| In order to ensure the yield and quality of apples,a large number of young apples are thinned and abandoned in orchards during the growth of apples every year,resulting in a lot of waste of resources.Young apples are rich in polyphenols,especially tannic acid(TA)and phlorizin(PH).In addition,polyphenols have attached researchers'attention due to their various physiological activities.Pervious research in our laboratory found that young apple polyphenols(YAP)have a strong inhibitory effect on ?-amylase,while there are few related researches on young apple polyphenols(YAP)inhibiting ?Glucosidase(?-G)and regulating gut microbiotas.Therefore,in this thesis,young apples(apple cultivar:'Jinshiji')was selected to extract and purify YAP by ethanol and macroporous resins,and the stability of YAP was predicted under simulated digestive environment in vitro.Then,the active compounds that effectively inhibit ?-G in YAP were screened,and their inhibitory mechanisms were explored.Finally,the effect of YAP on the gut microorganisms in mice was further investigated.The main research results were as follows:1.After extraction,purification and lyophilization,YAP was obtained in 1.76%±0.01%yield.HPLC results showed that the main components in YAP were PH,chlorogenic acid(CA),(-)-epigallocatechin(EGC)and(-)-epicatechin(EC).After in vitro simulated digestion,in oral or gastric digestion stage,there was no significant change observed in the composition and content of YAP,while the total polyphenols content was decreased?22%in intestinal stage.Among them,TA,quercetin-3-O-arabinoside,(+)-catechin(C),proanthocyanidin B1,4-p-coumaroylquinic acid,proanthocyanidin B2 and EGC lost more,and the remaining components have not changed significantly.Furthermore,after in vitro simulated digestion,the inhibitory ability of YAP on ?-G was reduced,and the halfinhibitory concentration(IC50)was increased from 15.00±0.18 ?g/mL to 67.10±1.34?g/mL.2.By screening the active components that effectively inhibit ?-G in YAP,it was found that TA,PH and EC were the main ?-G inhibitors,and the order of their inhibition capacity was TA>PH>EC.PH was a competitive ?-G inhibitor,YAP,TA,and EC were mixed ?-G inhibitors.Fluorescence quenching and circular dichrosm results showed that YAP,TA,PH and EC could significantly quench the fluorescence of ?-G,and the structure of ?-G was partially unfolded.Interestingly,contants indicating the interaction between?-G and these polyphenols,including the reciprocal of competitive inhibitors(1/Kic),fluorescence quenching contant(KFQ),and the binding energy(Eb),all showed the same order:TA>PH>EC,which was opposite to the order of IC50.This meant that the stronger the interaction of TA,YAP and EC to ?-G,the stronger their inhibitory effect;3.When exploring the effects of YAP and PH intake on the gut microbes in mice,it was found that YAP and PH could affect the composition of gut microorganisms and change the species diversity and abundance,and the effects of total polyphenols group(TP)and high dose phlorizin group(HP)are greater than low dose phlorizin group(LP)and the control group(CW).In terms of the diversity of gut microbes,by combining multiple analysis methods,it was found that the TP group and the HP group showed closer species composition and gene composition.In summary,it was found that YAP has a significant inhibitory effect on ?-G,and the active ingredients that effectively inhibit ?-G were TA,PH and EC.YAP,TA,PH and EC could interact with ?-G to change the secondary structure of ?-G,resulting in decreased ?-G activity.Further,their inhibitory mechanisms and the regulation of gut microbes in mice by YAP and PH were investigated.These results have proven that YAP can be regarded as a potential material to alleviate the symptoms of diabetes,laying a certain foundation for the development and diversified utilization of YAP in the future. |
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