3.6.3. Ferrous Ion (Fe2+) Chelating Activity

The Fe2+ chelating activity of *Em*-L, *Em*-A, *Es*-L, *Es*-A, and *Cv*-L extracts was estimated according to the method reported by Decker and Welch [40]. The samples were tested at different concentrations (0.0625–2 mg/mL). Briefly, different concentrations of each sample in 1 mL solvent were mixed with 0.5 mL of methanol and 0.05 mL of 2 mM FeCl2. The reaction was initiated by the addition of 0.1 mL of 5 mM ferrozine. Then, the mixture was shaken vigorously and left standing at room temperature for 10 min. The absorbance of the solution was measured spectrophotometrically at 562 nm. The control contained FeCl2 and ferrozine, complex formation molecules. Ethylenediaminetetraacetic acid (EDTA) was used as a reference. The percentage of inhibition of the ferrozine—(Fe2+) complex formation was calculated by the formula [(Ao − Ac)/Ao] × 100, where Ao is the absorbance of the control and Ac is the absorbance in the presence of the sample or standard. The results, obtained from the average of three independent experiments, are reported as mean inhibition of the ferrozine—(Fe2+) complex formation (%) ± SD and IC50 ± SD.
