论文标题:植物内生真菌抗真菌抗肿瘤活性的研究
Study on antifungal and anticancer activities of endophytes from plants
论文作者 李桂玲
论文导师 苏文金,论文学位 硕士,论文专业 微生物学
论文单位 厦门大学,点击次数 299,论文页数 61页File Size2729k
2001-06-01论文网 http://www.lw23.com/lunwen_1737782/ 植物内生真菌;抗真菌;抗肿瘤
endophytes;antifungal;anticancer
植物内生真菌是一个庞大的特殊真菌类群，次生代谢产物非常丰富，包括杀虫、抗菌、抗肿瘤等活性物质，是人们寻找新型抗菌、抗肿瘤药物的重要资源。本课题的工作主要包括南方红豆杉、三尖杉及香榧内生真菌的分离；内生真菌抗真菌、抗肿瘤活性菌株的筛选；以及对抗肿瘤高活性菌株H18-036的活性物质的初步研究。 从采集自福建省沙县、武夷山等地的三尖杉(Cephalotaxus Fortunei)、南方红豆杉(Taxus mairei)及香榧(Torreya grandis)的内皮层和韧皮部中，分离到172株植物内生真菌。抗真菌活性检测结果表明，90株内生真菌对一种或多种植物病原真菌——红色面孢霉(Neurospora Sp.)、木霉(Trichoderma sp.)、镰刀菌(Fusariumsp.)等有抑制作用，占供测真菌总数的52.3％。来自三尖杉、南方红豆杉和香榧的内生真菌的抗真菌活性菌株比例分别为40％，54.2％及57.1％。其中抑菌圈直径大于15mm的高抗菌株有35株。按Ainsworth等鉴定系统和方法进行鉴定，具有抗真菌活性的内生真菌主要分布于拟青霉属(Paecilomyces)、镰孢菌属(Fusarium)等17个属中。 利用抗肿瘤体外细胞毒筛选模型(MTT法)对内生真菌进行活性检测。结果表明，共有25株内生真菌(占总分离菌株的14.5％)对KB(人口腔上皮癌)或HL-60(人白血病)细胞具有显著的抑制活性。三尖杉、南方红豆杉及香榧内生真菌的抗肿瘤活性菌株占各供测菌株的3.3％，19.6％和8.6％。其中7个菌株的细胞毒ID_50在1,000以上，占总供测菌株的4.1％。菌株的初步鉴定结果显示，抗肿瘤活性菌株主要分布在拟青霉属及头孢霉属(Cephalosporium)中。 在抗肿瘤高活性菌株中，菌株H18-036对KB及HL-60细胞有明显抑制作用，初步鉴定为一种拟青霉(Paecilomyces sp.)。对其发摘要－11－酵液活性物质进行分离纯化，得到一个结晶及5个纯度较高的组分。该结晶与＊－036发酵液有机抽提所得结晶布雷菲德菌素A具有相同的Rr值、显色反应，初步判定为布雷菲德菌素A。5个纯度较高的组分经薄层层析检测及官能团显色反应，初步断定为生物碱类物质或含有氨（胺）基且易被氧化物质。MTT测定结果显示，至少有3个组分对HL－60细胞具有较高的抑制活性，其ID。。分别为800，＞2，000，800，具有深入研究的价值。
Study on antifungal and anticancer activities ofendophytes from plantsAbstractEndophytes constitute a large, special fungal group with abundant secondary metabolites, including insecticidal, antimicrobic and antitumor agents. They are important resources for new antimicrobic and anticancer drugs. It"s reported here the isolation results of endophytes from Taxus fflairei, Cepha]otaxus fortune] and Torreya grandis; the screening results of antifungal and anticancer strains; as well as the primary study of antituznor agents from anticancer strain H18-036, which has high activity.One hundred and seventy-two strains of endophytic fungi were isolated from Taxus zz~irei, Cephalo taxus fortune] and Torreya grandis. The result of the antifungal assay shows that ninety strains of the fungi have antagonism against one or more botanic pathogenic fungi, such as Neurospora sp., Trichoderiz~ sp. and Fusarium sp. etc. The percentage of antifungal strains to tested strains are as follows:40% Cephalotaxus fortune], 54.2% Taxus mairei, 57. 1% Torreya grandis Thirty-five strains have high antifungal activities with their inhibition zone diameter beyond l5rrm. The active endophytes are identified as seventeen genera, most of which belong to Paeci]omyces and Fusarium etc.Using the method of MTT(Methyl Thiazolyltetrazolium), these endophytes were screened for anticancer strains in vitro. The result shows that twenty-five strains(about 14.5% of the total strains isolated) can inhibit the growth of KB or HL-60 cells significantly. 3. 3% of endophytic fungi isolated from Cephalotaxus fortune], 19. 6% from Taxus n~ire], and 8. 6% from Torreya grandis have cytotoxic activities. Seven strains have their ID~ beyond 1,000, which constitute about 4. 1% of the total strains tested. The active strainsobtained mostly belong to Paecilowyces and CephalosporiuzizAmong all the highly active anticancer strains, strain H18-036, which was identified as Paeci]~yces, can inhibit the growth of HL-60 and KB cells significantly. Its culture product was isolated and purified, and one crystal with other five pure components were obtained. TLC(Thin Layer Chromatography) test results show that the crystal has the same R~ value and coloring reactions with crystal Brefeldin A, which was obtained by direct extraction from culture medium of strain H18-036. Through TLC and coloring reactions, another five components were initially identified as alkaloid or ainidocyanogen agents, easy to be oxidized. At least three of them are found to highly inhibit the growth of HL~-60 cells with the method of MTI7, and their ID~are as follows: 800, >2,000, 800. They are worthy of further study.
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