CN109180783A - 扇贝裙边来源的活性短肽 - Google Patents
扇贝裙边来源的活性短肽 Download PDFInfo
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- CN109180783A CN109180783A CN201811161961.5A CN201811161961A CN109180783A CN 109180783 A CN109180783 A CN 109180783A CN 201811161961 A CN201811161961 A CN 201811161961A CN 109180783 A CN109180783 A CN 109180783A
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Classifications
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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Abstract
本发明公开一种扇贝裙边来源的活性短肽，其氨基酸序列为Glu‑Phe‑Gln‑Ile‑Arg。该短肽具有明显的降血压活性，是一种由食品来源的、高安全性的、廉价的、具有可产业化的新型的ACE抑制剂，可以用于制备降血压药物或者保健食品。
Description
技术领域
本发明涉及一种生物来源的具有血管紧张素I转换酶抑制活性的小分子肽。
背景技术
高血压是最常见的心血管疾病之一，它能造成大脑、心血管、肾脏的损害，是引起脑卒中、心力衰竭和冠心病等的重要因素，严重威胁着人类的健康。因此，治疗和预防高血压对提高人类的健康水准，延长寿命有着重要的意义。
血管紧张素I转换酶在人体肾素-血管紧张素系统和激肽释放酶-激肽系统中，对血压调节起着重要的作用。ACE可以将血管紧张素I转换为血管紧张素Ⅱ，使周围小动脉、血管平滑肌收缩，同时刺激醛固酮分泌，促进人体肾脏对Na+、K+的重吸收，引起钠储量和血容量的增加，使血压升高；还可以使舒缓激肽失活，引起血压升高。
综上所述，ACE一方面产生使血压上升的血管紧缩素II，另一方面，使具有血管舒张作用的舒缓激肽失活，这都造成了血压的上升。所以，如果抑制了ACE的活性，就可以起到降压的作用。
现有的作为治疗高血压的合成物卡普托利就是ACE的抑制剂，但它有很多副作用，所以源于食品蛋白中的ACE抑制肽因其无毒副作用，同时具有其它疗效而被广泛应用，市场前景极好。
发明内容
本发明的目的在于提供一种由食品来源的、高安全性的、廉价的、具有可产业化的新型的ACE抑制剂。
本发明通过对扇贝裙边酶解液的分离，纯化得到氨基酸序列为EFQIR的新肽，具有ACE抑制活性，是有效的ACE抑制剂。
因此，本发明的首要，便在于提供一种扇贝裙边来源的活性短肽，其氨基酸序列为Glu-Phe-Gln-Ile-Arg(EFQIR)。该短肽具有明显的降血压活性，可以用于制备降血压药物或者保健食品。
本发明所述的活性短肽是从扇贝裙边胶原蛋白中分离得到的，具体通过下述方法：
(1)预处理：将冷冻的扇贝裙边于0～4℃条件下解冻后，按照1g:4ml的比例加入0～4℃蒸馏水浸泡，1h后换水一次；
(2)将预处理后的扇贝裙边与水按照质量比1:3匀浆，加入体系质量2.0％的木瓜蛋白酶，于60℃酶解1h，pH 7.0，得到酶解产物；
(3)步骤(2)所制备的酶解产物经超滤后，取分子量300-1000道尔顿的组分采用Sephadex LH-20进行分离，流动相为30％的甲醇水溶液，柱温为室温，检测波长为280nm，采集550min～600min组分，记为产物F5；采集800min～850min组分，记为产物F7；
(4)步骤(3)所获得的产物F5、F7分别以Hypersil C18柱分离：第一次分离流动相A液为：0.1％TFA+20％乙腈的水溶液，进样体积20μl，等度洗脱，分别采集3.3min～4min组分；所得组分再次分别经Hypersil C18柱分离，流动相B液为：0.1％TFA+10％乙腈的水溶液，进样体积20μl，等度洗脱，采集8min～8.2min组分，合并产物。
本发明进一步提供以上述活性短肽为原料、按照本领域的通常方法所制备的盐类。此处可举例但不限于由活性短肽与酸反应生成的盐类、与金属离子所形成的盐类，以及与有机基团所构成的胺盐类。其中，所述的与酸反应生成的盐类可举例但不限于由活性短肽与盐酸，硫酸，硝酸，磷酸等无机酸，以及蚁酸，乙酸，丙酸，甘氨胆酸，苹果酸，柠檬酸，酒石酸，琥珀酸等有机酸等形成的盐；所述的与金属离子形成的盐类可举例但不限于钠盐，钾盐，钙盐，铵盐；所述的胺盐类产物也可以举例但不限于由活性短肽与氨基乙醇，三乙氨，二环乙氨等形成的胺类盐。
本发明的上述活性短肽或其盐，可以通过口服、注射、皮肤、直肠等方式灵活给药。根据需要，与各种药用可接受载体配合制备成散剂，颗粒剂，片剂，胶囊，悬浮液，乳化液，喷剂，粉剂等。也可以直接或与可食用辅料配合添加至各种食品或保健品中，其产品形式可以根据市场灵活选择清凉饮料，乳酸饮料，调味品，汤类，奶酪，火腿，点心等。
附图说明
图1是五种蛋白酶酶解产物的ACE抑制活性筛选实验结果图。
图2扇贝裙边酶解物经葡聚糖凝胶LH-20分离及ACE活性测定结果图。
图3RP-HPLC一次纯化谱图。
图4RP-HPLC二次纯化谱图。
图5 F5-3-3LC-MS/MS一级质谱图。
图6 F5-3-3LC-MS/MS二级质谱图。
图7活性短肽对SHR大鼠的降血压效果实验结果图。
具体实施方式
下文以非限制性实施例的方式对本发明的技术方案和效果作出更加清楚的说明，但不应当理解为对本发明任意形式的限定。
如无特殊说明，本说明书中述及的木瓜蛋白酶、胃蛋白酶、酸性蛋白酶、中性蛋白酶及碱性蛋白酶均为天津市诺奥科技发展有限公司商品酶。
本说明书中采用反相高效液相色谱定量ACE与底物反应生成马尿酸的量进行ACE抑制活性测试(Biochemical Pharmacology,1971,20:1637-1648.)。
实施例1:筛选蛋白酶制备扇贝裙边中ACE抑制肽
(1)预处理：将冷冻的扇贝裙边于0～4℃条件下解冻后，按照质量体积比(g/ml)加入4倍体积的0～4℃蒸馏水浸泡，1h后换水一次，达到脱盐效果；
(2)酶解：预处理后的扇贝裙边与水按照质量比1:3混合匀浆，分别加入木瓜蛋白酶、胃蛋白酶、酸性蛋白酶、中性蛋白酶及碱性蛋白酶进行酶解，所述的酶解条件为：加酶量2.0％(w％)，酶解温度60℃，酶解时间1h，pH 7.0；灭活后，8000rpm离心20min，收集上清液，冷冻干燥，得到粉末状扇贝裙边蛋白肽，其ACE抑制活性测试结果如图1所示，木瓜蛋白酶酶解产物的ACE抑制率高达71.3％。
实施例2：酶解产物的分离纯化
分别以陶瓷膜、3kDa超滤膜、1kDa超滤膜及300Da超滤膜对实施例1所制得的未经过冷冻干燥的酶解液进行逐级分离，采用Sephadex LH-20分离，流动相为30％的甲醇溶液，柱温为室温，检测波长为280nm，结果如图2所示，采集550min～600min组分，记为产物F5；采集800min～850min组分，记为产物F7。进一步在RP-HPLC上用Hypersil C18柱洗脱组分F5和组分F7，第一次分离流动相A液为：0.1％TFA+20％乙腈的水溶液，进样体积20μl，采用等度洗脱(等度洗脱就是按照描述的流动相进行，没有梯度)，采集3.3min～4min组分，分别记为产物F5-3，F7-4，如图3所示。将F5-3和F7-4分别进行第二次分离，流动相B液为：0.1％TFA+10％乙腈的水溶液，进样体积20μl，等度洗脱；均采集8min～8.2min组分，合并，记为产物F5-3-3，如图4所示。
实施例3：扇贝裙边蛋白活性肽序列分析
对实施例2采集的各组分进行ACE抑制活性测定，结果显示组分F5-3-3活性最高，组分F5-3-3使用Orbitrap Q Exactive(Thermo Electron，San Jose，CA)进行质谱分析，该质谱仪配备有Dionex UltiMate 3000RSLCnano高效液相色谱分离仪(Thermo Scientific,USA)，用于肽段样品的反相液相分离，反相洗脱梯度设置如下：0～10min，3％B；10-11min，3％B～7％B；11-21min，7％B～45％B；22～32min，90％B；33～45min，2％B；其中流动相A为0.1％甲酸-水溶液，流动相B为含有0.1％甲酸的乙腈溶液。液相色谱-质谱系统的流速被控制为600nL/min，。
将F5-3-3收集浓缩后，用0.1％甲酸水溶液复溶，采用自动进样系统上样至质谱仪，自动上样系统包括一段4cm的毛细管捕集柱(200μm i.d.)，将毛细管分析柱的一端在高温下拉成内径约为5μm的喷针，用气压法将两根柱子都分别填充C18AQ填料(5μm,)。
Q Exactive质谱仪在正离子模式下进行肽段检测，所有的一级谱与二级谱的数据采集，都是采用数据依赖模式(DDA)，在静电场轨道阱(Orbitrap)内完成。离子传输毛细管的温度为250℃，电喷雾电压为2.0kV，高能碰撞诱导解离(HCD)池内的归一化碰撞能量为27.0。质谱扫描包含一次MS全扫描(m/z 100-1500)，分辨率为70000；然后选择其中强度最高的5个母离子峰采用高能碰撞诱导解离(HCD)的方式碎裂母离子，同时在静电场轨道阱(Orbitrap)内进行二级MS/MS碎片离子扫描分析，其分辨率为17500。其中，一级谱的自动增益控制(AGC)为3e6，最大离子注射时间设置为100ms，动态排除时间10s；二级谱的自动增益控制(AGC)为5e5，最大离子注射时间设置为120ms。系统的操作和数据的采集都使用Xcalibur软件(v2.1，Thermo公司)完成。结果如图5所示：该色谱峰的分子量为346.69Da，再结合其二级质谱图(如图6)中离子碎片分子量，得知此分子主要是以Y模式进行裂解，按离子碎片形式排列，判断出该色谱峰对应的氨基酸序列为Glu-Phe-Gln-Ile-Arg(EFQIR)。
实施例4：灌胃动物实验
选择30只自发性高血压大白鼠为实验模型，SPF级，体重240～280g，12周龄，心脏收缩血压均高于180mmHg；SHR适应环境3天后，如表1所示，随机分成空白对照组、阳性对照组和活性肽组，采用灌喂法进行灌喂。
表1
| 组别 | 只数 | 灌喂药品 | 灌喂剂量 | 饲料 |
| 空白对照组 | 10 | 蒸馏水 | 150mg/kg | 基础饲料 |
| 阳性对照组 | 10 | 卡托普利 | 20mg/kg | 基础饲料 |
| 活性肽组 | 10 | 活性短肽(实施例2) | 150mg/kg | 基础饲料 |
动物饲养条件：饲养房间要保持清洁，自然采光，温度要控制在25士2℃，自由摄食且取水，每五天更换一次垫料，每天早8:00～9:00和晚20:00～21:00，各灌喂一次，灌喂28天停药后，继续观察7天SHR的心脏收缩压变化。
采用尾套法测定SHR的心脏收缩血压。每天上午灌喂结束后，便开始检测SHR尾部动脉收缩压，血压检测前，先将机器预热，再将SHR固定在尾部测压仪上，当心率稳定时，方可测量其血压，分别在第0、1、3、5、9、14、23、26、28、30、32、35天时测定SHR的SBP，每只SHR血压测定三次，取平均值，同时记录整个饲养过程中SHR的体重。
大白鼠饲养一周后，将其分组，每日按大白鼠体重20-200mg/kg剂量EFQIR进行灌胃投入，如图7所示。可见：4周后对照组大白鼠的血压持续升高，而治疗组自发性高血压大白鼠的血压明显下降，高血压大白鼠的平均收缩压从190mmHg下降到170mmHg说明此肽具有明显的降血压效果。
Claims (4)
1.扇贝裙边来源的活性短肽，其氨基酸序列为Glu-Phe-Gln-Ile-Arg。
2.权利要求1所述的活性短肽，其特征在于，具有ACE抑制活性。
3.权利要求1所述的活性短肽的制备方法，包括如下步骤:
(1)预处理：将冷冻的扇贝裙边于0～4℃条件下解冻后，按照1g:4ml的比例加入0～4℃蒸馏水浸泡，1h后换水一次；
(2)将预处理后的扇贝裙边与水按照质量比1:3匀浆，加入体系质量2.0％的木瓜蛋白酶，于60℃酶解1h，pH 7.0，得到酶解产物；
(3)步骤(2)所制备的酶解产物经超滤后，取分子量300-1000道尔顿的组分采用Sephadex LH-20进行分离，流动相为30％的甲醇水溶液，柱温为室温，检测波长为280nm，采集550min～600min组分，记为产物F5；采集800min～850min组分，记为产物F7；
(4)步骤(3)所获得的产物F5、F7分别以Hypersil C18柱分离：第一次分离流动相A液为：0.1％TFA+20％乙腈的水溶液，进样体积20μl，等度洗脱，分别采集3.3min～4min组分；所得组分再次分别经Hypersil C18柱分离，流动相B液为：0.1％TFA+10％乙腈的水溶液，进样体积20μl，等度洗脱，采集8min～8.2min组分，合并产物。
4.权利要求1所述的扇贝裙边来源的活性短肽在制备防治高血压的药物或食品中的应用。
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