Preparation of the Plant Ethanolic Extracts

The plant materials will be cut into small pieces. Ethanol extraction will be performed by

soaking 250g of the cut materials in 95% ethyl alcohol for 24-48 hours. The mixture will

be filtered and the filtrate will be stored in an ambered- colored glass container. The

ethanol in the filtrate will be allowed to evaporate in a water bath.

Hydrogen Peroxide Scavenging Activity (Ruch,Cheng & Klauinig, 1989)

A solution of H2O2 (40ml) will be prepared in phosphate beffer (0.1 M,pH 7). Leaf

extracts at 10 mg/Ul ( 1 g of the crude extract per 1 ml of distilled water) will be added to

0.6ml of H2O2 solution to result to a total of volume of 3 Ml. the absorbance of the reaction

mixture will be recorded at 230 nm after 10 minutes in a spectrophotometer. A blank

solution containing phosphate buffer, without of H2O2 will be prepared. This will serve as

a control.

H2O2 scavenging activity(%) =A0 − A1 /A0 × 100%

Where: A0 was the absorbance of the control

A1 was the absorbance in the presence of the sample

The percentage scavenging of the plant extracts will be compared against tannic

acid which will serve as the positive control. The extent of H2O2 scavenging of tannic acid

will be calculated using the same equation. Three replicates will be made.
Phytochemical Screening (Raaman, 2006)

a. Test for Alkaloids

Ten milliliters of the filtrate will be evaporated to remove the ethanol and

the residue will be heated with 1 ml 2% hydrochloric acid in a water bath. After

cooling, the mixture will be filtered and treated with 5 drops of Mayer’s reagent. A

white creamy precipitate will indicate a positive result.

b. Test for Flavonoids

Surface of the magnesium strip will be cleaned using sand paper. It will be

cut into three equal parts for the replicates. Tem milliliters of the filtrate will

evaporated to remove the ethanol and will be mixed with 1 ml 10% hydrochloric

acid and magnesium metal. A reddish color will indicate the presence of flavonoids.

c. Test for Phytosterols

The extract will be subjected to Libermann-Burchard’s test. Ten milliliters of

the filtrate will be evaporated to remove the ethanol and will be dissolved in 2 mL

acetic anhydride. The mixture will be transferred to attest tube. Two drops of

concentrated sulfuric acid will be added slowly along the sides of the test tube. The

formation of blue green color will indicate the presence of phytosterols.

d. Test for Saponins

Ten milliliters of the filtrate will be evaporated to remove ethanol and will

be mixed with 5 mL distilled water in a test tube. It will be agitated for 10 minutes.
Formation of foam persistent for 1 minute or more will indicate the presence of

sapononins.

e. Test for Carotenoids

Ten milliliters of the filtrate will be evaporated to remove ethanol and will be

mixed with 5mL chloroform. The mixture will be transferred to a test tube. It will be

shaken vigorously. The resulting mixture will be filtered and will be added with 1

mL 85% sulfuric acid. A blue color at the interface will indicate the presence of

carotenoids.

f. Test for Tannins

Ten milliliters of the filtrate will be evaporated to remove ethanol. Five

milliliters of 45% ethanol will be added. The mixture will be cooled and filtered.

One milliliter of the filtrate will be diluted with 5mL distilled water and be added

with 2 drops of ferric chloride. A transient greenish to black color indicate the

presence of tannins.

g. Test for Terpenoids

Ten milliliters of the filtrate will be evaporated to remove ethanol. The

residue will be subjected to a test tube. Two drops of H 2SO4 will be added.

Formation of dark green color will indicate the presence of terpenoids.