Evaluation of oxidative stability of lipids by fluorescence spectroscopy and characterization of flavonoid antioxidant chemistry

Fluorescence spectroscopy is a sensitive and rapid technique that can be exploited for evaluation of the oxidative stability of lipids. In this study, two assays based on quenching of the fluorescence intensity of an extrinsic probe and an increase in its fluorescence anisotropy due to the free radicals generated during lipid peroxidation were developed for evaluation of antioxidant efficacy and for monitoring the progress of lipid peroxidation. The assays were used to evaluate the efficacies of metal chelating and free radical scavenging antioxidants.;Distinct structure-activity relationships were revealed for the antioxidant activities of flavonoids and isoflavonoids. Presence of electron-withdrawing groups enhanced activity whereas electron-donating groups decreased activity, with the substitution pattern on the B-ring of these compounds being an especially important determinant of activity. Among the isoflavonoids, the biological metabolites of these compounds had similar or superior antioxidant activities in comparison to the parent compounds, a finding that suggests that these compounds may retain their activity under in vivo conditions. An oxidation product of the reaction between the isoflavonoid genistein and peroxyl radicals was characterized and could serve as a marker for genistein antioxidant chemistry.;By use of the assays, the effects of chelators on metal-ion-induced peroxidations were found to be dependent on the type of metal ion used to initiate peroxidation and on the mole ratio of chelator relative to lipid. Flavonoids and isoflavonoids were the class of compounds selected to study prototypic, phenolic free radical scavenging antioxidants. These compounds were more effective at suppressing metal-ion-induced peroxidations than peroxidations induced by free radicals, an indication that metal chelation plays a larger role towards their antioxidant mechanism than has previously been believed. Additionally, fluorescence anisotropy measurements were conducted using a series of probes with the chromophore at varying levels of penetration into the membrane. The results suggest that the flavonoids and isoflavonoids partition preferentially into the hydrophobic interiors of membranes and decrease membrane fluidity in this region. The reduced fluidity could limit accessibility of free radical species generated during lipid peroxidation and stabilize the membrane, a structural mechanism of antioxidant action for these compounds.