【作者】 王莉；

【导师】 毕开顺； 屠鹏飞；

【作者基本信息】 沈阳药科大学 ， 药物分析学， 2010， 博士

【摘要】 心血管疾病是威胁人类健康的重大疾病之一。长期以来中医临床根据气血理论,采用益气活血方药治疗心血管疾病取得了较大的成效。为了阐明益气活血复方配伍理论,本课题组前期对复方配伍的药效协同作用进行了系统研究,证实了复方配伍的协同作用。为了进一步阐明复方配伍的作用机理,本研究以益气药人参的标准提取物—人参总皂苷(EPG)、不同药性的活血药丹参的标准提取物—丹参总酚酸(ESM)和红花的标准提取物—红花总黄酮(ECT)为研究对象,采用RRLC-ESI-MS技术分别对丹参标准提取物、人参标准提取物及其复方配伍在大鼠体内的药物动力学,包括吸收、分布和排泄进行了研究,旨在阐明丹参总酚酸和人参总皂苷在大鼠体内的吸收、分布和排泄规律,研究益气活血复方配伍对大鼠吸收、分布和排泄的相互影响。取得以下实验结果：1.建立了RRLC-ESI-MS法测定丹参和丹参标准提取物中9种酚酸(丹参素、原儿茶酸、原儿茶醛、香草酸、咖啡酸、阿魏酸、迷迭香酸、紫草酸和丹酚酸B)的含量,结果表明各成分在线性浓度范围内与峰面积线性关系良好(r≥0.9973),日内和日间精密度R.S.D.在2.4-5.7%之间,回收率在93.0-106.8%之间(R.S.D.≤9.6%),表明本法可以作为丹参及其制剂中酚酸成分的质量控制方法。2.建立了HPLC-CAD法测定人参和人参标准提取物中7种人参皂苷(人参皂苷Rg1,Re,Rb1, Rc, Rb2, Rb3, Rd)的含量,并与UV检测器和ELSD检测器进行灵敏度、线性和重复性的比较研究。结果表明,CAD检测器的灵敏度最高,线性和重复性结果与UV检测器接近,优于ELSD检测器,表明本法可以作为人参和人参标准提取物质量控制的有效方法。3.建立了RRLC-ESI-MS法测定大鼠血浆中5种酚酸(原儿茶酸、原儿茶醛、迷迭香酸、紫草酸、丹酚酸B)的含量,方法学研究结果表明,各项指标均符合生物样品分析方法指导原则的有关规定,该法可以用于准确测定大鼠血浆中5种酚酸的含量。分别利用本法对静脉注射ESM(50 mg·kg-1),EPG-ESM (50mg·kg-1:50mg·kg-1)后5种酚酸在大鼠血浆中的浓度和灌胃给药ESM (800 mg·kg-1), EPG-ESM (800 mg·kg-1:800mg·kg-1)后3种酚酸(PAC, PAL, SalB)在大鼠血浆中的浓度进行测定,并计算灌胃和静脉给药后各成分的药物动力学参数。结果表明,静脉给药,血浆中检测到PAC,PAL,RA,LA,Sal B5种成分；与EPG配伍后,与ESM相比,PAL,LA,Sal B3种成分的曲线下面积和达峰浓度均有不同程度的增加,而RA除曲线下面积呈小幅度的降低外,半衰期、达峰浓度和表观分布容积等均出现不同程度的增加；说明与EPG配伍后,对血浆中4种原型成分(PAL,RA,LA和Sal B)的吸收有一定的促进作用,并且增加了代谢产物PAC的达峰浓度。灌胃给药,血浆中检测到PAC, PAL, Sal B3种成分,PAL和Sal B的生物利用度分别为5.23%和4.12%；与EPG配伍后,PAL和Sal B的药动学参数均有不同程度的变化,AUC和Cmax等均有不同程度的增加,生物利用度分别提高至8.06%和5.61%,而代谢产物PAC的浓度也有明显增加。4.建立了HPLC-UV法同时测定大鼠组织脏器中3种酚酸(原儿茶酸、香草酸和丹酚酸B)的含量,准确度、精密度、回收率和稳定性考察结果均符合目前生物样品分析方法指导原则有关规定的要求。利用本法分别对灌胃给药ESM (800 mg·kg-1), EPG-ESM (800 mg·kg-1:800 mg·kg-1)后3种酚酸在大鼠组织(心脏、肝脏、脾脏、肺脏、肾脏)中的分布情况进行研究,结果表明,灌胃给药后,在心脏、肝脏、肺脏、肾脏中可以检测到原儿茶酸、香草酸和丹酚酸B三种活性成分,而脾脏中只检测到原儿茶酸和香草酸。灌胃复方配伍ESM-EPG后,与灌胃ESM相比,3种活性成分在心脏、肝脏、脾脏、肺脏、肾脏中的分布趋势基本一致,但含量均有不同程度的降低,表明配伍EPG后,对ESM中2种活性代谢产物(原儿茶酸、香草酸)和丹酚酸B原型在上述组织中的分布有一定的抑制作用。5.建立了大鼠尿液中原儿茶醛浓度的RRLC-ESI-MS测定方法,方法学研究结果表明,本法简便,灵敏度高,可以用于准确测定大鼠尿液中原儿茶醛的含量。采用本法分别对灌胃给药ESM (800 mg·kg-1), EPG-ESM (800 mg·kg-1:800 mg·kg-1)后原儿茶醛在大鼠尿液中的浓度进行测定。结果表明,尿液中原儿茶醛的累积排泄量很低,而粪便中没有检测到原儿茶醛,表明尿液可能是原儿茶醛的主要排泄途径,其余的原儿茶醛可能以代谢物的形式排泄。灌胃ESM后,原儿茶醛在尿液中的累积排泄率为1.99%,与EPG配伍后,累积排泄率降低至1.47%。结合前期血浆药物动力学结果,说明配伍EPG后,增加了原儿茶醛的生物利用度,降低了累积排泄率,促进原儿茶醛在大鼠体内的吸收。6.建立了大鼠粪便中丹酚酸B浓度的RRLC-ESI-MS测定方法,方法学研究结果表明,本法灵敏,专属性好,可以用于准确测定大鼠粪便中丹酚酸B的含量。采用本法分别对灌胃给药ESM(800 mg·kg-1),EPG-ESM (800 mg·kg-1:800 mg·kg-1)后丹酚酸B在大鼠粪便中的浓度进行测定。结果表明,粪便中丹酚酸B的累积排泄量较高,而尿液中没有检测到丹酚酸B,表明粪便可能是丹酚酸B的主要排泄途径,其余的丹酚酸B可能以代谢物的形式排泄。该结果也说明丹酚酸B口服吸收很差。灌胃ESM后丹酚酸B在粪便中的累积排泄率为60.36%,与EPG配伍后,累积排泄率降低至39.39%。结合前期血浆药物动力学结果,说明配伍EPG后,增加了丹酚酸B的生物利用度,降低了累积排泄率,促进丹酚酸B在大鼠体内的吸收。7.建立了大鼠血浆中6种人参皂苷(Rg1,Re,Rb1,Rc,Rb2,Rd)浓度的RRLC-ESI-MS测定方法,方法学研究结果表明,各项指标均符合生物样品分析方法指导原则的有关规定,表明本法可以用于准确测定大鼠血浆中6种人参皂苷的浓度。采用本法分别对静脉注射EPG(50 mg·kg-1),EPG-ESM(50 mg·kg-1：50 mg·kg-1)和EPG-ECT(50 mg·kg-1：50 mg·kg-1)后6种人参皂苷(Rg1,Re,Rb1,Rc,Rb2,Rd)在大鼠血浆中的浓度,和灌胃给药EPG(800 mg·kg-1),EPG-ESM(800 mg·kg-1：800 mg·kg-1)和EPG-ECT(800 mg·kg-1：800 mg·kg-1)后3种人参皂苷(Rb1,Rc,Rb2)在大鼠血浆中的浓度进行测定,并计算静脉注射与灌胃给药后各成分的药物动力学参数和灌胃给药后3种人参皂苷(Rb1,Rc,Rb2)的生物利用度。结果表明,静脉给药,血浆中可以检测到6种人参皂苷(Rg1,Re,Rb1,Rc,Rb2,Rd)。与ESM或ECT配伍后,与EPG相比,血浆中6种人参皂苷的浓度有不同程度的降低；药动学参数计算结果显示,6种成分的曲线下面积和达峰浓度也有不同程度的降低。灌胃给药,血浆中可以检测到3种人参皂苷(Rb1,Rc,Rb2)。灌胃EPG后,人参皂苷Rb1,Rc,Rb2的生物利用度分别为0.47%,0.67%和0.56%；与ESM配伍后,人参皂苷Rb1,Rc,Rb2的生物利用度分别提高至0.67%,0.93%和1.11%；与ECT配伍后,人参皂肾Rb1的生物利用度降低至0.39%,Rc,Rb2的生物利用度提高1.33%和0.92%。8.建立了RRLC-ESI-MS法同时测定大鼠尿液中6种人参皂苷(Rg1, Re, Rb1, Re, Rb2,Rd)的含量,方法学验证结果表明,本法准确,专属,重复性好,可以用于准确测定大鼠尿液中6种人参皂苷的含量。采用本法对灌胃给药EPG (800 mg·kg-1), EPG-ESM (800 mg·kg-1:800 mg·kg-1)和EPG-ECT (800 mg·kg-1:800mg·kg-1)后6种人参皂苷在大鼠尿液中的排泄情况进行研究。结果发现,6种成分在尿液中的累积排泄量相对较低。灌胃复方配伍EPG-ESM或EPG-ECT后,与灌胃EPG相比,改变了6种人参皂苷在尿液中排泄趋势,排泄量和累积排泄率均有明显降低。9.建立并验证RRLC-ESI-MS法同时测定大鼠粪便中6种人参皂苷的含量,方法学验证结果表明,本法灵敏、专属、准确,可以用于准确测定大鼠粪便中6种人参皂苷的含量。采用本法对灌胃给药EPG (800 mg·kg-1), EPG-ESM (800 mg·kg-1:800 mg·kg-1)和EPG-ECT (800 mg·kg-1:800 mg·kg-1)后6种人参皂苷在大鼠粪便中的排泄情况进行研究。结果发现,6种成分在粪便中的排泄率较高,说明粪便可能是其主要排泄途径。该结果也说明人参皂苷类成分口服吸收很差。复方配伍ESM或ECT后,与灌胃EPG相比,6种人参皂苷在粪便中的排泄趋势发生了改变,排泄量和累积排泄率均有不同程度的降低,结合前期血浆药物动力学试验结果和人参皂苷在尿液中的排泄结果,复方配伍后人参皂苷的生物利用度都有一定的提高,而在尿液和粪便中的排泄有不同程度的降低,说明复方配伍ESM或ECT后,对人参皂苷的吸收有一定的促进作用。更多还原

【Abstract】 For the purpose of investigating and clarifying the compatability principle for traditional Chinese medicine of tonifying Qi combined with activiting blood circulation, the standard extract of panax ginseng (EPG) for tonifying qi and different standard extract of Salvia miltiorrhiza (ESM) and Carthamus tinctorius (ECT) for activiting blood circulation were chosen for research, and the pharmacokinetics studies on the absorption, distribution and excretion of EPG, ESM and their combined compatibility in rat plasma, tissues, urine and feces were proceeded by RRLC-ESI-MS, and the synergistic reaction for combined compatibility of tonifying qi combined with activiting blood circulation for absorption, distribution and excretion in rat were investigated. The results were as follows:1. A rapid RRLC-ESI-MS method for simultaneous quantification of nine phenolic acids, namely danshensu, protocatechuic acid, protocatechuic aldehyde, vanillic acid, caffeic acid, ferulic acid, rosmarinic acid, lithospermic acid and salvianolic acid B in Danshen and ESM was established. All calibration curves showed good linear regression (r≥0.9973) within test ranges. The overall intra-day and inter-day variations (R.S.D.) ranged from 2.4 to 5.7%. The overall recoveries ranged from 93.0% to 106.8% with R.S.D. less than 9.6%. This developed RRLC-ESI-MS method provided a suitable quality control method for the determination of major bioactive constituents in Danshen and its preparations.2. A sensitive HPLC-CAD method was developd for simultaneous determination of seven major ginsenosides, namely ginsenosides Rg1, Re, Rb1, Rc, Rb2, Rb3 and Rd in Panax ginseng C. A. Meyer and EPG. This CAD method was evaluated in sensitivity, linearity and reproducibility compared to ELSD and UV. It was found the developed HPLC-CAD method had improved sensitivity, linearity and reproducibility compared to ELSD. This method was successfully applied to analyze seven ginsenosides in ten samples of Panax ginseng and three batches of EPG. The validation results indicated the improved method could be utilized as another approach for quality control of Panax ginseng and EPG.3. A RRLC-ESI-MS method was established for simultaneous quantification of five phenolic acids (PAC, PAL, RA, LA, Sal B) in rat plasma. The validated results of methodology were in accordance with the relevant regulations, and all data and results showed that this RRLC-ESI-MS method could be applied to determine the concentration of the five phenolic acids in rat plasma precisely.This method was successfully applied to the determination of the concentration of PAC, PAL, RA, LA and Sal B in rat plama after i.v. administration of ESM (50 mg·kg-1) and ESM-EPG (50 mg·kg-1:50 mg·kg-1), and PAL, PAC and Sal B in rat plasma after i.g. administration of ESM (800 mg·kg-1) and ESM-EPG (800 mg·kg-1:800 mg·kg-1), respectively, and the pharmacokinetic parameters of the analytes after i.g. administration and i.v. administration were also calculated.After i..v. administration, PAC, PAL, RA, LA and Sal B were detected; compared ESM-EPG compatibility to ESM, for PAL, LA and Sal B, Cmax and AUC increased differently, whereas for RA, except for slight reduction of AUC, t1/2, Cmax and Vz increased differently. It might be concluded that, after i.v. administration, compared ESM-EPG compatibility to ESM, there were some enhancement effects on the absoption of PAL, RA, LA and Sal B, and Cmax of the metabolite PAC also increased.After i.g. administration, PAC, PAL and Sal B were detected, and the bioavailability for PAL and Sal B were 5.23% and 4.12%; after combined with EPG, the pharmacokineteics parameters of PAL and Sal B changed differently, the AUC and Cmax increased in different extent, and the absolute bioavailability of PAL and Sal B were increased to 8.06% and 5.61%, respectively, furthermore for the metabolite PAC, Cmax also increased. 4. A HPLC-UV method was established for the determination of PAC, VAC and Sal B in rat tissues. The validated results of methodology were in accordance with the relevant regulations.This method was successfully applied to study the distribution of PAC, VAC and Sal B in rat tissues (heart, liver, spleen, lung and kidney) after i.g. administration of ESM (800 mg·kg-1) and ESM-EPG (800 mg·kg-1:800 mg·kg-1). The results indicated that after i.g. administration, PAC, VAC and Sal B could be detected in rat heart, liver, lung and kidney; while PAC and VAC in rat spleen.The distribution results indicated that, after i.g. administration, compared ESM-EPG compatibility to ESM, the concentrations of the three phenolic acids in rat tissues (heart, liver, spleen, lung and kidney) were decreased in different extent, which suggested that there were some depressant effects on the distribution of the two metabolites (PAC and VAC) and Sal B prototype.5. A RRLC-ESI-MS method was developed for the determination of PAL in rat urine. The validated results of methodology were in accordance with the relevant regulations, and all data and results showed that this RRLC-ESI-MS method could be applied to determine the concentration of PAL in rat urine precisely.This method was successfully applied to the determination of the concentration of PAL in rat urine after i.g. administration of ESM (800 mg·kg-1) and ESM-EPG (800 mg·kg-1:800 mg·kg-1). It was found that, the accumulative excretion dose of PAL was very low, whereas PAL was not detected in rat feces, which indicated that urine might be the main excretion path of PAL, and other amounts of PAL were proposed to be excreted as the metabolites of PAL.After i.g. administration, the accumulative excretion rate of PAL in rat urine was 1.99%, after combined with EPG, the accumulative excretion rate of PAL was decreased to 1.47%. Considering the former pharmacokinetic results in rat plasma, the bioavailability of PAL was improved, and the accumulative excretion rate was decreased, which indicated that EPG might promote the absorption of PAL. 6. A RRLC-ESI-MS method was developed for the determination of the concentration of Sal B in rat feces. The validated results of methodology were in accordance with the relevant regulations, and all data and results showed that this RRLC-ESI-MS method could be applied to determine the concentration of Sal B in rat feces.This method was applied to the determination of the concentration of Sal B in rat feces after i.g. administration of ESM (800 mg·kg-1) and ESM-EPG (800 mg·kg-1:800 mg·kg-1). It was found that, the accumulative excretion dose of Sal B in rat feces was relatively high, whereas Sal B was not detected in rat urine, which indicated that feces might be the main excretion path of Sal B prototype, and other amounts of Sal B were proposed to be excreted as the metabolites of Sal B. The results also indicated that the oral absorption of Sal B was bad.After i.g. administration, the accumulative excretion rate of Sal B was 60.36%, and after combined with EPG, the accumulative excretion rate of Sal B was decreased to 39.39%. Considering the former pharmacokinetic results in rat plasma, the bioavailability of Sal B was improved, the accumulative rate was decreased, which indicated that EPG might promote the absorption of Sal B.7. A RRLC-ESI-MS method was established for simultaneous determination of six ginsenosides (Rg1, Re, Rb1, Rc, Rb2 and Rd) in rat plasma. The validated results of methodology were in accordance with the relevant regulations, and all data and results showed that this RRLC-ESI-MS method could be applied to determine the concentration of six ginsenosides in rat plasma precisely.This method was successfully applied to the determination of ginsenosides Rg1, Re, Rb1, Rc, Rb2 and Rd in rat plama after i.v. administration of EPG (50 mg·kg-1), EPG-ESM (50 mg·kg-1:50 mg·kg-1) and EPG-ECT (50 mg·kg-1:50 mg·kg-1), and ginsenosides Rb1, Rc and Rb2 in rat plasma after i.g. administration of EPG (800 mg·kg-1), EPG-ESM (800 mg·kg-1: 800 mg·kg-1) and EPG-ECT (800 mg·kg-1:800 mg·kg-1), respectively, and the pharmacokinetic parameters of the analytes after i.g. and i.v. administration were also calculated. After i.v. administration, ginsenosides Rg1, Re, Rb1, Rc, Rb2 and Rd were detected in rat plasma. Compared EPG-ESM or EPG-ECT compatibility to EPG, the concentration of the six ginsenosides in rat plasma were decreased in differen extend; and the pharmacokinetics parameters results showed the AUC and Cmax of the six analytes were also decreased in different extend.After i.g. administration, ginsenosides Rb1, Rc, Rb2 were detected in rat plasma, the bioavalibility of them were 0.47%,0.67% and 0.56%. After combined with ESM, the absolute bioavailability of ginsenosides Rb1, Rc, Rb2 were increased to 0.67%,0.93% and 1.11%, respectively; whereas combined with ECT, the absolute bioavailability of ginsenosides Rb1 were decreased to 0.39%, and the absolute bioavailability of ginsenosides Rc, Rb2 were increased to 1.33% and 0.92%.8. A RRLC-ESI-MS method was established for simultaneous determination of six ginsenosides (Rg1, Re, Rb1, Rc, Rb2, Rd) in rat urine. The validated results of methodology were in accordance with the relevant regulations, and all data and results showed that this RRLC-ESI-MS method could be applied to determine the concentration of six ginsenosides in rat urine precisely.This method was successfully applied to the determination of six ginsenosides in rat urine after i.g. administration of EPG (800 mg·kg-1), EPG-ESM (800 mg·kg-1:800 mg·kg-1) and EPG-ECT (800 mg·kg-1:800 mg·kg-1), respectively. It was found that the acumulative excretion dose of the six ginsenosides in rat urine were relatively low.After i.g. administration, compared EPG-ESM or EPG-ECT compatibility to EPG, the excretion tendency of the six ginsenosides were changed, and the acumulative excretion doses of the six ginsenosides were decreased remarkably.9. A RRLC-ESI-MS method was established for simultaneous determination of six ginsenosides in rat feces. The validated results of methodology were in accordance with the relevant regulations, and all data and results showed that the RRLC-ESI-MS method could be applied to determine the concentration of six ginsenosides in rat feces precisely.This method was successfully applied to the determination of six ginsenosides in rat feces after i.g. administration of EPG (800 mg·kg-1), EPG-ESM (800 mg·kg-1:800 mg·kg-1) and EPG-ECT (800 mg·kg-1:800 mg·kg-1), respectively. It was found that the acumulative excretion doses of the six ginsenosides in rat feces were realtively high, which indicated that feces were the main excretion path of the six ginsenosides. The results also indicated that the oral absorption of the six ginsenosides was bad.After i.g. administration, compared EPG-ESM or EPG-ECT compatibility to EPG, the excretion tendency of the six ginsenosides were changed, the acumulative excretion doses of six ginsenosides of EPG in rat feces were decreased in different extent, which indicated that there were some inhibition effects on the excretion of the six ginsenosides in rat feces. Considering the pharmacokinetic results in rat plasma and the excretion results in rat urine, the bioavailabilities of the three ginsenosides were improved, and the excretion doses were decreased, which indicated that ESM or ECT might promote the absorption of ginsenosides.更多还原