3.3 Phytochemical analysis :
3.3.1. Test for steroids :
Salkowski test : Chloroform solution of the extract when shaken with concentrated sulphuric acid and on standing yields red colour.
3.3.2. Test for flavonoids:
Ferric chloride test : Alcoholic solution of the extracts mixed with few drops of neutral ferric chloride solution gives green colour.
3.3.3. Test of alkaloids :
The extract were mixed with ammonia and then extracted with chloroform solution. To this dilute hydrochloride acid was added. The acid layer was used for chemical tests for alkaloids.
Mayer’s test (Potassium Mercuric Iodide): The acid layer with few drops of Mayer’s reagent gives a creamy white precipitate.
3.3.4 Test for glycosides :
Borntrager’s test is employed for presences of anthraquinones. The drug is boiled with dilute sulphuric acid, filtrered 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 colour due anthraquinone glycosides
3.3.5 Test for saponins:
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.
3.3.6. 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
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 colour is obtained, tannins are present.
3.4. quantitative analysis:
3.4.1 Estimation of Flavonoids
Aluminium chloride forms an acid stable complex with the C3 or C5 hydroxyl group of flavones and flavonoids. In addition, aluminium chloride forms acid laible flavonoids .
The leaf eatracts and the standard rutin, were dissolved in the methanol separately for total flavonoids estimation. To each 1ml of extracts the 4 ml of water followed by 0.3 ml of 5% sodium nitrate were added. After 5 minutes, 0.3 ml of 10% aluminium chloride solution was added. After proper mixing absorbance was measured using a spectrophotometer at 510 nm and a standard graph was plotted. The absorbance value obtained from extracts were interpreted from the standard graph to get the total flavonoids content expressed as milligram equivalents of rutin.
3.4.2. Estimation of total Phenols :
phenols reacts with phsophomolybdic acid of folins- Ciocalteau reagent in alkaline medium and produce a blue coloured complex (molybdenum blue). The colour developed was read colorimetrically at 650 nm.
Taken 0.5 and 1 gm of the palnt sample and ground it with a pestle and mortar using 10 times volume of 80 % ethanol. Centrifuged the homogenate at 10000 rpm for 20 minutes and collected the supernatant. Re-extracted the residues with five times the volume of 80 % ethanol, centrifuged and pooled the supernatants. Evaporated the supernatant to dryness. Dissolved the dried residue in 5 ml of distilled water.
Pipetted out different aliquots (0.2-2) ml into test tubes. Made up the volume in each tube to 3 ml with water. Added 0.5 ml of Folin-Ciocaltean reagent. After 3 minutes, added 2 ml of 20 % sodium carbonate solution to each tube. Mixed thoroughly, placed the tubes in a boiling water bath for exactly one minute, cooled and measured the absorbance at 650 nm against a reagent blank. Plotted a standard curve using different concentration of catechol.