This document describes the DPPH radical scavenging assay method for determining the antioxidant potential of plant extracts. The DPPH assay involves adding varying concentrations of a test sample to the stable DPPH radical. Antioxidants in the sample will scavenge DPPH, reducing the purple color. The amount of color reduction is measured spectrophotometrically and used to calculate the sample's antioxidant capacity. A double dilution method is used to serially dilute the sample in microplate wells from 500 μg/ml to 1.95 μg/ml. The IC50 value, the concentration that causes 50% inhibition of DPPH, indicates the sample's antioxidant strength, with a lower IC50 reflecting higher antioxidant capacity.

1. ANTIOXIDANT ACTIVITY:
DPPH Radical Scavenging Assay
(Microtiter Plate Method)
By: RIPPIN

2. Free Radicals
• Group of atoms with one or more unpaired
electrons in outer orbital
• Highly unstable and very reactive species
• Types : ROS, RNS, metabolic intermediates
(metabolic activation of drugs, toxins,
pollutants etc).
• Examples : superoxide anion radical, hydrogen
peroxide, hydroxyl radical, nitric oxide radical
etc

3. Harmful effects of Free Radicals
React with biomolecules
Enzymes Nucleic acids
Carbohydrates
Cellular Disturbances
Tissue Injury
DISEASES

4. ANTIOXIDANTS
• Interact with and stabilize FREE RADICALS
• Prevent the cells from damage
• Prevent the development of various diseases

5. Determination of Anti- oxidative potential of
Antioxidants in samples
(Plant Extracts)
DPPH (2,2-Diphenyl-1-picrylhydrazyl) Assay
Diphenyl picryl hydrazine

6. Double Dilution Method (de torre et. al. 2019)
Reagents: 0.1 mM DPPH,
Methanol,
10mg/ml Ascorbic acid(positive control),
100µl DPPH+100µl methanol(control),
10mg/ml sample antioxidant
• 100 µl methanol in
each well
• 10 µl of extract in
1st well (500µg/ml)
+ 90 µl Methanol
• Transfer 100 µl of
mix from 1st to next
well and so on
• Discard 100 µl from
the last well
• 100 µl DPPH in each
well
• The final conc in
each well will
become half
• Repeat for positive
ctrl
• Add control in a
separate well
• Cover the plate with
foil and keep in
dark fro 30 mints
• Read at 517 nm via
microplate reader
Ctrl
DPPH+Methanol

7. Results
250 µg/ml
15.64µg/ml
7.8 µg/ml
3.9µg/ml
1.95µg/ml
125 µg/ml
62.5 µg/ml
31.25µg/ml
Scavenging effect (%) = {(Absorbance of control− Absorbance of
sample)/Absorbance of control} × 100

8. IC50 Value
• IC50 (half maximal inhibitory concentration)
the concentration of the antioxidant which
reduces DPPH to half i.e. 50 %.
• Lower the IC50 value, higher is the antioxidant
capacity of the antioxidant.

9. Thank you