Studies On Preparation Of Porphyra Haitanensis Antihypertensive Peptides By Enzyme Method And Antihypertensive Effect

Porphyra haitanensis can be used as a good material for researching and preparing alga antihypertensive peptides by analyzing and comparing the content of protein, the composition of amino acids and so on among five large-scale algae. Enzymatic method, Ultra-filtration, gelatin filtration, reversed-phase high performance liquid chromatography(RP-HPLC) and mass spectrum (MS) were employed to study the technological optimum conditions and methods of extracting antihypertensive peptides, while the alga antihypertensive peptides with high inhibitory activity against angiotensive converting enzyme (ACE) were separated, and model animal tests in vivo verified significant antihypertensive effect.Firstly, according to the component analysis, five algae in the coast of Fujian were found to be rich in protein, polysaccharides, minerals and other nutrients. Porphyra haitanensis contained most protein, which accounted for 28.78% dry weight, and more amino acids that were consistent with the pattern of antihypertensive peptide, revealing that Porphyra haitanensis was of suitable protein source for extracting antihypertensive peptide and would be the best material for preparing alga antihypertensive peptide.In this study, AS.1398 neutral protease, papain, trypsin, compound protease, alcalase and flavourzyme were used to hydrolyze Porphyra haitanensis, respectively. According to inhibitory activity against ACE of hydrolastes and economic benefit, AS.1398 neutral protease was selected for the proper enzyme to prepare ACE inhibitory peptides. In order to look for the optimal hydrolysis conditions of AS.1398 neutral protease, orthogonal experiment was conducted. The results showed that the optimal hydrolysis conditions were ascertained, the best hydrolysis temperature was 50℃, concentration of enzyme was 12000 U/g, concentration of substrate was 5%, the optimal pH was 7.5, reacting for 10 h. On these conditions, the hydrolysis ratio was 75.96%, and inhibitory percentage against ACE was 78.34%.The laver hydrolysates was separated and purified by ultra-filtration, Sephadex G-15 columniation, RP-HPLC, gaining a pure fraction III-E-6, with ACE inhibitory percentage 89.54%. Then the fraction was analyzed by MALDI-TOF/MS, speculating its molecular weight to be 860.17. Animal toxicological experiment validated the innocuity of laver antihypertensive peptides we've gained. Spontaneously Hypertensive Rat (SHR) and WKY (normal control) were taken as model of animal experiments in vivo. The laver antihypertensive peptides in different doses (500 mg/kg, 1000 mg/kg, 1500 mg/kg) and Captopril(20 mg/kg) were conducted. The once-oral administration result showed that the blood pressure of SHR dropped for 18.7 mmHg, 31.5 mmHg, 45.2 mmHg and 58.5 mmHg, respectively. The results of long-term oral administration showed that the blood pressure of SHR dropped for 21.2 mmHg, 28.0 mmHg, 37.0 mmHg and 47.0 mmHg respectively after 4 weeks administration. With the increase of dose, the blood pressure decreased quickly. Meantime, the results of control experiment in WKY revealed that WKY kept a normal blood pressure after administration with laver antihypertensive peptides, while the blood pressure dropped in the administration of Captopril. In addition, rats administrated by laver hypertensive peptides were found to put on weight significantly, indicating that laver hypertensive peptides not only controlled the lower blood pressure, but also promoted the growth of animals.