2.5. Ferrous Ion Chelating Assay

The antioxidant properties of iron were analyzed by the results of iron chelation; however, removal of iron ions is one of the things that can be achieved. In the medium of chelating agent, the structure cause complex was destroyed, which caused the complex ferrous ion to change from crimson restoration. The determination was based on the color reaction of the complex of ferrous ion and ferrozine at 562 nm. The lower the absorbance, the better the metal chelating activity. Then, 1 μL of CM of different concentrations (1–1000 μg/mL) was taken and mixed with 10 μL of FeCl2 (2 mM), let stand for 5 min, then 69 μL of methanol and 20 μL (5 mM) of ferrozine were added. The sample was vigorously shaken to start the reaction, later the mixture was kept at 25 °C for 10 min. The absorbance value of the mixture was measured at 562 nm. EDTA was used as a positive standard [15]. The calculation formula was:

where A_control = the absorbance of the control and A_sample = the absorbance of the sample.