2.8. Total Antioxidant Capacity (TAC)

TAC was measured using DPPH radical scavenging activity; oxygen radical absorbance capacity (ORAC); ABTS radical cation scavenging activity, and ferric reducing antioxidant power (FRAP). Relative antioxidant capacity index (RACI) was also calculated. All the analyses were carried out in duplicate.

Antioxidant activity against the DPPH radical was estimated according to the procedure described by Brand-Williams et al. [39] with some modifications. An amount of 100 µL of extracts was mixed with 400 µL of MilliQ water and 500 µL of DPPH working solution (120 µM using methanol as solvent) in a 96-well microplate. Absorbance at 515 nm was recorded for 30 min in a microplate reader (Fluostar Omega, BMG Ortenberg, Germany). Trolox was used as the standard (7.5–210 µM). The results were expressed as µmol Trolox equivalents (TE) 100 g⁻¹ d.m.

The procedure was based on a previously reported method with slight modifications [40]. The standard curve of Trolox (7.5–180 µM) and all the fractions were diluted in phosphate buffer (75 mM, pH 7.4). A volume of 150 μL fluorescein was placed in 96-well black polystyrene plates, and 25 μL of Trolox standard, sample, or phosphate buffer as blank were added, all in duplicate. Samples, standards, and blanks were incubated with fluorescein at 37 °C for 3 min before AAPH solution was added to initiate the oxidation reaction. Fluorescence was monitored over 100 min using a microplate reader (Fluostar Omega, BMG Ortenberg, Germany) at excitation and emission wavelengths of 485 and 528 nm, respectively. Results were obtained by external calibration, plotting the areas under the fluorescein decay curves as function of Trolox concentration. Data were expressed as µmol TE 100 g⁻¹ d.m.

The antioxidant capacity against diammonium salt of ABTS^•+ radical was evaluated according to the method first described by Re et al. [31], and lately [41], modified by Martin-Diana et al. [38]. First, 100 µL of diluted fractions were mixed with 1000 µL of ABTS^•+ working solution in an Eppendorf tube. The decay in absorbance at 734 nm was recorded over 60 min with a microplate reader. Trolox was used as the standard (7.5–210 µM). The absorbance was measured at 734 nm with a microplate reader (Fluostar Omega, BMG Ortenberg, Germany). Results were expressed as µmol TE 100 g⁻¹ d.m.

FRAP assay was performed following the protocol reported by Pereira et al. [42]. Absorbance at 700 nm was recorded in a microplate reader (Fluostar Omega, BMG Ortenberg, Germany). FeSO₄·7H₂O was used as a standard (2.4–3.8 mM). The results were expressed as µmol Fe²⁺ equivalents 100 g⁻¹ d.m.

RACI was used as an integral concept, which allows the comparison of antioxidant capacity derived from different chemical methods [43]. RACI values were determined through the following equation: (x − µ)/σ, where x is the antioxidant value, µ is the average value of the results of the corresponding method (ABTS, ORAC, DPPH, and FRAP), and σ is the standard deviation.