Ferric reducing antioxidant power assay

The ferric reducing antioxidant power (FRAP) assay was used as described previously (25) to assess the ability of the sample to reduce the Fe³⁺-2, 4, 6-tri(2-pyridyl)-s-triazine (TPTZ, Merck, Germany) complex to the ferrous form (Fe²⁺-TPTZ complex) at low pH, forming an intense blue- coloured complex with optimal absorbance at 593 nm. Briefly, TPTZ powder (0.00823 g) was dissolved in 2.5 ml HCl (40 mmol/L) to prepare the TPTZ solution, after which 25 ml of an acetate buffer solution (300 mmol/L, pH=3.6) and 2.5 ml of a FeCl₃. 6H₂O solution (20 mmol/L) were mixed with TPTZ solution to prepare fresh FRAP reagent. Then, 1.5 ml of fresh FRAP reagent was mixed with 150 μl tissue homogenates and incubated at 37˚C for 10 minutes. The absorption was measured at 593 nm using a spectrophotometer (Pharmacia, Novaspec II, Biochrom, England). FeSO₄ was used as a standard of FRAP assay and the results were expressed as µM/mg.