| Taxol(Paclitaxel) as natural diterpenoid was isolated from the plants of Taxus spp. It’s being thought as a new class of microtubule inhibitory activity to use as anticancer drug.The mechanism of taxolisunique, and its adaption to clinical cancer showed wider scope. Thus, taxol is considered as the most important discovery of anticancer drugs. Up to now, the commercialization of paclitaxel and its important semi-synthetic precursor 10-deacetyl baccatin Ⅲ(10-DAB Ⅲ) are mainly extracted from plants of the genus Taxus spp.But the content of these substances in plants is very low. Since an endophytic fungus obtained from the branches of the Pacific yew(T. brevifolia) has been found that could produce taxol, numerous investigations focused on endophytic fungi producing taxol and its analogs from Taxus spp and made greatly progress. Although the research reported on these endophytic fungi in China and abroad has gradually increased, it is still lack of stable, efficient and high yield strains which can be applied to industrial fermentation.Therefore, it is needed to found efficient endophytic fungi and optimize the fermentation conditions, to improvethe methods about the analysis and detection of taxol and its analogs in the fermentation broth of endophytic fungi, and to further search for craft of the separation and purification of taxol and its analogs in endophytic fungi.The purpose of this dissertation was to obtain high yield of fungiproducing taxol. Thus, we isolated the endophytic fungi from different parts(root, stem and leaf) of T. chinesis(Taihang yew) in different habitats and tested activity of these fungi producingthe taxol. As the results,186 strains of endophytic fungi were isolated from three parts of T. chinesis, in which the number of strains from roots, stems and leaves were 93, 67 and 26, respectively. Among them, 110 strains were isolated from wild T. chinesis, whereas 76 strains were isolated from wild liked T. chinesis.The endophytic fungi from the root of the wild type were more abundant and efficiency.The average content of taxol in the fermentation broth of endophytic fungi in root, stem and leaf were 248.57, 149.09 and 104.94 μg/L, respectively.The highest content of taxol in endophytic fungi was 622.75μg/L. Furthermore, detection methodsof the qualitative and quantitative of taxol in fermentation broth were studied.C18 solid phasecolumn(SPE) was used to extract the taxol, and then different concentrations of methanol and ammonium acetate was used to elute, at last high performance liquid chromatography(HPLC) was used to analyize the content of taxol.The results indicated that optimum conditions of HPLC analysis were the mobile phase of methanol: water: acetonitrile(V/V/V) = 20:45:35, flow rate and the detection wavelength for 0.70 m L/min and 227 nm, respectively; and the better effect and ability was 80% methanol, meanwhile, the recovery rate of taxol can be up to 87.6%.Therefore, this study established a fast, high efficiency and low cost SPE-HPLC method to qualitative and quantitative analysize the content of taxol in fermentation liquor of fungi.Then, the purification process of taxol in the fermentation liquor of endophytic fungi was studied.The nonpolar D4020 macroporous resin was used to test adsorption and separation effect of taxol in the fermentation liquor of endophytic fungui. The results showed that D4020 resin had better adsorption and separation effect. The purification process was optimized that the sample was dissolved in 50% methanol/water in room temperature, and the liquid in column was 1.5 m L/min when upload the sample, and 80% ethanol/water solution with a flow rate of 0.5 m L/minwas used to elute the sample in column, and the eluent volume was 7 times of resin volume, the recovery rate of taxol can be up to 90%.In this dissertation, endophytic fungi producing the 10-DAB Ⅲorigin from T. wallichiana var. mairei were investigated. 102 endophytic fungistrains were isolated and identified from root bark of T. wallichiana var. mairei. Though HPLC and LC-MS analyses one fungus strain named as Trichoderma sp. IRB54 producing 10-DAB Ⅲ was found. The content of 10-DAB Ⅲ in PDB cultivating of IRB54 was 187.56 μg/L. After that this chromatographic peak was isolated and purified from fermentation liquor of strain IRB54, and found that 1H NMR spectra data of this chromatographic peakwas the same with standard 10-DAB Ⅲ. It was confirmed that IRB54 can produce 10-DAB Ⅲ and provides a new resource of 10-DAB Ⅲ. The methods of rapid qualitative and quantitative analyses of Ⅲ 10-DAB in the fermentation liquor of endophytic fungi were also studied.The thin layer chromatography(TLC) with different proportions of three system: acetone/petroleum ether, hexane/chloroform/ethyl acetate/methanol/trimethylamine, and cyclohexane/ethyl acetate was used to analyze the quantitative content of 10-DAB Ⅲ. The HPLC analysis was combined to validate the content. It was found that the best condition for TLC analysis was the acetone/petroleum ether =4:1, and with the ethanol/sulfuric acid =9:1 as the chromogenic agent, the minimum detection limit was 0.3μg. Thus, a method of rapid qualitative and quantitative analysis of 10-DABⅢ in the fermentation liquor of endophytic fungus was established by TLC-HPLC. The content of 10-DABⅢ was compared with different conditions, including extraction from branches and the callus of T. wallichiana var. mairei, fermentation broth of Trichoderma sp.IRB54, fermentation of endophyte together with branches, and fermentation of endophyte together with callus. The results indicated that the content of 10-DAB Ⅲ in above mentioned conditions were 274.6, 154.3, 189.4, 887.5, and 684.7 μg/m L, in which the highest content of 10-DAB Ⅲ was up to 887.5μg/m Lin fermentation of endophyte together with branches. These studies laid the foundation for the development and utilization of endophytic fungiproducing Ⅲ 10-DAB.This paper also reviewed the discovery andresources of taxol, and summarized the research progress of endophytic fungiproducing taxol. Furthermore, the progress of terpenoids from endophytic fungi were reviewed, and summarized 274 terpenoids, including seven monoterpenoids(compounds 1-7), 69 sesquiterpenoids,(compounds 8-76), 38 diterpenoids(compounds 77-114), 160 meroterpenoids(compounds 115-274). And the biological activity of these terpenoids werealso summarized. In conclusion, this summary provide reference for the further research of terpenoids in endophytic fungi. |
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