Research On The Antioxidant Activity And ACE Inhibition Characteristics Of Two Peptides Enzyme Hydrolyzed From Volutharpa Ampullacea Perryi

Hypertension is the major risk factor for cardiovascular diseases.A major pharmacological target for the treatment of hypertensionis angiotensin-converting enzyme(ACE).The occurrence of hypertension is closely related to vascular endothelial dysfunction,in which oxidative stress-induced cell damage was the focus of recent research.Under the stimulation of outside environment,the vascular endothelial cells antioxidant mechanism was impaired,it leaded to imbalance of oxidative homeostasis in endothelial cells.Increased accumulation of intracellular reactive oxygen species(ROS),then the oxidation reaction takes place.In this study,two enzymatic hydrolysis peptides(amino acid sequence IVTNWDDMEK and VGPAGPRG)of Volutharpa ampullacea perryi were studied.The selective inhibition of ACE domain,structure-activity relationship,intervention of oxidative stress(OS)on endothelial cell injury,can activate the expression of Nrf2 signaling pathway-related factors and the stability of ACE inhibitory peptide were studied.To provide a theoretical basis for the development of functional foods for the prevention of hypertension and cerebrovascular diseases.The main research contents and results are as follows:(1)Through fluorescent enzyme labeling apparatus and ACE domain specific fluorescent substrates,to preliminary confirmation the two enzymatic peptides(IVTNWDDMEK and VGPAGPRG)inhibited ACE activity mainly by inhibiting the C-terminal domain of ACE.The inhibition rates were 76.34%and 39.65%(1 mmol/L),respectively.The inhibition effect was further validated by molecular simulation software.Discovery Studio software was used to simulate the action mode of ACE and two hydrolysis peptides from Volutharpa Ampullacea Perryi,and the results were consistent with the experimental results.By means of circular dichroism spectroscopy and Fourier transform infrared spectroscopy,it was preliminarily confirmed that the two enzymatic peptides could inhibit ACE activity by changing the conformation of ACE.The results showed that the two polypeptides could change the conformation of ACE.(2)To study the effects of two enzymatic hydrolysis peptides of of Volutharpa ampullacea perryi on oxidative stress(OS)induced by endothelial cell injury and the expression of Nrf2signaling pathway-related factor m RNA.The results showed that,after incubation of HUVECs for 24 h,48 h and 72 h,the two kinds of enzymatic hydrolysis peptides of Volutharpa ampullacea perryi were incubated respectively,and their were non-toxic to cells and could promote cell proliferation.They could promote NO release and reduce ET release.Furthermore,They affected the expression of ACE,ACE2,ET-1 and e NOS m RNA.It can repair oxidative stress damage caused by hydrogen peroxide.Reducing MDA content,increasing SOD activity and inhibiting ROS production.The expression of Nrf2,HO-1,NQO1,SOD1,SOD2 and CAT m RNA to Nrf2 signaling pathway was increased.(3)The thermal stability,digestion and absorption resistance,metal ions resistance,high salt and high sugar diet of the two kinds of enzymatic hydrolysis peptides of Volutharpa ampullacea perryi were determined by high performance liquid chromatography(HPLC).A quantitative analysis method for polypeptide was established.The results showed that the two peptides heated for 6 h at 100?,the reason may be that the long time heating at high temperature caused the breakage of the peptide bond,which affected its physiological activity.By simulating gastrointestinal digestion in vitro,the inhibitory rates of two kinds of polypeptides IVTNWDDMEK and VGPAGPRG in gastric juice at 2 h and intestinal juice at 6h were 30.47%,15.74%,28.01%and 12.33%,respectively.Peptides are decomposed into small molecules to some extent,but they still have ACE inhibitory activity after decomposition.In addition,the metal ions Zn²⁺and Fe³⁺could decreased the ACE inhibitory activity.High salt and sugar diet also decreased ACE inhibitory activity of enzymatic hydrolysates.Therefore,in the daily production and storage process,avoid high temperature and long time heating,avoid the addition of metal ions Zn²⁺and Fe³⁺,reduce salt and sugar intake,and prevent the occurrence of hypertension.This study provides a theoretical basis for the study of selective inhibition mechanism,protect endothelial cells from oxidative stress injury andstability of enzymatic hydrolysis peptides from Volutharpa ampullacea perryi.