Ferrous Ion Chelation and Reducing Power Assays

The chelating effect on ferrous ions of the prepared extracts was estimated by the method of Dinis with slight modifications [10 (link)]. Briefly, 100 μL of each test sample (1 mg/mL) was taken and raised to 3 mL with methanol. 740 μL of methanol was added to 20 μL of 2 mM FeCl₂. The reaction was initiated by the addition of 40 μL of 5 mM ferrozine into the mixture, which was then left at room temperature for 10 min and then the absorbance of the mixture was determined at 562 nm.
(i) Ferric Ion Reducing Antioxidant Power (FRAP Assay). FRAP activity was measured according to the method of Benzie and Strain [11 (link)]. Briefly, acetate buffer (300 mM, pH 3.6), TPTZ (2,4,6-tripyridyl-s-triazine) 10 mM in 40 mM HCl and FeCl₃·6H₂O (20 mM) were mixed in the ratio of 10 : 1 : 1 to obtain the working FRAP reagent. Test sample (0.5 mL) was mixed with 3 mL of working FRAP reagent and absorbance was measured at 593 nm after vortexing. Methanol solutions of FeSO₄·7H₂O ranging from 100 to 2000 μM were prepared and used for the preparation of the calibration curve of known Fe²⁺ concentration. The parameter equivalent concentration was defined as the concentration of antioxidant having a Ferric-TPTZ reducing ability equivalent to that of 1 mM FeSO₄·7H₂O.