4.4. Radical-Trapping Antioxidant Assay

ABTS radical scavenging assay was used to evaluate the possible RTA properties of tested compounds in cell-free conditions. The assay was done according to the described protocol by Lee et al. (2015) [48] with the following modification. First, the ABTS reagent was prepared by mixing ABTS (7  mM) with potassium persulfate (2.45  mM) at a 1:1 volumetric ratio, which was then allowed to generate free radicals (kept in the dark at room temperature for 16 h). Then, the solution was diluted with water to achieve absorbance between 0.4 and 0.6) at 734  nm. In the assay, 100  μL of the tested sample or vehicle was mixed with 100  μL of ABTS reagent in a 96-well microplate, and the mixture was then incubated at room temperature for 7–15  min. Upon incubation, the absorbance of the mixture was measured by a PowerWave XS microplate spectrophotometer (BioTek Instruments, Winooski, USA) at 734  nm. The ABTS radical scavenging property was measured using the following formula: