90种云南毒蕈体外抗癌活性评价及活性成分研究

【作者】 刘非燕；

【导师】 吴平；

【作者基本信息】 浙江大学 ， 植物学， 2006， 博士

【摘要】 高等真菌（蘑菇）是菌物界最为重要的成员之一,富含结构新颖且生物活性多样的化合物,因而成为新药开发的一种重要的天然资源,越来越引起人们的重视。为了系统地挖掘和开发我国高等真菌资源,特别是有毒高等真菌（毒蕈）资源,本论文开展了以下工作: 1.系统采集了我国云南省90种毒蕈,基本建立了其在云南的品种资源分布图谱和生态信息库。这些有毒真菌约占云南省毒蕈资源50%,隶属于2亚门:担子菌亚门、子囊菌亚门;2纲:层菌纲、核菌纲:3目:伞菌目、非菌褶目、肉座菌目:8科:鹅膏菌科、蘑菇科、牛肝菌科、丝膜菌科、红菇科、齿菌科、肉座菌科、陀螺菌科;14属:鹅膏菌属、蘑菇属、牛肝菌属、疣柄牛肝菌属、粉牛肝菌属、粘盖牛肝菌属、粉孢牛肝菌属、丝膜菌属、丝盖伞属、乳菇属、红菇属、肉齿菌属、菌寄生菌属、陀螺菌属。 2.详细评价了这些毒蕈的体外抗癌细胞毒性。采用四氮唑盐酶还原法（MTT）,从90种毒蕈的石油醚、乙酸乙酯、乙醇和水四种溶剂提取物中,发现了43种毒蕈的石油醚及（或）乙酸乙酯提取物对宫颈癌HeLa细胞株具有较强的细胞毒性(IC₅₀为5—100μg/ml),占所采毒蕈47.8%,其中8种毒蕈的提取物显示了强烈的抗癌细胞毒活性(IC₅₀<35μg/ml);而所有收集的毒蕈的乙醇和水提取物几乎没有显示抗癌细胞毒活性(IC₅₀>200μg/ml)。在有活性的43种毒蕈中共有35种毒蕈的体外抗癌细胞毒性均为首次报道,为进一步提取分离具有抗癌活性的先导化合物奠定了丰厚的材料基础。 3.分析鉴定了两种具有强烈抗癌细胞毒性的毒蕈——竹生肉球菌和黄赭鹅膏菌寄生菌的细胞毒性原因。论文首次报道轮枝胞菌素A是黄赭鹅膏菌寄生菌中的主要细胞毒性物质,而松胞菌素D是引起竹生肉球菌强烈细胞毒性的主要原因。经现代波谱技术确证了这两种物质的结构,论文也首次报道了轮枝胞菌素A晶体结构特征。 4.广泛探索了松胞菌素D和轮枝胞菌素A的体内外抗癌活性、抗真菌活性和杀虫活性。 松胞菌素D具有广谱抗癌细胞毒活性,对急性髓系白血病NB4、急性髓系白血病M3型HL-60、红白血病K562、急性髓系白血病单核型U937、急性髓系白血病慢粒型KU812,肝癌SMMC 7721,乳腺癌BCAP37,宫颈癌HeLa,口腔鳞癌SK等九个不同肿瘤株均有强烈的细胞毒活性(IC₅₀平均值约7.35×10^-6M),细胞毒性与紫杉醇接近(IC₅₀为2.0×10^-6M),并发现松胞菌素D可以通过诱导肿瘤细胞凋亡而抑制其增殖。论文首次报道松胞菌素D具有强烈的选择性抗作物病源真菌尖刀镰孢菌Fusarium oxysporium f.sp.活性（MIC为12.5μg/ml）和杀虫活性（对桃蚜、朱砂叶螨、小菜蛾均有一定的杀伤作用,但对朱砂叶螨、桃蚜的活性相对较高）。 轮枝胞菌素A也具有广谱抗癌活性,对红白血病K562,肝癌SMMC 7721、肝癌HepG2,乳腺癌BCAP37、乳腺癌MCF—7,宫颈癌HeLa,肺腺癌SPC—A1,前列腺癌DU145,大肠癌SW620,淋巴癌Jurkat等十个不同肿瘤株均有强烈的细胞毒活性,IC₅₀平均值为6.6×10^-8M,细胞毒性明显高于紫杉醇(2.0×10^-6M),轮枝胞菌素A可以通过诱导肿瘤细胞凋亡而抑制其增殖;体内抗癌结果表明0.05mg/kg的轮枝胞菌素A连续给药7天,对小鼠S180肉瘤的抑瘤率30.39%;论文首次发现轮枝胞菌素A具有强烈选择性抗作物病源真菌灰霉菌Botrytis cinerea作用,其MIC为10μg/ml,优于阳性对照灰黄霉素（31.25μg/ml）。 5.总结综述了毒蕈毒素的化学分类及其毒理。 所有这些研究对充分利用我国的高等真菌资源,进行系统的新药开发特别是抗肿瘤药物开发具有重要的意义。更多还原

【Abstract】 Natural compounds, e.g., derived from plants and fungi, which show various biological activities and chemical novelty, may be a source of potential drugs. In the search for antitumor leading compounds from high fungus, we screened the fruit bodies of 90 poisonous mushroom species of Basidiomycetes from Yunan, China, for the cytotoxic activity against human tumor cell lines by MTT bioassay.90 poisonous mushrom species belonging to fourteen genus such as Amanita, Agaricus, Boletus, Leccinum, Pulveroboletus, Suillus, Tylopilus, Cortinarius, Inocybe, Lactarius, Russula, Sarcodon, Hypomyces and Gomphus, eight families such as Amanitaceae, Agaricaceae, Boletaceae, Cortinariaceae, Russulaceae, Hydnaceae, Hypocreaceae and Gomphaceae, three Orders as Agaracales, Aphllophorales and Hypocreales, two classes as Hymenomycetes and Pyrenomycetes, two subphyla as Basidiomycotina and Ascomycotina were collected from Yunnan province, southwest of China. The results indicated that the most of lipid extracts including petroleum light and ethyl acetate extracts have significant cytotoxicity, whereas polar extracts including ethanol and water extracts have no clear cytotoxicity. It implied that the activity is associated with small molecular secondary metabolites not with high molecular weight. It was found that out of 90 investigated species 43 exhibited cytotoxic activity. Approximately 50% of screened crude extracts of mushroom showed cytotoxic activity while 8 species displayed strong cytotoxic activity with IC₅₀ （concentration that yields 50% growth of cancer cells）value of less than 35μg/ml. Among them, 35 species with cytotixicity were remained no chemical and biological reports till now. It appears therefore that further investigation of these groups of mushroom species is justified because they may constitute a potential source of chemotherapeutics.By bioassay-guided fractionation procedure using human tumor cell lines led to the isolation of two compounds from 2 species as Engleromyces geetzii P. Henn., Amanita flavorubescens affected by Hypomyces hyalinus. Structure elucidation of them have been identified as cytochalasin D and verticillin A, respectively. All of them showed strong cytotixicity against human tumor cell lines with average IC₅₀ values of 7.35 ×10^-6M and 6.6×10^-8M, respectively.In vitro test, Cytochalasin D showed antitumor activity against various human tumor cell lines shch as NB4, HL-60, K562, U937, KU812, SMMC 7721, BCAP37, HeLa, SK with an average IC₅₀ values of 7.35 ×10^-6M, antifungal properties against plant pathogenic fungi Fusarium oxysporium f.sp.with MIC values of 12.5μg/ml , insecticidal activity against Panonychus sp. And Myzus persicae （Sulzer）, whereas Veticillin A exhibited antitumor activity against various human tumor cell lines as K562, SMMC 7721, HepG2, BCAP37, MCF-7, HeLa, SPC-A1, DU145, SW620, Jurkat with an average IC₅₀ values of 6.6×10^-8M, antifungal properties against plant pathogenic fungi Botrytis cinerea with MIC values of 10μg/ml. NO insecticidal activity was found with Veticillin A. Both compounds can inhibit the proliferation of human cell lines through apoptosis.更多还原