DPPH Assay Protocol

Determination of Lambda Max and Linear Dynamic Range.

1. Weigh 0.0375g of solid DPPH and dissolve in 250 mL methanol using a 250-mL
volumetric flask to create a 150 ppm DPPH stock solution.
2. Through serial dilution, create series of concentration from 10 ppm to 100 ppm with 10-
ppm increment.
3. Using 20.00ppm DPPH solution, determine the Lambda max of the DPPH by determining
the absorbance of solution from 400 nm – 620nm using a double beam
spectrophotometer. The wavelength with the highest absorbance will serve as the
lambda max (515 nm).
4. Read the absorbance of the freshly prepared concentrations (10 ppm, 20ppm, 30 ppm,
40 ppm, 50 ppm, 60 ppm, 70 ppm, 80 ppm, 90 ppm, 100 ppm) using the previously
determined lambda max (515 nm).
5. Plot absorbance vs. concentration and determine the linear dynamic range.
6. Pick the concentration (70.00ppm) of the DPPH that shows high sensitivity (absorbance
< 1.500) but not beyond the linear dynamic range.

Determination of % Radical Scavenging Activity.

1. Create 20-mL of 1000 ppm stock solution of extract.

2. In a clean 10-mL screwcap tubes, pipette 0.053 mL, 0.27 mL, 0.53mL, 1.33 mL, and 2.67
mL from the stock extract solution. Prepare an additional 10-mL screwcap tube without
the extract solution for this will serve as the negative control. Create Three trials for
each volume of pipetted extract and the negative control.
3. From 150 ppm DPPH stock solution, pipette 1.87 mL into the 10-mL screwcap tubes
containing the extract solution and the negative control and immediately start the timer
for 30-minutes.
4. Dilute solutions containing the extract with DPPH and the negative control to 6-mL total
volume using methanol as diluent to prepare 10.00 ppm, 50.00ppm, 100.00ppm,
50.00ppm, and 500.00ppm of extract solution with 70.00ppm concentration of DPPH.
5. Allow the solution to stand for 30-minutes in a dark room at room temperature.
6. Immediately after 30-minutes, Read the absorbance of the solution at 515 nm
wavelength. The negative control will serve as the Acontrol and the extract with DPPH will
serve as the Asample.
7. Calculate the Percent Radical Scavenging Activity (%RSA) for each extract concentration
using the formula:

𝑨𝒄𝒐𝒏𝒕𝒓𝒐𝒍 − 𝑨𝒔𝒂𝒎𝒑𝒍𝒆
% RSA = x 100
𝑨𝒄𝒐𝒏𝒕𝒓𝒐𝒍
8. Plot the calculated %RSA vs. concentration of extract and determine the concentration
of extract that exhibits 100% RSA.
9. Repeat Steps 1 – 8. This time using Standard Ascorbic Acid as sample.
Data Analysis.

1. From the obtained concentration of the three extracts (Mayana, Dalupang, and Damung
Balang) that exhibits 100% RSA, Use T-test Case 2 to assess if there exist a significant
difference in the concentrations of the three extracts at 100% RSA.
2. Use T-test Case 1 to assess if there exist a significant difference in the concentration of the
standard ascorbic acid and the type of extract (Mayana, Dalupang, and Damung Balang) at
100% RSA.