2.9. Determination of the Antioxidant Capacity

The hydrophilic antioxidant capacity (HAC) was determined by the DPPH free-radical assay (2,2-diphenyl-1-picrylhydrazyl), as described by Rey et al. [44]. The assay was replicated twice and a curve of ascorbic acid was used as a standard. Results were expressed as mg of ascorbic acid equivalents (AsAE) per 100 g of fresh weight. DPPH-scavenging capacity was expressed as inhibition percentages by Formula (1):

The HAC was also determined in the same supernatant used in DPPH assay by using FRAP (ferric reducing antioxidant power) assay, as described by Benzie and Strain [45], with some modifications. First, a reagent was prepared with 10 mM TPTZ (2,4,6-tripyridy-Striazine, Sigma), 20 mM FeCl₃.6H₂O and 300 mM acetate buffer, pH 3.6, at 1:1:10, v:v:v and incubated in a bath at 37 °C for 10 min. In a 96-well plate 10 μL of each sample were mixed with 290 μL of FRAP reagent and incubated at 37 °C in darkness for 30 min. Thereafter, the absorbance change of the mixture was determined in a UV/Vis microplate spectrophotometer (Multiskan FC, Thermo Scientific, Madrid, Spain) at 595 nm. The assay was replicated twice and a curve of ascorbic acid was used as a standard. Results were expressed as mg of ascorbic acid equivalents (AsAE) per 100 g of fresh weight.

The ABTS (2,2′-azino-di-(3-ethylbenzthiazoline sulfonate) assay was used to determine antioxidant capacity in comparison to a standard antioxidant, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) (Sigma, Madrid, Spain). Total antioxidant capacity was quantified as described by Legua et al. [46], with slight modifications, which enabled us to determine antioxidant capacity due to both hydrophilic and lipophilic compounds in the same extraction. Absorbance of the extracts was determined with a UV/Vis microplate spectrophotometer (SPECTROstar^® Omega, BMG Labtech, Offenburg, Germany) at 730 nm. A calibration curve was drawn with Trolox (Sigma, Madrid, Spain) and results are expressed as Trolox equivalent antioxidant capacity (TEAC) per 100 g of fresh weight.