【作者】 孙立新；

【导师】 王敏伟； 毕开顺；

【作者基本信息】 沈阳药科大学 ， 药理学， 2005， 博士

【摘要】 白英是茄科（Solanaceae）植物Solanum lyratum Thunb．的干燥全草，主产我国长江以南各省区，具有清热解毒、祛风化痰、祛风湿等功效，用于治疗疟疾、黄疸、水肿、淋病、风湿关节炎、胆囊炎、癌症、子宫颈糜烂、白带、疔毒等。本文主要进行了白英粗提物体内外抗肿瘤作用、有效萃取物的筛选与体内抗肿瘤作用、活性追踪分离化学成分、活性单体化合物-16-妊娠双烯醇酮抗肿瘤作用的分子机制和白英中活性成分含量测定方法的建立等五个方面的研究。本文选取体外培养的人宫颈癌HeLa、人黑色素瘤A375、人乳腺癌MCF7、人肝癌Bel-7402、人胃腺癌SGC-7901、小鼠纤维肉瘤L929和人髓性白血病U937细胞为模型，以半数抑制浓度(IC₅₀)为指标，采用MTT法考察了白英醇提物和水提物对上述肿瘤细胞的生长抑制作用，结果表明，白英醇提物和水提物对上述肿瘤细胞有显著的增殖抑制作用，呈明显的浓度-效应依赖关系；白英醇提物和水提物对各种肿瘤细胞的敏感性不同，其中对SGC-7901、A375、Bel-7402、HeLa、L929细胞的抑制作用较强，对MCF7、U937细胞的抑制作用较弱；对同一种肿瘤细胞白英醇提物的抑制作用明显高于水提物。体内以小鼠移植性肿瘤肝癌H22和肉瘤S180为模型，以抑瘤率为指标，考察了白英醇提物和水提物的体内抗肿瘤作用，结果表明，白英醇提物对小鼠S180肉瘤和小鼠H22肝癌均有较强的抑制作用，高剂量组荷瘤小鼠的平均瘤重明显低于阴性对照组，且抑瘤率大于40％；白英水提物对小鼠S180肉瘤和小鼠H22肝癌均有一定的抑制作用，高剂量组荷瘤小鼠的平均瘤重明显低于阴性对照组，抑瘤率大于30％，可初步认为白英有一定的抗肿瘤作用，且醇提物比水提物的作用强。白英醇提物和水提物对小鼠的急性毒性均很低。本文选取体外培养的人宫颈癌HeLa、人黑色素瘤A375、人乳腺癌MCF7、人肝癌Bel-7402、人胃腺癌SGC-7901和小鼠纤维肉瘤L929细胞为模型，以半数抑制浓度(IC₅₀)为指标，采用MTT法对白英的石油醚萃取物、乙酸乙酯萃取物、正丁醇萃取物和水溶性萃取物进行了活性筛选，结果表明，白英乙酸乙酯萃取物和正丁醇萃取物对上述肿瘤细胞具有明显的增殖抑制作用，且呈浓度-效应依赖关系；白英乙酸乙酯萃取物和正丁醇萃取物对各种肿瘤细胞的敏感性不同，其中对A375和HeLa细胞的抑制作用较强，对SGC-7901和MCF7细胞的抑制作用次之，对Bel-7402和L929细胞的抑制作用较弱；对同一种肿瘤细胞乙酸乙酯萃取物的抑制作用明显高于正丁醇萃取物；石油醚萃取物和水溶性萃取物对肿瘤细胞生长无抑制作用。以小鼠移植性肿瘤肝癌H22和肉瘤S180为模型，以抑瘤率为指标，考察了体外活性萃取物的体内抗肿瘤作用，结果表明，白英乙酸乙酯萃取物有较强的体内抗肿瘤作用，正丁醇萃取物的体内抗肿瘤作用较弱。本文选取体外培养的人宫颈癌HeLa和人黑色素瘤A375为模型，以半数抑制浓度(IC₅₀)为指标，采用MTT法进行抗肿瘤活性追踪，利用硅胶柱色谱、葡聚糖凝胶柱色谱、聚酰胺柱色谱、制备薄层色谱和制备高效液相色谱等分离手段，从白英的乙酸乙酯萃取物分离出16个单体化合物，正丁醇萃取物分离出4个化合物，利用理化性质和光谱学技术，鉴定了15个化合物。其中2，8为新化合物，9为新天然产物，1，3为首次从该属植物中分离得到，4，7，12，14为首次从白英中分离得到。2个新化合物为：16-dehydropregnenolone 3-O-α-L-rhamnopyranosyl-（1→2）-β-D-glucopyranosiduronic acid（2），Diosgenin 3-O-β-D-glucopyranosiduronic acid methyl ester（8）；13个已知化合物为：16-妊娠双烯醇酮（16-Dehydropregnenolone）（1），5α-孕甾烯醇酮（3-hydroxy-5-pregn-16-en-20-one）（3），原儿茶酸（Protocatechuic acid）（4），香草酸（Vanillic acid）（5），咖啡酸（Caffeic acid）（6），薯蓣皂苷元（Diosgenin）（7），Diosgenin 3-O-β-D-glucopyranosiduronic acid（9），Diosgenin 3-O-α-L-rhamnopyranosyl-（1→2）-β-D-glucopyranosiduronic acid（10），Diosgenin 3-O-α-L-rhamnopyranosyl-（1→2）-β-D-glucuroniduronic acid methyl ester（11），槲皮素（Quercetin）（12），芦丁（Rutin）（13），β-谷甾醇（β-sitosterol）（14），6-甲氧基-7-羟基香豆素（Scopoletin）（15）。采用MTT法测定了13个化合物对体外培养的人宫颈癌HeLa、人黑色素瘤A375、人肝癌Bel-7402和人胃腺癌SGC-7901细胞的体外抗肿瘤活性，结果表明化合物1，7，10，11，12有中等强度的体外抗肿瘤活性，其它化合物无活性。化合物1，10，11的体外抗肿瘤活性为首次发现。本文综合应用了细胞形态学、生物化学和分子生物学的方法对16-妊娠双烯醇酮（16-Dehydropregnenolone）诱导HeLa细胞凋亡机制进行了初步探讨。光学显微镜下观察30μM 16-妊娠双烯醇酮作用于HeLa细胞24 h后细胞体积缩小变圆，产生凋亡小体。30μM 16-妊娠双烯醇酮处理的HeLa细胞经Hoechst 33258荧光染色，显微镜下观察正常细胞全部蓝染，细胞壁完整，16-妊娠双烯醇酮30μM作用细胞24 h后，发现部分细胞发生染色体凝集和质膜出泡，同时可见已破碎的核，有的已形成凋亡小体，对比正常细胞致密的核可推断细胞经历了凋亡性的细胞死亡。在琼脂糖凝胶电泳实验中，30μM 16-妊娠双烯醇酮作用24 h后呈“阶梯状”图谱（DNA ladder），说明16-妊娠双烯醇酮可诱导HeLa细胞发生明显的DNA片段化，即诱导细胞发生凋亡。流式细胞分析结果显示：16-妊娠双烯醇酮处理后的HeLa细胞被阻滞于G₀／G₁期，且呈时间和浓度依赖关系；16-妊娠双烯醇酮作用于HeLa细胞可以检测到亚二倍体峰（Sub-G₁），凋亡细胞数呈时间和浓度依赖关系，说明16-妊娠双烯醇酮是通过周期抑制和诱导细胞凋亡达到体外抗肿瘤作用。Western印迹实验结果表明：16-妊娠双烯醇酮作用于HeLa细胞后，caspase-3的酶原被降解，caspase-3抑制剂可部分抑制细胞死亡；16-妊娠双烯醇酮作用于HeLa细胞12h后caspase-3被活化，ICAD和PARP开始降解，24 h后DNA产生明显的有序化降解（DNA ladder），可知16-妊娠双烯醇酮诱导HeLa细胞凋亡时激活了caspase级联反应；在16-妊娠双烯醇酮诱导的HeLa细胞凋亡中，上调了促凋亡蛋白Bax的表达，下调了抗凋亡蛋白Bcl-2，使Bax／Bcl-2在药物作用后随时间延长而升高，此后caspase-3底物ICAD和PARP被降解，DNA发生片段化，表明16-妊娠双烯醇酮通过增加Bax／Bcl-2的比例，激活以线粒体为中心的信号转导途径而诱导细胞凋亡。本文以首次从白英中分离得到且具有抑制肿瘤细胞增殖活性的两个单体化合物薯蓣皂苷元和16-妊娠双烯醇酮为质控指标建立了高效液相色谱法。薯蓣皂苷元在0.04～0.2 mg·mL^-1范围内呈良好线性关系（r=0.9999），平均回收率为104.5％（RSD=1.4％）；16-妊娠双烯醇酮在5.06～45.54μg·mL^-1范围内呈良好线性关系（r=1.000），平均回收率为98.8％（RSD=1.7％）。该方法简便、快速、重复性好，可以作为白英质量控制的依据。更多还原

【Abstract】 The medicinal plant Solatium lyratum Thunb. belonging to the family of Solanaceae distributed widely in south areas of Changjiang River in China. The whole plant of Solatium lyratum has long been used as traditional Chinese folk medicine to treat malarias, jaundices, edemas, rheumatisms and furuncles. It was also used as an anti-tumor medicine in some folk remedy.This dissertation was divided into 5 aspects as following: （1） the anti-tumor activity in vitro and vivo of aqueous extract and ethanol extract of Solanum lyratum, （2） the screening of anti-tumor activity fractions and its anti-tumor effect in vivo, （3） the isolation of anti-tumor activity compounds by bioassay-directed fractionation and measurements of anti-tumor of isolated compounds, （4） the molecular mechanisms of anti-tumor effect of 16-dehydropregnenolone, （5） the establishment of quantitative methods of two active constituents, diosgenin and 16-dehydropregnenolone.The antiproliferative activities of aqueous extract and ethanol extract against human cervix epithelial cell line HeLa, human malignant melanoma cell line A375-S2, murine fibrosarcoma cell line L929, human breast cancer cell line MCF7, human hepatocarcinoma cell line BEL-7402, human gastric carcinoma cell line SGC-7901 and the myeloid leukemia cell line U937 were evaluated by MTT colorimetric assay in vitro. The results showed the aqueous extract and ethanol extract possessed the antiproliferative activity against different tumor lines in a dose-dependent manner. Compared with the cytotoxic activity of aqueous extract, the ethanol extract showed stronger cytotoxic activity. The inhibitory effects of aqueous extract and ethanol extract on tumor growth were observed by the models of implanted sarcoma 180 （S180） and hepatoma 22 （H22） in mice. Aqueous extract and ethanol extract restrained obviously the growth of tumor in mice. The inhibitory rates of aqueous extract on the growth of S180 and H22 at dose of 62.4 g·kg^-1 were 41.4% and 45.4%, respectively. The inhibitory rates of ethanol extract on the growth of S180 and H22 at doses of 62.4 g·kg^-1 were 39.0 % and 37.2 %, respectively.The anticancer activities of petroleum ether, EtOAc, n-BuOH and aqueous fraction were screened by MTT using human cervix epithelial cell line HeLa, human malignant melanoma cell line A375-S2, murine fibrosarcoma cell line L929, human breast cancer cell line MCF7, human hepatocarcinoma cell line BEL-7402, and human gastric carcinoma cell line SGC-7901. The results showed that EtOAc fraction exhibited strong antitumor activity; n-BuOH fraction possessed medium antitumor activity; petroleum ether fraction and aqueous fraction had no activity. In the meanwhile, the inhibitory effects of EtOAc fraction and n-BuOH fraction on tumor growth were observed by the models of implanted sarcoma 180 （S180） and hepatoma 22 （H22） in mice. EtOAc fraction was proved to be effective on the tumor-bearing mice of S180 and H22, and the inhibitory rate of the dose of 62.4g·kg^-1 were 42.7％and 46.0％, respectively, n-BuOH fraction exhibited weak effect on the tumor-bearing mice of S180, and the inhibitory rate of the dose of 62.4 g·kg^-1 were 33.3％. n-BuOH fraction exhibited no effect on the tumor-bearing mice of H22.Bioassay-directed fractionation of the EtOAC fraction and n-BuOH fraction led to isolation of 20 compounds from Solanum Lyratum with silica gel, sephadex LH20, polyamide, HPLC etc. Among them, 15 compounds were elucidated on the basis of physi-chemical and spectral methods. They identified as 16-dehydropregnenolone （1）, 16-dehydropregnenolone3-O-α-L-rhamnopyranosyl-（1→2）-β-D-glucpyranosid-uroni c acid （2）, 3-hydroxy-5-pregn-16-en-20-one （3）, protocatechuic acid （4）, vanillic acid （5）, caffeic acid （6）, diosgenin （7）, diosgenin 3-O-β-D-glucopyranosiduronic acid methyl ester （8）, diosgenin 3-O-β-D-glucopyranosiduronic acid （9）, diosgenin 3-O-α-L-rhamnopyranosyl-（1→2）-β-D-glucopyranosiduronic acid （10）, diosgenin 3-O-α-L-rhamnopyranosyl-（1→2）-β-D-glucuroniduronic acid methyl ester （11）, quercetin （12）, rutin（13）,β-sitosterol（14）, scopoletin （15）. Among them, compound 2 and 8 are new compounds, 9 is a new nature compound, 1 and 3 are isolated from the genus for the first time, and 4, 7, 12, 14 are isolated from the plant for the first time. The antiproliferative activities of 13 isolated compounds against human cervix epithelial cell line HeLa, human malignant melanoma cell line A375-S2, human hepatocarcinoma cell line BEL-7402 and human gastric carcinoma cell line SGC-7901 were evaluated by MTT. The result suggested compound 1, 7, 10, 11, 12 exhibited antiproliferative activity, and the anti-tumor activity of compound 1, 10, 11 are reported for the first time. In the present studies, We examined the molecular mechanism by which 16-dehydropregnenolone could induce apoptosis in HeLa cells. The characteristic changes of apoptosis, i.e., shrinkage of cells, blebbing nuclei and granular apoptotic bodies, were observed by phase contrast microscopy and Hoechst 33258 staining. When the DNA from HeLa cells incubated with 16-dehydropregnenolone was analyzed by agarose gel electrophoresis, it generated a characteristic "ladder pattern" of disconstinuous DNA fragments.Cell cycle analysis of HeLa cells was investigated by using FACS after PI staining. The results showed that 16-DHP-treated HeLa cells led to a marked accumulation in G₀/G₁ phase after 24h treatment and sub-G₁ apoptotic fraction was increased compared with control. It suggested that the growth inhibitory effect of 16-dehydropregnenolone was the result of arrest in G₀/G₁ phase and apoptosis.To determine whether Caspase-3 participates in 16-dehydropregnenolone induced apoptosis, HeLa cells were treated with 30μM 16-dehydropregnenolone for 24h in the absence or presence of caspase-3 inhibitor, z-DEVD-fmk. Z-DEVD-fmk effectively inhibited 16-dehydropregnenolone-induced HeLa cell death. In the following investigation, we assessed the expressions of caspase-3 substrates PARP and ICAD. The results showed that PARP （116 kDa） expression was down-regulated in a time dependent manner and minor 85 kDa fragment was increased, and ICAD was also down-regulated in time dependent manner, suggesting that caspases participated in 16-DHP induced HeLa cell death.Mitochondrial Bcl-2 family is a series of proteins that regulate apoptosis. Some of these proteins such as Bax and Bak, promote apoptosis, whereas others like Bcl-2 and Bcl-X_L inhibit apoptosis. After incubation with 16-dehydropregnenolone, expression of Bcl-2 protein was down-regulated, on the contrary, the level of Bax protein was increased. These results suggested that the mitochondrial pathway of cell death might be involved in HeLa cells death induced by 16-dehydropregnenolone.Diosgenin and 16-dehydropregnenolone, two anti-tumor activity constituents, were also determined by RP-HPLC. The linear range of them were 0.04-0.2 mg-mL^-1 （r=0.9999） and 5.06-45.54μg·mL^-1 （r=1.0000）, respectively. The average recoveries of of them were 104.5％（RSD=1.4％）and 98.8％（RSD=1.7％）, respectively. These methods were simple, rapid, accurate and provided a quantitative for the quality assessment of Solanum lyratum.更多还原