两种常用中草药多成分快速分析方法的研究

【作者】 乔艳玲；

【导师】 张金兰； 贺文义； 马辰； 张培成；

【作者基本信息】 中国协和医科大学 ， 药物分析， 2009， 硕士

【摘要】 中草药以多成分作为物质基础发挥药效作用,多成分的快速、准确、灵敏的分析检测技术和方法是中草药成分分析和质量评价的有力手段。RRLC（Rapid Resolution Liquid Chromatography）为一种新型的高分离度快速液相色谱技术,具有高分离度、高灵敏度和快速的特点,在复杂基质及多成分分析研究中体现出了较大的优势。本文第一部分采用RRLC分析技术,研究建立了2种常用中草药指纹图谱及主要成分定量测定的分析方法。乌头类药材在中国和一些东亚国家的民间用药中应用十分广泛。目前因服用乌头类药材而导致的中毒事件仍频繁发生。双酯型生物碱,如新乌头碱、乌头碱、次乌头碱等是乌头类药材中的剧毒成分,同时也是药效成分,该类成分的治疗窗很窄。如何快速、准确的评价和控制乌头类药材的质量,对于其安全用药至关重要。在第一部分的第二章,研究和建立RRLC方法用于乌头类药材的指纹图谱分析,并采用此方法同时测定各乌头药材中新乌头碱、乌头碱、次乌头碱的含量。该方法分析条件为:Zorbax Extend C₁₈色谱柱,流动相组成为甲醇-0.26%醋酸氨体系,梯度洗脱,RRLC方法分析每个样品的时间仅需10 min,比传统的HPLC方法的分析时间缩短了约6倍。采用RRLC-DAD/MS技术对乌头类药材指纹图谱中的11个主要成分进行了指认。新乌头碱的线性范围为4.93-492.50μg/mL,乌头碱的线性范围为5.18-517.50μg/mL,次乌头碱的线性范围为4.93-492.50μg/mL;四种生物碱的日内精密度的RSD在0.80-1.91%之间,日间精密度的RSD在0.19-4.93%之间;重复性的RSD小于2.19%;日内及日间稳定性的RSD分别小于3.23%、3.08%;回收率在98.37-101.50%之间,符合对上述三个生物碱的定量分析要求。因此,本文所建立的RRLC方法准确、快速、简便,既可以对大批量的乌头类药材进行常规的多成分指纹图谱分析,又可以同时对主要成分进行定量分析,为乌头类药材的质量评价提供参考信息。黄连为常用中药,传统的种植方式采用砍林搭建人工棚遮荫栽培黄连,消耗的木材量大,造成森林毁坏、水土流失;目前已经逐渐采用生态技术栽培黄连,包括林药间作、药药间作、粮药间作等生态技术新模式,提高了经济效益,也保护了生态环境。在第一部分的第三章,应用RRLC技术,研究和建立了灵敏度高、专属性好的RRLC-UV方法,用于评价不同种植条件、不同产地、不同炮制方法得到的黄连样品的质量,该方法不仅对四个主要生物碱（药根碱、黄连碱、巴马汀、小檗碱）进行含量测定,还能够同时进行黄连中多成分的指纹图谱分析。该方法分析条件为:Zorbax Eclipse Plus C₈色谱柱,流动相组成为乙腈-20 mmol/L KH₂PO₄体系,梯度洗脱,每个样品的分析时间仅需3.5 min,比传统的HPLC方法的分析时间缩短了近7倍。药根碱的线性范围为4.75-47.50μg/mL,黄连碱的线性范围为20.60-164.80μg/mL,巴马汀的线性范围为18.07-180.73μg/mL,小檗碱的线性范围为89.70-717.57μg/mL;4种生物碱的日内及日间精密度的RSD分别在0.4-1.5%及0.3-3.7%之间;重复性的RSD在0.6-1.2%之间;日内及日间稳定性RSD分别在0.4-0.5%及0.4-1.3%之间;回收率在96.30-104.10%之间,RSD小于3.3%,符合对上述四个生物碱的定量分析要求。药根碱、黄连碱、巴马汀、小檗碱的最低检测限分别为0.19 ng、0.21ng、0.15 ng、0.14 ng,最低定量限分别为57 ng,0.82 ng,0.55 ng,and 0.27 ng。方法准确、快速、简便,适用于黄连药材的日常质量控制。中药中的寡糖和多糖的药效作用越来越受到关注,寡糖分子量分布及多糖的单糖组成研究是评价其质量、提供理化性质和结构基本信息的重要内容,在本论文的第二部分第一章建立了毛细管凝胶电泳-激光诱导荧光法分析测定了疏血通注射液糖蛋白中N-连接寡糖链的分子量分布。首先用肽糖苷酶F（PNgase-F）释放出疏血通糖蛋白中N-端连接的寡糖,采用荧光试剂APTS标记后,以毛细管凝胶电泳-激光诱导荧光检测法（HPCE-LIF）分离和检测寡糖衍生物。分析条件为:eCAP^TMN-CHO中性涂层毛细管柱,分离介质为糖分离凝胶缓冲液,翻转电极,负极压力进样,正极检测,采用LIF检测器,激发波长488 nm,接收波长520 nm。寡糖衍生物按分子量由小到大的顺序出峰,通过与1-20个聚合度的葡聚糖衍生物电泳梯度图谱的比较,测得疏血通糖蛋白中N-连接寡糖链单糖、二聚葡萄糖、三聚葡萄糖、四聚葡萄糖。该方法是一种分析寡糖分子量分布的准确、有效的方法。第二部分第二章建立了一种1-苯基-3-甲基-5-吡唑啉酮（PMP）柱前衍生化HPLC同时测定5种中性糖（葡萄糖、甘露糖、半乳糖、鼠李糖、木糖）、2种糖醛酸（葡萄糖醛酸、半乳糖醛酸）的方法。PMP衍生物不易裂解,没有立体异构体,没有副产物产生。分析条件:采用常规的C₁₈色谱柱,梯度洗脱,DAD检测器240nm波长检测,几种单糖的PMP衍生物得到了很好的分离。用此方法对人工栽培的蛹虫草中多糖的单糖组成进行了分析。结果表明:本方法特异性好、简单、快速、准确、方便,可进一步推广应用于其他中草药多糖的单糖组成分析。更多还原

【Abstract】 The pharmacodynamic action of Traditional Chinese Medicines are based on the multicomponents existed in them.Rapid,precise and sensitive analytical technology and method are significant for components analysis and quality evaluation for Traditional Chinese Medicines.RRLC（Rapid Resolution Liquid Chromatography） is an advanced rapid resolution liquid chromatography.It has some dominance such as rapid,high resolution and high sensitivity.So it can be used to multiple-component analysis in complicated matrix appropriately.In the first part of the thesis,RRLC methods have been developed and validated for the fingerprint analysis and quantitation of principal constituents simultaneously of two commonly used Traditional Chinese Medicines.Aconite roots have been used widely in folk medicine throughout China and other East Asian countries.However poisoning induced by ingestion of aconite roots are still frequent and numerous.The diester-diterpene alkaloids especially such as mesaconitine, aconitine and hypaconitine are the main active components in aconite roots,however they are highly toxic and showed a narrow margin of safety between the therapeutic and toxic dosage.In the second chapter of the first part,a RRLC method coupled to diode array detection had been developed and validated for the fingerprint analysis of aconite roots and quantitation of mesaconitine,aconitine and hypaconitine simultaneously.The analysis was performed on a Zorbax Extend C₁₈ column and gradient elution.The mobile phase consisted of MeOH and water containing 0.26%ammonium acetate.The analysis run time was just 10 min shortened 6 times than that of traditional HPLC method for each sample of aconite roots.11 major chromatographic peaks were identified by RRLC-DAD/MS.The method showed good linearity within test ranges of 4.93-492.50μg/mL for mesaconitine,5.18-517.50μg/mL for aconitine,4.93-492.50μg/mL for hypaconitine.The RSDs of intra-day and inter-day precisions of the method were in the range of 0.80-1.91%and 0.19-4.93%for the three components.The repeatabilities were good with RSD less than 2.19%.It’s also showed good intra-day and inter-day stability by RSD less than 3.23%、3.08%respectively.The recoveries were in the range of 98.37-101.50%with RSD less than 3.41%for the three components.The method is rapid, accurate and suitable for fingerprint analysis and quantitation of the three components.It can provide reference information for quality evaluation of aconite roots. Rhizoma Coptidis is one of the most well-known and widely used herbs in traditional Chinese medicine.Traditional cultivation of Rhizoma Coptidis is carried out under artificial cover,a method that has been used for hundreds years.The deforestation and loss of water and soil that result from this method lead to damages to the natural environment.More recently,several ecological cultivation methods have been developed that shade Rhizoma Coptidis under economic crops.Such crops include fruit trees,Galla chinensis forests,corn,etc.In addition to protecting the environment,these new cultivation methods provide the additional economic benefits.In the third chapter of the first part,we developed and validated a sensitive and specific method using RRLC coupled with an ultra-visible spectrometric detector for analysis of Rhizoma Coptidis samples grown under different cultivation conditions,different habitats and processed with different methods.The method allows simultaneous quantitation of four mian alkaloids:berberine,palmatine,jatrorrhizine,and coptisine as well as fingerprint analysis for multicomponents in Rhizoma Coptidis.The analysis was carried out using a Zorbax Eclipse Plus C₈ reversed-phase column（4.6×50 mm,1.8μm） and gradient elution.The mobile phase consisted of acetonitrile and 20 mmol/L KH₂PO₄.It just needed 3.5 min shortened 7 times than that of traditional HPLC method for each sample of Rhizoma Coptidis.The method showed good linearity within test ranges of 4.75-47.50μg/mL for jatrorrhizine,20.60-164.80μg/mL for coptisine,18.07-180.73μg/mL for palmatine,and 89.70-717.57μg/mL for berberine.The RSDs of the intra-day and inter-day precisions were determined to be in the range of 0.4-1.5%and 0.3-3.7%for the four components. The repeatabilities were good with RSD in the range of 0.6-1.2%.It’s also showed good intra-day and inter-day stability by RSD in the range of 0.4-0.5%、0.4-1.3%respectively. The recoveries were in the range of 96.30-104.10%with RSD less than 3.3%for the four components.The lowest limit of detection was 0.19 ng for jatrorrhizine,0.21ng for coptisine,0.15 ng for palmatine,and 0.14 ng for berberine.The lowest limit of quantification was 57 ng for jatrorrhizine,0.82 ng for coptisine,0.55 ng for palmatine, and 0.27 ng for berberine.The method is accurate,rapid,and convenient,and is suitable for use in routine quality control of Rhizoma Coptidis.Oligosaccharides and polysaccharides have significant biological and pathological effects and have attracted more and more public attention.The research of size distribution of the oligosaccharides and monosaccharide composition is very elemental and important for quality evaluation,physico-chemical property investigation and structure investigation.In the first chapter of the second part,we developed a capillary electrophoresis method coupled with laser-induced fluorescence detector（LIF） to detect the size distribution of the oligosaccharides linked to the N-terminal of the glucoprotein in Shuxuetong injection.At first,N-oligosaccharides in glucoprotein were released by peptide N-glycoside F enzyme.Then oligosaccharides were derivatized with a sensitive fluorescent dye 8-aminopyrene-1,3,6-trisulfonate（APTS）,and all of the labeled oligosaccharides were separated well by capillary gel electrophoresis with laser-induced fluorescence detection.The analysis was carried out using an eCAP^TMN-CHO column. The separation buffer was eCAP^TM Carbohydrate separation gel buffer N.The polarity on the instrument was reversed before experiment.The sample was injected by pressure on negative electrode and detected on the positive electrode.The excitation wavelength was 488 nm,and the emission wavelength was 520 nm.The migration time of the labeled oligosaccharides was related to the size of the oligosaccharides and can be detected by the detector one by one.By comparing with the electrophoretogram of the labeled dextran（n=1-20） size marks,the size distribution of the oligosaccharides can be definited. Oligosaccharides linked to the N-terminal of the glucoprotein in Shuxuetong injection may have monosaccharide,disaccharide,trisaccharide and tetrasaccharide according to the results.This method is accurate and effective,can provide useful information for the study of relationship between structure and bioactivities of oligosaccharides.In the second chapter of the seond part,a simple and sensitive high performance liquid chromatographic method for simultaneous determination of five kinds of neutral monosaeeharides（glucose,mannose,galactose,rhamnose,xylose） and two kinds of uronic acid（glucuronic acid,galacturonic acid） by pre-column derivatization with 1-phenyl-3-methyl-5-pyrazolone（PMP） had been developed.The PMP-derivatives were stable and had no isomerides or by-products.The PMP-derivatives of the five neutral monosaccharides and two uronic acids were well separated by HPLC using a developed gradient elution process on a common C₁₈ column and monitored by DAD detector at 240 nm.We successfully applied this method to monosaccharide composition analysis of polysaccharides prepared from artificial Cordyceps militars.The method is specific, simple,rapid,precise,convenient and can be also used to analyze monosaccharide composition of polysaccharide isolated from Chinese herbal medicine.更多还原