Purification,identification And Hypoglycemic Mechanism Of Hypoglycemic Peptides From Walnut Protein Hydrolysate

According to the latest data announced by the International Diabetes Federation?IDF?,up to 2019,the number of diabetic patients in China is about 116.4 million,which has become the largest in the whole word.Type 2 diabetes mellitus?T2DM?,which accounts for up to 90% of the diabetes population,can induce complications such as cardiovascular disease,kidney disease,retinopathy,and neuropathy,and bring heavy economic burden to the patients' families and society.T2 DM is not a lifelong chronic disease,which can be effectively prevented and improved by proper dietary intervention.Therefore,it is of great practical significance to develop natural,safe,and efficient food functional factors to replace traditional medicines for dietary intervention in T2 DM.There are abundant walnut resources in China.Walnut protease hydrolysates?WHPs,3-10 kDa?isolated from walnut kernel protein have been proved to have hypoglycemic activity,but the structure of the active peptide and the mechanism of its action in the key regulation need to be further clarified,which will provide a scientific reference for the food derived peptide as a new type of food functional factor for the prevention and treatment of T2 DM.In this paper,the preparation,purification,structure identification and hypoglycemic activity of walnut active peptides were systematically studied.The main findings are as follows:?1?On the basis of the separation and purification of WHPs?3-10 kDa?Sephadex G25 and Sephadex G15 dextran gels,walnut peptides were further separated and purified by reversed-phase high performance liquid chromatography?RP-HPLC?,and a single component F1 obtained.Using electrospray ionization tandem mass spectrometry?ESI-MS/MS?technology for structural identification of component F1,432 active peptides with different amino acid sequences were obtained.According to the comparison between literatures report and BIOPEP database?http://www.uwm.edu.pl/biochemia?,10 kinds of potential active peptides were selected from which the amino acid sequences are Leu-Pro-Leu-Leu-Arg?LPLLR?,Leu-Val-Arg-Leu?LVRL?,Leu-Arg-Tyr-Leu?LRYL?,Pro-Asp-Leu-Thr-Arg-Ala-Leu-Arg?PDLTRALR?,Pro-Asp-Thr-Leu-Ala-Arg?PDTLAR?,Leu-Asp-Val-Val-Asp-His-Leu?LDVVDHL?,Val-Asp-Leu-Leu-Leu-Arg?VDLLLR?,Leu-Gly-Val-Leu-Leu-Arg?LGVLLR?,Leu-Val-Val-Asp-His-Leu?LVVDHL?,and Glu-Ala-Ala-Gly-Val-Leu-Glu-Val-Leu-Arg?EAAGVLEVLR?.The above 10 active peptides were synthesized using solid-phase synthesis technology to evaluate hypoglycemic activity and antioxidant activity.?2?Active peptides were primarily verified by three tests of ?-glucosidase inhibition rate,?-amylase inhibition rate,and oxygen free radical absorption capacity?ORAC?.The above results indicate that the activities of LPLLR,LRYL,LVRL,and EAAGVLEVLR are superior to other active peptides.Subsequently,the next cell verification test was carried out to verify the glucose consumption and antioxidant capacity of actives peptide in Insulin resistance?IR?hepatocellular carcinoma?HepG2?model.The conditions for the establishment of IR HepG2 cells model were as follows: 25 mmol/L high glucose concentration and 10^-6mol/L insulin concentration for 36 hours.The results show that LPLLR,LVRL,and LRYL can significantly improve the glucose consuming ability and enhance the activity of glutathione peroxidase?GSH-Px?and catalase?CAT?,and can effectively remove cells the content of internal reactive oxygen species?ROS?in IR HepG2 cells.However,EAAGVLEVLR cannot significantly improve the above effects.?3?To explore the effect and mechanism of LPLLR on glucose metabolism in IR HepG2 cells.The results show that LPLLR can promote glycogen synthesis by increasing the phosphorylation of GSK-3? at Ser9 and the expression of GS,and also can increase glucose uptake by increasing the expression and transport of GLUT4,and by down-regulating G6 Pase,PEPCK,TORC2,CREB,and PGC-1? expression inhibit gluconeogenesis.Mechanism research shows that LPLLR may mediate the activation of IRS-1/PI3K/Akt and AMPK signaling pathways to relieve IR,and then play the role of hypoglycemic.?4?The LPLLR stability analysis results show that: LPLLR has the characteristics of high temperature resistance,acid and alkali resistance and after simulating gastrointestinal digestion.It is found that LPLLR can still ensure structural integrity,indicating that LPLLR plays the role of hypoglycemic and antioxidant effects in IR HepG2 cells.