CN109265538A - 鲽鱼皮来源的活性二肽 - Google Patents
鲽鱼皮来源的活性二肽 Download PDFInfo
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- CN109265538A CN109265538A CN201811161941.8A CN201811161941A CN109265538A CN 109265538 A CN109265538 A CN 109265538A CN 201811161941 A CN201811161941 A CN 201811161941A CN 109265538 A CN109265538 A CN 109265538A
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Abstract
本发明公开一种鲽鱼皮来源的活性二肽，其氨基酸序列为Ser‑Trp。该二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。
本发明通过对鲽鱼皮胶原蛋白酶解液的分离、纯化得到氨基酸序列为SW的小分子肽，具有ACE抑制活性，是有效的ACE抑制剂。
因此，本发明的首要，便在于提供一种鲽鱼皮来源的活性二肽，其氨基酸序列为Ser-Trp(SW)。该二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。
本发明所述的活性二肽是从鲽鱼皮胶原蛋白中分离得到的，其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)将经预处理的鲽鱼皮(以干重计)与20mmol/mL的磷酸盐缓冲溶液按料液比1g:32.5mL混合，煮沸20min后，采用中性蛋白酶酶解9h；灭活后，8000rpm离心20min，收集上清液，得酶解液；
(3)步骤(2)所制得的酶解液经超滤后，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％的甲醇溶液，柱温为室温，检测波长为280nm，按时间收集480-570min的产物，记为F6；
(4)步骤(3)所获得的产物F6经RP-HPLC Hypersil BDS C18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；收集信号组分，监测并采集ACE抑制活性组分(第48-57管)。
本发明进一步提供以上述活性二肽为原料、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二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二肽与盐酸，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，琥珀酸等有机酸反应所形成的盐；所述的与金属离子形成的盐类可举例但不限于钠盐，钾盐，钙盐和铵盐；所述的胺盐类产物也可以举例但不限于由活性二肽与氨基乙醇，三乙氨，二环乙氨等形成的胺盐。
本发明的上述活性二肽或其盐，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、栓剂、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是用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是组分F6的高效液相分离图。
图3是组分F6-14的一级质谱图。
图4是组分F6-14的一级质谱图。
图5是鲽鱼皮胶原蛋白肽对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，本说明书中述及的中性蛋白酶等酶产品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：制备鲽鱼皮胶原蛋白肽
(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，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。
(2)酶解：将预处理后的鲽鱼皮(以干重计)与20mmol/mL磷酸盐缓冲溶液按料液比1：32.5(g/mL)混合，煮沸20min后，采用中性蛋白酶酶解9h，加酶量1000U/g，灭活后，8000rpm离心20min，收集上清液，得酶解液。
酶解液经过冷冻干燥，得到粉末状鲽鱼皮胶原蛋白肽。
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。将分子量为300-1000Da的胶原蛋白肽收集，采用Sephadex LH-20分离，流动相为超纯水溶液，柱温为室温，检测波长为280nm，结果如图1所示。进一步在RP-HPLC上用Hypersil C18柱洗脱第48-57管的组分F6，流动相A液为：0.1％TFA+0％乙腈的水溶液，用前超声脱气；流动相B液为:0.1％TFA+100％乙腈的水溶液，检测波长215nm，室温，进样体积20μl，采用梯度洗脱，洗脱方案为：
0～60min，A液100％～50％线性降低，B液0％～50％线性升高；
60～80min，A液0％，B液100％。
采集保留时间为39分钟的组分(F6-14)，结果如图2所示。
实施例3：胶原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F6-14活性最高。组分F6-14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。之后进入质谱分析，结果如图3所示：该色谱峰的分子量为292.13Da，再结合其二级质谱图(如图4)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Ser-Trp(SW)。
实施例4：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)；SW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5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。
Claims (4)
1.鲽鱼皮来源的活性二肽，其氨基酸序列为Ser-Trp(SW)。
2.权利要求1所述的活性二肽，其特征在于，具有ACE抑制活性。
3.权利要求1所述的活性二肽的制备方法，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)将经预处理的鲽鱼皮与20mmol/mL的磷酸盐缓冲溶液按料液比1g:32.5mL混合，煮沸20min后，采用中性蛋白酶酶解9h；灭活后，8000rpm离心20min，收集上清液，得酶解液；
(3)步骤(2)所制得的酶解液经超滤后，取分子量300-1000道尔顿的组分采用SephadexLH-20进行分离，流动相为30％的甲醇溶液，柱温为室温，检测波长为280nm，按时间收集480-570min的产物，记为F6；
(4)步骤(3)所获得的产物F6经RP-HPLC Hypersil BDS C18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；收集信号组分，监测并采集ACE抑制活性组分。
4.权利要求1所述的鲽鱼皮来源的活性二肽在制备防治高血压的药物或食品中的应用。
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