绞股蓝热处理产物抗NSCLC A549细胞有效成分研究

【作者】 吴倩；

【导师】 朴香兰；

【作者基本信息】 中央民族大学 ， 民族医学， 2012， 硕士

【摘要】 绞股蓝经高温、高压热处理得到的绞股蓝热处理产物与原药材相比,具有很强的抑制非小细胞肺癌(Non-small cell lung carcinoma, NSCLC)A549细胞的增殖活性。本研究目的：以绞股蓝热处理产物为研究对象,分离、鉴定其有效成分,并评价它们对A549细胞增殖的抑制作用。研究方法：分别通过100、110、120和130℃及加热1h、2h和3h,确立制备绞股蓝热处理产物的最佳条件；利用大孔树脂HP20吸附法,通过20%、50%及95%乙醇洗脱溶剂洗脱,筛选有效部位；采用硅胶柱色谱、凝胶色谱及反相C18色谱法,分离有效成分；通过NMR、Mass、UV等分析手段,鉴定有效成分的分子结构；运用MTT法检测绞股蓝热处理前后总提物及有效成分对A549细胞增殖的作用；利用UPLC-MS分析方法,测定不同热处理条件下的绞股蓝热处理产物及原药材中有效成分的含量。研究结果：(1)绞股蓝热处理产物的最佳制备条件为130℃、0.24MPa热处理3h,其乙醇提取物与原药材乙醇提取物相比,具有更强的抑制A549细胞增殖作用,其IC50±±SD值为198.13±7.60μg/mL,而原药材乙醇提取物为411.61±32.43μg/mL。(2)绞股蓝热处理产物在大孔树脂HP20的不同极性洗脱部位中,95%乙醇洗脱部位对A549细胞增殖具有较强的抑制作用。(3)从95%乙醇洗脱部位中分离、鉴定出4种达玛烷类皂苷,分别为达木林A(damulin A)、达木林B (damulinB)、绞股蓝它皂苷LI(gypenoside LI)及绞股蓝皂苷L(gypenoside L),其中达木林A与达木林B是绞股蓝热处理后新发现的化合物。(4)这四种化合物均具有较强的抑制A549细胞增殖作用,其IC50±SD值分别为26.984±0.51、4.56±0.58、50.96±9.55及34.94±4.23μg/mL,而阳性对照物人参皂苷Rg3(S)为39.26±2.05μg/mL。(5)四种达玛烷类皂苷在绞股蓝中的含量检测结果,在相同加热时间条件下,随着热处理温度的升高而增加；在同等温度条件下,随加热时间的增加而提高,最佳条件为130℃温度、0.24MPa压力、热处理3h,此时的绞股蓝热处理产物中含达木林A、达木林B、绞股蓝皂苷LI(gypenosideLI)及绞股蓝皂苷L(gypenoside L)分别为11902.19±0.18,5876.64±0.50,2462.52±0.21,6130.26±0.27μg/g。结论：热处理方法可以成为一种从天然物中获得更多活性成分的有效途径。更多还原

【Abstract】 Heat-processed Gynostemma pentaphyllum by high temperature and high pressure showed more inhibitory activity against non-small cell lung carcinoma (NSCLC) A549cells than that of the crude plant. The objectives of study were to isolate and identify the active constituents and evaluate their A549cell inhibitory activities from the heat-processed G. pentaphyllum. The best conditions of preparation of the heat-processed G. pentaphyllum were confirmed by steam-heating at100,110,120and130℃for1h,2h and3h, respectively. The active fractions were screened using sephadex HP20chromatography and20%,50%,95%ethanol elution. The active constituents were isolated by silica gel, sephadex and reverse phase C18column chromatography, and their chemical structures were identified with NMR, Mass and UV spectra data. The A549cell inhibitory activities of the before and after heat-processing G. pentaphyllum and their active constituents were measured using the3-(4,5-dimethyithiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. The contents of the active constituents of before and after heat-processed G. pentaphyllum were analyzed by UPLC-MS. The results showed that the best condition of preparation of the heat-processed G. Pentaphyllum was stream-heating at130℃,0.24MPa for3h and its ethanol extract appeared more A549cell inhibitory activity than that of the crude G. pentaphyllum. Its IC50value was198.13+7.60μg/mL, whereas for the crude plant it was411.61132.43μg/mL. Using HP20absorption, the95%ethanol elution fraction showed strong A549cell inhibitory activity. From the95%ethanol elution fraction, four dammarane-type saponins were isolated and identified as damulin A, damulin B, gypenoside LI and gypenoside L, respectively. And damulin A and damulin B were found from the heat-processed G. pentaphyllum at fist time. They all appeared strong A549cell inhibitory activities with IC50values of26.98±0.51,4.56±0.58,50.96±9.56and34.94±4.23μg/mL, respectively. Whereas for the positive control ginsenoside Rg3(S) it was39.26±2.05μg/mL. The contents of damulin A, damulin B, gypenoside LI and gypenoside L from the heat-processed G. pentaphyllum preparated at130℃,0.24MPa for3h were11902.19±0.18,5876.64±0.50,2462.52±0.21,6130.26±0.27μg/g, respectively. Heat-processing method was provided for preparation of more active constituents from natural products.更多还原