基于G-四链体为靶点的五种民族药用植物有效成分筛选和研究

发布时间：2018-04-23 08:28

本文选题：民族医药 + 民族药用植物　；参考：《中央民族大学》2016年博士论文

【摘要】：由于缺乏抗肿瘤特效药物,恶性肿瘤严重威胁着人类尤其是少数民族地区人们的生命健康和社会的经济发展,成为阻碍我国全面建设小康社会的一大障碍。少数民族医药学是我国少数民族几千年传统文化知识的结晶,它们不但在历史上为本民族的生存和繁衍昌盛做出过重大贡献,时至今日仍为本民族和其他民族防病治病和卫生保健事业发挥重要作用。寻找抗肿瘤特效药的希望就寄托在经过长期研究和临床实践及药理验证,对治疗肿瘤有着巨大优势和潜力的民族医药上。本研究是在对民族传统医药知识文化的认知和尊重的基础上,以资源丰富、具有抗肿瘤潜力的民族药物及民族药用植物为研究对象,以DNA G-四链体为抗肿瘤民族药物研发的新靶点,运用民族学、民族植物学、生物学、天然有机化学、仪器分析等多学科知识,从民族药物中寻找高效、低毒的抗肿瘤新型靶向制剂。本文开展了传统民族医药有效单体成分靶向结合G-四链体抗肿瘤活性研究,并基于上一部分实验材料和实验仪器适用范围的摸索,建立了CD结合NMR高效、快速的筛选民族药用植物中靶向G-四链体活性小分子化合物的方法。以畲药紫玉兰(Magnolia liliiflora Desr.)为民族植物学研究对象,CD结合NMR的方法靶向G-四链体追踪紫玉兰活性部位,进而分离得到具有抗肿瘤潜力的小分子化合物。研究结果不但能为抗肿瘤特效药物研发提供参考,而且能为传统民族医药的现代开发提供新思路,同时建立的活性筛选方法切实可行,能高效、快捷的筛选民族药物复杂体系中结构已知甚至是未知的活性化合物。研究思路和方法值得推广应用于其他民族医药的研究与开发。具体研究如下：一、开展了传统民族医药有效单体成分靶向结合G-四链体抗肿瘤活性研究。通过查阅民族医药文献发现,藏药掌叶大黄(Rheum palmatum L.)、维药茜草(Rubia cordifolia L.)及新疆紫草[Arnebia euchroma (Royle) Johnst.]、傣药木莲(Manglietia fordiana Oliv.)均被当地人用来清热、解毒,其有效单体成分芦荟大黄素、茜草素、紫草素、厚朴酚、和厚朴酚这些化合物在细胞水平上均具有一定抗肿瘤活性,但不明确是否作用于G-四链体达到抗肿瘤活性。小分子化合物空间上具有平面结构,理论上更容易靶向结合G-四链体。因此选择具有平面结构的上述民族医药有效单体成分为研究对象,研究靶向结合G-四链体活性,具体研究结果和结论如下：1、c-myc 2345序列形成了平行结构构型的G-四链体,可以用作民族药用植物与G-四链体相互作用研究的分子靶点。藏药掌叶大黄有效单体成分芦荟大黄素通过π-π堆积结合模式作用于c-myc 2345 G-四链体平面,芦荟大黄素与G-四链体以1：1形成络合物。研究结果表明藏药掌叶大黄有效单体成分芦荟大黄素具有靶向G-四链体抗肿瘤作用的潜力。2、维药茜草有效单体成分茜草素通过π-π堆积结合模式作用于c-myc 2345 G-四链体平面,茜草素与G-四链体以2：1形成络合物；维药新疆紫草有效单体成分紫草素能靶向结合c-myc 2345 G-四链体,形成2：1络合物,并通过沟槽结合和3’端的π-π堆积结合模式作用于c-myc 2345 G-四链体平面。研究结果表明维药茜草、新疆紫草有效单体成分茜草素、紫草素具有靶向G-四链体抗肿瘤作用的潜力。3、傣药木莲有效单体成分厚朴酚不能靶向结合c-myc 2345 G-四链体达到抗肿瘤作用,而其同分异构体和厚朴酚能够通过3’端π-π堆积和沟槽结合模式作用于c-myc 2345 G-四链体,和厚朴酚与G-四链体以2：1形成络合物。研究结果表明傣药木莲有效单体成分厚朴酚无靶向G-四链体抗肿瘤活性,和厚朴酚具有靶向G-四链体抗肿瘤作用的潜力。4、紫外吸收光谱和荧光光谱法是研究小分子与生物大分子相互作用的一种简单、常用方法,但其对小分子化合物的紫外吸收光谱性质要求较高,若小分子化合物与G-四链体的紫外吸收最大值波长处重合,则无法应用该方法进行实验。同时,紫外吸收光谱和荧光光谱法更善于发现π-π堆积结合模式的小分子化合物,NMR方法则能够发现弱结合及多种结合模式(沟槽结合、堆积结合)的小分子化合物。二、基于上一部分实验材料和实验仪器适用范围的摸索,建立了CD结合NMR高效、快速的筛选民族药用植物中靶向G-四链体活性小分子化合物的方法。以畲药紫玉兰(Magnolia liliiflora Desr.)为民族植物学研究对象,CD结合NMR的方法靶向G-四链体追踪紫玉兰活性部位,进而分离得到具有抗肿瘤潜力的小分子化合物,具体研究结果总结如下：1、CD变温实验最终确定95%乙醇为最适宜提取溶剂,其乙酸乙酯萃取层为分离的重点极性部位,进一步以石油醚-丙酮溶剂体系进行梯度洗脱后得到5个馏分,NMR实验确定馏分Fr.2(5：1)和Fr.3(3：1)为重点分离部位。2、根据靶向G-四链体追踪结果,利用常规硅胶柱层析(Silica gelCC),凝胶柱层析(Sephadex LH-20 CC)以及制备薄层层析(PTLC),共分离得到31个化合物。进一步利用UV、R、EI-MS、1-NMR、 13C-NMR以及2D-NMR (1H-1H COSY、HMQC、HMBC)等现代波谱技术,结合文献数据对比确定了31个化合物的结构,其中4个为新的木脂素化合物：liliflorin B (1), liliflorin C (2), liliflorin D (3), liliflorin E(4)。化合物5-22为已知的木脂素化合物,化合物23-27为已知的苯环化合物,化合物28-31为已知的倍半萜化合物。考虑到化合物的种类、新颖程度以及化合物的质量,选择了8个化合物为研究对象,利用NMR开展了其与G-四链体相互作用初步研究,发现化合物1,2,3,12,22具有靶向结合G-四链体的能力。3、化合物12(紫玉兰素A)质量足够,活性好,因此选择紫玉兰素A为与G-四链体相互作用的深入研究对象。研究结果表明紫玉兰素A(4.0μM)在K+缓冲液中可以将人端粒G-四链体的解链温度提高至69.93℃(△Tm=3.22℃),且不与DNA其他二级结构(双链DNA ds26、DNA发卡结构F10T、DNAG-四链体c-kit)相互作用,能够特异性以沟槽模式结合人端粒G-四链体,形成摩尔比1：1络合物。靶向G-四链体,从畲药紫玉兰中分离提取得到的紫玉兰素A具有靶向G-四链体抗肿瘤作用的潜力。论文的研究结果为传统民族医药靶向G-四链体抗肿瘤活性化合物的发现奠定物质基础,为高效、快速筛选民族药用植物靶向G-四链体的抗肿瘤活性小分子化合物提供了基础科学数据和技术支撑。论文所建立的研究方法能够应用于民族传统药物的二次开发,促进民族医药的现代利用,给少数民族人民带来经济效益,更好的促进我国民族地区经济和社会的发展。
[Abstract]:Due to the lack of antitumor drugs, malignant tumors seriously threaten people's life and health and social economic development, especially in ethnic minority areas. It has become a major obstacle to the overall construction of a well-off society in our country. Minority medical and pharmacy are the crystallization of traditional cultural knowledge of the ethnic minorities in China for thousands of years, and they are not only in history. It has made great contributions to the survival and prosperity of the nation, and today it still plays an important role in the prevention and treatment of diseases and health care for the nation and other nationalities. The hope of finding antitumor drugs is placed on the people who have a great advantage and potential in the treatment of cancer through long-term research, clinical practice and pharmacological validation. On the basis of the cognition and respect to the knowledge and culture of traditional medicine, this study is based on the rich resources, the national medicinal plants and ethnic medicinal plants with anti tumor potential, and the DNA G- four chain body is a new target for anti tumor national drug research and development, and it uses ethnology, ethnobotany, biology, and natural organic chemistry. In this paper, the anti-tumor activity of the effective single body component targeting G- four chain body was studied. Based on the exploration of the application range of the previous experimental materials and experimental instruments, the CD combined with NMR was established. A method for screening the active small molecules of G- four chain body in ethnic medicinal plants. Magnolia liliiflora Desr. was used as the object of Ethnological Study. CD combined with NMR was used to target G- four chain body to trace the active part of Magnolia, and then a small molecular compound with antitumor potential was obtained. It can not only provide reference for the research and development of antitumor drugs, but also provide new ideas for the modern development of traditional national medicine. At the same time, the active screening method is practical and effective. It can efficiently and quickly screen the known or unknown active compounds in the complex system of national drugs. The research and development of other ethnic medicine were studied as follows: first, the anti-tumor activity of the effective monomer targeting G- four chain body of traditional national medicine was studied. Through consulting the national medical literature, the Tibetan medicine Rheum palmatum L., the Rubia cordifolia L. and the euchroma (Royle) of Xinjiang purple grass [Arnebia (Royle) were found. Johnst.], Manglietia fordiana Oliv. is used by local people to remove heat and detoxify. The effective monomer components of aloe emodin, alizarin, herb, magnolol, and honokiol all have certain antitumor activity on the cell level, but it is not clear whether the G- four chain body can achieve the antitumor activity. Small molecule The compound has a plane structure in space, and it is more likely to target the binding of G- four chain body in theory. Therefore, to select the effective monomer components of the national medicine of the ethnic group as the research object, the target is combined with the activity of the G- four chain body. The specific results and conclusions are as follows: 1, c-myc 2345 sequence formed a parallel structure configuration of the G- four chain body, As a molecular target for the study of the interaction of ethnic medicinal plants with the G- four chain body. The effective monomeric aloe emodin of the palmatum palmatum of the palmatum palmatum of the Tibetan medicine is acted on the plane of the body of the c-myc 2345 G- four chain by the pion accumulation binding mode. The complex of the aloe emodin and the G- four chain body is formed by 1:1. The aloe emodin has the potential of anti tumor effect of the target G- four chain body.2, and the active monomer of the radix alizaris alizarin has the effect on the c-myc 2345 G- four chain body plane through the pion pion accumulation mode, and the alizarin and G- four chain body form the complex with 2:1, and the active monomer of the herb of Xinjiang purple herb can target c-myc 2345 G- four. The chain body forms the 2:1 complex and acts on the c-myc 2345 G- four chain body plane through the groove binding and the 3 'end pion accumulation binding mode. The results show that the Uygur herb, the active monomer of Alizarin Xinjiang, and the purple herb have the potential to target the anti-tumor potential of the G- four chain body, and the effective monomer magnolol of the Dai medicine Carpenter The antitumor effect was achieved by targeting the c-myc 2345 G- four chain body, and its isomers and honokiol can act on the c-myc 2345 G- four chain through the 3 'pion pion accumulation and the groove binding mode, and the complex of the magnolol and G- four chain body is formed by 2:1. The results show that the effective monomer magnolol of the Dai medicine is not targeted to the G- four chain body. The antitumor activity and the potential of magnolol to the anti-tumor effect of the target G- four chain body.4. The UV absorption spectrum and fluorescence spectrum are a simple and common method to study the interaction between small molecules and biological macromolecules, but the UV absorption spectra of small molecules are higher, if small molecules and the violet of the G- four chain body are purple. In addition, the method can not be used to experiment. At the same time, the UV absorption and fluorescence spectroscopy are better at finding small molecular compounds in the pion pion binding mode. The NMR method can find small molecular compounds with weak binding and a variety of binding modes (groove binding, heap binding). Two, based on the previous part The application range of experimental materials and experimental instruments was explored. A method of screening the active small molecular compounds targeting the G- four chain body in the ethnic medicinal plants with CD combined with NMR was established. The Magnolia liliiflora Desr. was used as the object of Ethnological Study. The CD junction NMR was targeted to the G- four chain to trace the activity of the Magnolia. The specific research results are summarized as follows: 1, the CD temperature experiment finally determines that 95% ethanol is the most suitable solvent, and the ethyl acetate extraction layer is the key polar part of the separation, and 5 fractions are obtained after the gradient elution of petroleum ether propanone solvent system, NMR The distillate Fr.2 (5:1) and Fr.3 (3:1) are the key separation sites.2. According to the tracking results of the target G- four chain bodies, 31 compounds are separated by conventional silica gel column chromatography (Silica gelCC), gel column chromatography (Sephadex LH-20 CC) and preparation of thin layer chromatography (PTLC). COSY, HMQC, HMBC) and other modern spectroscopy techniques, combined with literature and data comparison to determine the structure of 31 compounds, of which 4 are new lignan compounds: liliflorin B (1), liliflorin C (2), liliflorin D (3), liliflorin E (4). Compound 5-22 is known as lignan compound, compound 23-27 is known benzene ring compound, chemical combination, chemical combination, chemical compounds, compounds 23-27 Substance 28-31 is a known sesquiterpene compound. Considering the species, novelty and quality of the compound, 8 compounds are selected as the research object. A preliminary study on the interaction with the G- four chain body has been carried out by NMR, and it is found that compound 1,2,3,12,22 has the ability to target the binding of G- four chain bodies to.3, compound 12 (Magnolia A). It is sufficient and active, so the selection of Magnolia A is an in-depth study of the interaction with G- four chain body. The results show that the temperature of the A (4 M) in the K+ buffer can increase the solution chain temperature of the human telomere G- four chain body to 69.93 degrees C (delta Tm=3.22 C), and not with the other two structure of DNA (double stranded DNA ds26, DNA hairpin structure F10T). The interaction of four chain body c-kit) can form a mole ratio 1:1 complex by combining the trench pattern with the human telomere G- four chain body. The target G- four chain body and the extraction of the Magnolia A from the she medicine Magnolia Magnolia have the potential for the anti-tumor effect of the target G- four chain body. The research result of this paper is the traditional national medicine target to the G- four chain body. The discovery of tumor active compounds provides the material basis for the rapid screening of anti tumor active small molecule compounds targeting the G- four chain body by the national medicinal plant. The research method established in this paper can be applied to the two development of traditional national drugs and promote the modern utilization of national medicine. It will bring economic benefits to the minority people and better promote the economic and social development of our national regions.

【学位授予单位】：中央民族大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R29

【参考文献】