Enzyme Inhibitory Peptides Research Articles

Optimization of chemical and enzymatic hydrolysis for production of chicken blood protein hydrolysate rich in angiotensin-I converting enzyme inhibitory and antioxidant activity

Response surface methodology was adopted to optimize hydrolysis conditions for the production of antioxidant and angiotensin-I converting enzyme (ACE) inhibitory peptides from chicken red blood cells by both enzymatic and acid hydrolysis. During acid hydrolysis, temperature (P < 0.001) and acid concentration (P < 0.001) influenced the degree of hydrolysis (DH%) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity of the hydrolysate while ACE inhibitory activity of the hydrolysate was strongly influenced by acid concentration (P < 0.001). Temperature and time of hydrolysis had no effect (P > 0.05) on the ACE inhibitory activity of the hydrolysate. Acid hydrolysis conditions of 50°C, 32 h, and 0.03 N hydrochloric acid resulted in optimum DH% (33.1%), optimum DPPH scavenging activity (46%), and optimum ACE inhibitory activity (43.7%) of the hydrolysate. During enzymatic hydrolysis of chicken red blood cells, DH% was influenced by the temperature of hydrolysis (P < 0.001) and enzyme concentration (P < 0.001). DPPH scavenging of the hydrolysate was marginally (P < 0.05) influenced by the temperature of hydrolysis and ACE inhibitory activity of the hydrolysate was highly influenced by temperature (P < 0.001) and enzyme concentration (P < 0.001). Enzyme hydrolysis conditions of 60°C, 150 min, and 2.5% alcalase resulted in maximum DH% of 63.9%, while the highest DPPH scavenging activity (75%) of hydrolysate was observed under the hydrolysis conditions of 60°C, 30 min, and 2.5% of the enzyme. Optimum ACE inhibitory activity (45%) of the hydrolysate was achieved at hydrolysis conditions of 2.5% alcalase, 120 min of hydrolysis at 60°C. ACE inhibitory activity of the enzymatically hydrolyzed product was directly proportional to DH%, while DPPH activity was inversely proportional to DH%. DPPH scavenging activity of the acid hydrolysate was recorded at a lower range (34.8–56.9%) compared to the enzyme hydrolysate (40.4–77.4%), while ACE inhibitory activity of both the hydrolysates was observed in the same range (18.7–49.4 and 14.2–47.7% for acid and enzyme hydrolysate, respectively). This study indicated that chicken red blood cells could be successfully hydrolyzed by both chemical and enzymatic methods to obtain hydrolysates having antioxidant and ACE inhibitory activity.

Simulated in vitro gastrointestinal digestion of traditional Chinese Rushan and Naizha cheese: Peptidome profiles and bioactivity elucidation

Chinese Rushan and Naizha, the traditional acid coagulated cheese types produced from cow and yak milk, respectively, have been consumed for more than thousands of years. In this study, we aimed to characterise peptides of Rushan and Naizha in simulated in vitro gastrointestinal digestion using label-free based peptidomic. The identified peptide sequences were subjected to BIOPEP database driven bioactivity search. In total, 309 and 225 peptides were identified from Rushan and Naizha cheese, respectively, corresponding to 20 protein annotations. Analysis of label-free quantification found different protein digestibility, where casein was the primary source of peptides in Rushan, among which 62% represented β-casein by peptide count. The release of peptides was concentrated in specific residues 145–155 of β-casein in Rushan. In contrast, κ-casein and 7 minor milk proteins were dominant in digestion of Naizha cheese (p < 0.05). In particular, there were 11 peptides from digestion that were exact matches in databases to sequences with immunomodulatory, antibacterial, ACE-inhibition, DPP IV inhibition and antioxidant activities. Four novel angiotensin I-converting enzyme inhibitory (ACEI) activities peptides (YPFPGPIH, LKNWGEGW, RELEEIR, and HPHPHLS) were explored using molecular docking, chemically synthesized, and in vitro ACEI activity. The peptides had lower estimated free energy values (−5.34 to −7.66 kcal/mol), and exhibited the lowest IC50 value of 109.5, 77.7, 196.6, and 64.30 μM, respectively. Our study is the most comprehensive peptidomic analysis of Chinese Rushan and Naizha cheese to date.

Transport, In Vivo Antihypertensive Effect, and Pharmacokinetics of an Angiotensin-Converting Enzyme (ACE) Inhibitory Peptide LVLPGE

The angiotensin-converting enzyme (ACE) inhibitory peptide LVLPGE provides outstanding antihypertensive effects in vivo, with a maximum systolic blood pressure (SBP) drop of 39 mmHg at a dose of 10 mg/kg. We evaluated the gastrointestinal digestion, transport, and in vivo antihypertensive effects of LVLPGE at different doses. LVLPGE was resistant to gastrointestinal enzymes with a stability of 97.8% and a permeability Papp of (5.09 ± 0.94) × 10-7 cm/s. LVLPGE was mainly transported through the Caco-2 cell monolayer by the peptide transporter PepT 1 and passive-mediated transport. LVLPGE at doses of 30 and 50 mg/kg had a positive antihypertensive effect in vivo; 30 mg/kg had a more significant effect than 50 mg/kg. After oral administration, the pharmacokinetics of LVLPGE showed that the Cmax was 4.65 ng/mL at 2 min. The blood pressure-lowering effect increased as the concentration of LVLPGE increased in the plasma of spontaneous hypertensive rats (SHRs).

Activity and bioavailability of food protein-derived angiotensin-I-converting enzyme-inhibitory peptides.

Angiotensin-I-converting enzyme (ACE) inhibitory peptides are able to inhibit the activity of ACE, which is the key enzymatic factor mediating systemic hypertension. ACE-inhibitory peptides can be obtained from edible proteins and have the function of antihypertension. The amino acid sequences and the secondary structures of ACE-inhibitory peptides determine the inhibitory activities and stability. The resistance of ACE-inhibitory peptides to digestive enzymes and peptidase affect their antihypertensive bioactivity in vivo. In this paper, the mechanism of ACE-inhibition, sources of the inhibitory peptides, structure-activity relationships, stability during digestion, absorption and transportation of ACE-inhibitory peptides, and consumption of ACE-inhibitory peptides are reviewed, which provide guidance to the development of new functional foods and production of antihypertensive nutraceuticals and pharmaceuticals.

Stability and angiotensin converting enzyme inhibitory activity of peptide RVPSL-loaded graphene oxide

Previous work has demonstrated that the angiotensin converting enzyme (ACE) inhibitory peptide RVPSL is very unstable and loses its ACE inhibitory activity in the gastrointestinal tract. Thus, graphene oxide (GO) was used to improve the stability of RVPSL in the present work. The structure and morphology of RVPSL-GO were characterised using Fourier transform-infrared spectroscopy, X-ray photoelectron spectroscopy, scanning electron microscopy, and atomic force microscopy. Moreover, the cytotoxicity of RVPSL-GO was examined using HepG2 cells. RVPSL was successfully covalently bonded to GO and the RVPSL-loading capacities of RVPSL-GO were calculated to be 1.05 mg RVPSL/mg GO, and the loading efficiency value was 95.02%. The results showed that GO enhanced RVPSL ACE activity (at 0.026 mg/mL) from 26.47 to 39.70%. This may have caused a higher local concentration of RVPSL in the solution after GO was modified. Moreover, the stability of RVPSL was improved with protection from GO. The ACE inhibition rate of RVPSL-GO was 49.08%, while RVPSL was completely degraded after 2 h in simulated gastrointestinal digestion. In addition, RVPSL-GO displayed high viability for HepG2 cells with no significant cytotoxicity.

Accurate prediction for adsorption rate of peptides with high ACE-inhibitory activity from sericin hydrolysate on thiophene hypercross-linked polymer using CoMSIA in 3D-QSAR model

Efficient screening of angiotensin converting enzyme inhibitory (ACE-I) peptides from agricultural or edible sources attract increasing attention. However, their purification process from the complex natural system is still semi-empirical or even uncontrollable, which has seriously reduced their screening efficiency. Herein, inspired by the prediction of ACE-I activity, 3D-QSAR was proposed to predict the adsorption performance of peptides from sericin hydrolysate (SH) having high ACE-I activity on porous hypercross-linked polymers according to their molecular structures. Thiophene hypercross-linked polymer (T-HCP) possessing better screening capacity for ACE-I peptides was chosen as our research object in this work. The sequence and relative adsorption rate of 101 peptides in SH were analyzed by LC-MS and was used as a database to construct the relationship of peptide’s chemical structure and adsorption performance on T-HCP by Comparative molecular similarity indices analysis (CoMSIA) from 3D-QSAR. Optimum CoMSIA revealed that enhanced interaction of hydrophobicity and H-bond between T-HCP and the peptide was conducive to increase the adsorption performance of di- to hexa- peptides. Based on these relationships, the adsorption capability of 24 designed peptides with distinguished hydrophobic and H-bond fields was predicted on T-HCP by using optimum CoMSIA and the results of half of these were verified, which showed high consistency with their predicted adsorption rate. Interestingly, these peptides having higher adsorption capacities on T-HCP also possessed higher ACE-I activity. This can be attributed to the high concentration of aromatic surface with π-π interaction and weak-polar CSC group with H-bond interaction on T-HCP material, which is ideal for the selective adsorption of peptides with higher ACE-I activity from SH. This study provides important theoretical guidance for the industrial screening of bio-functional peptides from complex protein mixtures.

Purification, characterization and molecular docking study of angiotensin-I converting enzyme (ACE) inhibitory peptide from shortfin scad (Decapterus macrosoma) protein hydrolysate.

Hypertension is a threatening chronic disease, which become a global killer among the adult population. The mortality rate increasing day by day even several Angiotensin I-converting enzyme (ACE) inhibitor drugs were introduced. Bioactive peptides derived from aquatic resources exhibits potential ACE inhibitory activity. The objective of this work is to report the purification and molecular docking studies of angiotensin-I converting enzyme (ACE) inhibitory peptide isolated from shortfin scad (Decapterus macrosoma) waste protein hydrolysate (SWH), enzymatically prepared by using alcalase. The purification process included ultrafiltration, gel filtration and reverse phase high performance liquid chromatography (RP-HPLC). Results showed that ultra-filtered peptide fraction (< 3kDa) possessed the highest ACE inhibitory activity, followed by the fraction 14 by gel filtration. Fraction P obtained by RP-HPLC, with the amino acid sequence of RGVGPVPAA (IC50 = 0.20mg/ml) was identified. In terms of ACE inhibition, the Lineweaver-Burk plot showed that the SWH peptide obtained acted as a competitive ACE inhibitor. The molecular docking studies showed that the SWH peptide exhibit hydrogen bonds and Pi-interactions with ACE by Z-dock scores. These results showed that the purified peptide isolated from shortfin scad waste hydrolysate has potential antihypertensive properties which could potentially be used as functional food ingredients.

The production of collagen type I hydrolyzate derived from tilapia (Oreochromis sp.) skin using thermoase PC10F and its in silico analysis

ABSTRACT The main focus of this study was to produce the angiotensin-I converting enzyme (ACE) inhibitory peptides from collagen type I using new combination of Thermoase PC10F and tilapia skin as a protein collagen source. In silico evaluation of amino acids profile of collagen type I, ACE-inhibitory peptides and sensory profile of the hydrolyzate were performed using Protparam and BIOPEP, respectively, to initially predict the potential peptide produced. Hydrolysis conditions were studied based on the best preliminary design of degree hydrolysis (DH) using o-phthalaldehyde (OPA) method. The temperature of 60°C, pH of 5 and an enzyme to substrate ratio of 1:100 were observed as the best conditions to obtain the highest DH using Thermoase PC10F. Peptide chain length (PCL) showed a proportional decrease as DH increased. In silico study of alpha 1 and alpha 2 collagen type I indicated a significant amount of glycine and proline, which are known to stabilize the collagen structure. The profile of potential biological activity suggested that 247 ACE-inhibitory peptides could be obtained from the parent protein sequence of collagen type I alpha 1 and alpha 2. In silico hydrolysis by thermolysin is believed to release potent theoretical peptides with IC50 less than 5 μM for both collagen type I subunits with a high number of occurrences. Further analysis on its potential sensory peptides had shown that both alpha 1 and alpha 2 contained mainly sweet amino acid, bitter peptides, and bitter amino acids.

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.

A novel angiotensin-converting enzyme inhibitory peptide from rabbit meat protein hydrolysate: identification, molecular mechanism, and antihypertensive effect in vivo.

Bioactive peptides exhibiting angiotensin-converting enzyme (ACE) inhibitory effects and extracted from natural foods have potential as healthy and safe therapeutics for high blood pressure. The aim of this study was to isolate and purify ACE inhibitory peptides from rabbit meat protein hydrolysate, to explore the underlying mechanisms by molecular docking, and to evaluate the antihypertensive effects in vivo. A novel ACE inhibitory tetrapeptide Trp-Gly-Ala-Pro (WGAP) was identified and purified from a bromelain hydrolysate. WGAP acted against ACE in a non-competitive manner with an IC50 of 140.70 ± 4.51 μM. It was resistant to enzymatic degradation by pepsin and trypsin in vitro. Molecular docking analysis indicated that WGAP formed stable hydrogen bonds with ACE residues His353, Ala354 and ALA356. In vivo, 100 mg kg-1 WGAP significantly reduced systolic and diastolic blood pressure in hypertensive rats by up to 42.66 ± 2.87 and 28.56 ± 2.71 mmHg, respectively, 4 h after oral administration. ACE inhibitory peptides derived from rabbit meat have potential antihypertensive effects and provide a new route for the exploration of novel hypertension inhibitors and the utilization of rabbit meat.

Antihypertensive potential of fermented milk: the contribution of lactic acid bacteria proteolysis system and the resultant angiotensin-converting enzyme inhibitory peptide.

Hypertension has become an increasing health concern given that it is a major risk for cardiovascular disease. Synthetic antihypertensive drugs, including angiotensin-converting enzyme (ACE) inhibitors, effectively control high blood pressure but are associated with unpleasant side effects. Milk fermented by certain lactic acid bacteria (LAB) provides energetic contributions to the management of hypertension, especially the regulation of ACE. LAB are important food-grade microbial organisms that release ACE inhibitory peptides through their unique proteolysis system, which consists of cell-envelope proteinases (CEPs), transporter systems, and intracellular peptidases. Thus, the description of LAB proteolysis system genes and their contributions to ACE inhibitory peptide production is a challenging but promising study. This review provides a survey of LABs with potential ACE inhibitory activity and investigates the research progress of LAB proteolytic systems with an emphasis on the correlation of their components and ACE inhibitory activity. Subsequently, a depiction of the ACE inhibitory peptide action mechanism, structure-activity relationship and bioavailability is presented. The improved functional annotation of LAB proteolytic system genes will provide an excellent framework for future experimental validations of predicted ACE inhibitory activity in fermented milk.

Identification and Antihypertension Study of Novel Angiotensin I-Converting Enzyme Inhibitory Peptides from the Skirt of Chlamys farreri Fermented with Bacillus natto.

The aim of this study was to isolate the angiotensin I-converting enzyme (ACE) inhibitory peptides from the skirt of Chlamys farreri fermented with Bacillus natto and to explore the antihypertension effect through in vivo studies. ACE inhibitory peptides were purified from the fermentation mixture by ultrafiltration, gel filtration chromatography, and reversed-phase high-performance liquid chromatography sequentially. The amino acids' sequence of the five novel ACE inhibitory peptides were identified by liquid chromatography-tandem mass spectrometry. Animal experiments demonstrated that the novel ACE inhibitory peptides significantly reduced the blood pressure in spontaneously hypertensive rats after a single or long-time treatment. Potential mechanisms were explored, and the results indicated that the novel peptides could regulate the renal renin-angiotensin system, improve vascular remodeling, inhibit myocardial fibrosis, and rebalance the gut microbial dysbiosis. Our results suggest that the fermentation products of the Chlamys farreri skirt by B. natto are potential sources of active peptides processing antihypertension activities.

Identification of post-digestion angiotensin-I converting enzyme (ACE) inhibitory peptides from soybean protein Isolate: Their production conditions and in silico molecular docking with ACE

This study attempts to investigate natural angiotensin-I converting enzyme (ACE) inhibitors. Soybean protein isolated (SPI) hydrolysate (SPIH) was prepared by Alcalase from inexpensive SPI, and their ACE inhibitory peptides were obtained via membrane separation, ethanol precipitation and adsorption chromatography enrichment. Activated carbon was more suitable for peptide enrichment than eight macroporous resins. The peptide fraction yielded under optimal conditions (protein-active carbon mass ratio 2:1; adsorption pH 3.0 and time 2 h; desorption time 2 h) exhibited a 10.4 times higher ACE-inhibitory activity than SPIH. Novel peptides IY, YVVF, LVF, WMY, LVLL and FF (hydrophobicity values 10.51–12.87; activity scores 0.2373–0.999) might be the main contributors to SPIH’s ACE inhibition. IY had the lowest IC50 (0.53 ± 0.02 μM). YVVF had the greatest affinity (−9.8 kcal/mol) for 2OC2 (ACE’s C-domain receptor) via H-bonds. IY and WMY could be potent ACE inhibitors, and their ACE-inhibitory activities unaltered and increased after in vitro gastrointestinal digestion.

Safety evaluation of high-dose intake of casein-derived peptide Met-Lys-Pro in healthy adults: A randomized, double-blind, placebo-controlled trial.

Met‐Lys‐Pro (MKP) is a casein‐derived angiotensin I‐converting enzyme inhibitory peptide with the potential to cross the blood–brain barrier. It has shown preventive effects against high blood pressure (BP) and cognitive decline in animal models and human studies. MKP shows good water solubility and can be blended into a variety of foods. However, its ease of intake may contribute to the possibility of overdose. Therefore, we aimed to evaluate the safety of high‐dose intake of MKP in healthy adults by conducting a randomized controlled trial with 30 subjects. Participants were randomly allocated to the MKP (n = 15) or placebo (n = 15) group. Over 4 weeks, the MKP group received test powder containing a daily dose of 1,000 μg of MKP, five times the minimum effective dose for cognitive improvement, whereas the placebo group received dextrin powder containing no detectable MKP. No clinical problems were observed in anthropometric and BP measurements or in blood and urine parameters. No adverse events owing to MKP intake were observed. These findings suggest that consumption of MKP is safe, and that it may be applicable as a safe preventive measure against hypertension and cognitive decline in future.

Production of angiotensin‐converting enzyme inhibitory peptides in Iranian ultrafiltered white cheese prepared with Lactobacillus brevis KX572382

AbstractIn this study, ultrafiltered (UF) Iranian white cheese made with adjunct cultures including six Lactobacillus isolates (Lactobacillus brevis, L. casei and L. plantarum) from traditional Iranian Motal cheese. The peptide extract (&lt;5 kDa) of cheese samples were assessed for angiotensin‐converting enzyme (ACE)‐inhibitory activity during ripening (5 °C). Among the strains used, L. brevis KX572382 (M8) was selected because of the greater increase in (ACE)‐inhibitory activity in the cheese (P &lt; 0.05). The highest activity of M8 extract was observed on the 28th (71.72%) day of ripening (P &lt; 0.05). Proteolytic activity assessment and RP‐HPLC peptide profile of M8 water‐soluble extracts (WSEs) indicated the effect of M8 on further protein degradation due to secondary proteolysis. A total of 7 different peptide sequences, previously known in the literature for their ACE‐inhibitory activity, were tentatively identified by LC/ESI‐MS in 28‐day M8 peptide extract. Although the effect of M8 on pH and the proteolysis development in cheese was significant, no adverse effect was observed on the sensory properties. In conclusion, M8 strain can enhance the functional properties of Iranian UF white cheese.

Proteolysis of tilapia skin collagen: Identification and release behavior of ACE-inhibitory peptides

The structure–activity relationship (SAR) and release behavior of angiotensin I-converting enzyme inhibitory (ACEi) peptides obtained from enzymolysis of collagen influence the large-scale production of ACEi peptides. However, researchers have paid insufficient attention to these areas. In this study, we extracted collagen from tilapia skin, and hydrolyzed it using three proteases. A total of 270 peptides were released from the collagen parent protein. The SAR of these larger collagen ACEi peptides indicated that the presence of proline at position C2 of three C-terminal sequences has a greater effect on increasing the ACEi activity of the peptide than at position C1. The release behavior of these collagen peptides showed that bromelain and alcalase preferentially cleave the N-terminal region of the collagen α1 subunit and then the C-terminal region. These enzymes evenly cleave regions of the collagen α2 subunit. Collagenase preferentially cleaves the C-terminal region of the collagen subunit, followed by the N-terminal region, and then the middle region. The pattern of peptide release from different proteases and the SAR of larger collagen peptides can help guide food production processes to ensure food safety, and to produce high-quality active peptide products.

Identification and analysis of transepithelial transport properties of casein peptides with anticoagulant and ACE inhibitory activities

Casein is an excellent source for producing anticoagulant and angiotensin I-converting enzyme inhibitory (ACEI) peptides. Here, the anticoagulant and ACEI activities of the casein hydrolysate produced by in vitro simulated gastrointestinal (GI) digestion were evaluated. The casein hydrolysate showed potent anticoagulant activity by prolonging the thrombin time (TT) and activated partial thromboplastin time (APTT), and also presenting great ACEI activity, with an IC50 value of 0.52 mg mL−1. Subsequently, the transepithelial transport properties of the casein hydrolysate were analyzed by using the Caco-2 cell monolayer model. The peptides profile of the casein hydrolysate before and after it passed across the Caco-2 cell monolayer were identified by NanoLC-Q-TOF-MS/MS. The results showed that a total of 121 and 184 peptides were identified before and after casein hydrolysate moved through the Caco-2 cell monolayer, respectively. Eighty peptides were presented at both time points of the transport study. Among the 80 peptides, 26 of them were screened with a high possibility of exerting physiological roles after they were absorbed into the blood by in silico methods, and the physicochemical characteristics, e.g., hydrophobicity, net charge, and toxicity of the peptides also be evaluated. Our results provided a new prospect and approach for producing bioactive peptides from casein with anticoagulant and ACEI activities.

New angiotensin-converting enzyme inhibitory peptide from Coix prolamin and its influence on the gene expression of renin-angiotensin system in vein endothelial cells

Coix seed, which is a traditional Chinese medicine, has been used to treat hypertension for thousands of years. It has been shown that Coix prolamin peptides display high levels of angiotensin I converting enzyme (ACE) inhibitory activity. Hence, we purified the ACE inhibitory peptides from Coix prolamin hydrolysates and evaluated the influence of the most potent peptide on the renin-angiotensin system (RAS) genes expression in human umbilical vein endothelial cells (HUVECs). In this study, Coix prolamin peptides were sequentially separated by ultrafiltration, ion exchange chromatography, gel filtration chromatography and RP-HPLC, while the peptide structure was analyzed by mass spectrometry. Next, in silico proteolysis, pharmacophore and molecular docking were further applied to screen and optimize the structure of peptides. Finally, a novel ACE inhibitory peptide VDMF was obtained, in which its influence on the gene expression of RAS signaling pathway in AngⅡ-injury HUVECs was evaluated by quantitative real-time PCR. VDMF significantly down-regulated ACE, AngII type 1 receptor (AT1R) and ACE2 mRNA expression in comparation with model group, while up-regulating Mas gene expression. Hence, we obtained a novel antihypertensive candidate that was derived from the Coix peptides, which could involve a multi-modulation mechanism that regulates blood pressure.

Predictive Modeling of Angiotensin I-Converting Enzyme Inhibitory Peptides Using Various Machine Learning Approaches.

Food-derived angiotensin I-converting enzyme (ACE) inhibitory peptides could potentially be used as safe supportive therapeutic products for high blood pressure. Theoretical approaches are promising methods with the advantage through exploring the relationships between peptide structures and their bioactivities. In this study, peptides with ACE inhibitory activity were collected and curated. Quantitative structure-activity relationship (QSAR) models were developed by using the combination of various machine learning approaches and chemical descriptors. The resultant models have revealed several structure features accounting for the ACE inhibitions. 14 new dipeptides predicted to lower blood pressure by inhibiting ACE were selected. Molecular docking indicated that these dipeptides formed hydrogen bonds with ACE. Five of these dipeptides were synthesized for experimental testing. The QSAR models developed were proofed to design and propose novel ACE inhibitory peptides. Machine learning algorithms and properly selected chemical descriptors can be promising modeling approaches for rational design of natural functional food components.

ACE inhibitory peptide KYIPIQ derived from yak milk casein induces nitric oxide production in HUVECs and diffuses via a transcellular mechanism in Caco-2 monolayers

The purpose of this study was to explore the effects of KYIPIQ, an angiotensin I-converting enzyme (ACE) inhibitory peptide found in yak milk casein, on the production of nitric oxide (NO) in human vascular endothelial cells (HUVECs). Additionally, we also sought to study the transport pathway of this peptide across monolayers of the human epithelial colorectal adenocarcinoma cell line (Caco-2). Treatment with peptide KYIPIQ increased NO synthesis and expression of phosphorylated endothelial nitric oxide synthase (eNOS) in HUVECs. Our results demonstrate that KYIPIQ-induced eNOS phosphorylation is dependent on the protein kinase B (Akt) activation pathway. Furthermore, our data indicate that the peptide KYIPIQ can be transported across the Caco-2 cell monolayer and that paracellular transport is the main transcellular mechanism. Thus, our studies suggest that KYIPIQ can be potentially used as a therapeutic agent for the treatment of hypertension.