Study Of Inhibitory Effect Of Quercetin On Protein Glycation

Alpha-dicarbonyl compounds are the active carbonyl intermediate substances occur in non-enzymatic glycation reaction between reducing sugar and proteins,which can cause the cross-linking of proteins,destruction of the structure and function of proteins,damage of tissue,then lead to the formation of advanced glycation end products(AGEs).To develop a natural inhibitor of foodborne carbonyl compounds,quercetin is selected as the research object.Dicarbonyl compounds were detected by a new gas chromatography method to monitor the inhibition kinetics of quercetin on carbonyl compounds,thus quercetin metabolite in the reaction between quercetin with various concentrations of dicarbonyl compounds were monitored using of liquid chromatography-mass spectrometry technique.Fluorescence spectroscopy,Congo red dye,UV spectrophotometry and liquid chromatography-mass spectrometry techniques were used to monitor the inhibition ability of quercetin on bovine serum albumin glycosylation induced by dicarbonyl compounds.The main findings are as follows:1 Established a more agile and efficient detection method of MGO and GO.The optimum conditions were set as follows:DB:a-dicarbonyl compounds=67,derivatization time 10 min,acetaldehyde:DB=20,the extraction solvent methylene chloride,ultrasonic extraction 15 min,2 times,the injector was in 1:1 split mode,the 2.0 mL/min constant carrier gas(helium)flow rate was set.The GC oven temperature was programmed as follows:the initial oven temperature 40 ? with increased rate of 5 ?/min.MGO and GO minimum quantitation limits(s/n?10)were 0.4 ?g/mL and 0.8 ?g/mL.The detection limits(s/n ?3)were 0.1 ?g/mL and 0.2?g/mL,the method was sensitive and efficient.By the gas chromatography method above,the remaining MGO and GO were detected in quercetin-MGO/GO system at different time points,thus the inhibition kinetics changed by time were obtained.The results showed that quercetin have good inhibitory effect at the amount of 0.5 mM.2 In the analysis of the reaction of quercetin and MGO by HPLC-MS-MS,new compounds MM,DM-1,DM-2 and DM-3 were found by the comparison of molecular ion fragment peaks and secondary peaks,MM was an addition product of quercetin with one molecular MGO,and DM-1,DM-2,DM-3 as isomers were the addition products of quercetin with two molecular MGO.In the reaction of quercetin and GO,the products were found within 24 h at low content level.The reaction mechanism should be further verified.Further studied the inhibition ability of quercetin in BSA-MGO system by HPLC-MS-MS within 8 h,the Mono-,di-quercetin-MGO products were produced as in quercetin-MGO system while in the BSA-GO system,the MG-1,MG-2 products were produced within 8 h.3 To study the inhibitory effect of quercetin on the formation of advanced glycation end products(AGEs)in bovine serum albumin(BSA)systems caused by sugar or dicarbonyl compounds,BSA-MGO?BSA-GO and BSA-sugar reacting system were established.By using fluorescence spectrophotometry,the inhibitory abilities of quercetin on the formation of AGEs was investigated in three systems above.With the concentration of MGO?GO?sugar at 0.5?0.5?10 mM in three systems respectively,non-enzymatic glycation of BSA was obvious.Quercetin can effectively inhibit the produce of AGEs at the concentration of 0.25?0.25?1 mM respectively.The content of BSA glycation induced by MGO were detected in 0?168 h at 280 nm by UV-visible spectroscopy measurement,which indicated that MGO influenced the tyrosine and tryptophan primary structure of BSA.The glycation rate induced by GO is lower than by MGO.Congo red dye assay showed that the MGO-induced BSA glycation can make the secondary structure of BSA from a-helix to ?-pleeted sheet,and the absorbance had a positive correlation with the time.GO had a less obvious effect than MGO.With the addition of MGO and GO,the absorption values of BSA changed significantly,which means the increase of glycated fructosamine.The results showed that the amount of fructosamine reached equilibrium after the 72 hours and the fructosamine produced in BSA-MGO system was 3 times as high as in BSA-GO system.