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INTRODUCTION

INTRODUCTION INTRODUCTION TO HERBAL RESEARCH Nature always stands as a golden mark to exemplify the outstanding phenomenon of symbiosis. The biotic and abiotic elements of nature are all interdependent. The plants are indispensable to man for his life. The three important necessities of life – food, clothing and shelter – and a host of other useful products are supplied to him by the plant kingdom. Nature has provided a complete a store house of remedies to cure all ailments of mankind. The knowledge of drugs has accumulated over thousands of years as a result of man’s inquisitive nature so that we possess many effective means of ensuring health care 1. Some ancients as Assyrians, Arabs, Egyptians and Greeks left a legacy over a thousand years of medicinal plants use. They have discovered not only their medicinal properties, but also some dose-related effects of plants and their extracts. Despite the major advances in the modern medicine, the development of new drugs from natural products is still considered important. This seems to be even more relevant for the developing countries, where the costs to develop a drug are prohibitive. Since 1980, the World Health Organization has been encouraging countries to identify and exploit traditional medicine and phytotherapy 2. Scientific strategies for the study of natural products from plants have changed substantially in the past few years for a number of reasons, including advance in technology, new molecules of interest, changing ethical principles for organism collection and heightened awareness of the chemical and biological potential of the natural resources. Department of Pharmacognosy – PESCP

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The last couple of years have seen a resurgence interest in the use of herbal products. The World Health Organization (WHO) estimates that about 80% of the population living in the developing countries relies almost exclusively on traditional medicines; the medicinal plants play a major role and constitute the backbone of the traditional medicine. Indian materia medica includes 2000 natural products of therapeutic importance of which 400 are of mineral and animal origin and rest are of vegetable origin. India is perhaps the largest producer of medicinal herbs and is rightly called the “Botanical Garden of the World”. There are very few medicinal herbs of commercial importance, which are not cultivated or collected in this country. There are approximately 1250 Indian medicinal plants, which are used in formulating therapeutic preparation according to Ayurveda and other traditional system of medicine. Many screening programs of the natural resources have been initiated by various government and private agencies. Among these was the screening program by the Central Drugs Research Institute (CDRI) initiated in 1964 with a spanning period of 25 years and covered around 2500 plants with a variety of biological activities. Medicinal herbs have been in use for thousands of years, in one or another form under the indigenous system of medicine. The main Indian traditional system of medicine namely Ayurveda, Siddha and Unani are primarily plant based system. Since independence, India has made tremendous progress in Agro-technology, process technology, research and development, quality control and standardization of natural products.

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The evaluation of new drugs especially phytochemically obtained materials has again opened a vast area for research and development. Modern research in the herbal field is now focused on terms of standardization of plant material, extracts and specially prepared mixtures where the biological activity is used as a measure of their quality. Such a change has occurred for several different reasons, including the availability for biological activity, although some time it is difficult to measure because of interfering substance. Today plant based medicines are only 9.5% of the estimated 250,000 species worldwide. Therefore, the field is still wide open. Plants are recognized as rich source of medicines because they produce molecules that have intrinsic biological activity. This property is evolved as a defense against diseases or secondary metabolites can offer clues for the development of novel medicines. Compared with the synthetic drugs, however, the criteria and the approach for herbal drugs are much more complex. Several aspects which do not exist with synthetic drugs influence the quality. The problems specific to the phytopharmaceuticals are discussed bellow. •

Herbal drugs are mixtures of many constituents.

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The active principle(s) is (are) hardly known.

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Selective analytical methods may not yet exist.

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Reference compounds may not be available commercially.

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Chemical variability of plant materials.

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Natural variability or biodiversity.

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Chemovarieties (eg. Thyme) and chemocultivars (eg. Chamomile).

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Influence of harvest, drying and storage conditions.

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Influence of processing (different extracts: polarity of solvents, mode of extraction, instability of constituents). Fortunately, the interest and concern to discover novel compounds from

natural origins are ever on the increase. We should be aware of the fact that some global pharmaceutical companies have already engaged themselves in the intense and systematic research so as to develop new medicines from nature 3, 4.

ISOLATION OF ACTIVE CONSTITUENTS The investigation of the bioactive natural products has assumed a greater sense of urgency in response to the expanding human population and its subsequent demands for food, good health and increasing areas of land on which to live. Nature recognizes no artificial barriers such as those of the “academic disciplines” and thus it is no surprise to find investigators with quite different academic training studying various aspects of bioactive natural products. It is a multidisciplinary approach which not only facilitates the solution of specific problems such as those in plant toxicology but may also enhance the diversity and consequent value of bioactive natural product research.

Need for the isolation of natural products

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The use of herbal and other naturally based medicines has a long history. However the utilization of whole plant or other crude preparations for the therapeutic or experimental reasons can have several drawbacks, which include •

Variation in the amount of the active constituent with geographic areas, from one season to another, with different plant parts and morphology, and with climatic and ecologic conditions.

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Co-occurrence of undesirable compounds causing synergistic, antagonistic, or other undesirable, and possibly unpredictable, modulations of the bioactivity.

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Losses of bioactivity due to variability in collection, storage, and preparation of the raw materials. Thus the isolation of natural products that have biologic activity towards

organisms other than the source has several advantages, including •

Pure bioactive compounds can be in reproducible, accurate doses, with obvious benefits from experimental or therapeutic point of view.

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It can lead to the development of analytical assays for particular compounds or for classes of compounds.

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It permits the structural determination of bioactive compounds that may enable the production of synthetic material, incorporation of structural modifications and a rationalization of mechanism of action.

Extraction and isolation

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Extraction from the specific plant material is a trial and error exercise in which different solvents are tried under a variety of conditions such as time and temperature of extraction. The success or failure of the extraction process is monitored by the most appropriate assay. Once extracted from the plant, the bioactive component then has to be separated from the coextractives. This may involve simple crystallization of the compound from the crude extract or more usually it may involve further solvent partition of the coextractives and extensive chromatography, taking advantage of particular properties of the desired compound, such as acidity, polarity and molecular size. Final purification, to provide compounds of suitable purity for the structural analysis, may be accomplished by appropriate techniques such as recrystallization, sublimation, or distillation.

Structure Determination The process of structural determination involves accumulating data from numerous sources, each of which gives some structural information, and the assimilation of these data into a chemical structure that uniquely fits all the available structural information. A wide range of spectroscopic instrumentation, such as UV, IR, and visible absorption spectroscopies, NMR spectroscopy and Mass spectroscopy forms the backbone of the modern structural analysis 5.

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THE CONCEPT OF MARKERS Marker compounds are the constituents believed to be peculiar to the medicinal and aromatic plant, but which may not be the bioactive principles in the plant 6. According to WHO, a marker compound is “any characterizing physiological constituent of crude drug, which can be easily isolated and quantified whenever actual bioactive are unknown”. A good marker should have the following criteria •

It should be characteristic of (or unique to) the given crude drug or the herbal preparation.

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It should be a substance of established chemical structure.

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It should not be present in other herbs contained in the finished product.

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It should be present in sufficient quantities to develop a scientifically valid test method.

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It should be accessible to quantify with the common analytical equipments.

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It should be sufficiently stable and commercially available. Some compounds with a proven therapeutic activity can also be used as

markers for quality assurance if they meet the criteria for analytical markers (eg. Kavalactones in Kava kava or silybins/silydinins in Milk Thistle) 7. Department of Pharmacognosy – PESCP

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Table: 1 Some important plants and their Marker compounds 8, 9, 10

Name of the plant Catharanthus roseus Cissus quadrangularis Digitalis lanata Hibiscus rosasinensis Tarminalia arjuna Allium sativum Berberis aristata Dioscorea deltoidea Punica granatum Momordica charantia Coleus forskohlii Podophyllum hexandrum Wrightia tinctoria

Marker compound Vinblastine δ- Amyrin Digoxin Cyanidin-3,5-diglucoside Ellagic acid Diallyl sulphide Berberine Diogenin Punicaligin Stigmastadienol-β-D-glucoside Forskolin Podophyllotoxin Lupeol

The detection and quantification of a marker can ensure genuineness of the crude drugs, but it does not guarantee the bioactivity or efficacy of the crude drugs. Therefore the biomarkers based standardization; the process of activity guided fractionation is a pre-requisite. The process involves continual fractionation of the plant material monitored by a biological assay, till the compound responsible for the biological activity (in the bioassay used) is/are isolated in the pure form. If the bioassay used is general and nonspecific, then the isolated compounds can be labeled as “BIOMARKER” for the crude drug. But if the bioassay Department of Pharmacognosy – PESCP

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used is specific for a particular bioactivity, then the isolated compound can be called as the “BIO-ACTIVE MOLECULE” for that particular bioactivity 11.

NEED FOR STANDARDISATION Herbal medicines are in great demand in the developed as well as developing countries for primary healthcare because of their wide biological activities, higher safety margins and lesser costs. In India, the herbal drug market is about $ one billion and the export of plant based crude drugs is around $ 80 million. A need has therefore been generated for enforcing more stringent quality control on such products and for this, the plant material grown, harvested and processed for manufacturing should be as per the standardized protocols to ensure product uniformity, efficacy and safety. Standardization of herbal drugs is not an easy task. In order to obtain quality oriented herbal products, care should be taken right from the cultivation stage through collection, drying, identification, extraction, purification, packing and storage conditions. Standardization carried out by means of chemical, physical or analytical methods, may not correlate with the biological or pharmacological activities claimed, they should be standardized uniformly. The marker compound may or may not be biologically active compound, but it should be specific. Since standardization is carried out by means of analytical methods, the herbal products may not produce the claimed pharmacological activity. The problems in standardization arises due to •

Raw materials procured from different geological areas.

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Absence of proper authentication.

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Adulteration of crude drugs.

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Limited knowledge of active constituents.

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Complex composition of drugs.

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Lack of appropriate experimental models available for plant drugs.

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Lack of scientifically planned clinical trials for traditional medicines 3, 12.

Chromatographic fingerprinting has been in use for a long time for single chemical drug substances. The focus of fingerprinting in the classical pharmaceutical industry has been used to highlight impurity profiles, thereby giving indications of the source of the drug substance and the method of chemical preparation. Chemical and chromatographic techniques may also be used to aid in identification of herbal material or extract. Techniques such as HPLC, TLC, GC, CE have been used for identity tests. Spectroscopic methods such as IR, NMR, UV-Vis may also be used for fingerprinting. For standardization of natural product drugs, single chemical entities; “MARKER COMPOUNDS” may be used as potency standards in High Performance Liquid Chromatography (HPLC) analysis. Using well characterized marker compounds, conventional pharmaceutical manufacturing criteria for assay and content uniformity may be applied. These marker compounds may be used to help identify herbal materials, set specifications for raw materials, standardize botanical preparations during all aspects of manufacturing processes, and obtain stability profiles. Again the HPLC analysis for Department of Pharmacognosy – PESCP

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marker compounds may provide additional information in the form of chromatographic fingerprints 13. Traditionally Hemidesmus indicus (L.) R.Br. has been used in Ayurveda, Unani and Siddha system of medicines since a long time. The various literature surveys related to Hemidesmus indicus (L.) R.Br. shows that the major compounds (as seen on TLC/HPLC) which can be used for the quality control, are not highlighted and there is lacking of analytical methods also. The literature surveys also indicate that there is possibility of isolating new compounds from roots and other parts of the plant. Although Lupeol and Vanillin can be used as markers for this plant, but these phytoconstituents are not unique for this plant and they are found in large number of other plants also. Hence it was decided to carry out further research work in the field of isolation and standardization of markers from the roots of Hemidesmus indicus (L.) R. Br. (Asclepiadaceae). Here in the present study attempts had been made to isolate and standardize the constituents which are unique to this plant and characterize them as markers.

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