4.4.6. Determination of Hydroxyl Radical (OH•) Scavenging Activity

Hydroxyl (OH^•) radical scavenging activity was determined using a method from Chung et al. [93], with slight modifications, as previously described [49,94]. Specifically, 50 μL wine extracts at different concentrations (ranged from 25 to 800 μg/mL) were added to 225 μL of sodium phosphate buffer (0.2 M, pH 7.4), 75 μL of 2-deoxyribose (5 mM), 75 μL of FeSO₄-EDTA (10 mM), 250 μL of H2O, and 75 μL of H₂O₂ (10 mM), and the samples were incubated at 37 °C for 1 h. After incubation, 375 μL of TCA (2.8%) and και 375 μL of 2-thiobarbituric acid (1% dissolved in 50 mM NaOH) were added and samples were incubated at 95 °C for 10 min. Then, the samples were placed on ice for 5 min and centrifuged at 3000 rpm for 10 min at 25 °C. Absorbance was measured at 520 nm. In each experiment, one sample without H₂O₂ was used as blank and samples without wine extracts were used as control. All analyses were carried out in triplicate and at least two experiments were conducted. The OH^• 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 samples, respectively. The IC₅₀ value, defined as the concentration of the sample that led to a 50% decrease in the OH^• radical, was used to compare the radical scavenging efficiency of extracts. All analyses on tested samples were carried out in triplicate and at least two experiments were conducted.