Scavenging Activity Of Bound Polyphenols From Different Sources For Reactive Carbonyl Species And Its Mechanism

Reactive carbonyl species(RCS)is a group of reactive molecules with one or more carbonyl groups,including glyoxal,methylglyoxal,acrolein and malondialdehyde.RCS can be formed upon Maillard reaction and lipid oxidation during food processing and storage.Besides human diet,RCS in human body can also be constantly generated from the digestion and metabolism of lipids,carbohydrate and proteins.The abnormal accumulation of RCS favors carbonyl stress,promoting modification of tissue protein and DNA,and causing a series of diseases and chronic complications.Polyphenols can interact with macromolecules,including melanoidins and insoluble dietary fiber(IDF),via ester,ether and other chemical bonds to form bound phenolic compounds,which contribute to around 60% of the diet antioxidant capacity.Recently,growing attention has been focused on the ability of free polyphenols in scavenging RCS in vitro and in vivo.However,the RCS scavenging ability of bound polyphenols and its mechanism remain unknow.In this dissertation,bound-polyphenol rich soluble melanoidins and IDF were used to investigate the scavenging activity of bound polyphenols for typical RCS and the mechanism behind this scavenging process.Additionally,the bound polyphenols were applied to control the accumulation of RCS during food processing,storage and digestion process.This study lays the foundation for the research on protective effects of bound polyphenols on carbonyl threat,and provides new ideas for the development of functional foods rich in bound polyphenols.The main results are as follows:The carbonyl scavenging ability of soluble bound-polyphenol rich coffee and cocoa melanoidins and bound-polyphenol poor bread melanoidins were compared with each other under simulated physiological conditions.The results showed that glyoxal,methylglyoxal,acrolein and malondialdehyde were effectively scavenged by coffee and cocoa melanoidins when the concentration of melanoidins is higher than 0.5 mg/m L and the scavenging efficacy increased with the concentration,while only limited methylglyoxal and acrolein were quenched by bread melanoidins at the highest concentration(2.5 mg/m L),highlighting the predominant role of bound polyphenols in scavenging RCS.Coffee melanoidins(1.0 mg/m L)showed 68.0%,76.9%,83.1% and 35.8% scavenging capacity for glyoxal,methylglyoxal,acrolein and malondialdehyde,respectively,being more effective in scavenging glyoxal and malondialdehyde while less efficient in scavenging methylglyoxal and acrolein than cocoa melanoidins.Coffee melanoidins and its main bound polyphenols as chlorogenic acid and caffeic acid shared the similar RCS-scavenging kinetic profiles.Additionally,after reaction with RCS,the content of decreased free amino groups(0.01-0.05 ?mol)in coffee melanoidins was much lower than that of scavenged RCS(0.64 ?mol),indicating that coffee melanoidins scavenged RCS through their bound polyphenols not the free amino groups.Mass spectrometry data confirmed that caffeic acid could trap methylglyoxal under physiological conditions to form mono-and di-methylglyoxal-caffeic acid adducts,indicating that the formation of adducts between caffeic acid bound to the coffee melanoidins and RCS could be the molecular mechanism behind the RCS scavenging by coffee melanoidins.To further investigate the scavenging activity of bound-polyphenols rich macromolecules for RCS,the carbonyl scavenging reaction system of IDF was established to evaluate the carbonyl scavenging ability of bound-polyphenol rich blackberry,red cabbage and wheat bran IDF under simulated gastrointestinal conditions.Firstly,the phenolic compounds associated to blackberry,red cabbage and wheat bran IDF were characterized and quantified using UPLCQTOF-MS.The results showed that the content of total bound polyphenols in blackberry IDF was 1044.0 mg/100 g,with flavonoids,ellagitannins and hydroxybenzoic acid derivatives being the predominant ones;the bound polyphenols in red cabbage and wheat bran IDF mainly included hydroxycinnamic acid derivatives,reaching 623.3 and 469.9 mg/100 g,respectively.The carbonyl scavenging ability of three IDF was significantly influenced by the p H of the model system: all IDF nearly cannot scavenge RCS under p H 3.0,while glyoxal,methylglyoxal,acrolein and malondialdehyde were effectively scavenged by three IDF under p H 7.0.The scavenging efficacy was following the order: blackberry IDF > red cabbage IDF > wheat bran IDF.Upon simulated gastrointestinal digestion,three IDF still showed effective scavenging capacity for four RCS;more than 69.2%,57.8% and 56.5% of carbonyl scavenging ability was retained for blackberry,red cabbage and wheat bran IDF,respectively,after further simulated colon digestion though scavenging efficacy for four RCS was significantly decreased.To elucidate the mechanism behind the carbonyl scavenging ability of IDF,continuous alkaline and acidic hydrolysis was used to prepare bound-polyphenol poor IDF,and its carbonyl scavenging ability was compared with that of bound-polyphenol rich IDF.The results showed that after alkaline hydrolysis,more than 65.3%,54.5% and 70.1% of scavenging efficacy was reduced for blackberry,red cabbage and wheat bran IDF,respectively;nearly no carbonyl scavenging ability was retained after further acidic hydrolysis.The RCS adducts of the gallic acid,quercetin and catechin,as main bound polyphenols of blackberry IDF,in model systems were analyzed using UPLC-QQQ-MS/MS.Mono-methylglyoxal/acrolein-gallic acid adducts,mono-methylglyoxal/acrolein/glyoxal-quercetin adducts,di-methylglyoxal-quercetin adduct,mono-methylglyoxal/acrolein/glyoxal-catechin adducts and di-methylglyoxal-catechin adduct were identified.Using these formed adducts as standards,the alkaline extracts of blackberry IDF incubated with or without RCS were analyzed.There was no polyphenol-RCS adduct detected in the alkaline extracts of blackberry IDF while several gallic acid/quercetin/catechinRCS adducts were detected in the alkaline extract of blackberry IDF after the incaution with RCS,indicating the formation of bound polyphenol-RCS adducts.It further confirmed the hypothesis from the molecular level: IDF exerted their carbonyl scavenging ability through trapping RCS by their bound polyphenols.The inhibitory effect of carbonyl scavenging ability of cocoa melanoidins on protein glycation was evaluated using glyoxal/methylglyoxal-whey protein and glucose-whey protein systems as dairy mimicking models.The results showed that the carbonyl scavenging capacity and antiglycative activity of cocoa melanoidins,extracted from four groups of cocoa beans with different roasting degree,were positively correlated to their bound phenolic contents.In glyoxal/methylglyoxal-whey protein system,the addition of cocoa melanoidins effectively scavenged glyoxal and methylglyoxal,and reduced the formation of main advanced glycation end-products,with the highest inhibitory rate of 74.4% and 48.0% for carboxymethyllysine and methylglyoxal-hydroimidazolone,respectively,after 14 days of simulated storage.The results of sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis showed that cocoa melanoidins inhibited methylglyoxal-induced modification of whey protein.Looking at glucose-whey protein model,cocoa melanoidins inhibited the formation of carboxymethyllysine and methylglyoxal-hydroimidazolone,with the highest inhibitory rate of88.3% and 55.6%,respectively.Meanwhile,the formation of fructosyllysine(an Maillard reaction intermediate)was inhibited(11.3%-42.8%)in the presences of cocoa melanoidins,suggesting that cocoa melanoidins could exert antiglycative activity through not only scavenging RCS but also controlling the Maillard reaction in an early stage.Finally,the bound-polyphenol rich reconstituted crisps were prepared to study the inhibitory effect of bound polyphenols on the RCS accumulation during the in vitro digestion process of crisps.Black currant pomace,black chokeberry pomace and red cabbage were used to prepare bound-polyphenol rich reconstituted crisps by substituting 20% of potato flake.The total free phenolic content in reconstituted crisps dough substituted by black currant pomace,black chokeberry pomace and red cabbage was 874.7,1148.4 and 325.3 mg gallic acid equivalent/100 g,respectively,and the total bound phenolic content was 154.7,197.3 and 83.6mg gallic acid equivalent/100 g,respectively.After deep frying,the total free phenolic content in three crisps decreased by 79.9%,82.7% and 82.0%,respectively,while the total bound phenolic content increased to 446.3,569.0 and 251.7 mg gallic acid equivalent/100 g,indicating that a considerable part of free polyphenols was covalently linked to macromolecules in food matrix through chemical reactions during heating process.Compared with control crisps,the contents of main RCS in three bound-polyphenol rich reconstituted crisps significantly decreased.Furthermore,the inhibitory effect of bound polyphenols of crisps on RCS accumulation were evaluated during the in vitro digestion process of crisps.The non-extractable fraction of crisps and the same amount of frying oil were subjected together to in vitro digestion and the RCS concentrations were monitored.The results showed that the presence of nonextractable fraction of bound-polyphenol enriched crisps led to 44.0%-83.9% decrease in the concentrations of malondialdehyde and 4-hydroxy-2-nonenal,being more effective than Trolox(positive control)with the same antioxidant activity.This result indicated that bound polyphenols of reconstituted crisps could reduce the accumulation of RCS during the in vitro digestion of crisps as antioxidants and carbonyl scavengers.