Studies On Angiotensin-Converting Enzyme Inhibitory Peptides Derived From Food Proteins

Hypertension is a major risk factor for cardiovascular disease such as heart failure, stroke,coronary heart disease, and myocardial infarction. It is reported that more than 12,000thousands of people died from the cardio-and cerebro-vascular diseases resulted fromhypertension every year all over the world. Hypertension has now become the first killer ofpeople's health and the great public sanitary problem in the world. Angiotensin-convertingenzyme (ACE) inhibitors are the main drugs for the clinical treatment of hypertension. Foodprotein-derived ACE inhibitory peptides have received considerable attention in recent yearbecause of their lower side effects, higher safety and absorption. These food protein-derivedACE inhibitory peptides show great promise in the development of a novel physiologicallyfunctional food for preventing hypertension as well as for therapeutic purposes.In this study, the quantitative structure-activity relationships (QSAR) of ACE inhibitorytripeptides were firstly investigated. Alcalase, an alkaline protease for food industrial useproduced from Bacillus licheniformis, was selected to hydrolyze proteins based on the resultsobtained from structure-activity relationship study,and the ACE inhibitory activities of theenzymatic hydrolysates were measured at different hydrolysis times. The correlation betweenprotein compositions and ACE inhibitory activities of Alcalase hydrolysates of proteins wasinvestigated using multivariate regression statistical analysis. Alcalase hydrolysates of peanut,mung bean and rice protein isolates with potent ACE inhibitory activity were refinedlyprepared by desalinization and ultrafiltration and their antihypertensive activities wereevaluated. ACE inhibitory peptides were also isolated and characterized from proteinhydrolysates. The results were described as following:A direct spectrophotometric assay was developed for determination of angiotensinI-converting enzyme inhibitory activity of samples using hippuryl-L-histidyl-L-leucine as theACE-specific substrate. This method relies on previously published spectrophotometricdetermination of hippuric acid (HA) content in the urine, the method of which was based onthe specific colorimetric reaction of HA with benzenesulfonyl chloride in the presence ofquinoline, in which the absorbance was measured at 492 nm.This method proposed in thispaper was shown to be direct, sensitive, accurate, reproducible, and less expensive withouttedious separation of HA from the ACE reaction mixture, and can be used for the screening ofACE inhibitory peptides derived from food proteins.The quantitative structure-activity relationships of 40 ACE inhibitory tripeptides werestudied using the hydrophobic, steric, electronic descriptors of amino acid side chains and thetotal hydrophobicity of peptide for parameterizing the structural variability of ACE inhibitorytripeptides. A QSAR model was developed using ridge regression analysis method asY=5.6388+0.0071x1+0.2174x2+0.0052x3-0.0396x4-0.0522x5-0.0014x6-0.5687x7+0.2719x8+0.0047x9+0.7379x10 with the multiple correlation coefficient R and cross-validated Rcv2 of0.8597 and 0.6436, respectively, which showed this QSAR model possesses good predictiveability for the activity of ACE inhibitory peptides. The results from QSAR studies revealedthat the electronic charge index (ECI) of the antepenultimate amino acid residue from theC-terminal, which is negatively correlated to activity, is the most impotant factor affecting theACE inhibitory activity of tripeptide, followed by the total hydrophobicity of tripeptidepositively correlated to activity. The ECI of C-terminal amino acid and physicochemicalproperty of penultimate amino acid have little effects on activity of peptide. Highlyhydrophobic and bulky antepenultimate and C-terminal amino acids contribute to improvingthe activity of tripeptides.Alcalase was used to hydrolyze casein, bovine serum protein, zein, peanut proteinisolates, mung bean protein isolates and rice protein isolates, respectively, under the theoreticoptimum conditions of Alcalase, and the ACE inhibitory activities of the enzymatichydrolysates were measured at different hydrolysis times. The correlation between proteincompositions and ACE inhibitory activites of Alcalase hydrolysates of proteins wasinvestigated using partial least square (PLS) method. The unhydrolyzed proteins showed noinhibitory activity. Six kinds of protein hydrolysates generated with Alcalase displayeddifferent potential ACE inhibitory activities. The protein hydrolysates showed diverse ACEinhibitory activity, and the hydrolyzing time for obtaining the highest ACE inhibitory activitywas different in proteins because of different amino acid compositions of proteins. Thereexists an optimal degree of hydrolysis (or hydrolyzing time) in the preparation of foodprotein-derived ACE inhibitory peptides by enzymatic hydrolysis, obove which more ACEinhibitory peptides are degraded than new peptides are formed, thus decreasing the overallACE inhibitory activity. The contents of hydrophobic amino acids, aromatic amino acids,branched-side chains amino acids and average hydrophobicity of protein are positivelycorrelated to ACE inhibitory activity of Alcalase hydrolysates of proteins, while the contentsof uncharged-side chains amino acids, basic amino acids and acidic amino acids arenegatively related to activity. The conten of aromatic amino acids is the most impotant factoraffecting the ACE inhibitory activity of hydrolysates, followed by the average hydrophobicityof protein, and the content of uncharged-side chains amino acids has little effect on theactivity. The proteins with high contents of hydrophobic and aromatic amino acids are moresuitable sources for the preparation of ACE inhibitory peptides.Alcalase hydrolysates of peanut, mung bean and rice protein isolates with potent ACEinhibitory activity were refinedly prepared by desalinization and ultrafiltration. The saltcontents of the three kinds of proteins hydrolysates with high salt contents above 7% beforedesalinization were significantly decreased and the protein contents were increased afterdesalinization by ion exchange resin. The ACE inhibitory activities of the hydrolysates werenot changed by the treatment of desalinization, but increased by hollow fibre ultrafiltrationwith molecular weight cut-off 6000 Da, suggesting ultrafiltration is an effective technique toenrich for food protein-derived ACE inhibitory peptides. The 6000 Da permeates have highercontents of hydrophobic amino acids than the corresponding unhydrolyzed proteins.The impact of digestive proteases on ACE inhibitory activity of hydrolysates of peanut,mung bean and rice protein isolates were evaluated under simulated gastrointestinal digestionand the antihypertensive effects of these hydrolysates were investigated in spontaneouslyhypertensive rats (SHR). All of these three kinds of protein hydrolysates showedantihypertensive activities after single oral administration at a dose of 600 mg/kg of bodyweight. Mung bean protein isolates hydrolysate had the most potent antihypertensive activityand the least for peanut protein isolates hydrolysate among the three kinds of proteinhydrolysates. Maximum reduction of 30.8 and 19.7 mmHg in systolic blood pressure (SBP)was observed for mung bean and rice protein isolates hydrolysates 6 h afeter single oraladministration at a dose of 600 mg/kg of body weight, respectively, while the hydrolysate ofpeanut protein isolates was seen by a decrease of 13 mmHg 4 h after oral administration. Theinhibitory potencies of the three kinds of protein hydrolysates on ACE activity did notcorrelate with their antihypertensive activities found in SHR, suggesting that in vivoexperiments are necessary for the evaluation of the antihypertensive activities. There were nosignificant changes in the heart rate of SHR after oral administration of protein hydrolysates,suggesting that administration of the hydrolysates did not have a bad effect on the circulatorysystem of SHR.Good isolation performance was obtained under the following conditions when isolatingACE inhibitory peptides from hydrolysates of peanut, mung bean and rice protein isolates onSephadex G-10 column (1.8×60 cm): sample concentration, 100 mg/mL;injection volume,1.5 mL;flow rate, 0.4 mL/min;eluant, 0.02 mol/L sodium acetate-acetic acid buffer solution(pH 4.0). Seven kinds of novel ACE inhibitory peptides i.e. QGGSGMTLAFPLPK (frompeanut protein), KIFLRLS (from peanut protein), KNPLAGRDMDVF (from mung beanprotein), KDYRL (from mung bean protein), VTPALR(from mung bean protein),KLPAGTLF (from mung bean protein), and TQVY (from rice protein) with the IC50 values of10.6 μM, 36.6 μM, 8.3 μM, 26.5 μM, 82.4 μM, 134.1 μM, 18.2 μM, respectively, wereisolated and identified from hydrolysates of peanut, mung bean and rice protein isolates bysequential chromatographic method (Sephadex G-15 gel filtration chromatography, two-stepreverse-phase high performance liquid chromatography), and matrix assisted-laserdesorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS) andamino acid composition analysis.