合成二肽ACE抑制活性及抗高血压初步研究

目的:对合成二肽的ACE抑制活性进行系统地筛选,寻找具有高ACE抑制活性的二肽并研究其在动物体内的降血压作用。方法:利用可见光分光光度法对35种合成二肽的ACE抑制活性进行测定,并对具有高ACE抑制活性的二肽进一步测定其IC50值。选取ACE抑制效果最佳的二肽进行原发性高血压大鼠（SHR）降血压实验。实验采用灌胃方法分别以1、10、25、50、100 mg/kg的剂量溶于1 m L生理盐水进行给药,分别于给药后2 h和4 h测量大鼠血压变化。结果:研究发现大部分二肽都具有一定程度的ACE抑制活性,其中以氨基酸序列为半胱氨酸-丙氨酸（Cys-Ala,CA）和半胱氨酸-组氨酸（CysHis,CH）的二肽ACE抑制活性最高,其ACE抑制率分别达到84.38%和70.79%。经测定CA和CH的IC50值分别为23.22μmol/L和61.17μmol/L。SHR大鼠降血压实验表明CA在体内的降压效果显著。给药后4 h CA降压效果优于给药后2 h,给药后2 h CA降压效果与给药剂量呈较好的正相关性（R2>0.8）。结论:体外ACE抑制率测定及SHR大鼠降血压实验发现合成二肽CA具有较高的ACE抑制活性和较好的体内降血压效果,提示CA可以作为降血压保健食品的添加成分,值得进一步研究。      

高血管紧张素转换酶抑制活性的豆酱中优势霉菌的分离

以具有高血管紧张素转换酶(ACE)抑制活性的我国传统豆酱为研究对象,对发酵过程中的霉菌分布进行研究.酱曲中的霉菌数为10a cfu/g,后酵过程中仍保持在较高水平.分离两株优势霉菌,井根据其菌落形态、菌丝和孢子形态及产孢结构等鉴定为米曲霉原变种(Aspergillus oryzae)和灰蓝毛霉(Mucor griseo-cyanus),这两种霉菌在整个发酵过程中的存在对豆酱中ACE抑制剂的生成发挥着重要作用,为纯种发酵生产功能性调味品提供指导.     

Angiotensin I-converting enzyme inhibitory activities of Chinese fermented soypaste and estimation of the inhibitory substances

Angiotensin I-converting enzyme (ACE) inhibitory activities of aqueous extracts of Chinese commercial soypaste were investigated in this work. Six samples from northern China showed potent ACE inhibitory activities with IC₅₀ values less than 40.0 μg/mL. ACE inhibitors in the sample with the strongest activity were purified by ultrafiltration, solid-phase extraction and gradient RP-HPLC. According to spectroscopic methods, a compound (M328.1) was separated as C₁₅H₂₁NO₇. It was supposed to be a conjugate of phenylalanine and glucose generated by Maillard reaction during soypaste production, providing support on the contribution of Maillard reaction products to the ACE inhibitory activity of the sample. Results further indicated that the total ACE inhibition by the sample occurred from the combined function of various bioactive substances, suggesting that Chinese soypaste could be a good source of ACE inhibitors for exploring functional foods or ingredients with antihypertensive effect.     

Inhibition of angiotensin I-converting enzyme by wheat gliadin hydrolysates

A tryptic gliadin hydrolysate was fractionated into peptide fractions, which were assigned to either the central domain (CD) or terminal domains (TD) of gliadins. The domains were expected to contain amino acid (AA) sequences which, when released from the parent protein, inhibit the angiotensin I-converting enzyme (ACE), which plays a key role in regulating blood pressure. A proline (Pro) poor TD related fraction, containing the smallest peptides, showed the highest ACE inhibitory activity (IC₅₀ = 0.33 mg/ml). Additional peptidases were selected based on their in silico predicted ability to release ACE inhibitory peptides. Further hydrolysis of the tryptic hydrolysate fractions with thermolysin, Clarex, Alcalase and Esperase increased ACE inhibitory activities. Immobilised Ni²⁺-ion affinity chromatography (IMAC) purification of a TD related peptide fraction obtained by sequential hydrolysis with trypsin and thermolysin yielded a fraction with an IC₅₀ value of 0.02 mg/ml. This IMAC fraction was enriched in histidine and hydrophobic AA (Pro, Val, Ile, Leu and Phe).     

酱油对血管紧张素转换酶抑制作用的研究

考察了若干酱油样品的血管紧张素转换酶(ACE)抑制活性,发现所试样品均表现出ACE抑制活性,其IC50值范围为0.7405～3.0265mg/mL。样品的ACE抑制活性与其蛋白质降解程度、多肽含量及颜色值无明显相关性,应为多种活性物质综合作用的结果。对我国酱油产品中ACE抑制剂的结构表征可为潜在降血压功能性食品的研发提供指导。     

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.     

Inhibition properties of dipeptides from salmon muscle hydrolysate on angiotensin I-converting enzyme

We isolated Phe–Leu as an angiotensin I‐converting enzyme (ACE) inhibitor from hydrolysate of chum salmon muscle. The IC₅₀ value of this peptide was 13.6 μm , and it showed non‐competitive inhibition. The reverse sequence dipeptide Leu–Phe also showed ACE inhibitory activity. However, Leu–Phe is much less inhibitory than Phe–Leu with an IC₅₀ value of 383.2 μm . In addition, the inhibition mode was competitive. To investigate the relationship between dipeptide sequence and ACE inhibition properties, we further measured ACE inhibitory activity and inhibition mechanism using six Trp‐containing dipeptides, which had been identified from the same salmon muscle hydrolysate as ACE inhibitory peptides in a previous study. Peptides with Trp as the C‐terminal residue, Ala–Trp, Val–Trp, Met–Trp, Ile–Trp, Leu–Trp showed non‐competitive inhibition. On the other hand, reversed sequence peptides with Trp at the N‐terminal were competitive inhibitors, except Trp–Leu. These results indicate that the sequence of ACE inhibitory dipeptides can affect both inhibitory potency and the inhibition mechanisms.     

高效液相色谱法快速检测血管紧张素转换酶抑制活性

建立高效液相色谱法快速检测血管紧张素转换酶(ACE)抑制活性的方法。色谱条件为:以ODS C18柱(150mm×4.6mm,5μm)为色谱柱,乙睛-水(20:80,V/V,含0.5‰甲酸)为流动相,流速1mL/min,检测波长为228nm。结果表明:马尿酸在0.1～500μg/mL质量浓度范围内线性关系良好,相关系数r=0.9999;平均回收率为89.77%～99.57%,RSD为0.01%～1.52%。不同的ACE抑制剂检测结果证明该方法操作简便快速、重现性好、准确度高,可以作为ACE抑制剂体外活性的检测方法。     

我国豆酱血管紧张素转换酶抑制活性的比较

文章对我国三种市售豆酱的血管紧张素转换酶(ACE)抑制活性及其主要化学组成进行比较分析,所试样品的IC50值分别为1.373、0.876和1.691 mg/mL,活性最高样品中的多肽含量为24.09g/100g dry matter,不同样品间多肽分布无明显差异。样品的ACE抑制活性应为多种活性物质综合作用的结果,对其中ACE抑制剂构效关系的研究可为我国豆酱功能性的研发提供指导。     

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 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.     

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.     

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.     

Purification and characterisation of angiotensin I converting enzyme inhibitory peptides from lysozyme hydrolysates

Hen egg white lysozyme (HEWL) was hydrolysed with trypsin, papain and a combination of the two. The prepared hydrolysates exhibited ACE inhibitory activity. The hydrolysates were fractionated using ultrafiltration and reverse phase-high performance liquid chromatography (RP-HPLC). Three fractions, which showed the highest ACE inhibitory activities, were purified by RP-HPLC. They were the F₇ (from papain-trypsin hydrolysate), F₈ (from papain hydrolysate) and F₃ (from trypsin hydrolysate) fractions. The IC₅₀ values were 0.03, 0.155 and 0.23 mg/ml for F₇, F₈ and F₃, respectively. The F₇ fraction was the most potent ACE inhibitor peptide, and was composed of 12 amino acids, Phe-Glu-Ser-Asn-Phe-Asn-Thr-Gln-Ala-Thr-Asn-Arg (MW: 1428.6 Da). Lineweaver-Burk plots suggest that the F₇ peptide acts as an uncompetitive inhibitor against ACE. The kinetic parameters (K_m, V_max, and K_i) for the F₇ peptide were measured and compared to the control.     

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.     

乳酸菌盐溶性蛋白培养物中血管紧张素转化酶抑制活性肽的测定及分离

将16 株乳酸菌用MRS液体培养基3 代继代培养后,离心,并用灭菌的生理盐水制成乳酸菌悬浮液,接种于远东多线鱼肉盐溶性蛋白溶液（salt-soluble protein,SSP）,对其代谢产物进行血管紧张素转化酶（angiotensinconverting enzyme,ACE）抑制活性测定,筛选出ACE抑制活性较高的Pediococcus acidilactici ID7菌株（ACE抑制率为47.6%）和Lactobacillus plantarum 6214菌株（ACE抑制率为40.6%）。利用高效液相色谱（high performanceliquid chromatography,HPLC）对Pediococcus acidilactici ID7的盐溶性蛋白培养代谢物进行色谱分析,获得了两个峰,其相应成分对ACE抑制的IC50分别为1.21 μg/mL和1.07 μg/mL,利用凝胶过滤HPLC法对出现较高的ACE抑制活性峰的成分进行色谱纯化,获得了ACE抑制率为26.67%,分子质量为586.7 D以下的ACE抑制多肽。     

高效液相色谱法测定烟草中的淀粉含量

研究了用高效液相色谱法测定烟草中的淀粉含量,即先用酸将烟草中的淀粉水解为葡萄糖,再以甘露醇为内标物,用WatersSugar Pak1钙型阳离子交换柱为固定相,0 05g/LEDTA钙钠水溶液为流动相,示差折光仪为检测器测定淀粉水解产生的葡萄糖含量,由葡萄糖含量换算为淀粉含量。该方法的检测限为1 0mg/L,相对标准偏差为1 6%～2 1%,标准回收率在95%～105%之间。并用该方法测定了几种烟草样品中的淀粉含量。     

HPLC-ELSD法测定食品中异麦芽酮糖

建立了一种方便、准确、灵敏的方法以测定食品中异麦芽酮糖含量的高效液相色谱蒸发光散射检测器方法。使用Hypersil APS-2(NH2)(4.6 mm×250 mm,5μm)色谱柱分离,柱温40℃;以乙腈:水=88:12为流动相,流速1 m L/min;ELSD为检测器,雾化器温度50℃;蒸发器80℃;载气(氮气)流速1.6 L/min。结果:检出限为30μg/m L;在80~1200μg/m L内工作曲线线性良好,相关系数r2=0.9991,回收率85.6%~98.1%,精密度2.78%~2.92%。结果表明此方法前处理简单,易于操作,检测限低,在线性范围内线性良好,相关系数高,测量结果精确度高,是检测食品中异麦芽酮糖的有效方法。     

Identification of peptides in traditional and probiotic sheep milk yoghurt with angiotensin I-converting enzyme (ACE)-inhibitory activity

Two sets of traditional Greek sheep milk yoghurt were produced: the first one (YC) using normal yoghurt culture (Lactobacillus delbrueckii subsp. bulgaricus ?10.13 and Streptococcus thermophilus ?10.7) and the second (PR) with the same normal culture mixed with Lactobacillus paracasei subsp. paracasei DC412. YC and PR had similar physicochemical properties and proteolysis patterns throughout storage. Both products showed similar peptide profiles by RP-HPLC but quantitative differences were observed in respect to storage time. Single-strain cultures of the microorganisms used showed similar peptide profiles for both lactobacilli, yet L. delbrueckii subsp. bulgaricus was the most proteolytic of all three microorganisms. The peptide content and the ACE-inhibitory activity of the water-soluble extracts of yoghurts, YC and PR, increased throughout storage. Major peptides were identified from yoghurt PR and from the separate cultures of L. delbrueckii subsp. bulgaricus and L. paracasei subsp. paracasei. Most of these peptides were derived from β-casein. A peptide, β-CN f114-121, with well-established ACE-inhibitory and opiate-like activity was identified in yoghurt PR. Further identified peptides were regarded as potential ACE-inhibitors according to their sequence.