Isolation,Purification,Identification And Inhibition Mechanism Of ACE Inhibitory Peptides From Ginkgo Seeds

In recent years,natural food-derived angiotensin-converting enzyme?ACE?inhibitory peptides have potential applications in the food and pharmaceutical fields due to their high activity and good absorption,and have attracted more and more attention from researchers.In this study,Ginkgo biloba seeds was used as raw material to extract ginkgo protein.The hydrolysis product was obtained by alcalase hydrolysis.The preparation process was optimized by response surface methodology,and the ACE inhibitory activity peptide was obtained by purification.The effect of active peptide on human umbilical vein endothelial cells provides a theoretical basis for the research and development of ginkgo ACE inhibitor peptide.The main findings are as follows:1?Optimization of the preparation process of ginkgo ACE inhibitor peptide.The optimal enzymatic hydrolysis process of ginkgo ACE inhibitory peptide was obtained by single factor experiment and response surface optimization test with the degree of hydrolysis?DH?and peptide content as the index.The optimal conditions were:enzyme addition amount 2400 U,the temperature is 48°C,the ultrasonic power is 118 W,and the time is 23 min.Under these conditions,the polypeptide yield was 14.71%.2?Purification and identification of three ginkgo ACE inhibitor peptides.The ginkgo protein hydrolysate was coarsely separated by ultrafiltration technique and divided into five components:<1 kDa?1-3 kDa?3-5 kDa?5-10kDa and ginkgo protein total hydrolysate?GPHs?.The relationship between molecular weight,antioxidant activity in vitro and ACE inhibitory activity was studied.In addition,three potential ACE inhibitory peptides?TNLDWY?RADFY?RVFDGAV?were obtained by further purification with dextran gel chromatography,and structural identification by LC-MS/MS.The competitive patterns and interaction of these three ACE inhibitory peptides were studied.The results showed that the ginkgo peptide with low molecular weight had higher antioxidant and ACE inhibitory activity in vitro,the most in vitro ACE inhibitory activity of the identified peptide RVFDGAV,and its IC ₅₀ value was 1.286 mM,which showed a competitive inhibition mode.While TNLDWY,RADFY appears to be a non-competitive suppression mode.Molecular docking showed that the three ACE inhibitory peptides bind well to ACE and interact with each other.Therefore,the ginkgo peptide prepared by alcalase has an effective ACE inhibitory activity.3?The mechanism of action of ginkgo ACE inhibitor peptide.Human umbilical vein endothelial cells?EA.Hy 926?was used as a model to study the possible antihypertensive mechanism of ginkgo ACE inhibitory peptide.By observing cell viability and morphology,the amount of NO released,ET-1secretion and ROS level were analyzed.The level of inhibition of peptide expression on cells was studied from a hierarchical study of blood pressure-related gene expression.The results showed that ginkgo ACE inhibitory peptide may achieve antihypertensive effect by increasing NO release and decreasing secretion of ET-1 and ROS.The results of real-time PCR showed that the mRNA level of ACE gene was significantly decreased in RAAS system after treatment with ACE inhibitory peptide.The mRNA levels of MAPK1?MAPK3 and MAPK14 genes were significantly increased.The mRNA of NOSTRIN and NF-?B gene in vascular function was detected.The level was significantly decreased,and the mRNA levels of ECE1?eNOS?JAM1?GUCY1A1,and GUCY1B1 genes were significantly increased.