CN113444145A - 一种聚球藻血管紧张素转化酶抑制肽及其制备方法与应用 - Google Patents
一种聚球藻血管紧张素转化酶抑制肽及其制备方法与应用 Download PDFInfo
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Abstract
本发明涉及一种聚球藻血管紧张素转化酶抑制肽及其制备方法与应用。本发明的聚球藻血管紧张素转化酶抑制肽的序列为Val‑Thr‑Tyr(VTY)。本发明提供了一种聚球藻来源的新型血管紧张素转化酶抑制肽,该抑制肽结构新颖,分子量小,易被吸收并发挥作用,其对血管紧张素转化酶体外抑制IC50值为8.93μg/mL。本发明的聚球藻ACE抑制肽(Val‑Thr‑Tyr)在ACE抑制剂和高血压治疗相关的药品和保健品方面有广泛的应用前景。
Description
技术领域
本发明属于生物活性肽技术领域,具体涉及一种聚球藻血管紧张素转化酶(ACE)抑制肽及其制备方法与应用。
背景技术
生物活性肽(Bioactivitypeptides)是一类具有一定生理活性功能的多肽,又称功能肽,其分子量通常在10kDa以下,具有多种生物功能,包括抗肿瘤、抗菌、抗病毒、降血压、降血糖等活性,可以广泛应用于食品、保健品、化妆品和药物的开发。
高血压已成为当今世界死亡率较高的疾病之一,世界各国平均患病率在10%~12%,且人群分布较广。目前我国高血压患者超过1.2亿人,高血压及其并发症的威胁已严重影响人民的健康水平和生活质量。因此,高血压的防治可以最大限度降低心血管病的病残率和死亡率,具有重要的社会意义。
血管紧张素转化酶(ACE)是人体内调控肾素-血管紧张素系统(renin-angiotensin system,RAS)和激肽-缓激肽系统(kallikreo-bradykininsystem,KKS)的关键酶,高活性的血管紧张素转化酶(ACE)或者过量的血管紧张素转化酶(ACE)会造成血管收缩和血压升高,因此,通过抑制ACE的活性,能起到降低血压的效果,故ACE成为治疗高血压、心力衰竭等疾病的理想靶点。
目前国内外正式用于临床的ACE抑制剂有20多种,大多是合成类药物,服用时常会伴有许多毒副作用:如咳嗽、皮肤瘙痒、味觉紊乱或血压过低。因此,寻找和开发安全性高且无毒副作用的高效降压药物替代品是十分必要的。
海洋生物的多样性和海洋环境的复杂性造就了海洋天然活性化合物的新颖性、多样性以及独特性。自1960年以来,全世界大约有超过20000种天然化合物从海洋生物中获得,海洋天然产物越来越受到科学家的重视。
中国专利文献CN101906135A(申请号201010237795.X)公开一种口服有效的螺旋藻源新型降血压肽,所述降血压肽为一种三肽,序列为异亮氨酸-谷氨酰胺-脯氨酸(Ile-Gln-Pro,IQP),是一种血管紧张素转化酶(ACE)抑制肽,是ACE的非竞争性抑制剂,IC50值为5.77±0.09μmol/L。
中国专利文献CN103923177A(申请号201410172674.X)公开了一种新型的血管紧张素转化酶抑制肽,即从球等鞭金藻中制备得到的一种ACE酶抑制肽,其氨基酸序列为Tyr-Met-Gly-Leu-Asp-Leu-Lys,其体外ACE抑制IC50值为36.1μM。
聚球藻(Synechococcus sp.strainWH7805)是一种广泛分布于海洋环境中的单细胞蓝藻,富含蛋白质和其它活性组分,其主要蛋白组分藻胆蛋白具有抗氧化、抗肿瘤等多种功能,因此聚球藻可以作为活性肽的重要来源。
目前已经有多种从海洋藻类中筛选出的具有血管紧张素转化酶(ACE)抑制作用的多肽,如石莼、小球藻、紫菜等,但鲜有从聚球藻中分离得到ACE抑制肽的报道。
发明内容
针对现有技术的不足,本发明提供了一种聚球藻血管紧张素转化酶(ACE)抑制肽及其制备方法与应用。本发明以海洋聚球藻为原料,通过酶解和各种分离鉴定技术最终得到一种序列为Val-Thr-Tyr(VTY)的ACE抑制肽。本发明得到的特定序列的聚球藻ACE抑制肽在体外高效液相色谱法(HPLC)检测中显示有良好的ACE抑制活性。
本发明的技术方案如下:
一种聚球藻血管紧张素转化酶抑制肽,所述抑制肽的序列为Val-Thr-Tyr(VTY)。
优选的,所述抑制肽对血管紧张素转化酶体外抑制IC50值为8.93μg/mL。
本发明中所述序列为Val-Thr-Tyr(VTY)的血管紧张素转化酶抑制肽具有血管紧张素转化酶抑制活性,可从海洋聚球藻经酶解制备,也可以从其它蛋白经酶解制备,或经人工合成法制备。
在本发明一种优选的技术方案中,上述血管紧张素转化酶抑制肽的制备方法,包括如下步骤:
(1)收集聚球藻,加水混匀,藻水比0.03~0.05,单位g/mL,压力破碎后离心收集上清液,向上清液中加入thermolysin蛋白酶,thermolysin蛋白酶的加量为聚球藻质量的2~5%,70~75℃酶解2~4小时,沸水浴终止酶解,得酶解液;
(2)将步骤(1)酶解液离心收集上清液,经3kDa超滤膜超滤,得到肽分子量<3kDa的肽液;
(3)将步骤(2)得到的肽液利用TSK gel G2000 SWXL凝胶柱进一步分离纯化,ddH2O作为洗脱液,流速0.5mL/min,洗脱体积30mL,并在220nm处测定吸光度,收集洗脱体积在20-22mL的组分,得血管紧张素转化酶抑制肽。
优选的,步骤(1)所述thermolysin蛋白酶的酶活为30-175U/mg。
优选的,步骤(1)所述thermolysin蛋白酶的加量为聚球藻质量的2%。
优选的,步骤(1)所述酶解的条件为:温度70℃,pH 7.0~8.0,转速150rpm,酶解2~4小时。
优选的,步骤(1)所述沸水浴的时间为10~15min。
优选的,步骤(1)和(2)所述离心为10000~15000rpm离心10~15min。
上述血管紧张素转化酶抑制肽在制备治疗或预防高血压制品中的应用。
优选的,所述制品包括治疗或预防高血压的药品或保健品。
优选的,所述制品含有药理有效浓度的上述血管紧张素转化酶抑制肽。
有益效果:
本发明提供了一种聚球藻来源的新型血管紧张素转化酶抑制肽,氨基酸序列为Val-Thr-Tyr(VTY),该结构新颖,分子量小,易被吸收并发挥作用,其对血管紧张素转化酶体外抑制IC50值为8.93μg/mL。
本发明的聚球藻ACE抑制肽(Val-Thr-Tyr)在ACE抑制剂和高血压治疗相关的药品和保健品方面有广泛的应用前景。
附图说明
图1为聚球藻ACE抑制肽VTY的质谱鉴定图。
图2为不添加抑制肽时反应体系的HPLC色谱图。
图3为VTY终浓度为2μg/mL时反应体系的HPLC色谱图。
图4为VTY终浓度为4μg/mL时反应体系的HPLC色谱图。
图5为VTY终浓度为8μg/mL时反应体系的HPLC色谱图。
图6为VTY终浓度为12μg/mL时反应体系的HPLC色谱图。
图7为VTY终浓度为16μg/mL时反应体系的HPLC色谱图。
具体实施方式
通过以下具体实施例对本发明的技术方案作进一步的说明,但本发明的保护范围并不仅限于此。本发明的目的是以聚球藻为原材料分离筛选出具有特定序列的、活性好的ACE抑制肽,该抑制肽的序列为Val-Thr-Tyr(VTY),该多肽为含有3个氨基酸残基的直链多肽。该多肽在体外显示出良好的ACE抑制活性。
实施例中涉及的试剂及药品,若无特殊说明,均为普通市售产品。实施例中涉及的实验步骤,若无特殊说明,均为本领域常规操作。
thermolysin蛋白酶(EC 3.4.24.27):购自美国sigma公司,酶活为30-175U/mg。
实施例1:
聚球藻来源的血管紧张素转化酶抑制肽的制备:
(1)取聚球藻5g,用无菌海水清洗三次,加入150mL的水混匀,压力破碎后11000rpm离心15min,收集上清液,加入100mg的thermolysin蛋白酶,在恒温振荡水浴摇床中进行酶解,转速为150rpm,温度控制在70℃,溶液pH调节至7.0,酶解3h之后,沸水浴10min灭活酶终止酶解,得酶解液;
(2)将步骤(1)所得酶解液11000rpm离心15min去除杂质沉淀,收集上清液,经3kDa超滤膜超滤,得到肽分子量<3kDa的肽液,冷冻干燥;
(3)将步骤(2)冷冻干燥得到的样品利用TSK gel G2000 SWXL凝胶柱(7.8mm×30cm)进一步分离纯化,ddH2O作为洗脱液,流速控制在0.5mL/min,洗脱体积30mL,在220nm处测定吸光度,自动收集器收集洗脱样品,收集洗脱体积在20-22mL的组分,浓缩冻干。
将上述经凝胶柱分离纯化后收集到的组分使用LC-MS/MS进行鉴定,将质谱信息与聚球藻蛋白组信息进行比对,选择丰度前10且C端具有极性或较大基团残基的多肽进行人工合成,检测10个多肽的ACE抑制活性,得到序列为Val-Thr-Tyr(VTY)的多肽具有较好的ACE抑制活性。其中序列为Val-Thr-Tyr(VTY)的多肽的质谱鉴定图如图1所示,为本发明筛选出的血管紧张素转化酶抑制肽。
实施例2:
聚球藻来源的血管紧张素转化酶抑制肽的制备:
(1)取聚球藻5g,用无菌海水清洗三次,加入100mL的水混匀,压力破碎后11000rpm离心15min,,收集上清液,加入100mg的thermolysin蛋白酶,在恒温振荡水浴摇床中进行酶解,转速为150rpm,温度控制在70℃,溶液pH调节至8.0,酶解4h之后,沸水浴10min灭活酶终止酶解,得酶解液;
(2)将步骤(1)所得酶解液11000rpm离心15min去除杂质沉淀,收集上清液,经3kDa超滤膜超滤,得到肽分子量<3kDa的肽液,冷冻干燥;
(3)将步骤(2)冷冻干燥得到的样品利用TSK gel G2000 SWXL凝胶柱(7.8mm×30cm)进一步分离纯化,ddH2O作为洗脱液,流速控制在0.5mL/min,洗脱体积30mL,在220nm处测定吸光度,自动收集器收集洗脱样品,收集洗脱体积在20-22mL的组分,浓缩冻干。
将上述经凝胶柱分离纯化后收集的组分使用LC-MS/MS进行鉴定,将质谱信息与聚球藻蛋白组信息进行比对,选择丰度前10且C端具有极性或较大基团残基的多肽进行人工合成,检测10个多肽的ACE抑制活性,结果与实施例1相同,得到序列为Val-Thr-Tyr(VTY)的多肽具有较好的ACE抑制活性,为本发明筛选出的血管紧张素转化酶抑制肽。
实施例3:
根据实施例1和实施例2对血管紧张素转化酶抑制肽的序列鉴定,人工合成多肽(由强耀生物科技有限公司合成)Val-Thr-Tyr(VTY),纯度>98%,并对VTY的ACE抑制活性进行评估。采用高效液相色谱法(HPLC)测定多肽体外ACE抑制活性。本实施例所用的药品及仪器无特殊说明均可从商业途径获得。
高效液相色谱法(HPLC)测定ACE抑制活性的原理为:ACE可以将底物马尿酰-组氨酰-亮氨酸(Hip-His-Leu,HHL,美国Sigma公司)水解为马尿酸;当添加ACE的抑制剂时,马尿酸的生成量相应减少,通过检测紫外波长228nm处马尿酸的吸收峰面积就可以测定抑制剂对ACE的抑制率。
试剂:0.2U/mL的ACE溶液(缓冲体系为0.1M的硼酸缓冲液,pH 8.3,含0.4MNaCl);
12.5mM的HHL溶液(缓冲体系为0.1M的硼酸缓冲液,pH 8.3,含0.4M NaCl);
0.5mg/mL的小肽VTY溶液(ddH2O配制)。
实验方法:取2μL的小肽VTY溶液和20μL的ACE溶液混合,在37℃恒温水浴中预热5min,加入10μL的HHL溶液和33μL的0.1M硼酸缓冲液(pH 8.3,含0.4M NaCl)充分混匀以启动反应,37℃水浴中孵育60min之后加入100μL的1MHCl终止反应,上述反应体系13000rpm离心15min,过0.22μm的水相滤膜,使用液相系统测定228nm吸收波长下产物马尿酸的峰面积。
检测条件:HPLC连接C18柱(4.6×250mm,waters),流动相A为含有0.01%三氟乙酸(TFA)的超纯水,流动相B为含有0.01%三氟乙酸(TFA)的乙腈,洗脱方式为等度洗脱:50%B洗脱15min,流速为0.4mL/min。在波长228nm处检测马尿酸峰面积,ACE抑制率的计算公式如下:
I(%)=(Acontrol-Ainhibition)/Acontrol×100%
式中:Acontrol为不添加抑制肽时产生的马尿酸的峰面积,Ainhibition为添加抑制肽时产生的马尿酸的峰面积。
将ACE酶活反应体系中小肽VTY的终浓度调整为2、4、8、12、16μg/mL,按照上述实验方法测定VTY的ACE抑制率,每个浓度设置3个平行,计算小肽VTY的IC50值。IC50值为能抑制50%ACE活性时抑制肽的浓度。
其中,不同VTY终浓度条件下反应体系的HPLC色谱图如图2~7所示,HPLC色谱图中峰1为产物马尿酸,峰2为底物HHL,由于ACE的酶活反应体系中底物HHL是过量的,所以峰2是始终存在的;随着抑制剂VTY浓度的增加,产物峰峰1的峰面积由图2至图7逐渐减少,而底物峰峰2的峰面积则是逐渐增加,表明底物消耗减少,即随着抑制肽浓度的增加,其抑制作用在逐步增强。根据上述检测结果计算不同终浓度的VTY对ACE活性的抑制率,结果如表1所示。
表1.不同终浓度的VTY对ACE活性的抑制率
由表1的结果经拟合计算后,得到聚球藻ACE抑制肽VTY的IC50值为8.93μg/mL(23.39μM)。
以上结果表明:聚球藻ACE抑制肽VTY对ACE有较好的抑制活性,可在后续应用于高血压治疗相关的药品和保健品开发。
以上所述的实施例对本发明的技术方案进行了详细的说明,应理解的是以上所述仅为本发明的具体实施例,并不用于限制本发明,凡在本发明的原则范围内所做的任何修改、补充或类似方式替代等,均应包含在本发明的保护范围之内。
Claims (10)
1.一种聚球藻血管紧张素转化酶抑制肽,其特征在于,所述抑制肽的序列为Val-Thr-Tyr(VTY)。
2.如权利要求1所述的聚球藻血管紧张素转化酶抑制肽,其特征在于,所述抑制肽对血管紧张素转化酶体外抑制IC50值为8.93μg/mL。
3.权利要求1所述的血管紧张素转化酶抑制肽的制备方法,其特征在于,包括如下步骤:
(1)收集聚球藻,加水混匀,藻水比0.03~0.05,单位g/mL,压力破碎后离心收集上清液,向上清液中加入thermolysin蛋白酶,thermolysin蛋白酶的加量为聚球藻质量的2~5%,70~75℃酶解2~4小时,沸水浴终止酶解,得酶解液;
(2)将步骤(1)酶解液离心收集上清液,经3kDa超滤膜超滤,得到肽分子量<3kDa的肽液;
(3)将步骤(2)得到的肽液利用TSK gel G2000 SWXL凝胶柱进一步分离纯化,ddH2O作为洗脱液,流速0.5mL/min,洗脱体积30mL,并在220nm处测定吸光度,收集洗脱体积在20-22mL的组分,得血管紧张素转化酶抑制肽。
4.如权利要求3所述的制备方法,其特征在于,步骤(1)所述thermolysin蛋白酶的酶活为30-175U/mg;
优选的,步骤(1)所述thermolysin蛋白酶的加量为聚球藻质量的2%。
5.如权利要求3所述的制备方法,其特征在于,步骤(1)所述酶解的条件为:温度70℃,pH 7.0~8.0,转速150rpm,酶解2~4小时。
6.如权利要求3所述的制备方法,其特征在于,步骤(1)所述沸水浴的时间为10~15min。
7.如权利要求3所述的制备方法,其特征在于,步骤(1)和(2)所述离心为10000~15000rpm离心10~15min。
8.权利要求1所述的血管紧张素转化酶抑制肽在制备治疗或预防高血压制品中的应用。
9.如权利要求8所述的应用,其特征在于,所述制品包括治疗或预防高血压的药品或保健品。
10.如权利要求8所述的应用,其特征在于,所述制品含有药理有效浓度的权利要求1所述的血管紧张素转化酶抑制肽。
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