Purification and identification of ACE inhibitory peptides from Haruan (Channa striatus) myofibrillar protein hydrolysate using HPLC–ESI-TOF MS/MS

Haruan myofibrillar protein was hydrolysed with proteinase K and thermolysin to isolate Angiotensin converting enzyme (ACE) inhibitory peptides. The thermolysin hydrolysate of myofibrillar protein with the highest ACE inhibition activity (IC₅₀ = 0.033 mg/ml) was fractionated by ultrafiltration and size exclusion chromatography to three fractions. Fraction F2 with higher ACE inhibitory activity was separated into five fractions (A–E) using reversed-phased high performance liquid chromatography (RP-HPLC). Fraction C showed 81% inhibition activity and was subjected to HPLC coupled to electrospray ionisation-time-of-flight mass spectrometry (ESI-TOF MS/MS). Two peptide sequences for the most abundant fragments were identified as VPAAPPK (IC₅₀ = 0.45 μM) at 791.155 m/z and NGTWFEPP (IC₅₀ = 0.63 μM) at 1085.841 m/z. The presence of two proline residues at the C-terminal sequence is responsible for the high ACE inhibitory activity of these peptides. The results suggest that Haruan meat protein hydrolysate is a potent ACE inhibitor and may be used to decrease blood pressure.     

A novel angiotensin I converting enzyme inhibitory peptide from tuna frame protein hydrolysate and its antihypertensive effect in spontaneously hypertensive rats

Tuna frame protein was hydrolysed using Alcalase, Neutrase, pepsin, papain, α-chymotrypsin and trypsin. Peptic hydrolysate exhibited the highest ACE I inhibitory activity among them and was fractionated into three ranges of molecular weight (below 1, 1–5 and 5–10 kDa) using an ultrafiltration membrane bioreactor system. The 1–5 kDa fraction showed the highest ACE inhibitory activity and was used for subsequent purification steps. During consecutive purification, a potent ACE inhibitory peptide from tuna frame protein (PTFP), which was composed of 21 amino acids, Gly-Asp-Leu-Gly-Lys-Thr-Thr-Thr-Val-Ser-Asn-Trp-Ser-Pro-Pro-Lys-Try-Lys-Asp-Thr-Pro (MW: 2,482 Da, IC₅₀: 11.28 μm), was isolated. Lineweaver–Burk plots suggest that PTFP plays as a non-competitive inhibitor against ACE. Furthermore, antihypertensive effect in spontaneously hypertensive rats (SHR) also revealed that oral administration of PTFP can decrease systolic blood pressure significantly (P < 0.01). These results suggest that the PTFP would be a beneficial ingredient for nutraceuticals and pharmaceuticals against hypertension and its related diseases.     

Purification and identification of angiotensin I-converting enzyme inhibitory peptide from buckwheat (Fagopyrum esculentum Moench)

Angiotensin I-converting enzyme (ACE) inhibitory peptide was isolated and identified from buckwheat (Fagopyrum esculentum Moench). Buckwheat protein extract was prepared by stirring in water (pH 9.0) for 30 min, followed by centrifugation at 15,000g for 20 min. The protein extract was then filtered using an YM-10 membrane. An ACE inhibitor was purified using consecutive chromatographic methods including: ion-exchange chromatography, gel filtration chromatography, and reverse-phase high performance liquid chromatography. The ACE inhibitor was identified to be a tripeptide, Gly-Pro-Pro, having IC₅₀ value of 6.25 μg protein/ml, by protein sequencing system and electrospray-LC–mass spectrometry.     

Effect of angiotensin I converting enzyme inhibitory peptide purified from skate skin hydrolysate

Our objective was to evaluate the angiotensin I converting enzyme (ACE) inhibitory activity of skate skin protein hydrolysates and its corresponding fractions. The skate skin hydrolysates were obtained by enzymatic hydrolysis using alcalase, α-chymotrypsin, neutrase, pepsin, papain, and trypsin. Amongst the six hydrolysates, the α-chymotrypsin hydrolysate had the highest ACE inhibitory activity compared to other hydrolysates. The amino acid sequences of the purified peptides were identified to be Pro–Gly–Pro–Leu–Gly–Leu–Thr–Gly–Pro (975.38 Da), and Gln–Leu–Gly–Phe–Leu–Gly–Pro–Arg (874.45 Da). The purified peptides from skate skin had an IC₅₀ value of 95 μM and 148 μM, respectively, and the Lineweaver–Burk plots suggest that they act as a non-competitive inhibitor against ACE. Our study suggested that novel ACE inhibitory peptides derived from skate skin protein may be beneficial as anti-hypertension compounds in functional foods.     

Novel angiotensin I-converting enzyme inhibitory peptides derived from soya milk

Inhibitors of angiotensin I-converting enzyme (ACE) are useful in treating hypertension, and many have been derived from food products, including soybeans. Using the industrial protease PROTIN SD-NY10, we developed a processed soya milk (PSM) with enhanced ACE inhibitory activity. The ACE inhibitory activity of PSM (IC₅₀ = 0.26 μg/ml) was greater than that of regular soya milk (IC₅₀ = 8.75 μg/ml). Eight novel ACE inhibitory peptides were purified from PSM by reversed-phase chromatography: FFYY (IC₅₀,1.9 μM), WHP (4.8 μM), FVP (10.1 μM), LHPGDAQR (10.3 μM), IAV (27.0 μM), VNP (32.5 μM), LEPP (100.1 μM), and WNPR (880.0 μM). The IC₅₀ values of these peptides are comparable to those reported for other ACE inhibitory peptides derived from wheat, chicken, bonito, wine, etc. Due to the relatively low IC₅₀ values of several peptides identified in this study, they may serve as ideal base components of food supplements for patients with hypertension.     

Angiotensin-I-converting enzyme inhibitory activity and bitterness of enzymatically-produced hydrolysates of shrimp (Pandalopsis dispar) processing byproducts investigated by Taguchi design

The importance of water-to-substrate ratio, protease type, percent enzyme and incubation time on hydrolysates produced from shrimp processing byproducts was investigated using Taguchi’s L₁₆ (4⁵) experimental design. Protease type significantly (p < 0.05) influenced soluble yield, degree of hydrolysis (DH), angiotensin-I-converting enzyme (ACE) inhibitory activity and bitterness of hydrolysates, while percent enzyme only affected the DH. Hydrolysates produced by Alcalase and Protamex possessed strong ACE inhibitory activity (IC₅₀ = 100–200 μg/ml and 70 μg/ml, respectively), accompanied by high yield, high DH and strong bitterness. Furthermore, ACE inhibition was positively correlated (r² = 0.87) with bitterness of the hydrolysates. Fractionation by size-exclusion chromatography revealed that the bitter substances, which also showed strong ACE inhibition, were <3 kDa in size and contained many hydrophobic residues, including Tyr, Phe, Leu, Ile, Val and Lys. Despite the bitterness, these hydrolysates may have potential health benefits, arising from their potent ACE inhibitory activity.     

Chemical composition,angiotensin I-converting enzyme (ACE) inhibitory,antioxidant and antimicrobial activities of the essential oil from Periploca laevigata root barks

The present study describes the chemical composition, and antimicrobial, antioxidant and angiotensin I-converting enzyme (ACE) inhibitory activities of essential oil from Periploca laevigata root barks (PLRB), an aromatic plant widely distributed in Tunisia and used as a traditional medicinal plant. Gas chromatography/mass spectrometry was used to determine the composition of the PLRB oil. Forty-three components were identified in the essential oil and the main compounds were benzaldehyde (56%), methyl 4-methoxysalicylate (6.55%) and carvacrol (4.75%). The PLRB essential oil exhibited a dose-dependent manner of inhibitory activity toward ACE. The highest ACE inhibitory activity (54%) was observed at a concentration of 30 μg/ml. The PLRB oil was also found to possess antioxidant activities, as evaluated by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical method, β-carotene bleaching and reducing power assays. The antimicrobial activity of the essential oil was also investigated on several microorganisms. The inhibition zones and minimal inhibitory concentration (MIC) values of bacterial strains were in the range of 12–46 mm and 50–300 μg/ml, respectively. The inhibitory activity of the PLRB essential oil against Gram-positive bacteria was significantly higher than against Gram-negative. It also exhibited remarkable activity against several fungal strains.     

Tyr-Pro-Lys,an angiotensin I-converting enzyme inhibitory peptide derived from broccoli (Brassica oleracea Italica)

To investigate the naturally occurring angiotensin I-converting enzyme (ACE) inhibitor, broccoli (Brassica oleracea Italica) extracts were used for its isolation and identification. After treatment with 50% acetone for membrane breakdown, ethyl acetate, n-butanol, and water were used for the preparation of broccoli extracts. The water-soluble extract from broccoli had 76.9% ACE inhibitory activity, while those of other organic solvent extracts showed lower ACE inhibitory activities. An ACE inhibitory peptide was isolated using column chromatographic methods including: Amberlite XAD-4, Sephadex LH-20, and high performance liquid chromatography. The purified ACE inhibitory peptide was identified to be a tripeptide, Tyr-Pro-Lys, having an IC₅₀ value of 10.5 μg protein/ml.     

Hypotensive effect of a sweetpotato protein digest in spontaneously hypertensive rats and purification of angiotensin I-converting enzyme inhibitory peptides

We have investigated angiotensin I-converting enzyme (ACE) inhibitory activity in an enzyme digest of sweetpotato protein, the antihypertensive effect of the digest in spontaneously hypertensive rats (SHR), and the identification of an ACE inhibitory peptide. Protein was prepared from squeezed juice of sweetpotato by isoelectric focusing precipitation. Three kinds of proteases were selected for effective protein digestion. The digest, sweetpotato peptide (SPP), exhibited strong ACE inhibitory activity (IC₅₀: 18.2 μg/ml). SPP was orally administered by gavage to SHR at a dose of 100 mg/kg or 500 mg/kg. The systolic blood pressure and the diastolic blood pressure were measured at 0 (before administration), 2, 4, 8, and 24 h after administration. A dose-dependent decrease in systolic blood pressure in SHR was observed after oral administration of SPP. Significant differences between SPP-administered rats and control rats were observed 4 and 8 h after administration in the 500 mg/kg-administered group and 8 h after administration in the 100 mg/kg-administered group. Diastolic blood pressure also decreased in the SPP-administered groups, although the difference between SPP-administered rats and control rats was not significant. These results suggest that SPP may be useful in the prevention or treatment of hypertension. Peptides with ACE inhibitory activity were purified from SPP by absorption chromatography and preparative HPLC using an ODS column. The amino acid sequences of isolated peptides were I-T-P, I-I-P, G-Q-Y and S-T-Y-Q-T; their ACE inhibitory activities (IC₅₀) were 9.5 μM, 80.8 μM, 52.3 μM and 300.4 μM, respectively. In conclusion, I-T-P is a novel, strong ACE inhibitory peptide.     

α-Glucosidase inhibitors from Chinese Yam (Dioscoreaopposita Thunb.)

The inhibitory activities of crude extracts and purified constituents from the fresh tuberous rhizomes of Chinese Yam (Dioscorea opposita Thunb.), which is commonly called Huai Shan Yao in Chinese, were evaluated against yeast α-glucosidase in order to search for the active principals for treatment of diabetes. Bioassay-guiding isolation gave four compounds: trans-N-p-coumaroyltyramine (1) (IC₅₀ = 0.40 μM), 1,7-bis(4-hydroxyphenyl)heptane-3,5-diol (2) (IC₅₀ = 0.38 mM), 6-hydroxy-2,4,7-trimethoxyphenanthrene (3) (IC₅₀ = 0.77 mM) as α-glucosidase inhibitors, and cis-N-p-coumaroyltyramine (4), an isomer of compound 1, which showed no inhibitory activity against α-glucosidase. Furthermore, the separation and purification of compound 3 from Chinese Yam (Huai Shan Yao) was conducted by high-speed counter-current chromatography (HSCCC) using hexane–ethyl acetate–methanol–water (1:1:1:1, v/v/v/v). Compound 1, 2 and 4 were isolated from or detected in the Dioscoreaceae family for the first time.     

Amaranth seed protein hydrolysates have in vivo and in vitro antihypertensive activity

The objective of this work was to study the hydrolytic release of encrypted peptides with antihypertensive activity from storage proteins of Amaranthus mantegazzianus, as determined by in vitro assays, for the first time by in vivo studies in animal models, and by ex vivo assays. Hydrolysates with hydrolysis degree (DH) of 45% and 65% (IC50 0.12 mg/ml, equivalent to 300–600 μM) exhibited an angiotensin-I converting enzyme 1 (ACE) inhibitory activity equal or higher than the potential inhibitory of the average antihypertensive peptides registered in the BIOPEP database and of semi-purified Amaranthus hypochondriacus albumin and globulin protein fractions. Intragastric administration of hydrolysates with DH of 45% was effective in lowering blood pressure of male spontaneously hypertensive rats (SHR). Experiments performed in papillary muscles isolated from hearts and with isolated aortic smooth muscle of SHR suggest that the hypotensive effect could be attributed to a lowering of the peripheral resistance. We assume that the amaranth hydrolysates would be acting at the level of the local or autocrine renin–angiotensin system (RAS).     

A rapid,simple and sensitive fluorescence method for the assay of angiotensin-I converting enzyme

A fluorescent assay for angiotensin-I converting enzyme (ACE; EC 3.4.15.1) is described. It is based in the hydrolysis of the internally quenched fluorescent substrate o-aminobenzoylglycyl-p-nitrophenylalanylproline by the action of ACE. The fluorescence generated by the liberation of the product (the o-aminobenzoylglycine group) is read in a microtiter-plate multiscan fluorometer. The different conditions for the assay have been optimised for linearity, sensitivity and precision. Maximal enzyme activity was reached in the pH range 8.0–8.5 and 0.5–0.75 M NaCl concentration in the assay mixture. Kinetics of the enzyme reaction displayed a K_m = 109 μM, obtaining an optimal substrate concentration of 0.3 mM in the assay mixture. The assay was adequate for the study of ACE inhibition by captopril and several peptides and it also showed a very good correlation with a well-established method [Biochemical Pharmacology, 20(7) (1971) 1637]. The method has important advantages, being the availability of reagents, its simplicity and the capacity to process a high number of samples in a short time, most outstanding.     

Antioxidant triterpenoids from the stems of Momordica charantia

A new multiflorane triterpenoid and two new cucurbitane triterpenoids were isolated from the stems of Momordica charantia. The structures of the new compounds were elucidated by spectroscopic methods. These three new compounds, 1, 2 and 3 displayed ABTS radical cation scavenging activity with IC₅₀ values of 268.5 ± 7.9, 352.1 ± 11.5 and 458.9 ± 13.0 μM, respectively and an inhibitory effect on xanthine oxidase (XO) activity with IC₅₀ values of 142.3 ± 30.2, 36.8 ± 20.5 and 124.9 ± 8.3 μM, respectively. This finding indicated that 1–3 can be used as antioxidants.     

Comparison of analytical methods to assay inhibitors of angiotensin I-converting enzyme

The linearity, precision and repeatability of visible spectrophotometric (VSP) and high-performance liquid chromatography (HPLC) methods for analysis of inhibitory activity of angiotensin I-converting enzyme (ACE) were compared by using several inhibitors and Hip-His-Leu (HHL) as substrates. IC₅₀ values (concentration at which ACE activity is inhibited by 50%) of 0.00206 ± 0.00005 μg/mL for captopril, 192 ± 4.53 μg/mL for soybean peptides, and 153 ± 4.29 μg/mL for grass carp peptides determined by the VSP method, and these values were 1.07, 1.07, 1.18 and 1.44-fold, respectively, higher than those from the HPLC method. In addition, the inhibitory constant (K_i value) of captopril was determined to be 7.09 nM and 4.94 nM using VSP and HPLC method, respectively. These results showed that the HPLC method revealed a higher level of sensitivity and precision, suitable for assaying ACE inhibition activity of antihyper-sensitive peptides. In contrast, the VSP method can simultaneously measure several samples with simple operations, suitable for analysis of ACE inhibition activity of food protein enzymatic hydrolysates.     

Isolation and characterisation of an α-glucosidase inhibitory substance from fructose–tyrosine Maillard reaction products

An α-glucosidase inhibitory substance was isolated and characterised from fructose–tyrosine Maillard reaction products (MRPs) and the inhibition mode of the active substance determined. The ethyl acetate fraction of fructose–tyrosine MRPs showed strong α-glucosidase inhibitory activity; this fraction was isolated and purified using silica gel column chromatography and semi-preparative RP-HPLC. The structure of the purified compound was determined using spectroscopic methods. The isolated compound was identified as 2,4-bis (p-hydroxyphenyl)-2-butenal (C₁₆H₁₄O₃, HPB242). This is the first report of baker’s yeast α-glucosidase inhibitory activity of HPB242 isolated from fructose–tyrosine MRPs. The IC₅₀ value of HPB242 on α-glucosidase inhibition was 4.00 ± 0.09 μg/ml. Kinetic data revealed that HPB242 inhibits the p-NPG hydrolysing activity of baker’s yeast α-glucosidase noncompetitively with a K_i value of 0.870 mM.     

Flavonoids isolated from Syzygium aqueum leaf extract as potential antihyperglycaemic agents

Syzygium aqueum is a medicinal plant which is grown in tropical regions. In this study, the ethanolic extracts of S. aqueum leaf were investigated for its antihyperglycaemic activity. Our investigation revealed its effectiveness in inhibiting the carbohydrate hydrolysing enzymes, α-glucosidase (EC₅₀ = 11 μg/ml) and α-amylase (EC₅₀ = 8 μg/ml), at significant level than the commercial drug acarbose (EC₅₀ = 28 μg/ml, α-glucosidase; EC₅₀ = 12 μg/ml, α-amylase). In addition, the ethanolic leaf extracts were able to inhibit the key enzyme in the polyol pathway, aldose reductase (EC₅₀ = 0.03 μg/ml) and prevent the AGEs formation by 89%. Six flavonoid compounds, 4-hydroxybenzaldehyde (1), myricetin-3-O-rhamnoside (2), europetin-3-O-rhamnoside (3), phloretin (4), myrigalone-G (5) and myrigalone-B (6), were isolated from the ethanolic leaf extracts. Compounds (2) and (3) showed high inhibitory activities, with EC₅₀ values of 1.1 μM and 1.9 μM against α-glucosidase and EC₅₀ values of 1.9 μM and 2.3 μM against α-amylase, respectively. These findings provide a strong rationale to establish S. aqueum’s capability as an antihyperglycaemic agent.     

LC–MS/MS coupled with QSAR modeling in characterising of angiotensin I-converting enzyme inhibitory peptides from soybean proteins

The importance of soy products in reducing the risk of cardiovascular disease is well documented. Our previous computation study has indicated the presence of several potent ACE inhibitory peptides within soybean proteins which needs to be identified. The aim of the study was to identify ACE inhibitory peptides from soy proteins using LC–MS/MS coupled with quantitative structure–activity relationship (QSAR) model. Soybean protein hydrolysate digested by thermolysin showed an IC₅₀ value of 53.6 μg/mL, decreased slightly to 51.8 μg/mL after adding pepsin, and increased to 115.6 μg/mL after adding trypsin. A total of 34 peptides were characterised from LC–MS/MS. Five novel tripeptides, IVF, LLF, LNF, LSW and LEF, with predicted IC₅₀ values lower than 10 μM were synthesized and validated. The results showed that soybean is an excellent source of ACE inhibitory peptides.     

Angiotensin I-converting enzyme inhibitor derived from soy protein hydrolysate and produced by using membrane reactor

Five different proteolytic enzymes, including Alcalase, Flavourzyme, trypsin, chymotrypsin and pepsin were employed to hydrolyze isolated soy protein (ISP) to produce the hydrolysates, respectively. The result indicated that hydrolysis of ISP for 0.5–6 h with Alcalase produced the highest ACE inhibitory activity. Therefore, Alcalase was selected for further study on optimization of hydrolysis conditions. The optimum conditions for Alcalase to hydrolyze ISP to produce the lowest IC₅₀ value were: E/S = 0.01, hydrolysis temperature = 50 °C, pH 9.0 and hydrolysis time = 6 h. Under these conditions, the IC₅₀ value of ISP was significantly reduced from 66.4 to 0.67 mg protein/ml. The lower IC₅₀ value represented the higher the ACE inhibitory activity. Moreover, several membranes with molecular weight cut-offs (MWCFs) of 1000–30,000Da were used to filter the hydrolysate. The 10 kDa permeate obtained from the treatment of the hydrolysate by 10,000 Da MWCF membrane could further reduce its IC₅₀ value from 0.668 to 0.078 mg protein/ml with a peptide recovery of 67.5%. An operation stability study showed that the membrane reactor system could maintain a steady production of ISP hydrolysate for over 8 h. The in vitro effect of gastrointestinal protease on ACE inhibitory activity of 10 kDa permeate was also investigated. The results suggested that gastrointestinal proteases have very little effect on the ACE inhibitory activity of 10 kDa permeate.     

Angiotensin I-converting enzyme (ACE) inhibitory activities of sardinelle (Sardinella aurita) by-products protein hydrolysates obtained by treatment with microbial and visceral fish serine proteases

The angiotensin I-converting enzyme (ACE) inhibitory activities of protein hydrolysates prepared from heads and viscera of sardinelle (Sardinella aurita) by treatment with various proteases were investigated. Protein hydrolysates were obtained by treatment with Alcalase^®, chymotrypsin, crude enzyme preparations from Bacillus licheniformis NH1 and Aspergillus clavatus ES1, and crude enzyme extract from sardine (Sardina pilchardus) viscera. All hydrolysates exhibited inhibitory activity towards ACE. The alkaline protease extract from the viscera of sardine produced hydrolysate with the highest ACE inhibitory activity (63.2 ± 1.5% at 2 mg/ml). Further, the degrees of hydrolysis and the inhibitory activities of ACE increased with increasing proteolysis time. The protein hydrolysate generated with alkaline proteases from the viscera of sardine was then fractionated by size exclusion chromatography on a Sephadex G-25 into eight major fractions (P₁–P₈). Biological functions of all fractions were assayed, and P₄ was found to display a high ACE inhibitory activity. The IC₅₀ values for ACE inhibitory activities of sardinelle by-products protein hydrolysates and fraction P₄ were 1.2 ± 0.09 and 0.81 ± 0.013 mg/ml, respectively. Further, P₄ showed resistance to in vitro digestion by gastrointestinal proteases. The amino acid analysis by GC/MS showed that P₄ was rich in phenylalanine, arginine, glycine, leucine, methionine, histidine and tyrosine. The added-value of sardinelle by-products may be improved by enzymatic treatment with visceral serine proteases from sardine.     

Purification and characterization of angiotensin I converting enzyme inhibitory peptides from jellyfish Rhopilema esculentum

Two angiotensin I converting enzyme (ACE) inhibitory peptides were isolated from jellyfish Rhopilema esculentum protein hydrolysates prepared with pepsin and papain. Consecutive purification methods, including ion-exchange chromatography, size-exclusion chromatography and reverse-phase high-performance liquid chromatography, were used for isolation of ACE inhibitory peptides. The amino acid sequence of the peptides was identified as Gln-Pro-Gly-Pro-Thr and Gly-Asp-Ile-Gly-Tyr, respectively, and the IC₅₀ value of the purified peptides for ACE inhibitory activity was 80.67 μM and 32.56 μM. The purified peptides were evaluated for the antihypertensive effect in spontaneously hypertensive rats (SHR) after oral administration. Blood pressure decreased significantly after ingestion of the peptides. The results suggest that peptides derived from jellyfish R. esculentum may be beneficial as antihypertensive compounds in functional food resources.