Research Of Angiotensin-converting Enzyme (ACE) Inhibitory Peptide From Sardinops Melanosticta

Hypertension and its resulting cardiovascular diseases are the most important risk factors that cause human death.At present,the main drug for hypertension was synthetic angiotensin I-converting enzyme inhibitor,such as Captopril, Lisinopri, Fosinopril and Cilazpril.Although these synthetic ACE inhibitors had obvious antihypertensive effects, they also bring some undesirable side effects, for example, headache,cough,vertigo,fatigue and diarrhea.In recent years researchers pay more and more attention to ACE inhibitory peptides derived from food protein due to its high safety and nutrition.Sardinops melanostictais rich in proteins and familiar in the coastal region of China.Most of Sardinops melanosticta is processed into feed and fertilizer.In order to improve its added value,the main purpose of the paper is to research the ACE inhibitory activity in vitro,effect of antihypertensivein vivo and its structure and inhibition mechanism of Sardinops melanosticta by enzymatic hydrolysis.The main results were as following:(1)Sardinops melanosticta muscle was hydrolyzed using a combination of animal protein hydrolysis enzyme and flavor enzyme to obtain its hydrolysate coded as SMMH. SMMH was then ultrafiltered to yield 3 fractions named as A(MW>10 kDa),B(3kDa<MW<10kDa) and C(MW<3kDa) and measured their ACE inhibitory activity,the result suggested the fraction C exhibited the strongest ACE inhibitory activity in vitro.To further evaluate its antihypertensive effect,a single oral administration(short term) or continuous administration for 28d(long term) of fraction C were investigated,fraction C supplementation decreased the systolic blood pressure(SBP) in a dose-dependent manner.With the increase of dose,the decrease of systolic blood pressure in SHRs were significant.(2)In order to identity the effective antihypertensive peptide of the fraction C,the fraction C was separated into three fractions(p1,p2,p3) on a Sephacryl S-100 High resolution gel filtration column and measured their ACE inhibitory activity,the result suggested the p2 exhibited the strongest ACE inhibitory activity in vitro.Therefore,p2 was further separated into three fractions(p2-1,p2-2,p2-3) on a Q-Sepharose Fast Flow anion-exchange column and measured their ACE inhibitory activity,the result suggested the p2-2 exhibited the strongest ACE inhibitory activity in vitro.Lastly,fraction p2-2 was further purified using reverse phase HPLC,where five different fractions were separated;Among all fractions, p2-2-2 and p2-2-4 had the highest ACE inhibitory activities.Their amino acid sequences were identied as Lys-Val-Glu-Pro-Leu-Pro and Pro-Ala-Leu by ESI-MS/MS and their IC50 were 12.2 μM and 22.9μM, respectively.(3)To further evaluate Lys-Val-Glu-Pro-Leu-Pro and Pro-Ala-Leu antihypertensive effect,they were hydrolyzed by simulating gastrointestinal digestion, the result suggested they could good resistance tolerance to the enzymes in simulated gastrointestinal digestion.In addition,the inhibition type and mechanism of Lys-Val-Glu-Pro-Leu-Pro and Pro-Ala-Leu were investigated by studying on the catalysis of ACE on HHL to hippuric acid.The Linweaver-Burk plots suggested that Lys-Val-Glu-Pro-Leu-Pro and Pro-Ala-Leu were competitive inhibitors.