CN115116540A - 一种猫爪草提取物及其在癌症治疗中的应用 - Google Patents
一种猫爪草提取物及其在癌症治疗中的应用 Download PDFInfo
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Abstract
本发明公开一种猫爪草提取物及其在制备癌症治疗药物中的应用。所述猫爪草提取物中包含RTE的活性成分，主要作用靶点为AKT1、EGFR和HSP90AA1。本发明的猫爪草提取物的制备方法包括，将猫爪草粗粉用乙醇浸泡回流提取后，依次使用石油醚、乙酸乙酯和正丁醇进行萃取，选择乙酸乙酯萃取物。基于网络药理学方法，确认猫爪草的RTE的活性成分中的抗癌活性靶点为AKT1、EGFR和HSP90AA1活性靶点。本发明的RTE具有抗食管鳞癌作用，因此具有较广的应用价值。
Description
技术领域
本发明涉及植物提取物领域，具体涉及猫爪草提取物，更具体地，提供以AKT1、HSP90AA1、EGFR为抗癌主要作用靶点的猫爪草提取物及其用途。
背景技术
癌症是现今威胁人类健康的头号杀手，2020年全球有一千多万人死于癌症。癌症具有细胞分化和增殖异常、生长失去控制、浸润性和转移性等生物学特征，其发生是一个多因子、多步骤的复杂过程，分为致癌、促癌、演进三个过程，与吸烟、感染、职业暴露、环境污染、不合理膳食、遗传因素密切相关。目前的治疗方法中，对于早期或较早期实体肿瘤来说，手术切除仍然是首选的治疗方法，其次还包括化学治疗、放射线治疗等治疗方法，上述治疗方法具有较好的效果，但对患者身体素质具有一定的要求，其具有一定的承受力，能够承受上述治疗手段所带来的较大的生理病痛。中医药作为一种补充和替代药物，由于具有低副作用的优势，配合手术、放化疗可以减轻放化疗的毒副作用，促进患者恢复，增强对放化疗的耐受力，被广泛与抗肿瘤药物一起用于治疗各种形式的癌症。
猫爪草(学名：Ranunculus ternatusThunb.)是毛茛科，毛茛属一年生草本植物，主要分布于中国河南和安徽地区，中性植物，喜光，也耐阴，喜温暖湿润气候，生于丘陵、旱坡、田埂、路旁、荒地阴湿处，适应性强。对土壤要求不严，宜生长于疏松、适湿、肥沃的腐殖质壤土栽培，较耐水湿。猫爪草性温，味甘、辛，入肝、肺经，具有清热解毒、消肿散结、止咳祛痰等功效，临床用于治疗肺结核、淋巴结结核、咽喉炎、疟疾及各种癌症，但它们的活性成分和潜在作用靶点仍不清楚。
发明内容
针对猫爪草提取物在癌症治疗中的活性成分不明确、潜在作用靶点不清楚的问题，本发明通过网络药理学方法探讨RTE潜在作用靶点及诱导癌细胞凋亡的机制，并使用不同的溶剂萃取获得猫爪草醇提物不同部位，通过试验对猫爪草石油醚部位(R.ternatuspetroleum ether extract，RTP)和乙酸乙酯部位(R.ternatus ethylacetate extract，RTE)的抑制癌细胞增殖的作用加以证实。
本发明提供一种猫爪草提取物，其制备方法包括如下步骤：
步骤a：将猫爪草粗粉按比例加入乙醇浸泡、回流提取后过滤，重复提取多次后，合并滤液，减压浓缩至无乙醇味，得浓缩液；
步骤b：在浓缩液中加入超纯水溶解，依次加入石油醚、乙酸乙酯和正丁醇静置过夜，待分层后进行萃取，萃取一定次数后旋干、预冻，真空冷冻干燥后得到猫爪草醇提物不同部位(猫爪草石油醚部位、猫爪草乙酸乙酯部位和猫爪草正丁醇部位)后密封保存。
进一步的，所述步骤a中使用的乙醇是95％乙醇，所述猫爪草粗粉与乙醇的质量比为1:5-1:10，优选的，为1:10；所述浸泡、回流时间为1-3小时，优选的，所述浸泡时间为1小时，所述回流时间为3小时；所述重复提取次数为1-3次，优选的，为3次。
进一步的，所述步骤b中使用的超纯水与浓缩液的体积比为1:1-1:5，优选的，为1:1；所述萃取次数为3-5次，优选的，为3次；所述预冻的温度在-80℃，预冻时间为6-18h，优选的，为12h；所述真空冷冻干燥时间为24-72h，优选的为48h。
进一步的，使用时，将猫爪草醇提物不同部位加入DMSO，超声至完全溶解并定容为1ml，存于4℃备用。
本发明还提供一种基于网络药理学方法探讨如上述的猫爪草提取物诱导癌细胞凋亡的机制的方法，主要包括以下步骤：步骤(1)获取有效成分及匹配靶点，步骤(2)搜集癌症相关靶点，步骤(3)构建成分靶点网络图，步骤(4)构建靶点相互作用图，步骤(5)GO生物过程富集及KEGG信号通路分析，上述步骤中均需对应使用网络数据库和/或软件。
进一步的，所述网络数据库包括Pubchem(https://pubchem.ncbi.nlm.nih.gov/)、TCMSP(https://old.tcmsp-e.com/tcmsp.php)、SEA(http://sea.bkslab.org/)、TargetNet(http://targetnet.scbdd.com/)、PharmMapper(http://lilab-ecust.cn/pharmmapper/index.html)、SwissTargetPrediction(http://swisstargetprediction.ch/)、Uniprot(https://www.chemsrc.com/)、GeneCard(https://www.genecards.org/)、OMIM(https://www.omim.org/)、DAVID(https://david.ncifcrf.gov/)和STRING(https://string-db.org/)中的一种或多种；所述软件包括CAS(https://www.chemsrc.com/)、Venny 2.1.0(https://bioinfogp.cnb.csic.es/tools/venny/)和Cytoscape(Version3.7.2，http://www.cytoscape.org)中的一种或多种。
进一步的，所述步骤(1)包括：通过文献检索，收集RTE的化学成分，经Pubchem数据库及CAS网站验证和转化，通过TCMSP、SEA、TargetNet、Pubchem、PharmMapper、SwissTargetPrediction数据库，汇总每种活性成分的作用靶点及预测靶点，按度值(Degree值)筛选汇总后得到RTE的全部相关靶点，同时使用Uniprot数据库统一转换基因简称。
进一步的，所述步骤(2)包括：在GeneCard和OMIM数据库中，搜集癌症的相关靶点信息，将2个数据库所获得的数据，删除重复值，得到最终的疾病相关靶点；通过Venny2.1.0将RTE的相关靶点和癌症的相关靶点进行匹配映射，获得共同靶点，即为RTE抗癌的主要作用靶点。
进一步的，所述步骤(3)包括：将RTE的有效成分及抗癌的主要作用靶点导入Cytoscape软件中，构建药物-活性成分-主要靶点-疾病关系网络图(成分靶点网络图)，其中，以“节点”(Node)代表属性类型，如中药、化合物、靶点和疾病，“边”(Edge)代表交互关系，通过网络图中各节点的大小及颜色表示边的多少即度值的大小，将靶点及活性成分进行度值的排序，来展示整个网络中药物-活性成分-主要靶点-疾病之间的联系。
进一步的，所述步骤(4)包括：把RTE抗癌的主要作用靶点导入STRING数据库中，将研究物种选择为人类，删除没有相互作用关系的游离靶点，获得靶点相互作用网络图(protein-protein interaction，PPI)。
进一步的，所述步骤(5)包括：通过DAVID数据库对猫爪草核心作用靶点(成分靶点网络图大于平均度值的靶点和PPI大于平均度值的靶点的交集靶点)进行GO生物过程富集及KEGG信号通路分析，获得RTE发挥作用的主要信号通路及参与的生物过程。
本发明提供一种猫爪草提取物在制备癌症治疗药物中的应用。
进一步的，所述癌症是食管鳞癌。
进一步的，所述癌症治疗通过降低相关核心靶点的表达量来发挥抑制所述食管鳞癌的癌细胞增殖的功能。
进一步的，所述相关核心靶点为AKT1、EGFR和HSP90AA1。
进一步的，所述降低核心靶点的表达量通过调控MEK/ERK和PI3K/Akt通路和/或降低下游相关致癌因子HSP90和c-Myc的表达量来实现。
进一步的，所述食管鳞癌的癌细胞为TE-1、TE-13和/或EC-109。
进一步的，所述药物为口服剂型。
本发明通过文献检索获得RTE的活性成分，并通过网络药理学方法探讨RTE潜在作用靶点及诱导癌细胞凋亡的机制，发现AKT1、EGFR和HSP90AA1三个主要的抗癌作用靶点，同时本发明通过使用不同的溶剂萃取获得猫爪草不同提取物，并通过开展细胞实验对RTP和RTE抑制癌细胞(ESCC)增殖的作用加以证实。
附图说明
图1为RTE成分靶点网络图；
图2为RTE相关靶点的PPI网络图；
图3为RTE相关靶点GO富集分析结果图；
图4为RTE相关靶点KEGG富集分析结果图；
图5为RTE对相关核心靶点表达的影响实验结果图。
具体实施方式
为了使本发明要解决的技术问题、技术方案及有益效果更加清楚明白，以下结合实施例及附图，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。以下结合具体实施例对本发明作具体的介绍。
实施例1猫爪草不同有机溶剂提取物的制备：
称取猫爪草根的粗粉100g(药材公司购买)，加入10倍量95％乙醇浸泡1h，90℃回流提取3h，趁热过滤药液，再重复提取3次，合并滤液，60℃恒温减压浓缩至无乙醇味(可以通过闻来判定)，得浓缩液，在浓缩液中加入体积与浓缩液体积相同的超纯水溶解，依次使用石油醚(petrloeum ether)、乙酸乙酯(ethyl acetate)和正丁醇(n-BuOH)进行萃取，三种溶剂分别各萃取3次，将上述得到的猫爪草不同萃取液旋干后放于-80℃冰箱中预冻12h，置于真空冷冻干燥机低温升华干燥48h后，密封保存于-20℃，即得猫爪草醇提物石油醚部位(RTP)、猫爪草醇提物乙酸乙酯部位(RTE)和猫爪草醇提物正丁醇部位(RTB)。
实施例2基于网络药理学探讨RTE诱导癌细胞凋亡的试验：
(1)RTE的有效成分及匹配靶点的获取：
先分析RTE的全部活性成分，结果如表1：
表1.RTE的有效成分表
将表1中RTE的32个活性成分别输入TCMSP、Pubchem、SEA、TargetNet、PharmMapper、SwissTargetPrediction数据库中进行检索，把每个活性成分的相关靶点进行汇总及删除重复值，删除重复值后得到RTE相关靶点共472个。
(2)搜集癌症相关靶点
在Genecard和OMIM数据库获得搜集ESCC(食管鳞癌)的相关靶点信息，将2个数据库所获得的数据，删除重复值，得到最终的疾病相关靶点共4838个；将4838个ESCC相关靶点及472个RTE相关靶点导入Venny 2.1.0中，通过映射匹配韦恩图，得到ESCC与RTE重合靶点共281个，即为RTE抗癌的主要作用靶点。
(3)构建成分靶点网络图
将RTE的32个活性成分及281个抗癌的主要作用靶点导入Cytoscape软件中，构建药物-活性成分-主要靶点-疾病关系网络图(成分靶点网络图)，如图1所示，所有靶点节点用菱形表示，将网络图中所有靶点及活性成分进行度值的排序，化合物与靶点的平均度值为9.47，大于平均度值的有32个化合物和55个靶点(见表2)。
表2.RTE成分-靶点网络关键节点及其拓扑学特征
(4)构建靶点相互作用图
将281个抗癌的主要作用靶点上传至STRING数据库，设置Combined Score＞0.7，将研究物种选择为人类，隐藏没有联系的游离靶点，得到靶点相互作用网络图(PPI)，如图2所示，其中显示出的靶点有254个，边为1357条。将在STRING得到的PPI网络图导入Cytoscape3.7.2中，分析PPI网络图，计算平均度值为10.68，大于平均度值的节点有81个，见表3，排名前三位的靶点分别是AKT1、EGFR和HSP90AA1，后续实验取这三个靶点作为目标靶点。
表3.PPI关键节点排序表
(5)GO生物过程富集及KEGG信号通路分析
将PPI网络图大于平均度值的81个靶点，与成分靶点网络图中大于平均度值的55个靶点取交集，获得两组的交集靶点共14个(表4)，将14个核心靶点上传至DAVID数据库(2021年更新)进行GO生物过程富集分析。设置条件count≥2，P<0.05后筛选出GO生物过程52个、分子功能9个、细胞组分30个，选择前24个BP、前5个CC、前5个MF信息生成GO生物过程富集分析的可视化结果，如图3所示，从图中看出涉及信号转导、细胞因子介导的信号通路和药物应答等生物过程为RTE重要靶标干预的生物学过程。同样在DAVID数据库进行KEGG分析，设置条件count≥2，P<0.05后筛选出KEGG通路36条，生成气泡图如图4，从图中能看出涉及癌症通路、TNF信号通路、MAPK信号通路及PI3K/Akt通路等可能为RTE重要靶标干预的潜在信号通路，因此后续的实验选取这些细胞通路进行研究。
表4.核心靶点排名情况
| 序号 | 靶点 | 序号 | 靶点 |
| 1 | HSP90AA1 | 8 | PTGS2 |
| 2 | MAPK1 | 9 | CDK2 |
| 3 | RelA | 10 | F2 |
| 4 | ESR1 | 11 | MAPK10 |
| 5 | CASP3 | 12 | ESR2 |
| 6 | PPARG | 13 | PGR |
| 7 | AR | 14 | MMP3 |
实施例3细胞实验：
(1)RTE、RTP和RTB的抑制细胞增殖实验：
将不同浓度梯度的RTE(0，50，100，200，400，600和800μg/mL)、RTP(0，50，100，200，400，600和800μg/mL)和RTB(0，200，400，600，800，1000和2000μg/mL)分别作用于ESCC细胞株TE-1、TE-13和EC-109细胞株48h后，使用MTT法检测，结果见表5，从表5中可看出，RTE和RTP可显著抑制三种不同细胞株的增殖(P<0.001和P<0.05)，且两者在EC-109细胞株的IC50值(半抑制浓度)最小，但这两个不同试剂的提取物对三种细胞株的增殖抑制无显著差异(P>0.05)；而当RTB的浓度达2000mg/mL时对三种细胞株的增殖抑制不超过仍50％，说明RTB无抗肿瘤活性(结果未展示)。因此排除RTB提取物，可以采用RTE和RTP。
表5.RTE和RTP在三种不同ESCC细胞株的IC50值
由于RTP中的化学成分极性较差，限制了其在实际中的应用(用于制备药物)，因此下一步的实施例只选取RTE进行实验。
(2)RTE对相关核心靶点表达的影响实验：
将RTE分别作用于EC109和TE-13细胞株培养48h(对应为实验组和对照组)后，对多种细胞因子的蛋白表达或者状态水平利用western blotting进行检测，结果如图5所示。其中，与对照组相比，分别检测到p-ERK1/2(细胞外信号调节激酶1/2磷酸化状态，其表达与淋巴结转移和肿瘤分化程度密切相关)、p-Akt(又称作磷酸化蛋白激酶B或PKB，其表达与癌的分化程度、分期、淋巴结转移密切相关)、HSP90(热激蛋白90，是热休克蛋白的核心成员之一,它调控的很多客户蛋白(client protein)均与肿瘤等疾病的发生和发展关系密切)和c-Myc(c-myc基因既是一种可易位基因,又是一种多种物质调节的可调节基因,也是一种可使细胞无限增殖、获永生化功能、促进细胞分裂的基因)表达量降低，其中对于EC109细胞株，p-ERK1/2表达量降低得最为明显，其次是p-Akt，再次是c-Myc，HSP90表达量也降低，β-Actin表达量则未降低；对于TE-13细胞株，也是p-ERK1/2表达量降低得最为明显，其次是p-Akt、c-Myc和HSP90，β-Actin表达量则未降低；而总ERK1/2和Akt的表达量无显著变化(EC109和TE-13两个细胞株结果相同，均没有明显变化)，证实RTE通过调控MEK/ERK和PI3K/Akt通路，降低下游相关致癌因子HSP90和c-Myc的表达量，发挥抑制ESCC增殖的功能。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。
Claims (9)
1.一种猫爪草提取物的制备方法，其特征在于，包括如下步骤：
步骤a：将猫爪草根的粗粉加入乙醇浸泡、回流提取后过滤，重复提取多次后，合并滤液，减压浓缩得浓缩液；
步骤b：在所述浓缩液中加入水溶解，依次使用石油醚、乙酸乙酯和正丁醇萃取，选取乙酸乙酯萃取物。
2.如权利要求1所述的制备方法制备得到的猫爪草提取物。
3.如权利要求2所述的猫爪草提取物在制备癌症治疗药物中的应用。
4.如权利要求3所述的应用，其特征在于，所述癌症是食管鳞癌。
5.如权利要求4所述的应用，其特征在于，所述癌症治疗通过降低相关核心靶点的表达量来发挥抑制所述食管鳞癌的癌细胞增殖的功能。
6.如权利要求5所述的应用，其特征在于，所述相关核心靶点为AKT1、EGFR和HSP90AA1。
7.如权利要求5或6所述的应用，其特征在于，所述降低核心靶点的表达量通过调控MEK/ERK和PI3K/Akt通路和/或降低下游相关致癌因子HSP90和c-Myc的表达量来实现。
8.如权利要求7所述的应用，其特征在于，所述食管鳞癌的癌细胞为TE-1、TE-13和/或EC-109。
9.如权利要求3-6中任一项所述的应用，其特征在于，所述药物为口服剂型。
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| CB02 | Change of applicant information |
Country or region after: China Address after: 518055 No. 1068, Xue Yuan Avenue, University Town, Xili street, Nanshan District, Shenzhen, Guangdong Applicant after: SHENZHEN INSTITUTES OF ADVANCED TECHNOLOGY Applicant after: Shenzhen University of Technology Address before: 518055 No. 1068, Xue Yuan Avenue, University Town, Xili street, Nanshan District, Shenzhen, Guangdong Applicant before: SHENZHEN INSTITUTES OF ADVANCED TECHNOLOGY Country or region before: China Applicant before: Shenzhen University of Technology (preparatory) |