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: PREPARATION OF SAMPLE
Preparation of the sample is done using ethanol as the solvent of choice.
The leaves of Tarenna asiatica were collected manually, cut into smaller pieces, dried under shade for a specified period (approximately 14-16 days) and then ground into a course material (pulverization). This ground course plant material was used for the preparation of ethanolic extract.
Soxhlet apparatus is used for the extraction process. 200grams of dried course powder of Tarenna asiatica leaves was taken and extracted using enough quantity of ethanol (99.99%) for 3-4hrs while maintaining a temperature of 75-78.3oC.

Fig.3: Soxhlet apparatus used for preparation of ethanolic plant extract.

5.5: PHYTOCHEMICAL SCREENING 11
Phytochemical screening refers to the test or chemical reactions employed to identify the medicinally active compounds present in the given test sample to be examined. The crude extract was taken and used for phytochemical screening tests to evaluate the nature of the bioactive constituent’s present.

1. TEST FOR ALKALOIDS:
a) Dragendroff’s reagent: Potassium bismuth iodide solution is known as dragendroff’s reagent. Alkaloids react with this solution to give reddish brown precipitate.
b) Mayer’s reagent: Potassium mercuric iodide solution is mayer’s reagent. Alkaloids give cream colour precipitate when mixed with it.
c) Wagner’s reagent: Iodine-potassium iodide solution is wagner’s reagent. It reacts with alkaloids to give reddish brown precipitate.
d) Hager’s reagent: Chemically hager’s reagent is saturated solution of picric acid. In the presence of alkaloids, it gives yellow precipitate.
e) Tannic acid test: Alkaloids when mixed with tannic acid solution gives buff coloured precipitate.
f) Picrolonic acid test: Yellow coloured precipitate is formed by mixing alkaloids along with picrolonic acid.

2. TEST FOR AMINO ACIDS:
a) Millon’s test: 2ml of millon’s reagent is added to the test solution. Formation of white precipitate confirms the presence of amino acids in the test solution.
b) Ninhydrin test: In the presence of amino acids, the test solution when mixed with ninhydrin reagent results in the formation of violet colour.
3. TEST FOR CARBOHYDRATES:
a) Molisch’s test: The test solution is taken in a test tube to which few drops of alcoholic anaphthol solution is added along with few drops of concentrated sulphuric acid through the sides of the test tube. Presence of carbohydrates is confirmed by appearance of purple to violet colour ring at the junction.
b) Barfoed’s test: 1ml of test solution is taken along with 1ml of barfoed’s reagent and the resultant mixture is heated on a water bath. Formation of red cupric oxide confirms the presence of monosaccharides in the solution. Prolong heating (about 10mins) causes reduction owning to partial hydrolysis of disaccharides to monosaccharides.
c) Salivanoff’s test (test for ketones): Test solution is taken to which crystals of resorcinol are added along with equal volume of concentrated hydrochloric acid and the resultant mixture is heated on a water bath. Rose colour confirms the presence of ketones (e.g., fructose, honey etc).
d) Test for pentoses: Equal volumes of test solution and hydrochloric acid are taken to which small amount of phloroglucinol is added and the mixture is heated. Red colour produced confirms the test.
e) Osazone formation test: Test solution is taken to which phenyl hydrazine hydrochloride, sodium acetate and acetic acid is added. The mixture is heated, and resultant yellow crystals formed are examined under a microscope. These are characteristically shaped for sugars.

4. TEST FOR VOLATILE OIL:
a) A thin smear of the test solution is applied on a glass slide to which a few drops of SudanIII solution are added. Red coloured globules are formed in the presence of volatile oil.
b) Similar section of test solution is prepared on a glass slide as mentioned above, to this few drops of tincture alkane are added, red colour indicates the presence of volatile oil.

5. TEST FOR FLAVONIODS:
a) Shinoda test: Test solution was taken along with few magnesium turnings. To this concentrated hydrochloric acid was added drop wise. Pink, scarlet, crimson red or occasionally green to blue colour appears after few minutes.
b) Alkaline reagent test: Test solution is taken to which few drops of sodium hydroxide solution are added. Presence of flavonoids is confirmed by the addition of few drops of dilute acid which results in changing of intense yellow coloured solution to colourless.
c) Zinc hydrochloride test: A mixture of zinc dust and concentrated hydrochloric acid is added to the test solution. Red colour is produced after a few minutes.

6. TEST FOR GLYCOSIDES:
a) Fehling’s test: This test is characteristic (not specific) for detection of aldehydes (but not ketones) by reduction of copper-II solution (deep blue colour) to insoluble copper oxide (red precipitate). A positive test is either indicated by a green suspension or formation of red precipitate.
It is a highly sensitive test whereby the presence of a minute quantity of glucose (1mg) can be confirmed by formation of characteristic red precipitate.12
b) Borntrager’s test: 1ml sulphuric acid along with the test solution should be taken in a test tube and boiled/heated for five minutes and immediately filtered while hot. Let the filtrate cool before adding equal volume of dichloromethane or chloroform. The lower layer of dichloromethane or chloroform is to be separated and shaken with half its volume of dilute ammonia. Rose pink or red colour in the ammoniacal layer confirms the test.
c) Modified Borntrager’s test: 2ml of dilute sulphuric acid along with 200mg of test material is subjected to boiling. The solution is treated with 2ml of freshly prepared 5% aqueous ferric chloride solution for 5mins. Equal volume of chloroform is added, and the solution is shaken for a few minutes. The resultant solution is subjected to the test mentioned above.
Some plants contain anthracene aglycone in a reduced form, during the extraction process if ferric chloride is added, it results in the oxidation of anthraquinones, which shows response to Borntrager’s test.

7. TEST FOR TANNINS:
a) Goldbeater’s skin test:Goldbeater’s skin is an intestinal membrane that is obtained from ox which behaves like an untanned hide.
A piece of goldbeater’s skin is taken, 2% hydrochloric acid is added, rinsed with water and then placed in the test solution for 5 minutes. Remove the skin from the solution and wash with distilled water and transfer to a solution of 1% ferrous sulphate. Presence of tannins is indicated by appearance of black or brown coloured spots.
b) Ferric chloride test: The test extract is treated with ferric chloride solution; blue colour indicates presence of hydrolysable tannins whereas green colour indicates presence of condensed tannins.
c) Phenazone test: 5ml of aqueous test extract and 0.5gm of sodium acid phosphate is warmed and filtered. 2% phenazone solution is added to the resultant filtrate which results in the formation of a bulky precipitate which is often coloured.
d) Gelatin test: 1% gelatin solution containing 10% sodium chloride is added to the test solution resulting in precipitate formation.
e) Test for catechin: Catechins when treated with acids forms phloroglucinol which gives pink colour.
A matchstick is dipped in the test solution, dried and then moistened with concentrated hydrochloric acid. This treated matchstick was lit via a flame which resulting in changing of the wood colour to pink due to formation of phloroglucinol.

8. TEST FOR STEROIDS AND TRITERPENOIDS:
a) Libermannburchard test: Test extract is boiled with few drops of acetic anhydride and then cooled. To the solution concentrated sulphuric acid is added along the sides of the test tube resulting in formation of a brown ring at the junction of the two layers. If the upper layer turns green indicating the presence of steroids whereas formation of deep red colour occurs due to the presence of triterpenoids.
b) Salkowski test: Test extract is mixed with few drops of concentrated sulphuric acid. If the lower layer turns red in colour, indicates presence of steroids and formation of yellow colour results due to triterpenoids.
c) Sulphuric powder test: Small amount of sulphuric powder is added to the test solution.
The test is considered positive if the powder sinks to the bottom.

9. TEST FOR PROTEINS:
a) Warming test: Test solution is heated using a water bath resulting in coagulation of proteins.
b) Test for trichloroacetic acid: Precipitate formation occurs when trichloroacetic acid is added to the test solution.
c) Biuret test: Violet colour formation indicates the presence of proteins when (2ml) Biuret reagent is added to (2ml) of test solution.
d) Hydrolysis test: The test solution is hydrolysed using hydrochloric acid or sulphuric acid. Resultant solution is subjected to ninhydrin test to ascertain the presence of amino acids.
e) Xanthoproteic test: 1ml of concentrated nitric acid is added to 5ml of test solution and boiled which results in the formation of yellow precipitate. After cooling the solution, 40% sodium hydroxide solution is added which changes the colour of the solution to orange.

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