3.

7 Phytochemical analysis

3.7.1 Alkaloid analysis

3.7.1.1 Preliminary assay

The C. vulgaris extracts will be added with 5 mL of 2M hydrochloric acid

(HCl). It will be heated with constant stirring for about 5 minutes and then are to

be cooled. About 0.5 g sodium chloride will be added to the sample. It will be

then stirred and filtered. The residue will be washed with enough 2M HCl in order

to bring the filtrate to a volume of about 5 mL. One ml of the filtrate will be

separated and tested with 2 to 3 drops of Dragendorff’s reagent. Another 1 mL of

the filtrate will be separated and tested with 2 to 3 drops of Mayer’s reagent.

Slight turbidity, definite turbidity and heavy turbidity indicates a positive result.

3.7.1.2 Confirmatory test

The remaining 3 mL of the filtrate in the preliminary assay will be added

with a dropwise of 28% ammonia until the solution will be alkaline to litmus. The

alkaline solution will be extracted three times with small portions of less than 10

mL of chloroform. The lower chloroform extracts will be combined. The

chloroform extract will be evaporated to dryness under the hood and over a steam

bath. The residue will be mixed with 5 mL of 2M HCl, and will be stirred over a

steam bath for about 2 minutes and then cooled. The solution will be filtered and

the filtrate will be divided into two portions. One portion will be tested with

Mayer’s reagent and the other with Dragendorff’s reagent.

3.7.2 Terpenoids analysis

3.7.2.1 Preparation of the vanillin-sulfuric acid

5- mL of concentrated sulfuric acid will be added to 10 mL of 2.5%

vanillin ethanol.

3.7.2.2 TLC analysis

The prepared TLC plate will be spotted with the C. vulgaris extracts. The

plate will be developed in chloroform. The plate will be sprayed with vanillin-

sulfuric acid as the visualizing agent. The plate will be warmed over a hot plate

and will be observed for color changes until the maximum color is obtained. Red-

violet or purple spots, at about hRF of 60 to 70, will indicate the presence of

terpenoids.

3.7.3 Flavonoids analysis

3.4.5.1 Test tube screening methods

An equivalent of 10 g C. vulgaris sample will be evaporated to incipient

dryness over a steam bath. It will be cooled to room temperature and defatted by

taking up the residue with 9 mL hexane and water or with petroleum ether. The

hexane or petroleum ether extract will be discarded. The defatted aqueous layer

will be diluted with 10 mL of 80% ethyl alcohol. It will be filtered and the filtrate

will be divided into four test tubes. One portion will be separated as a control.
3.7.4 Saponins Analysis

3.7.4.1. The froth test

Two grams of the C. vulgaris sample will be placed in a test tube. 10 mL

of distilled water will be added to the test tube with stopper and will be shaken

vigorously for 30 seconds. It will be allowed to stand for 10 minutes and observed

for a ‘honeycomb’ froth.

If the ‘honeycomb’ froth greater than 2 cm height from the surface of the

liquid persists after 10 minutes the sample is considered positive for saponins.

3.7.5 Tannins Analysis

3.7.5.1 The test tube method

An equivalent of 10 g C. vulgaris will be taken from the stock extract and

it will be evaporated to incipient dryness over a steam bath. The residue will be

extracted with 20 mL of hot distilled water. 5 drops of 10% sodium chloride will

be added to the solution. The solution will be filtered and the filtrate will be

divided into three test tubes. One portion will be the control. The aqueous

solution of tannic acid will be the reference standard.

3.7.5.2 Gelatin test

An equal amount of 1% gelatin solution will be mixed with 10% sodium

chloride solution. One portion of the filtrate of the extract will be treated with

three drops of gelatin-salt reagent. The same will be done to the tannic acid

solution, the reference standard. It will be compared with the control and the

reference standard. The formation of a jelly-precipitate indicates the presence of

tannins.
3.7.6 Cyanogenic Glycosides Analysis

3.7.6.1 The Guignard Test

5 g sodium carbonate and 0.5 g picric acid will be dissolved in enough

water to make 100 mL sodium picrate solution.

Strips of filter paper (1 cm x 5 cm) will be dipped into a freshly prepared

solution of sodium picrate. The strips of filter paper will be dripped dry and then

finally dried between sheets of newsprints. Picrate paper will be prepared just

before use.

2 to 5 g of C. vulgaris will be placed in a test tube. It will be moisten with

enough water, then added with a few drops of chloroform to enhance enzyme

activity. One mL of 1% emulsion solution (B-glucoside) will also be added to

insure hydrolysis of the glycoside. The test tube will be stoppered with a cork

from which will be suspended a strip of yellow picrate paper. The paper strip

should not touch the inner side of the test tube. The test tube will be warmed at

35-40°C in a water bath or keep at room temperature for 2 to 3 hours. If no

change in color is observed after 3 hours, the test is considered negative.

Appearance of various shades of red within 15 min., when the tube is warmed,

gives the relative measure of the concentration of cyanogenic glycosides.