Interaction Of Several Flavonoids With Heme Proteins And Their Effects On Lipid Oxidation

In the presence of oxygen,light or endogenous hemoglobin proteins(e.g.hemoglobin,myoglobin),lipid oxidation is likely to occur,affecting the color and flavor of meat products.Therefore,many antioxidants are applied to conserve the red color and good quality of meat products.The synthetic antioxidants with some toxic side effects are usually used in meat products.The application of safe and efficient natural antioxidants in the meat industry has important theoretical and practical significance.This thesis is focused on the interactions between hemoglobin proteins and flavonoids and their effects on lipid oxidation.Firstly,the interactions between quercetin and bovine(or human)hemoglobin(Hb)were systematically investigated by fluorescence,circular dichroism,UV-vis absorption spectroscopy and molecular modeling,to demonstrate the structural mechanism by which quercetin affected Hb redox state and stability.Quercetin could bind to the central cavity of Hb with one binding site to form Hb-quercetin complex,and hydrophobic interaction played a major role in the binding process.After that,the binding of quercetin might increase the compactness of Hb molecule and narrow the crevice around the heme pocket,which contributed to the reduction of met-Hb to oxyHb and inhibition of hemin release from met-Hb.In addition,quercetin effectively inhibited Hb-mediated lipid oxidation in liposomes and washed muscle,which was ascribed to the conversion to oxy-Hb and decreased hemin dissociation from met-Hb.Consistent with its lower abilities to bind Hb and scavenge free radicals,rutin(i.e.,quercetin-3-rhamnosylglucoside)did not significantly influence the redox state of Hb nor reduce hemin release from Hb,and subsequently,it less effectively inhibited Hbinduced lipid oxidation than quercetin.Altogether,results herein provide significant insights into the interaction of quercetin with redox-active bovine Hb,which is beneficial to the application of natural quercetin in relative meat and blood products.Then,the bindings of epigallocatechin gallate(EGCG)or the endogenous antioxidant reduced glutathione(GSH)to bovine hemoglobin(Hb)and their effects on lipid oxidation were investigated.Similar to quercetin(Que),EGCG could interact with Hb to form an Hb-EGCG complex through electrostatic and hydrophobic forces in the central hydrophobic cavity of Hb,without significantly affecting the conformation of Hb.However,the binding constant of Que with Hb was higher than that of Hb-EGCG,which might be due to the larger steric hindrance of EGCG.EGCG also had the ability to reduce met-Hb to oxy-Hb,thereby inhibiting lipid oxidation by suppressing the release of free heme.GSH could not quench the intrinsic fluorescence of Hb and exhibit a negligible effect on reducing met-Hb,but it could inhibit Hb-mediated lipid oxidation.The abilities of EGCG,Que,and Rutin to reduce met-Hb and suppress heme release were compared,and the order of efficacy was found to be EGCG ≈ Que > Rutin,consistent with their abilities to inhibit lipid oxidation.Furthermore,the non-covalent interactions of three typical flavonoids,EGCG,Que,and Rutin,with myoglobin(Mb)were investigated.It was found that,the fluorescence quenching mechanism for these flavonoid-Mb complexes was static quenching.The main driving force in the binding process was hydrophobic interaction.EGCG and Que could effectively reduce met-Mb,while the reducing effect of Rutin was not significant.The abilities of these flavonoids to inhibit the release of free heme and lipid oxidation were found to be Que > EGCG > Rutin,consistent with their abilities to reduce met-Mb and scavenge free radicals.The ability to inhibit lipid oxidation was related to the free radical scavenging activity of flavonoids,Que had more hydroxyl groups on rings A and C than EGCG,which made it more effective in scavenging free radicals.Rutin contained a glycoside group,which weakened its ability to scavenge free radicals.In addition,Hb was more likely to release free heme than Mb because the tetramer Hb contained more heme groups than the monomeric Mb,and the Hb structure was more loosely packed.