2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity assay

The free radical scavenging activity of crude plant extracts was measured by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay [16] with some modifications. The plant extracts were diluted with dimethyl sulfoxide (DMSO) (Merck, Germany) to obtain twofold serial dilutions (1.562–400 μg/ml). The reaction mixture, containing 75 μl of plant extract and 150 μl of DPPH radical solution (0.1 mM in absolute ethanol), was added into a 96-well plate and incubated in the dark at room temperature for 30 min. L-ascorbic acid (1.562–100 μg/ml) was used as a positive control. The absorbance was measured at 517 nm by using a microplate reader. The assays were performed in triplicate. The percentage of the radical scavenging activity (%RSA) was calculated by using the formula below:

where Abs control is the absorbance of DPPH radical + ethanol and Abs sample is the absorbance of DPPH radical + plant extract.