2.3.1. Ferrozine Method

Ferrozine is a specific reagent that forms a magenta-colored complex with Fe²⁺. Hence, this methodology is principally used to determine the chelation of ferrous ions, but it can be also used for assessing total iron (Fe²⁺ and Fe³⁺) chelation after the reduction of residual unchelated ferric ions by HA, but only at pH 4.5.

Solutions of the dehydroflavonolignans in DMSO at various concentrations were mixed for 2 min with ferrous or ferric ions (50 µL, 250 µM) in acetate (pH 4.5 and 5.5) or HEPES (pH 6.8 and 7.5) buffers. HA (50 µL, 10 mM) was added at pH 7.5 to inhibit the oxidation of ferrous ions. To assess total iron chelation at pH 4.5, the same HA aqueous solution was added after the two-minute incubation of the ferric ions with the tested compounds to reduce the remaining Fe³⁺ ions to Fe²⁺ ions. Then, ferrozine (50 µL, 5 mM) was added and the absorbance of the purple complex ferrozine-Fe²⁺ was measured immediately and after 5 min at λ = 562 nm. To determine the degree of ferric ion reduction, solutions of the dehydroflavonolignans at various concentrations were mixed for 2 min with Fe³⁺ ions in buffers. Then, ferrozine was added, and the absorbance was measured again at λ = 562 nm immediately and again after 5 min. HA was used as a positive control (100% reduction) [28].