2.6.3. Determination of Ferrous Ion-Chelating (FIC) Activity

Since metal chelating capacity is claimed to be of the most important mechanisms which underpin antioxidant activity [28], an FIC activity assay was performed to better characterize the antioxidant activity of the samples. FIC activity was determined according to the method of Wang et al. [28] with some modifications [24]. The chelating ability of each freeze-dried extract, at various concentrations, was evaluated by measuring the inhibition of the Fe²⁺–ferrozine complex formation. An aliquot of 100 µL of each extract sample (mg/mL) was mixed with 135 µL of methanol plus 5 µL of 2 mM FeCl₂. The reaction was initiated by the addition of 10 µL of 5 mM ferrozine. After 10 min at room temperature, the Abs was measured at 562 nm. Methanol, instead of ferrozine solution, was used as a sample blank, which is required for error correction because of unequal color of the sample solutions. Methanol, instead of a sample solution, was used as a control. Results were expressed as relative iron chelating activity compared with the unchelated (without ferrozine) Fe²⁺ reaction, and EDTA was used as the reference standard. A lower Abs was indicative of a better FIC activity. The FIC activity was calculated as follows:

where A₀ was the Abs of the control, A₁ was the Abs of the sample or standard, while A₂ was the Abs of the blank.