蚕蛹蛋白及其水解产物中氨基酸组成分析

采用FMOC-OPA柱前衍生化-高效液相色谱法测定蚕蛹蛋白及水解液氨基酸含量,同时测定血管紧张素转化酶(ACE)抑制活性,研究蚕蛹蛋白水解后氨基酸组成的改变及对ACE抑制活性的影响。结果表明:水解液10kD以下组分(SN2)中疏水性氨基酸含量为49.15%,显著高于10kD以上组分(SN1)中的40.55%及蚕蛹蛋白中的45.21%,其中对ACE抑制活性影响较大的脯氨酸、芳香族氨基酸含量为7.75%和19.20%,高于SN1中的5.62%和13.59%及蚕蛹蛋白中的7.42%和15.81%,并且SN2中ACE抑制率为47.6%显著高于SN1和蚕蛹蛋白中的抑制率,表明水解后疏水性氨基酸组成的改变与ACE抑制活性的变化趋势相同,且ACE抑制活性主要集中于SN2。     

Systemic screening of milk protein-derived ACE inhibitors through a chemically synthesised tripeptide library

A tripeptide library consisting of 373 tripeptides (nine repeats) based on bovine α-S1 casein and β-lactoglobulin was synthesised chemically, purified by HPLC and the angiotensin-converting enzyme (ACE) inhibitory activities were assayed. Of the 364 tripeptides assayed, 144 showed a relative ACE inhibitory activity higher than 50% and 43 higher than 60%, at a set concentration of 2.5 mM. More potent tripeptides were found from α-S1 casein than from β-lactoglobulin. The high percentage of Pro/Tyr in caseins could be the reason for this. Fifteen tripeptides with relative activities higher than 50% were selected and assayed for their IC₅₀, using hippuryl-l-histidyl-l-leucine as the substrate. The most potent peptide showed an IC₅₀ of 0.85 μM.     

Purification of angiotensin converting enzyme inhibitory peptides from sunflower protein hydrolysates by reverse-phase chromatography following affinity purification

The purification of a peptidic fraction with angiotensin converting enzyme (ACE) inhibitory activity from sunflower protein hydrolysates by affinity chromatography was recently described. We now describe that reverse-phase HPLC fractionation of this product yields several fractions with IC₅₀ one order of magnitude higher than those previously purified by reverse-phase HPLC following gel filtration chromatography, showing that affinity chromatography is much more effective than gel filtration chromatography as a first step for purification of ACE inhibitory peptides. The amino acid composition of these fractions is presented, but attempts to determine their amino acid sequence failed, showing that these fractions contained more than one peptide.     

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.     

Isolation and characterisation of a novel angiotensin I‐converting enzyme‐inhibitory peptide derived from douchi,a traditional Chinese fermented soybean food

BACKGROUND: Douchi, a traditional fermented soybean food, has recently attracted a great deal of attention owing to its superior physiological activity. In the present study the angiotensin I‐converting enzyme (ACE)‐inhibitory activity of typical douchi procured from various regions of China was analysed. An ACE‐inhibitory peptide derived from the most potent douchi was also isolated and characterised. The pattern of ACE inhibition and resistance to hydrolysis by gastrointestinal proteases of this peptide are described. RESULTS: ACE‐inhibitory activities were detected in all douchi samples, with IC₅₀ values ranging from 0.204 to 2.011 mg mL^?1. Among the douchi samples, a Mucor‐type douchi exhibited the most potent ACE‐inhibitory activity (IC₅₀ = 0.204 mg mL^?1). A novel ACE‐inhibitory peptide was then isolated from this Mucor‐type douchi using ultrafiltration followed by Sephadex G‐25 column chromatography and reverse phase high‐performance liquid chromatography. The amino acid sequence of the purified peptide was identified by Edman degradation as His‐Leu‐Pro (IC₅₀ = 2.37 µmol L^?1). The peptide is a competitive inhibitor and maintained its inhibitory activity even after incubation with some gastrointestinal proteases. CONCLUSION: The present study shows that peptides derived from soybean fermentation during douchi processing could be the main contributor to the ACE‐inhibitory activity observed. Copyright © 2009 Society of Chemical Industry     

贮藏时间及缫丝工艺对桑蚕蛹蛋白质和脂肪酸组成及蛹油体外抗氧化活性的影响

以桑蚕蛹为对象,研究其在贮藏过程中及缫丝后蛋白质、脂肪酸变化及蚕蛹油的抗氧化活性,为缫丝副产物深加工提供理论支持。按照蚕蛹的形态变化,将贮藏期分为4 个阶段,对各阶段蚕蛹进行基本化学成分析;采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析桑蚕蛹蛋白质组分;气相色谱-质谱联用分析蚕蛹油脂肪酸组成;此外,对蚕蛹油1,1-二苯基-2-三硝基苯肼（1,1-diphenyl-2-picrylhydrazyl,DPPH）自由基和2,2’-联氮-二(3-乙基苯并噻唑啉-6-磺酸)（2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid),ABTS）阳离子自由基清除能力进行评价。结果表明,贮藏时间对蚕蛹基本化学成分总体有显著性影响（P＜0.05）。蚕蛹蛋白质主要分布在约30 kDa和约70 kDa处,且表达量随着蚕蛹贮藏时间延长而降低。蛹油含有16 种脂肪酸,相对含量较高的棕榈酸、硬脂酸、油酸、亚油酸和α-亚麻酸在贮藏期内变化显著（P＜0.05）。贮藏期蛹油DPPH自由基和ABTS阳离子自由基清除率的半抑制质量浓度范围分别为20.93～52.00 mg/mL和33.32～156.81 mg/mL,其体外抗氧化活性可能与其总酚含量（15.95～77.50 mg/kg）变化有关。经缫丝加工后,蚕蛹蛋白质和脂肪酸组成发生明显变化,且蚕蛹油体外抗氧化活性降低。综上,蚕茧的贮藏时间、缫丝工艺能够影响蚕蛹蛋白质、脂肪酸组成和蛹油中总酚含量,并对蚕蛹油的抗氧化活性产生较大影响。因此,可根据目的产物的表达水平或生物活性有针对性地收集各贮藏阶段的蚕蛹应用于加工生产中。     

Evaluation of angiotensin I-converting enzyme (ACE) inhibitory activities of smooth hound (Mustelus mustelus) muscle protein hydrolysates generated by gastrointestinal proteases: identification of the most potent active peptide

In this study, smooth hound protein hydrolysates (SHPHs), obtained by treatment with various gastrointestinal proteases, were analyzed for their angiotensin I-converting enzyme (ACE) inhibitory activities. Protein hydrolysates were obtained by treatment with crude alkaline enzyme extract, low molecular weight (LMW) alkaline protease, trypsin-like protease and pepsin from Mustelus mustelus, and bovine trypsin. All hydrolysates exhibited inhibitory activity toward ACE. Hydrolysate generated with alkaline protease extract displayed the highest ACE inhibitory activity, and the higher inhibition activity (82.6% at 2 mg/mL) was obtained with a hydrolysis degree of 18.8%. This hydrolysate was then fractionated by size exclusion chromatography on a Sephadex G-25 into five major fractions (P₁–P₅). ACE inhibitory activities of all fractions were assayed, and P₃ was found to display a high ACE inhibitory activity (62.24% at 1 mg/mL). P₃ was then fractionated by reversed-phase high-performance liquid chromatography (RP-HPLC) and ten fractions of ACE inhibitors were found (F₁–F₁₀). Sub-fraction F₃ showed the strongest ACE inhibitory activity, being able to suppress more than 60% of initial enzyme activity at a concentration of 100 μg/mL. The amino acid sequence of peptide F₃ was determined by ESI/MS and ESI–MS/MS as Ala-Gly-Ser, and the IC₅₀ value for ACE inhibitory activity was 0.13 ± 0.03 mg/mL. Further, purified peptide F₃ maintained inhibitory activity even after in vitro digestion with gastrointestinal proteases in order to demonstrate gastrointestinal stability digestion to enable oral application. These results indicate that smooth hound protein hydrolysate possesses potent antihypertensive activity.     

Inhibitory effect of polyphenol-rich extracts of jute leaf (Corchorus olitorius) on key enzyme linked to type 2 diabetes (α-amylase and α-glucosidase) and hypertension (angiotensin I converting) in vitro

Corchorus olitorius leaf is consumed in various parts of the world as leafy vegetable and folk remedy for the management of some degenerative diseases with dearth of information on its biochemical rationale. Therefore, this study sought to characterize the inhibitory action of polyphenol-rich extracts (free and bound) of C. olitorius on α-amylase, α-glucosidase and angiotensin I converting enzyme (ACE), as well as to identify the phenolic compound responsible for these activities. Our findings revealed that the extracts inhibited α-amylase and α-glucosidase (12.5–50.0 μg/mL), and ACE (10.0–50.0 μg/mL) in dose-dependently with free extracts having significantly (P < 0.05) higher α-amylase (17.5 μg/mL), α-glucosidase (11.4 μg/mL) and ACE (15.7 μg/mL) inhibitory activities as revealed by the IC₅₀. Reversed-phase HPLC analysis of the extracts revealed chlorogenic acid (7.5 mg/100 g) and isorhamnetin (51.1 mg/100 g) as the main phenolics in the free extract and caffeic acid (58.1 mg/100 g) in the bound extract. Therefore, the enzyme inhibitory activity of C. olitorius extracts may be attributed to the presence of caffeic acid, chlorogenic acid and isorhamnetin, thus justifying its use in folklore for the management of diabetes and hypertension.     

蚕蛹油α-亚麻酸对实验性高血脂大鼠的降脂作用

目的：研究蚕蛹油(silkworm pupa oil,SPO)α-亚麻酸(α-linolenic acid)对实验性高血脂大鼠的预防和治疗作用。方法：建立远交群(sprague dawley,SD)大鼠高脂血症模型,预防性及治疗性给予蚕蛹油α-亚麻酸,测定并分析对大鼠血清甘油三酯(TG)、总胆固醇(TC)、高密度脂蛋白胆固醇(HDL-C)、载脂蛋白A-I(apolipoprotein A-I,Apo A-I)及脂蛋白酯酶(lipoprotein lipase,LPL)和肝脂酶(hepatic lipase,HL)活性的影响。结果：蚕蛹油α-亚麻酸具有显著降低实验性高血脂大鼠血清TC、TG,升高HDL-C、Apo A-I的作用,同时可以增强LPL及HL活性。结论：蚕蛹油α-亚麻酸具有显著的预防和治疗高脂血症的作用。     

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.     

Characterisation of a new antihypertensive angiotensin I-converting enzyme inhibitory peptide from Pleurotus cornucopiae

This study describes the characterisation of a new angiotensin I-converting enzyme (ACE) inhibitory peptide from the fruiting body of Pleurotus cornucopiae which could be used as a functional food or nutraceutical compounds. After purification of the ACE inhibitor in an ultrafiltration, Sephadex G-25 column chromatography, successively C₁₈ and SCX solid-phase extraction and reverse-phase HPLC, two types of the purified ACE inhibitors with IC₅₀ values of 0.46 and 1.14 mg/ml were obtained. The two purified ACE inhibitors were analysed, showing two types of oligopeptides. The amino acid sequences of the two purified oligopeptides were found to be RLPSEFDLSAFLRA and RLSGQTIEVTSEYLFRH. The molecular mass of the purified ACE inhibitors was estimated to be 1622.85 and 2037.26 Da, respectively. Water extracts of P. cornucopiae fruiting body showed a clear antihypertensive effect on spontaneously hypertensive rats at a dosage of 600 mg/kg.     

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.     

Isolation of a novel peptide from silkworm pupae protein components and interaction characteristics to angiotensin I-converting enzyme

The hydrolysate of silkworm protein produced by acid protease (Asperqiius usamii NO. 537) contains angiotensin I-converting enzyme inhibitory activity. Four kinds of protein components were identified in silkworm pupae protein, albumin, globulin, prolamin and glutelin, and the albumin was in the highest quantity. The four component proteins were then further hydrolyzed by acid protease under the same conditions, and the hydrolysate of albumin was determined with the highest ACE inhibiting effect, followed by globulin. Albumin could be easily hydrolyzed under the aqueous conditions, and the overall albumin protein content was higher than the other three protein components. The peptide sequence “APPPKK” was determined by LC–MS/MS separation and X!Tandem software identification. The peptide inhibitory activity was 0.047 mg/mL in IC₅₀. The peptide was bonded to Asp⁴¹⁵, Asp⁴⁵³, Thr²⁸², His ³⁵³, Glu¹⁶² in hydrogen bond to ACE active pocket by flexible docking calculations. This study indicates that silkworm pupae protein may be a suitable candidate to explore functional foods with anti-hypertension bioactivity.     

Two Novel Bioactive Peptides from Antarctic Krill with Dual Angiotensin Converting Enzyme and Dipeptidyl Peptidase IV Inhibitory Activities

Inhibition of dipeptidyl peptidase IV (DPP‐IV) and angiotensin converting enzyme (ACE) are considered useful in managing 2 often associated conditions: diabetes and hypertension. In this study, corolase PP was used to hydrolyze Antarctic krill protein. The hydrolysate (AKH) was isolated by ultrafiltration and purified by size‐exclusion chromatography, ion exchange chromatography and reversed‐phase high‐performance liquid chromatography (RP‐HPLC) sequentially. The in vitro inhibitory activities of all AKHs and several fractions obtained against ACE and DPP‐IV were assessed. Two peptides, purified with dual‐strength inhibitory activity against ACE and DPP‐IV, were identified by TOF‐MS/MS. Results indicated that not all fractions exhibited dual inhibitory activities of ACE and DPP‐IV. The purified peptide Lys‐Val‐Glu‐Pro‐Leu‐Pro had half‐maximal inhibitory concentrations (IC₅₀) of 0.93±0.05 and 0.73±0.04 mg/mL against ACE and DPP‐IV, respectively. The other peptide Pro‐Ala‐Leu had IC₅₀ values of 0.64±0.05 and 0.88±0.03 mg/mL against ACE and DPP‐IV, respectively. This study firstly reported the sequences of dual bioactive peptides from Antarctic krill proteins, further provided new insights into the bioactive peptides responsible for the ACE and DPP‐IV inhibitory activities from the Antarctic krill protein hydrolysate to manage hypertension and diabetes.     

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.     

Isolation,purification and characterisation of angiotensin I‐converting enzyme–inhibitory peptides derived from catfish (Clarias batrachus) muscle protein thermolysin hydrolysates

The angiotensin I‐converting enzyme (ACE)‐inhibitory activities of catfish (Clarias batrachus) muscle protein hydrolysates were investigated. Thermolytic digests of C. batrachus sarcoplasmic and myofibrillar proteins exhibited inhibitory activity towards ACE and were purified with the aim of ultrafiltration, gel filtration and reversed‐phase high‐performance liquid chromatography (RP‐HPLC). The amino acid sequences of hydrolysates with the highest ACE‐inhibitory activities were determined using electrospray quadrupole time‐of‐flight tandem mass spectrometry (ESI‐TOFQ MS/MS). The sequences of GPPP (IC₅₀ = 0.86 μm ) and IEKPP (IC₅₀ = 1.2 μm ) corresponding to the fragments 986–989 and 441–445 of myosin‐I heavy chain were identified for the sarcoplasmic and myofibrillar protein hydrolysates, respectively. Peptide GPPP exhibited a mixed‐type inhibition whereas peptide IEKPP could only bind to the active sites of ACE. The results demonstrate that hydrolysates of C. batrachus muscle proteins obtained by thermolysin may contain bioactive peptides.     

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

Chickpea protein isolates and the protease alcalase were used for the production of protein hydrolysates that inhibit angiotensin I‐converting enzyme (ACE). The highest degree of inhibition was found in a hydrolysate obtained by 30 min of treatment with alcalase. This hydrolysate was used as starting material for the purification of ACE‐inhibitory peptides. After Biogel P2 gel filtration chromatography and HPLC C₁₈ reverse phase chromatography, four peptides with ACE‐inhibitory activity were purified. Two of them were competitive inhibitors of ACE, while the other two were uncompetitive inhibitors. These results show that chickpea proteins are a good source of ACE‐inhibitory peptides when hydrolysed with the protease alcalase. © 2002 Society of Chemical Industry     

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.     

Angiotensin I-converting enzyme inhibitor from Grifola frondosa

To investigate the hypotensive compounds from edible mushroom, extracts from 10 fruit bodies of mushrooms were screened for the inhibitory activity against angiotensin I converting enzyme (ACE). The most potent ACE inhibitory activity (58.7%) was detected in a cold water extract of Grifola frondosa, with an IC₅₀ of 0.95 mg. The ACE inhibitory activities of the cold and hot water extracts increased as the extraction time increased, but decreased slightly 15 h and 90 min, respectively, after extraction. After the purification of ACE inhibitory peptides with acetone fractionation and column chromatography, we obtained an active fraction with an IC₅₀ of 0.13 mg and a yield of 0.7%. The purified inhibitor showed competitive inhibition on ACE and this peptide maintained inhibitory activity even after digestion by intestinal proteases.     

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.