The Oxidative Behavior And Biological Effects Of Quercetin And Its Related Flavonoids

Flavonoids,a main category of natural polyphenols,are produced as secondary metabolic products with low molecular weights in physiological processes of plants.Flavonoids have been identified as an active ingredient in herbaceous plants and diets.The biological and pharmacological activities of flavonoids include antioxidant,anti-inflammatory,hypolipidemic,hypoglycemic,anticancer,and benefits to cardiovascular and amyloid disorders.Most of biological activities of flavonoids are believed to be related to their polyphenolic structures.More than 10000 species of flavonoids have been reported.In addition to antioxidant role,flavonoids have been evidenced to exhibit a pro-oxidant effect under certain conditions.The pro-oxidation produced by flavonoids induce possibly damages to biological molecules,particularly DNA and protein molecules.Natural flavonoids have a basic structure of C6-C3-C6 backbone,consisting of two aromatic and an oxygen-containing heterocyclic rings.The hydroxyl groups and other functional groups on these rings determine their physical and chemical properties,and also pharmacological activities.It is difficult to maintain a flavonoid compound in an intact formin the alkaline environment of circulatory system due to the weak acidic property of phenolic hydroxyls.However,numerous studies have so far attributedthe biological and pharmacological activities to the intact molecules of flavonoids.The poor stability and redox property of these chemicals under common experimental conditions havebeen neglected.In our earlier works,it has been found that some flavonoid compounds are oxidized quickly under in vitro physiological conditions(37?,pH 7.4).The biological activities,including the anti-and pro-oxidant activity and anti-amyloidogenic role of a flavonoid,are originated not only from the intact molecule,but also from the oxidant products of the chemical.Quercetin is one of the most abundant and most extensively studied flavonoids.In the present study,quercetin and other five flavonoids(kaempferol,myricetin,morin,luteolin and fisetin)have been selected to investigate the biological activities of the intact molecule and oxidative products under in vitro systems.The structure-activity relationship is also explored.Methods and resultsPart I The oxidation of flavonoids1.UV spectraUV spectra of the selected flavonoid compounds were scanned at 37? and pH 7.4.The results showed that the absorption peak of quercetin,myricetin,fisetin and kaempferol changed upon incubation,suggesting that these flavonoids were unstable under the condition used in this study.In contrast,the spectra of morin and luteolin were kept unchanged during the incubation.2.HPLC analysisHPLC analysis was performed to monitor the oxidation of flavonoids at different time points of incubation.The results demonstrated that the chromatographic peaks of quercetin,myricetin,fisetin and kaempferol decreased until disappeared,along with a new peak appeared at a different retention time.As similar to the UV spectra,the chromatographic peaks of morin and luteolin were kept unchanged during the incubation.3.ESI-MS analysis of oxidized quercetinThe sample of oxidized quercetin was subject to desalting prior to ESI-MS analysis in a negative-ion mode.The data suggested that there were three main oxidative products of quercetin with mass-to-charge ratios of 195,169 and 153.Possible degradation pathway and molecular structure of the oxidative species are suggested.Part ? The anti-and pro-oxidant properties of flavonoids1.The anti-and pro-oxidant activity of quercetin and its oxidized formHuman red blood cells were utilized as an in vitro model to investigate the anti-and pro-oxidant roles of quercetin and its oxidized species.The results indicated that,in the presence of oxidative stress,both quercetin and its oxidized form exhibited similar inhibitory roles on the AAPH-induced cell damage.In the absence of oxidative stress,however,both quercetin and its oxidized form exhibited pro-oxidant role on the cells,including inducing hemolysis and changes in cell morphology,and increasing the content of methemoglobin of the cells.By comparison,the pro-oxidant role of oxidized quercetin was weaker than that of the intact form.2.Effects of quercetin and myricetin on nonenzymatic glycosylationThe nonenzymatic glycosylation of a protein is closely related to oxidative stress.Both quercetin and its oxidized form exhibited an inhibitory role on glycosylation of bovine serum albumin(BSA)with a stronger effect of oxidized form than the native one.Of other flavonoids myricetin behaved a similar way as quercetin in the inhibition of glycosylation of BSA.The results of intrinsic fluorescence indicated that the binding of a flavonoid to BSA increased stability of the protein.3.Determination of pro-oxidant activity of flavonoids by derivative spectrometryOne of the pro-oxidant activities of flavonoids is to consume sulfhydryl groups of biological molecules.The traditional Ellman assay for sulfhydryl analysis is disturbed seriously by flavonoids due to the overlap of absorbent bands.Derivative spectrometry is a promising tool to eliminate the disturbance originated from the spectra of flavonoid molecules.The results showed that the more the hydroxyl group on the B ring,the stronger the ability to consume sulfhydryl group.However,the ability of oxidized flavonoid in consuming sulfhydryl groups was lower than the native form.Part ? The inhibitory role of flavonoids on protein amyloid fibrillation1.Effects of quercetin and its oxidized products on lysozyme fibrillationThe effects of quercetin and its oxidized products on lysozyme fibrillation were determined by ThT and ANS fluorescence,SDS-PAGE electrophoresis and circular dichroism.The results showed that the oxidation products of quercetin played a stronger inhibitory role than the intact molecules.2.Effects of flavonoids and their oxidation products on insulin fibrillationThe results indicated that oxidized quercetin increased the lag time of insulin fibrillation,although the overall role in amyloid inhibition was lower than the native form.Of the 6 flavonoids oxidized myricetin has the strongest inhibitory role on insulin fibrillation.The oxidative products of morin,luteolin,fisetin and kaempferol shared a similar effect as their native form in the inhibition of insulin fibrillation.ConclusionIn the present study the oxidation and biological activities of quercetin and other five flavonoids have been investigated under in vitro systems.The data suggested that the oxidation of a flavonoid was closely related to the number and position of hydroxyl group on the polycyclic structure.The flavonoids with a catechol or pyrogallic structure are highly susceptible to oxidation;whereas the flavonoids with a resorcinol structure can keep intact under the condition of this study.The biological activities of a flavonoid are also closely related to its molecular structure.As same as its oxidative property,a flavonoid with a catechol or pyrogallic structure exhibits strong biological activities,such as the roles in anti-and pro-oxidation,anti-amyloidogesis and inhibition of glycosylation.These actions are possibly attributed to its susceptibility to oxidation and transformation into quinonoid intermediates.In comparison with the intact molecule,an oxidized flavonoid exhibits a stronger role on amyloid fibrillation and a weaker role in pro-oxidation.The inside molecular mechanism is unclear and remains further exploration.