【作者】 张莲莲；

【导师】 谈锋；

【作者基本信息】 西南大学 ， 生物化学与分子生物学， 2007， 硕士

【摘要】 本试验主要以曼地亚红豆杉（Taxus media cv．Dark Green Spreader）为原料，研究了从曼地亚红豆杉（Taxus media cv．Dark Green Spreader）枝叶中提取分离紫杉醇的工艺，并对其产紫杉醇的内生真菌进行了筛选。1)曼地亚红豆杉枝叶中紫杉醇分离纯化工艺研究以曼地亚红豆杉（Taxus media cv．Dark Green Spreader）的枝叶为原料，采用渗漉法制备得到纯度为0.184％的紫杉醇浸膏，回收率为81.8％。再以获得的浸膏进行柱色谱纯化的研究，分别筛选了大孔离子交换树脂和硅胶柱层析的条件，使紫杉醇粗品的纯度分别达到2.01％、7.24％，回收率分别为95.1％、96.8％。利用高速逆流色谱对紫杉醇粗品进行了精制，采用的溶剂系统为正己烷：乙酸乙酯：甲醇：水=4：5：4：5，经过一次分离，由纯度为7.24％的紫杉醇粗品制备得到纯度为78.3％的紫杉醇，回收率为97.6％。进一步优化溴加成的方法，使色谱行为十分近似的紫杉醇和三尖杉宁碱得到分离，紫杉醇的纯度达到84.6％，通过脱色和重结晶技术，纯度达到98.5％以上，达到出口美国的标准。对从曼地亚红豆杉（DGS）枝叶中提取到的紫杉醇纯品进行质谱分析，发现其分子离子峰[M+Na]⁺=876.5，经计算其分子量与紫杉醇标准品（853.9）基本吻合，并对其进行生物活性检测，发现其能够诱导HaLe细胞凋亡，其IC₅₀值(1.43×10³ ng·mL^-1)与紫杉醇标准品的IC₅₀值(1.47×10³ ng·mL^-1)基本上一致。由于曼地亚红豆杉的DGS品种枝叶中紫杉醇含量明显高于Hicksii品种，后者作为紫杉醇原料已经通过美国FDA认证，前者尚未通过美国FDA认证，我们经过上述实验可以确定从曼地亚红豆杉DGS品种中提取分离的样品为紫杉醇，为紫杉醇生产中选用一个高含量品种提供了理论依据。2)产紫杉醇内生真菌的研究从曼地亚红豆杉中筛选到两株产紫杉醇的内生真菌，分别为XMB-3和XML-2，紫杉醇产量分别为10.87μg·L^-1和109.7μg·L^-1，经过形态学分析，得知它们分属于黑葱花霉属（Periconia Tode．ex Schw）和长梗串孢霉属（Monilochaetes Halst．ex Harter）。将XML-2进行大规模培养，提取得到其紫杉醇的纯品，对其进行质谱分析，发现其分子离子峰[M+Na]⁺=877.7，经计算得知，其分子量与紫杉醇标准品的分子量（853.9）仅相差0.8，初步确定为紫杉醇。对从内生真菌中提取分离的紫杉醇采用MTT法进行药理试验，发现可以诱导HaLe细胞的凋亡，其IC₅₀值为1.53×10³ ng·mL^-1，略高于紫杉醇标准品的IC₅₀值(1.47×10³ ng·mL^-1)，说明具有与紫杉醇类似的生物活性，为利用内生真菌发酵生产紫杉醇及其类似物提供了科学依据。更多还原

【Abstract】 The studies focused on the technology of preparation of taxol and selecting taxol-producing endophytic fungus from Taxus media cv. Dark Green Spreader.1 Studies on separation and purification of taxol from breaches and leaves of Taxus media cv. Dark Green SpreaderThe raw material, branches and leaves of Taxus media cv. Dark Green Spreader was extracted by percolation, and the content percent of taxol was 0.184% in the extraction. Its recovery was 81.8%. Then the extraction was purified by column chromatography, including the condition selection of macro porous ion-exchange resins and the silica-gel column. The purity were increased 2.01% and 7.24% respectively, with the corresponding recovery 95.1% and 96.8%.Refining the crude simple by high-speed counter-current chromatography （HSCCC） with a solvent system composed of n-hexane-ethyl acetate-methanol-water （4:5:4:5, V/V）, in a single operation, the purity of taxol increased from 7.24% to 78.3%, the recovery was 97.6%. By the optimal bromination technology, cephalomannine whose chromagraphy action is similar to taxol, and taxol were isolated, and taxol at 84.6% purity was gained, then following decoloration and recrystallization, the purity of taxol was over 98.5% quality for exporting to USA.The identification of taxol from Taxus media cv.Dark Green Spreader was analyzed by the mass spectrum. Its molecular ionic peak [M+Na]⁺was 876.5, which was the same as taxol standard（853.9）. Then investigated the effect of taxol on apoptosis in HeLa cell, the result indicted that the taxol of IC₅₀ value (1.43×10³ ng·mL^-1) was correspond to taxol standard (1.47×10³ ng·mL^-1).To be summarize, our research confirmed that the sample was taxol, extracted from Taxus media cv. Dark Green Spreader whose taxol content was higher than Taxus media var. Hicksii obviously, which was authenticated by FDA. That provided a theoretic evidence to select a high-content variety of producing taxol.2 Studies on taxol-producing endophytic fungusTwo endophytic fungus, XMB-3 and XML-2 have been already isolated from Taxus media, and the taxol content were 10.87μg·L^-1 and 109.7μg·L^-1 respectively, through the morphological analysis, each of them belonged to Periconia Tode. ex Schw and Monilochaetes Halst. ex Harter.The taxol sample was extracted from endophytic fungi of XML-2 which was cultivated cosmically, and its molecule ionic peak （[M+Na]⁺=877.7） was identified by mass spectrum, that was discrepant to taxol standard. Consequently, the sample was judged as taxol primarily.Subsequently, we investigated the effect of taxol on apoptosis of HeLa cell via MTT method, the result showed that it could induce the apoptosis of HeLa cell and its IC₅₀ value was 1.53×10³ ng·mL^-1, higher than taxol standard’s (1.47×10³ ng·mL^-1). This research manifested that the taxol sample refined from endophytic fungi had similar bio-activity to taxol standard, and it may provided a scientific evidence to produce taxol and its analogs by endophytic fungi.更多还原