CN116098967B - 一种平潜补利汤及其有效成分和应用 - Google Patents
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Abstract
本发明涉及一种平潜补利汤及其有效成分，所述平潜补利汤是由白术11‑13份、茯苓11‑13份、钩藤11‑13份、刺蒺藜9‑11份、黄芪19‑21份、牛膝11‑13份、天麻9‑11份、杜仲9‑11份、夏枯草14‑16份、泽泻29‑31份组成。实验通过筛选平潜补利汤中的有效成分以及高血压病的治疗靶点，其有效成分为山柰酚(Kaempferol)、β‑谷甾醇(beta‑sitosterol)、槲皮素(Quercetin)、β‑胡萝卜素(beta‑carotene)；关键靶点为ALB、IL6、VEGFA、MYC、Caspase‑3。在自发性高血压大鼠(SpontaneouslyHypertensiveRat，SHR)体内验证平潜补利汤治疗高血压的作用机制，因此平潜补利汤可用于高血压病的治疗。
Description
技术领域
本发明涉及医药领域，具体涉及一种平潜补利汤及其有效成分和应用。
背景技术
高血压(hypertension)是一种动脉血压升高的慢性病，是过早死亡和致残的可控危险因素，与心肌梗死、心力衰竭、缺血性或出血性中风以及终末期肾脏疾病的风险增加密切相关[1-2]。2013-2014年中国流行病学调查显示，27.8％(2.92亿)的成年人患有高血压，较2002年的发病率(18.8％)增加32％，给中国的医疗系统带来巨大的负担[3]。
高血压病已成为影响人们健康的主要病症之一，高血压病多表现为头痛、眩晕、心悸失眠，重者可发展为中风，属中医的头痛、眩晕、中风等病症范畴。由于不良的饮食习惯，生活节奏的加快、工作竞争的激烈，均可能引起人体的血压升高。还有生存的压力、工作的压力、竞争的压力等可导致人体的精神紧张、植物神经失调，也容易引起血压升高。目前，市场治疗高血压的药物，无论中药还是西药，都没有显著及长期的效果，难以治愈，更不能根除。
中国专利CN：201210408948.1，公开了一种降血压的中药组合物。其是由紫丹参25-40份，怀牛膝12-18份，夏枯草22-35份，丹皮10-18份，马兜铃26-34份，钩藤10-20份，刺蒺藜12-16份，代赭石25-35份，白术20-40份，党参15-25份，茯苓15-25份，菊花10-20份，泽泻16-28份。用于治疗高血压、高血压引起的头晕、头痛，疗效十分明显。但却未见如本申请关于治疗高血压药物疾病的报道。
发明内容
本发明的目的是针对现有技术中的不足，本发明的第一方面，提供一种治疗高血压的中药组合物，所述的中药组合物是由白术11-13份、茯苓11-13份、钩藤11-13份、刺蒺藜9-11份、黄芪19-21份、牛膝11-13份、天麻9-11份、杜仲9-11份、夏枯草14-16份、泽泻29-31份组成。
优选地，所述的中药组合物是由白术12份、茯苓12份、钩藤12份、刺蒺藜10份、黄芪20份、牛膝12份、天麻10份、杜仲10份、夏枯草15份、泽泻30份组成。
本发明的第二方面，提供如上任一所述的中药组合物在制备治疗高血压药物中的应用。
优选地，所述中药组合物通过抑制炎症反应和RAAS系统，改善血管内皮功能，减少心肌和血管内细胞凋亡，从而降低血压，改善心肌和胸主动脉重构。
本发明的第三方面，提供一种治疗高血压的中药组合物，所述中药组合物的活性成分山柰酚、谷甾醇、槲皮素和β-胡萝卜素。
本发明的第四方面，提供如上活性成分在制备治疗高血压药物中的应用。
优选地，山柰酚、谷甾醇、槲皮素和β-胡萝卜素通过上调ALB、VEGFA的表达，下调IL6、MYC、CASP3的表达，进而激活PI3K-Akt信号通路，从而降低血压。
本发明的第五方面，提供山柰酚、谷甾醇、槲皮素和β-胡萝卜素在制备升高Pi3k/Akt信号通路激活药物中的应用。
本发明优点在于：
1.平潜补利汤由天麻钩藤饮化裁而来，去除黄芩、栀子等清热泻火之药防止长期服用苦寒伤胃，加用杜仲、黄芪等健脾补肾之药以补虚填髓。
2.本研究提示山柰酚、β-谷甾醇、槲皮素、β-胡萝卜素是平潜补利汤发挥药理作用的关键成分。山柰酚可以增加内皮细胞一氧化氮(nitric oxide,NO)的释放、拮抗血管平滑肌中的钙离子通道，进而降低血压[4-8]。β-谷甾醇具有抗炎、抗氧化应激的作用，在抗高血压和保护靶器官过程中发挥关键作用[9-11]。槲皮素在临床上拥有确切的降压疗效，可以改善内皮功能、抑制肾素-血管紧张素-醛固酮系统(Renin-angiotensin-aldosterone System，RAAS)和调节血管平滑肌细胞(vascular smooth muscle cell，VSMC)的收缩[12-15]。研究表明血清低水平的β-胡萝卜素与高血压、冠状动脉疾病等发病相关，超剂量β-胡萝卜素治疗可以降低SHR的血压[16-17]。
3.本研究的WB验证结果表明平潜补利汤可以激活PI3K-Akt信号通路，PI3K-Akt信号通路亦位于KEGG通路富集分析的前20位。PI3K-Akt参与高血压所导致的内皮损伤、心肌重构和肾脏损伤等进程，可调控一氧化氮合酶(nitric oxide synthas，NOS)活性，促进NO的合成和释放，达到调控血压的作用[18-19]。IL-6、MYC、VEGF等关键靶点均位于PI3K-Akt的信号通路上，说明这些靶点可能是平潜补利汤激活PI3K-Akt信号通路的媒介，最终增加NO的合成和释放，发挥降压疗效。
附图说明
图1为平潜补利汤成分作用靶点与高血压病靶点交集维恩图。
图2为中药-成分-靶点网络图。
图3为蛋白-蛋白互作网络图。
图4为GO功能分析和KEGG富集分析图。
图5为平潜补利汤主要活性成分-关键靶标对接的可视化展示图；注：A：β-谷甾醇-IL6；B：槲皮素-IL6；C：槲皮素-Caspase3；D：山柰酚-Caspase3；E:槲皮素-ALB；F：山柰酚-ALB。
图6为分子对接热图；
图7为12周内各组大鼠血压变化趋势图(n＝6)；注：与空白组比较，*P＜0.05；与模型组比较#P＜0.05。
图8为各组大鼠胸主动脉和心脏病理变化；A：大鼠胸主动脉(HE，×4)；B：大鼠胸主动脉(HE，×40)；C；大鼠心脏(HE，×40)；图B箭头代表弹力纤维走形不规则，图C箭头代表心肌细胞排列紊乱伴见水肿。
图9为平潜补利汤对各组大鼠胸主动脉关键基因mRNA表达影响(n＝3)；注：与空白组比较，*P＜0.05；与模型组比较#P＜0.05。
图10为平潜补利汤对PI3K-Akt信号通路的影响(n＝3)；注：与空白组比较，*P＜0.05；与模型组比较#P＜0.05。
具体实施方式
下面结合附图对本发明提供的具体实施方式作详细说明。
实施例1治疗高血压的中药组合物(一)
白术12份、茯苓12份、钩藤12份、刺蒺藜10份、黄芪20份、牛膝12份、天麻10份、杜仲10份、夏枯草15份、泽泻30份。
实施例2治疗高血压的中药组合物(二)
白术12份、茯苓11份、钩藤13份、刺蒺藜10份、黄芪19份、牛膝13份、天麻10份、杜仲9份、夏枯草16份、泽泻30份。
实施例3治疗高血压的中药组合物(三)
白术11份、茯苓13份、钩藤12份、刺蒺藜9份、黄芪21份、牛膝12份、天麻9份、杜仲11份、夏枯草15份、泽泻29份。
实施例4治疗高血压的中药组合物(四)
白术13份、茯苓12份、钩藤11份、刺蒺藜11份、黄芪20份、牛膝11份、天麻11份、杜仲10份、夏枯草13份、泽泻31份。
实施例5有效成分与作用靶点的筛选
1.方法
1.1网络药理学分析
1.1.1有效成分的收集与作用靶点筛选
平潜补利汤由白术、茯苓、钩藤、刺蒺藜、黄芪、牛膝、天麻、杜仲、夏枯草、泽泻配伍而成。利用2014年5月更新的TCMSP数据库(Traditional Chinese Medicine SystemsPharmacology Database and Analysis Platform，http://tcmspw.com)检索平潜补利汤的有效活性成分，并使用符合口服生物利用度(OB)≥30％及类药性(DL)≥0.18条件的活性成分及其作用靶点，通过文献对具有较强活性(其不能同时满足二者筛选条件)的成分进行补充。通过2022年4月29日更新的UniProt(https://www.uniprot.org/，)数据库将预测出的靶点蛋白名转换为基因名。
1.1.2高血压病靶点的筛选
以“hypertension”为检索词，利用5.10版本的Gene Cards数据库(https://www.genecards.org/)、2021年3月更新的人类孟德尔遗传数据库(Online MendelianInheritance in Man，OMIM，http://www.omim.org/)检索疾病靶点，剔除重复数据。使用维恩图对中药活性成分靶点以及高血压病靶点取交集。
1.1.3D-C-G网络构建
将筛选出的中药活性成分、作用靶点和药物信息等，通过Cytoscape 3.7.1软件构建D-C-G网络图，依据“NetworkAnalyzer”板块相关的度值、紧密度等拓扑数据进行网络拓扑学分析。
1.1.4GO和KEGG功能分析
使用R包“ClusterProfiler”[20]对交集基因进行功能注释，以求全面探讨这些基因的功能相关性。P值和q值均小于0.05的GO和KEGG富集通路被认为是显著性类别。
1.1.5蛋白-蛋白互作网络(ProteinProtein Interactionnetwork,PPI)构建
PPI数据来自于2021年8月12日更新的11.5版本的String数据库(https://string-db.org/)，物种限定为“Homo sapiens”，置信度>0.4。隐藏游离节点，其余参数保持不变，文件导出为TSV格式。将TSV文件导入Cytoscape3.7.1，利用NetworkAnalyzer插件进行拓扑学分析，依据节点度值大小构建PPI网络，进行可视化分析。
1.1.6分子对接验证
将D-C-G网络图中Degree值大于20的有效成分与PPI网络中得到的Degree值位于前5的靶点进行分子对接。从2019年3月更新的Pubchem(pubchem.ncbi.nlm.nih.gov/)下载有效成分的3D结构，再使用ChemBio3D转换成.mol文件。通过PDB数据库(www1.Rcsb.org)得到核心靶点的pdb结构文件，再使用Pymol软件去除核心靶点的pdb结构文件中的水分子和小分子配体得到蛋白受体文件。将小分子配体文件和蛋白受体文件导入AutoDockTools1.5.6软件计算每对小分子配体和蛋白受体文件最佳结合的区域，通过AutoDockVina软件进行半柔性分子对接计算得到每对小分子和作用靶点的亲和力值。使用Pymol软件绘制有效成分与靶点的结合构像图并标注出结合位点。
2.结果
2.1活性化合物的筛选与靶标预测
共筛选出138种有效成分，其中白术3种，刺蒺藜8种，杜仲24种，茯苓6种，钩藤31种，黄芪16种，牛膝16种，天麻17种，夏枯草10种，泽泻7种。删除重复数据后，最终123种有效成分纳入进一步分析。从TCMSP数据库中获取122个平潜补利汤活性靶标。Degree值≥16的有效成分的信息见表1。
表1部分活性成分信息表
2.2高血压病靶点的筛选
以“hypertension”为关键词，检索GeneCards数据库和OMIM数据库共获取疾病靶点2827个。高血压病靶点与活性成分靶点交集靶点为84个，通过Venny2.1.0绘制Venn图，见图1。
2.3D-C-G网络的构建
为了更加整体和直观地反映平潜补利汤活性化合物与潜在靶标之间的相互作用，本研究将123个活性化合物映射到122个对应的潜在靶标，构建了D-C-G网络。如图2所示，网络由339个节点(10个草药节点、123个有效化合物节点和122个化合物相关目标节点)和1417个相互作用边组成。筛选度值大于20的成分，即山柰酚(Kaempferol)、β-谷甾醇(beta-sitosterol)、槲皮素(Quercetin)、β-胡萝卜素(beta-carotene)。
2.4PPI网络的分析
通过STRING在线数据库分析84个交集靶点的蛋白互作关系，并通过Cytoscape进行可视化。根据Cytoscape插件计算了每个基因的平均连接度(degree)，选取degree值排名前5的靶点作为关键靶点，分别是白蛋白(Albumin，ALB)、白介素-6(Interleukin-6，IL6)、血管内皮生长因子A(Vascular Endothelial Growth Factor，VEGFA)、原癌基因(Proto-oncogene,MYC)、半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)。PPI网络图见图3。
2.5GO富集和KEGG通路富集分析
如图4所示，进一步对84个交集靶点进行富集分析，GO分析表明主要的生物学过程为对类固醇激素的反应、氧化应激反应等；主要的细胞组分为膜筏、膜微区等；主要的分子功能为类固醇结合、RNA聚合酶Ⅱ转录因子结合等；主要富集的KEGG通路包括129条信号通，其中与高血压病关联较大的信号通路为TNF信号通路、PI3K-Akt信号通路、Apoptosis信号通路等。
2.6分子对接结果
通过分子对接验证发现，平潜补利汤中的核心有效成分山柰酚(Kaempferol)、β-谷甾醇(beta-sitosterol)、槲皮素(Quercetin)、β-胡萝卜素(beta-carotene)等与ALB、IL6、VEGFA、MYC、CASP3等关键靶点的亲和力值大部分小于-5kcal/mol。说明这些有效成分与核心靶点结合稳固[21]，能够改善或治疗高血压病。选择亲和力值排名前6的关键蛋白和活性分子绘制对接模式图见图5，分子对接热图见图6。
实施例6动物实验
1.实验材料
1.1实验动物
8周龄SPF级SHR 12只，Sprague Dawley(SD)大鼠6只，所有大鼠均为雄性，体质量160-180g，均由北京维通利华公司提供(中国北京，证号SCXK(京)2020-0011)。所有大鼠均在上海中医药大学动物实验中心饲养(实验动物使用许可证号:SYXK(沪)2020-0009)，在SPF级室内，温度为20℃，湿度为50-65％，光/暗循环12h。实验前适应性喂养2周。本研究使用的所有实验方案均按照上海中医药大学机构动物护理与使用委员会的规定进行(伦理号：PZSHTUCM220110013)。
1.2仪器与试剂
平潜补利汤中药配方颗粒(江阴天江药业有限公司，批号2109321)；BCA蛋白浓度检测试剂盒、SDS-PAGE凝胶快速配制试剂盒、蛋白Marker、Western一抗二抗洗脱液(上海碧云天生物技术有限公司，批号0701018181104、P0012AC、061418181119、P0025-250mL)；RNA抽取试剂盒、HiScriptⅢRT SuperMix for qPCR(+gDNAwiper)试剂盒、TaqPro UniversalSYBR qPCRMaster Mix试剂盒(南京诺唯赞生物科技有限公司，批号RC112、R323-01、Q712-02/03)；GAPDH(Proteintech公司；美国，批号051201)；PI3K(1:1000)、p-Akt(Ser473，1:2000)、Akt(1:1000)抗体(CST生物科技公司；美国)，批号17366、4060、4691)；二抗(合肥白鲨生物科技有限公司，批号69110200)；RT-qPCR引物(生工生物工程(上海)股份有限公司，批号H001)。VIVID彩色心脏多普勒超声仪(GE公司；美国)；SDS-PAGE电泳仪、湿转转膜仪(Bio-Rad公司；美国)；5430R型高速冷冻离心机(Eppendorf公司；德国)；BP-2006A大鼠无创尾动脉血压计(北京软隆生物技术有限公司)；StepOne Plus实时荧光定量PCR仪(AppliedBiosystems公司；美国)。
1.3平潜补利汤的制备和干预措施
本研究所使用的中药配方颗粒是单味饮片煎煮剂净膏粉简单的物理混合，没有复方中药的共煎流程。每剂中药颗粒相当于中药汤剂剂量的天麻10g、钩藤12g、夏枯草15g、刺蒺藜10g、泽泻30g、牛膝12g、杜仲10g、黄芪20g、白术12g，茯苓12g。正常成年人的体质量为60kg，按照体表面积法计算，200g大鼠的剂量设定为人类的6.3倍，即15g/kg。将每剂中药配方颗粒充分溶于96mL蒸馏水中，即得到1.5g/mL质量浓度的中药原液。各组大鼠适应性喂养2周后随机分为3组，平潜补利汤组和模型组各包括SHR6只，分别使用平潜补利汤中药原液和同等剂量的蒸馏水灌胃治疗；空白组包括SD大鼠6只，不予以任何处理。各组均治疗12周后处死。
1.4大鼠尾动脉血压的测量
本研究所使用的血压测量，使用大鼠无创尾动脉血压计测量所有大鼠的血压，固定每周的某天上午8～10h测量，每只测量5次取平均值，每周测量1次，连续测量12周。收缩压(Systolic Pressure，SBP)≥140mmHg符合实验要求。
1.5心脏彩超
采用彩色心脏多普勒超声仪，运用二维和M型超声，Teich法测定连续3个以上心动周期大鼠的左室射血分数(left ventricular ejection fraction，LVEF)、舒张末期室间隔厚度(Interventricular Septal Thickness at Diastole，IVSd)、左室舒张末期内径(left ventricular internal diameter at end-diastole，LVIDd)、左室收缩末期内径(left ventricular internal diameter at end-systole，LVIDs)、左室后壁舒张末期厚度(Left ventricularposteriorwall diastole，LVPWd)。
1.6心脏和胸主动脉组织病理学形态观察
首先使用体积分数为4％多聚甲醛固定各组大鼠的胸主动脉和心脏组织，经石蜡包埋，切片，HE染色后，显微镜下观察各组大鼠胸主动脉和心脏组织病理学形态改变。
1.7RT-qPCR检测相关基因的表达
本研究采用qRT-PCR检测PPI网络中关键靶点的mRNA表达。胸主动脉中总RNA使用RNA抽取试剂盒分离，使用HiScriptⅢRT SuperMix for qPCR(+gDNAwiper)试剂盒，反应体系为20μL；使用Taq Pro Universal SYBR qPCR Master Mix试剂盒进行qPCR反应，遵照说明书提供的条件，进行逆转录和qPCR反应。每个RNA样本设置3次重复，结果用GAPDH归一化。采用2ΔΔCT法进行相对定量分析。引物序列见表2。
表2引物序列
1.8蛋白质印迹法(WesternBlot，WB)检测PI3K、p-Akt和Akt的表达
取100mg大鼠胸主动脉组织，用1mL RIPA裂解液和10μL苯甲磺酰氟(PMSF)混合。用研磨机充分研碎组织，在4℃、12000r/min条件下离心20min，收集上清，使用BCA蛋白测定试剂盒测定总蛋白浓度。使用SDS-PAGE电泳仪、湿转转膜仪，共分离50μg变性总蛋白样品，并转移到硝化纤维素膜上。用5％脱脂奶粉阻断膜1h，然后与一抗在4℃孵育过夜。将印迹洗净，与二抗室温孵育1h，用化学发光法检测免疫反应条带。
1.9统计分析
通过SPSS 22.0软件处理数据，符合正态性及方差齐性，组间比较采用单因素方差分析；不符合正态性和(或)方差齐性，则组间比较使用非参数检验。P＜0.05表示差异有统计学意义。
2.结果
2.1平潜补利汤对SHR血压水平的降低作用
如图7所示，10周龄时，治疗组和模型组的SBP、舒张压(Diastolic Pressure，DBP)和平均动脉压(MeanArterial Pressure，MAP)无差异(P＞0.05)，但均高于空白组，且SBP＞140mmHg，说明高血压模型构建成功。在13～14周时，治疗组的SBP、DBP和MAP较模型组降低(P＜0.05)。在21周时，治疗组的SBP、DBP和MAP低于模型组(P＜0.01)。说明平潜补利汤可以延缓SHR大鼠的血压升高。
2.2心脏彩超评价心肌重构
如表3，各组在治疗12周后，模型组和平潜补利汤组的IVSd、LVIDd、LVPWd均较空白组升高(P＜0.05)，但是，平潜补利汤组的IVSd、LVIDd低于模型组(P＜0.05)；三组的LVIDs和LVEF无差异(P＞0.05)。
表3各组大鼠心功能等指标(n＝6)
注：与空白组比较，*P＜0.05；与模型组比较#P＜0.05。
2.3各组大鼠心肌和胸主动脉组织病理学
如图8A～8B所示，与空白组相比，模型组的大鼠胸主动脉内径增大，中膜增厚，弹力纤维走形不规则；内膜不光滑，内皮细胞不完整且排列无序，胞体核小而深染，萎缩。与模型组比较，使用平潜补利汤干预后主动脉内径减小，中膜厚度降低，弹力纤维走行趋于规则；内膜光滑，细胞核基本恢复正常，内皮细胞亦有不同程度改善。
如图8C所示，与空白组比较，模型组大鼠心肌细胞细胞核深染、排列紊乱，且部分心肌细胞水肿肥大。使用平潜补利汤干预后，心肌细胞形态趋于正常，心肌形态学变化较模型组改善。
2.4平潜补利汤对各组大鼠胸主动脉相关基因mRNA的表达影响
如图9所示，实验结果表明，空白组的VEGFA、ALB mRNA表达高于模型组和平潜补利汤组(P＜0.05)，IL-6、Caspase-3、MYC的mRNA表达低于模型组和平潜补利汤组(P＜0.05)。相较于模型组，平潜补利汤组VEGFA、ALB mRNA表达上调(P＜0.05)，IL-6、Caspase-3、MYC的mRNA表达降低(P＜0.05)。
2.5平潜补利汤对PI3k-Akt信号通路的影响
如图10所示，平潜补利汤组和模型组与空白组对比，胸主动脉的PI3K和p-Akt/Akt的蛋白表达升高(P<0.05)，说明PI3k-Akt信号通路可能处于抑制状态；而平潜补利汤组与模型组相比，PI3K和p-Akt/Akt的蛋白表达增高(P<0.05)，表明平潜补利汤可以激活PI3k-Akt信号通路。
3.结论
本研究表明平潜补利汤可以降低SHR血压，改善心肌和胸主动脉重构，其机制可能是上调ALB、VEGFA的表达，下调IL6、MYC、CASP3的表达，进而激活PI3K-Akt信号通路，验证了网络药理学的预测结果。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员，在不脱离本发明方法的前提下，还可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。
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Claims (4)
1.一种同时改善心肌和胸主动脉重构的治疗高血压的中药组合物，其特征在于，所述的中药组合物是由白术11-13份、茯苓11-13份、钩藤11-13份、刺蒺藜9-11份、黄芪19-21份、牛膝11-13份、天麻9-11份、杜仲9-11份、夏枯草14-16份、泽泻29-31份组成。
2.根据权利要求1所述的中药组合物，其特征在于，所述的中药组合物是由白术12份、茯苓12份、钩藤12份、刺蒺藜10份、黄芪20份、牛膝12份、天麻10份、杜仲10份、夏枯草15份、泽泻30份组成。
3.根据权利要求1-2任一所述的中药组合物，其特征在于，所述中药组合物的活性成分为山柰酚、谷甾醇、槲皮素和β-胡萝卜素。
4.根据权利要求1-3任一所述的中药组合物在制备治疗高血压药物中的应用。
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