4.4.5. Determination of Superoxide Radical (O2•−) Scavenging Activity

Superoxide (O₂^•−) radical scavenging activity was determined using a slightly modified protocol from Gülçin et al. [91]. According to this protocol, superoxide anion (O₂^•−) was generated in a phenazine methosulfate and reduced nicotinamide adenine dinucleotide (PMS-NADH) system through NADH oxidation, and it reduced nitroblue tetrazolium (NBT²⁺; yellow) to formazan (blue) [92]. Briefly, in 625 μL of Tris-HCl (16 mM, pH 8.0), 125 μL of NBT (300 μM), 125 μL of NADH (468 μM) and 50 μL of each wine extract (ranging from 3.1 to 400 μg/mL) were added at different concentrations. The reaction was started by adding 125 μL of PMS (60 μM). The samples were incubated for 5 min and absorbance was measured at 560 nm. In each experiment, the samples without PMS were used as blanks and the samples without wine extracts were used as controls. The superoxide (O₂^•−) radical scavenging activity was calculated according to the equation:

where Abs_control and Abs_sample were the values of absorbance from control and tested wine extract samples, respectively. Moreover, the percentage inhibition and the IC₅₀ value, defined as the concentration of the sample that led to a 50% decrease in the O₂^•− radical, were used to compare radical scavenging efficiency among extracts. All analyses on tested samples were carried out in triplicate and at least two experiments were conducted.