2.5. DPPH radical scavenging activity assay

The percentage of antioxidant scavenging activity for each MAA was determined as follows, each MAA (62.5 μM, 117.7 μM and 210.5 μM concentrations) and an ascorbic acid positive control (3.9 μM, 7.8 μM, 15.6 μM, 31.3 μM, 62.5 μM and 312.5 μM concentrations) were prepared in DMSO. For each sample, the reaction mixture consisted of 0.1 mL of the test sample and 1.5 mL of 70 μM DPPH in methanol. The colour change from violet to yellow, when DPPH is reduced upon reaction with an antioxidant, was recorded at 515 nm using a UV/VIS spectrophotometer (model 7315, Jenway Ltd., Stone, Staffordshire, UK) after 30 min at room temperature, with reaction mixtures shielded from light. The mixture of DMSO (0.1 mL) and DPPH (1.5 mL) served as the reaction blank. The percentage of DPPH radical scavenging activity was calculated as: % scavenging activity = 100 x (A_blank – A_sample)/A_blank. The DPPH scavenging activity was proportional to the MAA concentration (for levels examined), and the 515 nm absorbance of the fully reduced DPPH was set to zero. Experiments were performed in technical triplicates with three replicates, and % scavenging activities were plotted as the mean of the 9 triplicate/replicate values against test sample concentrations (μM). IC₅₀ values were estimated from linear regression of the data.