3.6.5. Ferrozine Iron Metal Chelation Assay

The transition metal ion Fe²⁺ has the ability to sustain the formation of free radicals through electron gain or loss. As a result, the chelation of metal ions with chelating agents can reduce the formation of reactive oxygen species [69]. Sample EDT was prepared at the concentration of 0.5 mg/mL in methanol, while the other samples were prepared at 2.5 mg/mL. An EDTA stock solution of 0.1 mM was prepared in water, and 11 serial dilutions were prepared in the concentrations of 5, 10,15, 20, 25, 30, 35, 40, 50, 60, and 70 μM (Figure 3). The assay was carried out according to the method of Santos [70], with minor modifications to be carried out in microplates; briefly, 20 μL of the freshly prepared ferrous sulphate (0.3 mM) were mixed with 50 μL of the sample/compound in 96-well plates (n = 6). Afterwards, 30 μL of ferrozine (0.8 mM) was added to each well. The reaction mixture was incubated at room temperature for 10 min. At the end of incubation time, the decrease in the produced color intensity was measured at 562 nm. Data were represented as means ± SD according to the following equation: (1) percentage inhibition = ((average absorbance of blank − average absorbance of the test)/(average absorbance of blank)) × 100. The results were recorded using a FluoStar Omega microplate reader (BMG LABTECH, Germany).