3.4.4. ABTS Assay

Antioxidant activity was quantified using ABTS solution (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) following the method described by Re et al. [53] and Rufino et al. [50] ABTS with potassium persulfate is reduced in the presence of hydrogen-donating antioxidants. This assay measures their relative ability to scavenge ABTS, and consequently, the antioxidant activity of the sample. The radical ABTS solution was prepared with 88 µL of potassium persulfate (37.8 g/L) mixed with 5 mL of ABTS stock solution (3.8 g/L) and placed in the dark for 16 h. The obtained ABTS solution was diluted in ethanol until an absorbance value of approximately 0.70 was read at 734 nm. The extract was diluted 1:100 in acetone 70% (v/v), and 30 µL of the solution was mixed with 3 mL of ABTS solution and incubated for 6 min. Then, the absorbance was measured at 734 nm using ethanol as a blank for the control.

The antioxidant activity (DPPH, FRAP and ABTS) was determined using standard calibration curves prepared with Trolox (6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) and the results were expressed as Trolox Equivalent Antioxidant Capacity [TEAC (µmol Trolox/g dry mass)]. For each determination method, the samples were analyzed in triplicate.