Study On Purification And Structure-Activity Relationship Of ACE Inhibitory Peptides From Perilla Seeds

Perilla can be used for medicinal and spice purposes,and its seeds can be extracted for oil,which has high nutritional value.So far,the research on active peptides of perilla seeds at home and abroad has mainly focused on antioxidant,antitumor,anti-inflammatory and anti-allergic activities,as well as the preparation of perilla seed protein presenting peptides,while there are almost no reports on antihypertensive peptides.In this study,perilla seed meal was used as raw materials to obtain ACE inhibitory peptides by enzymatic hydrolysis,and the peptides were separated and purified,amino acid sequence identification,cellular assay verification,and preliminary study of its possible mechanism of action.The research content of this dissertation is as follows:Study on the process optimization and stability of perilla seed ACE inhibitory peptide prepared by enzymatic hydrolysis.Alkaline extraction and acid precipitation method was used to obtain perilla seed protein.The ACE inhibitory peptide was prepared by the enzymatic hydrolysis method,and the ACE inhibition rate was used as a screening index,and the optimal hydrolase of perilla seed protein was preferably as neutral protease.Single factor test combined with response surface optimization,the optimal conditions for preparing ACE inhibitory peptides by enzymatic hydrolysis of perilla seed protein with neutral protease:the amount of enzyme added is 7100 U/g,the temperature is 55℃,the p H is 6.5,the substrate concentration is 1%,and the digestion time is 2 h.Under this condition,the ACE inhibition rate of the digestion peptides（1mg/m L）is 88.55±3.3%.Stability experiments found that perilla seed ACE inhibitory peptide has good stability to acid,alkali,heat,and some metal ions.The peptides also showed good stability in the experiment of simulating gastrointestinal digestion.Isolation,purification and identification of ACE inhibitory peptides from Perilla Seed.Sephadex G-25 combined with RP-HPLC was used to separate and purify the enzymatic hydrolysis products of perilla seeds.LC-ESI-MS/MS was used to identify the amino acid sequence of the active peptides.Screened with existing ACE inhibitor peptides in the database and discovered 5 new ACE inhibitor peptides.The amino acid sequences and molecular weights are:LLRPPSH（818.96 Da）,VGAY（408.45 Da）,LLRNP（611.73 Da）,VLKPPSY（802.95 Da）,FRQL（562.66 Da）.In vitro ACE inhibitory activity results showed that their IC₅₀ were 3.731 mg/m L,0.861 mg/m L,1.231 mg/m L,1.194 mg/m L,1.730 mg/m L,respectively.Preliminary study on the possible mechanism of perilla seed ACE inhibitory peptide VGAY.VGAY is a competitive inhibitor of ACE.The cytotoxicity of CCK-8showed that when the concentration of VGAY was 1 mg/m L,it had no significant toxicity to HUVEC cells.At concentration of 0.05 mg/m L,VGAY can stimulate the release of NO in HUVEC cells significantly.The molecular docking results showed that VGAY could bind to part of the active sites of ACE through hydrogen bonds,thereby achieving the effect of inhibiting ACE.After the C-terminal tyrosin residue was mutated to Histine,the ACE inhibitory activity of VGAH decreased significantly,indicating that the C-terminal tyrosin residue play a great role in the ACE inhibitory activity of VGAY.In addition,retain the C-terminal Tyr to study the effect of Trp on the ACE inhibitory activity.Compared with VGAY,the ACE inhibitory activity of VGWY is increased by about 50%.