DPPH Radical Scavenging Assay

The radical scavenging activity of all compounds was tested through 2,2-diphenyl-1-picryhydrazyl (DPPH) assay which determines the EC₅₀ values as previously described [42 (link)]. The test consists of adding 190 µL of DPPH solution (200 µM) in ethanol to 10 µL of potential antiradical molecule solution at different concentrations (from 800 to 12.5 µM). The reaction was carried out in 96-well microplates and the disappearance of DPPH radicals was monitored at 520 nm every 5 min during 7.5 h in a multiplate Biotek (Winooski, VT, USA), Epoch 2 spectrophotometer. The EC₅₀ values, corresponding to the concentration needed to reduce half of the initial population of DPPH radicals, were provided by the crossing point of the curves of %DPPH and %reduced DPPH. Curves dots were obtained using an average of the last six measurements for each concentration in Regressi^® software version 3.99.