CN113801195B - 一种金枪鱼鱼卵降血压肽及其应用 - Google Patents
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Abstract
本发明公开了一种具有血管紧张素转化酶(ACE)抑制活性的金枪鱼鱼卵降压肽及其制备方法和用途，该降压肽的氨基酸序列为Trp‑Pro‑Phe‑Ala‑Cys‑Ser(WPFACS)，ESI/MS检测分子量为709.8Da。本发明制备的金枪鱼鱼卵降压肽具有显著的血管紧张素转化酶(ACE)抑制活性，可应用于治疗高血压相关的药物或保健食品。本发明以金枪鱼鱼卵为原料，利用酶解工艺和色谱制备技术制备得到了一种具有ACE抑制的降血压肽，为金枪鱼鱼卵高值化利用提供技术支持。
Description
技术领域
本发明涉及多肽技术领域，具体涉及一种金枪鱼鱼卵的降血压肽及其应用。
背景技术
高血压是一种常见病和多发病。此病一般起病缓慢，患者早期常无症状，或仅有头晕、头痛、心悸、耳鸣等症状，表面上看是一种独立的疾病，实际上是引发心、脑血管和肾病变的一个重要的危险因素，如果治疗不当就会病变成为较严重的脑中风、心肌梗死和肾功能衰竭等这些常见高血压并发症。血管紧张素转化酶(ACE)又称为肽基-羧基肽酶或者激肽酶Ⅱ，分子量为150,000Da，属血管内皮细胞膜结合酶。ACE可以将十肽的血管紧张素Ⅰ水解成八肽的血管紧张素Ⅱ，促使血管进一步收缩，导致血压升高。同时，ACE也可通过作用于肾上腺皮质而促进醛固酮的分泌。因此，ACE是肾素-血管紧张素-醛固酮(RAS)的重要成分。另外，ACE还可以催化具有降压作用的缓激肽水解而使其失去活性。因此，寻找合适的抑制体内ACE活性的物质，就可以达到降低或者控制血压的目的。血管紧张素转化酶抑制剂(ACEI)是一类发展迅速的抗高血压药物，其主要通过抑制ACE活性，致使血管紧张素Ⅰ不能转换为血管紧张素Ⅱ，同时抑制ACE对缓激肽的灭活作用，从而产生降压效应。
发明内容
本发明以金枪鱼鱼卵为原料，利用酶解工艺和色谱制备技术制备得到了一种具有ACE抑制的降血压肽，为金枪鱼鱼卵高值化利用提供技术支持。
一方面，本发明提供一种金枪鱼鱼卵降血压肽，其氨基酸序列为：Trp-Pro-Phe-Ala-Cys-Ser，ESI-MS测定其分子量为709.8Da。
另一方面，本发明提供了一种金枪鱼鱼卵降血压肽的制备方法，其包括以下步骤：将金枪鱼鱼卵微波和超声预处理、酶解、超滤、凝胶色谱分离和反相高效液相色谱制(RP-HPLC)纯化，得到降压肽Trp-Pro-Phe-Ala-Cys-Ser。
具体地，一种金枪鱼鱼卵降血压肽包括以下步骤：
1)金枪鱼鱼卵的预处理：将金枪鱼鱼卵解冻、除杂，然后加入双蒸水混合、匀浆，放置于电脑微波超声组合成/萃取仪中，在微波功率500W条件下，处理5min，取出冻干；冻干后的粉末加入乙酸乙酯混合，于室温3000W超声处理25min，离心出去上清液，得金枪鱼鱼卵粉末；
2)金枪鱼鱼卵酶解液的制备：将上述金枪鱼鱼卵粉末与甘氨酸-NaOH缓冲液混合，调节混合液pH值至7.0，调温度至50℃，加入中性蛋白酶(5.0×104U/g)，酶解3.5h；酶解液放于沸水种10min后，灭酶活，得金枪鱼鱼卵酶解液(STH)；
3)金枪鱼鱼卵酶解液的超滤分级：将金枪鱼鱼卵酶解液(STH)采用1kDa，3.5kDa和5kDa超滤膜进行超滤处理，收集分子量小于1kDa(STH1)，得超滤酶解物；
4)金枪鱼鱼卵降压肽的制备：将超滤酶解物(STH1)依次经Sephadex G-15凝胶色谱和反相高效液相色谱(RP-HPLC)纯化，得金枪鱼鱼卵降压肽。
作为优选，所述Sephadex G-15凝胶色谱分离步骤如下：将超滤酶解物(STH1)配成50μg/mL溶液，经过葡聚糖凝胶G-15柱层析分离，用双蒸水进行洗脱，根据220nm下的吸光度曲线收集洗脱组分，测定各组分的血管紧张素转化酶(ACE)抑制活性，具有最高ACE抑制活性的组分即为凝胶层析酶解物。
作为优选，所述RP-HPLC纯化步骤如下：将凝胶层析酶解物配成45μg/mL溶液，利用RP-HPLC(条件为：进样量为10μL；色谱柱为Zorbax C18(250×4.6mm，5μm)；流动相为50％乙腈；洗脱速度为0.5mL/min；紫外检测波长为220nm)进行纯化，测定各分离组分的血管紧张素转化酶(ACE)半数抑制率(IC50)，选择最优的组分，测定其氨基酸序列和分子量，得氨基酸序列为Trp-Pro-Phe-Ala-Cys-Ser(WPFACS)，ESI/MS检测分子量为709.8Da。
作为优选，所述的步骤1)中的金枪鱼为鲣鱼Katsuwonus pelamis。
作为优选，所述步骤1)中解冻除杂后的金枪鱼与蒸馏水的重量体积比g/ml为1:5。
作为优选，所述步骤1)中冻干后的粉末与乙酸乙酯的重量体积比g/ml为1:4。
作为优选，所述步骤2)中中性蛋白酶的添加量为金枪鱼鱼卵粉末重量的0.8％～1.5％。
作为优选，所述步骤2)中金枪鱼鱼卵粉末与甘氨酸-NaOH缓冲液的重量体积比g/ml为1:8。
另一方面，本发明提供了一种金枪鱼鱼卵降血压肽用于制备降血压相关疾病的药物中的用途。
本发明公开了一种具有血管紧张素转化酶(ACE)抑制活性的金枪鱼鱼卵降压肽及其制备方法和用途，该降压肽的氨基酸序列为Trp-Pro-Phe-Ala-Cys-Ser(WPFACS)，ESI/MS检测分子量为709.8Da。本发明采用的是一种可控和对环境友好的生物酶法，易通过对酶解过程的监控，使金枪鱼鱼卵降压肽最大程度地释放出来，提高了原料的利用率；同时所制备的降压肽安全、无毒副作用；本发明制备的降压肽具有显著的血管紧张素转化酶(ACE)抑制活性，可应用于治疗高血压相关的药物或保健食品。
附图说明
图1实施例中金枪鱼鱼卵酶解物(STH)及其超滤分级组分(STH1～STH5)在5mg/mL浓度下对血管紧张素转化酶(ACE)抑制率(％)。
图2为实施例中葡聚糖凝胶G-15柱层析分离后各组分的吸光度曲线。
图3为实施例中金枪鱼鱼卵超滤酶解物(STH1)及其凝胶色谱分离组分(STH1A～STH1D)在5mg/mL浓度下对血管紧张素转化酶(ACE)抑制率(％)。
图4是本发明实施例用Zorbax C18(250×4.6mm，5μm)柱进行纯化时所获得的色谱图。
图5是本发明实施例用RP-HPLC制备的各组分对ACE的半数抑制率(IC50)。
图6是本发明实施例金枪鱼鱼卵降压肽Trp-Pro-Phe-Ala-Cys-Ser的结构图。
图7是本发明实施例金枪鱼鱼卵降压肽Trp-Pro-Phe-Ala-Cys-Ser的质谱图。
具体实施方式
下列实施例用于进一步解释说明本发明，但是，它们并不构成对本发明范围的限制或限定。本发明实验所用的金枪鱼为鲣鱼(Katsuwonus pelamis)，由宁波今日食品有限公司提供。
实施例
1)金枪鱼鱼卵的预处理：将金枪鱼鱼卵解冻、除杂，称取1000g，然后与双蒸水按照1g：5mL的重量体积比混合、匀浆，放置于电脑微波超声组合成/萃取仪中，在微波功率500W条件下，处理5min，取出冻干；冻干粉末与乙酸乙酯按照1g：4mL的重量体积比混合，于室温3000W超声处理25min，离心出去上清液，得金枪鱼鱼卵粉末；
2)金枪鱼鱼卵酶解液的制备：将上述金枪鱼鱼卵粉末与甘氨酸-NaOH缓冲液按照1g：8mL的重量体积比混合，调节混合液pH值至7.0，调温度至50℃，按照金枪鱼鱼卵粉末质量的1.5％加入中性蛋白酶(5.0×104U/g)，酶解3.5h；酶解液放于沸水种10min后，灭酶活，得金枪鱼鱼卵酶解液(STH)；
3)金枪鱼鱼卵酶解液的超滤分级：将金枪鱼鱼卵酶解液(STH)采用1kDa，3.5kDa和5kDa超滤膜进行超滤处理，分别收集分子量小于1kDa(STH1)、1-3.5kDa(STH2)、5-5kDa(STH3)、3.5-10kDa(STH4)、大于10kD(STH5)的组分，测定各组分在5mg/mL浓度下对血管紧张素转化酶(ACE)抑制率(％)(见图1)，STH1具有最高ACE抑制活性，即为超滤酶解物；
4)金枪鱼鱼卵降压肽的制备：将超滤酶解物(STH1)依次经Sephadex G-15凝胶色谱和反相高效液相色谱(RP-HPLC)纯化，得金枪鱼鱼卵降压肽；
Sephadex G-15凝胶色谱纯化步骤：将超滤酶解物(STH1)配成50μg/mL溶液，经过葡聚糖凝胶G-15柱层析分离，用双蒸水进行洗脱，根据220nm下的吸光度曲线收集洗脱组分(见图2)，测定个组分(STH1A～STH1D)5mg/mL浓度下对血管紧张素转化酶(ACE)抑制率(％)(见图3)，其中，STH1D具有最高ACE抑制活性，即为凝胶层析酶解物。
RP-HPLC纯化步骤：将凝胶层析酶解物(STH1D)配成45μg/mL溶液，利用RP-HPLC(条件为：进样量为10μL；色谱柱为Zorbax C18(250×4.6mm，5μm)；流动相为50％乙腈；洗脱速度为0.5mL/min；紫外检测波长为220nm)进行纯化(见图4)，测定各分离组分(STP1～STP6)的血管紧张素转化酶(ACE)半数抑制率(IC50)(见图5)，STP5具有最低的ACE半数抑制率(IC50)，测定STP5的氨基酸序列和分子量。
结构检测：收集ACE半数抑制率(IC50)最低的组分，，最低组分(STP5)的降压肽经检测为单一峰，利用蛋白/多肽序列分析仪测定氨基酸序列为Trp-Pro-Phe-Ala-Cys-Ser(WPFACS)(见图6)，ESI/MS检测分子量为709.8Da(见图7)。
Claims (2)
1. 一种金枪鱼鱼卵降血压肽，其特征在于所述降压肽的氨基酸序列为：Trp-Pro-Phe-Ala-Cys-Ser，ESI-MS测定其分子量为709.8 Da。
2.如权利要求1所述一种金枪鱼鱼卵降血压肽用于制备降血压相关疾病的药物中的用途。
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