黄连—吴茱萸药对主要成分的溶出变化和药动学研究

【作者】 邓雅婷；

【导师】 毕开顺； 陈晓辉； 谢智勇；

【作者基本信息】 沈阳药科大学 ， 药物分析学， 2008， 硕士

【摘要】 不同比例的黄连—吴茱萸药对具有不同的药效,但其物质基础和配伍机理尚未阐明,本研究以化学成分的体外溶出和体内吸收为主线,采用液相色谱和液相色谱—质谱联用技术系统的研究了药对在体外的化学成分及其溶出变化,在体内的动力学过程和代谢产物,为从化学成分的角度阐明配伍机理提供依据。1.黄连—吴茱萸药对主要成分的研究采用U6（6³*3）均匀设计表安排实验,以17个色谱峰和小檗碱含量的综合评分为指标,优化了黄连—吴茱萸药对半仿生提取的最佳工艺条件:三次提取的pH依次是1.6,6.0和7.0,总提取时间为2 h。以Hypersil BDS C₁₈为色谱柱,乙腈—水（含0.5%甲酸,0.2%氨水）为流动相梯度洗脱,综合分析不同样品的色谱峰保留时间、紫外光谱特征,一级和二级全扫描质谱图,归属了黄连—吴茱萸（6∶1）半仿生提取液中的17个色谱峰:8个来自吴茱萸,9个来自黄连,半仿生提取无新的物质生成;指认了4个色谱峰分别为药根碱、羟基吴茱萸碱、巴马汀和小檗碱;推断了另3个色谱峰分别为表小檗碱、非洲防己碱和黄连碱。建立了全面反映黄连—吴茱萸药对半仿生提取液化学成分的RP-HPLC法,在该条件下对黄连—吴茱萸（1∶0,1∶1,1∶2,1∶6,6∶1,2∶1,0∶1）的半仿生提取液进行色谱分析,考察了17个组分与黄连—吴茱萸配伍之间的关系。黄连—吴茱萸不同配伍比例对各主要组分溶出的影响不同。其中,来自吴茱萸药材的8个成分随着配伍黄连比例的增大溶出增加:来自黄连药材的色谱峰7,9,10随着配伍吴茱萸比例的增大溶出增加;来自黄连药材的小檗碱型化合物随着配伍吴茱萸比例的增大溶出减少。2.黄连—吴茱萸药对中黄连主要成分在大鼠体内的药动学研究建立了大鼠血浆中小檗碱、巴马汀和药根碱同时含量测定的LC-MS/MS方法。血浆样品经沉淀蛋白法预处理后,以乙腈—水（含0.1%甲酸）（70∶30,v/v）为流动相,经Hypersil BDS C₁₈柱分离后,采用电喷雾离子源（ESI）,在选择反应监测（SRM）模式下测定待测物的含量。用于定量分析的离子反应分别为m/z 336→320（小檗碱）、m/z 352→336（巴马汀）、m/z 338→322（药根碱）和m/z 356→192（内标:四氢巴马丁）。三种待测物均在1-250 ng/mL范围内线性关系良好,定量下限均为1 ng/mL。该法简单、快速、灵敏,并成功应用于大鼠灌胃给予黄连—吴茱萸（6∶1）药对（左金丸）后三种成分的药动学研究。小檗碱、巴马汀和药根碱的药—时曲线均呈现多峰现象。采用非房室模型计算各成分的主要药动学参数,其中小檗碱C_max为121.1±11.9 ng/mL,AUC_0-∞为28341.5±5241.2 ng·min/mL,t_1/2为336.9±46.5 min;巴马汀C_max为35.2±9.5 ng/mL,AUC_0-∞为6345.5±133.9 ng·min/mL,t_1/2为215.7±33.5 min;药根碱C_max为219.9±12.8 ng/mL,AUC_0-∞为43576.9±4767.8 ng·min/mL;t_1/2为325.3±8.0 min,达峰时间均为90 min。3.黄连—吴茱萸药对中黄连主要成分在大鼠体内代谢产物的初步研究采用LC-MS/MS技术,通过对给药前后大鼠血浆样品的分析,初步推断了大鼠灌胃黄连—吴茱萸（6∶1）后黄连主要成分在体内的Ⅰ相和Ⅱ相代谢物,结果表明Ⅰ相代谢物主要来自母药的O-去烷基化反应,药根碱可以形成Ⅱ相代谢物葡萄糖醛酸结合物,小檗碱和巴马汀则不能与葡萄糖醛酸直接结合,需经Ⅰ相代谢后才能形成Ⅱ相代谢物。但由于没有对代谢物结构作进一步确证及缺乏对照品,以上所做的工作只能是一个初步的推测。更多还原

【Abstract】 The pharmacologic actions of coptis-evodia herb couple changed with the proportion of each herb,but the chemical foundation and involved mechanisms have not been revealed.The thesis investigated the chemical composing of the herb couple and their dissolution rates in vitro and the fate in vivo by applying liquid chromatography-mass spectrometry（LC-MS）and high performance liquid chromatography（HPLC）.It would offer some references for elucidating the mechanisms of coptis-evodia herb couple based on chemical composing.1.Study on the chemical composing of coptis-evodia herb coupleThe Semi-bionic Extraction was optimized by homogenous design while the score of total area of 17 peaks together with the quantity of berberine was chosen as marker. The optimized conditions as follows:pH values for three extraction processes were 1.6, 6.0,7.0 respectively and total extraction time was 2 h.A comprehensive HPLC-DAD-MS method was developed to study the chemical composing of semi-bionic extracts of coptis-evodia herb couple.The extract was separated on a Hypersil BDS C₁₈column（4.6 mm×200 mm,5μm,Elite,China）using acetonitrile-ammonium formic buffer as mobile phase by gradient elution.Detection was performed on DAD and MS equipped with an electrospray ionization（ESI）source by full scan and product full scan on positive mode.The chromatogram of coptis-evodia showed 17 main peaks,eight of which were from evodia while the others were from coptis,and it suggested that no new component was created in the semi-bionic extract of the herb couple.By comparison of the retention time,the on-line UV spectra and MS spectra,four peaks were identified as jatrorrhizine,hydroxevodiamine,palmatine and berberine,and three peaks were deduced as epiberberine,columbamine and coptisine.Seven samples with different proportions of coptis and evodia（1:0,1:1,1:2,1: 6,6:1,2:1,0:1）were extracted and then analyzed by RP-HPLC,in order to study the rule of solubility changes with the content of the copfis-evodia herb.Seventeen components were investigated in our experiment.Dissolution rate of the components from evodia elevated with the increasing of coptis;nine components were from coptis, three of which increased while the others decreased with the increasing of evodia.The proportion had different influence on the rule of solubility of different components in the herb.2.Pharmacokinetic studies of main components from coptis after oral administration of coptis-evodia herb couple in ratsA rapid and sensitive liquid chromatography/tandem mass spectrometry （LC-MS/MS）method was developed to simultaneously determine berberine,palmatine and jatrorrhizine in rat plasma.After mixing with the internal standard（IS） tetrahydropalmatine,plasma samples were pretreated by protein precipitation. Chromatographic separation was carried out on a Hypersil BDS C₁₈column using a mixture of water（containing 0.1%formic acid）and acetonitrile（30:70,v/v）as mobile phase.Detection was performed by selected reaction monitoring（SRM）mode via electrospray ionization（ESI）source operating in the positive ionization mode.The method was linear over the concentration range of 1-250 ng/mL for all components. The fully validated LC-MS/MS method has been successfully applied to the pharmacokinetic studies of berberine,palmatine and jatrorrhizine in rat plasma after oral administration of coptis-evodia（6:1,g/g）herb couple.Three peaks were observed in both individual and mean plasma-concentration curves of berberine,palmatine and jatrorrhizine.The concentration-time data was analyzed by non-compartmental methods in order to obtain the pharmacokinetic parameters:C_maxfor berberine,palmatine and jatrarrhizine is 121.1±11.9 ng/mL,35.2±9.5 ng/mL and 219.9±12.8 ng/mL, respectively.AUC_0-∞for berberine,palmatine and jatrarrhizine is 28341.5±5241.2 ng·min/mL,6345.5±133.9 ng·min/mL and 43576.9±4767.8 ng·min/mL,respectively. t_1/2for berberine,palmatine and jatrarrhizine is 336.9±46.5 min,215.7±33.5 min and 325.3±8.0 min,respectively,t_maxis 90 min for each component.3.Presumption of the metabolites of main components from coptis after oral administration of coptis-evodia herb couple in ratsThe present study characterized the metabolic profile of main components from coptis after oral administration of coptis-evodia herb couple in rats by analyzing plasma samples before and after dosing with the aid of LC-MS/MS.The phaseⅠmetabolic position and pathways were supposed as O-dealkylation.The phaseⅡmetabolites were identified as glucuronide conjugate of jatrorrhizine and phaseⅠmetabolites.But it was a kind of supposition for lack of further validation and absence of compounds.更多还原