血管紧张素转换酶抑制肽的分离

采用截留分子量分别为30 ku、10 ku、6 ku、3 ku的中空纤维超滤膜对扇贝酶解产物进行分级分离,并结合凝胶柱层析分离技术测定了各分离组分的分子量分布,同时还测定了各分离组分对血管紧张素转换酶(ACE)的抑制活性。实验结果表明,不同截留分子量超滤膜可以实现酶解产物按其分子量大小的分级分离,不同分离组分的ACE抑制力活性差异较大,总趋势是截留分子量相对较小的超滤膜透过液的ACE抑制活性较强。其中,截留分子量为3 ku超滤膜的透过液具有最强的ACE抑制活性,其ACE抑制率I(%)=96.17%,半抑制浓度IC_(50)= 0.078 mg·mL~(-1)。通过对截留分子量为3 ku超滤膜的透过液依次经过Sephadex G-25及Sephadex G-15凝胶柱层析分离纯化后,分离出了ACE抑制活性最强的2个组分活性肽,其平均分子量分别为1 300 u和900 u左右,其IC_(50)分别为0.026 mg·mL~(-1)和0.012 mg·mL~(-1)。     

血管紧张素转换酶（ACE）的提取与活性验证

在从紫菜中提取制备降血压肽的研究中,为筛选能够抑制血管紧张素转换酶（ACE）活性的功能肽,需要大量ACE进行体外检测。但ACE成本昂贵,本文利用硫酸铵分级盐析法提取猪肺组织中的ACE,测其酶活力为15．45×10^-3U／mg,比活力为33．80×10^-3U／mg。通过卡托普利来验证其抑制效果,抑制率达49．15％。采用本实验室初步制得的紫菜降血压肽对自制ACE和商品ACE的活性进行抑制,测定IC50发现该抑制剂对2种ACE的抑制效果差异不大,且活性较为稳定,因此所提取的ACE粗酶液可直接用于降血压肽的体外检测实验。     

血管紧张素转化酶抑制肽研究进展

为研究鳗鱼加工副产物的综合利用,以鳗鱼骨胶原蛋白为原料,采用酶解法制备血管紧张素转化酶 (Angiotensin-converting enzyme, ACE) 抑制肽。以ACE抑制活性和水解度为评价指标,通过单因素和响应面试验确定最佳的酶解制备条件,并分析酶解产物的分子质量分布和氨基酸组成。结果显示,碱性蛋白酶为最适水解酶,最佳酶解条件为：温度50 ℃,质量浓度15 g·L^–1,酶解时间5.25 h,加酶量3.1% (质量分数),pH 9.2。在此条件下,鳗鱼胶原蛋白肽的ACE抑制活性为70.33%,与预测值接近;酶解产物中分子质量小于1 kD的肽占57.02%,1~3 kD的肽占36.55%;氨基酸组成分析表明,酶解产物中与ACE抑制活性有关的疏水性氨基酸 (如脯氨酸、缬氨酸、异亮氨酸、亮氨酸、苯丙氨酸) 含量增加。     

3942中性蛋白酶酶解中国毛虾蛋白制备血管紧张素转移酶抑制肽

采用3942中性蛋白酶酶解中国毛虾(Acetes chinensis)蛋白，制备具有抑制血管紧张素转移酶(ACE)活性的多肽。采取正交实验研究料水比、酶量、反应温度、反应时间4个因素对酶解中国毛虾蛋白产物的ACE抑制活性的影响。结果显示，在料水比1：3(体积比)，酶量0．5％，温度45℃，时间8h的水解条件下得到的酶解产物F7的ACE抑制活性最高，其抑制率达到92．86％。用层析柱Sephadex G-25、SP-Sephadex C-50对F7进行分离纯化，选取ACE抑制活性最高的峰进一步用反相高压液相色谱进行纯化，得到单一组分FⅠ。经飞行时间质谱和碰撞诱导裂解分析确定，FⅠ为新型的ACE抑制肽Ser-Pro，IC50值为272μmol／L。     

中国毛虾ACE抑制肽的初步研究

对以中国毛虾为原料酶法制备具有抑制血管紧张素转换酶(ACE)活性的酶解产物的方法作了探讨。以体外活性(ACE抑制率)为指标,通过正交试验确定胃蛋白酶的最佳酶解条件为pH2.4、温度41℃、酶量900U·g-1底物、底物浓度8%;酶解产物的IC50为0.65mg·mL-1,再分别利用SephadexG 25和SephadexG 15对其进行进一步分离提纯,IC50降至0.084mg·mL-1和0.046mg·mL-1,活性组分中的疏水性氨基酸含量增高,最终活性产物的分子量分布在700～1900。     

罗非鱼肉双酶分步控制酶解制备血管紧张素转化酶抑制肽的研究

本实验以罗非鱼肉为材料，利用Trypsin、Alcalase2．4L、Pratamex两两复合后，分步控制酶解制备血管紧张素转化酶抑制肽（ACEIP）。以ACE抑制率（Ⅰ值）、氨基酸态氨含量（Cn值）、和TCA可溶性蛋白（短肽）含量（Cp值）为指标对酶解过程进行分析，说明双酶分步控制酶解过程以及产物的特点，指出以单酶控制酶解为基础的双酶分步控制酶解法优于单酶控制酶解法。Trypsin与Alcalase2．4I．、Trypsin与Protamex及Alcalase2．4L与Protamex双酶分步控制酶解产物经稀释40倍后对ACE的最大抑制率分别可达到82．9％、82．9％和91．91％，具有较好的抑制效果。     

鳄鱼血蛋白酶解产物抗氧化特性和血管紧张素转化酶抑制活性研究

研究了鳄鱼血蛋白酶解产物的抗氧化特性和对血管紧张素转化酶（ ACE）的抑制活性。利用木瓜蛋白酶酶解鳄鱼血浆蛋白和血球蛋白,用分光光度法测定了酶解产物的抗氧化能力和用高效液相色谱（ HPLC）测定其ACE的抑制率。结果显示：鳄鱼血浆和血球蛋白酶解产物的亚铁离子螯合能力差异性不显著（ P＞0．05）;在0～5 mg/mL的浓度范围内,血球蛋白酶解产物清除ABTS自由基的能力大于血浆蛋白酶解产物,且在浓度为1 mg/mL时,两者清除ABTS自由基的能力差异性极显著（ P＜0．01）;血浆蛋白酶解产物清除DPPH自由基的能力在0～5 mg/mL的浓度范围内随着蛋白浓度的增加而升高,血球蛋白酶解产物在蛋白浓度为4 mg/mL处达到最大清除率,之后下降;在0～20 mg/mL的浓度范围内,两种酶解产物的还原力随着蛋白浓度的提高显著升高,但两者还原力的差异性不显著（ P＞0．05）;鳄鱼血浆和血球蛋白酶解产物对ACE具有良好的抑制力,其最大抑制率可分别达到75．56％和86．42％。研究表明,鳄鱼血蛋白酶解产物在体外具有抗氧化和抑制ACE的活性。     

利用水产蛋白开发酶解降血压肽

据国家卫生部2002年8月在全国范围内开展的“中国居民营养与健康状况调查”显示,我国目前成人高血压患病率为18．6％,全国现有高血压患者1．6亿,比1991年增加7000万,但目前平均的治疗率和控制率分别为24．7％和6．1％,仍处于较低水平。而且随着人民生活水平的提高和社会节奏的不断加快,高血压患者有年轻化趋势且数量正逐年增加,这一严峻形势引起了社会各方的共同关注。     

虾头内源酶酶解虾肉产物的ACE抑制活性及其降低原发性高血压老鼠血压的效果

虾头内源酶酶解虾肉产物的ACE抑制活性及其降低原发性高血压老鼠血压的效果=Angiotensin I converting enzyme inhibitory activity of shrimp meat hydrolysate by endogenous enzymes from shrimp head and its antihypertensive effects on spontaneously hypertensive rats［刊,中］/洪鹏志（广东海洋大学食品科技学院,湛江524088）,曹文红,郝更新,章超桦//水产学报,—2007,31(1).-76～83 在pH 7.35、温度57.2 ℃、水解时间4 h、虾肉/虾头为1∶1,底物浓度为20%（W/V）的条件下,利用虾头的内源酶酶解虾肉（南美白对虾）,制备了具有血管紧张素转化酶（ACE）抑制活性的酶解产物,其对ACE的抑制率为41.9%（2 mg·mL-1）。通过凝胶过滤层析测定出酶解产物的分子量分布较广,在7.4×104至29.7之间。分子量在2.7×103以下的凝胶层析组分占酶解产物氨基酸总量的83.1%。收集的高活性组分分子量在959～338之间,其IC50为0.32 mg·mL-1苯丙氨酸、疏水氨基酸（亮氨酸、异亮氨酸和缬氨酸）和芳香族氨基酸（脯氨酸、酪氨酸和色氨酸）占氨基酸总量的42.33%。体内试验的结果显示,南美白对虾的虾肉虾头内源酶酶解产物经灌喂原发性高血压老鼠后显示较强的降血压活性。 图4表2参13 关键词：南美白对虾;内源酶;血管紧张素转化酶抑制活性;抗高血压作用 E-mail:zhangch@gdou.edu.cn     

源于海洋贝类蛋白的ACE抑制肽研究现状

ACE抑制肽在血压调节方面起着重要作用,而海洋贝类含有丰富的蛋白质,并具有开发ACE抑制肽的潜能。本文主要简述了源于海洋贝类蛋白源的ACE抑制肽的制备、分离纯化、活性评价及构效关系等方面,以期为海洋贝类蛋白的开发利用提供参考。     

Identification of angiotensin I-converting enzyme inhibitory peptides derived from salmon muscle and their antihypertensive effect

ABSTRACT:   The salmon peptide digested from salmon muscle showed a strong inhibitory activity against the angiotensin I-converting enzyme (ACE). The antihypertensive effect of the salmon peptide on spontaneously hypertensive rats (SHR) was examined. After the single intravenous administration of the salmon peptide at a dose of 30 mg/kg body weight, the systolic blood pressure (SBP) was significantly reduced against the control. Further, a double-blind, placebo-controlled, parallel-group study determined the efficacy of the salmon peptide in mild hypertensive subjects. The SBP, after a 1.0 g of salmon peptide intake, was significantly reduced at 4 weeks after the intake, and 2 weeks after the intake finished, compared to the value before ingestion. Bioassay-guided separation of the salmon peptide, using a combination of column chromatographic techniques, led to the identification of 20 active di- and tri-peptides, including Ile-Val-Phe and Phe-Ile-Ala as two new ACE inhibitory tripeptides. Ile-Trp had the strongest ACE inhibitory activity (IC₅₀ = 1.2 μM) in vitro , and contributed 5.2% to the total ACE inhibitory activity. The salmon peptide and Ile-Trp showed a digestive resistance by in vitro assay, which mimicked the digestive organ, and had no affinity for factors related to blood pressure regulation, except for the ACE inhibitory activity.     

Angiotensin-converting enzyme I inhibitory activity of phlorotannins from Ecklonia stolonifera

ABSTRACT:   As part of our study of the isolation of antihypertensive agents derived from natural marine products, the bioactivity of 10 edible Korean seaweeds were screened by angiotensin converting enzyme (ACE) inhibitory and peroxynitrite assays. Among the crude extracts of selected seaweeds, including five Phaeophyta ( Ecklonia stolonifera , E. cava , Pelvetia siliquosa , Hizikia fusiforme , and Undaria pinnatifida ), four Rhodophyta ( Gigartina tenella , Gelidium amansii , Chondria crassicaulis , and Porphyra tenera ) and one Chlorophyta ( Capsosiphon fulvescens ), the ethanol extracts of E. stolonifera , E. cava , P. siliquosa , U. pinnatifida , and G. tenella exhibited significant inhibitory properties against ACE at more than 50% inhibition at a concentration of 163.93 µg/mL. Phloroglucinol 1 , eckstolonol 2 , eckol 3 , phlorofucofuroeckol A 4 , and dieckol 5 had been isolated previously, and triphlorethol-A 6 and fucosterol 7 were isolated for the first time from E. stolonifera. Also, the ACE inhibitory and peroxynitrite scavenging properties of phlorotannins 1–6 were evaluated, along with fucosterol 7 obtained from E. stolonifera . Among profound peroxynitrite scavenging compounds 1–6 , phlorotannins 3 , 4 and 5 were also determined to manifest marked inhibitory activity against ACE, with 50% inhibition concentration (IC₅₀) values of 70.82 ± 0.25, 12.74 ± 0.15, and 34.25 ± 3.56 µM, respectively.     

降血压肽的研究进展

本文主要从降血压肽的作用机理、制备方法、分离纯化技术及活性测定手段等方面,结合国内外的相关研究,综述了降血压肽领域的研究进展。并在此基础上,展望了食源性降血压肽于医疗保健方面的发展前景。旨在为降血压肽的进一步深入研究提供依据,为其产业化生产及市场推广提供参考,为降血压治疗提供新的选择。     

Potential Precursor of Angiotensin-I Converting Enzyme (ACE) Inhibitory Activity and Structural Properties of Peptide from Peptic Hydrolysate of Cutlassfish Muscle

ABSTRACT

Peptic hydrolysates were prepared by digesting the cutlassfish muscle protein using pepsin for 1, 3, and 6 h, and their inhibitory activity against angiotensin-I converting enzyme (ACE) was studied. The ACE-inhibitory effect of the peptic hydrolysate of cutlassfish muscle generated at the 3 h time point exhibited the strongest activity. After identifying the optimal hydrolysate, the active peptide was isolated by ultrafiltration, gel permeation, and high performance liquid chromatography (HPLC). The resulting purified peptide was characterized using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/TOF MS/MS) and was identified to be a 496.44 Da pentapeptide (Phe-Ser-Gly-Gly-Glu). The ACE-inhibitory activity of the active peptide exhibited an IC₅₀ value of 0.033 ± 0.003 mg/ml. A molecular docking program was used to simulate the interaction between the peptide and ACE, which revealed that the inhibitory effect was mainly due to the hydrogen bonds between ACE and the peptide. Based on the ACE-inhibitory properties and the molecular docking study of the resulting active peptide, we demonstrated an increase in nitric oxide (NO) production in a dose-dependent manner. In conclusion, cutlassfish protein hydrolysate and the resulting active peptide could be used as active ingredients in functional food as anti-hypertensive agents.     

酶联免疫法测定水产品中呋喃唑酮代谢物AOZ的残留

运用酶联免疫法对水产品中呋喃唑酮代谢物残留测定的方法进行研究。结果表明,稀释倍数对回收率影响显著,而乙酸乙酯提取次数、光照条件和放置时间对测定结果无显著影响。向样品中分别添加0.60、2.00、6.00 μg·kg^-1 3个浓度水平的呋喃唑酮代谢物时,平均回收率分别为99.2%、96.0%和100.0%;批内变异系数为1.76%～12.57%,批间变异系数为5.43%～8.58%,最低检测限为0.3 μg·kg^-1。该方法灵敏度高,重复性较好, 适用于水产品中呋喃唑酮代谢物残留的筛选。