K2o8s2

The radical inhibition activity (AIR) of 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS^•+) was analyzed according to the initial principles proposed by Miller et al. [42 (link)] with the reaction conditions modified by Re et al. [43 (link)]. The method is based on the ability of substances to eliminate the cationic ABTS^•+ radical, a blue-green chromophore with maximum absorption at 734 nm, resulting in the formation of the stable, colorless ABTS product.
Initially, ABTS (7 mM.L⁻¹; Sigma–Aldrich; A1888; São Paulo/SP, Brazil) and potassium persulfate (140 mM.L⁻¹; K₂O₈S₂; Sigma–Aldrich; 216224; São Paulo/SP, Brazil) were mixed and left in the dark for 16 h to form the ABTS^•+ radical (2.45 mM.L⁻¹). Then, the radical was diluted with phosphate-buffered saline until reaching an absorbance of 0.700 ± 0.020 in an 800XI spectrophotometer (Femto; São Paulo/SP, Brazil) at 734 nm. Then, 30 μL of sample or standard was added to the solution (in triplicate), and after 5 min, the final absorbance was read. In addition, Trolox^® (1 mM.L⁻¹; Sigma–Aldrich; 23881-3; São Paulo/SP, Brazil) was used as a standard antioxidant. We calculated the inhibition activity according to the following equation:
where A_control represents the absorbance of the ABTS^•+ radical (2.5 mM.L⁻¹), and A_sample represents the absorbance of the sample.

The ABTS assay was determined according to the methodology adapted from Miller et al. [42 (link)] and modified by Re et al. [43 (link)]. ABTS^•+ 2.45 mM L⁻¹ (Sigma-Aldrich; A1888; São Paulo, Brazil) was prepared using 7 mM.L⁻¹ ABTS^•+ and 140 mM L⁻¹ of potassium persulfate (K₂O₈S₂; Sigma Aldrich; 216224; São Paulo, Brazil) incubated at room temperature without light for 16 h. Then, the solution was diluted with phosphate-buffered saline until it reached an absorbance of 0.700 (± 0.02) at 734 nm.
To measure the antioxidant capacity, 2.97 mL of the ABTS^•+ solution was transferred to the cuvette, and the absorbance at 734 nm was determined using a spectrophotometer 800 XI (Femto; São Paulo, Brazil). Then, 0.03 mL of the sample was added to the cuvette containing the ABTS^•+ radical and, after 5 min, the second reading was performed. The synthetic antioxidant Trolox was used as a standard solution for the calibration curve (y = 0.4162x − 0.0023, where y represents the value of absorbance, and x the value of concentration, expressed as mM.L⁻¹; R² = 0.9789). The results were expressed as mM L⁻¹. The values found for the samples were compared to the Trolox standard (1 mM L⁻¹).