知母皂苷B-Ⅱ的药代动力学及代谢机制研究
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摘要
中药在我国有着悠久的使用历史，经历了几千年的临床验证，历久弥新。大量基础与临床研究证明其在某些病症有着独特的治疗作用，由于其相对低毒、不良反应发生率低、程度较轻等特点，受到越来越多的关注，近年来，以中药为代表的天然药物在世界各地的使用大幅增长，但是大量的天然药物成分存在吸收利用度低、代谢过程不清楚的情况，这成了新药创制过程中的“拦路虎”。
本课题以临床常用中药知母中提取的天然有效成分知母总皂苷及其主要有效成分知母皂苷B-II为主要研究对象，在建立知母总皂苷半成品、成品的质量标准的基础上，运用LC-MS/MS联用分析技术为主要检测手段，建立知母皂苷B-II体内定性定量分析方法，深入探究其体内代谢动力学特征及体内外代谢机制，内容包括：
1、分别建立注射用知母总皂苷半成品、成品的质量标准。方法采用薄层色谱法对知母皂苷B-II进行薄层鉴别，结果薄层鉴别斑点清晰、分离度好、专属性强、阴性对照无干扰；采用高效液相色谱法非别对知母皂苷B-II、知母皂苷E1、知母皂苷B进行含量测定。结果表明方法简便、可靠、重现性好,可作为知母总皂苷半成品、成品的质量控制方法。
2、分别静注给药5.4、1.8和0.9mg/kg三个剂量的知母皂苷B-II，AUC0-t分别为12560.62±2304.23ng.h/mL、2363.64±184.44ng.h/mL、843.70±34.28ng.h/mL，Cmax分别为48996.55±9472.31ng/mL、12295.36±816.69ng/mL、3876.93±189.20ng/mL，其Cmax及AUC0-t均与给药剂量呈正相关，表现为线性药代动力学特征。Cmax和AUC0-t与给药剂量呈正相关，表明知母皂苷B-II静注后在大鼠体内为线性药代动力学特征。以180mg/kg、90mg/kg和30mg/kg三个剂量组分别对大鼠灌胃知母皂苷B-II后的AUC0-t分别为2291.38±1294.56ng.h/mL、331.01±218.25ng.h/mL、123.64±124.92ng.h/mL，Cmax分别为1654.56±1342.83ng/mL、442.69±353.38ng/mL、56.60±39.44ng/mL，显示Cmax和AUC0-t与给药剂量相关性不太强，AUC0-t随剂量增加较快。另外，给药知母皂苷B-II后，Beagle犬药时曲线图也存在双峰现象或多峰现象，推测可能存在肠肝循环现象。分别口服与静注给药，得到各自的药时曲线下面积，由此计算出相对生物利用度值在0.75%左右，结果表明，知母皂苷B-II在SD大鼠体内的的口服生物利用度比Beagle犬略高，但都相对较低，也进一步显示知母皂苷B-II原型药物在胃肠道中吸收很弱。
知母皂苷B-II在SD大鼠体内组织分布结果显示：大鼠静注给药后消除速度较快；6小时后在各脏器中基本原形无药物蓄积，各脏器组织中的药物浓度均明显低于血浆药物浓度，其中肝、心、肺组织药物浓较高，大脑最少。这可能与其吸收代谢过程缓慢有关。药物的主要吸收部位是小肠，所以药物驻留而达到最高浓度。
知母皂苷B-II原型药物在SD大鼠体内排泄研究显示：该药0~30h内从胆汁排泄的药物量占总给药量的1.27%，0~96h内从尿液中排泄的药物量为占总给药量的1.55%，96h内累积粪便中排泄量为给药剂量的0.048%。
3.采用平衡透析法测定知母皂苷B-Ⅱ在人血浆中的蛋白结合率，结果表明，与血浆蛋白结合率实验结果显示，知母皂苷B-II药物浓度与血浆蛋白结合率之间无显著性差异（p＞0.05），而透析时间和血浆蛋白结合率之间则存在一定差异（p＜0.05），72h后，药物在血浆和透析中达到平衡。结果表明，72h后，药物在血浆和透析中达到平衡，本品在血浆和透析中达到平衡，知母皂苷B-Ⅱ的蛋白结合率大于85%，提示药物进入体内后，与血浆中的蛋白相结合，而发挥作用的是游离态的药物所以可能会影响药物向组织分布。
4、利用LC-ESI-MS/MS技术，对知母皂苷B-II在大鼠体内的代谢进行了初步探索。结果在胃肠内容物样品中共检测到M1~M6等6种代谢产物;尿液样品中检测到M1、M2、M4等3种代谢产物;在粪便样品中检测到M1、M4等2种代谢产物。三种样品共同的代谢产物为M1知母皂苷AIII和M4知母皂苷元。
采用人肝微粒体体外孵育实验，通过IC50值判断知母皂苷B-Ⅱ对CYP3A4、CYPlA2、CYP2C9、CYP2C19、CYP2E1和CYP2D6这6种酶活性是否产生作用，从而影响经人体的6种代谢酶的底物经药物代谢酶的代谢。实验结果表明知母皂苷B-II对上述药物代谢酶的抑制作用较弱，不太可能在临床上导致因其抑制药物代谢酶而产生的药物相互作用。
Traditional Chinese medicine (TCM) which has a long history in China,has beenfound and proven to have some unique advantages in some complex diseases in view ofnumbers of evidence from basic and clinical research.It’s advantage Relatively low toxicityand fewer adverse reactions are observed and gradually taken seriously by more and morecountry. In recent years, the usage of TCM is increased dramatically, But there are a lotof"obstacles"faced by natural medicines including TCM, such as low bioavailability,unsharp components, unclear metabolic process and so on,in their morden process of drugdevelopment.
The main research objects in this topic are total anemarrhenae saponins andtimosaponin B–II,an active ingredient, overall extracted from rhizoma anemarrhenae,ancommonly used TCM in clinic.On basis of quality standards for semi-finished productsand finished products of the total anemarrhenae saponins, a kind of qualitative andquantitative analysis method was developed with HPLC and LC-MS/MS/MS technologyas a main monitoring way. The pharmacokinetical characteristics of timosaponin B-II andits metabolic mechanism in vitro and in vivo were researched, which includes:
1. To establish the quality standards of the total saponins semi-finished products andfinished products for injection, a TLC was employed to identify Timosaponin B-II. TheTLC spots were clear,well-separated and specific yet without interference from negativecontrol. While the content of Timosaponin B-II, Timosaponin E1, Timosaponin B,in thetotal saponins semi-finished products and finished products was determined by HPLC,the methods can be used for the quality control simply, reliably.
2. After a intravenous administration with5.4,1.8and0.9mg/kg three doses oftimosaponin B-II in SD rats, AUC0-tof timosaponin B-II was12560.62±2304.23,2363.64±184.44,843.70±34.28ng.h/mL and Cmaxof B-II was48996.55±9472.31ng/mL,12295.36±816.69ng/mL,3876.93±189.20ng/mL respectively. The area under theconcentration-time curve and peak concentration with dose performance positivecorrelation and show linear pharmacokinetic characteristics. Dose Cmaxand AUC0-twaspositively correlated, indicating that Timosaponin B-II intravenous linear pharmacokineticcharacteristics in rats. After oral administration of the timosaponin B-IIwith180mg/kg,90mg/kg,30mg/kg three dose groups, respectively, the AUC0-twas2291.38±1294.56ng.h/mL,331.01±218.25ng.h/mL,123.64±124.92ng.h/mL, Cmaxwas1654.56 ±1342.83ng/mL,442.69±353.38ng/mL,56.60±39.44ng/mL respectively. It indicated thatthe dependency of Cmaxand AUC0-ton dosage was not strong.
Theconcentration-time curves for timosaponin B-II in Beagle dogs showed bimodalphenomenon or multimodal phenomenon,which indicated existence of enterohepaticcyclings potentially. Oral and intravenous administration, respectively, getting thebioavailability which is about0.75%, the timosaponin B-II in SD rats in vivo oralbioavailability is slightly higher than Beagle dogs, but are relatively low, and furthershowed that the timosaponin B-II weak absorption in the gastrointestinal tract as aprototype drug.
The tissue distribution of Timosaponin B-II in SD rats: It was eliminated fast afterintravenous administration; there was no prototype drug accumulation after6hours, theconcentration of the drug in various organs and tissues in various organs were significantlylower than plasma concentrations, of which the drug concentrations in liver, heart and lungtissue were higher and lowest in brain. This may be related to absorption of slow metabolicprocesses. Drug absorption site is in the small intestine, with the highest concentrationbecause of the drug reside.
Timosaponin B-II excretion study as a prototype drug: the drug from biliary excretionwas1.27%of total dose of in30hours, the urine excretion of drugs accounted for the totaldose of1.55%among0to96h, and accumulated excretion in feces was0.048%within96hours.
3. The equilibrium dialysis method was used for the determination of timosaponin B-IIin human plasma protein binding, the results show that there was no significant difference(p>0.05) between drug concentration and plasma protein binding rate, but there are somedifferences between the duration of dialysis and plasma protein binding rate (p <0.05) after72hours. The results showed that the protein binding rate of timosaponin B-II of was morethan85%after72hours and there were a balance of the drug in the plasma and dialysisequilibrium, which indicated that the most of drugs were binded with plasma protein in thebodies. It may affect drug distribution to the organization Because of the free drugs waslow.
4, LC-ESI-MS/MS was used to study the Timosaponin B-II metabolism in ratsmajor metabolites.Six metabolites including M1to M6were detected in the gastrointestinalcontents; M1, M2, M4were detected in the urine;2kinds of metabolites (M1、M4) detectedin the feces sample. The common metabolite samples were M1(timosaponinAIII) and M4.
The parts of human livers were used as a source of microsomes in vitro incubationexperiments to verdict whether Timosaponin B-II has activity effects on CYP3A4theCYPlA2, CYP2C9and CYP2C19, CYP2E1and CYP2D6, thus affecting enzymessubstrate metabolism by drug-metabolizing enzymes. The results show that thetimosaponin B-II’s weak inhibition of drug metabolizing enzymes, which is unlikely tolead to clinical drug interactions because of its inhibition of drug-metabolizing enzymes.
本课题以临床常用中药知母中提取的天然有效成分知母总皂苷及其主要有效成分知母皂苷B-II为主要研究对象，在建立知母总皂苷半成品、成品的质量标准的基础上，运用LC-MS/MS联用分析技术为主要检测手段，建立知母皂苷B-II体内定性定量分析方法，深入探究其体内代谢动力学特征及体内外代谢机制，内容包括：
1、分别建立注射用知母总皂苷半成品、成品的质量标准。方法采用薄层色谱法对知母皂苷B-II进行薄层鉴别，结果薄层鉴别斑点清晰、分离度好、专属性强、阴性对照无干扰；采用高效液相色谱法非别对知母皂苷B-II、知母皂苷E1、知母皂苷B进行含量测定。结果表明方法简便、可靠、重现性好,可作为知母总皂苷半成品、成品的质量控制方法。
2、分别静注给药5.4、1.8和0.9mg/kg三个剂量的知母皂苷B-II，AUC0-t分别为12560.62±2304.23ng.h/mL、2363.64±184.44ng.h/mL、843.70±34.28ng.h/mL，Cmax分别为48996.55±9472.31ng/mL、12295.36±816.69ng/mL、3876.93±189.20ng/mL，其Cmax及AUC0-t均与给药剂量呈正相关，表现为线性药代动力学特征。Cmax和AUC0-t与给药剂量呈正相关，表明知母皂苷B-II静注后在大鼠体内为线性药代动力学特征。以180mg/kg、90mg/kg和30mg/kg三个剂量组分别对大鼠灌胃知母皂苷B-II后的AUC0-t分别为2291.38±1294.56ng.h/mL、331.01±218.25ng.h/mL、123.64±124.92ng.h/mL，Cmax分别为1654.56±1342.83ng/mL、442.69±353.38ng/mL、56.60±39.44ng/mL，显示Cmax和AUC0-t与给药剂量相关性不太强，AUC0-t随剂量增加较快。另外，给药知母皂苷B-II后，Beagle犬药时曲线图也存在双峰现象或多峰现象，推测可能存在肠肝循环现象。分别口服与静注给药，得到各自的药时曲线下面积，由此计算出相对生物利用度值在0.75%左右，结果表明，知母皂苷B-II在SD大鼠体内的的口服生物利用度比Beagle犬略高，但都相对较低，也进一步显示知母皂苷B-II原型药物在胃肠道中吸收很弱。
知母皂苷B-II在SD大鼠体内组织分布结果显示：大鼠静注给药后消除速度较快；6小时后在各脏器中基本原形无药物蓄积，各脏器组织中的药物浓度均明显低于血浆药物浓度，其中肝、心、肺组织药物浓较高，大脑最少。这可能与其吸收代谢过程缓慢有关。药物的主要吸收部位是小肠，所以药物驻留而达到最高浓度。
知母皂苷B-II原型药物在SD大鼠体内排泄研究显示：该药0~30h内从胆汁排泄的药物量占总给药量的1.27%，0~96h内从尿液中排泄的药物量为占总给药量的1.55%，96h内累积粪便中排泄量为给药剂量的0.048%。
3.采用平衡透析法测定知母皂苷B-Ⅱ在人血浆中的蛋白结合率，结果表明，与血浆蛋白结合率实验结果显示，知母皂苷B-II药物浓度与血浆蛋白结合率之间无显著性差异（p＞0.05），而透析时间和血浆蛋白结合率之间则存在一定差异（p＜0.05），72h后，药物在血浆和透析中达到平衡。结果表明，72h后，药物在血浆和透析中达到平衡，本品在血浆和透析中达到平衡，知母皂苷B-Ⅱ的蛋白结合率大于85%，提示药物进入体内后，与血浆中的蛋白相结合，而发挥作用的是游离态的药物所以可能会影响药物向组织分布。
4、利用LC-ESI-MS/MS技术，对知母皂苷B-II在大鼠体内的代谢进行了初步探索。结果在胃肠内容物样品中共检测到M1~M6等6种代谢产物;尿液样品中检测到M1、M2、M4等3种代谢产物;在粪便样品中检测到M1、M4等2种代谢产物。三种样品共同的代谢产物为M1知母皂苷AIII和M4知母皂苷元。
采用人肝微粒体体外孵育实验，通过IC50值判断知母皂苷B-Ⅱ对CYP3A4、CYPlA2、CYP2C9、CYP2C19、CYP2E1和CYP2D6这6种酶活性是否产生作用，从而影响经人体的6种代谢酶的底物经药物代谢酶的代谢。实验结果表明知母皂苷B-II对上述药物代谢酶的抑制作用较弱，不太可能在临床上导致因其抑制药物代谢酶而产生的药物相互作用。
Traditional Chinese medicine (TCM) which has a long history in China,has beenfound and proven to have some unique advantages in some complex diseases in view ofnumbers of evidence from basic and clinical research.It’s advantage Relatively low toxicityand fewer adverse reactions are observed and gradually taken seriously by more and morecountry. In recent years, the usage of TCM is increased dramatically, But there are a lotof"obstacles"faced by natural medicines including TCM, such as low bioavailability,unsharp components, unclear metabolic process and so on,in their morden process of drugdevelopment.
The main research objects in this topic are total anemarrhenae saponins andtimosaponin B–II,an active ingredient, overall extracted from rhizoma anemarrhenae,ancommonly used TCM in clinic.On basis of quality standards for semi-finished productsand finished products of the total anemarrhenae saponins, a kind of qualitative andquantitative analysis method was developed with HPLC and LC-MS/MS/MS technologyas a main monitoring way. The pharmacokinetical characteristics of timosaponin B-II andits metabolic mechanism in vitro and in vivo were researched, which includes:
1. To establish the quality standards of the total saponins semi-finished products andfinished products for injection, a TLC was employed to identify Timosaponin B-II. TheTLC spots were clear,well-separated and specific yet without interference from negativecontrol. While the content of Timosaponin B-II, Timosaponin E1, Timosaponin B,in thetotal saponins semi-finished products and finished products was determined by HPLC,the methods can be used for the quality control simply, reliably.
2. After a intravenous administration with5.4,1.8and0.9mg/kg three doses oftimosaponin B-II in SD rats, AUC0-tof timosaponin B-II was12560.62±2304.23,2363.64±184.44,843.70±34.28ng.h/mL and Cmaxof B-II was48996.55±9472.31ng/mL,12295.36±816.69ng/mL,3876.93±189.20ng/mL respectively. The area under theconcentration-time curve and peak concentration with dose performance positivecorrelation and show linear pharmacokinetic characteristics. Dose Cmaxand AUC0-twaspositively correlated, indicating that Timosaponin B-II intravenous linear pharmacokineticcharacteristics in rats. After oral administration of the timosaponin B-IIwith180mg/kg,90mg/kg,30mg/kg three dose groups, respectively, the AUC0-twas2291.38±1294.56ng.h/mL,331.01±218.25ng.h/mL,123.64±124.92ng.h/mL, Cmaxwas1654.56 ±1342.83ng/mL,442.69±353.38ng/mL,56.60±39.44ng/mL respectively. It indicated thatthe dependency of Cmaxand AUC0-ton dosage was not strong.
Theconcentration-time curves for timosaponin B-II in Beagle dogs showed bimodalphenomenon or multimodal phenomenon,which indicated existence of enterohepaticcyclings potentially. Oral and intravenous administration, respectively, getting thebioavailability which is about0.75%, the timosaponin B-II in SD rats in vivo oralbioavailability is slightly higher than Beagle dogs, but are relatively low, and furthershowed that the timosaponin B-II weak absorption in the gastrointestinal tract as aprototype drug.
The tissue distribution of Timosaponin B-II in SD rats: It was eliminated fast afterintravenous administration; there was no prototype drug accumulation after6hours, theconcentration of the drug in various organs and tissues in various organs were significantlylower than plasma concentrations, of which the drug concentrations in liver, heart and lungtissue were higher and lowest in brain. This may be related to absorption of slow metabolicprocesses. Drug absorption site is in the small intestine, with the highest concentrationbecause of the drug reside.
Timosaponin B-II excretion study as a prototype drug: the drug from biliary excretionwas1.27%of total dose of in30hours, the urine excretion of drugs accounted for the totaldose of1.55%among0to96h, and accumulated excretion in feces was0.048%within96hours.
3. The equilibrium dialysis method was used for the determination of timosaponin B-IIin human plasma protein binding, the results show that there was no significant difference(p>0.05) between drug concentration and plasma protein binding rate, but there are somedifferences between the duration of dialysis and plasma protein binding rate (p <0.05) after72hours. The results showed that the protein binding rate of timosaponin B-II of was morethan85%after72hours and there were a balance of the drug in the plasma and dialysisequilibrium, which indicated that the most of drugs were binded with plasma protein in thebodies. It may affect drug distribution to the organization Because of the free drugs waslow.
4, LC-ESI-MS/MS was used to study the Timosaponin B-II metabolism in ratsmajor metabolites.Six metabolites including M1to M6were detected in the gastrointestinalcontents; M1, M2, M4were detected in the urine;2kinds of metabolites (M1、M4) detectedin the feces sample. The common metabolite samples were M1(timosaponinAIII) and M4.
The parts of human livers were used as a source of microsomes in vitro incubationexperiments to verdict whether Timosaponin B-II has activity effects on CYP3A4theCYPlA2, CYP2C9and CYP2C19, CYP2E1and CYP2D6, thus affecting enzymessubstrate metabolism by drug-metabolizing enzymes. The results show that thetimosaponin B-II’s weak inhibition of drug metabolizing enzymes, which is unlikely tolead to clinical drug interactions because of its inhibition of drug-metabolizing enzymes.
引文
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12G.C. Kite, E.A. Porter, M.S.J. Simmonds, Chromatographic behaviour of steroidal saponins studiedby high-performance liquid chromatography-mass spectrometry[J], Journal of Chromatography A,2007,1148(2):177-183.
13高守红.虎杖苷的体内分析及临床前药代动力学研究[D].上海,第二军医大学,2005
14王德娟,王栋.北豆根中蝙蝠葛碱在小鼠体内的组织分布研究[J].中医药学报,2012,40(6):31-33
15刘睿,谢跃生,潘桂湘等.药物血浆蛋白结合率测定方法的研究进展[J].天津中医药,2007,24(6):526-528
16李俊娟,吴阳,张英丰.平衡透析法测定丹参素血浆蛋白结合率[J].河南中医,2012,32(6):763-764
17马一鸣,范昭泽,陈旭等. TJ0711盐酸盐的血浆蛋白结合率及其在大鼠体内的组织分布[J].中国医院药学杂志,2011,31(15):1235-1238
18单进军.祖师麻主要活性成分的口服吸收及代谢研究[D].南京中医药大学,南京,2009
19邱峰.浅谈中药成分体内代谢研究[J].国际药学研究杂志,2010,37(5):321-328
20姜鹏.麝香保心丸代谢组学和代谢动力学研究[D].第二军医大学,上海,2012
21廖晓慧.抗肿瘤活性成分SYS330的小肠吸收机制研究[D].华中农业大学,武汉,2007
22钟大放,药物代谢（M）,中国医药科技出版社,北京,1996.
23S. Rendic, Summary of information on human CYP enzymes: human P450metabolism data[J], Drugmetabolism reviews,2002,34(1-2):83-448.
24A. Aminimanizani, P. Beringer, R. Jelliffe, Comparative pharmacokinetics and pharmacodynamics ofthe newer fluoroquinolone antibacterials[J], Clinical pharmacokinetics,2001,40(3):169-187.
25M. Hao, Y. Zhao, P. Chen, H. Huang, H. Liu, H. Jiang, R. Zhang, H. Wang, Structure-activityrelationship and substrate-dependent phenomena in effects of ginsenosides on activities ofdrug-metabolizing P450enzymes[J], PLoS One,2008,3(7):259-274.
26M.F. Paine, H.L. Hart, S.S. Ludington, R.L. Haining[J], A.E. Rettie, D.C. Zeldin, The humanintestinal cytochrome P450“pie”, Drug Metabolism and Disposition,2006,34(5):880-886.
27徐斌,赵刚,位华等.20味中成药对5个人肝微粒体酶活性的影响[J].药学实践杂志,2009,,27(5):353-356
28何雅君.基于CYP3A4代谢的中药麝香保心丸多成分相互作用研究[D].上海交通大学,上海,2012
29李海云.常用中药成分对药物代谢酶CYP3A4活性的影响[D].第二军医大学,上海,2009
30H.J. KIM, E.J. CHANG, S.H. CHO, S.K. CHUNG, H.D. PARK, S.W. CHOI, Antioxidative activityof resveratrol and its derivatives isolated from seeds of Paeonia lactiflora[J], Bioscience, biotechnology,and biochemistry,2002,66(9):1990-1993.
31S.A. Testino, High-throughput inhibition screening of major human cytochrome P450enzymes usingan in vitro cocktail and liquid chromatography-tandem mass spectrometry [J], Journal ofpharmaceutical and biomedical analysis,2003,30(5):1459-1467.
32J.M. Margolis, R.S. Obach, Impact of nonspecific binding to microsomes and phospholipid on theinhibition of cytochrome P4502D6: implications for relating in vitro inhibition data to in vivo druginteractions[J], Drug Metabolism and Disposition,2003,31(5):606-611.
33T.D. Bjornsson, J.T. Callaghan, H.J. Einolf, V. Fischer, L. Gan, S. Grimm, J. Kao, S.P. King, G. Miwa,L. Ni, The conduct of in vitro and in vivo drug-drug interaction studies: a PhRMA perspective[J], TheJournal of Clinical Pharmacology,2003,43(5):443-469.
34T.H. Tran, L.L. von Moltke, K. Venkatakrishnan, B.W. Granda, M.A. Gibbs, R.S. Obach, J.S.Harmatz, D.J. Greenblatt, Microsomal protein concentration modifies the apparent inhibitory potency ofCYP3Ainhibitors[J], Drug Metabolism and Disposition,2002,30(12):1441-1445.
35M.E. Barecki, C.N. Casciano, W.W. Johnson, R.P. Clement, In vitro characterization of the inhibitionprofile of loratadine, desloratadine, and3-OH-desloratadine for five human cytochrome P-450enzymes[J], Drug Metabolism and Disposition,2001,29(9):1173-1175.
36H.S. Brown, A. Galetin, D. Hallifax, J.B. Houston, Prediction of in vivo drug-drug interactions fromin vitro data: factors affecting prototypic drug-drug interactions involving CYP2C9, CYP2D6andCYP3A4[J], Clinical pharmacokinetics,2006,45(10):1035-1050.
37S.C. Uhn, Eun, Y.P., Mi, S.D., Bum, S.P., Keehyuk, K., Kyung, H.K., Tight-binding inhibition byα-naphthofavone of human cytochrome P4501A2[J], Biochim Biophys Acta,2003,1648(1-2):195-202.
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2倪梁红,秦民坚.知母资源化学及药理研究进展.中国野生植物资,2005,24,4:16-20
3陈万生;孙连娜;郑水庆等.知母商品药材中的皂苷类成分含量测定.中国医院药学杂志,2006,26(6):656-658
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8T.J. Li, Y. Qiu, P.Y. Yang, Y.C. Rui, W.S. Chen, Timosaponin B-II improves memory and learningdysfunction induced by cerebral ischemia in rats[J], Neuroscience letters,2007,421(2):147-151.
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10王莉,知母皂苷B-II在大鼠体内药代动力学研究[J],安徽医科大学,合肥,2010,pp.23-36.
11蔡飞.滋肾丸药代动力学及其配伍机制研究[D],上海,第二军医大学，2011
12G.C. Kite, E.A. Porter, M.S.J. Simmonds, Chromatographic behaviour of steroidal saponins studiedby high-performance liquid chromatography-mass spectrometry[J], Journal of Chromatography A,2007,1148(2):177-183.
13高守红.虎杖苷的体内分析及临床前药代动力学研究[D].上海,第二军医大学,2005
14王德娟,王栋.北豆根中蝙蝠葛碱在小鼠体内的组织分布研究[J].中医药学报,2012,40(6):31-33
15刘睿,谢跃生,潘桂湘等.药物血浆蛋白结合率测定方法的研究进展[J].天津中医药,2007,24(6):526-528
16李俊娟,吴阳,张英丰.平衡透析法测定丹参素血浆蛋白结合率[J].河南中医,2012,32(6):763-764
17马一鸣,范昭泽,陈旭等. TJ0711盐酸盐的血浆蛋白结合率及其在大鼠体内的组织分布[J].中国医院药学杂志,2011,31(15):1235-1238
18单进军.祖师麻主要活性成分的口服吸收及代谢研究[D].南京中医药大学,南京,2009
19邱峰.浅谈中药成分体内代谢研究[J].国际药学研究杂志,2010,37(5):321-328
20姜鹏.麝香保心丸代谢组学和代谢动力学研究[D].第二军医大学,上海,2012
21廖晓慧.抗肿瘤活性成分SYS330的小肠吸收机制研究[D].华中农业大学,武汉,2007
22钟大放,药物代谢（M）,中国医药科技出版社,北京,1996.
23S. Rendic, Summary of information on human CYP enzymes: human P450metabolism data[J], Drugmetabolism reviews,2002,34(1-2):83-448.
24A. Aminimanizani, P. Beringer, R. Jelliffe, Comparative pharmacokinetics and pharmacodynamics ofthe newer fluoroquinolone antibacterials[J], Clinical pharmacokinetics,2001,40(3):169-187.
25M. Hao, Y. Zhao, P. Chen, H. Huang, H. Liu, H. Jiang, R. Zhang, H. Wang, Structure-activityrelationship and substrate-dependent phenomena in effects of ginsenosides on activities ofdrug-metabolizing P450enzymes[J], PLoS One,2008,3(7):259-274.
26M.F. Paine, H.L. Hart, S.S. Ludington, R.L. Haining[J], A.E. Rettie, D.C. Zeldin, The humanintestinal cytochrome P450“pie”, Drug Metabolism and Disposition,2006,34(5):880-886.
27徐斌,赵刚,位华等.20味中成药对5个人肝微粒体酶活性的影响[J].药学实践杂志,2009,,27(5):353-356
28何雅君.基于CYP3A4代谢的中药麝香保心丸多成分相互作用研究[D].上海交通大学,上海,2012
29李海云.常用中药成分对药物代谢酶CYP3A4活性的影响[D].第二军医大学,上海,2009
30H.J. KIM, E.J. CHANG, S.H. CHO, S.K. CHUNG, H.D. PARK, S.W. CHOI, Antioxidative activityof resveratrol and its derivatives isolated from seeds of Paeonia lactiflora[J], Bioscience, biotechnology,and biochemistry,2002,66(9):1990-1993.
31S.A. Testino, High-throughput inhibition screening of major human cytochrome P450enzymes usingan in vitro cocktail and liquid chromatography-tandem mass spectrometry [J], Journal ofpharmaceutical and biomedical analysis,2003,30(5):1459-1467.
32J.M. Margolis, R.S. Obach, Impact of nonspecific binding to microsomes and phospholipid on theinhibition of cytochrome P4502D6: implications for relating in vitro inhibition data to in vivo druginteractions[J], Drug Metabolism and Disposition,2003,31(5):606-611.
33T.D. Bjornsson, J.T. Callaghan, H.J. Einolf, V. Fischer, L. Gan, S. Grimm, J. Kao, S.P. King, G. Miwa,L. Ni, The conduct of in vitro and in vivo drug-drug interaction studies: a PhRMA perspective[J], TheJournal of Clinical Pharmacology,2003,43(5):443-469.
34T.H. Tran, L.L. von Moltke, K. Venkatakrishnan, B.W. Granda, M.A. Gibbs, R.S. Obach, J.S.Harmatz, D.J. Greenblatt, Microsomal protein concentration modifies the apparent inhibitory potency ofCYP3Ainhibitors[J], Drug Metabolism and Disposition,2002,30(12):1441-1445.
35M.E. Barecki, C.N. Casciano, W.W. Johnson, R.P. Clement, In vitro characterization of the inhibitionprofile of loratadine, desloratadine, and3-OH-desloratadine for five human cytochrome P-450enzymes[J], Drug Metabolism and Disposition,2001,29(9):1173-1175.
36H.S. Brown, A. Galetin, D. Hallifax, J.B. Houston, Prediction of in vivo drug-drug interactions fromin vitro data: factors affecting prototypic drug-drug interactions involving CYP2C9, CYP2D6andCYP3A4[J], Clinical pharmacokinetics,2006,45(10):1035-1050.
37S.C. Uhn, Eun, Y.P., Mi, S.D., Bum, S.P., Keehyuk, K., Kyung, H.K., Tight-binding inhibition byα-naphthofavone of human cytochrome P4501A2[J], Biochim Biophys Acta,2003,1648(1-2):195-202.
[1]杨招萍,金麟根.我国中药产业比较优势及出口发展战略研究[J].时代经贸,2007,5(73):103~105
[2]段金廒,范欣生,宿树兰,等.中药及方剂量效关系的研究进展与思考[J].南京中医药大学学报,2009,25(4):241~245
[3] Chan E. Interactions between traditional Chinese medicines and Western therapeutics[J].Current opinion in drug discovery&development,2010Jan;13(1):50~65
[4]宋景春,郭文怡.中药复方的临床药动学进展[J].中国医药学报,2001,16(1):20~22
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