2.4.4. Ferrous Ion Chelating Ability

The Fe2<sup>+</sup>-chelating activity (FCA) was determined according to the method of Boyer (1988) [26] with slight modifications. Samples were dissolved in water in ratio 1/1 and centrifuged at 2500× *g* for 5 min; 100 μL of the supernatant were mixed with 50 μL of 2 mM ferrous chloride and 100 μL of 5 mM ferrozine for 30 min at room temperature. The absorbance was read at 560 nm using a microplate reader (POLARStar OPTIMA, BMG Labtech, O ffenburg, Germany). The metal chelating activity was calculated as follows:

$$\text{Chelating activity} \left( \% \right) = \left( \frac{\text{Ablank} - \left( \text{Assample} - \text{Acontrol} \right)}{\text{Ablank}} \right) \times 100\%$$

where Ablank, Asample, and Acontrol are the absorbance of the blank, the sample, and the control at 520 nm, respectively.
