METHODOLOGY

Research Design

The research design used was Completely Randomized Design (CRD).

The experimental group was composed of seven treatments and one replicate was

done for each treatment. The control group was composed of positive control

(ascorbic acid) and a negative control (ethanol).

GENERAL PROCEDURE

Preparation of leaf extract

Two (200.00) grams of Selliguea taeniata leaf were gathered from

Longlong, La Trinidad, Benguet, while Elaeagnus philippinensis were gathered

from Tuap, Puguis, La Trinidad, Benguet. These were removed from the stems

and washed carefully. Then the leaf were cut into tiny pieces then underwent air

drying for an hour. These were then immersed separately in 95% ethanol for 48

hours as done by Augustine al (2005). After 48 hours, these were filtered using a

filter paper.

The filtered liquids were transferred into an evaporating dish for the

process of evaporation. The evaporation of the solvent was done through a water

bath until the liquid became syrupy to obtain a concentrated extract. The volumes

of the extracts were obtained using a graduated cylinder. Aliquots were taken to

make different dilutions.

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Preparation of DPPH Solution

One and two tenths (1.2) milligrams of DPPH were dissolved in one

milliliter of 70% ethanol and subsequently diluted again with ten milliliters of 95

% ethanol.

DPPH (2-2Diphenyl-1-Picryhydrazyl) Assay

The antioxidant activity of the various dilutions was mixed with 2 mL of

30 µm DPPH solution. It was then incubated at 37 degrees Celsius for 30 minutes.

After 30 minutes, the treatment was observed at 517 nm using a

spectrophotometer.

Interpreting results in DPPH assay

Absorbance reading of each treatment was interpreted using a

spectrophotometer at 517 nm. Inhibition of the free radical by the sample in

percent was calculated in the following formula:

𝐴𝑏𝑠𝑏𝑙𝑎𝑛𝑘 −𝐴𝑏𝑠𝑠𝑎𝑚𝑝𝑙𝑒
Free radical inhibition = × 100%
𝐴𝑏𝑠𝑏𝑙𝑎𝑛𝑘

Where Absblank is the absorbance of the blank sample and Abssample is the

absorbance of the test samples. Inhibition of the free radical means how much free

radical was scavenged.

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Statistical Analysis of data

The Single analysis of variance ANOVA was used to determine if there is

a significant difference among the different treatments. All hypotheses were

tested at 0.05 level of significance. When the computed value of F was greater

than the tabulated value, Duncan’s test was used to determine which treatments

are significantly different between the different concentrations and the positive

and negative control.

Disposal of Materials

Aseptic technique was used in the disposal of test organisms. The used

materials were autoclaved for 30 minutes with a temperature or 212 degrees

Celsius with a pressure of 15 psi. The autoclaved materials were cooled before

disposing them properly.

Figure 2 shows the flowchart of how the methods and procedures of this study

was organized and conducted.

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 Gathering of Materials

Preparation of Materials

Preparation of Elaeagnus philippinensis and Selliguea taeniata leaf extracts

Preparation of DPPH solution

Mixing of Elaeagnus philippinensis and Selliguea taeniata leaf extracts and the
controls to the DPPH solution

Incubation of the treatments mixed with DPPH solution at 37° C for 30

minutes

Absorbance reading through spectrophotometer at 517 nm

Interpreting results in DPPH assay

Statistical Analysis

Conclusion

Figure 2. Flowchart of Methods and Procedures

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