Methodology

Phytochemical screening

Preparation of Plant extract

Dry plant materials thoroughly at room temperature. This will take several days to

completely dry plant materials. If not use immediately, the dried materials can then be

stored for reasonable length of time. After air drying, reduce the size of the plant

materials by cutting it with scissor and/or ground it using mill, and powderized using

food processor or blender. Placed the ground materials in a clean and dry container

for the next step which is the simple extraction with alcohol or by serial extraction

with solvents of increasing polarity. Hexane, dichiromethane, acetone and alcohol:

(methanol or ethanol).

Simple extraction with alcohol

Weigh about 200 grams of the ground materials into the 1000mL Erlenmeyer flask.

Add enough ethanol until the alcohol level is about 1inch above the surface of the

plant materials. Soaking will be done for 48 hours to ensure that 100% of extractable

components will be extracted. After the 48 hours of extraction, remove the alcoholic

extract from the plant materials by filtration. Suction filtration is more preferred to

evaporation at a temperature below 50°C. When about 90% of the solvent will be

removed, stopped the rotary evaporation and recover the crude extract for

phytochemical screening and alcohol solvent for further purification.

Saponins, using Froth test method

Add equal volume of water to about two mL of the extract in the test tube and shake

for 30 seconds. A positive result for saponins will be indicated by the formation of

froth, which is about 2cm in height that would last in 30 minutes.

Cyanogenic glycosides

Place into 20 mL test tube about one ml of the extract and add 4-5 drops of

chloroforms. Suspend a picrate paper (a filter paper soaked with picrate solution) just

above the solution (ensure that the paper will not touch the wall of the test tube) and

placed the test tube hot water bath. Cover the tube with a dropper placed in an

inverted position. A positive result will be indicated by the immediate formation of

red color at the surface of the picrate paper.

Alkaloids

Transfer sufficient amount of the extract into an evaporating dishand evaporate it to

almost dryness under boiling bath. When almost dry, remove the dish from the bath

and cool. With stirring, add into the dish about 5-10mL of 2M HCI and 0.5 grams of

sodium chloride crystal and then place it under boiling water bath for about five

minutes. Cool the resulting solution and filter. Wash the residue in the filter paper

with few mL of 2M HCI. Collect the combined filtrate and washing and divide into

three equal parts. Add into the first part 3-5 drops of Wagner’s reagent, the second

part with Mayer’s reagent and the third part serves as the control. A positive result is

indicated by the formation of brown precipitate, with Wagner’s reagent and white

precipitate with Mayer’s reagent. Express the result as follows: (-) for absence of

precipitate ; (+) if the solution becomes turbid only, (++) if the precipitate form is

only moderate amount and (+++) if the precipitate form is heavy.

Flavonoids

Transfer enough amount of crude extract in an evaporating dish. Evaporate to almost

dryness under boiling water bath. Cool then be defat with hexane until the hexane

washing becomes almost clear. Dissolve the defatted extract with 10mL of 80%

alcohol, filter and divide the filtrate into two equal parts. To the first part, add 0.5mL

of 12M HCI and the second part serves as a control. Place the two test tubes in a hot

water bath and observe the change color. Positive result will be indicated by the

development of red color of solution. Sometimes development of red color is very

slow, so this reaction should be observed in two (2) hours. The result will be recorded

as follows: (-) no change of colors occurs; (+) if the red coloration is very light, (++)

if the red coloration is moderate and (+++) if dark red color is produced.

Tannins

Transfer enough amount of crude extract in an evaporating dish. Evaporate to almost

dryness under boiling water bath. Cool then add 20mL of boiling water followed the

addition with 2-3 drops of 10% NaCI solution. Filter the resulting solution and wash

the residue with water if necessary. Recover the combined filtrate and washing and

divide into three equal. To the first part add 3-5 drops of 1% Ferric chloride, the

second part will be added with 3-5 drops of Gelatin-salt reagent and the third part

serves as a control. A positive result will be indicated by the formation of black or

blue-black color of precipitate with ferric chloride test and white precipitate with

Gelatin-sodium chloride test. Recorded the result as follows (-) for absence if the

solution becomes turbid only, (++) if the precipitate form is only on moderate amount

and (+++) if the precipitate form is heavy.

Steroids

Transfer enough amount of crude extract in an evaporating dish and evaporate to

almost dryness under boiling water bath. Cool the dried crude extracted defat with

hexane. Add into the extract with 3-5mL of Ferric chloride reagent and filter. Divide

the filtrate into two (2) equal parts. Slowly add into the first part with one (1) mL

concentrated sulfuric acid thru the wall of the test tube. The formation of brown (or

sometimes blue or green) ring at the boundary region of the aqueous extract and

sulfuric acid, indicates the presence of 2-deoxy sugars.

Anthraquinones

Transfer enough amount of crude extract in an evaporating dish and evaporate to

almost dryness under boiling water bath. Cool and defat with hexane. Add into the

dish 10mL of distilled water, stir and the filter the resulting solution. Extract the

filtrate twice with 5mL of benzene. Stand for few minutes to allow complete

separation of the aqueous of benzene layer. Separate the benzene layer using transfer

pipet and place in a test tube containing one (1) mL of ammonia reagent. Shake for

few seconds and observe the color in aqueous color. The development of reddish pink

color in the aqueous layer of solution indicates the presence of anthraquinones.

Answer sheet

Record your result as follows: (-) for negative result; (+) for trace amount which is

indicated only by turbid formation and/or very light color of solution; (++) for

moderate amount and (+++) for results indicated by formation of heavy precipitate

and/or dark color of solution. Fill this in the table below