3.4. Ferrozine Iron Chelating Assay

A Ferrozine assay was used to measure the ability of seed coat extracts to chelate Fe²⁺ using the indirect colorimetric method reported by Carter [71] and modified by Santos et al. [31]. Seed coat extracts were diluted in 10% methanol as necessary to obtain absorbance readings in the linear range. Serial dilutions (1–50 µg/mL) of disodium ethylenediamine tetra-acetic acid (EDTA-Na₂) were used to generate a standard curve. Distilled water and dilution solvent replaced the Ferrozine reagent and the seed coat sample in the blank and control samples, respectively. In a 96-well plate, 50 µL of each sample, control (solvent only), blank or standard (EDTA-Na₂) solution were mixed with 160 µL of 50 mM ammonium acetate buffer (pH 6) and 20 µL of 0.3 mM FeSO₄ solution. The plate was incubated for 5 min to allow ferrous chelation by polyphenols in the samples. A volume of 30 µL of 1 mM Ferrozine solution was added to each well to react with any free ferrous ions remaining in the reaction mixture forming a blue-colored complex that was monitored after 15 min at 562 nm in a microplate reader. A decrease in the absorbance (A) indicated an increase in iron chelating ability of the sample. Results are expressed as mg EDTA equivalents per mg dry weight of seed coat.