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3.6. ANTIOXIDANT ASSAY:
Antioxidant Assays :
DPPH assay (2, 2-diphenyl-1-picrylhydrazyl) The radical scavenging activity of different extracts was determined by using DPPH assay according to Chang et al. (2001). The decrease in the absorption of the DPPH solution after the addition of an antioxidant was measured at 517nm. Ascorbic acid (10mg/ml DMSO) was used as reference.
Principle :
1, 1 Diphenyl 2- Picryl Hydrazyl is a stable (in powder form) free radical with red color which turns yellow when scavenged. The DPPH assay uses this character to show free radical scavenging activity. The scavenging reaction between (DPPH) and an antioxidant (HA) can be written as,
(DPPH)+ (H-A) ? DPPH-H+(A)
Antioxidants react with DPPH and reduce it to DPPH-H and as consequence the absorbance decreases. The degree of discoloration indicates the scavenging potential of the antioxidant compounds or extracts in terms of hydrogen donating ability.
Reagent preparation :
0.1mM DPPH solution was prepared by dissolving 4mg of DPPH in 100ml of ethanol.

Working procedure :
Different volumes (2 – 20µl) of plant extracts were made up to 40µl with DMSO and 2.96ml DPPH (0.1mM) solution was added. The reaction mixture was incubated in dark condition at room temperature for 20 min. After 20 min, the absorbance of the mixture was read at 517 nm. 3ml of DPPH was taken as control. The % radical scavenging activity of the plant extracts was calculated using the following formula,
% RSA = abs control- abs sample / abs control ×100
Where, RSA is the Radical Scavenging Activity; Abs control is the absorbance of DPPH radical + ethanol; Abs sample is the absorbance of DPPH radical + plant extract.

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