Screening And Inhibition Mechanism Of Oat Source ?-glucosidase Inhibitory Peptide And DPP-? Inhibitory Peptide

Diabetes is a relatively common chronic disease,and its threat to human health cannot be ignored.Type II non-insulin dependent diabetes mellitus?T2DM?accounts for 90% of all diabetic patients.Treatment of T2DM usually involves lifestyle changes and medications.Some drugs approved for the treatment of hyperglycemia in type?diabetes are not only expensive but also have serious side effects.In this study,the key enzymes in the process of carbohydrate digestion,?-glucosidase?AG?,and dipeptidyl peptidase IV?DPP-IV?in insulin secretion were used as research targets,and oat protein was used as raw material to prepare by enzymatic hydrolysis.The peptides inhibiting activity and DPP-IV inhibitory activity of the AG;separating,purifying and identifying the obtained polypeptide;using molecular mimic docking means to study its inhibition mechanism and verify its inhibitory activity,providing a safe and effective new way for the treatment of T2DM.Oat protein isolate was obtained by alkali-soluble acid precipitation method,and its protein content was 86.25±1.12%.Enzymatic hydrolysis of oat protein isolate?2%,w/v?by alkaline protease,trypsin and papain was used to inhibit AG activity.DPP-IV inhibitory activity and degree of hydrolysis as indicators to optimize the enzymatic conditions.The polypeptide solution obtained by hydrolysis of oat protein isolate by alkaline protease for 5.0 h has good AG inhibitory activity and DPP-IV enzyme inhibitory activity,and the inhibition rates are respectively 93.85±0.65% and 92.92±0.13%,and the degree of hydrolysis at this time was 15.53±0.02%.The prepared oat peptides were separated and purified by ion exchange chromatography DEAE-52,and the obtained five components,among which H1 had better AG inhibition rate and DPP-IV enzyme inhibition rate,were respectively 52.13%±3.17% and 19.18%±3.56%,61.42% of the peptides of this fraction are concentrated in the molecular weight range of 180-5000 Da.The fraction was further separated and purified by Sephasdex-G25 chromatography.The component H1-1 has better DPP-IV enzyme inhibitory activity,and its inhibition rate is 18.50±3.01%.Component H1-2 has good AG inhibitory activity and its inhibition rate is 35.84±0.14%.In order to understand the digestive properties of the peptide,this experiment investigated the effect of polypeptide digestion on AG inhibitory activity and DPP-IV inhibitory activity after gastrointestinal mimic digestion.The results showed that the DPP-IV inhibitory activity of component H1-1 was observed in the stomach.Significantly increased from the original 63.78±1.54% to 91.94±5.18%?P<0.05?,after the intestinal phase,the phase decreased to 44.54±3.22%.The AG inhibitory activity of component H1-2 decreased slightly to 32.14±0.95% after passing through the gastric phase,but after the intestinal phase,the inhibitory activity increased significantly from the original35.98±1.8% to 90.47±2.48%?P<0.05?.The amino acid sequences of the polypeptides of the components H1-1 and H1-2were determined by tandem mass spectrometry.H1-1 gave 11 peptides and H1-2 gave 41peptides.In order to screen out peptides with better inhibitory activity,the binding of NNANQLEPR?NN9?to AG was calculated to be-5.70 kcal/mol,and the AG inhibitory activity was IC₅₀=89.97±1.43 ?mol/L.The inhibitory effect was significantly higher than that of the positive control acarbose(IC₅₀=157.06±1.87?mol/L,P<0.05).The substrate binding domain of NN9 and AG is combined by hydrogen bonding interaction to competitively inhibit the binding of the substrate to AG,thereby exhibiting good inhibitory activity.SPVAEVPFLR?SP10?,with DPP-IV binding energy-6.11 kcal/mol,with DPP-IV enzyme inhibitory activity,its IC₅₀ value is 115.92±2.10?mol/L,the inhibition effect is higher than the crude peptide before purification,but lower than Positive control sitagliptin(IC₅₀=1.01±0.12?mol/L).SP10 mainly combines the hydrophobic interaction and van der Waals force with the catalytic structure of DPP-IV to change the spatial conformation of DPP-IV enzyme,thereby reducing the activity of DPP-IV.