CN116082444A - 一种马面鲀鱼鳔降压肽及其制备方法和应用 - Google Patents
一种马面鲀鱼鳔降压肽及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种具有血管紧张素转化酶(ACE)抑制活性的马面鲀鱼鳔降压肽及其制备方法和用途,该降压肽的氨基酸序列为Ser‑Pro‑Gly‑Phe‑Met(SPGFM),ESI/MS检测分子量为537.6Da。本发明马面鲀鱼鳔经微波和超声预处理、酶解、超滤、凝胶色谱纯化和反相高效液相色谱制(RP‑HPLC)纯化,得降压肽Ser‑Pro‑Gly‑Phe‑Met(SPGFM)。本发明制备得到的活性肽Ser‑Pro‑Gly‑Phe‑Met(SPGFM)具有显著的血管紧张素转化酶(ACE)抑制活性和人脐静脉内皮细胞(HUVEC)保护作用,可用于高血压治疗相关的药物。
Description
技术领域
本发明属于生物工程技术领域,具体涉及一种马面鲀鱼鳔降压肽及其制备方法和应用。
背景技术
高血压是指在静息状态下动脉收缩压和/或舒张压增高(≥140/90mmHg),可伴有心、脑、肾等器官的功能或器质性损害的临床综合征。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,实际上是导致中风、高血压性心脏病和肾功能衰竭等多种严重并发症的“祸首”。
血管紧张素转换酶(Angiotensin-I-Converting Enzyme,ACE)是血管紧张素系统中的一种关键酶,可以催化血管紧张素Ⅰ转化为具有强大的血管收缩作用的血管紧张素Ⅱ,并且使具有降血压作用的缓激肽失活。因此,可以通过抑制ACE活性来治疗高血压。血管紧张素转换酶抑制剂(ACEI)已被广泛研究用于预防和控制高血压。但人工合成的ACEI具有不良副作用,例如持续性干咳、皮疹和味觉障碍等。
发明内容
基于此,本课题以马面鲀鱼鳔为原料,利用酶工程技术制备一种降压五肽Ser-Pro-Gly-Phe-Met(SPGFM),显示出显著的降压效果,可作为药物或者辅助药物用于高血压的治疗。
本发明所要解决的第一个技术问题是针对上述的技术现状提供一种马面鲀鱼鳔降血压五肽,该五肽对血管紧张素转化酶(ACE)具有显著的抑制作用,对人脐静脉内皮细胞(HUVEC)具有显著的保护作用。
本发明所要解决的第二个技术问题是提供一种具有ACE抑制作用的马面鲀鱼鳔降血压五肽的制备方法。
本发明所要解决的第三个技术问题是提供一种具有ACE抑制活性的马面鲀鱼鳔降血压五肽在制备治疗高血压药物或保健品方面的应用。
本发明为解决上述第一个技术问题所采取的技术方案为:一种马面鲀鱼鳔降血压五肽,该降血压五肽的氨基酸序列为Ser-Pro-Gly-Phe-Met(SPGFM),ESI-MS测定分子量为537.6Da。
本发明为解决上述第二个技术问题所采取的技术方案为:一种马面鲀鱼鳔降压肽的制备方法,其特征在于包括以下步骤:
1)马面鲀鱼鳔的预处理:将马面鲀鱼鳔解冻、除杂、组织捣碎机中匀浆至糊状,然后加入到NaOH溶液于4℃下浸泡9~12h,过滤,用蒸馏水将NaOH冲洗干净,干燥,加入乙酸乙酯,于室温42KHZ、300W超声处理15~20min,离心除去上清液,得马面鲀鱼鳔粉末;
2)马面鲀鱼鳔酶解液的制备:将上述马面鲀鱼鳔粉末加入到甘氨酸-NaOH缓冲液中混合,调节混合液pH值至6.5~7.5,调温度至45~55℃,加入中性蛋白酶(5.0×104U/g),酶解3~5h;酶解液放于沸水种5~10min后,灭酶活;调节混合液pH值至9.5~10.5,调温度至40~45℃,加入碱性蛋白酶(5.0×104U/g),酶解3~5h;酶解液放于沸水种5~10min后,灭酶活;得马面鲀鱼鳔酶解液;
3)马面鲀鱼鳔酶解液的超滤分级:将马面鲀鱼鳔酶解液采用1kDa,5kDa和10kDa超滤膜进行超滤处理,分别收集分子量小于1kDa、1-5kDa、5-10kDa、大于10kD的组分,测定个组分的血管紧张素转化酶(ACE)抑制活性,最高ACE抑制活性组分即为超滤酶解液,冻干,得超滤酶解物。
4)马面鲀鱼鳔降压肽的制备:将超滤酶解物依次经羟丙基葡聚糖凝胶(SephadexLH-20)柱层析和反相高效液相色谱(RP-HPLC)纯化,得马面鲀鱼鳔降压肽。
在本发明的一些实施方式中,所述步骤1)中马面鲀为绿鳍马面鲀(Thamnaconusmodestus)。
在本发明的一些实施方式中,所述步骤1)中NaOH溶液的浓度为0.05mol/L,马面鲀鱼鳔与NaOH溶液的重量体积比为1g:10~15mL。
在本发明的一些实施方式中,所述步骤1)中马面鲀鱼鳔与乙酸乙酯的重量体积比为1g:8~10mL。
在本发明的一些实施方式中,所述步骤2)中甘氨酸-NaOH缓冲液的浓度为0.05mol/L,pH为9.5;所述步骤2)中马面鲀鱼鳔粉末与甘氨酸-NaOH缓冲液的重量体积比为1g:6~8mL。
在本发明的一些实施方式中,所述步骤3)中中性蛋白酶的添加量为马面鲀鱼鳔粉末质量的1.0~2.0%。
在本发明的一些实施方式中,所述步骤3)中碱性蛋白酶的添加量为马面鲀鱼鳔粉末质量的1.0~2.0%。
在本发明的一些实验方式中,所述步骤4)中的羟丙基葡聚糖凝胶(Sephadex LH-20)色谱纯化和RP-HPLC纯化为:
羟丙基葡聚糖凝胶(Sephadex LH-20)色谱纯化:将超滤酶解物配成45~55μg/mL溶液,经过羟丙基葡聚糖凝胶(Sephadex LH-20)柱层析分离,用双蒸水进行洗脱,根据214nm下的吸光度曲线收集洗脱组分,其中,具有最高ACE抑制活性的峰为凝胶层析酶解物,冻干。
RP-HPLC纯化:将上述马面鲀鱼鳔凝胶层析酶解物用双蒸水配成90~100μg/mL的溶液,利用RP-HPLC进行纯化,根据制备寡肽的ACE抑制活性得1个高活性寡肽Ser-Pro-Gly-Phe-Met(SPGFM),ESI-MS测定分子量为537.6Da。
进一步地,所述RP-HPLC条件为:进样量180~200μL;色谱柱Hypersil 300A C18(250mm×10.0mm,10μm);流动相:60%乙腈;洗脱速度1.5~2.0mL/min;紫外检测波长214nm。
本发明为解决上述第三个技术问题所采取的技术方案为:一种马面鲀鱼鳔降压肽的应用,本发明马面鲀鱼鳔降压肽Ser-Pro-Gly-Phe-Met(SPGFM),对ACE具有显著的抑制作用,半数抑制率(IC50)为25.78±1.36μM;另外,Ser-Pro-Gly-Phe-Met(SPGFM)对人脐静脉内皮细胞(HUVEC)无明显毒性,并能促进HUVEC细胞中内源性舒张因子一氧化氮(NO)的释放和抑制内源性收缩因子内皮素-1(ET-1)的生成,本发明Ser-Pro-Gly-Phe-Met(SPGFM)对HUVEC细胞具有一定的降压和调节功能,可应用于制备治疗高血压相关的药物或保健食品领域。
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,使马面鲀鱼鳔降压肽最大程度地释放出来,提高原料的利用率,本发明所制备的降压肽是马面鲀鱼鳔经酶水解制得,安全、无毒副作用,ACE抑制活性显著,对高血压患者具有降压作用。
本发明的制备得到的SPGFM可以作为药品或保健食品,本发明工艺科学合理,操作简单,具有较强的工业实施性。较之现有的制备方法,本发明同时融合了原料的微波和超声预处理、中性蛋白酶酶解、超滤、凝胶色谱和RP-HPLC多种手段,技术完善,而且得到的降压肽具有较高的活性。
附图说明
图1是本发明实施例中马面鲀鱼鳔酶解物(TMPH)及其超滤分级组分(TMPH-I~TMPH-IV)在5mg/mL浓度下对血管紧张素转化酶(ACE)抑制率(%)。
图2是本发明实施例中用羟丙基葡聚糖凝胶(Sephadex LH-20)柱(2.6cm×120cm)对TMPH-I进行分离纯化时所获得的色谱图。
图3是本发明实施例马面鲀鱼鳔超滤酶解物(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Hypersil 300A C18(250mm×10.0mm,10μm)柱对TMPH-IC进行分离纯化时所获得的色谱图。
图5是本发明实施例用RP-HPLC制备组分对ACE的半数抑制率(IC50)。
图6是本发明实施例马面鲀鱼鳔降压肽Ser-Pro-Gly-Phe-Met(SPGFM)的结构图。
图7是本发明实施例马面鲀鱼鳔降压肽Ser-Pro-Gly-Phe-Met(SPGFM)的质谱图。
图8是本发明实施例马面鲀鱼鳔降压肽Ser-Pro-Gly-Phe-Met(SPGFM)对人脐静脉内皮细胞(HUVEC)的活力影响。
图9是本发明实施例马面鲀鱼鳔降压肽Ser-Pro-Gly-Phe-Met(SPGFM)对人脐静脉内皮细胞(HUVEC)内一氧化氮(NO)含量的影响。
图10是本发明实施例马面鲀鱼鳔降压肽Ser-Pro-Gly-Phe-Met(SPGFM)对人脐静脉内皮细胞(HUVEC)内内皮素-1(ET-1)含量的影响。
具体实施方式
以下结合附图实施例对本发明作进一步详细描述。本发明所使用的溶剂没有特别的限制,可采用商购的常规溶剂。
正常组:生长在培养基中的未做任何处理的人脐静脉内皮细胞(HUVEC)。
CP组:阳性对照药物卡托普利处理的人脐静脉内皮细胞(HUVEC)。
NE组:阴性对照药物去甲肾上腺素处理的人脐静脉内皮细胞(HUVEC)。
SPGFM组:寡肽SPGFM处理的人脐静脉内皮细胞(HUVEC)。
实施例
一种马面鲀鱼鳔降压肽的制备方法,制备流程如下:绿鳍马面鲀鱼鳔→组织破碎→微波和超声预处理→酶解→超滤→凝胶色谱纯化→RP-HPLC纯化→马面鲀鱼鳔降压肽。
1)绿鳍马面鲀鱼鳔的预处理:将绿鳍马面鲀鱼鳔解冻、除杂、组织捣碎机中匀浆至糊状,然后按照料液比1g:13mL加入到NaOH溶液(0.05mol/L)于4℃下浸泡11h,过滤,用蒸馏水将NaOH冲洗干净,干燥。按照1g:9mL加入乙酸乙酯,于室温42KHZ、300W超声处理19min,离心出去上清液,得绿鳍马面鲀鱼鳔粉末。
2)绿鳍马面鲀鱼鳔酶解液的制备:将上述绿鳍马面鲀鱼鳔粉末与0.05mol/L,pH9.5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7.2,调温度至48℃,按照绿鳍马面鲀鱼鳔粉末质量的1.2%加入中性蛋白酶(5.0×104U/g),酶解5h;酶解液放于沸水种10min后,灭酶活;调节混合液pH值至10,调温度至45℃,按照绿鳍马面鲀鱼鳔粉末质量的1.2%加入碱性蛋白酶(5.0×104U/g),酶解3.5h;酶解液放于沸水种8min后,灭酶活;得绿鳍马面鲀鱼鳔酶解液(TMPH)。
3)绿鳍马面鲀鱼鳔酶解液的超滤分级:将绿鳍马面鲀鱼鳔酶解液(TMPH)采用1kDa,5kDa和10kDa超滤膜进行超滤处理得4个组分,分别为TMPH-I(MW<1kDa)、TMPH-II(1<MW<5kDa、TMPH-III(5<MW<10kDa、TMPH-IV(MW)10kDa)的组分,参照文献[汤海霞,王爽爽,郝果,宋宇轩,张磊,葛武鹏。一种新型绵羊乳酪蛋白ACE抑制肽结构鉴定及分子结合机制分析[J]。食品工业科,43(1):110-118],测定个组分的血管紧张素转化酶(ACE)抑制活性(见图1),最高ACE抑制活性组分TMPH-I即为超滤酶解液,冻干,得超滤酶解物。
4)绿鳍马面鲀鱼鳔降压肽的制备:将超滤酶解物(TMPH-I)依次经羟丙基葡聚糖凝胶(Sephadex LH-20)柱层析和反相高效液相色谱(RP-HPLC)纯化,得绿鳍马面鲀鱼鳔降压肽。
①羟丙基葡聚糖凝胶(Sephadex LH-20)色谱纯化:将超滤酶解物配成50μg/mL溶液,经过羟丙基葡聚糖凝胶(Sephadex LH-20)柱层析分离,用双蒸水进行洗脱,根据214nm下的吸光度曲线收集洗脱组分(TMPH-IA~TMPH-ID)(见图2),测定组分TMPH-IA~TMPH-ID的血管紧张素转化酶(ACE)抑制活性(见图3),其中,TMPH-IC具有最高ACE抑制活性,即为凝胶层析酶解物。
②RP-HPLC纯化:将凝胶层析酶解物(TMPH-IC)配成95μg/mL溶液,利用RP-HPLC(条件为:进样量180μL;色谱柱Hypersil 300A C18(250mm×10.0mm,10μm);流动相:60%乙腈;洗脱速度2.0mL/min;紫外检测波长214nm)进行纯化(见图4),测定各分离组分(TMHP1~TMHP5)的血管紧张素转化酶(ACE)半数抑制率(IC50)(见图5),TMHP3具有最低的ACE半数抑制率(IC50),测定其氨基酸序列和分子量。
③结构检测:收集ACE半数抑制率(IC50)最低组分(TMHP3),利用蛋白/多肽序列分析仪测定氨基酸序列为Ser-Pro-Gly-Phe-Met(SPGFM)(见图6),ESI/MS检测分子量为537.6Da(见图7)。
将上述制得的绿鳍马面鲀鱼鳔降压肽Ser-Pro-Gly-Phe-Met(SPGFM)进行ACE抑制活性实验。实验结果表明:该多肽的半数抑制率(IC50)为25.78±1.36μM。
参照文献[Shuo-Lei Zheng,Qian-Bin Luo,Shi-Kun Suo,Yu-Qin Zhao,Chang-Feng Chi,Bin Wang.Preparation,identification,molecular docking study andprotective function on HUVECs of novel ACE inhibitory peptides from proteinhydrolysate of skipjack tuna muscle[J].Mar.Drugs 2022,20,176]评价Ser-Pro-Gly-Phe-Met(SPGFM)对人脐静脉内皮细胞(HUVEC)及其相关指标的影响,结果证明:Ser-Pro-Gly-Phe-Met(SPGFM)对HUVEC无明显毒性(见图8),并能促进HUVEC细胞中内源性舒张因子一氧化氮(NO)的释放和抑制内源性收缩因子内皮素-1(ET-1)的生成(见图9和图10),表明Ser-Pro-Gly-Phe-Met(SPGFM)对HUVEC细胞具有一定的降压和调节功能。
最后,还需要注意的是,以上列举的仅是本发明的一个具体实施例。显然,本发明不限于以上实施例,还可以有许多变形。本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变形,均应认为是本发明的保护范围。
Claims (10)
1.一种马面鲀鱼鳔降血压五肽,其特征在于所述降血压五肽的氨基酸序列为Ser-Pro-Gly-Phe-Met(SPGFM),ESI-MS测定分子量为537.6Da。
2.如权利要求1所述一种马面鲀鱼鳔降压肽的制备方法,其特征在于包括以下步骤:
1)马面鲀鱼鳔的预处理:将马面鲀鱼鳔解冻、除杂、组织捣碎机中匀浆至糊状,然后加入到NaOH溶液于4℃下浸泡9~12h,过滤,用蒸馏水将NaOH冲洗干净,干燥,按照1g:8~10mL加入乙酸乙酯,于室温42KHZ、300W超声处理15~20min,离心除去上清液,得马面鲀鱼鳔粉末;
2)马面鲀鱼鳔酶解液的制备:将上述马面鲀鱼鳔粉末加入到0.05mol/L,pH9.5甘氨酸-NaOH缓冲液中混合,调节混合液pH值至6.5~7.5,调温度至45~55℃,加入中性蛋白酶(5.0×104U/g),酶解3~5h;酶解液放于沸水种5~10min后,灭酶活;调节混合液pH值至9.5~10.5,调温度至40~45℃,加入碱性蛋白酶(5.0×104U/g),酶解3~5h;酶解液放于沸水种5~10min后,灭酶活;得马面鲀鱼鳔酶解液;
3)马面鲀鱼鳔酶解液的超滤分级:将马面鲀鱼鳔酶解液采用1kDa,5kDa和10kDa超滤膜进行超滤处理,分别收集分子量小于1kDa、1-5kDa、5-10kDa、大于10kD的组分,测定个组分的血管紧张素转化酶(ACE)抑制活性,最高ACE抑制活性组分即为超滤酶解液,冻干,得超滤酶解物。
4)马面鲀鱼鳔降压肽的制备:将超滤酶解物依次经羟丙基葡聚糖凝胶(Sephadex LH-20)柱层析和反相高效液相色谱(RP-HPLC)纯化,得马面鲀鱼鳔降压肽。
3.如权利要求2所述一种马面鲀鱼鳔降压肽的制备方法,其特征在于所述步骤1)中马面鲀为绿鳍马面鲀(Thamnaconus modestus)。
4.如权利要求2所述一种马面鲀鱼鳔降压肽的制备方法,其特征在于所述步骤1)中NaOH溶液的浓度为0.05mol/L,马面鲀鱼鳔与NaOH溶液的重量体积比为1g:10~15mL;所述步骤1)中马面鲀鱼鳔与乙酸乙酯的重量体积比为1g:8~10mL。
5.如权利要求2所述一种马面鲀鱼鳔降压肽的制备方法,其特征在于所述步骤2)中甘氨酸-NaOH缓冲液的浓度为0.05mol/L,pH为9.5;所述步骤2)中马面鲀鱼鳔粉末与甘氨酸-NaOH缓冲液的重量体积比为1g:6~8mL。
6.如权利要求2所述一种马面鲀鱼鳔降压肽的制备方法,其特征在于所述步骤3)中中性蛋白酶的添加量为马面鲀鱼鳔粉末质量的1.0~2.0%。
7.如权利要求2所述一种马面鲀鱼鳔降压肽的制备方法,其特征在于所述步骤3)中碱性蛋白酶的添加量为马面鲀鱼鳔粉末质量的1.0~2.0%。
8.如权利要求2所述一种马面鲀鱼鳔降压肽的制备方法,其特征在于所述步骤4)中的羟丙基葡聚糖凝胶(Sephadex LH-20)色谱纯化和RP-HPLC纯化为:
羟丙基葡聚糖凝胶(Sephadex LH-20)色谱纯化:将超滤酶解物配成45~55μg/mL溶液,经过羟丙基葡聚糖凝胶(Sephadex LH-20)柱层析分离,用双蒸水进行洗脱,根据214nm下的吸光度曲线收集洗脱组分,其中,具有最高ACE抑制活性的峰为凝胶层析酶解物,冻干;
RP-HPLC纯化:将上述马面鲀鱼鳔凝胶层析酶解物用双蒸水配成90~100μg/mL的溶液,利用RP-HPLC进行纯化,根据制备寡肽的ACE抑制活性得1个高活性寡肽Ser-Pro-Gly-Phe-Met(SPGFM),ESI-MS测定分子量为537.6Da。
9.如权利要求8所述一种马面鲀鱼鳔降压肽的制备方法,其特征在于所述RP-HPLC条件为:进样量180~200μL;色谱柱Hypersil 300A C18(250mm×10.0mm,10μm);流动相:60%乙腈;洗脱速度1.5~2.0mL/min;紫外检测波长214nm。
10.如权利要求1所述一种马面鲀鱼鳔降压肽在制备治疗高血压药物或保健品方面的应用。
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