4.8. Total Antioxidant Capacity: ABTS and FRAP Assays

For both assays, skin samples were dissected and homogenized into ice-cold buffer containing 1.15% KCl. Homogenates were then centrifuged (1000× g in 4 °C for 10 min) [16]. A stock solution of ABTS (7mM in water) was mixed with 2.45 mM potassium persulfate (final concentration) to obtain ABTS⁺. Prior to the use, ABTS⁺ working solution was further diluted in phosphate buffer pH 7.4 to reach an absorbance of 0.8 (±0.02) at 730 nm. Briefly, the supernatant (7 μL) was added to 200 μL of the diluted ABTS⁺ solution; samples were vortex-mixed and allowed to stand for 6 min. Reading was performed at 730 nm. For the FRAP assay, the supernatants (30 μL) were mixed with the FRAP reagent. Prior to the use, FRAP reagent was prepared as follow: 0.3 mM acetate buffer pH 3.6, 10 mM TPTZ in 40 mM hydrochloride acid, and 20 mM ferric chloride. Reading was performed at 595 nm in a microplate reader. All results were compared to a standard curve of trolox (concentration ranging 0.01–20 nmol). Results are presented as nmol trolox equivalent per mg of skin tissue.