2.3.1. DPPH Radical Scavenging Assay

The DPPH assay was carried out in a 96-microwell plate according to a previously described protocol [62]. In brief, DPPH solution (60 µM in MeOH) was added to studied compounds or MeOH as the control. The 96-microwell plate was incubated (in the dark, at r.t. for 1 h) and the absorbance values were read at 517 nm by a SPECTROstarNano (220–1000 nm) UV–Vis spectrophotometer, Ortenberg, Germany. During the first screening, all compounds were tested ad 100 µM, then for compounds 2a–c the EC₅₀ (effective concentration), the concentration of substrate that causes a 50% reduction in the DPPH colour, was calculated.

All experiments were made in triplicate. The percentage of the antioxidant activity (%AA) of the compounds was determined according to Equation (1):

Abs_DPPH is the absorbance of DPPH solution, and Abs_sample is the absorbance of DPPH solution containing the tested compound.

The DPPH radical scavenging ability of the most active compounds 2a–c was expressed as Trolox equivalent antioxidant capacity (TEAC) value by using Trolox standard regression curve [63]. Each concentration was tested in triplicate and the EC₅₀ value was reported as the mean ± the standard error of the mean (SEM) of three independent experiments.