| Taxanes are a group of specific diterpenoid compounds with a pentamethyl [9.3.1.0] 3,8 tricycopentadecane taxane skeleton including taxol and taxotere, a type of the most effective anticancer drugs. There are over 350 members in this family, classified according to their structural differences. Each class of the taxanes has different polar and pharmaceutical properties and special biological activities. It is very difficult to synthesize taxanes by chemical means. The increasing clinical demands for active taxanes and the exhausted supply of natural Taxus, initial source of taxanes, have spurred to produce these drugs or useful precursors by means of large-scale Taxus cell culture and Taxus planting, which however were hampered by low and unstable taxol yields. Presently, the manipulation and regulation of the metabolic pathway at molecular level for the Taxus cell culture system are believed to be a promising approach to increase the yields of taxol and its related taxanes. Any attempt to manipulate genes of the metabolic pathway requires a full understanding of taxol biosynthesis, including key enzymes, reactions, occurrent sites, and mechanisms of exudation, transport and storage after syhthesis. The dissertation presented here emphasized the study of taxol biosynthesis on metabolic profiling analysis of Taxus chinensis , and the new approaches to improve the yields of taxol and related taxanes in plant cuttings as well as in extracts by incubating clipplings and selective oxidation-hydrolysis were also studied. Firstly, the methods to determine minim taxanes which present in methanol extracts of Taxus chinensis plant or cell cultures were established. Solid-phase extraction (SPE) method was used for sampling after plant materials were ground with the help of liquid nitrogen and then extracted by methanol. And the chromatographic conditions of a C18 column (Hypersil ODS2 , 250 mm ×4.60 mm, 5 μm), the mobile phase being composed of acetonitrile-methanol-water, which eluted isocratically at flow rate 1 ml/min) and detection at 227 nm were employed. The calibration curve showed a good linearity in the mass concentration range of 2.0～180 mg/L for taxol (r= 0.9993), 2.0～166.0 mg/L for 10 –DAB-III(r=0.9975), 6~170 mg/L for B III (r=0.9937), 7.0～200.0 mg/L for B-VI(r=0.9921), 1.0～130 mg/L for DAB-I(r=0.9945), respectively. The detection limits of this method were 0.5～4.5 mg/L. The recovery for taxol was 98% with relative standard deviation (RSD) 1.5%.The inter days RSD of this method was 1.8% and between days 2.5%. The characteristic chromatograms including taxane compounds as more as possible were obtained following the above sampling methods and the chromatographic conditions. The metabolic profiling of the taxanes presented in Taxus chinensis cell culture extracts were characterized. About 13 taxane compounds were identified or characterized by means of liquid chromatography mass. Eight of them were identified as taxuyunanine C , yunnanxane , 2α,5α,10β-triacetoxy-14β-propionyloxytaxa-4(20),11-diene, 2α,5α,10β-triacetoxy-14β-(2-methyl)butyryloxytaxa-4(20) 11-diene , taxol , and Baccatin III, DAB-I, B-VI, respectively. Others were given the molecular weight, taxane skeletons, types and numbers of substituents. The dynamic profiling analysis of the taxanes by methyl jasmonate (MJ) elicitation and fungal elicitor revealed that the cell lines had the capability of production of a large assortment of other compounds possessing positionally different acylation patterns, especially formation of taxanes acetylation at C14-side, which may lead to the low production of taxol. The metabolism and accumulation of taxanes in plants was also investigated. It was found that (1) the needle of yew was a important organ or part to biosynthesize taxanes; (2) the synthesis of taxanes seemed to be non-tissue specificity; (3) taxanes accumulation tended to be polar trend, the non-polar taxanes preferred to accumulation in the sites of plant where have thick cell walls such as bast, in contrast, and polar taxanes could be detected frequently in leaves; (4) the distribution of taxanes in a part of plant related to the changes of seasons; (5) high content taxol often were found in the areas where Taxus good-growth. The variations of taxane contents in plant after harvest were also studied. It was observation that the accumulation of taxol in needles of the clippings was found increase in the initial stages of the stored period and then decreased gradually, the variation was related to the manner of preservation, timing and plant tissue, indicating taxol biosynthesis in response to mechanical wounding of harvest. Based the results, a novel method for in vitro incubating Taxus clippings to improve taxol yields was proposed. An application of selective oxidation-hydrolysis reactions was conducted to convertxyloside or glucoside taxanes into corresponding free taxanes for enhancement of taxol and it related taxane yields. With the new method, recovery of 168% could be achieved for taxol, 142% for 10-DAT, indicating the significance and potential of the method to make full use of plant sources.
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