Angiotensin I-converting Enzyme Inhibitory Properties Research Articles

Characterization, ACE Inhibitory and Antioxidative Properties of Peptide Fractions Obtained from White Shrimp (Litopenaeus vannamei)

Background: Aquatic organisms are considered to be an important source of bioactive peptides with a high antioxidant and antihypertensive capacity. Therefore, the objective of this study was to hydrolyse peptide fractions from white shrimp (Litopenaeus vannamei) muscle by Alcalase and Protamex and to evaluate the angiotensin I-converting enzyme (ACE) inhibitory and the antioxidant activities. Methods: Protein hydrolysates of White shrimp were obtained by enzymatic hydrolysis using Alcalase and Protamex until the degree of hydrolysis reached 10% and 20%. Peptide fractions were obtained from White shrimp protein hydrolysates by ultrafiltration using membranes with sizes of 10 and 3 kDa. The antioxidant activity was evaluated for the three peptide fractions (F1: >10 kDa, F2: 3-10 kDa and F3: <3 kDa). To measure the antihypertensive activity, fractions with molecular sizes of less than 3 kDa were used. Results: The fractions obtained with Alcalase showed greater inhibitory effects on the ACE. In general, the molecular weight of the fractions influenced the antioxidant activity, with fractions smaller than 3 kDa having a high capacity for sequestering the DPPH radical, while peptide fractions with a size greater than 10 kDa presented higher reducing power. However, in capturing the ABTS radical, a high antioxidant capacity was observed for both fractions. Conclusion: The results suggest white shrimp would be an attractive raw material for the manufacture of antioxidant and anti-hypertensive nutraceutical ingredients.

Antioxidant properties, phenolic and mineral profiling, assessment of angiotensin I converting enzyme (ACE) inhibitory potential of Elsholtzia communis (Collett & Hemsl.) Diels from North East India

IntroductionElsholtzia communis (Collett & Hemsl.) Diels, (family: Lamiaceae) is a wild edible plant consumed as a vegetable by many tribes of the North-Eastern states of India. The objective of our study was to explore the antioxidant capacity, phenolic and mineral composition and angiotensin-I converting enzyme (ACE) inhibitory properties of E. communis. MethodsThe leaf, stem, and root extract of E. communis were screened for their antioxidant and ACE inhibitory properties. Quantification of ten phenolics was conducted using reverse phase HPLC-DAD analysis and the five phenolics were isolated using conventional column chromatography and correlated with antioxidant activities using Pearson's coefficient. An assessment of dietary minerals present in the leaf, stem, and root of this herb was carried out using ion chromatography. ResultsThe leaf exhibited the most potent DPPH scavenging and ACE inhibitory activity with IC50 value of 20.38± 0.061 µg/mL and 20.34± 0.025 µg/mL respectively. Leaf was recognized as the most prominent source of TPC, TFC, K+, Na+, Ca2+ and Mg2+. The HPLC analysis had established the stem as a rich source of gallic acid (1.08±0.002 mg/g DW), quercetin (0.26±0.001 mg/g DW) and p-coumaric acid (0.17± 0.001 mg/g DW), while catechol (0.19±0.005 mg/g DW) and vanillic acid (0.07±0.005 mg/g DW) contents were found to be highest in the leaf. NMR and HRMS data confirmed the structures of the isolated phenolics. ConclusionThus, diverse pharmacological behavior and distinctive presence of nutraceuticals and important dietary minerals suggest that this herb may be useful in the future development of herbal formulations.

In vitro antioxidant and angiotensin I-converting enzyme inhibitory properties of peptides derived from corn gluten meal

Corn gluten meal (CGM) was hydrolyzed by Alcalase 2.4L and Protex 7L to obtain low-molecular-weight peptides. The antioxidant and angiotensin-I-converting enzyme (ACE) inhibitory activities of the peptides were determined by 2,2′-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) free radical scavenging activity, the JHH7 cell oxidative stress model, and the ACE inhibition assay, respectively. Corn peptides (CPs) had strong antioxidant capacity to scavenge ABTS radicals (1.44 ± 0.11 mmol/g), and significantly reduced reactive oxygen species and malondialdehyde levels in oxidized JHH7 cells, increasing superoxide dismutase, catalase, and glutathione peroxidase activities by 26.20%, 147.52%, and 43.20%, respectively, at the concentration of 800 μg/mL. CPs had high ACE inhibitory activity, with an IC50 value of 0.27 ± 0.05 mg/mL. Next, the CPs were separated by reverse-phase high-performance liquid chromatography. Twelve fractions were collected, and their ABTS free radical scavenging and ACE inhibitory activities were determined. Fractions 1, 2, 3, 4, and 9 showed higher ABTS free radical scavenging and ACE inhibitory activities. Twelve peptides were identified from active fractions by liquid chromatography–tandem mass spectrometry (LC–MS/MS) as Leu-Ser-Pro-Tyr, Leu-Asn-Ser-Pro-Ala-Tyr, Tyr-Gly-Pro-Gln, Pro-Pro-Tyr, Ala-Tyr-Pro-Glu, Thr-Tyr-Ser-Gly-Pro-Lys, Ala-Tyr-Pro-Gly-Pro-Gln, Leu-Ala-Tyr-Pro-Gln, Ala-Tyr-Leu-Gln-Gln-Gln, Ser-Ala-Pro, Asn-Ala-Pro, and Val-Asn-Ala-Pro. These peptides exhibited good ABTS free radical scavenging and ACE inhibitory activities. Therefore, CPs may have potential applications in the food industry as functional material additives.

Production of GABA-enriched idli with ACE inhibitory and antioxidant properties using Aspergillus oryzae: the antihypertensive effects in spontaneously hypertensive rats.

The present study aimed to develop a fermented food (idli) with enhanced γ-aminobutyric acid (GABA) and angiotensin I-converting enzyme (ACE) inhibitory properties using a GABA-producing fungus. Aspergillus oryzae NSK fermented idli batter and GABA was maximized (451.7 mg kg-1) in 120 h. The ACE inhibitory, 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) free radical scavenging and nitric oxide radical scavenging activities increased to 41.8%, 1.9 and 0.6 μmol trolox equivalent antioxidant capacity (TEAC) per gram in 120 h, respectively. In contrast, phytic acid and trypsin inhibitor activities decreased to 3.01 g kg-1 and 30.8 mg kg-1, respectively. The systolic blood pressure of spontaneously hypertensive rats in the fermented idli diet group was lower than those fed a plain idli diet. Lipid peroxidation in the plain idli diet group was significantly higher, whereas superoxide dismutase and glutathione reductase activities were significantly lower. The expression of genes ET-1, HSP70, NF-κB and iNOS in the aorta of SHRs that received GABA-containing diets was down-regulated between 2.2 and 3.8 fold. The production of GABA-enriched foods can be a promising approach to lower the blood pressure of spontaneously hypertensive rats.

Antihypertensive and anticoagulant properties of glycosaminoglycans extracted from the sturgeon (Acipenser persicus) cartilage

Abstract Large amounts of valuable waste are produced during sea food processing. This has a great potential for conversion to biologically active proteins and polysaccharides. Among these compounds, sulfated polysaccharides have been considered due to their many biological properties. The present work was conducted to study anticoagulant activities and angiotensin-I converting enzyme (ACE) inhibitory effects of glycosaminoglycans (GAGs) extracted from the cartilage of sturgeon (Acipenser persicus). The enzymatic extraction of sturgeon cartilage was performed in the presence of cetylpyridinium chloride salt. The structure was characterized via electron microscope and Fourier transform infrared spectroscopy (FTIR) analysis. Herein, ACE inhibitory and anticoagulant properties of extracted GAGs were determined. The amount of GAGs was 6.8±1.3% of cartilage dry weight. GAGs showed good activity in ACE inhibitory – with a highest level of 85.7%. The derived anticoagulant activity indexes, APPT (activated partial thromboplastin time) and TT (Thrombin time) of the extracted polysaccharide showed a prolonging of clotting time, compare to control. The results of this study revealed that the cartilage extracted GAGs possess promising ACE inhibitory properties and anticoagulant effects. Thus, the product can be substituted for blood reducing drugs and antithrombotic agents at least in laboratory conditions.

Ageing-Time Dependent Changes of Angiotensin I-Converting Enzyme-Inhibiting Activity of Protein Hydrolysates Obtained from Dry-Cured Pork Loins Inoculated with Probiotic Lactic Acid Bacteria

This study aimed to determine the ageing-time dependent changes of the angiotensin I-converting enzyme (ACE)-inhibition of protein extracts obtained from LAB-inoculated dry-cured pork loins over 360 days of ageing and their hydrolysates obtained after in vitro hydrolysis and absorption. The increasing ageing time was accompanied by a growth in the ACE inhibitory activity of the water-soluble and salt-soluble protein extracts. Based on the hierarchical cluster analysis, 180 days was indicated as the optimal time for ageing of dry-cured pork loins in terms of ACE inhibition. The effect of the strain used on the ACE inhibition varied over long-term aging. The peptides generated during in vitro hydrolysis have between 6 and 22 amino acids in a sequence; the Pro, Ala, Lys, and Glu molecules comprise the largest share. This study demonstrated that pork muscle proteins may lead to production of numerous peptides with ACE inhibitory properties.

Antioxidative sulphated polygalactans from marine macroalgae as angiotensin-I converting enzyme inhibitors

Antioxidant and antihypertensive potential of the sulphated polygalactans isolated from the marine macroalgae Kappaphycus alvarezii and Gracilaria opuntia were assessed by utilising different in vitro systems. The galactans isolated from K. alvarezii possessed significantly greater antioxidative properties as determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH IC90 0.97 mg/mL) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS.+ IC90 0.72 mg/mL) scavenging activities than those isolated from G. opuntia (DPPH IC90 1.2 mg/mL and ABTS 0.86 mg/mL). The sulphated polygalactan →4)-4-O-sulphonato-(2-O-methyl)-β-D-galactopyranosyl-(1→4)-3,6-anhydro-(2-O-methyl)-α-D-galactopyranan from K. alvarezii showed greater angiotensin-I-converting enzyme (ACE) inhibitory activity (IC50 0.02 μg/mL) than →3)-4-O-sulphonato-(6-O-acetyl)-β-D-galactopyranosyl-(1→4)-3,6-anhydro-(2-O-sulphonato)-α-D-galactopyranosyl-(1→3)-4-O-sulphonato-(6-O-acetyl)-β-D-xylosyl-(1→3)-4-O-sulphonato-(6-O-acetyl)-β-D-galactopyranosyl-(1→4)-3,6-anhydro-(2-O-sulphonato)-α-D-galactopyranan motif extracted from G. opuntia (IC50 0.70 μg/mL). Structure activity correlation studies displayed that the ACE inhibitory properties of titled polygalactans were directly proportional to their electronic properties and inversely with the steric and hydrophobic characteristics. Putative ACE inhibitory mechanism of action of sulphated galactans from marine macroalgae corroborated the structure bioactivity correlation analysis.

GC–MS based metabolite profiling and angiotensin I-converting enzyme inhibitory property of black tea extracts

Angiotensin I-converting enzyme inhibitors are used as therapeutic agents for the treatment of hypertension. Regular consumption of black tea (Camellia sinensis (L.) Kuntze, Theaceae) has been reported to lower blood pressure. The aims of the present work were to compare chemical composition and angiotensin I-converting enzyme inhibitory properties of infusion and decoction of four samples of black tea. GC/MS based metabolomics approach helped in identification of fifty-one metabolites including ten organic acids, one inorganic acid, sixteen amino acids, two sugars, five sugar alcohols, fifteen phenols and flavonoids, two fatty acids from infusions and decoctions of four black tea samples. Partial least squares discriminant analysis and orthogonal partial least squares discriminant analysis models showed good classification among the two groups, diffusion and infusion, based on metabolites. Both infusion and decoction inhibited the enzyme. However, the activity differed with samples. Multivariate analysis also segregated extracts on the basis of activity. Thearubigin, theaflavin, catechin inhibited the enzyme. Epicatechin, epigallocatechin gallate, gallic acid, caffeine showed lower activity.

A comparative study on antihypertensive and antioxidant properties of phenolic extracts from fruit and leaf of some guava (Psidium guajava L.) varieties

Investigation of antihypertensive and antioxidant properties of fruit and leaf extracts from four varieties (giant white, small white, stripped and pink) of guava (Psidium guajava L.) in vitro was the focus of this study. Consequently, methanol/1 M HCl (20:1 v/v) extraction of fruit and leaf of the guava varieties were carried out. Thereafter, the extracts were assayed for their angiotensin I converting enzyme (ACE) inhibitory effect, total phenol and flavonoid contents, reducing property, radicals (DPPH, ABTS•+, hydroxyl and nitric oxide) scavenging ability, Fe2+ chelating ability, and inhibition of Fe2+ and Sodium nitroprusside (SNP) induced lipid peroxidation reactions (in vitro). Furthermore, the phenolic constituents of the extracts were characterised with gas chromatography (GC). The results showed that all the extracts significantly (P < 0.05) inhibited ACE activity, scavenged (DPPH, ABTS•+, nitric oxide and hydroxyl) radicals, chelated Fe2+ and also inhibited Fe2+ and SNP induced lipid peroxidation in rat heart (in vitro). Nevertheless, the pink guava variety had the highest ACE inhibitory and antioxidant properties. In addition, Rosmarinic acid, eugenol, carvacrol, catechin and caffeic acid were the dominant phenolics found in the extracts. The ACE inhibitory effects and antioxidant properties of the guava extracts, which correlates significantly with their phenolic constituents, could largely contribute to their antihypertensive properties as obtained in traditional medicine.

Characterization, antioxidative and ACE inhibitory properties of hydrolysates obtained from thornback ray (Raja clavata) muscle

Thornback ray muscle hydrolysates (TRMHs) prepared by treatment with proteases from Bacillus subtilis A26 (TRMH-A26), Raja clavata crude alkaline protease extract (TRMH-Crude), Alcalase (TRMH-Alcalase) and Neutrase (TRMH-Neutrase) were elaborated and their antioxidant properties and angiotensin I-converting enzyme (ACE) inhibitory activities were tested. TRMHs showed different degrees of hydrolysis (DH from 11 to 22%) and hydrophobic/hydrophilic peptide ratio. Protein content varied from 71 to 74%. Gly, Pro, Asp and Asn were the most prominent amino acids, while hypoxanthine was the major nucleotide related compound present. The antioxidant activity was assayed using various tests. TRMH-Neutrase exhibited the highest antioxidant activity in DPPH scavenging, reducing power and inhibition of β-carotene bleaching tests. However in the total antioxidative efficacy, TRMH-Crude exhibited the highest activity. TRMH-Crude and TRMH-Neutrase were the most potent to prevent DNA oxidation by Fenton reagent. Concerning anti-ACE activity, TRMH-A26 and TRMH-Neutrase exhibited the highest activity with 87% at 5mg/ml. The results revealed that TRMHs could be employed as a protein source in food additive processing or diets for aquatic organisms and other farmed animals. Biological significanceThe present study explores for the first time the elaboration of enzymatic hydrolysates from thornback ray R. clavata. The hydrolysates are well characterized and showed an interesting protein content as well as the presence of nucleotide related compounds, essential amino acids and taurine, which make them an interesting source of fish meal in aquaculture feeds. The hydrolysates were found to exhibit ACE inhibitory activity and antioxidant activity. The hydrolysates could serve also as a potential protein source for functional foods.

Angiotensin I-converting enzyme inhibitory activity and hypocholesterolemic effect of some fermented tropical legumes in streptozotocin-induced diabetic rats

Cardiovascular disease (CVD) is the common cause of morbidity and mortality in diabetes. Any antidiabetic management strategy should therefore include the control of CVD risk factors. Thus, this study describes the effect of fermented legume condiment-supplemented diets on lung angiotensin I-converting enzyme (ACE) and lipid profile in streptozotocin (STZ)-induced diabetic rats. Adult male Wistar rats were used for the study and diabetes was induced in them by a single intraperitoneal injection of STZ (35 mg/kg b.w.). After the diabetic status had been confirmed, the animals were randomly divided into six groups consisting of six animals each and were fed with diets supplemented with fermented Bambara groundnut, locust bean, and soybean. The study lasted for 14 days after which the plasma was analyzed for the lipid profile and the rats’ lungs were assayed for ACE activity. Elevated ACE activity, plasma total cholesterol, triglyceride, and LDL-cholesterol showed significant (P < 0.05) reduction in the rats treated with fermented legume condiment-supplemented diets, with a concomitant increase in plasma HDL-cholesterol as compared with the diabetic control. This study therefore concludes that fermented legume condiment-supplemented diets could attenuate diabetes-induced dyslipidemia, a major coronary disease risk factor, in experimental rats. This health benefit of the condiment diets could be associated with their ACE inhibitory and antihypercholesterolemic properties. However, the diet supplemented with fermented locust bean condiment appeared to be the most potent.

Structural, functional, and ACE inhibitory properties of water-soluble polysaccharides from chickpea flours.

The present study aimed to characterize and investigate the functional and angiotensin-I converting enzyme (ACE) inhibition activities of chickpea water-soluble polysaccharides (CPWSP). Physico-chemical characteristics were determined by nuclear magnetic resonance spectroscopy (NMR), Fourier transform-infrared spectroscopy (FT-IR) analysis, and X-ray diffractometry (XRD). Functional properties (water holding capacity: WHC, water solubility index: WSI, swelling capacity: SC, oil holding capacity: OHC, foaming, and emulsion properties) and ACE activities were also investigated using well-established procedures. The FT-IR spectra obtained for the CPWSP revealed two significant peaks, at about 3500 and 500 cm(-1), which corresponded to the carbohydrate region and were characteristic of polysaccharides. All spectra showed the presence of a broad absorption between 1500 and 670 cm(-1), which could be attributed to CH, CO, and OH bands in the polysaccharides. CPWSP had an XRD pattern that was typical for a semi-crystalline polymer with a major crystalline reflection at 19.6 °C. They also displayed important techno-functional properties (SWC, WSI, WHC, and OHC) that can be modulated according to temperature. The CPWSP were also noted to display good anti-hypertensive activities. Overall, the results indicate that CPWSP have attractive chemical, biological, and functional properties that make them potential promising candidates for application as alternative additives in various food, cosmetic, and pharmaceutical preparations.

Investigation of Azorean macroalgae for angiotensin I-converting enzyme (ACE) inhibitory peptides. Extraction, purification and antihypertensive activity evaluation

Recently, much attention has been paid to the potential of marine organism constituents for the treatment of hypertension. Hypertension is one of the major independent risk factors for cardiovascular diseases and is estimated to affect about 25% of the world's adult population [1]. It is well known that the angiotensin I-converting enzyme (ACE) plays an important role in the regulation of blood pressure [2] by converting angiotensin I to angiotensin II, a potent vasoconstrictor. Generally, ACE synthetic inhibitors cause a number of undesirable side effects. For this reason, there is a trend towards searching for natural ACE inhibitors as alternatives to synthetic drugs. Taking into consideration the low level of pollution in Azorean seawater [3], this present study aims to contribute to: (i) the investigation of twelve Azorean marine macroalgae by the determination, for the first time, of their in vitro ACE inhibitory properties, (ii) the discovery of novel marine ACE inhibitory peptides with potential impact on the hypertensive patients. This investigation was carried out by optimizing the protein enzymatic hydrolysis followed by the peptides purification and ACE inhibitory evaluation. The results show that half of the selected marine macroalgae: Pterocladiella capillacea, Porphyra sp., Fucus spiralis, Ulva compressa, Osmundea pinnatifida and Ulva rigida presented an ACE inhibition activity of 48.73%, 50.96%, 56.77%, 59.80%, 62.59% and 65.68%, respectively, using pepsin followed by bromelain hydrolysis. The U. rigida (UV) was further purified by size exclusion and reverse-phase C18 chromatography showing four fractions: UV1, UV2, UV3 and UV4 with 2.37%, 32.40%, 64.25% and 31.28% of ACE inhibition activity, respectively. The isolated novel peptides will consequently have impact on public health, particularly on the hypertensive patients, in the regional economy and also will provide an incentive for the marine diversity [4] conservation.

Hydrolysis and purification of ACE inhibitory peptides from the marine microalga Isochrysis galbana

Response surface methodology (RSM) was employed to optimize enzymatic hydrolysis conditions for the production of angiotensin I-converting enzyme (ACE) inhibitory peptides from the marine microalga Isochrysis galbana. A hydrolysis temperature of 55.64 °C, a substrate concentration of 5.46 g (100 mL)−1, and a trypsin enzyme/substrate ratio (E/S) of 6.27 % were found to be optimal for obtaining the highest ACE inhibitory activity of 47.62 %. The protein hydrolysates were then separated using Sephadex G-25 column chromatography and an AKTA purifier (Inertsil ODS-3 C18, 10 × 250 mm). Using tandem mass spectrometry, the active form of the purified peptide with the most potent ACE inhibitory activity was identified as Tyr-Met-Gly-Leu-Asp-Leu-Lys, with an IC50 of 36.1 μM. This study improves our understanding of the ACE inhibitory properties of I. galbana protein hydrolysate and may be useful in further identification of ACE inhibitory peptides in other marine algae.

Winged bean [Psophorcarpus tetragonolobus (L.) DC] seeds as an underutilised plant source of bifunctional proteolysate and biopeptides

Hypertension is one of the major causes of cardiovascular-related diseases, which is highly associated with angiotensin-I-converting enzyme (ACE) activity and oxidative stress. In this study, winged bean seed (WBS), a potential source of protein, was utilised for the production of bifunctional proteolysate and biopeptides with ACE inhibitory and antioxidative properties. An enzymatic approach was applied, coupled with pretreatment of shaking and centrifuging techniques to remove endogenous ACE inhibitors prior to proteolysis. ACE inhibition reached its highest activity, 78.5%, after 12 h proteolysis while antioxidative activities, determined using assays involving DPPH˙ radical scavenging activity and metal ion-chelating activity, reached peaks of 65.0% and 65.7% at 8 h and 14 h, respectively. The said bioactivities were proposed to share some common structural requirements among peptides. A two-dimensional approach was employed for characterisation of effective peptides based on hydrophobicity, using RP-HPLC, and isoelectric property, using isoelectric focusing technique. Results revealed that acidic and basic peptides with partially higher hydrophobicity provided higher ACE inhibition activity than did neutral peptides. Finally, by using Q-TOF mass spectrometry, two peptide sequences (YPNQKV and FDIRA) with ACE inhibitory and antioxidative activities were successfully matched with a database. This study indicates that the WBS proteolysate can be a potential bifunctional food ingredient as the identified biopeptides demonstrated both ACE inhibitory and antioxidative activities in vitro.

Angiotensin I converting enzyme inhibitory peptides obtained after in vitro hydrolysis of pea (Pisum sativum var. Bajka) globulins.

Pea seeds represent a valuable source of active compounds that may positively influence health. In this study, the pea globulins were digested in vitro under gastrointestinal condition and potentially bioaccessible angiotensin I converting enzyme (ACE) inhibitory peptides were identified. The degree of hydrolysis after pepsin, 14.42%, and pancreatin, 30.65%, were noted. The peptides with the highest ACE inhibitory properties were separated using ion exchange chromatography on DEAE-cellulose. Thirteen peptides fractions were obtained but only four showed potential antihypertensive properties. The highest inhibitory activity was determined for the fraction F8 (IC50 = 0.0014 mg/mL). This fraction was separated on Sephadex G10 and two peptide fractions were obtained. The peptides fraction (B) with the highest ACE inhibitory activity (IC50 = 0.073 mg/mL) was identified by ESI-MS/MS. The sequences of ACE inhibitory peptides were GGSGNY, DLKLP, GSSDNR, MRDLK, and HNTPSR. Based on Lineweaver-Burk plots for the fraction B, the kinetic parameters as K m, Vmax, and K i and mode of inhibition were determined. This fraction belongs to uncompetitive inhibitor of ACE activity. The seeds of pea are the source of precursor protein, which releases the ACE inhibitory peptides as a result of enzymatic hydrolysis.

Purification and characterization of angiotensin I-converting enzyme inhibitory peptides of small red bean (Phaseolus vulgaris) hydrolysates

Angiotensin I-converting enzyme (ACE) inhibitory activity was investigated for small red bean (Phaseolus vulgaris) protein hydrolysate produced by sequential digestion of Alcalase, papain followed by in vitro gastrointestinal simulation. The hydrolysate had ACE inhibitory activity with IC50 of 67.2±1.8μg protein/mL. Peptides responsible for potent ACE inhibitory activity were isolated by a three-step purification process, including ultrafiltration, gel filtration and preparative reverse phase high performance chromatography (RP-HPLC). The fraction obtained after RP-HPLC fractionation with the highest activity yielded an IC50 of 19.3±1.4μg protein/mL. Enzymatic kinetic studies using this fraction demonstrated competitive inhibition with Ki of 11.6±1.7μg protein/mL. Mass spectrometric characterization identified for the first time the octapeptide PVNNPQIH which demonstrated an IC50 value of 206.7±3.9μM. The results expand the knowledge base of ACE inhibitory properties of small red bean protein hydrolysate and should be useful in further identification of specific ACE inhibitory peptides in beans.

Angiotensin I-converting enzyme inhibitory properties of Phaseolus vulgaris bean hydrolysates: Effects of different thermal and enzymatic digestion treatments

ACE inhibitory activity was investigated for protein hydrolysates derived from protein isolates of three Phaseolus vulgaris varieties, namely, navy bean, black bean and small red bean. Hydrolysis was performed using sequential digestion of Alcalase/Flavourzyme (AF) or Alcalase/papain (AP). Samples of the protein isolates were heated prior to hydrolysis and the results (i.e., degree of hydrolysis (DH), electrophoresis profiles, and ACE inhibitory activities) were compared to unheated samples. For all varieties, significantly higher DH values (P<0.05) were obtained for the AF hydrolysates compared to the AP hydrolysates. Heat treatments improved the DH values of navy bean and small red bean, however, lower values were observed for the heated black bean samples. For all unheated samples, phaseolins were degraded slowly to fragments with an estimated molecular mass around 20–24kDa which were resistant to further AF or AP digestions. Extensive degradations were observed for heat-treated samples and phaseolins were hydrolyzed into smaller peptides at the initial stage of digestions. AP hydrolysates for all investigated varieties presented higher ACE inhibitory activity than the AF hydrolysates. The highest ACE inhibitory activities were obtained after 100min of AP digestion for heated samples from navy bean, and 95min of AP digestion for heated samples from both black and small red beans, which yielded an IC50 of 68±5μgprotein/ml, 83±13μgprotein/ml and 78±7μgprotein/ml, respectively. Stabilities of these hydrolysates were tested by simulated in vitro gastrointestinal digestion followed by ACE inhibition studies, and lower IC50 values were determined for all three hydrolysates.

Isolation of novel bioactive regions from bovine Achilles tendon collagen having angiotensin I-converting enzyme-inhibitory properties

Collagen, a structural biopolymer of the extracellular matrix, is known to conceal several bioactive peptides which, when excised, can display physiological actions including angiotensin II-converting enzyme (ACE) inhibition. ACE is a key protease controlling the blood pressure (BP) by cleaving dipeptides from an inactive propeptide to produce angiotensin II, a potent BP regulator. Natural inhibitors of ACE, though less potent, have the advantage of being biocompatible and non-toxic. This study was undertaken to identify such cryptic regions from bovine Achilles tendon collagen. Bacterial collagenase was used to hydrolyze collagen and the hydrolysate was subjected to separation through ion-exchange column chromatography. Fractions were subjected to ACE inhibition assays and further purified by gel permeation chromatography. Two biologically active cryptic peptides were obtained displaying potent inhibition abilities; D1 and E2. The peptides were in the mass range of 1.5–3.5kDa and the inhibition was found to be competitive. Sequence analysis confirmed a relatively higher % occurrence of amino acids A and N in comparison to collagen and a hydrophobic C-terminal with P as the terminus. Both peptides were found to retain 80% of activity, even after digestive enzyme treatment. IC50 values revealed D1 to be the most potent inhibitor. Docking studies revealed that both peptides were using the C-terminal to interact with ACE-binding site. A comparison with other peptides displaying competitive inhibition hinted at the presence of a unique sequence GX′Y′ where X′ is often P, L, I or A and Y′ often P as the probable C-terminal for effective inhibition.

Antioxidant and angiotensin I-converting enzyme inhibitory properties of oligopeptides derived from black-bone silky fowl (Gallus gallus domesticus Brisson) muscle

Black-bone silky fowl (Gallus gallus domesticus Brisson) muscle was hydrolyzed with Alcalase 2.4L and papain, and treated by multistage separation. The black-bone silky fowl muscle peptides (BSFP) with low molecular weight obtained was tested for antioxidant and angiotensin I-converting enzyme (ACE) inhibitory activities. BSFP was then separated by reversed-phase high performance liquid chromatography. Five major fractions were collected and their 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging and ACE inhibitory activities were assayed. Fractions 3–5 displaying higher activities were subjected to mass spectrometry to identify the active peptides. A total of 29 peptide sequences were identified, and 11 peptides were synthesized for further ABTS radical scavenging and ACE inhibitory activity analysis. Finally, one potent antioxidative peptide (Leu-Trp-Arg, 3.25±0.11mmol Trolox equivalents/g sample) and two novel potent ACE inhibitory peptides (Leu-Glu-Arg, IC50=45.62±2.40μM; Gly-Ala-Gly-Pro, IC50=253.07±6.66μM) were found. Their activities were approximately 6.9, 14.1 and 2.8 times higher than that of BSFP, respectively.