壮药棵矮瓤乙酸乙酯部位化学成分研究及抗肝癌活性初探

发布时间：2018-08-08 16:22

【摘要】：目的:壮药棵矮瓤系菊科植物馥芳艾纳香Blumea aromatica(Wall.)DC.的干燥全草,别名黄药子、山风、香艾、大开门、艾纳香等,具有活血止痒、祛风消肿的功效,主要用于湿疹,皮肤瘙痒,风湿性关节炎等症。壮药棵矮瓤的化学成分主要有黄酮类、挥发油类、萜类、甾体类化合物等,目前只有少量文献对其化学成分和药理活性进行研究和报道。课题组前期对壮药棵矮瓤组分进行了活性筛选,发现乙酸乙酯部分具有较好抗肝癌活性,因此,本课题对其乙酸乙酯部位的化学成分进行了研究,并对分离出来的新化合物进行了体外抗肝癌活性的初步筛选研究。方法:称取壮药棵矮瓤药材50 kg粉粗粉,用500 L 80%乙醇浸泡7 d,过滤,药渣再用400 L 80%的乙醇浸提2次,每次7 d,合并3次乙醇提取液,旋蒸回收乙醇,得到3.12 kg壮药棵矮瓤乙醇提取物浸膏。热水混悬所得乙醇浸膏后用石油醚和乙酸乙酯依次萃取,回收有机相,得到602.78 g乙酸乙酯提取部位浸膏。多次反复采用硅胶色谱法、葡聚糖凝胶色谱法、ODS、Sephadex LH-20、MCI等柱层析以及重结晶法等现代分离方法和精制纯化技术对壮药棵矮瓤乙酸乙酯部位提取物进行分离纯化,利用~1H-NMR、~(13)C-NMR、MS及文献参考等方法对分离提纯的单体化合物进行结构鉴定。采用MTT方法检测新化合物4和14对肝癌细胞株(Hep3B和HepG2)的增殖作用。化合物4和14设置9个药物组(1、5、10、30、70、100、150,200μmol/L)、顺铂组(75μmol/L)以及空白对照组,分别于给药24 h、48 h、72 h后,根据试剂说明说操作,用酶标仪检测OD值,并计算肝癌细胞增殖抑制率和IC50值。结果:壮药棵矮瓤乙酸乙酯部位共分离纯化得到16个单体化合物,分别鉴定为山奈酚⑴、5,7-二羟基-4',6-二甲氧基黄酮醇⑵、木犀草素⑶、(20R)-2α,3α,20-三羟基-16β-乙酰孕甾烷⑷(新)、(20R)-5α-2α,3α,16β,20-四羟基孕甾烷⑸、3,5,7,4’-四羟基-6-甲氧基黄酮醇⑹、β-谷甾醇⑺、β-胡萝卜苷⑻、3,5,3',4'-O-四甲基槲皮素⑼、3',4',7-三甲氧基槲皮素⑽、艾纳香素⑾、没食子酸乙酯⑿、没食子酸⒀、7-hydroxyspruceanol⒁(新)、芹菜素⒂、香豆酸⒃。化合物4对Hep3B和HepG2在48 h的IC50分别为78.15、151.24μmol·L-1,72h分别为55.27μmol·L-1和126.64μmol·L-1。化合物14对Hep3B和HepG2在48 h的IC50分别为135.80μmol·L-1和223.78μmol·L-1,72 h分别为121.44μmol·L-1和193.82μmol·L-1。结论:分离鉴定的16个化合物中,有10个化合物是首次从棵矮瓤中分离得到,它们分别是5,7-二羟基-4',6-二甲氧基黄酮醇⑵、(20R)-2α,3α,20-三羟基-16β-乙酰孕甾烷⑷(新)、(20R)-5α-2α,3α,16β,20-四羟基孕甾烷⑸、3,5,7,4-四羟基-6-甲氧基黄酮醇⑹、3,5,3',4'-O-四甲基槲皮素⑼、3',4',7-三甲氧基槲皮素⑽、没食子酸乙酯⑿、7-hydroxyspruceanol⒁(新)、芹菜素⒂、香豆酸⒃。其中(20R)-2α,3α,20-三羟基-16β-乙酰孕甾烷⑷和7-hydroxyspruceanol⒁为新化合物;在一定浓度梯度下化合物4和14对肝癌细胞(Hep3B、HepG2)的增殖均有一定程度的抑制作用,并且呈现浓度依赖性抑制作用。
[Abstract]:Objective: strong medicine plant dwarf flesh of the Compositae Blumea aromatica (Wall.) DC. dry whole grass, alias xanthate, mountain wind, fragrant Artemisia, big door, aromania and so on, has the effect of activating blood to stop itching, dispelling wind and swelling, mainly used in eczema, skin itching, rheumatic arthritis and other diseases. Oil, terpenoids, steroid compounds and so on, at present only a small amount of literature on the chemical composition and pharmacological activities of the research and report. A preliminary screening study was carried out on the anti hepatoma activity of the isolated new compounds in vitro. Methods: the crude powder of 50 kg powder of strong medicinal herbs was called, and 7 d was soaked with 500 L and 80% ethanol. The residue was filtered by 400 L 80% ethanol for 2 times, 7 d each time, combined with 3 ethanol extracts, and distilled to recover ethanol, and 3.12 kg strong herbs were obtained. The low pulp of 3.12 kg strong medicine was obtained. The extract of ethanol extract was extracted with petroleum ether and ethyl acetate in order to recover organic phase and get the extract of 602.78 g ethyl acetate extract. Many modern separation methods and refining methods, such as silica gel chromatography, dextran gel chromatography, ODS, Sephadex LH-20, MCI and other column chromatography and recrystallization, were repeatedly used. The purification technology was used to isolate and purify the extract of ethyl acetate from the dwarf pulp of Zhuang medicine. The structure identification of the isolated monomers was carried out by ~1H-NMR, ~ (13) C-NMR, MS and literature reference. The proliferation effect of new compound 4 and 14 on the hepatocellular carcinoma cell lines (Hep3B and HepG2) was detected by MTT method. Compound 4 and 14 set up 9 drugs. Group (1,5,10,30,70100150200 mu mol/L), cisplatin group (75 mu mol/L) and blank control group, after administration of 24 h, 48 h, 72 h respectively, according to the reagent description operation, using enzyme labeling instrument to detect the OD value, and calculate the proliferation inhibition rate and IC50 value of liver cancer cells. Results: 16 monomer compounds were isolated and purified from the ethyl acetate fraction of the low flesh of the strong medicine tree, respectively. 5,7- two hydroxyl -4', 6- two methoxy flavonol (2), luteolin 3, (20R) -2 a, 3 alpha, three hydroxyl -16 beta acetosterosterane (New), (20R) -5 alpha -2 a, 3 alpha, 16 beta, 20- four hydroxy gestosterane, 3,5,7,4' - four hydroxy methyl methoxy flavonol, beta carotene, beta carotene, four methyl quercetin 3', 4', 7- trimethoxy quercetin, artemisin, gallic acid, 7-hydroxyspruceanol (New), apigenin, and coumaric acid. Compound 4 to Hep3B and HepG2 in 48 h IC50 are 78.15151.24 micron mol / L-1,72h, respectively, 55.27 Mu mol. C50 was 135.80 Mu mol. L-1 and 223.78 mol. L-1,72 h respectively 121.44 Mu mol. L-1 and 193.82 micron L-1. respectively. Of the 16 compounds identified, 10 compounds were isolated from the low pulp for the first time. They were 5,7- two hydroxyl -4', two methoxy, 3 A and three hydroxy three hydroxy beta acetyl gestopanes. (New) (New) (20R) -5 alpha -2 alpha, 3 alpha, 16 beta, 20- four hydroxyl sterane, 3,5,7,4- four hydroxy -6- methoxy flavonol, 3,5,3', 4'-O- four methyl quercetin, 3', 4', 7- trimethoxy quercetin, ethyl gallate, 7-hydroxyspruceanol (New), apigenin, 3 alpha, three hydroxyl, beta acetyl gestophanus And 7-hydroxyspruceanol as a new compound; under a certain concentration gradient compound 4 and 14 have a certain inhibitory effect on the proliferation of liver cancer cells (Hep3B, HepG2), and present a concentration dependent inhibitory effect.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R29

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