2.6. In Vitro Antioxidant Activity Assays

2,2′-diphenyl-1-picrylhydrazide (DPPH) assays were carried out on phenolic acid sample extracts (see above) using a Jasco V-630 double beam spectrophotometer, as described elsewhere [33]. For the setup of calibration curves, 950 µL of 11 µM DPPH in methanol and 50 µL of methanolic Trolox (Tx) solution at different concentrations (0.05–2.00 mM), or 50 µL of methanol (blank solution), were thoroughly mixed in a 2-mL polypropylene vial. For sample analysis, the Trolox solution was replaced with 50 µL of suitably diluted sample. The vial was incubated in the dark at room temperature for 24 h, then the absorbance of the solution was read at 515 nm. Calibration curves were set up plotting the discoloration ratio (i.e., [ABS_{without TX}/ABS_{with TX}] − 1) as a function of Tx concentration. Trolox equivalents (TE) of the samples were calculated interpolating on the calibration curve.

Oxygen radical absorbance capacity (ORAC) assays were carried out on the same extracts (see above) using a Perkin Elmer (Turku, Finland) Viktor X3 multilabel plate reader, essentially as described by Ou et al. [34]. TE were calculated from the relative area under the curve of the emission intensity vs. time plot.