Chapter III

Methodology

A. Collection and Preparation of Plant Samples

Terminalia catappa Linn samples were collected at Buanoy, Balamban, Cebu and were

submitted to Ms. Rhea Charisse Capus. The samples were washed with tap water to remove dirt

and dust, were dried under the heat of the sun, and were grind for suitable extraction.

B. Extraction of Crude Ethanolic Extract

The collected samples were cut or grind into pieces with a mass of 178.17 grams. It was

macerated or soaked in a 600ml absolute ethanol for 168 hours. The extract was filtered and

concentrated to syrupy consistency using the distillation process. The extract was then added

with distilled water.

C. Phytochemical screening

The diluted ethanolic extract was the subject in the phytochemical screening for the

secondary metabolites.
Table 1.1. Phytochemical Screening: Test Performed, Reagent Used, and Expected Results

Secondary
Test Performed Reagent Expected Results
Metabolites
1% dilute HCl and Brown/Reddish
Alkaloids Wagner’s Test
IKI Precipitate
Alkaline Reagent Few drops of NaOH From intense yellow
Flavonoids
Test and dilute acid color to colorless
2ml H2O and diluted Dark Green or Blue
Tannins FeCl3 Test
FeCl3 Solution Green Color
Concentrated H2SO4
Quinones H2SO4 Test Red Color
Solution
Carbohydrates Iodine Test Iodine Solution Blue Black Color
Phenols FeCl3 Test FeCl3 Solution Bluish Black Color

D. Determination of bioactive compounds

The diluted ethanolic extract was used for the determination of bioactive compounds present

in Terminalia catappa. There were at least 18 test tubes that were used in order to put the diluted

ethanolic extract with a volume of about 2 ml. Each determined bioactive compounds was

recorded using table showing its result gathered by the researchers.

Test for Alkaloids

Basing from the table, the test that was performed was the Wagner's test. The diluted

ethanolic extract was treated with few drops of Wagner's reagent (IKI). The formation of

brown/reddish precipitate indicated the presence of Alkaloids.

Test for Flavonoids

 Few drops of NaOH and dilute acid to the diluted ethanolic extract took place in order to

perform the Alkaline Reagent test. The appearance of yellow color to colorless indicated the

presence of alkaloids.

Test for Tannins

To determine the presence of tannins, 1ml ferric chloride solution and 2ml distilled H 2O

were treated twice to the diluted extract to perform the Ferric chloride test. A blue green or dark

green color indicated the presence of tannins.

Test for Quinones

A Sulfuric Acid Test was performed in order to determine the presence of quinones. A

red color indicated its presence after 1 ml of concentrated H 2SO4 was poured into the test tube of

the extract.

Test for Carbohydrates

Few drops of Iodine an aqueous solution was treated to the diluted extract in order to

perform Iodine Test and blue black indicated its presence.

Test for Phenols

The test that was performed to determine Phenols in the diluted extract was the Ferric

Chloride (FeCl3) test. Few drops of FeCl3 solution was put or dropped to the diluted extract. A

bluish black color forming indicated the presence of phenols.

 SCHEMATIC DIAGRAM

COLLECTION AND PREPARATION OF PLANT

SAMPLES

EXTRACTION OF CRUDE ETHANOLIC EXTRACT

PHYTOCHEMICAL
SCREENING

DETERMINATION OF BIOACTIVE
COMPOUNDS