Source Of Angiotensin I-converting Enzyme Inhibitory Peptides Research Articles

A novel ACE inhibitory peptide from Douchi hydrolysate: Stability, inhibition mechanism, and antihypertensive potential in spontaneously hypertensive rats

Angiotensin I-converting enzyme (ACE) regulates blood pressure through the renin-angiotensin system. Douchi, a traditional fermented soybean condiment, may have antihypertensive effects, but research on ACE inhibitory peptides from Douchi hydrolysates is limited. We hypothesized that enzymatic treatment could enhance ACE inhibitory peptide diversity and efficacy. We tested ten single enzymes and four combinations, finding pepsin-trypsin-chymotrypsin most effective. Hydrolysates were purified using Sephadex G-15 and reversed-phase HPLC, and peptides were identified via LC-MS/MS. Five peptides (LF, VVF, VGAW, GLFG, NGK) were identified, with VGAW as the most potent ACE inhibitor (IC50 46.6 ± 5.2 μM) showing excellent thermal and pH stability. Lineweaver-Burk plots confirmed competitive inhibition, and molecular docking revealed eight hydrogen bonds between VGAW and ACE. In hypertensive rats, VGAW significantly reduced blood pressure at 12.5, 25, and 50 mg/kg. These findings highlight Douchi as a source of ACE inhibitory peptides and suggest VGAW as a promising functional food ingredient.

Preparation and identification of novel angiotensin-I-converting enzyme inhibitory peptides from Moringa oleifera leaf

Moringa oleifera (M. oleifera) is considered a medicinal and edible plant with significant health benefits for humans and animals. It is speculated that the antihypertensive effect of M. oleifera leaf is attributed to the inhibition action of protein-derived bioactive components on Angiotensin-I-Converting Enzyme (ACE). In view of this, combined enzymatic hydrolysis was adopted instead of individual enzymatic treatment to prepare ACE inhibitory peptides, and the hydrolysis conditions were optimized from the perspective of ACE inhibitory activity. Following ultrafiltration, QExactive identification, and in silico screening, four novel ACE inhibitory peptides (IPPAYSK, ILVDR, FFFPK, and LLDPR) were collected. The IC50 values of these peptides ranged from 81.25 to 510.67 μM. In addition, molecular docking simulation and inhibition kinetics were analyzed to explain the inhibition mechanisms. Our study confirms that M. oleifera leaf is a superior source of ACE inhibitory peptides and has potential to serve as ingredients in functional foods for the prevention or management of hypertension.

Novel ACE Inhibitory Peptides Derived from Simulated Gastrointestinal Digestion in Vitro of Sesame (Sesamum indicum L.) Protein and Molecular Docking Study.

The aim of this study was to isolate and identify angiotensin I-converting enzyme (ACE) inhibitory peptides from sesame protein through simulated gastrointestinal digestion in vitro, and to explore the underlying mechanisms by molecular docking. The sesame protein was enzymatically hydrolyzed by pepsin, trypsin, and α-chymotrypsin. The degree of hydrolysis (DH) and peptide yield increased with the increase of digest time. Moreover, ACE inhibitory activity was enhanced after digestion. The sesame protein digestive solution (SPDS) was purified by ultrafiltration through different molecular weight cut-off (MWCO) membranes and SPDS-VII (< 3 kDa) had the strongest ACE inhibition. SPDS-VII was further purified by NGC Quest™ 10 Plus Chromatography System and finally 11 peptides were identified by Nano UHPLC-ESI-MS/MS (nano ultra-high performance liquid chromatography-electrospray ionization mass spectrometry/mass spectrometry) from peak 4. The peptide GHIITVAR from 11S globulin displayed the strongest ACE inhibitory activity (IC50 = 3.60 ± 0.10 μM). Furthermore, the docking analysis revealed that the ACE inhibition of GHIITVAR was mainly attributed to forming very strong hydrogen bonds with the active sites of ACE. These results identify sesame protein as a rich source of ACE inhibitory peptides and further indicate that GHIITVAR has the potential for development of new functional foods.

In Silico Analysis of Relationship between Proteins from Plastid Genome of Red Alga Palmaria sp. (Japan) and Angiotensin I Converting Enzyme Inhibitory Peptides.

Plastid proteins are one of the main components in red algae. In order to clarify the angiotensin I converting enzyme (ACE) inhibitory peptides from red alga Palmaria sp. (Japan), we determined the plastid genome sequence. The genome possesses 205 protein coding genes, which were classified as genetic systems, ribosomal proteins, photosystems, adenosine triphosphate (ATP) synthesis, metabolism, transport, or unknown. After comparing ACE inhibitory peptides between protein sequences and a database, photosystems (177 ACE inhibitory peptides) were found to be the major source of ACE inhibitory peptides (total of 751). Photosystems consist of phycobilisomes, photosystem I, photosystem II, cytochrome complex, and a redox system. Among them, photosystem I (53) and II (51) were the major source of ACE inhibitory peptides. We found that the amino acid sequence of apcE (14) in phycobilisomes, psaA (18) and psaB (13) in photosystem I, and psbB (11) and psbC (10) in photosystem II covered a majority of bioactive peptide sequences. These results are useful for evaluating the bioactive peptides from red algae.

Production of angiotensin I converting enzyme inhibitory (ACE-I) peptides during milk fermentation and their role in reducing hypertension

ABSTRACTFermented milk is a potential source of various biologically active peptides with specific health benefits. Angiotensin converting enzyme inhibitory (ACE-I) peptides are one of the most studied bioactive peptides produced during milk fermentation. The presence of these peptides is reported in various fermented milk products such as, yoghurt, cheese, sour milk, etc., which are also available as commercial products. Many of the ACE-I peptides formed during milk fermentation are resistant to gastrointestinal digestion and inhibit angiotensin converting enzyme (ACE) in the rennin angiotension system (RAS). There are various factors, which affect the formation ACE-I peptides and their ability to reach the target tissue in active form, which includes type of starters (lactic acid bacteria (LAB), yeast, etc.), substrate composition (casein type, whey protein, etc.), composition of ACE-I peptide, pre and post-fermentation treatments, and its stability during gastrointestinal digestion. The antihypertensive effect of fermented milk products has also been proved by various in vitro and in vivo (animal and human trials) experiments. This paper reviews the literature on fermented milk products as a source of ACE-I peptides and various factors affecting the production and activity of ACE-I peptides.

ACE-I inhibitory properties of hydrolysates from germinated and ungerminated Phaseolus lunatus proteins

Phaseolus lunatus protein concentrates and the proteases Alcalase(R) and Pepsin-Pancreatin were used for the production of protein hydrolysates that inhibit angiotensin-I converting enzyme (ACE). Protein concentrate obtained from germinated and ungerminated seeds flour was hydrolyzed with Alcalase(R) at enzyme/substrate ratio (E/S) 1/10 and during 0.5 and 2.0 h, respectively. On the other hand, protein concentrate obtained from ungerminated (E/S: 1/10) and germinated (E/S: 1/50) seeds flour was sequentially hydrolyzed with Pepsin-Pancreatin during 1.0 and 3.0 h, respectively. Peptide fractions with ACE inhibitory activity in a range of 0.9 to 3.8 µg/mL were obtained by G-50 gel filtration chromatography and high- performance liquid chromatography C18 reverse phase chromatography. The observed amino acid composition suggests a substantial contribution of hydrophobic residues to the peptides’ inhibitory potency, which potentially acts via blocking of angiotensin II production. These results show that P. lunatus seed proteins are a potential source of ACE inhibitory peptides when hydrolyzed with Alcalase(R) and Pepsin-Pancreatin.

Production of angiotensin‐I‐converting enzyme inhibitory peptides from β‐lactoglobulin‐ and casein‐derived peptides: An integrative approach

Angiotensin I-converting enzyme (ACE) inhibition is one of the mechanisms by which reduction in blood pressure is exerted. Whey proteins are a rich source of ACE inhibitory peptides and have shown a blood pressure reduction effect i.e. antihypertensive activity. The aim of this work was to develop a simplified process using a combination of adsorption and microfiltration steps for the production of hydrolysates from whey with high ACE inhibitory activity and potency; the latter was measured as the IC50, which is the peptide concentration required to reduce ACE activity by half. This process integrates the selective separation of β-lactoglobulin- and casein-derived peptides (CDP) from rennet whey and their hydrolysis, which results in partially pure, less complex hydrolysates with high bioactive potency. Hydrolysis was carried out with protease N "Amano" in a thermostatically controlled membrane reactor operated in a batch mode. By applying the integrative approach it was possible to produce from the same feedstock two different hydrolysates that exhibited high ACE inhibition. One hydrolysate was mainly composed of casein-derived peptides with IC50=285 μg/mL. In this hydrolysate we identified the well-known potent ACE-inhibitor and antihypertensive tripeptide Ile-Pro-Pro (IPP) and another novel octapeptide Gln-Asp-Lys-Thr-Glu-Ile-Pro-Thr (QDKTEIPT). The second hydrolysate was mainly composed of β-lactoglobulin derived peptides with IC50=28 μg/mL. This hydrolysate contained a tetrapeptide (Ile-Ile-Ala-Glu) IIAE as one of the two major peptides. A further advantage to this process is that enzyme activity was substantially increased as enzyme product inhibition was reduced.

Purification and characterization of angiotensin I-converting enzyme inhibitory peptides from enzymatic hydrolysate of hen egg white lysozyme

The aims of this study were to explore new functional bioactivity of hen egg white lysozyme (HEWL) protein hydrolysate and to identify potential angiotensin I-converting enzyme (ACE) inhibitory peptides from HEWL. The protein was hydrolyzed by gastrointestinal enzymes, including pepsin, α-chymotrypsin and trypsin, under simulated physiological conditions, and the hydrolysate exhibited potent ACE inhibitory activity with an IC50 value of 15.6±1.4μg/mL. Three novel ACE inhibitory peptides, specifically, Met-Lys-Arg, Arg-Gly-Tyr and Val-Ala-Trp with IC50 values of 25.7±0.2, 61.9±0.1 and 2.86±0.08μM, respectively, were isolated from the hydrolysate of HEWL in two stages of reverse-phase high-performance liquid chromatography (RP-HPLC), and their sequences were analyzed by UPLC–MS/MS. Kinetics studies suggested that the purified peptides were all competitive inhibitors; preincubation experiments implied that the peptide Arg-Gly-Tyr was a true substrate and that the other two peptides were true ACE inhibitors. These results indicate that HEWL may become an effective source of ACE inhibitory peptides. This study reveals the antihypertensive bioactivity of HEWL protein hydrolysate and provides further evidence that eggs are an excellent source of health-promoting food.

Purification of angiotensin I-converting enzyme inhibitory peptides from a cowpea ( Vigna unguiculata) enzymatic hydrolysate

Angiotensin I-converting enzyme (ACE) inhibitory peptides were isolated and characterized from cowpea ( Vigna unguiculata L. walp). Cowpea protein isolate was prepared by wet fractionation, extensively hydrolyzed with Flavourzyme ® and filtered through four molecular weight cut-off (MWCO) membranes in a high performance ultrafiltration (UF) cell. The ultrafiltered fraction with the highest ACE inhibitory activity was purified using gel filtration chromatography followed by reverse-phase high performance liquid chromatography (RP-HPLC). The cowpea peptide inhibition (IC 50) values were similar to those reported for many other natural ACE inhibitory peptides. The <1 kDa ultrafiltered fraction exhibited the highest biological activity. The observed amino acid composition suggests a substantial contribution of hydrophobic residues to the peptides’ inhibitory potency, which potentially acts via blocking of angiotensin II production. When hydrolyzed with the protease Flavourzyme ®, cowpea V. unguiculata protein is a good source of ACE inhibitory peptides with potential applications in physiologically functional foods with antihypertensive activity.

Analysis of Novel Angiotensin I-Converting Enzyme Inhibitory Peptides from Enzymatic Hydrolysates of Cuttlefish (Sepia officinalis) Muscle Proteins

The angiotensin I-converting enzyme (ACE) inhibitory activities of protein hydrolysates prepared from cuttlefish (Sepia officinalis) proteins by treatment with various bacterial proteases were investigated. The hydrolysate generated by the crude enzyme from Bacillus mojavensis A21 displayed the highest ACE inhibitory activity, and the higher inhibition activity (87.11 +/- 0.92% at 2 mg/mL) was obtained with hydrolysis degree of 16%. This hydrolysate was fractionated by size exclusion chromatography on a Sephadex G-25 into eight major fractions (P(1)-P(8)). Fraction P(6), which exhibited the highest ACE inhibitory activity, was then fractionated by reversed-phase high performance liquid chromatography (RP-HPLC). Eleven ACE inhibitory peptides were isolated, and their molecular masses and amino acids sequences were determined using ESI-MS and ESI-MS/MS, respectively. The structures of the most potent peptides were identified as Ala-His-Ser-Tyr, Gly-Asp-Ala-Pro, Ala-Gly-Ser-Pro and Asp-Phe-Gly. The first peptide displayed the highest ACE inhibitory activity with an IC(50) of 11.6 microM. The results of this study suggest that cuttlefish protein hydrolysates are a good source of ACE inhibitory peptides.

Mung-bean Protein Hydrolysates Obtained with Alcalase Exhibit Angiotensin I-converting Enzyme Inhibitory Activity

Mung-bean protein isolates were hydrolysed by two proteases alcalase and neutrase commercially available for food industry use, and the angiotensin I-converting enzyme (ACE) inhibitory activities of the enzymatic hydrolysates were measured at different hydrolysis times. The non-hydrolysed protein showed no inhibitory activity. Hydrolysates generated with neutrase displayed very low ACE inhibitory activity, while those obtained with alcalase exhibited high inhibitory activity. The highest ACE inhibitory activity with the IC50 value of 0.64 mg protein/mL was found in the hydrolysate obtained with alcalase at 2h of hydrolysis time. These results indicated that mung-bean protein is a good protein source of ACE inhibitory peptides when hydrolysed with the protease alcalase. The mung-bean protein hydrolysates prepared with alcalase might be utilised for physiologically functional foods with antihypertensive activity.

Utilisation of rapeseed protein isolates for production of peptides with angiotensin I-converting enzyme (ACE)-inhibitory activity

ACE activity is related to increased arterial pressure and coronary diseases. A rapeseed protein isolate was hydrolyzed with the protease Alcalase in order to investigate the possible presence of ACE inhibitory peptides in the resulting hydrolysates. Hydrolysis for 30 min yielded a hydrolysate with the highest ACE inhibitory activity. Two fractions of this hydrolysate obtained by Biogel P2 gel filtration chromatography were used for further purification of ACE inhibitory peptides. Three fractions with ACE inhibitory activity were purified by reverse-phase HPLC of Biogel P2 f ractions. This demonstrates that rapeseed protein hydrolysates represent a good source of ACE inhibitory peptides .

Hypotensive Peptides from Milk Proteins

Hypertension is the major controllable risk factor associated with cardiovascular disease (CVD) events such as myocardial infarction, stroke, heart failure, and end-stage diabetes. A 5 mm Hg decrease in blood pressure has been equated with approximately 16% decrease in CVD. In the U.S. alone current annual antihypertensive drug costs are approximately dollars 15 billion. The renin-angiotensin-aldosterone system is a target for blood pressure control. Cleavage of angiotensinogen by renin produces angiotensin I which is subsequently hydrolyzed by angiotensin-I-converting enzyme (ACE) to angiotensin II (a potent vasoconstrictor). Various side effects are associated with the use of ACE inhibitory drugs in the control of blood pressure including hypotension, increased potassium levels, reduced renal function, cough, angioedema, skin rashes, and fetal abnormalities. Milk proteins, both caseins and whey proteins, are a rich source of ACE inhibitory peptides. Several studies in spontaneously hypertensive rats show that these casokinins and lactokinins can significantly reduce blood pressure. Furthermore, a limited number of human studies have associated milk protein-derived peptides with statistically significant hypotensive effects (i.e., lower systolic and diastolic pressures). The advent of effective milk protein based functional food ingredients/nutraceuticals for the prevention/control of blood pressure therefore has the potential to significantly reduce global healthcare cost.

Purification of an ACE inhibitory peptide after hydrolysis of sunflower (Helianthus annuus L.) protein isolates.

Sunflower protein isolates and the proteases pepsin and pancreatin were used for the production of protein hydrolysates that inhibit angiotensin-I converting enzyme (ACE). Hydrolysates obtained after 3 h of incubation with pepsin and 3 h with pancreatin were studied. An ACE inhibitory peptide with the sequence Phe-Val-Asn-Pro-Gln-Ala-Gly-Ser was obtained by G-50 gel filtration chromatography and high-performance liquid chromatography C18 reverse phase chromatography. This peptide corresponds to a fragment of helianthinin, the 11S globulin from sunflower seeds, which is the main storage protein in sunflower. These results show that sunflower seed proteins are a potential source of ACE inhibitory peptides when hydrolyzed with pepsin and pancreatin.

A quantitative in silico analysis calculates the angiotensin I converting enzyme (ACE) inhibitory activity in pea and whey protein digests

Angiotensin I converting enzyme (ACE) inhibitory peptides can induce antihypertensive effects after oral administration. By means of an ACE inhibitory peptide database, containing about 500 reported sequences and their IC 50 values, the different proteins in pea and whey were quantitatively evaluated as precursors for ACE inhibitory peptides. This analysis was combined with experimental data from the evolution in ACE inhibitory activity and protein degradation during in vitro gastrointestinal digestion. Pea proteins produced similar in silico scores and were degraded early in the in vitro digestion. High ACE inhibitory activity was observed after the simulated stomach phase and augmented slightly in the simulated small intestine phase. The major whey protein β-lactoglobulin obtained the highest in silico scores, which corresponded with the fact that degradation of this protein in vitro only occurred from the simulated small intestine phase on and resulted in a 10-fold increase in the ACE inhibitory activity. Whey protein obtained total in silico scores of about 124 ml/mg, compared to 46 ml/mg for pea protein, indicating that whey protein would be a richer source of ACE inhibitory peptides than pea protein. Although β-lactoglobulin is only partially digested, a higher ACE inhibitory activity was indeed found in the whey (IC 50 = 0.048 mg/ml) compared to the pea digest (IC 50 = 0.076 mg/ml). In silico gastrointestinal digestion of the highest scoring proteins in pea and whey, vicilin and albumin PA2, and β-lactoglobulin, respectively, directly released a number of potent ACE inhibitory peptides. Several other ACE inhibitory sequences resisted in silico digestion by gastrointestinal proteases. Briefly, the quantitative in silico analysis will facilitate the study of precursor proteins on a large scale and the specific release of bioactive peptides.