Studies On The Technology Of Preparation Of Taxol And Taxol-producing Endophytic Fungus From Taxus Media Cv. Dark Green Spreader

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.