多组分中药物质组溶出及药代动力学评价方法研究
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摘要
中药具有多组分，多靶点的特点，组分含量、性质差异大。受种类繁多的单一标准品难于获得以及研究思路的限制，药物浓度法、药效法和毒效法等传统分析方法无法全面反映中药内在质量与整体药效。针对上述问题，本论文提出以中药提取物作为“全组分标准”定量新思路。以临床疗效确切的升麻口服片剂作为模型药物，建立适合多组分中药特点的高通量、高灵敏度、高选择性的UPLC-MS/MS定量分析新方法。建立释放介质中希明婷片各组分的LC-MS/MS分析方法，进行希明婷片多组分同步体外释放评价。采用LC-ELSD-MS/MS法对希明婷原料升麻异丙醇中12个中药组分进行大鼠体内的药代动力学评价，针对体外含量和体内/外暴露、释放结果归纳中药多组分作用特点。
通过本论文的研究，将会丰富和发展UPLC-MS/MS对于复杂组分定量的基础理论和研究思路，有助于揭示和阐明中药的复杂作用机制及其科学内涵，为应用现代评价方法研究中药提供方法学的支撑。
Throughout history, traditional Chinese medicine(TCM) hasplayed an importantrole in China for theprevention and treatment of various human ailments. The use ofherbal medicines continues to expand rapidly in both developing anddevelopedcountries bringing with it increased concerns about their safety.As pointedout in “General Guidelines for Methodologieson Research and Evaluation ofTraditional Medicines (WorldHealth Organization,2000)”,“The quantity andqualityof the safety and efficacy data on traditional medicineare far from sufficient to meetthe criteria needed to supportits use world-wide. The reasons for lack of researchdataare due not only to health care policies, but also to a lackof adequate or acceptedresearch methodology for evaluatingtraditional medicine”. Our research is aimed atdeveloping this research methodology.
The curative effect of traditional Chinese medicine (TCM) is often the result ofintegrated contributions from and dynamic interactions between a number ofbioactive compounds. The problem of obtaining pure standards of each constituentemerges as a major issue exacerbated by the fact that TCM constituents arenotoriously unstable and subject to degradation during attempts to extract them, sothe drug concentration,pharmacodynamics and toxicity oftraditionalanalysis methodcan not fullyreflectthe intrinsicquality andthe overallefficacyof TCM. To circumventthe difficulty, we use the raw TCM itself as “a mixed external standard”for thedevelopment and validation of an LC-MS/MS assay. Ximingting oraltabletsandYangshenye ginsenoside freeze-dried powder were used as a model drug, and thehigh-throughput, high sensitivity, high selectivityUPLC-MS/MSmulti-componentquantitativeanalysis method was developed.
1.The isolation of cimicifugoside H-1,cimicifugoside H-2，23-epi-26-deoxyactein,cimigenolxyloside and25-O-acetylcimigenoside were isolated from Shengma extract and characterization by DAD，MS and1H NMR. The purity ofthe urisomer swas determined by HPLC，and the results showed that the Purity of thefive monomer was up to98%，which met the requirements of reference standards.
2.Pharmacokinetic study of cimicifugoside H-1, cimicifugoside H-2，23-epi-26-deoxyactein, cimigenolxyloside and25-O-acetylcimigenoside in rats. AnLC-MS/MS assay was developed for the determination ofcimicifugoside H-1,23-epi-26-deoxyactein, cimigenolxyloside and25-O-acetylcimigenoside obtainedfrom Cimicifuga foetida L. and applyed to investigate their pharmacokinetic (PK)properties and bioavailabilities through oral administration of single oral doses of aCimicifuga foetida L. extract (12.5,25and50mg/kg) and single intravenous (i.v.)doses (5mg/kg) of the individual cimicifugosides in rat.PK parameters wereestimated by non-compartmental analysis.PK parameters forcimicifugoside H-1,cimicifugoside H-2，23-epi-26-deoxyactein, cimigenolxyloside and25-O-acetylcimigenoside following i.v. administration of individual cimicifugosideswere respectively: t1/20.30，1.13，2.36，5.67and0.34h；AUC0-t307，389，10633，19751and2037ng﹒h/mL；Cmax1126，808，5683，2757，4231ng/mL；PK parameterscimicifugoside H-1, cimicifugoside H-2，23-epi-26-deoxyactein, cimigenolxylosideand25-O-acetylcimigenoside afteroral administration of individual cimicifugosideswere respectively: t1/21.68，2.04，3.47，9.59and12.6h；AUC0-t91.0，20.3，10595，26051and1305ng﹒h/mL；Tmax0.75，1.17，0.82，4.59and7.3h。
The results suggest that although the chemical structure of the fivecimicifugoside was similar, the pharmacokinetics characteristics was different. Themaximum value of the plasma concentration and mean retention time was severaltimes higher than the minimum. There may be large difference in tissue distributionbetween different cimicifugoside.
3. Studies on dissolution test method of traditional Chinese patent medicine-dissolution rate of Ximingting Patent: To establish a high-throughput analysis methodfor the multi-index components dissolution rate in Tradition Chinese Patent Medicine(TCPM). The herbal extract was used as was “Overall Component Reference”, and ahigh-performance liquid chromatography with electrospray ionization tandem mass spectrometry (HPLC-MS/MS) was developed. In this study, isopropanol extract ofCimicifuga was the “overall component reference” and was used for the preparationof standard series solution to establish the standard curve. Fourteen components ofextract were simultaneousquantified by LC-MS/MS. The method was successfullyemployed in a dissolution study of fouteen components of Ximingting. This paperdeveloped a novel method to research multi-component dissolution test method oftraditional Chinese medicine and laid the foundation for evaluating the process ofmedicine in vitro.
4. Quantitation of Traditional Chinese Medicines: Assay and PharmacokineticStudies of Cimicifuga foetida L. extract: Rapid and sensitive bioanalytical methodsare essential to support safety and efficacy research on traditional Chinese medicine(TCM). However TCM are complex mixtures of many constituents for which purestandards are usually not available. In this report, a ‘relative reference approach’ thatherb extracts, instead of purified standards acted as the reference substance. Thisapproach is well suited for the analysis of the compounds without standards. On thisbasis, Liquid chromatography evaporative light scattering detector/tandem massspectrometry (LC-ELSD-MS/MS) was applied to provide a chemical profile ofCimicifuga foetida L. extract. The assay was applied to a pharmacokinetic study ofthe extract in rat which showed that the relative levels of the12constituents inplasma were different from the levels expected based on the relative amounts in theextract.PK parameters for compound A-L following oral administration of the extractwere respectively:t1/27.70，7.0，7.5，11.6，10.9，6.14，8.5，10.3，7.62，8.43，6.08and5.5h；AUC0-t143,22.4,0.8,5.49,100,112,90.8,6.82,315,553,4.07and20.6E μg﹒h/mL；Tmax为2.8,4.30,11.4,10.9,6.17,2.7,4.02,7.67,15.0,11.5and6.0h。
PK studies on TCMs containing many constituents indicate that both animalsand humans are exposed to some but not all constituents and that the PK profiles, likethe pharmacological activities, can be unexpected. Thusknowledge of the PK andbioavailability of individual constituents can provide a link between the effects ofconsumption of a particular TCM and its pharmacological effects.In the present study, we compared systemic exposure to the putatively active cimicifugosides inCimicifuga foetida L. extract to illuminate the factors governing exposure toindividual compounds. The results indicate that differences in clearance on the onehand and interconversion of cimicifugosides on the other are primary factorscontrolling exposure to the cimicifugosides. Further studies are needed to characterizethe bioavailability and pharmacokinetics of herbal medicinal products in order to takefull advantage of their therapeutic potential.
通过本论文的研究，将会丰富和发展UPLC-MS/MS对于复杂组分定量的基础理论和研究思路，有助于揭示和阐明中药的复杂作用机制及其科学内涵，为应用现代评价方法研究中药提供方法学的支撑。
Throughout history, traditional Chinese medicine(TCM) hasplayed an importantrole in China for theprevention and treatment of various human ailments. The use ofherbal medicines continues to expand rapidly in both developing anddevelopedcountries bringing with it increased concerns about their safety.As pointedout in “General Guidelines for Methodologieson Research and Evaluation ofTraditional Medicines (WorldHealth Organization,2000)”,“The quantity andqualityof the safety and efficacy data on traditional medicineare far from sufficient to meetthe criteria needed to supportits use world-wide. The reasons for lack of researchdataare due not only to health care policies, but also to a lackof adequate or acceptedresearch methodology for evaluatingtraditional medicine”. Our research is aimed atdeveloping this research methodology.
The curative effect of traditional Chinese medicine (TCM) is often the result ofintegrated contributions from and dynamic interactions between a number ofbioactive compounds. The problem of obtaining pure standards of each constituentemerges as a major issue exacerbated by the fact that TCM constituents arenotoriously unstable and subject to degradation during attempts to extract them, sothe drug concentration,pharmacodynamics and toxicity oftraditionalanalysis methodcan not fullyreflectthe intrinsicquality andthe overallefficacyof TCM. To circumventthe difficulty, we use the raw TCM itself as “a mixed external standard”for thedevelopment and validation of an LC-MS/MS assay. Ximingting oraltabletsandYangshenye ginsenoside freeze-dried powder were used as a model drug, and thehigh-throughput, high sensitivity, high selectivityUPLC-MS/MSmulti-componentquantitativeanalysis method was developed.
1.The isolation of cimicifugoside H-1,cimicifugoside H-2，23-epi-26-deoxyactein,cimigenolxyloside and25-O-acetylcimigenoside were isolated from Shengma extract and characterization by DAD，MS and1H NMR. The purity ofthe urisomer swas determined by HPLC，and the results showed that the Purity of thefive monomer was up to98%，which met the requirements of reference standards.
2.Pharmacokinetic study of cimicifugoside H-1, cimicifugoside H-2，23-epi-26-deoxyactein, cimigenolxyloside and25-O-acetylcimigenoside in rats. AnLC-MS/MS assay was developed for the determination ofcimicifugoside H-1,23-epi-26-deoxyactein, cimigenolxyloside and25-O-acetylcimigenoside obtainedfrom Cimicifuga foetida L. and applyed to investigate their pharmacokinetic (PK)properties and bioavailabilities through oral administration of single oral doses of aCimicifuga foetida L. extract (12.5,25and50mg/kg) and single intravenous (i.v.)doses (5mg/kg) of the individual cimicifugosides in rat.PK parameters wereestimated by non-compartmental analysis.PK parameters forcimicifugoside H-1,cimicifugoside H-2，23-epi-26-deoxyactein, cimigenolxyloside and25-O-acetylcimigenoside following i.v. administration of individual cimicifugosideswere respectively: t1/20.30，1.13，2.36，5.67and0.34h；AUC0-t307，389，10633，19751and2037ng﹒h/mL；Cmax1126，808，5683，2757，4231ng/mL；PK parameterscimicifugoside H-1, cimicifugoside H-2，23-epi-26-deoxyactein, cimigenolxylosideand25-O-acetylcimigenoside afteroral administration of individual cimicifugosideswere respectively: t1/21.68，2.04，3.47，9.59and12.6h；AUC0-t91.0，20.3，10595，26051and1305ng﹒h/mL；Tmax0.75，1.17，0.82，4.59and7.3h。
The results suggest that although the chemical structure of the fivecimicifugoside was similar, the pharmacokinetics characteristics was different. Themaximum value of the plasma concentration and mean retention time was severaltimes higher than the minimum. There may be large difference in tissue distributionbetween different cimicifugoside.
3. Studies on dissolution test method of traditional Chinese patent medicine-dissolution rate of Ximingting Patent: To establish a high-throughput analysis methodfor the multi-index components dissolution rate in Tradition Chinese Patent Medicine(TCPM). The herbal extract was used as was “Overall Component Reference”, and ahigh-performance liquid chromatography with electrospray ionization tandem mass spectrometry (HPLC-MS/MS) was developed. In this study, isopropanol extract ofCimicifuga was the “overall component reference” and was used for the preparationof standard series solution to establish the standard curve. Fourteen components ofextract were simultaneousquantified by LC-MS/MS. The method was successfullyemployed in a dissolution study of fouteen components of Ximingting. This paperdeveloped a novel method to research multi-component dissolution test method oftraditional Chinese medicine and laid the foundation for evaluating the process ofmedicine in vitro.
4. Quantitation of Traditional Chinese Medicines: Assay and PharmacokineticStudies of Cimicifuga foetida L. extract: Rapid and sensitive bioanalytical methodsare essential to support safety and efficacy research on traditional Chinese medicine(TCM). However TCM are complex mixtures of many constituents for which purestandards are usually not available. In this report, a ‘relative reference approach’ thatherb extracts, instead of purified standards acted as the reference substance. Thisapproach is well suited for the analysis of the compounds without standards. On thisbasis, Liquid chromatography evaporative light scattering detector/tandem massspectrometry (LC-ELSD-MS/MS) was applied to provide a chemical profile ofCimicifuga foetida L. extract. The assay was applied to a pharmacokinetic study ofthe extract in rat which showed that the relative levels of the12constituents inplasma were different from the levels expected based on the relative amounts in theextract.PK parameters for compound A-L following oral administration of the extractwere respectively:t1/27.70，7.0，7.5，11.6，10.9，6.14，8.5，10.3，7.62，8.43，6.08and5.5h；AUC0-t143,22.4,0.8,5.49,100,112,90.8,6.82,315,553,4.07and20.6E μg﹒h/mL；Tmax为2.8,4.30,11.4,10.9,6.17,2.7,4.02,7.67,15.0,11.5and6.0h。
PK studies on TCMs containing many constituents indicate that both animalsand humans are exposed to some but not all constituents and that the PK profiles, likethe pharmacological activities, can be unexpected. Thusknowledge of the PK andbioavailability of individual constituents can provide a link between the effects ofconsumption of a particular TCM and its pharmacological effects.In the present study, we compared systemic exposure to the putatively active cimicifugosides inCimicifuga foetida L. extract to illuminate the factors governing exposure toindividual compounds. The results indicate that differences in clearance on the onehand and interconversion of cimicifugosides on the other are primary factorscontrolling exposure to the cimicifugosides. Further studies are needed to characterizethe bioavailability and pharmacokinetics of herbal medicinal products in order to takefull advantage of their therapeutic potential.
引文
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