In Vitro Digestion And Fermentation Of Polyphenols From Porphyra Haitanensis And Their Effect Of Blood Glucose Regulation

Type 2 diabetes is a metabolic disease characterised by chronic hyperglycaemia,which is a major threat to human health worldwide.Chronic hyperglycaemia can lead to a variety of complications,resulting in reduced quality of life and increased mortality.The chemically synthetic agents currently used to lower blood glucose in clinical practice have caused certain damage to the human body during the treatment of hyperglycemia,thus the search for and development of bioactive substances of natural origins as hypoglycemic agents or functional foods has become a hot research topic for scholars at home and abroad.Porphyra haitanensis is an important economic seaweed in Fujian Province,which has rich nutritional and medicinal values.According to previous research,the polyphenols of P.haitanensis have strong in vitro α-glucosidase inhibitory and antioxidant activities,which have potential hypoglycemic effects.However,the effect of metabolic transformation during digestion on its hypoglycemic activity and mechanism are still unclear.Therefore,this study was conducted to analyze the phenolic transformation and hypoglycemic activity during the digestion of P.haitanensis polyphenols using an in vitro digestion model based on widely targeted metabolomics and UPLC-ESI-MS/MS techniques;to investigate the regulatory effect of P.haitanensis polyphenols on gut microbiota and anti-diabetic function using an in vitro colonic fermentation model and 16 S r RNA sequencing technology;to establish a small intestinal epithelial monolayer using Caco-2 cells and to investigate the hypoglycemic mechanism of P.haitanensis polyphenols by Western Blot.The main findings are as follows:(1)After oral,gastric and small intestine digestion,the total polyphenol content(TPC),α-glucosidase inhibitory activity and oxygen radical absorption capacity(ORAC)of P.haitanensis polyphenols were significantly higher at different harvest periods,reaching a peak after small intestine digestion.In addition,TPC,α-glucosidase inhibitory activity and ORAC of P.haitanensis polyphenols at all stages of digestion increased with the increasing harvest periods,reaching a maximum at the fourth harvest.A total of 143 phenolic metabolites were identified during the digestion of P.haitanensis polyphenols,containing 32 phenolic compounds with identified hypoglycaemic activity.Citrus flavonoids such as naringenin,orangiferin and naringenin were the main phenolics that distinguished the first and fourth P.haitanensis,conferring higher hypoglycaemic activity to the fourth harvest.Moreover,the digestion process promotes the biotransformation of phenolic compounds in the P.haitanensis polyphenols.After digestion,hypoglycaemic compounds such as citrus flavonoids,calycosin,apigenin and genistein can still be present in large quantities.(2)The TPC and ORAC of the P.haitanensis polyphenols increased and then decreased with the increase of fermentation time,reaching a maximum at 12 h.The inhibitory activity of α-glucosidase decreased gradually with the increasing of fermentation time.Moreover,P.haitanensis polyphenols lowered the p H and significantly increased the content of short-chain fatty acids(P(27)0.05)during colonic fermentation.At the phylum level,P.haitanensis polyphenols increased the relative abundance of Bacteroidetes and decreased the relative abundance of Firmicutes;at the genus level,P.haitanensis polyphenols decreased the relative abundance of Enterococcus and Streptococcus as well as increased the relative abundance of Bacteroides and Phascolarctobacterium in the intestine,preventing and regulating type 2 diabetes through antiobesity and lowering blood glucose.(3)Using the Caco-2 cell model,it was found that P.haitanensis polyphenols could significantly reduce α-glucosidase activity,with an α-glucosidase inhibitory activity of 64.39 ±4.05 % at a concentration of 2 mg/m L.The P.haitanensis polyphenols significantly inhibited the diffusing ability of glucose and the ability of Caco-2 cells to take up glucose,and the ability to take up 2-NBDG was significantly reduced by 60.82 ± 3.60 % compared to the blank control.By establishing a monolayer model of Caco-2 cells,it was found that P.haitanensis polyphenols had a significant inhibitory effect on small intestinal glucose transport.In addition,Western Blot revealed that P.haitanensis polyphenols significantly down-regulated the expression of SGLT1 and GLUT2 transport proteins in Caco-2 cells,inhibiting glucose uptake in the small intestine and exerting a hypoglycemic effect by suppressing the expression of small intestinal glucose transport proteins.The results of this thesis provide a scientific basis for the development of effecient natural hypoglycaemic drugs and functional foods using P.haitanensis polyphenols,as well as a theoretical reference for the high value application of P.haitanensis.