CN114989250B - 一种来源于海水珍珠的血管紧张素转化酶抑制多肽及其应用 - Google Patents
一种来源于海水珍珠的血管紧张素转化酶抑制多肽及其应用 Download PDFInfo
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Abstract
本发明属于海洋生物小分子活性肽领域，具体涉及一种来源于海水珍珠的血管紧张素转化酶抑制多肽及其应用，其氨基酸序列为Lys‑Lys‑Cys‑His‑Phe‑Trp‑Pro‑Phe‑Pro‑Trp。本发明通过将合浦珠母贝所产无核珍珠粉碎，脱钙制备珍珠基质蛋白，酶解珍珠基质蛋白，将酶解物通过高效液相色谱分离纯化得到该小分子活性肽并进行活性验证。结果表明本发明的活性肽具有很好的ACE抑制活性，IC50值为4.17μM，可用于高血压疾病的保健和治疗，可用于保健品及生物医药制品的开发，具有广阔的应用前景。
Description
技术领域
本发明涉及一种从合浦珠母贝珍珠基质蛋白中分离纯化得到的血管紧张素转化酶抑制多肽，属于生物技术领域。
背景技术
血管紧张素转化酶(Angiotensin Converting Enzyme,ACE)是一种广泛存在于哺乳动物组织中的二肽羧基肽酶，在人体内参与血压的调节。ACE可以水解血管紧张素Ⅰ将其末端的组氨酸亮氨酸去除，转化为血管紧张素Ⅱ。血管紧张素Ⅱ作用于动脉血管平滑肌，使其收缩引起血压迅速升高。因此血管紧张素转化酶抑制剂成为临床上重要的降血压药物，如开托普利、赖诺普利等。但此类化学合成降压药物往往要长期服用，毒副作用的累积效应不容忽视。食物来源的蛋白制备的ACE抑制肽具有生理活性高和毒副作用小的优点成为研究的热点。
珍珠，由95％的碳酸钙和5％的有机基质组成，其中有机基质包括基质蛋白、几丁质等多糖和极少量的脂类，基质蛋白是有机基质的主要成分。在中国古代，珍珠不仅用作装饰品或首饰，还是一味珍贵的中药材，《本草纲目》记载：珍珠无毒，镇心点目珍珠涂面，令人润泽好颜色，可坠痰，除面斑，止泻除小儿惊热，安魂魄止遗精白浊，解痘疗毒，令光泽洁白。并且可以将珍珠磨成珍珠粉直接内服，如中国古代武则天、慈禧、和珅具有内服珍珠粉并对健康有益的历史记载，在古埃及也有埃及艳后将珍珠溶解服用的故事，可见珍珠粉可以看作一种特殊的保健食品，是安全无毒有益的，只是在古代比较奢侈昂贵而只有少数人能享用。目前珍珠基质蛋白ACE抑制肽未见报道。
发明内容
本发明针对上述问题、现状和发展前景，提供一种来源于海水珍珠的血管紧张素酶抑制活性肽及应用，该活性肽具有ACE抑制活性，分子量小，活性高，易于分离纯化，可用于高血压相关疾病的保健及治疗，在生物医药领域有广阔的应用前景。
本发明为实现上述目的提供的技术方案为：
一种从海水珍珠中分离纯化的新型血管紧张素酶抑制活性肽，其氨基酸序列为Lys-Lys-Cys-His-Phe-Trp-Pro-Phe-Pro-Trp。
以合浦珠母贝珍珠为例，一种从合浦珠母贝珍珠基质蛋白中分离纯化的新型血管紧张素酶抑制活性肽，其制备方法包括以下步骤：
(1)珍珠基质蛋白的提取：将合浦珠母贝无核珍珠粉碎，收集珍珠粉，置于烧杯中，加入五倍体积的1.5M乙二胺四乙酸(EDTA)脱钙，离心收集上清液透析，然后冷冻干燥获得珍珠基质蛋白。
(2)珍珠基质蛋白的酶解：取珍珠基质蛋白冻干粉加入胰蛋白酶(trypsin)进行酶解，胰蛋白酶的加入量为珍珠基质蛋白重量的5％，酶解体系的pH为8.0，反应温度为50℃，酶解时间为2小时，沸水浴灭活后离心收集上清液，抽滤后使用蛋白浓缩离心管进行离心超滤，截留分子量的规格为3KD，收集分子量小于3KD的滤过液，浓缩后冷冻干燥获得酶解好的珍珠基质蛋白多肽混合物。
(3)珍珠基质蛋白ACE抑制多肽的分离纯化：使用高效液相色谱对酶解好的珍珠基质蛋白多肽混合物进行纯化，色谱柱为C18，流动相A为含有0.1％三氟乙酸的去离子水，流动相B为含有0.1％三氟乙酸的乙腈，流速为1ml/min，紫外检测波长为280nm，收集保留时间为14min的样品，浓缩后冷冻干燥即得到序列为Lys-Lys-Cys-His-Phe-Trp-Pro-Phe-Pro-Trp的血管紧张素转化酶抑制多肽。
本发明提出的活性肽可以用于制备血管紧张素转化酶抑制剂、制备具有调节血压作用的药物或者制备保健品。
本发明的有益效果：
(1)本发明获得了一种从合浦珠母贝珍珠基质蛋白中分离纯化的新型血管紧张素酶抑制活性肽，该活性肽具有很好的ACE抑制活性，ACE抑制IC50值为4.17μM，可用于开发治疗高血压的相关保健品或者药物，在保健品及医药领域有广阔的应用前景。
(2)本发明为珍珠的进一步精深技工和医药开发提供了可靠依据。
附图说明
图1为本发明小分子活性肽的ACE抑制HPLC图，其中：A，对照；B，多肽终浓度0.025mg/ml(17.63μM)；C，多肽终浓度0.05mg/ml(35.26μM)；D，多肽终浓度0.1mg/ml(70.52μM)；E，多肽终浓度0.15mg/ml(105.78μM)；F，多肽终浓度0.2mg/ml(141.04μM)；G，多肽终浓度0.25mg/ml(176.3μM)。
图2为本发明小分子活性肽的分子式；
图3为本发明小分子活性肽的ACE抑制活性图。
具体实施方式
下面结合说明书附图和具体实施例进一步阐明本发明。
实施例1珍珠基质血管紧张素转化酶抑制多肽的制备
(1)珍珠基质蛋白的提取
将产自广东湛江的合浦珠母贝无核珍珠洗净、晾干，使用中药粉碎机充分粉碎，在粉碎过程中及时停顿，给粉碎容器降温，防止粉碎容器过热，破坏珍珠活性成分。收集珍珠粉，置于烧杯中，加入五倍体积的1.5M EDTA，置于4℃层析冷柜中搅拌脱钙，24小时后装入50ml高速离心管离心(10000rpm)。收集上清液，用0.2μm微孔滤膜抽滤，滤过液置于4℃层析冷柜中低温透析48h，期间多次换水，然后冷冻干燥获得珍珠基质蛋白。
(2)珍珠基质蛋白的酶解
取珍珠基质蛋白冻干粉，加入trypsin进行酶解。胰蛋白酶的活性为10000U/g，胰蛋白酶的加入量为珍珠基质蛋白重量的5％，酶解体系的pH调整为8.0，在50℃恒温水浴锅中酶解2小时，然后放入沸水浴中灭活，装入50ml高速离心管离心(10000rpm)。收集上清液，用0.2μm微孔滤膜抽滤，滤过液使用Millipore蛋白浓缩离心管进行离心超滤(4000rpm)，内置低吸附的Ultracel-PL超滤膜截留规格为3KD，收集分子量小于3KD的滤过液，浓缩后冷冻干燥获得酶解好的珍珠基质蛋白多肽混合物。
(3)珍珠基质蛋白ACE抑制多肽的分离纯化
使用高效液相色谱对酶解好的珍珠基质蛋白多肽混合物进行纯化，采用C18色谱柱，流动相A为含有0.1％三氟乙酸的去离子水，流动相B为含有0.1％三氟乙酸的乙腈。紫外检测波长为280nm，流速为1ml/min，收集保留时间为14min的样品，浓缩后冷冻干燥即得到序列为Lys-Lys-Cys-His-Phe-Trp-Pro-Phe-Pro-Trp的血管紧张素转化酶抑制多肽。
实施例2：珍珠基质血管紧张素转化酶抑制多肽的ACE抑制活性测定
使用高效液相色谱法测定ACE抑制多肽对ACE的抑制活性。
将得到的ACE抑制多肽溶于0.1M硼酸盐缓冲溶液(含0.3M NaCl，pH8.3)中制成1mg/ml的样品溶液，将血管紧张素转化酶及其底物马尿酰组胺酰亮氨酸HHL(Hip-His-Leu)溶于0.1M硼酸盐缓冲溶液(含0.3M NaCl，pH8.3)中，配成浓度200U/L的ACE酶液和浓度5mM的HHL底物溶液。
实验组共六组，取HHL底物溶液80μl，分别加入5μl、10μl、20μl、30μl、40μl和50μl样品溶液，用0.1M硼酸盐缓冲液补足至180μl，对照组用0.1M硼酸盐缓冲液代替样品液，混匀后放入37℃恒温水浴中保温5min，再加入20μlACE酶液启动反应，以120r/min频率37℃振荡水浴保育30min。加入0.2ml的1M HCl终止反应。将溶液置于高效液相色谱仪中测定其中马尿酸的含量。
液相条件如下：流速为每分钟0.5ml，流动相A为纯乙腈，流动相B为含0.5％三氟乙酸的去离子水，二者比例为1:3，检测波长为228nm。由于ACE可以稳定降解马尿酰组胺酰尿氨酸并生成马尿酸，一旦ACE的活性受到抑制，样品中马尿酸的生成量也随之减少。底物的ACE抑制活性可按以下公式计算：
ACE抑制率＝(As-Ad)/Ad
上式中，As为样品组中马尿酸检测峰面积；Ad为空白组中马尿酸检测峰面积，并计算IC50值。
实验结果见图1和图3，序列为Lys-Lys-Cys-His-Phe-Trp-Pro-Phe-Pro-Trp的多肽体外血管紧张素转化酶抑制IC50值为4.17μM，该多肽具有很好抑制血管紧张素转化酶的活性。
实施例3：珍珠基质血管紧张素转化酶抑制多肽的序列分析
取实施例1中所制备得到的珍珠基质血管紧张素转化酶抑制多肽，经基质辅助激光解吸电离飞行时间质谱(MALDI-TOF/TOF-MS)测定，其分子量为1417.5Da，其氨基酸序列为：Lys-Lys-Cys-His-Phe-Trp-Pro-Phe-Pro-Trp(如SEQ ID NO:1所示)。
以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明披露的技术范围内，根据本发明的技术方案及其发明构思加以同等替换或改变，都应涵盖在本发明的保护范围内。
序列表
<110> 浙江清荣生物科技发展有限公司
浙江清华长三角研究院
<120> 一种来源于海水珍珠的血管紧张素转化酶抑制多肽及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 10
<212> PRT
<213> 合浦珠母贝(Pinctada fucata)
<400> 1
Lys Lys Cys His Phe Trp Pro Phe Pro Trp
1 5 10
Claims (4)
1.一种来源于海水珍珠的血管紧张素转化酶抑制多肽，其特征在于，所述血管紧张素转化酶抑制多肽的氨基酸顺序为Lys-Lys-Cys-His-Phe-Trp-Pro-Phe-Pro-Trp。
2.根据权利要求1所述的血管紧张素转化酶抑制多肽在制备血管紧张素转化酶抑制剂中的应用。
3.根据权利要求1所述的血管紧张素转化酶抑制多肽在制备具有调节血压作用的药物中的应用。
4.根据权利要求1所述的血管紧张素转化酶抑制多肽在制备保健品中的应用。
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