2.4. Antioxidant Activity Determined by DPPH•, ABTS and CUPRAC Assays

Free-radical scavenging activity was evaluated employing the DPPH^• and ABTS assays [31]. 2,2-Diphenyl-1-picrylhydrazyl radical (DPPH^•) was dissolved in ethanol (150 μL, 140 μM) and added to 150 μL ethanol solution of the test sample (62.5–500 μM of SIM-53B) or ethanol (negative control) in each well of a flat-bottomed 96-well microliter plate (TPP, Tissue Culture Test Plates). The reaction between DPPH^• and SIM-53B was then monitored at λ = 517 nm by using a Synergy H4 Hybrid Multi-Mode Microplate Reader (Bio-Tek Instruments, Inc) at T = 20 °C in the dark for 180 min. Each set of experiments was performed in triplicate. For the reduction kinetics of the DPPH^•, absorbance was measured using an Agilent Cary 3500 UV-Vis spectrophotometer with the Compact Peltier UV-Vis Module, set at a wavelength of 517 nm. Spectrophotometric disposable (2 mL capacity and 1 cm path-length) cuvettes were used. The absorbance of a sample and a blank were measured simultaneously at a sampling rate of one point per second. Automatic acquisition of data was set for a reaction time of 90 min 5 s after mixing DPPH^• (70 μM) with SIM-53B solution in methanol. All samples were analyzed in duplicate at temperature (20.0 ± 0.1) °C.

For the ABTS assay, a slightly modified procedure described in [32] was used. To 10 mL of 7 mM stock solution of 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid ammonium salt) (ABTS) was added 178 µL 140 mM solution of potassium persulfate. Working solution was allowed to react for 16 h at room temperature in the dark. The solution was than diluted by mixing 1 mL ABTS^•+ with 64 mL of ethanol (96%) to obtain an absorbance of 1.1 units at 734 nm. The solution of SIM-53B and solutions of standards (Trolox and resveratrol) were freshly prepared in 96% ethanol at 1 mM concentration. To a test tube were added ethanol [(2.00−x) mL] and solution of SIM-53 (or standard) (x mL) and finally 2 mL of ABTS^•+ so as to make the final volume 4.0 mL. The tubes were closed by parafilm, and the mixtures were vortexed and incubated for 90 min at room temperature in a dark condition. Absorbance at λ = 734 nm was recorded against a blank using the mentioned UV–Vis spectrophotometer. The calibration curve obtained can be found in the Supplementary material.

Trolox equivalent antioxidant capacity (TEAC_CUPRAC) of SIM-53B was determined using its Cu²⁺ reducing capability in the presence on neocuproine by the CUPRAC method [33]. The solution of SIM-53B and solutions of standards (Trolox and resveratrol) were freshly prepared in 96% ethanol at 1 mM concentration. To a test tube were added 1 mL each of CuCl₂ (10 mM in water), neocuproine (7.5 mM in 96% ethanol) and ammonium acetate buffer (pH 7, 1 mM in water) solutions. SIM-53 (or standard) solution (x mL) and water [(1.10−x) mL] were added to the mixture so as to make the final volume 4.1 mL. The tubes were closed by parafilm, and the mixtures were vortexed and incubated for 60 min at room temperature (or in a water bath at T = 50 °C for 20 min). Absorbance at 450 nm was recorded against a reagent blank using the mentioned UV–Vis spectrophotometer. The molar absorptivity (ε) for each antioxidant was calculated from the slope of the calibration line by plotting absorbance versus concentration (the calibration curve obtained can be found in the Supplementary material). TEAC_CUPRAC was calculated by dividing the molar absorptivity of SIM-53B or resveratrol by that of Trolox.