【作者】 孙磊；

【导师】 袁继超； 范巧佳；

【作者基本信息】 四川农业大学 ， 作物栽培学与耕作学， 2009， 硕士

【摘要】 川牛膝(Medicinal Cyathula Root)为苋科(Amaranthaceae)杯苋属(CyathulaBlume)多年生草本植物川牛膝(Cyathula officinalis Kuan)的干燥根,为川产道地常用中药材,以四川雅安的天全、宝兴和乐山的金口河区产量最大。近来由于品种的退化和混杂、栽培年限及栽培方法的改变等原因,导致产品质量变异较大,正确评价川牛膝药材质量对指导品种选育与提纯复壮、科学栽培和正确用药均有重要意义,为此本文通过试验研究,建立了一套方便快捷的川牛膝药材HPLC指纹图谱构建方法,并用其对不同品种类型、不同产地、不同栽培年限、产品不同部位等样品的整体质量进行了评价,结果如下:1.以金口河4年生川牛膝作为供试样品,对不同色谱柱、测定波长、流动相、梯度洗脱程序、柱温、流速等色谱条件进行了比较研究,得出了优化的色谱检测条件;同时比较分析了不同提取溶剂、提取方法和条件等对样品提取效果的影响,得出了样品的最佳提取方法。对试验得出的样品提取方法和优化的色谱条件进行方法学验证的结果表明,各项试验的主要共有峰的保留时间和相对峰面积均无显著变化,RSD均分别小于0.5%与3.9%,系统稳定性和重复性良好,仪器精密度较高。2.从雅安天全县、宝兴县、荥经县、峨眉金口河区等川牛膝主产区收集了27批不同来源(包含不同栽培地点、品种类型和栽培年限)的川牛膝样品,并从成都大丰药材市场购入4批川牛膝药材和饮片,以上述31个样品为材料,根据优化后的样品提取方法和色谱条件,检测得到了川牛膝药材指纹图谱,确定其中10个共有峰作为指纹图谱的特征组分,并对其中的杯苋甾酮色谱峰进行了鉴别,建立了其共有模式特征指纹图谱。31个样品的色谱峰分离效果好,符合指纹图谱技术有关规定,其全谱与共有模式指纹图谱的相似度较高,在0.791～0.969之间。3.对31个样品的HPLC指纹图谱进行系统聚类,结果表明,川牛膝的品质具有一定的地域性特征,来自同一地区的川牛膝样品大多聚在一群之中。但天全两路乡的6批样品分别聚于3群中,表明其种源混杂严重。4.26个参试样品中,天全三年生大白牛膝T17的品质最高。宝兴一年生大白牛膝T11的品质最低。就杯苋甾酮含量而言,三年生白牛膝以金口河品质最高、宝兴次之、荥经最低;三年生大白牛膝和红牛膝均以天全产最高、宝兴次之、荥经最低;就所有检出成分的总量而言,则三年生白牛膝和大白牛膝均以天全产最高、宝兴第二、荥经最低;三年生红牛膝以荥经最高、宝兴第二、天全最低。5.就所有检出成分的总量的含量而言,宝兴和天全两地均以白牛膝最佳,其次为大白牛膝,红牛膝最差;但如果仅以杯苋甾酮含量作为品质衡量标准,则宝兴产川牛膝以白牛膝最好、红牛膝最差,而天全产川牛膝则以大白牛膝为最佳。6.川牛膝根不同部位的质量有差异,从总体上看,两年生牛膝以侧根的杯苋甾酮含量和所有检出成分的总量较高,其次为主根的两端,中部的杯苋甾酮含量略低。三年生川牛膝杯苋甾酮含量以主根下部为最高。7.将不同产地(居群)的种子集中同时种植于天全县两河乡新沟村时,其两年生川牛膝中杯苋甾酮含量和所有检出成分的总量均以种子来源为天全紫石乡(居群)和天全县小沟村(居群)的最高;通过比较分析还发现,川牛膝对环境的适应性不强,栽培环境的变化会造成质量的波动。更多还原

【Abstract】 Medicinal Cyathula Root is the dry root of Cyathula officinalis Kuan Plant,which is one kind of Perennial herb,Amaranthaceae,Cyathula Blume.Cyathula officinalis Kuan mainly distributes in Si’chuan,Yun’nan and Gui’zhou province.No matter wild or cultivated,most of them are growing in the districts above 1500m altitude.And especially the the areas of Si’chuan Tian’quan,Bao’xing,Han’yuan and Jin’kouhe own the maximun output.Recently,as a result of the degradation and hybridization of varieties,planting limit, changes of cultivation methods and other factors,there are huge heteromorphosis in the product qualities.Exact evaluation of the CyathuIa offcinalis Kuan plays a sinificant role in guiding scientific cuItivation,breeding purification and rejuvenation.Therefore,this paper established HPLC fingerprint building method for checking the quality of Cyathula offcinalis Kuan just by experimentation,and evaluated the whole quality of the samples, which are quite different in varieties,producing areas,planting limit,and so on.The evaluating results are as follows:1.Set the 4-year-old Cyathula officinalis Kuan from Jin’kouhe as a sample,the paper compared and optimized the chromatographic method which consist of column,detection wavelength,mobile phase,elution program,column temperature,and flow rate.And also obtained the best sample extraction method through analyzing the impact led by the different extraction solvents,method and conditions.As a result of the method validation, the retention times and relative peak areas of the main shared peaks had no significant changes in the test.Their RSDs were all less than 0.5%and 3.9%respectively.And the System stability and repeatability,equipment precision are all satisfactory.2.Using the optimized extraction methods and chromatographic conditions,studied 31 batchs of samples,which including 27 batchs collected from Tianquan,Baoxing, Yingjing,Jinkouhe and 4 batchs bought from dafeng market in chengdu.Using the optimized extraction methods and chromatographic conditions,studied 31 batchs of samples,which including 27 batchs collected from Tianquan,Baoxing,Yingjing,Jinkouhe and 4 batchs bought from dafeng market in chengdu.The chromatogram showed that the peaks were separated well and accorded with the fingerprint of the criterion.Among 31 samples 10 shared peaks were indentified,which also are special fingerprint peaks.Then, the author using the optimized extraction methods and chromatographic conditions,studied 31 batchs of samples,which including 27 batchs collected from Tianquan,Baoxing, Yingjing,Jinkouhe and 4 batchs bought from dafeng market in chengdu.The chromatogram showed that the peaks were separated well and accorded with the criterion of the Fingerprint,and the similarities are between 0.791 and 0.969,satisfactory.Among 31 samples 10 shared peaks were indentified,which also are special fingerprint peaks.And then,the Cyasterone peak was been located and the HPLC Fingerprint was been built up.3.The recult of the cluster analysis during the 31 batchs of samples shown,The quality of Cyathula officinalis Kuan certain regional characteristics,and most of samples from the same region are gathered in the same group.However,the 6 batchs from Lianglu distributed in 3 groups,maybe,their provenance were serious mixed.4.Cyasterone as a mark,in three-year Cyathula officinalis Kuan,the Bainiuxi (vernacular name,a kind of Cyathula officinalis Kuan) from Jingkouhe is the best, Baoxing followed,Yingjing is poor;the best Dabainiuxi and Hongniuxi are from Tianquan, the weakers are from Baoxing,and the worsts are from Yingjing.On all of the methanol-soluble composition,the three-year Bainiuxi and Dabainiuxi from Tianquan are the best,Baoxing followed,the worsts are from Yingjing;the three-year Hongniux from Yingjing has the best quality,followed by Baoxing,Tianquan is the most bad.5.All of the methanol-soluble composition as a mark,in Baoxing and Tianquan,the Bainiuxi is best,followed by Dabainiux,Hongniuxi is in the end;However,set Cyasterone as a mark,Binniux from Baoxing and Dabainiuxi from Tianquan are the best.6.Also,the qualities are different among different parts of the Cyathula officinalis Kuan root.Overall,in two-years root,Cyasterone and all of the methanol-soluble composition in lateral roots are the richest,followed by the two ends of the main root,the middle is slightly lower.In three-years root,Cyasterone in the bottom of main root is the highest.7.The author planted the seeds from different populations together at Xingou Tianquan.And the best quality appears from the Zishi(Tianquan) and Xiaogou(Tianquan) populations;after comparing and analyzing,the author also found that their ecological adaptability are so weak.And the living environments’ change can cause the quality swinging.更多还原