2.3. Radical Scavenging Capacity: DPPH and ABTS Tests

Leaf alcoholic extract was tested at 200, 100, 50, 25, 12.5, 6.25, and 3.125 μg/mL (final concentration levels) towards ABTS [2,2′-azinobis-(3-ethylbenzothiazolin-6-sulfonic acid)] radical cation and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical.

ABTS radical cation was generated as previously reported [18]. The ABTS^●+ solution was diluted with Phosphate-buffered saline (PBS; pH 7.4) until an absorbance of 0.7 at 734 nm was read. The extract at different doses was directly dissolved in the ABTS^●+ solution, and after 6 min the absorbance was measured by a Victor3 spectrophotometer (Perkin Elmer/Wallac, Waltham, MA, USA) in reference to a blank, in which the samples were replaced with solvent [18].

DPPH^● scavenging capability was estimated as previously reported [18], and the absorption at 517 nm was measured on the Victor3 spectrophotometer (Perkin Elmer/Wallac, Waltham, MA, USA) in reference to a blank, in which the samples were replaced with the solvent.

Trolox (4, 8, 16, 32 µM) was used as positive standard, and Trolox Equivalent Antioxidant Capacity (TEAC) of beech-leaf extract was calculated, based on both ABTS and DPPH tests. For each antiradical test, three replicate measurements for three samples (n = 3) of the extract (in total, 3 × 3 measurements) were performed. All data were expressed as mean ± standard deviation (SD).