3.4.2. Oxygen Radical Absorbance Capacity Assay (ORAC)

The ORAC assay measures the scavenging of the peroxyl radical AAPH by antioxidant compounds. The fluorometric method described by de la Fuente et al. [18] was applied. Sodium fluorescein (0.015 mg/mL), AAPH radical solution (120 mg/mL), and Trolox standard solution (100 μM) were prepared with phosphate buffer (75 mM, pH 7). Adequate diluted extracts were required. The operating conditions for the final reaction consisted of 50 μL of diluted extract, Trolox standard or phosphate buffer (blank), 50 μL of fluorescein, and 25 μL of AAPH incubated at 37 °C in a Multilabel Plate Counter VICTOR3 1420 (PerkinElmer, Turku, Finland). Fluorescence filters for an excitation wavelength (485 nm) and an emission wavelength (535 nm) were selected. The fluorescence was recorded every 5 min over 60 min, where the fluorescence in the assay was less than 5% of the initial value. Differences of areas under the fluorescence decay curve (AUC) between the blank and the sample over time were compared and the results were expressed as μM Trolox Equivalents.