3.3.1. DPPH Assay

DPPH is a molecule containing a stable free radical. The reduction of DPPH (purple) in ethanol solution takes place in the presence of a hydrogen-donating antioxidant due to the formation of the non-radical form of DPPH-H (yellow). This transformation results in color change from purple to yellow, which can be measured spectrophotometrically. Thus, the decrease in absorbance is proportional to the decrease in concentration of DPPH (free radical) and discoloration of reaction mixture indicates the scavenging of free radicals by a tested antioxidant.

In order to determine antioxidant capacity, 20 μL of the compound solutions (dissolved in 96% ethanol) was mixed with 180 μL of 0.3 mM ethanolic DPPH solution. The change in the absorbance was detected at 517 nm after 30 min of incubation. Results were expressed as percentage decrease in absorbance of the tested sample, compared to the sample containing the solvent + reaction mixture (blank with maximum concentration of the DPPH radical). L-ascorbic acid was used as a reference compound. Calculations were made using MS Excel, from the proportion where the change of absorbance in the vitamin C (vit. C) group was treated as 100%, according to Equation (1):

Calculation of % Vit. C activity: ΔAb-the change in the absorbance of the test sample and ΔAVit. C is change in the absorbance of reference sample, or with vit. C.