3.5.3. Oxygen Radical Absorbance Capacity (ORAC) Assay

The ORAC assay was performed according to the method described by Prior et al. [67] with slight modifications. In a black 96-well plate, 12 μL of the Trolox® working solutions (83 μM, 167 μM, 250 μM, 333 μM, and 417 μM prepared with phosphate buffer at pH 7.4) and EO sample (2.0 mg/mL) were added in triplicate (n = 3). Subsequently, 138 μL of fluorescein solution was added followed by 50 μL of freshly prepared by dissolving 2,2'-Azobis (2-methylpropionamidine) dihydrochloride (AAPH) (Sigma-Aldrich®, Cat no. 440914) in phosphate buffer (150 mg of AAPH in 6 mL buffer). (-)-Epigallocatechin gallate (EGCG) (Sigma-Aldrich®, Cat no. E4143) was used as a positive control. For the blank, the phosphate buffer was added in place of the sample. The total volume of the assay was 200 μL and the temperature was set at 37 ◦C. Readings of the EO/EGCG samples (2.0 mg/mL) and Trolox® working standard solutions were taken using the excitation wavelength set at 485 nm and the emission wavelength at 530 nm for 2 h at 1 min reading interval. After analysis, the data points of the blank, EO sample, EGCG sample, and Trolox® working standards were summed up over time to obtain the area under the fluorescence decay curve (AUC). The ORAC values were calculated using the linear regression (R2 = 0.9861) equation (Y = aX + c) between Trolox® concentration (Y) (μM) and the net area (blank-corrected) under the fluorescence decay curve (X). The results were expressed as the mean (±SD) of triplicate measurements in <sup>μ</sup>mol of Trolox® equivalents per liter of the sample tested (μmol TE/L).
