【作者】 刘新；

【导师】 王长云；

【作者基本信息】 中国海洋大学 ， 药物化学， 2009， 博士

【摘要】 中药复方是中医用药的主要形式,是基于辩证观点,按照君臣佐使的组方原则选择药物定量配伍而成,是中医辩证论治理论的具体体现。由于复方化学成分和药效作用的复杂性,以及有效成分对机体的多靶点协同作用,使复方效应物质基础研究十分困难。如何对中药复方药效成分及其药效作用机理做出科学的诠释,是本领域亟待解决的重要学术问题。本研究选择临床有效的抗肿瘤复方海藻制剂,采用整方研究策略,从多成分、多靶点、多途径阐释复方的药效物质基础,初步探讨了复方的抗肿瘤作用机制。中药复方海藻制剂是由海藻、黄芪、女贞子等传统中药,依据中医药组方原理配伍而成的抗肿瘤复方制剂。本研究采用整方研究策略,以体外免疫调节、细胞毒、抗氧化等抗肿瘤相关的活性为指导,运用色谱分离技术(硅胶柱层析、凝胶柱层析、制备薄层色谱和制备高效液相色谱等技术)和波谱分析技术(1H NMR, 13C NMR, 2D NMR, MS, IR, UV等技术),从复方水煎液的乙酸乙酯相和正丁醇相中分离得到60个单体化合物,鉴定了49个化合物的结构,其中2个为新化合物,1个为新天然产物,包括黄酮苷类、黄酮类、酚类、木脂素类、甾体类、萜类、内酯类等近10种结构类型。运用GC/MS分析技术从石油醚相中鉴定了15个脂肪酸类化合物,6个倍半萜及甾体类等化合物。从水相中分离获得以葡萄糖为主构成的杂多糖,运用GC分析技术鉴定出单糖组成主要为葡萄糖,并有少量的鼠李糖、岩藻糖、甘露糖及半乳糖。以与肿瘤密切相关的多个靶点,分别在细胞水平(细胞增殖,细胞毒)和分子水平(细胞因子IL-2, IL-4, INF-γ)上,探讨了复方化学成分的抗肿瘤作用。发现,复方海藻制剂是一个化学成分复杂的有机整体,通过多种途径产生抗肿瘤作用。黄酮苷类、黄酮类、酚类和多糖类成分是复方抗肿瘤作用的有效分子群。黄酮及其苷类化合物具有显著的免疫激活作用;黄酮及其苷类以及咖啡酸衍生物还显示强抗氧化作用;多糖类化合物则表现出对免疫细胞功能起调节和促进作用。这4类有效分子群相互配合,协同作用,加上复方其他化学成分的辅助作用(如脂肪酸类及倍半萜类化合物弱的细胞毒作用),体现综合效力,从而发挥整体复方的多成分、多靶点、多途径的抗肿瘤作用。为追踪复方化合物的来源,从君药海藻海蒿子Sargassum pallidum (Turn.) C. Ag.中分离得到了22个单体化合物,鉴定了18个化合物的结构,发现了与复方化合物结构相似的黄酮类及甾体类化合物。从臣药女贞子中分离鉴定了7个化合物,发现复方酚苷类化合物β-D-glucopyranoside, 2-(4-hydroxyphenyl), 6-acetate (44)来源于女贞子,不是复方合煎的产物;推测复方内酯类化合物(3-Ethylidene-2-oxo-tetrahydro-pyran-4-yl)-acetic acid methyl ester (42)和[5-(1-Hydroxyl-ethyl)-4-hydroxymethylene-2-oxo-tetrahydro-pyran-3-yl]-acetic acid methyl ester (43)可能是女贞子中的裂环环烯醚萜类化合物在复方煎煮过程中分解产生的。本研究的创新之处:采用整方研究策略,对化学成分复杂、药理作用多样的复方药效物质基础进行了探索性研究;根据复方化合物结构类型和生物活性的特点,提出复方多成分、多靶点、多途径的有效分子群协同作用模式。本研究结果为复方海藻制剂临床应用及其现代化研究开发提供了基础资料,对中药复方药效物质基础研究具有启迪和借鉴意义。更多还原

【Abstract】 Tradititonal Chinese medicine preparation (TCMP) was a main form for Chinese medical practice. TCMP was composed according to the compatibility theory and dialectical view. It was very difficult to study on the pharmacodynamic material bases of TCMP due to the complexity of chemical constituents and pharmacodynamic effects. It is significant to elucidate the effective constituents and function mechanisms of TCMP. In this paper, the pharmacodynamic material bases of Seaweed Complex Preparation (SCP), a clinical traditional Chinese medicine preparation with antitumor effects, was investigated by a“whole prescription”research strategy in a view of multicomponents, multitargets and mutiapproaches.SCP was mainly composed by several traditional Chinese herbs, such as seaweed Sargassum pallidum (Turn.) C. Ag., the radix of Astragalus membranaceus (Fisch.) Bge. and the fruits of Ligustrum lucidum Ait. As a clinical preparation, it has been used to treat lung cancer, liver cancer and digestive cancer. The pharmacodynamic material bases of SCP was studied by the“whole prescription”method under the guidance of bioactivities of immunomodulatory, cytotoxic, antioxidant and antibacterial activities in vitro. More than 60 compounds were isolated from the ethyl acetate fraction and n-BuOH fraction of the preparation by chromatography on silica gel, Sephadex LH-20, PTLC and PHPLC. Structures of 49 compounds including 2 novel compounds and 1 new natural product were identified by spectroscopy such as 1H NMR, 13C NMR, 2D NMR, MS, IR and UV. More than 10 structure types were found, such as flavonoid glycoside, flavonoid, phenolic compound, lignan, steroid, terpene and lactone. The petroleum ether fraction was analyzed by GC/MS resulting in the identification of 15 acyclic compounds, 6 other chemical constituents including terpenoid and sterol. The polysaccharide isolated from the water fraction was mainly composed of glucose with a small quantity of rhamnose, mannose, fructose and galactose.The antitumor effects of the chemical constituents of SCP was investgated at cell level for cell proliferation inhibition and cytotoxicity (cell lines P388, BEL7402, KB) and molecule level for immunomodulatory activites (cytokines IL-2, IL-4 and INF-γ). It was found that SCP was an organic unity of complicated chemical constituents with multiple antitumor approaches. Four molecular communities including flavonoid glycosides, flavonoids, phenolic compounds and polysaccharides could be involved in antitumor effects. Flavonoid glycosides and flavonoids exhibited immunomodulatory activites. Flavonoid glycosides and caffeic acid derivatives showed remarkable antioxidant activities. And polysaccharides showed immunological enhancement effects. Synergism of these four molecular communities together with auxiliary effect of other components (such as the weak cytotoxic activities of sesquiterpenes and lipids) embodied the muticompenent and mutitarget features of SCP.The chemical constituents of monarch drug S. pallidum (Turn.) C. Ag. were investgated in order to trace the origin of the compounds obtained from SCP. More than 22 compounds were obtained from the whole plant. Structures of 18 compounds were identified including dipeptides, coumarins, flavonoids and sterols, among which several flavonoids and sterols were the same or similar to that of SCP. The chemical constituents of the fruits of L. lucidum Ait. was also studied to investigate the origin ofβ-D-glucopyranoside, 2-(4-hydroxyphenyl), 6-acetate (44). It was indicated that this compound existed in L. lucidum Ait.. The possible producing approaches of (3-Ethylidene-2-oxo-tetrahydro-pyran-4-yl)-acetic acid methyl ester (42) and [5-(1-Hydroxyl-ethyl)-4-hydroxymethylene- 2-oxo-tetrahydro-pyran-3-yl]-acetic acid methyl ester (43) were deduced as products generated from decoction of the preparation.The acting-pattern of effective molecular communites with muticomponents, mutitargets and mutiapproaches was proposed by using“whole prescription”strategy. The results obtained from our research should provid elemental information for clinical application and further development of SCP. And the strategy and methods used in our experiments should also have an enlightenment and reference significance to the research on pharmacodynamic material bases of traditional Chinese medicine prescription.更多还原