【作者】 王慧；

【导师】 董小萍；

【作者基本信息】 成都中医药大学 ， 中药学， 2008， 博士

【摘要】 蒲葵子为棕榈科植物蒲葵Livistona chinensis R.Br.的干燥成熟果实。性味甘、淡、涩,平,具有软坚散结、收敛止血之功效,适用于癥瘕结气,崩漏,跌打损伤,眼底出血等症。现代研究表明,蒲葵子具有抗癌之功效,民间广泛用于治疗各种癌症,如食道癌、鼻咽癌、恶性葡萄胎、白血病、绒毛膜上皮癌、肺癌。但对蒲葵子有效部位药理研究及化学成分研究未见有公开的报道。为了进一步合理开发利用蒲葵子药材,寻找其有效成分,揭示其抗肿瘤作用物质基础,并为药材质量标准的制定提供科学依据,本课题以蒲葵子为研究对象,对其药效物质基础进行了系统研究。化学成分预试验结果表明蒲葵子中可能含有酚类、蒽醌类、香豆素、甾体三萜类、皂苷类、氨基酸类等成分。本课题针对蒲葵子的功效及临床应用,选择适宜的肿瘤细胞株对蒲葵子进行了体外抗肿瘤活性部位初步筛选。首先对蒲葵子进行部位分离,对其石油醚提取物、乙酸乙酯提取物、正丁醇提取物和水提取物,采用MTT法测定了它们各自对人慢性粒细胞白血病细胞株K562、人卵巢癌细胞株SKOV3、人结肠癌细胞株HT-29、人膀胱癌细胞株T24等四种瘤株的生长抑制情况;采用ELISA法检测了它们各自对肿瘤细胞分泌血管内皮生长因子（VEGF）水平的影响;以活性部位作用于VEGF诱导后的人脐静脉内皮细胞株（HUVEC）,采用RT-PCR和Western blot方法分析内皮细胞Flk-1 mRNA和蛋白的表达变化。结果表明,蒲葵子乙酸乙酯提取物可选择性地抑制结肠癌细胞株HT-29和膀胱癌细胞株T24肿瘤细胞的生长,其抑制率可分别达74.66%和86.52%,并可显著降低各肿瘤细胞株分泌VEGF的水平。石油醚提取物对HT-29和T24生长也有一定的抑制作用,其最高抑制率分别为43.80%和38.67%,并可降低各肿瘤细胞分泌VEGF水平,但作用强度均不及乙酸乙酯部位。水和正丁醇提取物未表现抗肿瘤活性。对VEGF诱导内皮细胞表达Flk-1 mRNA和蛋白,乙酸乙酯提取物表现出明显的抑制作用。药理研究结果表明蒲葵子乙酸乙酯提取物有较好的抗肿瘤作用,是抗肿瘤作用的主要活性部位,石油醚提取物也显示一定的活性。对体外抗肿瘤试验筛选出的活性部位,采用柱色谱方法进一步进行系统的化学成分分离和纯化,运用熔点测定、UV、IR、MS、¹H-NMR、¹³C-NMR等方法进行结构鉴定。从蒲葵子乙酸乙酯部位分离得到10个化合物,鉴定了其中5个化合物,分别为:（-）表缅茄儿茶精（（-）-epiafzelechin,Ⅰ）、β-胡萝卜苷（daucosterol,Ⅱ）、β-谷甾醇（β-sitosterol,Ⅲ）、1,6,8-三羟基-3-甲基蒽醌（emodin,Ⅵ）、5,7,4’三羟基-3’,5’-二甲氧基黄酮（tricin,Ⅶ）,其中1,6,8-三羟基-3-甲基蒽醌为首次从该植物中分得。采用GC-MS分析鉴定蒲葵子的石油醚提取部位脂肪酸成分,鉴定出其中11个化合物,占总脂肪酸的94.54%,其中棕榈酸（37.10%）、亚油酸（27.90%）、油酸（20.96%）相对含量较高,不饱和脂肪酸相对含量达到52.28%,并含有ω-3多不饱和脂肪酸:α-亚麻酸、二十碳五烯酸。本课题首次对蒲葵子药材进行了质量控制的研究。建立了小麦黄素对照品的标准;采用高效液相色谱法测定了蒲葵子药材中小麦黄素的含量;并对蒲葵子药材进行了指纹图谱的研究,研究结果表明,该方法稳定、可靠、重现性好,10批药材得到的色谱指纹图谱共标定了31个共有峰,其共有峰的相对保留时间基本一致,不同产地批次蒲葵子药材化学组成相似,其相对比例较稳定。综上所述,本课题首次确定了蒲葵子的乙酸乙酯提取物是其体外抗肿瘤作用的主要活性部位,石油醚提取物也有一定的活性。从乙酸乙酯提取物中分离鉴定了5个化合物,从石油醚提取物中鉴定了11个化合物。同时对蒲葵子药材进行了指纹图谱研究。本课题为最终揭示蒲葵子抗肿瘤作用及其物质基础,建立药材质量的科学评价方法,实现药材可持续开发利用及抗肿瘤中药新药开发提供了科学依据。更多还原

【Abstract】 Fructus Livistonae Chinensis is the dried ripe fruit of Livistona chinensis R.Br.It is sweet,tasteless,astringent in flavour and mild in nature.Its actions include softening and dissolving hard masses,stopping bleeding by astringency.It can be used for abdominal lumps,stagnation of qi,metrorrhagia,Traumatic injury,subhyaloid hemorrhage et al. Modern research had delicated that Fructus Livistonae Chinensis possessed the effect of anti-tumor,and widely used for the treatment of cancer in the folk,such as esophagus cancer,nasopharyngeal cancer,chorioadenoma,leukaemia,choriocarcinoma,lung cancer. But researches on the effective part about chemical constituents and pharmacology haven’t been reported.In order to obtain a more comprehensive understanding of its effective physiological constituents,reveal the substantial foundation of anti-tumor effect and provide scientific basis of producing medicine reasonably,a phytochemical study has been carried out.The fore-experiments of its chemical components showed that it contained phenols, anthraquinones,coumarin,steroids,triterpenoids,saponin,amino acids,et al.The 70%alcoholic extracts obtained from the dried fruits were divided into four portions including petroleum ether extracts,ethyl acetate extracts,n-Butyl alcohel extracts and water extracts for the first time.MTT assay and ELISA were used to detect the proliferation and the secretion of vascular endothelial growth factor（VEGF） of chronic myelogenous leukemia K562,ovarian neoplasm SKOV3,colon carcinoma HT-29 and bladder cancer T24 cell lines respectively.The expression of fetal liver kinase（Flk-1） protein and mRNA in endothelial cells induced by VEGF was analyzed by Western blotting and RT-PCR.The results showed that the ethyl acetate part could selectively inhibit the proliferation of HT-29 and T24 cell lines in a dose-dependent manner（the maximum inhibitory rate were 74.66%and 86.52%）,and could significantly inhibit the secretion of VEGF protein of the four tumor cell lines.The petroleum ether part had similar inhibitory effect as well as the ethyl acetate part,but its potency is inferior to the ethyl acetate part（the maximum inhibitory rate were 43.80%and 38.67%）,The water-soluble and n-butanol parts had no effect on different tumor cell lines.The ethyl acetate part could inhibit significantly the expression of Flk-1 protein and mRNA of HUVEC induced by VEGF.The ethyl acetate part has fine anti-tumor activity,and its mechanism might be associated with reducing VEGF protein secretion and inhibiting the expression of Flk-1 mRNA and protein.Effective parts were seperated and purified by chromatographic methods. Compounds were identified by determining melting point,UV,IR,MS,¹H-NMR, ¹³C-NMR,et al.10 compounds had been isolated from ethyl acetate extract of Fructus Livistonae Chinensis,and 5 compounds were identified:（-） -epiafzelechin（Ⅰ）, daucosterol（Ⅱ）,β-sitosterol（Ⅲ）,emodin（Ⅵ）,tricin（Ⅶ）.The compoundⅥwere obtained from the plant for the first time.For the first time,we analyzed the chemical constituents of petroleum ether extracts of Fructus Livistonae Chinensis and the constituents were separated and identified by GC-MS.The results showed that 11 compounds were identified. Unsaturated fatty acids constituted 52.28%of the total fatty acids,which includeω-3 Polyunsaturated fatty acid:α-linolenic acid,EPA.For the first time,the content of tricin in Fructus Livistonae Chinensis was determined by HPLC.The results showed the HPLC method was sentives and specifive. Finger printing research on Fructus Livistonae Chinensis was conducted.The results showed that 31 peaks could denote the fundamental substance of Fructus Livistonae Chinensis,the research method was advanced and feasible,which indicated the finger printing is somehow meanmingful as a measure of quality control when the chemical components are unknown.The research provided the scientific fundation to reveal the substantial foundation of anti-tumor effect,establish scientific evaluating method of quality control,achieve medicine material continuously reasonable exploitation,exploit antitumor new medicine of Chinese herbal medicine.更多还原