Hydroxyl Radical Scavenging Activity

The hydroxyl radical-scavenging activity was conducted using the 2-deoxyribose method (Halliwell et al., 1987). Briefly, the assay mixture contained 2.8 mM 2-deoxyribose, 20 μM ferrous ion solution, 100 μM EDTA, and different sample concentrations (10–100 μM) in a total volume of 1 ml of 10 mM potassium phosphate buffer (pH 7.4). All the components were dissolved in 10 mM phosphate buffer (pH 7.4). The ferrous iron solution and EDTA were premixed before they were added to the assay mixture. The reaction was started by the addition of a mixture of 1.42 μM H₂O₂ and 100 μM ascorbate. The mixture was incubated at 37°C for 30 min. At the end of the incubation time, 1 ml of 1% (w/v) TBA in 50 mM sodium hydroxide and 1 mL of 2.8% (w/v) TCA were added and the mixture was heated for 30 min in a boiling water bath, cooled, and the absorbance at 532 nm was measured, which corresponds to deoxyribose damage. BHT and gallic acid were used as a positive control. All experiments were conducted in triplicate.

The inhibition percentage (I%) of the radical-scavenging capacity was calculated using the following equation:

Where Ahydroxyl is the absorbance of the hydroxyl solution, Ablank is the absorbance of methanol instead of the hydroxyl solution, As-hydroxyl is the absorbance of the hydroxyl solution in the presence of sample, and As-blank is the absorbance of methanol in the presence of the sample. IC₅₀ values, which represent the concentration of the sample that caused 50% hydroxyl radical-scavenging activity, were calculated from the plot of inhibition percentage against sample concentration.