CN105017122A - 一种抗高血压活性肽、其制备方法及应用 - Google Patents
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Abstract
本发明涉及一种抗高血压活性肽、其制备方法及应用,活性肽的氨基酸序列为:茶氨酸-γ-氨基丁酸-脯氨酸。本发明提出了一种不同的活性肽,该活性肽的ACE抑制活性能够达到64.5%,采用2mg/kg剂量给药5天后血压值能够基本稳定在165mmHg左右。
Description
技术领域
本发明涉及一种抗高血压活性肽、其制备方法及应用。
背景技术
高血压是我国最常见的心血管疾病,其发生率在成年人中高达20%,高血压患者由于动脉血压长期高于正常血压,不仅能引起头痛、头昏、心悸等症状,而且能引起患者心、脑、肾等器官损害,导致出血性脑卒中、心肌梗死、心力衰竭和脑血栓并发症,使患者偏瘫或死亡,明显降低患者生活质量。高血压病的病因复杂,已知体内许多系统与血压的调节有密切关系,抗高血压药物可以作用于影响血压调节的任一环节而使血压下降。当前常见的治疗高血压的药物有以下六种:利尿剂(可能引起低血钾症,影响糖代谢,使糖耐量下降),β-阻滞剂(可带来中枢神经系统、消化系统以及血管系统的不良反应),α-阻滞剂、钙拮抗剂(会导致水肿、头痛、潮红、多尿、低血压以及心脏传导受阻),血管紧张素II受体阻滞剂(可引起干咳、血钾高、甚至血管性水肿)和血管紧张素转换酶抑制剂(ACEI)。由于现有大部分药物都有较强的副作用,所以科学家们一直致力于开发高效低毒的药品或食品,含有抑制血管紧张素转换酶(ACE)活性的小肽的功能食品或药品,使高血压患者得益,研究表明,该类药物优于一般血管扩张药,在使血压下降时,心、肾血流量不下降;优于钙拮抗剂,不会引起水钠潴留,不加快心率;优于α-阻滞剂,不会引起体位性低血压;优于老一代降压药,无中枢作用;优于硝酸酯类药物,无耐药性、无肝脏首过效应和无停药反跳现象[吴金珊,李佳.抗高血压药物的研究进展和临床应用[J].中国实用医药,2009,4(23):231-232.]。专利CN200710047836.7通过水解酪蛋白得到的抗高血压活性三肽Asn-Pro-Trp,其具有一定的降血压效果,具体地,该活性三肽对ACE抑制活性为85.5%,其采用5mg/kg的剂量给药5天后,大鼠的血压稳定在176mmHg,并且后续血压有所回升;亦有专利CN201210247822.0通过合成方法制备得到抗高血压活性三肽:缬氨酸-异亮氨酸-脯氨酸,其ACE抑制活性为仅为36.58±4.66%。
发明内容
本发明所要解决的技术问题是提供一种结构不同且具有良好的ACE抑制活性和明显的降血压效果的抗高血压活性肽、其制备方法及应用。
为解决以上技术问题,本发明采取如下技术方案:
一种抗高血压活性肽,所述的活性肽的氨基酸序列为:茶氨酸-γ-氨基丁酸-脯氨酸。
一种抗高血压活性肽的制备方法,其通过将脯氨酸二氯树脂和芴甲氧羰基γ-氨基丁酸在偶联剂和有机溶剂的存在下,进行偶联得到中间产物;再将所述的中间产物和芴甲氧羰基茶氨酸在偶联剂和有机溶剂的存在下,进行偶联,经纯化后得到所述的抗高血压活性肽。
具体地,一种抗高血压活性肽的制备方法,其通过将脯氨酸二氯树脂和芴甲氧羰基γ-氨基丁酸在偶联剂和有机溶剂的存在下,进行偶联50~70分钟,然后经洗涤得到中间产物;再将所述的中间产物和芴甲氧羰基茶氨酸在偶联剂和有机溶剂的存在下,进行偶联50~70分钟,经洗涤、纯化后得到所述的抗高血压活性肽。
进一步地,所述的偶联剂为O-苯并三氮唑-N,N,N',N'-四甲基脲四氟硼酸,所述的有机溶剂为N,N-二异丙基乙胺。
进一步地,经所述洗涤得到所述的中间产物的具体方法为:将所述的脯氨酸二氯树脂和所述的芴甲氧羰基γ-氨基丁酸进行偶联反应后,用体积比为1:4~6的哌啶和二甲基甲酰胺混合液洗涤多次,然后用六氢吡啶进行脱保护,脱保护结束后再用体积比为1:4~6的哌啶和二甲基甲酰胺混合液洗涤多次,得到所述的中间产物。
进一步地,经所述的洗涤、纯化后得到所述的抗高血压活性肽的具体方法为:将所述的中间产物和所述的芴甲氧羰基茶氨酸进行偶联反应后,用体积比为1:4~6的哌啶和二甲基甲酰胺混合液洗涤多次,然后用六氢吡啶进行脱保护,脱保护结束后再用体积比为1:4~6的哌啶和二甲基甲酰胺混合液洗涤多次,然后经抽干、纯化后得到所述的抗高血压活性肽。
进一步地,采用半制备型反相高效液相色谱进行所述的纯化。
更进一步地,所述的半制备型反相高效液相色谱的反相柱为:VYDAC-C18柱;流动相为:溶液A为溶解于分析纯级乙腈的0.05%~0.15%(v/v)的三氟乙酸,溶液B为溶解于纯水的0.05%~0.15%(v/v)的三氟乙酸;洗脱梯度为:0min~25min:5%~30%的溶液A,95%~70%的溶液B;26min~55min:100%的溶液A;流速:0.5~1.5毫升/分钟。
此外,本发明还涉及该抗高血压活性肽在制备抗高血压的药品或食品中的应用。
此外,本发明还涉及一种抗高血压的药物组合物,其包括有效成分和辅料,所述的有效成分包括如权利要求1所述的抗高血压活性肽。
其中,所述的辅料包括常规的药用辅料或食品添加剂。
由于以上技术方案的采用,本发明与现有技术相比具有如下优势:
本发明提出了一种不同的活性肽,该活性肽的ACE抑制活性能够达到64.5%,采用2mg/kg剂量给药5天后血压值能够基本稳定在165mmHg左右。
附图说明
附图1为抗高血压大鼠实验检测结果图。
具体实施方式
下面结合具体的实施例对本发明做进一步详细的说明,但本发明不限于以下实施例。以下实施例中,在没有特别说明时,“%”均指质量百分比。
实验材料:
新西兰大白兔:上海交通大学药学院动物实验中心提供;
20周龄雄性高血压模式大鼠(SHR):上海实验动物中心提供;
微量移液器(100~1000μL,20~200μL,10~100μL,0.5~10μL),EppendorfLtd;
过滤器(Φ50mm),上海医药工业研究所;
微孔滤膜(Φ50mm,孔径为0.22μm),上海摩速科学器材有限公司;
Centrifuge 5415D小型高速离心机,Eppendorf Ltd;
10层析系统,Superdex 30prep grade层析柱(GE Healthcare,USA);
Zorbax SB-C18反相色谱柱(Agilent);
摩尔超纯水机,上海摩勒科学仪器有限公司;
GL-22M高速冷冻离心机,上海卢湘仪离心机仪器厂;
JY2002型电子天平,上海良平仪器仪表有限公司;
手提式压力蒸汽灭菌器,上海医用核子仪器厂;
HWS26型电热恒温水浴锅,上海一恒科技有限公司;
Lab Dancer试管振荡器,IKA Works Guangzhou;Electrolux BCD-252T型冰箱,伊莱克斯(中国)电器有限公司;
DW-HW138型超低温冰箱,中科美菱低温科技有限责任公司;
分析天平,Meitelei-tolido,German;
大鼠血压计:SoftronBP-98A,北京软隆生物技术有限公司。
实施例1 茶氨酸-γ-氨基丁酸-脯氨酸的合成
首先在脯氨酸二氯树脂上接入芴甲氧羰基γ-氨基丁酸,同时加入TBTU(O-苯并三氮唑-N,N,N',N'-四甲基脲四氟硼酸)和DIEA(N,N-二异丙基乙胺)进行偶联,偶联时间为60分钟,并用体积比为20%的哌啶/二甲基甲酰胺洗涤3次去除末端芴甲氧羰基基团,然后用六氢吡啶进行脱保护,脱保护结束后再用体积比为20%的哌啶/二甲基甲酰胺洗涤6次。抽干后加入芴甲氧羰基茶氨酸,TBTU和DIEA进行偶联,偶联时间为60分钟,并用20%的哌啶/二甲基甲酰胺洗涤3次,用六氢吡啶进行脱保护,再用体积比为20%的哌啶/二甲基甲酰胺洗涤6次抽干,最后通过半制备型反相高效液相色谱进行纯化(反相柱:VYDAC-C18柱(4.6×250mm,5μm);流动相:溶液A(溶解于分析纯级乙腈的0.1%(v/v)三氟乙酸),溶液B(溶解于纯水的0.1%(v/v)三氟乙酸)。洗脱梯度如下:0min~25min:5%~30%A,95~70%B;26min~55min:100%A,0%B。
流速:1.0毫升/分钟;分离收集洗脱峰,冻干后备用。
结构测定:
C16H28N4O5 Mw:356.2
1H-NMR(CD3OD,500MHz)δ:1.20(3H,t)3.24(2H,m),2.18(2H,m),2.07(2H,m),3.56(1H,m),3.20(2H,m),1.83(2H,m),3.51(1H,m),2.02(1H,m),2.33(1H,m),4.33(1H,t)。
13C-NMR(CD3OD,125MHz)δ:172.7,30.3,30.5,53.4,171.2,39.4,26.7,31.4,174.7,61.2,28.6,22.6,46.0,174.9,34.2,15.1。
具体结构式如式1所示:
式1:茶氨酸-γ-氨基丁酸-脯氨酸
实施例2 抗高血压体外试验
马脲酰组氨酰亮氨酸(hippuryl-L-histidyl-L-leucine,HHL)在ACE酶的催化下快速分解产生马尿酸(Hippuric Acid,HA)和二肽histidyl-leucine(HL),加入ACE酶抑制剂后,ACE酶的活性受到抑制,HA和HL的生成量减少,本实施例中ACE通过兔肺提取,酶活力为0.76mU/mL,通过RP-HPLC方法(色谱柱:ZorbaxSB-C18柱(5μm,4.6mm×250mm;流动相:20%(v/v)分析纯乙腈+0.1%(v/v)三氟乙酸;流速:1.0毫升/分钟))测定228nm下HA的生成量,按下列公式评价ACE酶抑制剂对ACE酶的抑制率。
ACE抑制活性(%)=[(B-A)/(B-C)]×100%
A:样品组HA峰面积
B:对照组HA峰面积
C:空白组HA峰面积
具体反应体系和条件见表1:
表1
本实施例方法测得本发明的活性三肽对ACE抑制活性为64.5%(茶氨酸-γ-氨基丁酸-脯氨酸)。
实施例3 抗高血压动物体内试验
采用SoftronBP-98A型大鼠血压仪进行尾静脉测定血压的方法,测定大鼠的收缩压(SBP)。
1、在SHR大鼠(自发性高血压大鼠)清醒状态下,首先将老鼠放在鼠袋中,保持恒温,采用SoftronBP-98A型大鼠血压仪进行尾静脉测定血压的方法,测定大鼠的收缩压(SBP)。于实验前一周开始隔天测定大鼠的血压,待大鼠稳定适应后开始实验记录。先测定灌胃前的大鼠血压,然后2mg/kg剂量(根据大鼠的体重灌胃)样品(活性三肽)灌胃,对照组灌胃纯水同样剂量,连续灌胃样品10天,测定灌胃样品后的第1d、3d、5d、7d、9d、10d大鼠血压。每个测试点均测定2次大鼠的血压,2次测定的间隔时间约1min,取2次测定值的平均值作为该测试点大鼠的血压(参见图1)。
图1为对照组大鼠及两种活性肽灌胃给药后的血压变化情况,其测得数据用SPSS系统软件进行系统处理,采用t检验方法。由实验结果可知,与对照组相比,两种活性肽灌胃给药1天后,SHR高血压大鼠在后血压值均有显著下降,给药5天后,茶氨酸-γ-氨基丁酸-脯氨酸样品组血压值基本稳定在165mmHg左右。说明本发明活性肽有较好的降血压效果。
以上对本发明做了详尽的描述,其目的在于让熟悉此领域技术的人士能够了解本发明的内容并加以实施,并不能以此限制本发明的保护范围,凡根据本发明的精神实质所作的等效变化或修饰,都应涵盖在本发明的保护范围内。
Claims (4)
1. 一种抗高血压活性肽,其特征在于:所述的活性肽的氨基酸序列为:茶氨酸-γ-氨基丁酸-脯氨酸。
2. 一种如权利要求1所述的抗高血压活性肽的制备方法,其特征在于:其通过将脯氨酸二氯树脂和芴甲氧羰基γ-氨基丁酸在偶联剂和有机溶剂的存在下,进行偶联得到中间产物;再将所述的中间产物和芴甲氧羰基茶氨酸在偶联剂和有机溶剂的存在下,进行偶联,经纯化后得到所述的抗高血压活性肽。
3. 如权利要求1所述的抗高血压活性肽在制备抗高血压的药品或食品中的应用。
4. 一种抗高血压的药物组合物,其包括有效成分和辅料,其特征在于:所述的有效成分包括如权利要求1所述的抗高血压活性肽。
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