CN109232731A - 源于鲽鱼皮胶原蛋白的ace抑制肽及其制备方法 - Google Patents
源于鲽鱼皮胶原蛋白的ace抑制肽及其制备方法 Download PDFInfo
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Abstract
本发明公开一种源于鲽鱼皮胶原蛋白的ACE抑制肽，其氨基酸序列为Gly‑Trp。该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
本发明属于生物来源活性物质的制备，尤其涉及生物来源的活性物质的提取分离方法。
背景技术
肽的消化吸收理论的进展证实了机体可直接吸收小分子肽类物质，肽类物质不但可以作为营养的有益补充，有些具有一定序列的肽还具有特殊的生理活性――即生物活性肽。来源于食物蛋白质中的小分子生物活性肽具有如下优势：分子质量小，易于消化吸收，不易引起免疫原性；空间构效关系简单，易于进行活性机理的研究；来源于食物蛋白质，不易引起毒副作用。
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(ACE)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。因此抑制ACE活性，能够起到降血压功效。
目前市场上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ACE抑制肽。基于此，本发明首先提供一种源于鲽鱼皮胶原蛋白的ACE抑制肽，其氨基酸序列为Gly-Trp(GW)。
另一方面，本发明提供上述源于鲽鱼皮胶原蛋白的ACE抑制肽的制备方法，包括如下步骤：
(1)预处理：鲽鱼皮去鳞去肉，洗净，剪碎成1cm×1cm的小块，4℃条件下，按料液比1g:10mL加入0.1mol/L的NaOH溶液搅拌24h，每12h更换NaOH溶液(除去鲽鱼皮中非胶原蛋白和色素)；然后用4℃水冲洗鱼皮至中性，沥干；
(2)经预处理后的鲽鱼皮，按料液比1g:30ml加入0.5mol/L的冰乙酸水溶液，在4℃低速搅拌24h，4℃条件下10000r/min离心15min，取上清液；将未提取彻底的鱼皮再次用等体积的0.5mol/L冰乙酸水溶液提取，取上清液；合并2次提取所得的上清液；加入NaCl溶液搅拌，至最终盐浓度为0.9mol/L；静置过夜后，体系于4℃条件下5000r/min离心20min，弃上清液；所得沉淀溶于0.5mol/L冰乙酸水溶液，8000r/min离心20min除去不溶性杂质；继而经3次盐析后，将沉淀先用0.1mol/L的冰乙酸水溶液透析2天，每12h换一次透析液；再用超纯水进行透析3天，每12h换一次超纯水；最后对经透析处理的溶液进行冷冻干燥，得到酸溶性胶原蛋白(ASC)；
(3)将步骤(2)所制备的酸溶性胶原蛋白(ASC)按照2g/100ml的浓度加入至0.05mol/L的磷酸缓冲液中，加入500-2000U/g中性蛋白酶，pH 7-7.5和45±2℃条件下搅拌反应8～10h，得到酶解液；
(4)对步骤(3)所制备的酶解液，首先分别利用陶瓷膜、3kDa超滤膜、1kDa超滤膜及300Da超滤膜进行逐级分离，取300-1000Da组分进行Sephadex LH-20凝胶层析柱分离，流动相为30％的甲醇溶液，流速为0.5mL/min，上样体积为10mL，样品浓度为0.2g/mL，检测波长为280nm；台式纪录仪记录速度为0.2mm/min，电压10mv，电流2安培；采集第58-68管的组分(F7)；
(5)采用RP-HPLC Hypersil BDS C18对上述步骤(4)所采集的组分(F7)进一步分离，上样体积20μl，上样浓度10mg/mL，流速1mL/min；
梯度洗脱：流动相A液：0.1％的TFA水溶液；流动相B液：乙腈；
0min：A，100％；B 0％；
40min：A，0％；B，100％；
50min：A，0％；B，100％；
检测波长为215nm；监测ACE抑制活性，采集保留时间为39min的组分(F7-17)。
上述获得的本发明的鲽鱼皮胶原蛋白的ACE抑制肽经检测获知其氨基酸序列，为Gly-Trp。该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。因此，本发明再一方面的目的在于提供所述的活性肽，在制备防治高血压的药物或食品中的应用。
附图说明
图1是五种蛋白酶媒介产物的ACE抑制活性筛选实验图
图2(A)是LH-20凝胶色谱对鱼皮胶原蛋白酶解产物的分离结果图。
图2(B)LH-20凝胶色谱分离组分ACE抑制活性效果图。
图3组分F7的高效液相色谱图。
图4组分F7-17的一级质谱图。
图5组分F7-17的二级质谱图。
图6鲽鱼皮胶原蛋白肽对SHR大鼠的降血压效果实验结果图。
图7鲽鱼皮胶原蛋白肽对SHR大鼠RAS系统相关指标影响实验结果图：其中：a.血清中ACE含量；b.血清中Ang II含量；c.血清中血管紧张素原酶(ATG)含量；d.血清中醛固酮(ALD)含量。
具体实施方式
本发明涉及一种源于鲽鱼皮胶原蛋白的ACE抑制肽，即氨基酸序列为GW活性短肽的制备方法。从鲽鱼皮中制备酸性胶原蛋白，进而对胶原蛋白进行酶解。本发明也包括使用中性蛋白酶对鲽鱼皮行酶解的步骤，分离纯化方式及序列确定。
对酶解用蛋白酶的筛选在研发的前期进行了详尽的研究。在用于测试的酸溶性胶原蛋白(ASC)中加入酸性蛋白酶(pH 3.0和45℃)、碱性蛋白酶(pH 10.0和45℃)、中性蛋白酶(pH 7.2和45℃)、木瓜蛋白酶(pH 6.0和55℃)和胃蛋白酶(pH 2.0和37℃)五种蛋白酶对鲽鱼皮进行酶解。每种酶的加酶量为250U/g，酶解时间为1h、3h、5h、7h、9h、12h、24h、36h、48h，料液比为1:40g/mL。酶解完成后灭活，8000rpm离心20min，收集上清液，冷冻干燥，得到粉末状鲽鱼皮胶原蛋白肽。测定其ACE抑制活性，中性蛋白酶酶解产物的ACE抑制活性最高(如图1)。通过单因素和正交实验，中性蛋白酶酶解鲽鱼皮胶原蛋白的最优条件为：酶解时间9h，加酶量1000U/g，料液比1:32.5g/mL。
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.)。
下述非限制性实施例用于对本发明做进一步说明，不应被理解为对本发明内容任意形式的限定。
实施例1：鲽鱼皮胶原蛋白的制备
鲽鱼皮去鳞去肉，洗净，剪碎成1cm×1cm的小块，4℃条件下，以料液比1:10(g/ml)加入0.1mol/L的NaOH溶液搅拌24h(NaOH溶液每12h更换一次)，除去鲽鱼皮中非胶原蛋白和色素，然后用4℃水冲洗鱼皮至中性，沥干，-20℃冷冻保存。
取处理后的鲽鱼皮，以料液比1∶30(g/ml)加入0.5mol/L的冰乙酸溶液，在4℃低速搅拌24h，4℃条件下10000r/min离心15min，取上清液；将未提取彻底的鱼皮再次用等体积的0.5mol/L冰乙酸溶液提取。用冰乙酸溶液提取2次后，混合上清液，加入一定量NaCl溶液搅拌，至最终盐浓度为0.9mol/L。静置过夜，4℃条件下5000r/min离心20min后弃上清液。将沉淀溶于0.5mol/L冰乙酸溶液，8000r/min离心20min除去不溶性杂质。经3次盐析后，继而将沉淀先用0.1mol/L的冰乙酸溶液透析，每12h换一次透析液，透析2天；然后再用超纯水进行透析，每12h换一次超纯水，透析3天；最后对透析过的胶原蛋白溶液进行冷冻干燥，得到酸溶性胶原蛋白(ASC)。
实施例2：酶解产物的制备
以实施例1制备的酸溶性胶原蛋白为原料进行酶解条件试验，考察中性蛋白酶、对其进行酶解的方案，对酶解时间、加酶量、料液比进行正交试验分析。通过三个单因素试验结果选出最优条件，即加酶量为1000U/g，时间为9h，料液比为1:35g/mL，设定三因素三水平如下表1和表2：
表1：三因素三水平设计表
表2：三因素三水平L933正交表
依据正交表进行正交试验，结果如下表3：
表3：正交试验结果
通过上述正交试验对酶解时间、加酶量和料液比进行筛选，由结果可知，鲽鱼皮酶解的最优条件为：酶解时间9h，加酶量1000U/g，料液比1:32.5g/mL。
影响因素的主次：料液比＞时间＞加酶量。
实施例3：酶解产物的分离纯化
采用上述实施例2检测所得的最优选条件对实施例1所制备得到的产物进行酶解，即酶解时间9h，加酶量1000U/g，料液比1:32.5g/mL，酶解产物按照下述步骤进行分离纯化：
分别利用陶瓷膜、3kDa超滤膜、1kDa超滤膜及300Da超滤膜对酶解液进行逐级分离，得到不同分子量分布的酶解液，并进行ACE抑制活性测试，得到300-1000Da的胶原蛋白肽ACE抑制率最高为72.67％，IC50值为1.72mg/ml。将该组分进行Sephadex LH-20凝胶层析柱分离，流动相为30％的甲醇溶液，流速为0.5mL/min，上样体积为10mL，样品浓度为0.2g/mL，检测波长为280nm；台式纪录仪记录速度为0.2mm/min，电压10mv，电流2安培；以20min/管的速度收集1～100管组分，得到9个峰，分别是F1-F9，如图2所示；以肽含量及ACE抑制活性为标准采集保留第58-68管的组分(F7)进一步分析；
采用RP-HPLC Hypersil BDS C18对所收集的58～68管产物(F7)进一步分离，上样体积20μl，上样浓度10mg/mL，流速1mL/min；
梯度洗脱：流动相A液：0.1％的TFA水溶液；流动相B液：乙腈；
0min：A，100％；B 0％；
40min：A，0％；B，100％；
50min：A，0％；B，100％；
通过ACE抑制活性监测，在上述条件下，39分钟出峰的组分活性最高，标记为F7-17。
实施例4：胶原蛋白活性肽序列分析
F7-17经反高效液相色谱洗脱，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，梯度洗脱：0-10min，3％B；10-11min，3％～7％B；11-21min，7％～45％B；22-32min，90％B；33-45min，2％B。之后进行质谱分析。结果如图4，该色谱峰的分子量为262.12Da，再结合其二级质谱图(如图5)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Gly-Trp(GW)
实施例5：降血压动物实验
健康雄性自发性高血压大鼠30只，随机分成3组：空白对照组；卡托普利对照组(30mg/kg)；GW灌胃组(300mg/kg)。适应性暂养后，利用尾部动脉测定法检测大鼠的原始血压值，实验开始后每12h间隔灌喂两次，每日测定血压一次，连续投喂28d后停止灌胃，继续观察7d大鼠的血压变化。利用SPSS软件对其显著性进行分析，灌胃15d后，如图6所示，GW灌胃组与空白组相比，自发性高血压大鼠的血压明显下降，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。
实施例6：SHR血清中RAS系统指标测定
自发性高血压大鼠在实施例5灌胃结束36h后活体尾部采血，2500r/min离心10min，取出上清液，于-80℃冰箱保存备用。RAS系统中主要成分血管紧张素转化酶(ACE)、血管紧张素转化酶Ⅱ(Ang II)、血管紧张素原(ATG)、醛固酮(ALD)分别使用大鼠血管紧张素转化酶酶联免疫吸附测定试剂盒，大鼠血管紧张素II酶联免疫吸附测定试剂盒，血管紧张素原酶联免疫吸附测定试剂盒和醛固酮酶联免疫吸附测定试剂盒进行测定。结果如图7所示，灌喂28天后各组大鼠血液中的ACE、AngⅡ、ATG和ALD的含量均有变化。其中鲽鱼皮胶原蛋白肽组和阳性对照组大鼠血液中ACE、AngⅡ、ALD的含量均比空白组的大鼠血液中含量低；鲽鱼皮胶原蛋白肽组和阳性对照组大鼠血液中ATG的含量均比空白组的大鼠血液中含量高，说明鲽鱼皮胶原蛋白肽有降血压效果。
Claims (3)
1.源于鲽鱼皮胶原蛋白的ACE抑制肽，其氨基酸序列为Gly-Trp。
2.权利要求1所述的ACE抑制肽的制备方法，包括如下步骤：
(1)预处理：鲽鱼皮去鳞去肉，洗净，剪碎成1cm×1cm的小块，4℃条件下，按料液比1g:10mL加入0.1mol/L的NaOH溶液搅拌24h，每12h更换NaOH溶液；然后用4℃水冲洗鱼皮至中性，沥干；
(2)经预处理后的鲽鱼皮，按料液比1g:30ml加入0.5mol/L的冰乙酸水溶液，在4℃低速搅拌24h，4℃条件下10000r/min离心15min，取上清液；将未提取彻底的鱼皮再次用等体积的0.5mol/L冰乙酸水溶液提取，取上清液；合并2次提取所得的上清液；加入NaCl溶液搅拌，至最终盐浓度为0.9mol/L；静置过夜后，体系于4℃条件下5000r/min离心20min，弃上清液；所得沉淀溶于0.5mol/L冰乙酸水溶液，8000r/min离心20min除去不溶性杂质；继而经3次盐析后，将沉淀先用0.1mol/L的冰乙酸水溶液透析2天，每12h换一次透析液；再用超纯水进行透析3天，每12h换一次超纯水；最后对经透析处理的溶液进行冷冻干燥，得到酸溶性胶原蛋白；
(3)将步骤(2)所制备的酸溶性胶原蛋白按照2g/100ml的浓度加入至0.05mol/L的磷酸缓冲液中，加入500-2000U/g中性蛋白酶，pH 7-7.5和45±2℃条件下搅拌反应8～10h，得到酶解液；
(4)对步骤(3)所制备的酶解液，首先分别利用陶瓷膜、3kDa超滤膜、1kDa超滤膜及300Da超滤膜进行逐级分离，取300-1000Da组分进行Sephadex LH-20凝胶层析柱分离，流动相为30％的甲醇溶液，流速为0.5mL/min，上样体积为10mL，样品浓度为0.2g/mL，检测波长为280nm；台式纪录仪记录速度为0.2mm/min，电压10mv，电流2安培；采集第58-68管的组分；
(5)采用RP-HPLC Hypersil BDS C18对上述步骤(4)所采集的组分进一步分离，上样体积20μl，上样浓度10mg/mL，流速1mL/min；
梯度洗脱：流动相A液：0.1％的TFA水溶液；流动相B液：乙腈；
0min：A，100％；B 0％；
40min：A，0％；B，100％；
50min：A，0％；B，100％；
检测波长为215nm；监测ACE抑制活性，采集保留时间为39min的组分。
3.权利要求1所述的活性肽，在制备防治高血压的药物或食品中的应用。
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