Evaluation of angiotensin I-converting enzyme (ACE) inhibitory activities of hydrolysates generated from byproducts of freshwater clam

Solid wastes of freshwater clam in food processing, including mainly mantle, were used as a raw material for the recovery of bioactive peptides related to angiotensin I-converting enzyme (ACE) inhibitor. Among the primary hydrolysates of dried mantle (DM), the peptides corresponding to hydrolysates using 2 crude peptidases exhibited a strong ACE inhibitory activity (IC₅₀, 0.23 mg/mL), and recovery efficiency of soluble materials and their protein content were considerably large with 42.65% and 468.6 mg/g, respectively. The ACE inhibitory activity of all secondary hydrolysates digested by pepsin and trypsin was significantly increased as compared to primary hydrolysates. Furthermore, the peptic secondary hydrolysates were fractionated by gel filtration and reverse phase-HPLC (RP-HPLC) and characterized by matrix-assisted laser desorption-ionization time of flight/mass spectrometry (MALDI-TOF/MS). These peptides with molecular weight of less than 1 kDa possessed the stronger ACE inhibitory effect, and their inhibitory pattern was found to be competitive. The results showed that the DM hydoplysates might be utilized as a rich source of bioactive peptide.     

Antioxidant and free radical-scavenging activities of smooth hound (Mustelus mustelus) muscle protein hydrolysates obtained by gastrointestinal proteases

We have investigated the antioxidative activity of five hydrolysates from smooth hound (Mustelus mustelus) meat obtained by various gastrointestinal proteases: crude enzyme extract, low molecular weight (LMW) alkaline protease and trypsin-like protease from M. mustelus intestine, pepsin from M. mustelus stomach, and bovine trypsin.     

Three novel angiotensin I-converting enzyme (ACE) inhibitory peptides from cuttlefish (Sepia officinalis) using digestive proteases

The angiotensin I-converting enzyme (ACE) inhibitory activities of protein hydrolysates prepared from muscle of cuttlefish (Sepia officinalis) by treatment with various digestive proteases were investigated. The most active hydrolysate was obtained with the crude protease extract from the hepatopancreas of cuttlefish (64.47 ± 1.0% at 2 mg of dry weight/ml) with a degree of hydrolysis of 8%. By gel filtration on Sephadex G-25 and RP-HPLC on C18 column, three novel peptides with high ACE-inhibitory activity were purified and their molecular masses and amino acid sequences were determined. The three peptides Val-Tyr-Ala-Pro, Val-Ile-Ile-Phe and Met-Ala-Trp with IC₅₀ values of 6.1, 8.7 and 16.32 μM, respectively, were novel ACE-inhibitory peptides. Lineweaver–Burk plots suggest that the three purified peptides act as non-competitive inhibitors against ACE. These results suggest that some peptides from cuttlefish could be a beneficial ingredient for nutraceuticals against hypertension.     

Isolation and characterisation of a novel angiotensin I-converting enzyme (ACE) inhibitory peptide from the algae protein waste

A hendeca-peptide with angiotensin I-converting enzyme (ACE) inhibitory activity was isolated from the pepsin hydrolysate of algae protein waste, a mass-produced industrial by-product of an algae essence from microalgae, Chlorella vulgaris. Edman degradation revealed its amino acid sequence to be Val-Glu-Cys-Tyr-Gly-Pro-Asn-Arg-Pro-Gln-Phe. Inhibitory kinetics revealed a non-competitive binding mode with IC₅₀ value against ACE of 29.6 μM, suggesting a potent amount of ACE inhibitory activity compared with other peptides from the microalgae protein hydrolysates which have a reported range between 11.4 and 315.3 μM. In addition, the purified hendeca-peptide completely retained its ACE inhibitory activity at a pH range of 2–10, temperatures of 40–100 °C, as well as after treatments in vitro by a gastrointestinal enzyme, thus indicating its heat- and pH-stability. The combination of the biochemical properties of this isolated hendeca-peptide and a cheap algae protein resource make an attractive alternative for producing a high value product for blood pressure regulation as well as water and fluid balance.     

Evidence of in vivo satietogen effect and control of food intake of smooth hound (Mustelus mustelus) muscle protein hydrolysate in rats

Protein hydrolysates are of a significant interest, due to their potential application as a source of bioactive peptides in nutraceutical and pharmaceutical domains. The present study was focused on the effect of protein hydrolysate from smooth hound (Mustelus mustelus) (SHPH) in the regulation of components of the food intake control such as satiety. SHPH was produced by intestinal digestive proteases from the same species. The amino acid analysis by GC/MS showed that the hydrolysate was rich in leucine, alanine, glycine, threonine, serine, lysine and glutamate. The molecular weights of peptides in SHPH were estimated by ESI-MS to be between 200 and 2500 Da. Biological in vivo capacities of SHPH in rats were evaluated by determination of the CCK-like peptides and insulin content using a clinical human radioimmunoassay. The food intake and the body weight of rats were measured during the period of treatment. Rats treated with SHPH showed a significant decrease in body weight at the end of treatment, as well as a decrease of food intake. Our findings revealed a possible mechanism of the beneficial effects of SHPH in appetite regulation, and this might be important to prevent the risk of a number of medical conditions including type II diabetes.     

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.     

Identification of novel angiotensin I-converting enzyme inhibitory peptide from collagen hydrolysates and its molecular inhibitory mechanism

The fish collagen protein was hydrolysed and further fractionated into four molecular weight ranges by ultrafiltration. Subsequently, the peptide fraction with the potent angiotensin I-converting enzyme (ACE) inhibitory activity was identified. The potential inhibitory mechanism of the peptide was clarified by molecular docking. As a result, FCPH-Ⅳ with molecular weight between 600 and 1000 Da exerted the high ACE inhibitory activity and was identified by de novo peptide sequencing. The peptide GHVGAAGS exhibited significant ACE inhibitory activity with the IC₅₀ value of 407.28 ± 3.55 μm . In addition, the docking results showed the interactions between the amino acids at the four positions closest to the C-terminal site of GHVGAAGS and the major active residues (GLN281, HIS353, LYS511 and HIS513) of ACE lead to the conformational change in ACE. This work indicates that fish collagen could be utilised to produce ACE inhibitory peptides and develop health products.     

Angiotensin I-converting enzyme (ACE) inhibitory peptide derived from Tenebrio molitor (L.) larva protein hydrolysate

Short peptides from different sources have been proven to be very efficient inhibitors of angiotensin I-converting enzyme (ACE), an enzyme with a major role in the regulation of blood pressure. In order to obtain ACE-inhibitory peptides from Tenebrio molitor (L.) larva, enzymatic hydrolysis was performed using Alcalase. The hydrolysate (DH 20 %) with the highest ACE inhibition was fractionated into four peaks according to their relative molecular weight by gel filtration on Sephadex G-15. The IC₅₀ value of peak 2 (with the relative molecular weight 180–500 Da) was 0.23 mg/mL, while it was 0.39 mg/mL of hydrolysate before fractionation determined by reversed-phase high performance liquid chromatography (RP-HPLC). Antihypertensive activity of peak 2 was proven by single oral administration to spontaneously hypertensive rats (SHR). Multiple dose oral administration (100, 200, and 400 mg/kg body weight) to SHR led to a significant decrease in blood pressure for peak 2. The reduction of systolic blood pressure even reached 27 mm Hg at 4 h post-administration at a dose of 400 mg/kg body weight. Additionally, further separation and purification of the ACE-inhibitory peptides from peak 2 were performed using RP-HPLC on C₁₈ column. Then a novel peptide Tyr–Ala–Asn was identified by tandem mass spectrometry with an IC₅₀ value of 0.017 mg/mL. The result of research indicates that Tenebrio molitor (L.) larva protein may be a suitable candidate to prepare ACE-inhibitory peptides which could be used for food industry and nutraceuticals.     

A HPLC-UV method for the determination of angiotensin I-converting enzyme (ACE) inhibitory activity

To determine the angiotensin-converting enzyme (ACE) inhibitory activity of a fish hydrolysate, different methods were tested. Finally, a sensitive, extraction-free HPLC method using N-(3-[2-furylacryloyl)-Phe-Gly-Gly (FAPGG) as substrate was preferred. This method relies on the UV-titration of the peptide 2-furylacryloyl-l-Phe (FAP) resulting from the hydrolysis of the FAPGG after a chromatographic separation on a reverse phase column. The experimental conditions (enzyme/substrate ratio, incubation time, NaCl concentration) were optimised for linearity, sensitivity and precision. The assay was adequate for the study of ACE inhibition by Captopril, used as reference, and several peptides. Captopril and the fish hydrolysate had IC₅₀ values, respectively of 0.19 ng and 43 μg with standard deviations of 0.09 ng and 5 μg. Afterwards, the determination of the Hill coefficient sustained the hypothesis that active peptides present in the fish hydrolysate were low-molecular weight molecules. This result was confirmed by the activity measurement of the fish hydrolysate fractions obtained by gel filtration.     

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.     

Angiotensin I-converting enzyme inhibitory and hypocholesterolemic activities: Effects of protein hydrolysates prepared from Achatina fulica snail foot muscle

The angiotensin I-converting enzyme (ACE) inhibitory activity and hypocholesterolemic effect of Achatina fulica snail foot muscle protein hydrolysates (SFMPH) and its hydrolysates were studied. The SFMPHs were prepared at a temperature of 121°C for 60 min. To obtain the enzymatic hydrolysates, the SFMPHs were further hydrolysed with three proteases (papain, trypsin, or alcalase). Among all the hydrolysates, alcalase hydrolysate showed the highest degree of hydrolysis and was dominated by a small molecular size fraction (189–686 Da). The SFMPH treated by alcalase was effective in disintegrating intact cholesterol micelles. Furthermore, alcalase hydrolysate with a hydrolysis time of 60 min showed a strong ACE inhibitory activity in vitro with an IC₅₀ of 0.024 mg/mL. Therefore, alcalase hydrolysate may be a promising ingredient for the use in functional foods.     

Effect of angiotensin I-converting enzyme (ACE) inhibitory peptide purified from enzymatic hydrolysates of <Emphasis Type="Italic">Styela plicata</Emphasis>

Angiotensin I-converting enzyme (ACE) inhibitory peptide was isolated from Styela plicata. The S. plicata was hydrolyzed with various proteases including Protamex, Kojizyme, Neutrase, Flavourzyme, Alcalase, trypsin, α-chymotrypsin, pepsin, and papain. The hydrolysate prepared with Protamex had the highest ACE inhibitory activity compared to the other hydrolysates. We attempted to isolate ACE inhibitory peptides from hydrolysate prepared with Protamex using ultra-filtration, gel filtration on a Sephadex G-25 column and reversed-phase high-performance liquid chromatography (RP-HPLC) on an ODS column. IC₅₀ value of the purified ACE inhibitory peptide was 24.7 μM, and Lineweaver–Burk plots suggest that the purified peptide from S. plicata acts as mixed-type inhibitor against ACE. Amino acid sequence of the purified peptide was identified as Met-Leu-Leu-Cys-Ser, with a molecular weight 566.4 Da. The results of this study suggest that peptides derived from S. plicata may be beneficial as anti-hypertension compounds in functional foods resource.     

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 ± 1.8 μg 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 ± 1.4 μg protein/mL. Enzymatic kinetic studies using this fraction demonstrated competitive inhibition with K_i of 11.6 ± 1.7 μg protein/mL. Mass spectrometric characterization identified for the first time the octapeptide PVNNPQIH which demonstrated an IC₅₀ value of 206.7 ± 3.9 μM. 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.     

Free radical scavenging, angiotensin I-converting enzyme (ACE) inhibitory, and in vitro anticancer activities of ramie (Boehmeria nivea) leaves extracts

The objectives of this study were to evaluate the antioxidant activity, angiotensin I-converting enzyme (ACE) inhibitory activity, and anticancer activity of ramie (Boehmeria nivea) leaves (RL). The RL was extracted with 70%(v/v) ethanol (RLE) and fractionated with the solvents of hexane, chloroform, ethylacetate, and aqueous. The ethylacetate fraction (EF) contained the highest phenolic contents of 651.65 mg/g, followed by RLE of 148.72 mg/g. The EF showed strong 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, superoxide radical scavenging activity, and hydroxyl radical scavenging with a 50% inhibition concentration (IC₅₀) of 0.097, 0.129, and 0.191 mg/mL, respectively. The ACE-inhibitory activity of EF was 80.32% at a concentration of 0.1 mg/mL. The EF showed growth-inhibitory effect of 67.21% at 0.25 mg/mL on the LoVo cell line, and 56.08% at 0.25 mg/mL on the NCI-H460 cell line, respectively. Therefore, it was suggested that the RL were potential materials for use as functional food and medicine.     

利用鲫鱼加工下脚料酶解制备ACE抑制肽的工艺优化

以鲫鱼加工下脚料为原料,用酶解法制备ACE抑制肽,并以血管紧张素转化酶(angiotension converting enzyme,ACE)抑制率为指标,从碱性蛋白酶、风味蛋白酶、中性蛋白酶、胰蛋白酶、木瓜蛋白酶、胃蛋白酶中筛选出最佳酶解蛋白酶为胃蛋白酶;以单因素试验为基础,应用Box-Benhnken中心组合设计原理和响应面分析法,探讨各自变量及其交互作用对ACE抑制率的影响,通过模拟得到的二次多元式方程预测模型,确定最佳酶解条件为:料液比14.4(mV)、加酶量[E]/[S]=521U/g、酶解时间5.3h。以优化条件制备的酶解产物ACE实际抑制率可达75.79%,与理论预测值76.17%相差不大。     

Angiotensin converting enzyme (ACE) inhibitory,antihypertensive and antihyperlipidaemic activities of protein hydrolysates from Rhopilema esculentum

Angiotensin-converting enzyme (ACE) inhibitory, antihypertensive and antihyperlipidaemic activities of protein hydrolysates (RPH) from the jellyfish Rhopilema esculentum were investigated. R. esculentum was hydrolysed sequentially with pepsin and papain, and then the hydrolysate was ultrafiltered with a 2000 Da cut-off membrane. It was found that RPH contained high levels of Gly, Glu, Pro, Asp and Ala, having potential ACE inhibitory activity in vitro with an IC₅₀ of 1.28 mg/ml. It was also found that systolic blood pressure was reduced markedly in spontaneously hypertensive rats after single and chronic oral administration of RPH, indicating that RPH had an antihypertensive effect. In addition, oral administration of RPH decreased total serum cholesterol and triglyceride, and increased high-density lipoprotein cholesterol in rats fed with high-fat diet. These results indicate that RPH may prove to be a promising functional food for the prevention and treatment of hypertension and hyperlipidaemia.     

Impact of processing on stability of angiotensin I-converting enzyme (ACE) inhibitory peptides obtained from tuna cooking juice

In this study, angiotensin I-converting enzyme (ACE) inhibitory peptides, which had previously been identified in an active gelfiltration fraction from tuna cooking juice, were examined for the stability of their inhibitory properties and composition changes during processing and in the presence of gastrointestinal proteases. Results indicated that ACE inhibitory peptides reserved almost the same composition before and after various temperatures (20–100 °C), levels of pressure (50–300 MPa) and pH (2–10) treatments. ACE inhibitory peptides retained 95–99% activity after simulated digestion. High Performance Liquid Chromatography (HPLC) chromatograph peptide mappings exhibited slight differences before and after temperature (100 °C), pressure (300 MPa) and pH (2, 10) treatments. Our results indicate that tuna cooking juice-derived ACE inhibitory peptides possess some degree resistance to the influence of temperature, pressure, pH treatments, and gastrointestinal proteases.     

胃蛋白酶水解甘薯蛋白制备血管紧张素转化酶抑制肽的研究

采用胃蛋白酶水解甘薯蛋白制备血管紧张素转化酶(ACE)抑制肽,通过四元二次回归正交旋转组合设计,考察底物浓度、酶与底物浓度比、pH值、温度对ACE抑制率的影响,并确定最优酶解工艺参数,最终建立了ACE抑制率与各影响因素的回归模型。在此基础上,确定了胃蛋白酶水解甘薯蛋白的最适条件为:底物浓度2.3%、酶与底物浓度比3.7%、pH2.3、温度37℃、时间8h,采用该优化工艺,得到ACE抑制率最大为78.37%的水解产物。为开发防治高血压的保健食品提供了理论依据。     

Identification and characterization of a novel angiotensin I-converting enzyme inhibitory peptide (ACEIP) from silkworm pupa

The angiotensin I-converting enzyme inhibitory peptide (ACEIP) was isolated and characterized from silkworm pupae and purified using Sephadex G-25 gel filtration. The structure and physicochemical properties of pupa ACEIP were analyzed. The α-P₃ fraction exhibited the most potent ACE inhibitory activity. After purification via semi-preparative reverse-phase HPLC (RP-HPLC) and HPLC, the α-P₃-6-b component was revealed to have the highest ACE inhibitory activity (IC₅₀=28.3 μg/mL). Edman degradation revealed a Val-Glu-Ile-Ser amino acid sequence in which novel active sequences were identified. Physicochemical property testing showed that purified pupa ACEIP exhibits good solubility, heat resistance, and acid resistance that all indicate ACEIP derived from silkworm pupa is an excellent food-derived ACEIP.     

分子排阻色谱纯化核桃蛋白源ACE抑制肽条件优化

为了得到新的ACE抑制肽,采用分子排阻色谱对核桃蛋白碱性蛋白酶水解物进行分离、纯化。优化了分子排阻色谱分离、纯化核桃蛋白水解物的洗脱剂、洗脱流速、上样体积和样品浓度,比较了其纯化前后ACE抑制活性变化、分析了不同组分RP–HPLC图谱及其氨基酸组成。结果表明,以Sephadex G–15为分离介质的色谱优化条件为:超纯水为洗脱剂、洗脱流速0.4 m L/min、上样体积1.5 m L、样品浓度150 mg/m L。在此条件下,经过分子排阻色谱纯化后,得到4个主要组分,其中活性最高组分的ACE抑制率达90.35±0.25%,其IC50值为0.158 mg/m L。经过RP–HPLC进一步分离,分子排阻色谱纯化后高活性组分保留时间主要集中于10 min、13 min、17 min和21 min,并且其疏水性氨基酸的含量得到显著富集,其中芳香族氨基酸Tyr、Phe的含量分别达10.46%和20.15%。