【作者】 任彦荣；

【导师】 陈国平；

【作者基本信息】 重庆大学 ， 生物医学工程， 2008， 博士

【摘要】 青蒿素是举世闻名的抗疟药,虽然临床应用多年,但仍存在一些缺点,尤其水溶性和脂溶性均很低,造成生物利用率低、复燃率高。目前青蒿素及其衍生物的抗肿瘤活性成为研究的热点,有希望被开发成为新型植物广谱抗癌药,同样存在溶解性问题。因此,开展青蒿素的修饰及抗肿瘤活性的研究对其综合利用具有重要的意义。为了改善青蒿素的水溶性,提高其抗肿瘤活性,本研究通过化学制药和生物制药方法对中药单体青蒿素进行糖基化修饰,并进行结构表征和抗肿瘤活性研究,最后对现有的青蒿素类药物构效关系进行探讨,以对后续研究工作提供指导。主要研究内容和结论如下:①化学法合成糖基化青蒿素衍生物及体外抗肿瘤活性研究1)首次采用溴代乙酰化一锅法对青蒿素进行糖基化修饰,产物经1H NMR、13C-NMR、LC-MS和IR等方法表征,获得几个新的糖类修饰的青蒿素衍生物。该方法与曾报道的方法相比,反应条件相对温和、操作简便,不需要额外的脱保护步骤。2)水溶性实验表明,青蒿素经糖基修饰后,水溶性均得到提高,有利于药物的吸收和生物利用率的提高。3)MTT法对正常细胞及肿瘤细胞增殖抑制活性实验表明,糖基修饰的青蒿素对正常细胞的增殖抑制降低,而抑瘤作用没有降低或者提高,其中青蒿素葡萄糖苷对Hela细胞作用最好。4)采用倒置显微镜和透射电镜手段直观观察到青蒿素葡萄糖苷作用Hela细胞后,细胞形态发生变化,细胞数量减少,能诱导Hela细胞产生凋亡。5)利用流式细胞仪和western印迹探讨了青蒿素葡萄糖苷诱导Hela细胞凋亡的机制,可能是通过上调bax基因的表达,下调bcl-2基因的表达,来促进线粒体细胞色素C的释放,从而达到上调caspase-3基因,激活caspases系统的级联效应,最终导致Hela细胞凋亡,凋亡机制为非p53基因依赖途径。②酶催化合成糖基化青蒿素衍生物及体外抗肿瘤活性研究1)在国内外首次通过酶催化方法对青蒿素进行糖基化修饰,其中以半乳糖作为给体效率最高,达到68%。该方法与化学方法相比,具有产物效率高、底物专一性强等特点。2)对酶催化法的反应条件进行摸索和优化,得到最优化的操作条件。3)鉴于酶催化法的特点,将半乳糖基化青蒿素处理后首先借助生物分析手段进行结构分析。采用Tricine-SDS-PAGE电泳从分子量角度,确定了取代糖基数目;通过圆二色谱检测取代位置为青蒿素羟基位置,非内酯环,糖苷键的构型为β型;通过荧光分析,进一步确定糖基连接到青蒿素分子上。其次,采用红外、核磁光谱等化学分析手段对结构进行确证。最后,采用ArgusLab软件模拟了糖基化青蒿素分子的立体结构。4)MTT法对正常细胞及肿瘤细胞Hela实验表明,半乳糖基化青蒿素对正常细胞生长抑制降低,抑瘤能力增强。Hoechst33342/PI细胞核染色进一步说明半乳糖基化青蒿素能诱导肿瘤细胞的细胞核凋亡。利用流式细胞仪和western印迹探讨了半乳糖基化青蒿素诱导细胞凋亡的机制,与化学方法制备的产物机制相似。③糖基化青蒿素体内抗肿瘤活性研究成功建立了小鼠宫颈癌模型,对体外筛选得到的糖基化青蒿素进行初步的体内抗肿瘤活性研究。结果表明,与生理盐水对照组相比,青蒿素及其糖基化衍生物均可有效抑制肿瘤的生长和延长小鼠寿命,毒性实验表明属于低毒类物质。与青蒿素组相比,糖基修饰产物各个指标均有提高,以酶催化法制备的半乳糖-青蒿素效果最好。④从分子对接和三维定量构效关系角度对青蒿素及其衍生物的生物活性进行了初步的探讨,通过理论模型研究了非键效应(静电、立体和疏水)对Fe2+介导青蒿素过氧桥断裂及其抗疟活性的影响。1)基于Autodock对青蒿素与血红素作用模式进行调查,寻找潜在的活性构象。2)将有机分子中常见的原子按基本的化学性质进行分类,通过计算不同种类原子之间的非键作用模型,得到用于描述分子的三维场描述子,即三维全息作用矢量(3D-HoVAIF)。3)在青蒿素活性构象基础上搭建一系列具有药效活性的青蒿素衍生物空间构象,并使用3D-HoVAIF对这些分子结构信息进行表征。4)采用遗传-偏最小二乘回归(GA-PLS)方法建立青蒿素及其衍生物的3D-HoVAIF描述子与其生物活性之间隐含关系的线性模型,并使用内部和外部验证的方法确认了模型的统计意义。该模型具有稳定性和预测能力。5)通过统计模型对青蒿素及其衍生物的生物活性进行分析和解释,此举为青蒿素结构修饰和改造提供了有益的帮助。总之,通过研究发现糖基修饰青蒿素能提高其水溶性、降低毒副作用,并且某些糖修饰后其抗肿瘤活性得到提高。这些研究将对青蒿素的综合利用具有实际价值,为青蒿素的修饰提供新的思路,也为其他药物的糖基修饰提供借鉴和参考。更多还原

【Abstract】 Artemisinin, serving as antimalarial, is generally accepted in the world. However, there are still some defects, e.g. low water-solubility and liposolubility that leads to low bioavailability and high recrudescence rate. The anticancer activites of artemisinin and its derivates may become the hot of research, and it is potent to develop a new broad-spectrum anticancer drug. Therefore, artemisinin modification and exploration of antineoplastic activity are extremely meaningful to its multiple utilizations.This project proposed pro-drug’s glycosylation modification, traditional Chinese herb monomer artemisinin was glycosylated, then performed structural characterization and investigation of anticancer activites, finally exploring structure-activity relationship for presently available artemisinin analogues. Main woks are presented as following:①Synthesizing glycosylated artemisinin by chemical methods, and studying on anti-tumor activites of the compound in vitro.1)Preparing a few new sugar-residue products of artemisinin by acetylated protection one-pot.Measuring and analyzing products in 1H NMR, 13C-NMR ,LC-MS and IR methods, and ascertaining the target products. Compared this synthetic methods and methods previously reported, the methods had relatively mild reaction conditions, which was not only simpler, but also needn’t extra deprotection steps.2)It showed that the water-solubility was enhanced and profited its absorption and raising the utilization rate of biology.3)MTT test showed that glycosylation artemisinin derivates had larger ability of inhibiting growth in cancer cells and lowered toxicity in normal cells. In particular, artemisinin-glucoside had the best activity on Hela cells.4)Directly observing the effection of artemisinin-glucoside on Hela cells by inverted microscope and transmission electron microscope, which the cells shape changed and the number of cells reduced. It showed that artemisinin-glucoside can induce apoptosis of Hela cells.5)Detected apoptosis by flow cytometry and western blotting for approaching mechanism of artemisinin-glucoside, which could be up-regulated bax gene and down -regulated bcl-2 gene, so as to release the cyc.Therefore, it can increase the expression caspase-3 gene and activate the caspases cascading effect, so resulted the apoptosis, the mechanisms on Hela cells was non-p53 gene way. ②Synthesizing glycosylated artemisinin by enzyme catalysis methods, and then studying on activites of anti-cancer in vitro.1)Modifying artemisinin with sugar-residue by enzyme catalysis methods at the first time in the world, and found that galactose had the maximum of efficiency, which can up to 68%. Compared enzyme catalysis and chemical methods, it was found that enzyme catalysis reaction had the characteristics of higher efficiency and stronger specific.2)Groping and optimizing the reaction condition of glycosylation by enzyme catalysis, and then obtaining the best operating conditions.3)Firstly, respecting the feature of enzyme catalysis, analyzing structure of galactosylated artemisinin with bioanalysis methods after treating it.Determining the number of sugar substitute in galactosylated artemisinin by Tricine-SDS-PAGE electrophoresis from the perspective of molecular weight. Detecting the artemisinin hydroxy position by Circular Dichroism, which showed that the position was replaced at hydroxide radical. The glucosidic bond isβ-type. Fluorescence method confirmed further that the artemisinin-residue had sugar molecule. Then confirming the structure by NMR and IR methods. At last, simulating the stereochemical structure of glycosylation artemisinin by Arguslab soft.4)MTT test showed that the galactosylated artemisinin had larger ability of inhibiting growth in cancer cells and lowered toxicity in normal cells. Moreover, it was demonstrated that galactosylated artemisinin can induce apoptosis in cancer cells by Hoecst33342\PI stain. Approaching the apoptosis mechanism by flow cytometry and western blotting, it was similar to that of the product by chemical methods.③Studying on anti-tumor activites of glycosylated artemisinin in vivo. Establishing mouse cancer of the cervix model,then studying on anti-tumor activites of glycosylated artemisinin in vivo. Compared with normal sodium, the results showed artemisinin and its glycosylated derivates can suppress growth of tumor and increase in life span of mouse.The products belonged to low-toxicity materials.Otherwise, compared with artemisinin, each index of glycosylated products raised, galactose- artemisinin by enzyme catalysis methods was best.④Base on molecular docking and three-dimensional structure-activity relationship, bioactivities of artemisinin and analogues were primarily explored, and via theoretical modeling, nonbonding effects (referring to electrostatic, steric and hydrophobic interactions) had been investigated for their influences on Fe2+-mediated artemisinin peroxy-bridge breakage and antimalarial activity.1)With Autodock, artemisinin-haemoglobin interaction modes were investigated, expecting to find potential active conformations.2)Common atoms in organic molecules were classified in terms of their fundamental chemical properties, and three-dimensional field descriptor (referring to three-dimensional holographic vector, 3D-HoVAIF) was yielded via calculating nonbonding interactions among different types of atoms.3)Based upon the active conformation of artemisinin, spatial conformations for a series of derivatives of pharmacological activities were constructed, with their structures characterized by 3D-HoVAIF.4)Genetic algorithm-partial least square (GA-PLS) was employed to build up relationship between 3D-HoVAIF descriptors and the bioactivities, and meanwhile internal-external dual-testes were used to confirm statistical significance of SAR models. This model possessed stability and ability of forecasting.5) That analyzing and interpreting bioactivities of artemisinin and analogues by the statistical model benefits structural modification of artemisinin.In sum, artemisinin modified with glycon can enhance water solubility, lower the toxicity. Moreover, some glycon modified artemisinin can raise the activity of anticancer. These works not only has actual value to artemisinin multiple utilizations, but also provide new path for artemisinin modification and reference for other drugs modified with glycon.更多还原