ACE Inhibitive Peptides Prepared From Pilose Antler Blood Hydrolysate Of Tianshan Wapiti

Hypertension is one main dangerous factor which causes cardiovascular disease such as myocardial failure,blood-stroke,coronary artery disease as well as myocardial infarction and has become one of the main diseases which harm human health.Inhibitors of ACE have been widely used in clinical treatment as effective anti-high blood pressure drug.These inhibitors mainly include a couple of chemical synthetic drugs which remains some kinds of by-effect such as dysgeusia,angioedema and nonproductive cough and so on.ACE inhibitive peptide has become high light of bioactive peptides research region according to their high safety,little by-effect and good absorption.Pilose antler blood of Tianshan wapiti contain abundant proteins,amino acids, immunoglobulin,trace element,polysaccharides,enzymes as well as vitamins.In ad'dition,the blood also contain a kinds of physiological active substances such as blood testosterone,estrone,adnephrin and so on.These proteins contain many potemtial ACE inhibitive peptides which could be released generously by suitable enzymolysis technology.Research on ACE inhibitive peptides from the blood expand'ed the comprehensive usage.The enzymolysis of blood was made by neutral and alkaline proteinase respectively.Alkaline proteinase Alcalase was choosen for preparation of ACE inhibitiory peptides according to the activity of its hydrolyzate.Four important characters of enzymolysis were optimized by single factor test and orthogonal experiment.The best enzymolysis characters of alkaline proteinase was as follows: six percentage concentration of substrate,40μl/g enzyme,pH 8.0,55℃.The blood degree of hydrolysis was 28.0%while the ACE inhibitive rate was 93.55%in above condition.The Alcalase hydrolyzate of blood were separated by molecular sieve chromatographic column Sephadex G-15.The best separation condition was follow charaters:gel column,1.8×60 cm;concentration of sample,50 mg/ml;injection volume,1.5 ml;flow,0.4 ml/min;eluting reagent,0.02 mol/L HAc-NaAc buffer(pH 4.0).Single active component was gained by twice RP-HPLC in use of the ACE inhibitive peptides component produced by gel filtration.It was identified as a new ACE inhibitive peptide Pro-Pro-Leu-Tyr(PPLY) by LC/MS,which was purified from Alcalase hydrolyzate of the blood,its IC50 was identified as 11.5μM.Model of PPLY was built by drug design software InsightⅡand docked with ACE.The PPLY binding site which was consisted of residues Y62,N66,Y69,L140, E143,H353,A354,F359,Y360,K368,H383,E384,H387,F391,Y392,R402,G404, H410,E411,F512,H513,P519,R522 and Y523 in ACE was identified.It was located in ACE active-site groove and taken ACE catalytic site.The binding energy between PPLY and ACE was -31.9821 kcal/mol which was consist of electrostatic energy(-24.4872 kcal/mol) and vw energy(-6.4944 kcal/mol).The binding energy was lower than the energy between lisinopril and ACE which suggested that PPLY was easily bound on ACE than lisinopril.