Potent Angiotensin-converting Enzyme Inhibitory Peptides Research Articles

Release of an encrypted, highly potent ACE-inhibitory peptide by enzymatic hydrolysis of moth bean (Vigna aconitifolia) protein.

Dietary approaches for the regulation of blood pressure are the need of the hour. Hence, identifying the foods possessing such activity is gaining importance. With this aim, moth bean (Vigna aconitifolia), an underutilized pulse, was explored for the presence of antihypertensive activity in terms of angiotensin converting enzyme (ACE)-inhibition bioactivity. Defatted moth bean protein concentrate was hydrolyzed by using different proteases including Alcalase, papain, and trypsin, to identify the enzyme producing highly potent ACE inhibitory peptides. The hydrolysate showing the highest ACE inhibitory activity was further fractionated using an ultrafiltration membrane (10, 3 and 1 kDa) based on ACE inhibitory activity. The active fraction was further subjected to the ion-exchange chromatography followed by RP-HPLC and LC-MS/MS analysis for the enrichment and identification of ACE inhibitory peptides. Finally, based on the bioinformatic analysis, few peptides were synthesized and evaluated for ACE inhibitory activity, followed by docking study and molecular dynamic simulation of a peptide with the highest ACE inhibitory activity. Out of the three proteases, Alcalase-derived hydrolysate showed the highest (~59%) ACE inhibition activity. Molecular weight-based fractionation revealed that <1 kDa fraction possessed the highest ACE inhibitory activity. Activity guided separation of 1 kDa fraction using ion-exchange chromatography, RP-HPLC and LC-MS/MS showed the presence of about 45 peptides. Based on the bioinformatic analysis, 15 peptides were synthesized and evaluated for ACE inhibitory activity. Among these, a novel octapeptide FPPPKVIQ showed the highest ACE inhibitory activity (93.4%) with an IC50 of 0.24 μM. This peptide retained about 59% activity post gastrointestinal digestion simulation. A Dixon plot as well as docking studies revealed the uncompetitive inhibitory nature of this peptide with a Ki value of 0.81 μM. Molecular dynamic simulation studies till 100 ns ensured the stability of the ACE-peptide complex. Thus, present study identified a novel potent ACE inhibitory peptide from moth bean that can be incorporated in a functional dietary formulation for regulation of hypertension.

A Review on Factors and Processing Methods Affecting Antihypertensive Properties of Legumes, and Antihypertensive Properties of Selected Legumes

Hypertension is one of the major risk factors which leads to cardiovascular diseases, and the typical treatment for hypertension is drug therapy. However, as there are side effects to drug therapy, there has been an increase in research on legume proteins as biopeptides have shown antihypertensive effects. Many species of legumes have been cultivated for consumption as they are a good source of protein, carbohydrates, minerals, vitamins, and dietary fiber. Bioactive compounds of legumes potentially improve factors that affect antihypertensive properties, such as angiotensin-converting enzyme (ACE) inhibition activity, renin inhibition activity, and γ-aminobutyric acid (GABA) production. The processing method of enzymatic hydrolysis can improve ACE inhibition activity, as can be seen with horse gram, pigeon pea, and lentil hydrolysates containing potent ACE inhibitory peptides of Thr-Val-Gly-Met-Thr-Ala-Lys-Phe, Val-Val-Ser-Leu-Ser-Ile-Pro-Arg, and Asn-Ser-Leu-Thr-Leu-Pro-Ile-Leu-Arg-Tyr-Leu, while pigeon pea and kidney bean hydrolysates have shown good renin inhibition activity. Fermentation can also be used to process legumes as potent ACE inhibitory peptide Val-Val-Ser-Leu-Ser-Ile-Pro-Arg was identified from fermented pigeon pea, while kidney beans and lentils demonstrated good GABA production through natural fermentation and fermentation with microbial cultures. The germination processing method could also help improve ACE inhibition activity as horse gram has shown good inhibition activity. In vivo study of pigeon pea, kidney bean, and lentils showed potential antihypertensive properties as a significant lowering of systolic blood pressure of test subject was observed. Research done on the structure and function of antihypertensive properties of legumes can help in the development of functional food products which will be beneficial to human health.

Two novel potent ACEI peptides isolated from Pinctada fucata meat hydrolysates using in silico analysis: identification, screening and inhibitory mechanisms.

The aim of this study was to discover potent angiotensin-converting enzyme (ACE) inhibitory (ACEI) peptides from Pinctada fucata (P. fucata) for treating hypertension and to characterize them using in silico analysis. The P. fucata proteins were hydrolyzed by Alcalase®, a serine endopeptidase with broad selectivity, at various times (0, 2, 4, 6, 8, 10 h). The degree of hydrolysis (DH) and ACEI activity of the different hydrolysates were measured. Considering the molecular weight and ACEI activity, the 10 h hydrolysate was purified by a series of traditional separation methods, including ultrafiltration, gel G-25 chromatography, and reversed-phase high-performance liquid chromatography (RP-HPLC), with ACEI activity as a guide. The results showed two fractions, C17 and C18, eluted by means of semi-preparative RP-HPLC, and showed the highest ACEI activities of 80.33 ± 2.70% and 81.66 ± 0.29%, respectively, at 1 mg mL−1. The two fractions were then identified using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) and their MS/MS spectra data were subjected to de novo sequencing. Subsequently, the potential ACEI peptides were screened by in silico methods, namely, to analyze the average local confidence (ALC) value obtained from the sequencing software and the P-value from the Pepsite 2. In total, 13 potential ACEI peptide sequences were obtained and identified from the two fractions by LC-ESI-MS/MS, and two novel tetrapeptides, FRVW (607.3314 Da) and LPYY (555.2881 Da), were screened for synthesis according to the in silico analysis. The in vitro ACEI tests indicated that FRVW and LPYY had IC50 values of 18.34 and 116.26 μM, respectively. The Lineweaver–Burk plot showed that FRVW was a noncompetitive inhibitor, and LPYY was shown to be a mixed-mode type inhibitor. A stability study against ACE indicated that both peptides were hydrolyzed by ACE to some extent, the higher ACEI activity following incubation with ACE indicating that they should be classified as pro-drug substrates. Molecular docking results showed that hydrophobic amino acids (HAAs) within peptides formed vital interactions including hydrogen bonds, electrostatic forces, van der Waals forces and Pi–Pi interactions with ACE residues, which stabilized the enzyme–peptide complex. Furthermore, the docking results accorded with the inhibition kinetic mode. Our study demonstrated that FRVW and LPYY isolated from P. fucata have potential applications as antihypertensive agents.

In Silico Evaluation of Food Derived Bioactive Peptides as Inhibitors of Angiotensin Converting Enzyme (ACE)

Hypertension is declared as the major risk factor of cardiovascular diseases and stroke, and the leading cause of premature deaths. ACE is a zinc dependent dipeptidyl peptidase and plays key role in controlling blood pressure via renin angiotensin system (RAS), and hence serves as the promising target for antihypertension drugs. Many food derived antihypertensive peptides have been identified recently. However, their ACE inhibitory activity, interactions and stability are not fully evaluated. Our work focused on combination of modern bioinformatics techniques for efficient evaluation of potent ACE inhibitory food peptides and understanding of interactions between ACE and inhibitory peptides. We reported novel antihypertensive peptide IQDVPS, LQPGS, VIP from date, salmon and soybean proteins respectively. Food proteins were digested in-silico to release peptides. Molecular docking studies revealed high binding affinities and interactions with ACE active site. MD simulations and Alanine Scanning were carried out to study the stability of these ACE-peptide complexes in cell like environment. The results showed that the suggested peptides competitively inhibit ACE by tightly binding to its active site, meanwhile maintaining the structural stability of the complex. ACE-LQPGS (Salmon) was found to have best binding with least structural fluctuations.

Development of a Soy Protein Hydrolysate with an Antihypertensive Effect.

In this study, we combined enzymatic hydrolysis and lactic acid fermentation to generate an antihypertensive product. Soybean protein isolates were first hydrolyzed by Prozyme and subsequently fermented with Lactobacillus rhamnosus EBD1. After fermentation, the in vitro angiotensin-converting enzyme (ACE) inhibitory activity of the product (P-SPI) increased from 60.8 ± 2.0% to 88.24 ± 3.2%, while captopril (a positive control) had an inhibitory activity of 94.20 ± 5.4%. Mass spectrometry revealed the presence of three potent and abundant ACE inhibitory peptides, PPNNNPASPSFSSSS, GPKALPII, and IIRCTGC in P-SPI. Hydrolyzing P-SPI with gastrointestinal proteases did not significantly affect its ACE inhibitory ability. Also, oral administration of P-SPI (200 mg/kg body weight) to spontaneous hypertensive rats (SHRs) for 6 weeks significantly lowered systolic blood pressure (−19 ± 4 mm Hg, p < 0.05) and controlled body weight gain relative to control SHRs that were fed with physiological saline. Overall, P-SPI could be used as an antihypertensive functional food.

Molecular interactions, bioavailability, and cellular mechanisms of angiotensin-converting enzyme inhibitory peptides.

Food-derived angiotensin-converting enzyme inhibitory (ACEi) peptides have gained substantial interest as potential alternatives to synthetic drugs in the management of hypertension. Peptide size and sequence are two critical factors that determine their potency, bioavailability, and cellular mechanisms. Molecular interaction studies between ACE and ACEi peptides support that potent ACEi peptides are generally composed of hydrophobic, positively charged, and aromatic or cyclic amino acid residues at the third, second, and first position from the C-terminus, respectively. Small peptides containing N-terminal Tyr and/or C-terminal Pro could improve their stability against enterocyte peptidases, thus their bioavailability. Different ACEi peptides can reduce aberrant cellular proliferation, excessive inflammation, and oxidative stress but through different mechanisms. Further understanding the structure-activity-bioavailability relationships will help design novel potent ACEi peptides with improved bioavailability and in vivo efficacy. PRACTICAL APPLICATIONS: ACEi peptides have the potential for uses as functional food ingredients against hypertension.

Identification and molecular docking study of novel angiotensin-converting enzyme inhibitory peptides from Salmo salar using in silico methods.

In order to circumvent some challenges of the classical approach, the in silico method has been applied to the discovery of angiotensin-converting enzyme (ACE) inhibitory peptides from food proteins. In this study, some convenient and efficient in silico tools were utilized to identify novel ACE inhibitory peptides from Salmo salar. Collagen from Salmo salar was digested in silico into hundreds of peptides. Results revealed that tetrapeptides PGAR and IGPR showed potent ACE inhibitory activity, with IC50 values of 0.598 ± 0.12 and 0.43 ± 0.09 mmol L-1 , respectively. The molecular docking result showed that PGAR and IGPR interact with ACE mostly via hydrogen bonds and attractive charge. Peptide IGPR interacts with Zn+ at the ACE active site, showing high inhibitory activity. Interaction with Zn+ in ACE may lead to higher inhibitory activity of peptides, and Pi interactions may promote the effect of peptides on ACE. The in silico method can be an effective method to predict potent ACE inhibitory peptides from food proteins. © 2018 Society of Chemical Industry.

Novel angiotensin-converting enzyme inhibitory peptides from caseins and whey proteins of goat milk

Angiotensin-converting enzyme (ACE) plays a central role in blood pressure regulation by producing the vasoconstrictor angiotensin II. The inhibition of ACE with natural inhibitors, as alternatives to avoid the side effect of synthetic drugs, is a major target in the prevention of hypertension. In this study, we examined the separated caseins and whey proteins of goat milk for the presence of ACE inhibitory peptides. Digestion of isolated whey proteins and caseins of goat milk by gastric pepsin generated soluble hydrolysates exhibiting significant inhibition of ACE compared to weak inhibition by undigested proteins. The hydrolysates were fractionated by size exclusion chromatography, Sephacryl S-100 column, into four fractions (F1–F4). The late-eluting fraction (F4) of either whey or caseins exhibited greater ACE inhibition. Peptides in both F4 fractions, isolated by RP-HPLC, exhibited variable ACE inhibitory activities with the hydrophobic peptide peaks being the most potent ACE inhibitors. MALDI-TOF MS/MS resulted in identification of three potent ACE inhibitory peptides: PEQSLACQCL from β-lactoglobulin (residues 113–122), QSLVYPFTGPI from β-casein (residues 56–66), and ARHPHPHLSFM from κ-casein (residues 96–106). The peptides from whey and caseins exert significant ACE inhibitory activities comparable to that of captopril, an antihypertensive drug, exhibiting IC50 values of 4.45μM and 4.27μM, respectively. The results introduce, for the first time, new potent ACE-inhibitory peptides that can be released by gastric pepsin of goat milk whey and caseins and thus may pave the way for their candidacy as anti-hypertensive bioactive peptides and prevention of associated disorders.

High-level expression of angiotensin converting enzyme inhibitory peptide Tuna AI as tandem multimer in Escherichia coli BL21 (DE3)

Food-derived angiotensin converting enzyme inhibitory peptides (ACEIP) show a great promise for prevention and treatment of hypertension. Tuna muscle derived peptide, Tuna AI (PTHIKWGD), is a potent ACEIP with high activity and stability. To improve the expression level of Tuna AI in Escherichia coli, multimer genes of Tuna AI were assembled and expressed as fusion proteins by constructing the vectors of pET30-4nTunaAI (n=1, 2, 4 and 8). The results showed the multimer genes were successfully expressed in the constructed system, and their expression level improved significantly with increasing repeat degree. The E. coli harboring pET30-32TunaAI was selected for production of peptide based on its highest expression level and its fusion protein was suit for urea-washing purification. The peptide monomer was released from the fusion protein by formic acid cleavage, and successively purified by membrane filtration, cation exchange chromatography and size exclusion chromatography, with a final yield of 218.9mg/L. The purified monomeric peptide was confirmed by mass spectrometry analysis, N-terminal sequencing, and activity measurement. This peptide resulted in a significant decrease of systolic blood pressure by 36.5mmHg in rats at 4h. These results suggest our strategy is an economic solution for the mass production of antihypertensive peptides.

Effect of power ultrasound pretreatment on peptidic profiles and angiotensin converting enzyme inhibition of milk protein concentrate hydrolysates

The use of power ultrasound as a pretreatment to enhance the hydrolysis of milk protein concentrate (MPC) and subsequent angiotensin converting enzyme (ACE) inhibitory activity has been studied. Liquid chromatography was used to analyse peptide profiles of Neutrase-derived MPC hydrolysates after pretreatment at 0, 1, 3, 5 and 8 min at an ultrasound power level of 800 W. The peptide profiles indicated an increase in number of peptides when ultrasound pretreatment was applied. There was also an increase in the degree of hydrolysis of MPC hydrolysates. The profiles indicated that new small peptides in ultrasound pretreated samples (1-5 min) which were not present in the control samples and 8 min pretreated samples, could be responsible for increased ACE inhibitory activity. These small peptides were digested in the 8 min pretreated samples. Ultrasound pretreatment of MPC increases the ACE inhibitory activity of the hydrolysates because of the production of new small peptides. This can be used as a means to derive potent ACE inhibitory peptides at industrial scale in complex protein sources.

Functional Properties and Bioactivities of Pine Nut (Pinus gerardiana) Protein Isolates and Its Enzymatic Hydrolysates

The functional properties and bioactivities of the pine nut protein isolates (PPI) and its enzymatic hydrolysates (PPH) prepared with Alcalase at 5 %, 10 %, 15 % and 25 % degree of hydrolysis (DH) were studied. The solubility of PPH significantly increased (p < 0.05) with the increase of the DH, while the foaming capacity of PPH was only improved at a low DH. However, enzymatic hydrolysis reduced the emulsifying capacity of PPH. The DPPH radical scavenging and inhibition of linoleic acid oxidation activities of PPH were significantly improved by a low DH (5 %) compared with those of PPH with a higher DH and the original PPI (p < 0.05). The reducing power of PPH at all DH decreased in comparison to that of the original PPI. Potent angiotensin-converting enzyme (ACE) inhibitory peptides could be generated by hydrolysis with Alcalase, and the ACE inhibitory activity of PPH increased (p < 0.05) with the DH. These results revealed that a low degree of enzymatic hydrolysis was appropriate to obtain PPH with improved functional properties and good antioxidant activities, while a high degree of hydrolysis was essential to obtain highly potent ACE inhibitory peptides from PPI. These results suggest that the control of the DH may be an effective strategy to modify specific functional and bioactive properties of PPH, and PPH has potential as a functional food ingredient for related functional and health benefits.

Enzyme Hydrolysates from Stichopus horrens as a New Source for Angiotensin-Converting Enzyme Inhibitory Peptides.

Stichopus horrens flesh was explored as a potential source for generating peptides with angiotensin-converting enzyme (ACE) inhibitory capacity using 6 proteases, namely alcalase, flavourzyme, trypsin, papain, bromelain, and protamex. Degree of hydrolysis (DH) and peptide profiling (SDS-PAGE) of Stichopus horrens hydrolysates (SHHs) was also assessed. Alcalase hydrolysate showed the highest DH value (39.8%) followed by flavourzyme hydrolysate (32.7%). Overall, alcalase hydrolysate exhibited the highest ACE inhibitory activity (IC50 value of 0.41 mg/mL) followed by flavourzyme hydrolysate (IC50 value of 2.24 mg/mL), trypsin hydrolysate (IC50 value of 2.28 mg/mL), papain hydrolysate (IC50 value of 2.48 mg/mL), bromelain hydrolysate (IC50 value of 4.21 mg/mL), and protamex hydrolysate (IC50 value of 6.38 mg/mL). The SDS-PAGE results showed that alcalase hydrolysate represented a unique pattern compared to others, which yielded potent ACE inhibitory peptides with molecular weight distribution lower than 20 kDa. The evaluation of the relationship between DH and IC50 values of alcalase and flavourzyme hydrolysates revealed that the trend between those parameters was related to the type of the protease used. We concluded that the tested SHHs would be used as a potential source of functional ACE inhibitory peptides for physiological benefits.

Angiotensin I Converting Enzyme Inhibitory Peptides from Simulated in Vitro Gastrointestinal Digestion of Cooked Eggs

Egg proteins are an excellent source of bioactive peptides. The purpose of this work was to study the effect of cooking methods on the production of angiotensin converting enzyme (ACE) inhibitory peptides. Boiled or fried eggs (in the forms of whites, yolks, and whole eggs) were digested by gastrointestinal tract proteases at simulated gut conditions. Fried egg digests showed more potent activity than those of boiled egg digests; the fried whole egg digest had an IC(50) value of 0.009 mg protein/mL. This hydrolysate was further purified by cation exchange chromatography and gel filtration chromatography. Seven peptides, Val-Asp-Phe (IC(50): 6.59 microM), Leu-Pro-Phe (10.59 microM), Met-Pro-Phe (17.98 microM), Tyr-Thr-Ala-Gly-Val (23.38 microM), Glu-Arg-Tyr-Pro-Ile (8.76 microM), Ile-Pro-Phe (8.78 microM), and Thr-Thr-Ile (24.94 microM), were identified by liquid chromatography-mass spectrometry (LC-MS/MS), and their IC(50) values were predicted by using our previously reported structure and activity models. The presence of several tripeptides from in vitro simulated gastrointestinal egg digest indicates that these peptides may be absorbed into the body and exert an in vivo antihypertensive activity, although in vivo study is needed to confirm this assumption. Our results showed that in vitro digestion of cooked eggs could generate a number of potent ACE inhibitory peptides which may have implications for cardiovascular disease prevention, including hypertension.

Identification of novel angiotensin‐converting enzyme‐inhibitory peptides from ovine milk proteins by CE‐MS and chromatographic techniques

In this report, we present the use of CE-MS as complement to RP separation for the identification of novel angiotensin-converting enzyme-inhibitory (ACEI) peptides from a complex milk protein hydrolysate. As preliminary step, fast protein LC (FPLC) was used to isolate the different casein fractions from raw ovine milk. Enzymatic hydrolysis of these fractions was performed by using proteolytic enzymes of gastrointestinal origin. The most active hydrolysate, corresponding to kappa-casein hydrolyzed with pepsin, chymotrypsin, and trypsin, was fractionated by RP-HPLC and the peptides contained in the active fractions were sequenced by CE coupled to IT-MS (CE-MS). The use of CE-MS allowed the identification of short peptides with ACEI activity included in the scarcely retained fraction obtained by semipreparative RP-HPLC. Among the identified peptides, those with hydrophobic or positively charged residues at the C-terminal tripeptide were chemically synthesized to determine their ACEI activity. This procedure allowed us to identify four novel potent ACEI peptides from kappa-casein with sequences IAK, YQQRPVA, WQVLPNAVPAK, and HPHPHLSF. These active sequences could be obtained by enzymatic hydrolysis either of the individual kappa-casein fraction or the total casein fraction from ovine milk.

Inhibitory effect of tuna peptide on endothelin production in cultured endothelial cells.

The effect of tuna muscle-derived angiotensin-converting enzyme (ACE) inhibitory peptide (tuna AI) on the production of potent contracting factor, endothelin-1 (ET), in the cultured bovine aorta endothelial cells (BAECs) has been investigated. ET production in the culture medium was measured by radioimmunoassay. BAECs spontaneously produced ET in a time-dependent manner for 4 to 24 h after incubation. Addition of tuna AI to the medium resulted in a time- and concentration-dependent decrease in the production of ET. The tuna AI-induced inhibition of ET production was blocked by B-3824 (bradykinin (BK) B2-receptor antagonist), NG-monomethyl-L-Arg and indomethacin, but not by angiotensin II receptor antagonist. While BK, sodium nitroprusside and platelet-derived growth factor (PDGF) significantly inhibited ET production, angiotensin II and interleukin-1 did not. Potent ACE inhibitory peptides among tuna AI analogues did not always inhibit ET production. The results suggest that tuna AI inhibits ET production at least in part via potentiation of PDGF expression in addition to potentiation of BK pathway.