八种中草药中抗肿瘤活性物质体外筛选及其机制初探
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摘要
本实验进行了抗肿瘤活性物质体外筛选及其作用机制的研究。首先用MMT法对芸香草、半边莲、蜜蒙花、甘草、地榆、柴胡、旱芹和莪术等8种中草药作用于癌细胞进行筛选。结果表明，浓度为1.0-1.5mg／ml的甘草乙醇浸提液(ESCG)、莪术乙醇浸提液(ESCC)和柴胡乙醇浸提液(ESCB)对体外培养的肝癌细胞SMMC-7721和肺癌细胞SPC-A1有较强的抑制增殖作用。在一定范围内，抑制作用随着浓度的加大而加强，并且随着时间的延长，细胞成活率呈现下降趋势。在浓度1.0-1.5mg／ml时，6-12小时是ESCB和ESCG对肿瘤细胞的最佳抑制时间，而ESCC最佳作用时间则是在12-24小时。曲线抑制率的计算还表明：活性物质对肿瘤细胞的作用，更大地依赖于活性物质本身，而不是肿瘤细胞株。
将1.5mg／ml ESCG、ESCC和ESCB分别作用于癌细胞，光镜下72小时内可见具“凋亡”形态的细胞：胞质浓缩，膜起小泡，出现凋亡小体，膜仍具完整性。用Hoechst33342及PI荧光染料对活性物质作用下的细胞进行联合染色，可以看到，不仅有发出红色荧光的坏死细胞和发出蓝色荧光的正常细胞，还有发出草蓝色荧光的凋亡细胞。提取细胞DNA进行琼脂糖凝胶电泳，出现明显的“DNA ladder”，表明一定浓度的ESCG、ESCC和ESCB在体外能诱导肺癌细胞SPC-A1和肝癌细胞SMMC-7721凋亡。凋亡曲线证明了肿瘤细胞的凋亡率随着活性物质浓度的增大而增大。以上结果表明，在体外ESCG、ESCC和ESCB对肝癌细胞SMMC-7721和肺癌细胞SPC-A1具有显著的抗增殖作用，它们能够诱导癌细胞的凋亡。
In this essay, screening of antitumor active substances in vitro and mechanism of them were investigated. The inhibitory effect of Cymbopogon distans, Lobelia chinensis, Buddleja offlcinalis, Glycyrrhiza uralensis Sanguisorba offlcinalis, Bupleurum chinense, Apium graveolen and Curuma zedoaria on the growth of two cancer cell line was tested with the MTT assay. The result indicated that ethanol soluble constituent of G. uralensis (ESCG), ethanol soluble constituent of C. zedoaria (ESGC) and ethanol soluble constituent of B. chinense (ESCB) can inhibit proliferation of SMMC-7721 and SPC-A1 cells at the concentration of 1.0-1.5mg/ml. The inhibitory rate increased in the agents concentration. The inhibitory rate of the cell line also increased with time in the range of the test. The inhibition rate of SMMC-7721 by them were : 51.6%. 48.5% and 52.9% respectively. And the inhibition rate of SPC-A1 by them were : 53.2%-. 47.9% and 58.2% respectively. At the concentration of 1.0-1.5mg/ml, the ESCB and ESCG have the
best effect after 6-12 hours of treatment, and the ESCC has the best effect after 12-24 hours. The curve results suggested that the effect of ESCB, ESCG and ESCC exerting depends rather on active materials than on tumor cells.
Within 72 hours obviously apoptosis such as: condensation of chromatin, apoptotic bodies in morphology can be seen in 1.5mg/ml ESCB-treating, ESCG-treating and ESCC-treating tumor cells. Blue fluorescence and red fluorescence of cells turn up by Hoechst33342 and PI staining. Also the apoptosis in tumor cells was conformed by DNA ladder formation on gel electrophoresis. The results showed that ESCG, ESCC and ESCB exhibited a marked antiproliferative effect on SMMC-7721 cells and SPC-A1 cells, and they can induce apoptosis in vitro.
将1.5mg／ml ESCG、ESCC和ESCB分别作用于癌细胞，光镜下72小时内可见具“凋亡”形态的细胞：胞质浓缩，膜起小泡，出现凋亡小体，膜仍具完整性。用Hoechst33342及PI荧光染料对活性物质作用下的细胞进行联合染色，可以看到，不仅有发出红色荧光的坏死细胞和发出蓝色荧光的正常细胞，还有发出草蓝色荧光的凋亡细胞。提取细胞DNA进行琼脂糖凝胶电泳，出现明显的“DNA ladder”，表明一定浓度的ESCG、ESCC和ESCB在体外能诱导肺癌细胞SPC-A1和肝癌细胞SMMC-7721凋亡。凋亡曲线证明了肿瘤细胞的凋亡率随着活性物质浓度的增大而增大。以上结果表明，在体外ESCG、ESCC和ESCB对肝癌细胞SMMC-7721和肺癌细胞SPC-A1具有显著的抗增殖作用，它们能够诱导癌细胞的凋亡。
In this essay, screening of antitumor active substances in vitro and mechanism of them were investigated. The inhibitory effect of Cymbopogon distans, Lobelia chinensis, Buddleja offlcinalis, Glycyrrhiza uralensis Sanguisorba offlcinalis, Bupleurum chinense, Apium graveolen and Curuma zedoaria on the growth of two cancer cell line was tested with the MTT assay. The result indicated that ethanol soluble constituent of G. uralensis (ESCG), ethanol soluble constituent of C. zedoaria (ESGC) and ethanol soluble constituent of B. chinense (ESCB) can inhibit proliferation of SMMC-7721 and SPC-A1 cells at the concentration of 1.0-1.5mg/ml. The inhibitory rate increased in the agents concentration. The inhibitory rate of the cell line also increased with time in the range of the test. The inhibition rate of SMMC-7721 by them were : 51.6%. 48.5% and 52.9% respectively. And the inhibition rate of SPC-A1 by them were : 53.2%-. 47.9% and 58.2% respectively. At the concentration of 1.0-1.5mg/ml, the ESCB and ESCG have the
best effect after 6-12 hours of treatment, and the ESCC has the best effect after 12-24 hours. The curve results suggested that the effect of ESCB, ESCG and ESCC exerting depends rather on active materials than on tumor cells.
Within 72 hours obviously apoptosis such as: condensation of chromatin, apoptotic bodies in morphology can be seen in 1.5mg/ml ESCB-treating, ESCG-treating and ESCC-treating tumor cells. Blue fluorescence and red fluorescence of cells turn up by Hoechst33342 and PI staining. Also the apoptosis in tumor cells was conformed by DNA ladder formation on gel electrophoresis. The results showed that ESCG, ESCC and ESCB exhibited a marked antiproliferative effect on SMMC-7721 cells and SPC-A1 cells, and they can induce apoptosis in vitro.
引文
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2．于尔辛，汤钊道．1993．现代肿瘤学．上海：上海医科大学出版社，1-5．
3．卫东，姜芸珍，赵知中．1990．三尖杉酯碱类似物的合成及其抗肿瘤活性．药学学报，25(9)：677-683．
4．马维勇，张椿年．1997．鬼臼毒素抗肿瘤药物的研究进展[J]．中国医药工业杂志，28(2)： 65
5．王玉露．2003．核糖体失活蛋白抗肿瘤作用的研究．Trait Pharmaceutical Journal，154：1-4．
6．王岳五等．2000．甘草残渣中多糖GPS抗肿瘤作用的研究．南开大学学报，33(4)：15-18．
7．王建锋，苏文金．2000．紫杉醇诱导细胞调亡机制研究进展．国外医学：肿瘤学分册，27(4)：204-207．
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