1. PHYTOCHEMICAL SCREENING OF UNKNOWN DRUGS

2. Phytochemistry is mainly concerned with enormous varieties of secondary plant metabolites which are biosynthesized by plants. Most of the best plant medicines are the sum of their constituents. The beneficial physiological and therapeutic effects of plant materials typically result from the combinations of these secondary products present in the plant. The information on the constituents of the plant clarifies the uses of the plants but only a small percentage have been investigated for their phytochemicals and only a fraction has undergone biological or pharmacological screening. In phytochemical evaluation the powdered leaves were subjected to phytochemical screening for the detection of various plant constituents, characterized for their possible bioactive compounds, which have been separated and subjected to detailed structural analysis.

3. Phytochemical analysis procedure:  After authentication, the fresh, healthy plant dry in shade for 2-3weeks then pulverize in a blender, sieve and use for further studies.  Subject the powdered drugs under investigation to the following tests:

4. 1.Detection of alkaloids: Dissolve the extract individually in dilute Hydrochloric acid and filter. 1.a) Mayer’s Test: Treat the filtrate with Mayer’s reagent (Potassium Mercuric Iodide). Formation of a yellow colored precipitate indicates the presence of alkaloids. 1.b) Wagner’s Test: Treat the filtrate with Wagner’s reagent (Iodine in Potassium Iodide). Formation of brown/reddish precipitate indicates the presence of alkaloids. 2.c) Dragendroff’s Test: Treat the filtrate with Dragendroff’s reagent (solution of Potassium Bismuth Iodide). Formation of red precipitate indicates the presence of alkaloids. Solvent free extract, 50 mg was stirred with few ml of dilute hydrochloric acid and filtered. The filtrate was tested carefully with various alkaloidal regents as follows.

5. 2. Detection of carbohydrates: Dissolve the extract individually in 5 ml distilled water and filter. Use the filtrate to test for the presence of carbohydrates. Molisch’s Test: Treat the filtrate with 2-3 drops of 1% alcoholic α-naphthol and 2ml of concentrated sulphuric acid was added along the sides of the test tube. Formation of the violet ring at the junction indicates the presence of Carbohydrates. Benedict’s test: Treat the filtrate with Benedict’s reagent and heated gently. Orange red precipitate indicates the presence of reducing sugars. Fehling’s Test: Treat the filtrate with dil. HCl, neutralize with alkali and heated with Fehling’s A & B solutions. Formation of red precipitate indicates the presence of reducing sugars.

6. 3- Detection of anthraquinone glycosides: Extracts were hydrolyzed with dil. HCl, and then subjected to test for glycosides. Modified Borntrager’s Test: Treat the filtrate or powder with dil. HCl, ferric chloride solution and immerse in boiling water for about 10 minutes. Cool and extract with equal volumes of benzene or chloroform. The benzene layer will be separated and treated with ammonia solution. Formation of rose-pink colour in the ammonical layer indicates the presence of anthranol glycosides. 4- Detection of cardiac glycosides (Keller Kelliani’s test): 2ml of filtrate will be treated with 2ml of glacial acetic acid in a test tube and a drop of ferric chloride solution will be added to it. This will be carefully underlayed with 1ml concentrated sulphuric acid. A brown ring at the interface indicated the presence of deoxy sugar characteristic of cardenolides. A violet ring may appear below the ring while in the acetic acid layer, a greenish ring may form.

7. 5- Detection of saponins glycosides: Froth Test: 1 gm of powder will be diluted with distilled water to 6 ml and this will be shaken in a graduated cylinder for 15 minutes. Formation of 1 cm layer of foam indicates the presence of saponins. Foam Test: 0.5 gm of powder will be shaken with 2 ml of water. If foam produced persists for ten minutes it indicates the presence of saponins. 6- Detection of unsaturated sterols and/or triterpenes: Dissolve the residue in 10 ml of Petroleum ether or anhydrous chloroform and filter. Divide the filtrate into two equal portions and test as follows: 1.Salkowski’s Test: Filtrate will be treated with with 2 ml of sulphuric acid, shaken and allowed to stand. Appearance of golden yellow color changing to orange and then to red, indicates the presence of unsaturated sterols and/or triterpenes. 2.Libermann Burchard’s test: To the chloroform solution, add 1 ml of acetic anhydride followed by the addition of sulfuric acid down the walls of the test tube to form a layer, the formation of a reddish-violet color at the junction of the two layers, and a green color in the chloroform solution indicates the presence of unsaturated sterols and/or triterpenes.

8. 7- Detection of phenols: Ferric Chloride Test: Extracts will be treated with 3-4 drops of ferric chloride solution. Formation of bluish black color indicates the presence of phenols. 8- Detection of flavonoids Alkaline Reagent Test: 2ml of extract will be treated with few drops of 20% sodium hydroxide solution. Formation of intense yellow colour, which becomes colourless on addition of dilute hydrochloric acid, indicates the presence of flavonoids. Lead acetate Test: Extracts were treated with few drops of lead acetate solution (10 %). Formation of yellow color precipitate indicates the presence of flavonoids.

9. 10-Detection of tannins: Extract about 1 g of the powdered drug with 5 ml of alcohol and filter. Test the clear alcoholic filtrate as follows: a.Add ferric chloride, a blue color indicates the presence of pyrogallol (hydrolyzable) tannins, and a green color indicates the presence of catechol (condensed or non-hydrolyzable) tannins. b.To 5 ml of the alcoholic extract add 2 ml of vanillin-hydrochloric acid solution, a rose red color indicates the presence of gallic acid. c.To 5 ml of the alcoholic extract add 2 ml of freshly prepared bromine water, the formation of a precipitate indicates the presence of tannins of non-hydrolysable type. 11- Detection of lignin: 2 ml of filtrate will be treated with alcoholic solution of phloroglucinol and concentrated hydrochloric acid. Appearance of red colour shows the presence of lignin. 12- Detection of proteins and amino acids: Ninhydrin Test: (1% ninhydrin solution in acetone): 2ml of filtrate will be treated with 2-5 drops of ninhydrin solution placed in a boiling water bath for few minutes and observed for the formation of blue to purple color. Formation of the color indicates the presence of amino acid.