【作者】 曹越；

【导师】 罗颂平；

【作者基本信息】 广州中医药大学 ， 中医妇科学， 2010， 博士

【摘要】 目的：对补肾安胎的基本方剂,源自清代名医张锡纯《医学衷中参西录》的寿胎丸,经过不同提取方法的考察,不同纯化方法的考察,经过薄层图谱的预试,明确提取液的主要化学成分,进行高效液相色谱研究,对不同流动相、洗脱梯度、色谱柱、检测波长、仪器等条件进行筛选和优化,完成了系统的方法学考察,建立了寿胎丸成品的HPLC质量控制方法,成功构建寿胎丸指纹图谱。方法与结果：实验一：根据寿胎丸处方的药物组成,各类药物的有效成分,考察乙醇提取、蒸馏水提取、乙醇提取加蒸馏水提取、乙醇提取后蒸馏水提取四种方法,用高效液相色谱的方法分析各提取液供试品。结果：结合药效及图谱信息综合分析,醇提加水提液之指纹图谱色谱峰数量,大小与峰形较好,提取的寿胎丸组方的有效成分更完整,同种成分的含量更多,因此,选择醇提加水提为寿胎丸指纹图谱的提取方法。实验二：通过薄层液相色谱TLC法,对寿胎丸供试品及单味药材菟丝子、桑寄生、续断进行分析,通过薄层色谱的方法对提取液的化学成分进行粗略监控,明确其主要成分,针对成品中所含有效成分的结构特点及理化特征,摸索高效液相的色谱条件。结果：按照寿胎丸模拟生产流程及寿胎丸供试品溶液的制备项制备的寿胎丸供试液及单味药材菟丝子、桑寄生、续断,以芦丁,槲皮素,槲皮苷,金丝桃苷,山柰酚,川续断皂苷Ⅵ,齐墩果酸等对照品作为参考,在薄层色谱TLC预试中均能看到与大多数对照品相对应的斑点,可证明寿胎丸及单味药材含有以上对照品的成分,因此可根据上述成分的性质大致选择合适的液相条件,为指纹图谱的研究提供物质基础。实验三：采用高效液相色谱法分析寿胎丸供试液,对供试品的预处理方法进行比较研究,考查甲醇(乙腈)—水、甲醇(乙腈)—醋酸水、甲醇(乙腈)—磷酸水多种色谱系统和梯度洗脱程序,以出峰多、分离度好,并确保主要化学成分在指纹图谱中出现为标准,来最终确定了HPLC的洗脱溶液及洗脱梯度。选取了210nm、230nm、325nm、355nm、256nm在60min切换为210nm5个波长进行检测,根据寿胎丸中主要成分的结构及UV紫外扫描光谱图进行分析和各波长的色谱峰检测信息,确定检测波长。对比Dikma Platisil ODS 5μm 250×4.6mm ser#:5031458, UltimateTM XB-C18 5μm, 4.6×250mm ser#:210802004, Agilent HC-C18 Analytical 4.6×250mm 5-MicronP.N.518905-902的分离效果,选择最佳的分离效果色谱柱作为建立高效液相指纹图谱的色谱柱。考察柱温25℃、30℃、35℃对色谱峰分离的影响,选择最佳柱温。综合以上因素,建立寿胎丸指纹图谱,经过精密度考察,稳定性考察,重复性考察,中间精密度考察(包括不同日期,不同人员,不同仪器的对比实验)等方法学考察,考察建立的方法是否科学可行,对10批成品分析,考察相似度是否符合要求。结果：根据各色谱峰的紫外光谱图,可知寿胎丸供试品溶液中主要含有黄酮类、有机酸、三萜皂苷类、环烯醚萜类、内酯类五大类物质；并对寿胎丸指纹图谱中峰面积≥3进行归属。利用寿胎丸的指纹图谱与相应药材的指纹图谱及阴性对照,通过色谱峰的保留时间与光谱图进行对比确定,以提高确定归属的准确性。所得供试品溶液的色谱图中36个色谱峰中,检出与处方中菟丝子、桑寄生、续断药材参照指纹图谱相同的色谱峰共36个,其色谱峰如下：归属于菟丝子的色谱峰应有8个,归属于桑寄生的色谱峰有7个,归属于续断的色谱峰应有21个,12#峰为一混合峰,归属于菟丝子和桑寄生。寿胎丸色谱峰与各对照品色谱峰的保留时间、PAD光谱图比较,可得到寿胎丸中的已知物质有7个,占总峰面积27%,分别为绿原酸、芦丁、金丝桃苷、槲皮苷、槲皮素、山柰酚、续断皂苷Ⅳ,其中绿原酸为有机酸,芦丁、金丝桃苷、槲皮苷、槲皮素、山柰酚为黄酮类物质,续断皂苷Ⅳ为皂苷类物质。其中金丝桃苷、山柰酚为菟丝子所含成分,芦丁、槲皮素、槲皮苷为桑寄生所含成分,绿原酸、续断皂苷Ⅳ为续断所含成分,较深入地明确了该产品的内在成分。经过一系列方法学考察：采用《中药色谱指纹图谱相似度评价系统2004A版》进行评价,相似度均大于0.900。对10批寿胎丸样品,菟丝子,桑寄生,续断3味原料中药材也分别进行了指纹图谱研究,对其相似度进行评价,相似度均大于0.900。证明本研究具有良好的专属性,科学适用。结论：1.以高效液相色谱法建立寿胎丸复方指纹图谱,得到寿胎丸中的已知物质7个,分别为绿原酸、芦丁、金丝桃苷、槲皮苷、槲皮素、山柰酚、续断皂苷Ⅳ。2.以寿胎丸复方指纹图谱与相应药材的指纹图谱对照,初步确定金丝桃苷、山柰酚为菟丝子所含成分,芦丁、槲皮素、槲皮苷为桑寄生所含成分,绿原酸、续断皂苷Ⅳ为续断所含成分。本研究的完成为寿胎丸的建立了完善的高效液相色谱指纹图谱方法；为寿胎丸的深入的临床应用提供了科学依据；为开发寿胎丸及建立寿胎丸的质量控制标准打下基础。更多还原

【Abstract】 Purpose:Shou Taiwan is the basic Prescription of reinforcing Kidney and antiabortion. It was created by Zhang Xichun in Qing period. It has very effect for protecting growth of the fetus. In this study, It established the chromatographic fingerprint of water and ethanol soluble component of shoutaiwan by using HPLC for identifying and evaluating its quality. We analyse 10 batch of finished products and herbs with HPLC. We choose the best condition from different preparation method, solid-mobile phase, elution gradients, detect wave-length, etc. We use the "Chinese medicine Chromatographic Fingerprint Similarity Evaluation Software" to identify it eligible or not. Finally, we established the HPLC chromatographic fingerprint of Shoutaiwan succefully.Methods and results:Experiment One:Based on the constitude and active ingredient of Shou Taiwan, this study reviews four extracting methods.Results:Combine with the aggregate analysis of pesticide effect and atlas, water and ethanol soluble component of shoutaiwan is bast in number, size and shape of the peak, also the active ingredient of the Shou Taiwan is most perfect and content of the component is most. Hence the water and ethanol soluble component of shoutaiwan is the best way to extract.Experiment Two:Analyse the Shou Taiwan and semen cuscutae, Taxillus chinensis, Dipsacus asper Wall, this study roughly monitors the the chemical constituents by thin layer chromatography method, clears the main composition and tries to find chromatographic condition of HPLC, aiming at the structure characteristic and rationalized features of the active ingredient of the end product.Results:According the simulation production flow of Shou Taiwan and refer the solution of Shou Taiwan, semen cuscutae, Taxillus chinensis and Dipsacus asper Wall with Rutin, quercetin, Quercitrin, hyperin, kaempferol, Dipsacaceae saponinsⅥ, oleanolic acid, finding spot corresponding to a lot of control article in thin layer of chromatographic (TLC), it proves that Shou taiwan and medicinal materials contain the component of the control article. So according the above character to choose suitable liquid condition, this study provide material basis for fingerprint.Experiment Three:Analyse the Shou Taiwan solutions for testing with HPLC, compare the different ways to deal with samples. Checking different chromatogram system and gradient elution procedure, taking the peak to be many and resolution to be good, and guaranteed the main chemical conponents occurs in the standard fingerprint, then finally the best solution and procedure. We chose 210nm、230nm、325nm、355nm、256nm switching to 210nm, and define the detece wavelength as the information of Chromatographic Peak. The research compares the results in different environment, such as Dikma Platisil ODS 5μm 250×4.6mm ser#:5031458, UltimateTM XB-C185μm,4.6×250mm ser#:210802004, Agilent HC-C18 Analytical 4.6×250mm 5-Micron P. N.518905-902, then chooses the best one to be the chromatographic column in HPLC fingerprint. Comparing different column temperature (25℃、30℃、35℃) effects to the abruption of chromatographic peak to choose the best temperature of chromatographic column. Above all, based on the Shou Taiwan fingerprint, checking the precision, stability, repeatability and intermediate precision (including different date, different people, different instrument) to find the scientific and feasibility of the methods. The study analyses ten groups of end products to find out if the precision is ok.Results:In this study, we used acetonitrile(A) with a-(0.05mol/L) KH2PO4-H3PO4 buffer solution pH=3.0(B) gradient mobile system. The detecting wavelength was set at 220 nm and the flow rate was 1.0ml/min.36 characteristic peaks was selected.We have known that there are five types materials in the sample solution of Shoutaiwan.They are flavonoids, organic acids, triterpenoid saponins, iridoids and andrograpuolide. We identificatied the chromatographic peaks which the peak area is bigger than 3. It was detected 36 chromatographic peaks. The number of chromatographic peaks which was belong to semen cuscutae was eight, The number of chromatographic peaks which was belong to Taxillus chinensis was seven, The number of chromatographic peaks which was belong to Dipsacus asper Wall was twentyone, the twelfth chromatographic peak was mixing peak. there are seven coupounds:Asperosaponin, Rutin, Quercetin, Quercitrin Hyperoside, Kaempferol, Chlorogenic Acid. Chlorogenic Acid is organic acids. Rutin, Quercetin, Quercitrin Hyperoside, Kaempferol are flavonoids. Asperosaponin is triterpenoid saponins. Hyperoside and Kaempferol are in the herb of semen cuscutae. Rutin, Quercetin, Quercitrin are in the herb of Taxillus chinensis. Asperosaponin and Chlorogenic Acid are in the herb of Dipsacus asper Wall. It defined the composition of Shoutaiwan deeply. It also established the Chromatographic Fingerprint of HPLC of semen cuscutae, Taxillus chinensis, Dipsacus asper Wall. The similiarity degree of three herbs were more than 0.900.Conclusion:First, seven kinds of materials detected by the fingerprint of Shou taiwan estabilished by HPLC respectively are:Asperosaponin, Rutin, Quercetin, Quercitrin Hyperoside, Kaempferol, Chlorogenic Acid.Second, compared the fingerprint of Shou taiwan with the fingerprint of corresponding herbs, primarily identifies Hyperoside and Kaempferol as the main component of semen cuscutae, Rutin, Quercetin and Quercitrin as the main component of taxillus chinensis, Asperosaponin and Chlorogenic Acid as the main component of dipsacus asper WallThis method shows high precision and good repeatability. So it can be used as the means to assess the quality of Shoutaiwan.更多还原