【作者】 李兴；

【导师】 史清文；

【作者基本信息】 河北医科大学 ， 药物化学， 2008， 硕士

【摘要】 蟛蜞菊(Wedelia chinensis),异名马兰草、蟛蜞花、路边菊,是菊科蟛蜞菊属植物蟛蜞菊的全草。主要分布于中国南部的福建、广东、广西等省[1]。蟛蜞菊性甘、微酸、凉,具有清热解毒、化痰止咳、凉血平肝、祛瘀消肿等功效[2],主要用于治疗白喉、百日咳、痢疾、痔疮、跌打损伤。南方各省民间常用于治疗咽喉炎、喉痹、喉蛾等,还可用于预防麻疹。现临床上用于治疗肝炎、口腔炎等,效果较好[3]。但目前对该植物化学成份的研究还并不深入,研究表明已从中分离出了有机酸类、倍半萜类、三萜类、三萜苷类、黄酮类和甾类等化合物[4]。文献还报道蟛蜞菊化学成份具有广泛的药理作用,如保肝作用、解热镇痛作用和抗炎作用,但报道的多为提部位的药理活性,而其作用的物质基础尚未明确。目的:本研究旨在以蟛蜞菊(地上部分)为研究对象,运用硅胶柱色谱、聚酰胺柱色谱、葡聚糖凝胶色谱、制备薄层色谱、高效液相制备色谱以及重结晶等分离纯化技术对其地上部分的提取物进行系统研究,从中分离得到单体化合物,并应用UV、IR、MS、1H-NMR、13C-NMR、1H-1H COSY、HMQC、HMBC、NOESY等现代化手段完成单体化合物分子的平面结构和立体结构的鉴定,再对分离得到的单体化合物进行初步的抗肿瘤活性实验,以期寻找到其活性成分或活性部位,以开辟中药资源,丰富中药的多样性和完善用药的合理性。方法:干燥的蟛蜞菊属植物蟛蜞菊地上部分(3.0 Kg)适当粉碎后,用95%乙醇连续回流提取3-4次,趁热活性炭脱色、过滤,合并滤液减压浓缩至粘膏状(600 g),即得到蟛蜞菊地上部分的提取物;将其溶于盐水中(约相当于1 g/mL的水溶液,低温水浴加热助溶),形成悬浊液,依次用石油醚、二氯甲烷、水饱和的醋酸乙酯萃取,继而得到石油醚部位(24.5 g)、二氯甲烷部位(15.0 g)和醋酸乙酯部位(13.0 g)。本研究先后对石油醚部位、二氯甲烷部位和醋酸乙酯部位采用硅胶柱色谱进行初步分离,析出的固体和洗脱液浓缩母液,经薄层色谱检识后(薄层色谱检识板:由GF254薄层色谱硅胶与0.5% CMC-Na混匀后铺制于玻璃板上,自然晾干而成。样品点样于该类薄板上,晾干,适当展开剂展开,挥尽展开剂后先在254 nm紫外灯下观察是否有荧光,再浸入10%的硫酸-乙醇液中,取出后经140℃加热显色),进一步用硅胶柱色谱、聚酰胺柱色谱、葡聚糖凝胶色谱、制备薄层色谱、高效液相制备色谱反复分离纯化得到单体化合物。并运用化学方法和多种波谱技术(UV, 1H-NMR, 13C-NMR, 1H-1H-COSY, HMQC, HMBC等)对这些单体化合物进行检测,依据测定的图谱数据分析确定单体化合物的平面结构和立体结构。再对得到的单体化合物进行抗肿瘤活性的初步研究。将HeLa等肿瘤细胞株悬液接种于96孔培养板,分别加入不同浓度单体化合物,培养箱中培养2天,于培养结束前4小时,各培养孔加入5 mg/ml四氮唑盐(MTT) 10μl,培养结束后,用酶联免疫检测仪检测各孔吸光度(OD)值,测定波长λ= 570 nm,参考波长λ= 630 nm,并计算肿瘤细胞生存率。结果:对蟛蜞菊属植物蟛蜞菊地上部分的化学成分进行了系统研究,具体采用了硅胶柱色谱、聚酰胺柱色谱、葡聚糖凝胶色谱、制备薄层色谱、高效液相色谱等多种分离手段或方法,从中得到13个单体化合物,并运用UV、1H-NMR、13C-NMR、1H-1H COSY、HMQC、HMBC等现代化手段对其进行结构鉴定,已确定了4个化合物的结构,该4个化合物的结构名称分别为:化合物D为24-甲叉基-9,19-环木菠萝烷醇;化合物F为β-谷甾醇;化合物H为齐墩果酸;化合物K为齐墩果酸-11,13(18)-二烯-3-O-b-D-葡萄糖醛酸甲酯。其它9个化合物的结构还在鉴定中,它们分别是:化合物A, B, C, E, G, I, J, L, M。对其中6个化合物(compound A, D, H, I, J, M)进行了抗肿瘤活性筛选,结果显示:化合物A、I和M对妇科肿瘤细胞,尤其对人子宫内膜癌细胞增殖有较强的抑制活性;化合物A和M对人脑神经胶质瘤细胞显示强的抑制活性;化合物H对人子宫颈癌细胞、人卵巢囊腺癌细胞和人黑色素瘤细胞的增殖有较好的抑制活性。结论:通过系统提取分离植物蟛蜞菊(Wedelia chinensis)地上部分,共分离得到13个化合物,并且运用了多种现代波谱手段及其相关知识对该13个化合物进行了结构鉴定,已确定了4个化合物的结构,其它9个化合物的结构还在鉴定中,其中24-甲叉基-9,19-环木菠萝烷醇(化合物D)和齐墩果酸(化合物H)为首次从该属植物中分离得到。对其中6个化合物进行了活性筛选发现有4个化合物具有显著抗肿瘤活性,其中化合物A、I和M对人的妇科肿瘤细胞,尤其对人子宫内膜癌细胞增殖有较强的抑制活性;化合物A和M对人脑神经胶质瘤细胞显示强的抑制活性;化合物H对人子宫颈癌细胞、人卵巢囊腺癌细胞和人黑色素瘤细胞的增殖有较好的抑制活性。更多还原

【Abstract】 Wedelia chinensis is one of the genus Wedelia plant belong to Compositae, which mainly distributed in south China, including Fujian, Guangdong and Guangxi [1]. It is used for the treatment of diphtheria, chincough, diarrhea, hemorrhoids, injuries from falls, faucitis, measles as a folk herbal medicine [2]. Now it is preferably used for the treatment of hepatitis and stomatitis in clinic [3]. But a few phytochemical investigation of W. chinensis has been reported, and recent studies showed that organic acids, sesquiterpenes, triterpenoids, triterponoid saponins, flavonoids and steroids were isolated from the plants of this genus [4]. Extensive bioactivitie studies have shown that crude extract of Wedelia plants exhibited hepatoprotective effects, antipyretic-analgesic effects and antibacterial effects, and the mechanism of action of pure compounds is waiting to be investigated.Object: To develop the natural resources, search for its active components, extend the clinical effects and try to find out a new drug from Traditional Chinese Medicine. We studied the components in the air-dried aerial parts of Wedelia chinensis with Silica gel column chromatography to fractionate the crude extracts; with preparative pre-TLC and pre-HPLC to separate and purify integrates and to get compounds in pure form. Using spectroscopic methods including UV, IR, HR-FAB-MS, 1H-NMR, 13C-NMR, 1H-1H-COSY, HMQC, HMBC and NOESY to establish the structures of the isolated pure compounds. The pure compounds are tested for antitumor activity against several human tumor cell lines.Methods: The air-dried aerial parts of Wedelia chinensis (3.0 Kg) were pulverized and extracted with 95% alcohol for three or four times, filtered, and eliminated chlorophyll with activated carbon. The alcohol extract was concentrated to yield the total crud extract (600 g). The total crud extract was suspended in salt water (1:1) and re-extracted with petroleum ether, dichloromethane and ethyl acetate in order. Accordingly three sections were obtained: the petroleum ether section (24.5 g), the dichloromethane section (15.0 g), and the ethyl acetate section (13.0 g). Each section was applied to silica gel column chromatography for preliminary fractionation, and every fraction eluted from column chromatography was monitored with TLC, the developed TLC was detected by UV light at 254 nm and followed by color reaction with spraying 10% H2SO4-ethanol reagent and heating at 140℃. Similar fractions were pooled and subjected to silica gel column chromatography, polyamide column chromatography and sephadex LH-20 column chromatography. Combined some similar subfractions and applied to preparative TLC and/or reversed phase preparative HPLC for further purification at a flow rate of 3 mL/min. At last, pure compounds were obtained and the chemical and spectroscopic methods (UV, 1H-NMR, 13C-NMR, 1H-1H-COSY, HMQC, HMBC) were used for their identification of chemical structure.Preliminary antitumor activitys are test against several human tumor cell lines. Logarithmically proliferating cells were plated into 96-well plates (1×104 cells/well) with the medium containing the test compounds at the indicated doses, followed by being cultured for 2 days. After the culture, the activity of mitochondrial succinic dehydrogenase was measured by further incubation of the cells with 5 mg/ml MTT (Sigma) for 4 h, followed by estimation of absorbance at 570 nm with a reference wavelength of 655 nm. Cell viability was calculated by absorbance as a percentage of the survival cells.Result: Column chromatography, preparative TLC and preparative HPLC were employed to isolate and purify the components in the ethanol extract of Wedelia chinensis, and 13 compounds were isolated from above plant. Variety of spectroscopic methods was used to elucidate the structures of the compounds. Among them, the structures of four compounds were identified on the basis of chemical and spectral analysis as: compound D 24-methylene-9,19-cycloartanol; compound Fβ-sitosterol; compound H oleanolic acid; compound K Oleanolic acid-11,13(18)-dien-3-O-β-D-glucuronopyranosyl methylester. Remain 9 compounds are undergoing spectral elucidation. Antitumor activity of six pure compounds were tested, and the result showed that compounds A, I, and M were active against gynecological tumor cell lines, especially endometrial adenocarcinoma cell line (HEC-1); Compounds A and M also exhibited strong inhibitory effect against glioma cell line (T-98); Compound H showed moderate antibacterial activity against cervical cancer cell line (HeLa), Ovarian clear-cell carcinoma cell line (HAC-2) and melanoma cell line (HMV-1).Conclusion: Separation of extract of the air-dried aerial parts of Wedelia chinensis by all kinds of methods including chromatography, preparative TLC and/or reversed phase preparative HPLC yielded 13 compounds. Among them 24-methylene-9,19-cycloartanol (compound D) and oleanolic acid (compound H) are isolated from genus Wedelia for the first time. Activity screening showed that compounds A, I, and M were active against gynecological tumor cell lines, especially to HEC-1; Compounds A and M also exhibited strong inhibitory effect against T-98; Compound H showed moderate antibacterial activity against HeLa, HAC-2 and HMV-1.更多还原