, M. S. 2004, J. Nat. Prod., 67, 2141. 38. Lotsch, J., Geisslinger, G. 2001, Clin. Pharmacokinet., 40, 485. 39. Okouneva, T., Hill, B. T., Wilson, L., Jordan, M. A. 2003, Mol. Cancer. Ther.,

2, 427. 40. Cragg, G. M., Newman, D. J. 2004, J. Nat. Prod., 67, 232. 41. Yang, S. S., Cragg, G. M., Newman, D. J., Bader, J. P. 2001, J. Nat. Prod.,

64, 265. 42. Yu, D., Suzuki, M., Xie, L., Morris- Natschke, S. L., Lee, K. H. 2003, Med. Res.

Rev., 23, 322. 43. Creagh, T., Ruckle, J. L., Tolbert, D. T., Giltner, J., Eiznhamer, D. A., Dutta, B.,

Flavin, M.T., Xu, Z. Q. 2001, Antimicrob. Agents. Chemother., 45, 1379. 44. Gordaliza, M., Garcia, P. A., del Corral, J. M., Castro, M. A., Goomez-Zurita, M.

A. 2004, Toxicon., 44, 441. 45. Horwitz, S. B. 2004, J. Nat. Prod., 67, 136. 46. Pisha, E., Chai, H., Lee, I. S., Chagwedera, T. E., Farnsworth, N. R., Cordell, G.

A., Beecher, C. W., Fong, H. S., Kinghorn, A. D., Brown, D. M., Wani, M. C., wall, M. E., Hieken, t. J., Das Gupta, T. K., Pezzuto, J. M. 1995, Nat. Med., 1, 1046.

47. Silva, G. L, Cui, B., Chavez, D., You, M., Chai, H. B., Rasoanaiyo, P., Lynn, S. M., O’ Neill, M. J., Lewis, J. A., Besterman, J. M., Monks, A., Farnsworth, N. R., Cordell, G. A., Pezzuto, J. M., Kinglhorn, A. D. 2001, J. Nat. Prod., 64, 1514.


48. Hwang, B. Y., Su, B. N., Chai, H., Mi, q., Kardono, L. B., Afriastini, J. J., Riswan, S., Santarsiero, B. D., Mesecar, A. D., Wild, R., Fairchild, C. R., Vite, G. D., Rose, W. C., Farnsworth, N. R., Cordell, G. A., Pezzuto, J. M., Swanson, S. M., Kinghorn, A. D. 2004, J. Org. Chem., 69, 3350.

49. Ley, S. V., and Baxendale, I. R. 2002, Nat. Rev. Drug. Discov., 1, 573. 50. Geysen, H. M., Schoenen, F., Wagner, R. 2003, Nat. Rev. Drug. Discov., 2, 222. 51. Lombardino, J. G., Lowe III, J. A. 2004, Nat. Rev. Drug. Discov, 3, 853. 52. Clardy, J., Walsh, C. 2004, Nature., 432, 829. 53. Peterson, E. A., and Overman., L. E. 2004, Proc. Natl. Acad. Sci. USA.,

101, 11943. 54. Koehn, F. E., and Carter, G. T. 2005, Nat. Rev. Drug. Discov., 4, 206. 55. Lee, M. L., and Schneider, G. 2001, J. Comb. Chem., 3, 284. 56. Feher, M., and Schmidt, J. M. 2003, J. Chem. Inf. Comput. Sci., 43, 218. 57. Piggott, A. M., and Karuso, P. 2004, Comb. Chem. High. Throughput. Scr.,

7, 607. 58. Burke, M. D., Berger, E. M., Schreiber, S. L. 2004, J. Am. Chem. Soc., 126,

14095. 59. Ganesan, A. 2004, Curr. Opin. Biotech., 15, 584. 60. Tan, D. S. 2004, Comb. Chem. High. Throughput. Scr., 7, 631. 61. Mattijis, K. J. et al. 2006, Biomol. Eng., 23, 265. 62. Oka, H., Yamamoto, S., Kuroki, T., et al. 1995, Cancer., 76, 743. 63. Fan, X. H., Cheng, Y. Y. 2004, Chem. J. Chinese. U., 25, 2004. 64. Wang, J., Ha, w. Y., Ngan, F. N., et al. 2001, Planta Med., 67, 781. 65. Techen, N., Khan, I. A., Pan, Z. 2006, Planta Med., 72, 241. 66. Kumar, S., Prasad, K. V., Choudhary, M. L. 2006, Curr. Sci., 90, 1108. 67. Croston. G. E. 2002, Trends. Biotechnol., 20, 110. 68. Smith, T., Biotani, L., Bibby, c., Brackett, D., Corsi, F., da Fonesca, G. A. B.,

Gascon, C., Dixon, M. G., Hilton-Taylor, c., Mace, G., Mittermier, R. A., Rabinovich, J., Richardson, B. J., Rylands, A., Stein, B., Stuart, S., Thomsen, J., Wilson, C. 2000, Science, 289, 2073.

69. Balick, M. J. and Cox, P. A. 1996, Plants, People, and Culture: The Science of Ethnobotany, Scientific American, New York.

70. Thomas, M. B., Lin, N., and Beck, H.W. 2001, Pharm. Biol., 39 Supplement, 41. 71. Vaidya, A. D. B., Vaidya, R. A., and Nagaral, S. I. 2001, J. Assoc. Physicians.

India., 49, 534. 72. Verpoorte, R., Kim, H. K., Choi, Y. H. 2005, International conference on

promotion and development of botanicals with International coordination: exploring quality, safety, efficacy and regulations, P. K. Mukherjee (Ed.), Allied Book Agency, Kolkata, 10.

2. ethno medicine in complementary therapeutics - · pdf fileethno medicine in complementary...

Documents