| Radix Angelicae Dahuricae, the dried root of Angelica dahurica (Fisch. ex Hoffm)Benth. et Hook. f. and Angelica dahurica (Fisch. ex Hoffm) Benth. et Hook. f. var.formosana (Boiss.) Shan et Yuan, first recorded in the Shennong’s classic of MateriaMedica, is a well-known traditional Chinese medicine which has been widely used inChina for over2000years as an antipyretic and analgesic for the treatment of colds,headaches, toothaches, coryza andreduction of swelling and pain from sores and wounds.At present, the study of A.dahurica was confined to the traditional quality control,process optimization and pharmacological effects and there was no report about theapplications of modern techniques, in-depth discussion on effective compounds andcomprehensive study in vivo for A.dahurica. In this study, the investigation on the qualitycontrol in vitro and metabolic process in vivo of A.dahurica were comprehensivelyperformed basd on the development of plantform on extraction, separation, identificationand preparation of A.dahurica, using multidimensional chromatography-massspectrometry based on advanced pretreatment technology as research tools. Initially, anefficient accelerated solvent extraction (ASE) was optimized to maximize the extractionyields of compounds from A.dahurica using response surface methodology, andmeanwhile, a reliable high-performance-liquid-chromatography (HPLC) coupled withmass spectrometry was employed to identify the compounds in A.dahurica extracts.Subsequently, the pharmacodynamic effects of the compounds groups with differentpolarities separated by macroporous resin were evaluated according the analgesic test ofrats. After the active ingredients were found, a high-speed counter currentchromatography was adopted for the large-scale preparation of the active monomer fromA.dahurica extracts. In all, the study on the extraction, separation, identification andseparation of A.dahurica provide the technical, informational and material support for theanalysis on the chemical fingerprints in vitro and metabolic fingerprints in vivo. With thesupport, the study was further carried out from three aspects. First, based on the optimalextraction method and the development of chemical fingerprint, the quanlity control ofA.dahurica was comprensively performed coupled with chemometric methods andquantitative analysis of multi-compounds, to ensure safe and effective herb use. Second,the coumarins prototypes and their metabolites was detected in plasma, bile, urine andbrain of rats after oral administration of A.dahurica extracts, to explore the metabolic characteristic of active compounds of A.dahurica in vivo. Third, the pharmacokineticstudies of seven main coumarins was performed in rat plasma after oral administration ofA.dahurica extracts to provide clinical references. The investigation on the material basisand metabolic characteristics of A.dahurica is of great significance for the clarification ofpharmacological ingredients and mechanisms of action. The detailed results werepresented as follows：1. Optimal extraction and qualitative fingerprint analysis of Angelica dahurica byaccelerated solvent extraction and high performance liquid chromatographicanalysis with photodiode array and mass spectrometry detectionsInitially, after five different extraction methods were compared to maximize theextraction yields of coumarins from A.dahurica, it was found that the extractionefficiency of ASE with good reproducibility was five times more than that of otherconventional extraction methods. Therefore, ASE was chosen to extract components fromA.dahurica due to its economic, efficient and automatic merits. Subsequently, a responsesurface methodology was employed to optimize the extraction parameters yielding theoptimum conditions of ASE (ethanol as extraction solvent, extraction temperature174℃,extraction time13min and two static cycles) based on the results by single-factorexperiments. At last, with the abundant constituents in the high content obtained in theA.dahurica extracts, a total of24components were identified by the high-performanceliquid chromatography coupled with photodiode array, electrospray ionization ion traptandem mass spectrometry and time of flight mass spectrometry(HPLC-PDA-ESI-ITMSn/TOF-MS). In addition, the identification pattern was revealedfor the characterization of isomers. The chapter provided the technical and informationalfoundation for quality control and metabolism analysis of A.dahurica.2. Acquirement and preparation of effective compounds using preparationtechnology combined with analgesis efficacyA.dahurica is widely used for the treatment of migraine and dysmenorrhea, but theeffective compounds were not fully elucidated. Initially, using macroporous resin, theA.dahurica extracts were separated into three groups according to their polarities,including the compounds groups with large polarity, middle polarity and small polarity.Meanwhile, the monomer of isoimperatorin was also obtained. Then, by the rat model of formalin-induced pain, it was found that the compounds groups of A. dahurica withmiddle polarity and small polarity exhibited analgesic activity. Especially, the compoundsgroups with small polarity showed not significant analgesic activity but also calmingeffect. Based on the fact that the effective compounds groups of A. dahurica were found,analytical high speed counter current chromatography (HSCCC) was employed toprepare effective monomers of A. dahurica. Six monomers were large-scale obtained,which were imperatorin, phelloptorin, isoimperatorin, xanthotoxol, hydrateoxypeucedanin and isooxypeucedanin. In all, the combination of multidimensionalcolumn, HSCCC and HPLC based on the analgesic efficacy provided the material supportfor the analysis of the chemical fingerprints in vitro and metabolic fingerprints in vivo.3. Quality control of multi-compounds for A.dahurica using quantitative fingerprintanalysis coupled with pattern recognitionThe origins of A.dahurica were different, such as Hang-A.dahurica,Chuan-A.dahurica, Yu-A.dahurica and Qi-A.dahurica. The contents of each componentfrom A.dahurica vary significantly with different source, which may affect the clinicalefficacy. In the chapter, the chromatographic fingerprints and quality of13A.dahuricasamples from different regions in China were constructed and estimated based on thedeveloped ASE and HPLC methods. At first, According to the results of analgesisefficacy, the peaks representing large polarity were removed because those may affect theresults of quality assessment. And then, similarity analysis, hierarchical cluster analysisand discriminant analysis were employed to distinguish the different A.dahurica sampleson various aspects and search common and discriminate peaks that may impact thequality of herbs. The common and discriminate peaks were selected as quantitativemarkers. At last, amounts of the nine quantitative markers were simultaneouslydetermined for the quality control of13batches of A.dahurica samples. Collectively, thecombination of fingerprint analysis of TCMs and pattern recognition methods couldcomprehensively evaluate the quality of TCMs and also provide the evidences to selectthe quantitative markers. Meanwhile, the quantitative analysis of multi-compounds isvery essential for the quality control of TCMs in detail. And the information fromquantitative analysis of multi-compounds indicated that HCA was more accurate than SAon the quality evaluation of herbs. 4. The study of metabolism fingerprints after oral administration of A.dahuricaextracts in rat plasma, bile, urine and brainThe study on metabolism fingerprints of TCMs in vivo is crucial for the elucidationof mechanism of action. In this chapter, the coumarins prototypes and their metaboliteswere analyzed in rat plasma, bile, urine and brain after oral administration of A.dahuricaextracts. After the comparisions of different sample preparation techniques, liquid liquidextraction and solid phase extraction were chosen. Then, the HPLC-PDA-ESI-ITMSnmethod was adopted for the determination of the coumarins prototypes and theirmetabolites. The results showed that twelve prototypes were detected in the rat plasma,bile, urine and brain, namely byakangelicol, xanthotoxol, hydrate oxypeucedanin,apaensin, xanthotoxin, bergapten, oxypeucedanin, isooxypeucedanine,5-methoxy-8-hydroxypsoralen, imperatorin, phelloptorin and isoimperatorin, andmeanwhile, thirteen metabolites were also detected. Moreover, it is indicated that thecoumarins prototype and metabolites are excreted from urine, becaused that most of themwere detected in urine of rats.5. The pharmacokinetic study of multi-compounds after oral administration ofA.dahurica extracts in rat plasmaIn the chapter, based on the coumarins from A.dahurica detected in the rats after oraladministration of A.dahurica extracts, seven prototypes were selected and determined inthe rat plasma for the pharmacokinetic study of multi-compounds, which are xanthotoxol,hydrate oxypeucedanin, xanthotoxin, bergapten, imperatorin, phelloptorin andisoimperatorin. A high performance liquid chromatographic method-tandem massspectrometry (HPLC-MS/MS) method, coupled with liquid-liquid extraction addingacetic acid has been developed for the determination of the seven coumarins in rat plasma.These analytes were simultaneously detected by triple quadrupole mass spectrometer inthe multiple reaction monitoring mode with a chromatographic run time of11.0min. Withthe establishment of plasma concentration-time curve profiles, the pharmacokineticparameters were evaluated. The results showed that absorption of the seven coumarinswith maximum concentration occurred approximately2h and the elimination occurredwithin12h after oral administration of A.dahurica extracts. The pharmacokinetic studyprovide basis for the clinical applications of A.dahurica. In all, with modern techniques and analgesic test, the platform was efficient, reliableand comprehensive for the study of mainly active coumarins from A.dahurica containingextraction, separation, identification, preparation, quality control and metabolic analysis.The study is meaningful for the investigation of pharmacodynamic material basis and themechanisms of action of A.dahurica. |
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