Hypoglycemic Mechanism Of Proanthocyanidins From Bayberry Leaves Via Regulating Starch Digestion And Absorption Together With Hepatic Glucose Metabolism

In recent years,some proanthocyanidins extracted from natural plants have been reported to have hypoglycemic activity and low side-effects,but the relevant mechanism studies are still incomplete and lack of systematic.In addition,the current research on proanthocyanidins focuses on the procyanidins category with catechins as the structural unit,such as grape seed procyanidins,which have limitations such as cumbersome access to raw materials and high price.In our previous work,proanthocyanidins from bayberry(Morella rubra Sieb.et Zucc.)leaves(BLPs)were isolated and extracted,which belong to prodelphinins category with EGCG as the structural unit.Among them,the structural unit of gallate accounts for more than 98%.Comparatively speaking,BLPs have the advantages of abundant sources and being easily accessible.The unique structure of BLPs may make it have stronger hypoglycemic activity,which remains to be studied.Therefore,this thesis used the in vitro simulated digestion model,the Caco-2 cell and Hep G2 cell model,and the high-sugar diet induced insulin-resistant Drosophila model to systematically investigate the hypoglycemic effect and related mechanism of BLPs in the process of starch digestion-small intestinal glucose absorption/transport-hepatic glucose metabolism,from the two perspectives of reducing blood glucose production and increasing blood glucose utilization.The main findings are as follows:(1)Effects of proanthocyanidins from bayberry leaves on the physicochemical properties,structure and digestibility of starchBased on the in vitro simulated digestion model,it was found that 0.5%-5.0%of BLPs could inhibit the in vitro starch hydrolysis rate,reduce the RDS content from34.58%to 5.50%-12.44%,and increase the RS content from 31.29%to56.84%-69.71%.BLPs have stronger inhibitory ability on starch digestibility than the other three commercially available proanthocyanidins extracted from grape seed,peanut skin and pine bark.Furthermore,it was confirmed that BLPs could bind to starch especially amylose through the iodimetry and static rheological experiments.The results of several instrumental analyzes by DSC,XRD,FTIR,SEM and CLSM showed that,the non-covalent interaction between BLPs and starch reduced the thermal stability and molecular order of starch,changed the distribution and arrangement of starch chain,as well as destroyed the crystalline structure of starch,thereby affecting the starch digestibility.(2)Effects of proanthocyanidins from bayberry leaves on activities of starch digestive enzymes and their interactionsThe results of in vitro enzyme kinetic experiments showed that BLPs were mixed inhibitors of?-amylase and?-glucosidase,and the acarbose equivalents of IC₅₀ values were 2.92 mmol AE/g and 517.01 mmol AE/g,respectively.The fluorescence spectra and thermodynamic analysis confirmed the static fluorescence quenching mechanism and non-covalent interaction between BLPs and the two digestive enzymes.Furthermore,in the experiments of synchronous fluorescence spectroscopy,three-dimensional fluorescence spectroscopy,circular dichroism,FT-IR and molecular docking,the results showed that BLPs could bind to the amino acid residues(Tyr and Trp)in digestive enzymes,thereby altering the microenvironment polarity and the secondary structure of enzymes(increased?/?ratio),increasing the probability of polypeptide chains misfolding,and destroying the native conformation of enzymes,which comprehensively explained why BLPs reduce the activity of the two digestive enzymes.(3)Regulation and molecular mechanism of proanthocyanidins from bayberry leaves on glucose absorption and transport in small intestineUsing the dialysis method and the glucose absorption model of Caco-2 cells,it was found that BLPs significantly inhibited the glucose diffusion capacity and the glucose uptake in Caco-2 cells.Furthermore,the self-differentiated Caco-2 cell monolayer model was established to simulate the process of glucose transmembrane transport in the small intestine under fasting and postprandial conditions.After 120min of transport,BLPs(100?g/m L)reduced the amount of glucose transport by30.41%and 41.49%under low and high glucose conditions,respectively.The results of q PCR and western-blot analysis showed that BLPs significantly down-regulated the gene and protein expression of GLUT2 and SGLT1,as well as the transcriptional levels of PLC and PKC.It suggested that the inhibition effect of BLPs on the small intestinal glucose absorption and transport was attributing to inhibiting the expression and activity of glucose transporters,which is possibly related to PLC-PKC pathway.(4)Regulation and molecular mechanism of proanthocyanidins from bayberry leaves on hepatic glucose metabolismUsing the insulin-resistant Hep G2 cell model and molecular biological methods to explore the molecular regulation mechanism of BLPs on hepatic glucose metabolism.The results revealed that BLPs promoted hepatic glycogen synthesis via the PI3K/AKT-GSK3?-GYS2 pathway.The intervention of BLPs(100?g/m L)not only increased the glycogen content in normal Hep G2 cells by 50.06%,but also restored the glycogen production in IR-Hep G2 cells to normal level.For another,BLPs inhibited hepatic gluconeogenesis via the PI3K/AKT-FOXO1-PEPCK/G6Pase pathway.Compared with the blank control group and IR model control group,the intervention of BLPs(100?g/m L)reduced the PEPCK activity by 37.69%and49.90%,while reduced the G6Pase activity by 51.64%and 48.03%,respectively.In conclusion,BLPs can enhance the glucose utilization capacity of liver cells and improve insulin resistance.(5)Verification of hypoglycemic effect of proanthocyanidins from bayberry leaves using high-sugar diet induced insulin-resistant Drosophila modelFeeding fruit flies with a high-sugar diet containing 30%sucrose for 21 days could successfully induce insulin-resistant Drosophila model(denoted as HSD flies).After BLPs intervention(1-5 mg/m L)for 21 days,the body weight of HSD flies was reduced by 10.73%,the total body glucose content and triglyceride content were respectively reduced by 46.78%and 59.01%,the?-amylase and?-glucosidase activities were the largest decreased by 27.79%and 51.55%,and the over-expression of glucolipid metabolism related genes were ameliorated(i.e.,the gene expression of dilp2,dilp3,In R,d AKT,d TOR,d FOXO,PEPCK,MAPK,SREBP,FAS,E78 and LSD were down-regulated).These results indicated that BLPs can effectively alleviate a series of adverse symptoms in HSD flies,such as the disorder of physiological markers,digestive dysfunction and dysglycemia.The above findings comprehensively elucidated the hypoglycemic mechanism of BLPs,laying a theoretical foundation for the subsequent research and development of natural drugs or functional food for lowering blood sugar,thereby enhancing the reuse of bayberry by-products.