Preparation And Mechanism Of ACE-inhibitory Peptides From Urechis Unicinctus

In this project,Urechis Unicinctus was used as a raw material,and ACE inhibition was used as the screening index,and a controllable enzymolysis technology was used to prepare peptides with antihypertensive activity.A series of separation,purification and identification techniques,such as degreasing,drying,enzymolysis,ultrafiltration,Sephadex G-15 gel column chromatography,reversed-phase high-performance liquid chromatography?RP-HPLC?,and mass spectrometry,have been performed to obtain seven peptides with ACE inhibitory activity,and their hypotensive activity and related activities are studied at the chemical level and the cellular level.Through the technology of enzyme kinetics and computer-simulated molecular docking,we explore the mode of action of active peptides and ACE.The main research contents and results are as follows:?1?Preparation of ACE inhibitory peptides:Screening for protease and enzymatic hydrolysis conditions using ACE inhibitory rate as an indicator.First,the defatted Urechis Unicinctus was digested under the optimal conditions of pepsin,neutrase,alcalase,trypsin,and papain,and the neutrase solution obtained had a higher ACE inhibition rate.Through the single factor experiment,the conditions of enzymatic hydrolysis were further optimized,and the optimal enzymatic hydrolysis conditions were finally determined as follows:neutral protease,45?,pH 7.0,4%enzyme addition,feed-liquid ratio 1:30,enzymolysis time 4 h.Enzymatic hydrolysis was performed under the optimal enzymatic conditions,and ultrafiltration was performed.The activity evaluation results showed that the ACE inhibition rate of the enzymatic hydrolysate components of<1 kDa was the best.This component was sequentially subjected to Sephadex G-15 gel chromatography and reversed-phase high performance liquid chromatography?RP-HPLC?Isolation and purification yielded 7 oligopeptides?F1-F7?.The sequence was determined by mass spectrometry and amino acid sequence analysis:Gln-Pro-Met-Thr-Phe?F1?,Ile-Val-Lys-His-Phe?F2?,Phe-Pro-Tyr-Lys-His?F3?,Trp-Pro-Gly-Lys-Ala?F4?,Gly-Leu-Thr-Lys-Pro?F5?,Met-Pro-Gln-Gly-Ile-Val-Val?F6?,Ile-Val-Met-Arg-Ile?F7?.?2?ACE inhibitory activity of peptides derived from U.Unicinctus:Screening of ACE inhibitory rate for oligopeptides F1-F7,the results show that F2,F3,F4,F5 and F7 have better ACE inhibitory activity and are significantly higher than other peptides.The IC₅₀values were 0.47 mg/mL,0.71 mg/mL,0.32 mg/mL,0.29 mg/mL,and 0.38 mg/mL.And analyzed the structure-activity relationship of its activity difference.Enzyme kinetics and computer-simulated molecular docking technology have found that oligopeptides F2,F4,F5,F7 and ACE are competitive inhibition,while F3 is non-competitive inhibition.?3?Functional evaluation of the effects of U.unicinctus-derived ACE activity inhibitory peptide on human umbilical vein endothelial cells?HUVEC?:F2,F3,F4,F5,and F7 with good ACE inhibitory activity were selected to act on HUVEC cells.The experimental results show that F2,F3,F4,F5,and F7 have no toxic side effects on HUVEC cells within a certain concentration range?50-200?M?;These five oligopeptides can not only inhibit the generation of endogenous contraction factor endothelin?ET-1?,promote the release of diastolic factor nitric oxide?NO?,but also activate endothelial nitric oxide synthase?eNOS?to a certain extent,inhibit the activity of induced nitrogen monoxide synthase?iNOS?,and thus release more NO,improves endothelial cell dysfunction caused by norepinephrine.?4?Effects of ACE inhibitory peptide from monocyclic spiny weeds on blood pressure regulators in Vascular Endothelial Cellsoxidatively:F4 and F5 with better clearance activity were selected based on the clearance rate of superoxide anion?O₂^-?for subsequent experiments.H₂O₂ induced HUVEC cells were used to establish an oxidative damage model,the results showed that the concentration of hydrogen peroxide was 700?M,and the model was successfully established at 4 hours after injury.Both peptides can promote the release of NO and increase the activity of SOD and CAT in cells,and reduce the content of MDA and reduce the content of LDH in the extracellular culture medium.DCFH-DA staining observations show that U.unicinctus-derived peptides?F4 and F5?can reduce the increase of reactive oxygen species in HUVEC cells induced by H₂O₂,and the activity increased in a concentration-dependent manner.Therefore,F4 and F5 have a certain degree of protection against oxidative damage to HUVEC cells induced by hydrogen peroxide.In summary,the peptides digested by neutral protease were purified by ultrafiltration,gel chromatography,and high-performance liquid chromatography.Seven peptides were identified by ESI-MS,and peptides F2,F3,F4,F5,and F7 has a better ACE inhibitory effect,and peptides F4 and F5 have the effect of protecting oxidatively damaged HUVEC cells.