2.11. Determination of Antioxidant Activity by the DPPH Method

DPPH is a stable free radical that has a deep violet colour in methanol or ethanol and has maximum absorbance at 517-520 nm. When an antioxidant neutralises DPPH, the solution becomes colourless or pale yellow and absorbance is reduced. As antioxidant activity increases, the absorbance of the DPPH solution decreases. The antioxidant activity of samples (Pecel vegetables, peanuts, etc.) was evaluated according to the method described by Rezaie et al. [18] and optimised by Murwani (unpublished) using 60 μM DPPH in ethanol (control radicals). We obtained the maximum absorbance for the DPPH solution at 517 nm. Antioxidant activity was determined by adding 0.1 g of each sample to 5 mL of 60 μM DPPH. The reaction mixture was homogenised using a vortex and incubated for 1 h at room temperature in the dark. The absorbance of the mixture was recorded against a blank (ethanol) at 517 nm using a UV-Vis spectrophotometer (Spectroquant®Pharo300). This absorbance is designated as sample absorbance (A_sample), and each sample was analysed in triplicate. Antioxidant activity was expressed as percent inhibition of DPPH radicals using the following equation:

where A_control is the absorbance of the 60 μM DPPH solution and A_sample is the absorbance of the sample.