【作者】 杨宪；

【导师】 杨水平；

【作者基本信息】 西南大学 ， 森林培育， 2009， 硕士

【摘要】 南方红豆杉（taxus chinensis vaF.mairei）,又称美丽红豆杉,为裸子植物门（Gymnospermae）红豆杉科（Taxaceae）红豆杉属（Taxus）植物。为红豆杉属植物中分布最广泛的一种,主要分布于长江流域、南岭山脉山区及河南、陕西、甘肃、台湾等省的山区或溪谷,它是亚热带常绿阔叶林、常绿落叶阔叶混交林的特征种,常与其他阔叶树、竹类以及针叶树混生,分布于海拔800～1600米,资源储量相对较红豆杉其它各种多。自1971年从红豆杉中分离出紫杉醇后,它已成为研究抗肿瘤天然药物的重点,南方红豆杉更成为我国紫杉醇药物生产的主要树种。南方红豆杉中紫杉醇含量甚微,其含量范围仅仅在0.003%～0.008%之间,含有大量的植物腊、色素和树胶等杂质,特别是其中共存许多分子结构和理化性质与紫杉醇极其相近的紫杉烷系列化合物,而这些成分会严重干扰紫杉醇的反相高效液相色谱法（RP-HPLC）测定。为了能准确测定紫杉醇含量,试验优选了RP-HPLC测定紫杉醇含量的色谱条件,即:用SHIMADZU C₁₈色谱柱（250mm×4.6mm ID,5μm）;甲醇-水-乙腈（22:32.5:45.5）为流动相,紫外检测波长为227nm。紫杉醇对照品溶液浓度在0.0004376mg.mL^-1～0.004376mg.mL^-1范围内,线性方程为:Y=2.152968×107X+1773（r=0.9999）（n=5）;相关系数r=0.9999（n=5）时,与峰面积线性关系良好;通过检测限（紫杉醇进样量为0.001050μg时）和定量限（紫杉醇进样量为0.003501μg时）,确定了紫杉醇对照品溶液最小浓度也符合RP-HPLC的检测要求。对南方红豆杉人工林材样品制备优化处理如下:将采集的南方红豆杉整株样品干燥,用6倍量（W/V）的乙醇浸提两次,合并提取液,减压浓缩至干;再用6倍量（W/V）的二氯甲烷液液萃取三次,合并萃取液,减压浓缩至干,残渣加色谱甲醇溶解,制成南方红豆杉样品溶液;用加样回收方法考察了该方法的可行性。同时随机采集了10批南方红豆杉药材样品,进一步验证了建立的RP-HPLC检测紫杉醇含量方法的可行性,结果表明该方法快速、简便、可靠,重现性好,能作为人工种植南方红豆杉中紫杉醇含量检测方法。在对重庆市忠县的南方红豆杉人工林材各部位紫杉醇含量变化规律研究过程中,以紫杉醇含量为指标,采用正交试验,通过4因素5水平交表（L16（4⁵））,对南方红豆杉影响因素年龄、季节（以随机选取各季节的一个月份作为代表）、不同部位3因素进行了优选试验。结果表明:南方红豆杉中紫杉醇含量主要影响因素是年龄,其次是不同部位,采收季节对紫杉醇的含量影响也是显著的;通过多重比较,确定T9（A₃B₁C₄）效果最好,即在1月份时采用6年生人工种植南方红豆杉叶,为优化选出的紫杉醇最佳采集时机,再结合红豆杉生产实际情况,采集6年生的枝叶作为紫杉醇的试验对象。干燥方法对南方红豆杉中紫杉醇含量的影响研究中,采用自然风干、减压干燥和微波干燥3种干燥方法,综合各干燥方法中的不同干燥条件,试验结果表明:减压干燥宜选用60℃;微波干燥低火;而自然风干和其它干燥方法可能造成紫杉醇显著降解,均不宜取。优化并建立红豆杉枝叶中紫杉醇的提取工艺,充分考虑了工业化生产的可行性。以使用RP-HPLC测定的紫杉醇含量为指标,采用正交试验法对提取过程中乙醇浓度、乙醇用量、提取次数、提取时间4因素进行优选试验。结果表明:从红豆杉中提取紫杉醇的最主要影响因素是乙醇浓度及乙醇用量,高浓度、适当多的用量有利于提取;提取时间和次数也极为重要;单因素效果分别以每次提取2h和共提取2次为最佳。最终T9（A₃B₃C₂D₁）和T8（A₃B₂C₁D₃）,即采用8倍量（W/V）95%乙醇提取2次,每次提取2小时,或采用6倍量（W/V）95%乙醇提取1次,提取4小时,均为优化选出的紫杉醇最佳提取工艺条件。在中药的开发过程中,色谱指纹图谱研究工作是国内中药现代化研究的热点之一,为中药材建立相应的指纹图谱是现阶段控制中药内在质量的有效手段。试验中共收集到人工种植南方红豆杉枝叶药材11批、研究过程中考察了色谱条件的优化,包括色谱柱、检测波长和流动相的优化选择;同时参照国家食品药品监督管理局对中药材指纹图谱的要求,对色谱条件进行了方法学验证,即仪器精密度试验、样品重复性和稳定性试验;在此基础上构建了色谱指纹图谱,包括:共有峰的标定、参照峰的选择、共有峰相对保留时间及积分相对比值的确定、非共有峰占总峰面积的百分比、共有峰的相似度计算,色谱条件的优化结果表明,选用C₁₈柱,检测波长为227nm,以乙腈和水为流动相进行梯度洗脱,为最佳色谱条件。根据色谱条件的优化结果,对仪器精密度、样品重复性和稳定性进行试验,高效液相色谱测定结果中,共有峰相对保留时间和相对峰面积的RSD都小于3.0%,表明采用高效液相色谱法进行色谱指纹图谱的研究是切实可行。通过色谱指纹图谱的构建,确定了17个共有峰,以9号峰作为参照峰;共有峰相对保留时间和相对峰面积的平均RSD都小于2.0%,说明试验结果可靠;共有峰面积百分比在4.1%～8.3%,符合指纹谱研究规定小于10%的检测要求;采用相关系数法和夹角余弦法对共有峰的相似度进行了计算,两种计算方法结果全图谱相似度均≥93%,表明所建立指纹图谱的技术指标稳定。选定的指纹图谱可对人工种植南方红豆杉枝叶药材组分群体特征的一致性作出可靠的判断。更多还原

【Abstract】 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.更多还原