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474 PHG Screening, separation and analysis for plant constituents Dr. Areej M. Al-Taweel Required Text book: - Wagner, H., Bladt, S. & Zgainski, E. M, Plant Drug Analysis. - Richard J. P. Cannnell, Natural Product Isolation. - Robert M. Silverstein and Francis X. Webster, Spectrometric Identification of Organic Compounds. Introduction The use of plant-derived medicinal dates back many centuries although it is still under estimation in modern medicine. Plants remain the most important source of natural drugs. - More than 30% of prescription drugs are natural products. - More than 60% of anticancer and anti-infective drugs are natural products. The main sources of drugs are as follows: 1- Natural substances: From plants, microorganisms, animals,- etc. (totally obtained from nature). 2- Semisynthetic substances: These are drugs that are manufactured by partial synthesis. 3- Synthetic substances: These are drugs which are manufactured by total synthesis (i.e. complete synthetic process or processes). Medicinal plants information sources: Information, however can be obtained from one or more of the following sources: 1- Herbals 2- Medical botany 3- Ethnobotany 4- Herbaria (herbarium) 5- Field exploration 6- Phytopharmacological surveys Phytochemical Screening To carry phytochemical screening the following points must be fulfilled: 1- Selection of promising plant materials. 2- Proper collection of selected plants. 3- Authentication of plant material. 4- Drying of plant materials. 5- Grinding of the dried plants. 6- Garbling of the dried plants 7- Packing, storage and preservation 8- Extraction and fractionation of constituents. 9- Methods of separation and purification. 10- Methods of identification of isolated compounds (structure elucidation e.g. UV, IR, MS, H-NMR and C-NMR). 1. Selection of promising plant materials: Before investing time, effort and money in phytochemical screening it is very important to select a promising plant. The choice of promising plant depends upon the following: 1- A plant which have a biological activity. 2- A plant used in folk medicine. 3- A plant which show a particular toxicities. 2. Proper collection of selected plants Drug may be collected from: 1- Wild plants. 2- Cultivated plants. Wild plant Cultivated plant Disadvantage Advantage 1- Scattered in large or unlimited area Present in limited area. 2- Difficult to reach Easy to reach 3- The collector must be highly skilled botanists The collector must not be skillful person 4- Deficiency may occur due to continuous collection Continuous supply The following precautions should be considered during stage of collection: a- The proper time of the day, time of the year and maturity stage of collection is particularly important because the nature and quantity of constituents may vary greatly in some species according to the season and time of collection. b- The collected plant should be free from any contamination. c- Collecting plants which are free from diseases (i.e. which are not affected by viral, bacterial, fungal infection). 3- Authentication of plant material This may be confirmed by: 1- Establishing the identity by a taxonomy experts. 2- Collection of a common species in their expected habitat by a field botanist. 3- By comparing the collecting plant with a voucher specimen ( herbarium sheet) 4- Drying of plant materials Aim of drying: 1- Ease of transport. 2- Ease of grinding 3- Inhibit the growth of microorganisms. 4- Preservative of active constituents. Drying is done in: -Shade and in sunlight (Natural drying). - Hot air drying or by freeze-drying (Artificial drying). Changes may occur during the drying: 1- Size and weight 2- Shape and appearance 3- Color 4- Odor 5- Taste 6- Active constituent: Slow drying of vanilla pods lead to obtain vanillin from glucovanillic alcohol. CH3O CH2OH CH3O Enz H2O Glu-O Glucovanillic alcohol (glucoside) CHO CH2OH CH3O Enz [O] HO HO Vanillin 5- Grinding of the dried plants 6- Garbling of the dried plants 7- Packing, storage and preservation 8- Extraction and fractionation of constituents There is no general (universal) method for the extraction of plant materials. The precise mode of extraction depends on: 1- The texture of the plant material. 2- The water content of the plant material. 3- The type of substances to be extracted or nature of active constituents. Extraction: is the separation of medicinally active portion of plants or animal tissues through the use of selective solvent and suitable methods extraction. The principal methods of extraction are: 1- Maceration 2- Percolation 3- Infusion 4- Decoction 5- Digestion 6- Continuous hot extraction technique (Soxhlet extraction process). 7- Liquid-liquid extraction 8- Solvent-solvent ppt. 9- Distillation Continuous hot extraction technique (Soxhlet extraction process) Choice of solvent As a general empirical rule: Non polar solvents (petroleum ether and hexane) will dissolve non-polar compounds (fats and waxes). While polar solvents (methanol, ethanol and water) dissolve polar compound (alkaloid salts and sugars). (that mean like dissolve like) The affinity of the solute for the organic phase may be greatly increased by using mixture of solvents instead of single ones (sometimes used mixtures of solvent to increase the solubility). Example: solublization of an aliphatic carboxylic acid in ethanol, acetone and a mixture of both. O------------H-O-CH 2-CH3 In ethanol -R-C Hydrogen bond O-H O In acetone R-C Hydrogen bond CH3 O-H---------O=C CH3 In a mixture of acetone and ethanol O HO-C 2H5 (ethanol) CH3 R-C OH O=C (acetone) CH3 Screening of Drugs After a new substance (expected to be a new drug) has been developed. -The next step is to subject it to a specified set of procedures in experimental animals. The aim is to determine whether or not the compound possesses the desired action. Phytopharmacological screening: ● Antimicrobial activity Cinnamomum verum Thymus vulgaris Lavendula officinalis (Eugenol) (Thymol) (Linalool) ● Antineoplastic activity Catharanthus roseus Taxus brevifolia (Vincrestine, vinblastin) (Taxol) ● Antimalarial: Cinchona succirubra Artemisia annua (Quinine) (Artemisinin) Phytopharmacological screening: ● Hypoglycemic: Garlic (Allium sativum) (Allicin) ● Cardiotonic Digitalis purpurea Strophanthus kombe (Digoxin and Digitoxin) (K-strophanthoside) ● Antiarrythemic Cinchona succirubra (Quinidine) Type of pharmacological screening: 1-Blind screening 2- Simple screening 3- Programmed screening The organization of blind screening Screening for pharmacological activities proceeds from general observation on intact animals to the more specific techniques using isolated organs. It might include the following tests: A- Preliminary observations on the behaviour of conscious animals (neuropharmacological tests) B- The cardiovascular test. D- Isolated organ preparations especially the isolated guinea pig ileum.