Preliminary Phytochemical Screening of Individual Crude Drugs
Khandelwal 2000; Farnsworth et al., 1969; Segelman et al.
The plant may be considered as biosynthetic laboratory for multitude of compounds like alkaloid, glycoside, volatile oils, tannins, saponins and flavonoids etc. These compounds are termed as secondary metabolites and are responsible for therapeutic effects. To check the presence or absence of primary and secondary metabolites, all the extract were subjected to battery of chemical tests.
TEST FOR STEROLS
1) Salkowski Test
Few mg of residue of each extract was taken in 2 ml of chloroform and in it 2 ml of concentrated sulphuric acid was added from the side of the test tube. The test tube was shaken for few minutes. The development of red color in the chloroform layer indicates the presence of sterols.
2) Liebermann-Burchard Reaction
Few mg of residue was dissolved in chloroform. To this, few ml of acetic anhydride was added. Then two drops of conc. sulphuric acid was added from the side of the test tube. The greenish transient color indicates the presence of sterols.
TEST FOR GLYCOSIDES
1) Borntrager’s Test
Borntrager’s test is employed for presences of anthraquinones. The drug is boiled with dilute sulphuric acid, filtered and to the filtrate benzene, or ether or chloroform is added and shaken well. The organic layer is separated to which ammonia is added slowly. The ammoniacal layer shows pink to red color due to presences of anthraquinone glycosides.
2) Legal Test
The test is employed for digitoxose containing glycosides. The
extract of drug is dissolved in pyridine, sodium nitroprusside solution is added
to it and made alkaline, pink or red color is produced.
TEST FOR ALKALOIDS
Few mg of the residue of each extract was taken separately in 5 ml of 1.5 % v/v hydrochloric acid and filtered. These filtrates were then used for alkaloid detection.
1) Dragendorff”s Reagent
It was prepared by mixing solution A (17 g of bismuth sub nitrate + 200 g of tartaric acid + 800 ml distilled water) and solution B (160 g potassium iodide + 400 ml distilled water).
Above Dragendorff”s reagent was sprayed on Whatman no. 1 filter paper then the paper was dried. The test filtrate after basification with dilute ammonia was extracted with chloroform and this extract was applied on the filter paper, impregnated with Dragendorff”s reagent with the help of capillary tube. Development of an orange red color on the paper indicates the presence of alkaloids.
2) Mayer’s Reagent
1.36 g of mercuric chloride was dissolved in 60 ml water and 5 g of potassium iodide dissolved in 10ml of distilled water, solution were mixed and diluted to make up volume 100 ml. To a little of each extract taken in dilute hydrochloric acid in a watch glass, few drops of the reagent was added, formation of cream colored precipitate shows the presence of alkaloids.
3) Wagner’s Reagent
1.27 g of iodine and 2 g of potassium iodide were dissolved in 5 ml of water and the solution was diluted to 100 ml with water. When few drops of this reagent were added to the test filtrate, a brown color precipitate was formed indicating the presence of alkaloids.
4) Hager’s Reagent
A saturated aqueous solution of picric acid was employed for this test. When the test filtrate was treated with this reagent, an orange yellow precipitate was formed indicating the presence of alkaloids.
TEST FOR SAPONINS
1) Foam Test
A few mg of the test residue was taken in a test tube and shaken vigorously with small amount of sodium bicarbonate and water. If stable, characteristic honeycomb like froth is obtained, saponins are present.
TEST FOR TANNINS
The test residue of each extract was taken separately in water, warmed and filtered. Tests were carried out with the filtrate using following reagent-
1) Ferric Chloride Test
A 5 % solution of ferric chloride in 90 % alcohol was prepared. Few drops of this solution was added to a little of the above filtrate. If dark green or deep blue color is obtained, tannins are present.
2) Lead Acetate Test
A 10 % w/v solution of basic lead acetate in distilled water was added to the test filtrate. If precipitate is obtained, tannins are present.
3) Potassium Dichromate Test
If on an addition of a solution of potassium dichromate in test filtrate, dark color is developed, tannins are present.
TEST FOR FLAVONOIDS
A small quantity of test residue was dissolved in 5 ml of ethanol (95 % v/v) and treated with few drops of concentrated hydrochloric acid and 0.5 g of magnesium metal. The pink, crimson or magenta color is developed within a minute or two, if flavonoids are present.
TEST FOR PROTEINS
1) Biuret Test
A few mg of the residue was taken in water and 1 ml of 4 % copper sulphate was added to it. Violet or pink color is formed, if proteins are present.
2) Xanthoproteic Test
A little residue was taken with 2 ml of water and 0.5 ml of concentrated nitric acid was added to it. Yellow color is obtained, if proteins are present.
TEST FOR AMINO ACID
The ninhydrin reagent is 0.1 % w/v solution of ninhydrin in n-butanol. A little of this reagent was added to the test extract. A violet or purple color is developed, if amino acids are present.
TEST FOR SUGARS
1) Molisch’s Test
The Molisch’s reagent was prepared by dissolving 10 g of α- naphthol in 100 ml of 95 % alcohol. A few mg of the test extract was placed in a test tube containing 0.5 ml of water, and it was mixed with two drops of Molisch’s reagent. To this solution, was added 1 ml of concentrated sulphuric acid from the side of the inclined test tube, so that the acid formed a layer beneath the aqueous solution without mixing with it. If a red brown ring appears at the common surface of the liquids, sugars are present.
2) Barfoed’s Test
This reagent was prepared by dissolving 13.3 g of crystalline neutral copper acetate in 200 ml of 1 % acetic acid solution. The test residue dissolved in water and heated with a little of the reagent. If a red precipitate of cuprous oxide is formed within two minutes, mono-saccharides are present.
GPAT 2012 >