By Akshay Anand

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Pharmacognosy : Chapter 2-B CHEMOTAXONOMY

Chemotaxonomy is a recent approach in the study of Crude-Drug Classification, where in Taxonomy and Biogenesis is given equal importance. Chemotaxonomy recognises that the plants having similar constituents share similar medicinal properties and nature. (Eg.: all species of Cinchona contain Quinine, all are useful in treatment of Malaria) Taxonomy based on conventional methods and chemical constituents is known as Chemotaxonomy. It is defined as a scientific investigation of chemical characters for the study of problems of Plant Taxonomy and Plant Phytogeny (origin of races) Chemical taxonomists regard the various chemical constituents of plants and characters to be considered along with other features of the plants traditionally used in taxonomy.

HISTORY:
In the early years of 15th century, Herbalists arraged the plants according to their Vernas or Local Names. At the beginning of 18th century, Botanists James Petiver and Nehemiah Grew recognised that some groups of plants which possessed Therapeutic Properties also coincided with Morphological Characters. (Eg.: Labiatae, Umbelliferae, Cruciferae etc.) A.P. de Candolle laid the foundation for the study of Chemotaxonomy. He considered characters like Bitter Flavour, Susceptibility of plants to Insect-Attacks etc.. Grieshoff, a pharmacist considered readily detectable compounds like Tannins as criteria for classification. Mervoir studied the comparative phytochemistry of fixed oils and employed to demonstrate phytogenetic relationships.

PRINCIPLES OF CHEMOTAXONOMY:
The Principles of Chemotaxonomy were elaborated in the past century by A.P. de Candolle and Grieshoff. Two Postulates were put forward by them. 1. Plant Chemotaxonomy will be the most useful guide to man who is in search of new Industrial and Medicinal plants. 2. Chemical characters of plants will be most valuable to taxonomy in the future. While the first postulate proved to be extremely useful. The second one came to be accepted very slowly. Examples: 1. Starting material for hormone synthesis, Search for suitable sugars was essentially guided by taxonomical concepts Diascorea, Lilliaceae 2. Search for Pregnane Derived Alkaloids (Genus Holarrhena): Many plants of family Apocynaceae are found containing this alkaloid. 3. Steroidal Alkaloids are found in rich, in family Buxaceae. 4. Cardiac Glycosides are found in rich, in Asclepiadaceae and Apocynaceae. 5. Diterpenoids are found in rich, in family Euphorbiaceae.

By Akshay Anand

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CHEMICAL CHARACTERS AS GUIDES FOR CLASSIFICATION:

In conventional method, plants are classified on the basis of many morphological characters. Varied interpretations by different taxonomists result in disagreement regarding classification. In such instances, economists look for characters other than morphological characters. In such cases, chemical characters become very useful guides for taxonomists. 1. There are two different concepts regarding families Liliaceae and Amaryllidaceae. Liliaceae is characterised by the presence of Hypogynous Flowers where as Amaryllidaceae is characterised by Epigynous Flowers. However Hutchison believes that the most essential character of Amaryllidaceae is their Umbelliferae Inflorescence with Involucral Bracts. If these characters are considered, Genus Allium and Iulated Genera are to be transferred to Amaryllidaceae. In such instances, Chemical Characters aid Taxonomists. 2. Highly characterised alkaloids of Amaryllidaceae are lacking in Genera Allium and Steroidal Saponins which are not generally found in Amaryllidaceae, are found in Allium, which are wide spread in Liliaceae. So Allium is placed under Liliaceae. 3. Even though chemotaxonomy is useful in certain aspects, is still in Infancy Stage. A classification similar to taxonomical classification at present is beyond the capacity of the chemists. 4. Conventional Classification is based on the comparative studies of the genetically controlled morphological characters and few anatomical characters of plants. Some of the characters are of large systematic categories like divisions, classes and orders. Others are left general and used to classify plants into families, genera, species etc.. The main advantage here is simplicity. It does not require any special equipment. Hence almost all plants are studied and classified. 5. The disadvantages are due to different interpretation. Sometimes, plants are classified into different groups by different botanists. In nature, evolution is constant and progressive. Evolution depends upon a combinations of internal and external factors such as mutation, selection etc.. 6. Convergence: During evolution, it may so happen that the dissimilar plants may give rise to morphologically similar plants known as Convergence. 7. Divergence: Sometimes, related plants may give rise to very dissimilar plants known as Divergence. Such phenomenon causes considerable taxonomic difficulties.

CHEMOTAXONOMY:
It is based on the pattern of presence of chemical constituents. These characters are genetically controlled and can be exactly described in terms of definite structural and configuration chemical forms. Chemotaxonomy can be elaborated under the following headings: 1. Descriptive Chemotaxonomy, the study of chemical constituents and comparison. 2. Dynamic Chemotaxonomy, the study of biosynthetic pathways and comparisons of these pathways on the basis of taxonomy. 3. Serotaxonomy, the study of pattern of Enzymes. 4. DNA-Hybridization, the elucidation of chemical structures of genes.

By Akshay Anand

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1. DESCRIPTIVE CHEMOTAXONOMY:
Plants generally contain various types of chemical constituents. These substances which are common in occurrence, such as amino acids, sugars, fatty acids etc. are of little or no taxonomic importance. Similarly compounds found in only Single Species (rare occurrence) are also not important. Hence compounds of Intermediate Distribution only are of Taxonomic Importance. In the study of Descriptive Chemotaxonomy of plants, Primary and Secondary metabolites provide more information. a. Primary Metabolites: These are the plants of vital metabolise pathways and most of them are universal in occurrence. They lack systemic significance and sometimes quantities may vary. In such cases, excess of primary metabolite is stored. This can also serve Chemotaxonomy. Examples: In Blue-Green algae, Cyanophycean Starch is stored in excess. In Xanthophyta, Leucecin is stored in excess. b. Secondary Metabolites: They perform no vital function in plants. Therefore they are less wide spread. The most well known groups are alkaloids, glycosides, terpenoids, fixed oils, waxes, tannins, flavonoids, etc.. The secondary metabolites are large molecules with many side groups which can be variously substituted thus allowing a wide range of possible types of molecules. There is a relation between complexity of the substance and its Taxonomic Significance. Greater the complexity of the molecule, greater is the number of steps required for biosynthesis, therefore narrower is its distribution and hence greater is the Taxonomic Significance. Eg.: Morphine; Biosynthetically complex alkaloids of Morphine occur in two species of genus Papaver i.e. Papaver somniferum and Papaver setigerum. But the simple alkaloid Reserpine is found in all plants of Poppy family. The phenolic compounds have proved that maximum taxonomists are required for chemotaxonomy. The main reason for its population is that they are quickly and easily extracted from plant material, easily separated by Paper Chromatography and are readily identified by Location Reagents. Eg.: Flavonoids which have relatively common nucleus with great variety of side groups which characterise individual compounds. Flavones, Flavanones, Isoflavones, Flavanols, Hydroquinones, Chalcones and Biflavonyls form examples.

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2. DYNAMIC CHEMOTAXONOMY:
Different plants sometimes contain same constituents but are formed by different pathways. Such plants probably contain similar enzyme system and the compounds may indicate a relationship between the related plants. Example 1: Benzoic acid in plants is formed by three ways,a. by Shikimic acid b. by degradation of large molecules c. by cylisation Though the Benzoic acid formed is chemically same, formations by different pathways are considered to constitute different objects. Example 2: Lysine can be formed from,a. - diamino pimeric acid (DAP) (DPA) b. amino adipic acid (AAA) These compounds are biologically different compounds. Alkaloids arising from different Lysines are biologically different.

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3. SEROTAXONOMY:
The results from proteins in taxonomy can be classified as a main heading corresponding to three main methods employed. Serology Electrophoresis / Immunoelectrophoresis (Gel Diffusion) Amino-Acid Sequence

SEROLOGY:
The technique of Serology relies under immunological reaction shown by mammals when they are invaded by foreign proteins. The plant extract containing protein (antigen) is injected to the mammals (usually Rabbits). After some days, mammals form Antibodies specific to the Antigens. These antibodies are extracted and can be used as Standard for tests against other plant extracts. These relationships has to be ascertained with first plant and the amount of Coagulation it causes, measures the similarity of species. The protein extract of a plant when relationship has to be ascertained, is mixed with antibodies of the first plant. If precipitation is formed even at higher dilution of the Serum, it indicates that the two plants are closely related. If precipitate is not formed, it indicates that they are not related.

ELECTROPHORESIS:
Electrophoretic separation of proteins depend on Amphoteric Properties, where they are charged Positively (+) or Negatively (-) to various extent according to pH of the medium. A porous material like filter paper, cellulose acetate strip pr starch gel moistened with buffer supports protein mixture. When current is passed, the proteins migrate on this supporting medium and separate into distinct bands on it. The bands can be cut and selected using a suitable solvent to separate proteins. Alternatively they can be stained using suitable dyes like Bromophenol blue.

IMMUNOELECTROPHORESIS (GEL DIFFUSION):

Comparison between protein mixtures are used for possible chemotaxonomic grouping.

The antibody of specific antigen is placed in a trough on a thin agar slab. The various proteinous (antigen) mixtures are kept in the position as shown in the figure. After some time, antigen and antibody components diffuse radially from their positions on the agar. When antibody meets antigen, precipitation occurs as an arc which represents the toxin of the original plant. Ranunculaceae and Phaseolus plants are studied more for their chemotaxonomic relationship. In number of instant considerations, correlation between serotaxonomy and chemical serotaxonomic schemes is obtained.

By Akshay Anand AMINO-ACID SEQUENCE:

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Amino-acids from polypeptide chain can be broken one by one and identified by chromatographic method and the sequence is determined.

4. DNA-HYBRIDIZATION:
It is possible to break a double helix DNA into two complementary compounds or strands. The single strands consist of purine and pyramidine bases, (adenine, guanine, cytosine and thymine). Under suitable conditions, two strands come together again and combine to form a complete molecule (reassociated DNA). Reassociation depends on collision rate and concentration (purine adenine always combines with pyrimidine thymine and pyramidine cytocine always combines with purine guanine). Single stranded DNA (unlabelled) in low concentration is mixed with single strand of unknown DNA (labelled). Under suitable conditions the two strands associate wherever they are common. The unpaired single strands are removed by suitable means and radio activity is measured. The amount of activity indicates the extent of Hybridization. PROCEDURE: DNA exists as a double helix. The two complementary strands can be separated (dissociated) from each other by heating to 100 0C and cooling quickly. The known Species-A (long single strand) DNA are immobilized in agar. DNA from Species-B (whose relationship has to be ascertained) is radio labelled and then broken into short lengths by pressure. The short lengths of double helix get dissociated and react with the immobilized single strands of Species-A. After sometime the unreacted short lengths of Species-B DNA get washed away. The amount of labelled Species-B DNA

By Akshay Anand

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which has been hybridized with Species-A DNA is measured by removing the labelled polynucleotides and measuring the removed labels.

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B.Pharm Pharmacognosy Notes Typeset and Illustrations by Akshay Anand M