Analysis And Extraction Of Paclitaxel And Study On Fingerprint Chromatograph From Cultivated Taxus Chinensis Var. Mairei

The plant of taxus chinensis var.mairei,who was also named taxus mairei,originated from gymnospermae and taxaceae and taxus.It is distributed as one of the most wide plant in the taxus. Taxus chinensis var.mairei distributed mainly in Yangtze River Basin,nanling Mountains,Henan Province,Shaanxi Province,Gansu Province and Taiwan Province etc.in mountains or valley.And it was too characteristic species in subtropical evergreen broadleaved forest and evergreen and deciduous broadleaved mixed forest.Taxus chinensis var.mairei mixed and grew in other broadleaved tree,bamboo and conifer.Taxus chinensis var.mairei distributed at 800m～1600m altitude,resources reserves of taxus chinensis var.mairei.was more than the other species. Paclitaxel was isolate from taxus in 1971 year old.It became the emphasis on antitumor national drug.Taxus chinensis var.mairei became the main tree species which contained paclitaxel.Paclitaxel was very little in taxus chinensis var.mairei.The content range of its was 0.003%-0.008%,it include the impurities of plant rap,pigment and balsam etc.Especially many taxoids were similar with paclitaxel in molecular structure and physicochemical properties.They is a serious interference to determination of paclitaxel by Reversed-Phase High Performance Liquid Chromatography.In order to accurately determine the contents of paclitaxel in taxus chinensis var.mairei.Optimization of chromatographic conditions was follow:SHIMADZU C₁₈ column (250mm×4.6mm ID,5μm),Using methanol- water-acetonitrile as mobile phase(22:32.5:45.5) and ultraviolet detection wavelength 227nm,Linear Equation of paclitaxel was Y=2.152968×107X+1773(r=0.9999)(n=5).It presented a good linear relation with the peak area in the range of 0.0004376mg·.mL^-1～0.004376mg·mL^-1(n=5).The detection limit for paclitaxel was 0.001050μg.And the limit of quantification was 0.003501μg.Results show that the minimum concentration of paclitaxel can meet the requirement of RP-HPLC determination.The optimized method was used to handle the sample of the taxus chinensis var.mairei,which was planted in artificial system.After the collected samples of taxus chinensis var.mairei,were dried.They were extracted 2 times with 6 times(W/V) amount of ethanol..then extract solution was obtained and merging.After it was concentrated.They were solvent extraction 3 times with 6 times(W/V) amount of dichloromethane,then extract solution was diluted by methanol.The method was verified by recovery rate.at the same time 10 batches samples were determined by RP-HPLC.The results showed that this method was rapid,Simple and reliable.Variational rule of the paclitaxel who existed in different parts of taxus chinensis var.mairei. artificially planted.Paclitaxel was regarded as the parameter of quantitative determination.By using four factors five levels orthogonal experiment(L16(4⁵)).The test of age,season(a random month of four seasons was selected) and different parts were optimized and chosen in taxus chinensis var.mairei.The result showed that age was the most important factor,different parts was secondly important factor,seasonal influence was also significant(P=0.05).Multiple comparison showed that T9(A₃B₁C₄) was the best.When 6-year- old leaves of taxus chinensis var.mairei. Which were collected in January was the best choice.To evaluate the content of paclitaxel in Taxus chinensis var.mairei processed by different drying methods,natural drying,decompression drying and microwave drying were adopted.By the comprehensive survey of drying methods and various drying condition.The results showed that suitable temperature of decompression drying was 60℃.The best choice of microwave drying was low fire.However the obvious degradation of paclitaxel was caused by the using of method of natural drying,or the other drying methods.They should not be selected.To optimize and establish the extractive technology of paclitaxel from taxus chinensis var.mairei..Methods RP-HPLC was applied to determine the content ofpaclitaxel.The orthogonal test was adopted to examine the effects of the 4 factors consisting of ethanol concentration,ethanol volume,extractive times and extractive time.Results Ethanol concentration and ethanol volume were essential factors influencing the extraction of paclitaxel.High concentration and amount of ethanol facilitated the extraction.Extractive times and extractive time were also important.By single 2 factorial effect,2 h and 2 times in each extraction of taxolwere the best.Conclusion The optimum extractive condition is T9(A₃B₃C₂D₁) and T₈(A₃B₂C₁D₃).During the development the traditional Chinese medicine.The research of chromatographic fingerprint is one of the hot spots of the traditional chinese medicine modernization,in the process of test.11 batches artificial planting of taxus chinensis var.mairei was collected,studied the chromatography condition optimizing included column,detection wavelength and mobile phase. And verified the methodology of chromatographic conditions such as instrument precision, repeatability and stability.At the basis,constructed chromatographic fingerprint,Including calibration of common peak,selection of standard peak,determination of the relative retention time as well as that of the relative peak area.The optimized chromatographic Conditions showed that SHIMADZU C₁₈ column(250mm×4.6mm ID,5μm),ultraviolet detection wavelength 227nm.Using acetonitrile-water-as mobile phase and gradient elution.According to the result of chromatographic conditions,the instrument precision,sample epeatability and stability was tested.In the result of high efficacy liquid chromatography,the RSD of the relative retention time as well as that of the relative peak area of the shared peak was lower than 3.0%,showed that it was feasible measures.According to chromatographic fingerprint,17 shared peaks were determined,and NO.9 was the reference peak.The RSD of the relative retention time as well as that of the relative peak area of the shared peak was lower than 2.0%,The method attained the reliable results.The percentage of shared peak area was more than 4.1%and less than 8.3%, coincided with the requirements.The similarity was analysis by the included angle cosine method and correlation coefficient method,and were all≥93%,showed that the technical index of fingerprint established was stability,selected chromatograms can make a relaible judgement to the group characteristics of the leaves and branches of artificial planting of taxus chinensis var.mairei.