2.4.4. Oxygen Radical Absorbance Capacity (ORAC Assay)

The ORAC assay measures the antioxidant scavenging function against peroxyl radical-induced by AAPH at 37 °C [7,27]. In the ORAC assay, fluorescein sodium salt is used as a fluorescent probe with an excitation wavelength of 485 nm and an emission wavelength of 528 nm. The loss of fluorescein fluorescence indicates the extent of its reaction with the peroxyl radicals. All solutions were prepared in a phosphate buffer (75 mM, pH 7.4). The sample (25 μL) and fluorescein solution (8.68 × 10⁻⁵ mM, 150 μL) were mixed and incubated at 37 °C for 30 min. After the incubation, 25 μL AAPH (153 mM) was added quickly to start the reaction. Microliter plate fluorescence was recorded every minute for 120 min. Blank was phosphate buffer (75 mM, pH 7.4), and various concentrations of trolox (6.25–200 μM) were used as standards. The final ORAC values were calculated by standards or samples and the net area under the curve (AUC), subtracting the AUC of the blank. The results are expressed as mM trolox equivalents (TE) per gram of sample (mM TE/g).