Interaction of diosmetin, diosmin and diosmetin-7-O-glucoside with human erythrocytes, their model membrane, hemoglobin and redox-active metal ions.

Diosmin, a flavonoid with documented vascular-protective properties, together with the closely related compounds diosmetin and diosmetin-7-O-glucoside, has not previously been subjected to a systematic comparative biophysical characterization. In this study, we performed an integrated biophysical comparison of these three structurally related flavonoids to delineate how structural differences translate into distinct patterns of metal-ion coordination, membrane interaction and hemoglobin engagement, thereby providing a mechanistic context for their behavior in complex erythrocyte- and simplified hemoglobin-based redox systems. All three compounds exhibited low direct radical-scavenging activity in the DPPH• assay and showed differences in predicted lipophilicity and metal-ion interactions. Diosmetin, the most lipophilic derivative, selectively chelated Cu2+/Cu+, while diosmin showed the strongest chelation of both Fe2+ and Fe3+; none of the tested flavonoids reduced iron ions. Diosmetin inhibited rat intestinal α-glucosidase, and both diosmetin and diosmin inhibited lipoxygenase with comparable potency. In intact human erythrocytes, all flavonoids did not cause hemolysis and did not alter osmotic resistance within the concentration range tested, but they differentially affected cell morphology and response to Fe3+-induced oxidative stress. Diosmin and diosmetin-7-O-glucoside predominantly induced echinocyte formation, whereas diosmetin favored stomatocytes and, at the highest concentration, partially reduced Fe3+-induced hemolysis. Measurements in erythrocyte-mimetic liposomes showed no effect of any compound on acyl chain order, while diosmetin induced a pronounced decrease in membrane dipole potential, in contrast to the modest changes observed for diosmin, indicating different modes of interaction with the lipid headgroup region. All three flavonoids quenched the hemoglobin tryptophan fluorescence via a static mechanism, with a similar quenching efficiency but differing binding constants. ATR-FTIR spectroscopy did not reveal detectable alterations in the overall secondary-structure content of hemoglobin. Collectively, the results demonstrate systematic differences among diosmetin, diosmetin-7-O-glucoside and diosmin in metal-ion coordination, membrane-related effects and hemoglobin interactions, providing a comparative biophysical basis for distinguishing their activities. Based on these results, diosmetin appears to be a particularly interesting candidate for further biophysical and functional investigation.