【作者】 王丽；

【导师】 裴妙荣；

【作者基本信息】 山西中医学院 ， 中药学， 2015， 硕士

【摘要】 目的利用现代的提取、分离、分析手段对中华苦荬菜正丁醇部位的化学成分进行提取、分离、纯化和结构鉴定。在Hep G2细胞葡萄糖消耗模型上,初步确定木犀草苷和木犀草素的降血糖作用机制。方法乙醇加热回流提取经过鉴定的中华苦荬菜全草,回收乙醇,得浸膏。浸膏加水混悬,依次用石油醚、乙酸乙酯和正丁醇萃取,回收溶剂后,得石油醚部位、乙酸乙酯部位、正丁醇部位和水部位。将正丁醇部位通过大孔树脂纯化,硅胶柱层析粗分离后,用葡聚糖凝胶层析、聚酰胺层析等分离方法进行分离,重结晶得到不同的单体,并通过HPLC、NMR、TLC、UV等方法进行化合物结构鉴定。分别设置木犀草苷和木犀草素的高、中、低、极低四个药物浓度,以二甲双胍和罗格列酮作为阳性对照组,在Hep G2细胞葡萄糖消耗模型实验上,通过结合MTT实验结果和细胞消耗葡萄糖浓度实验结果,综合确定木犀草苷和木犀草素的最佳药物浓度。分别设置不同浓度葡萄糖的培养基,用最佳药物作用浓度进行干预,实验结束后依据细胞消耗葡萄糖浓度实验结果,确定药物促进细胞消耗葡萄糖时对葡萄糖浓度的依赖性。分别设置空白组、高浓度胰岛素组、低浓度胰岛素组、药物组、药物组加高浓度胰岛素组、药物组加低浓度胰岛素组进行试验,实验结束后依据细胞消耗葡萄糖浓度实验结果,确定促进细胞葡萄糖消耗时对胰岛素依赖性。结果从中华苦荬菜中分离得到8个化合物,苯并香豆素、对甲氧基苯甲酸首次从中华苦荬菜中分离得到;3’-羟基-7-O-β-D-葡萄糖-黄酮苷、5,8,3’-3-羟基-7-O-β-D-葡萄糖-黄酮苷为新化合物;其余为芹菜素-7-O-β-D-葡萄糖苷木犀草素-7-O-β-D-葡萄糖苷乙酸酯、木犀草素-7-O-β-D-葡萄糖苷、木犀草素。1μmol/L时木犀草苷和1μmol/L木犀草素对细胞的毒性最小,并且促进细胞消耗葡萄糖的能力最强。药物促进细胞消耗葡萄糖在低浓度(<12.8mmol/L)时,对葡萄糖浓度有依赖性;药物促进细胞消耗葡萄糖在高浓度(>12.8mmol/L)时,对葡萄糖浓度无依赖性。木犀草苷和木犀草素促进细胞消耗葡萄糖时不需要胰岛素的共同作用,在有胰岛素共同作用时细胞消耗葡萄糖的能力反而有所降低。结论从中华苦荬菜中分离得到8个化合物,其中有两个新化合物,两个首次从中华苦荬菜中分离得到。木犀草苷和木犀草素在具有促进Hep G2细胞消耗葡萄糖的能力,初步确定它们是胰岛素增敏剂,可通过减少肝脏葡萄糖输出,改善胰岛素抵抗等作用降低血糖。更多还原

【Abstract】 Purpose The part n-butyl alcohol was extracted, separated, purified from the Ixeris Chinens, and the structure of the obtained product was analyzed using the latest methods of analysis of the chemical compositions.The Hep G2 Cell glucose consumption model has been used to determine the underlying mechanism of decreasing blood sugar by galuteolin and luteolin extracted from Ixeris Chinens.Methods The extraction process was started by refluxing with heating the identified Ixeris Chinens, followed by recovering the ethanol through rotary evaporation instrument. Then mixing the distilled water with the extraction, and adding the petroleum ether, ethyl acetate and n-butanol separately and obtained the corresponding part after the extraction had been dissolved. The n-butanol part was purified with macroporous resin, column chromatography filled by silicone or polyamide. The products were identified after recrystallized by HPLC, NMR, TLC, etc. The concentrations of galuteolin and luteolin were optimized according to the MTT results and the glucose consumed by Hep G2 after the Hep G2 was subjected to the different concentrations of galuteolin and luteolin.The cells were cultivated in the substrate with different glucose concentrations, and the optimized galuteolin and luteolin were added into the substrate to interfere the process of cell consuming glucose. According to the alternations of glucose consumption, the effect of glucose concentration in the substrate on the amount of glucose consumed by cell had been determined.The effect of insulin on the glucose consumption had been investigated after the cell subjected to the blank, high concentration insulin, low concentration insulin, galuteolin and luteolin, galuteolin and luteolin with high insulin concentration, as well as galuteolin and luteolin with low insulin concentration.Results Eight compounds were isolated from Ixeris Chinens, coumarin and methoxy benzoic acid were first isolated from the Ixeris Chinens; 3’-OH-7-O-β-D-glu-flavonoidglycoside、5,8,3’-3-OH-7-O-β-D-glu-lavonoidglycoside, are the new constituents isolated from the Ixeris Chinens; the other constituents are Apigenin-7-O-β-D-glucopyranoside, Luteonin-7-O-β-D-glucopyranoside acetate, Luteolin-7-O-β-D-glucopyranoside and Luteolin.1μmol/L galuteolin and 1μmol/L luteolin had the minimum cytotoxicity and greatest promotion effect for the glucose consumption process among the four used concentrations.The amounts of glucose consumed by the cell showed an increased tread as a function of the glucose concentration in the substrate and reached the peak valued at 11mmol/L, then no obvious variations of cell consuming glucose ability had been observed.The promotion of glucose consumption process by galuteolin and luteolin would be decreased slightly under the insulin. Thus, it is speculated that the underlying mechanism of decreasing the blood sugar by galuteolin and luteolin would be the same as the metformin hydrochloride.Conclusion Eight compounds were isolated from Ixeris Chinens. Two new compounds were first found and the other two compounds were obtained for the first time. Also, Galuteolin and luteolin are insulin-sensitizer and could decrease the blood sugar via reducing the amount of glucose output by liver and decreasing the insulin resistance effect.更多还原