Application Of Modern Analytical Techniques In Identification Of Active Components In Traditional Chinese Medicinal Herbs

The study of pharmaceutical components in Chinese medicinal herbs demands not only the extract quantitative analysis of target compounds,but also the characterization of the compounds,and eventually control and evaluate the quality of Chinese medicinal herbs. It demands not only the differentiation of chemical compounds,but also the characterization of the compounds in plasma,and the possible pharmaceutical components were detected.It demands not only the differentiation of active compounds in plasma,but also metabolic and action mechanism of the compounds in plasma,and the possible pharmaceutical components were detected,which evaluate the pharmaceutical action and select as pharmaceutical components.It was necessary for pharmaceutical analysis to use multiple LC-MS techniques, because that the single analytical technique could not fulfill the demand of Chinese medicinal herbal analysis for its complexity and entirety.In this research,based on modern analytical techniques,from the two Chinese medicinal herbs of Sehisandra chinensis and Baizhi,we developed several method and strategies of characterization of chemical components in Chinese medicinal herbs and preliminary screening of pharmaceutical active components,established the fingerprints of chemical components in vivo and in vitro,and apply these method and related analytical techniques in whole quality control of Chinese medicinal herbs and multiple components pharmacokinetics.And baed on the analysis of pharmaceutical active components,multiple analytical techniques were compared in analysis of pharmaceutical components in vivo and in vitro according to different requirement.In consideration of complex chemical components and ambiguous chromatographic peaks,we developed a combinative method and stragetry by using HPLC-DAD, HPLC-TOF/MS and HPLC-QIT/MS according to the feature of multiple analytical techniques,which could be applied in non-target screening and identification of multi-component in vivo and in vitro of Chinese medicinal herbs.The extracting conditions of Sehisandra chinensis were systematic optimized in the experiments.The crude drugs of Sehisandra chinensis was selected basing on the indexes of six target components including extracting time,extracting solvents and extracting times. The Sehisandra chinensis samples were prepared by sonication in methanol for 30 min. There were 21 lignan components in Sehisandra chinensis identified based on the characterization and structure elucidation,and unambiguous fingerprints in vitro were developed.The similar UV spectra of the identified peaks indicated that they possessed the same skeletal structure,the dibenzocyclooctadien system.As in the analysis of the lignin compounds in the methanol extract of S.chinensis(Turcz.) Baill.fruits by LC/MSⁿ,some compounds of the identified peaks showed the intense ion[M+H-18]⁺,which is due to the fact that these compounds all have a hydroxyl at their C-6 or C-7.So it is concluded that if there is a hydroxyl at the lignan compound's C-6 or C-7,it will be easy to give an ion as a result of the loss a molecule of water.Similarly,some compounds of the identified peaks that have an acyloxy group showed the ion[M+H-RCOOH]⁺,and on the basis of this information the type of acyloxy groups can be identified simultaneously.The similarity of structure results in fragmentation in a similar pathway.There are some lignin compounds that have the same molar mass,and are pairs of positional isomers or stereoisomers that can possibly be coeluted.By LC/MSⁿ analysis,the isomers' retention time,[M+H]⁺ and the diagnostic product ions can be obtained.On the basis of these data the differentiation of those isomers can be achieved.Three pairs of isomers in Sehisandra chinensis were differentiated in this experiment.Furocoumarins in Angelica dahurica were identified using three analytical techniques. Fifteen furocoumarins were characterized by HPLC-TOF/MS,and elucidated by HPLC-QIT/MS.For mono-oxysubstituting furocoumarins,if the isopentenoxy group was at C-5 or C-8,they could produce fragment ions at m/z 203 in MS or MS².The fragment ions(m/z 203) as the precursor subjected to the next stage ESI-MS analysis,produced ions at m/z 175[203-CO]⁺ and 159[203-CO₂]+.If isopentenoxy group was at C-5,the relative abundance of m/z 159 was larger than that of m/z 175.However,if isopentenoxy group was at C-8,the result was on the contrary.This was probably attributed to different conjugation extensions.It was most likely easy for conjugation moiety losing CO at C-8 and non-conjugation moiety losing CO₂ at C-2.That is to say,when the isopentenoxy group was at C-8,it simultaneously had two fragmentation patterns to lose CO(C-8 and C-2), and the relative abundance of m/z 175(loss of CO) was larger than that of m/z 159(loss of CO₂);but when the isopentenoxy group was at C-5,only one fragmentation pathway of losing CO(C-2) was present,and the relative abundance of m/z 175(loss of CO) was smaller than that of m/z 159(loss of CO₂).If the alkoxy moieties were both at C-5 and C-8, its fragmentation pattern was loss of CO,but loss of CO₂ was not observed.The reason why neutral loss of CO was the main fragmentation behavior was probably because its carbonyl groups at C-5 and C-8(m/z 217) not only formed conjugation with carbonyl group at C-2,but also formed homo-quinone conjugation.Forty-seven volatile oils were identified successfully searching against formula mass databases by GC-MS.Bsed on the characterization of chemical components of Chinese medicinal herbs,11 lignans were identified successfully using this method after administration of the WWZ extract.A quantitative method of six lignans in Sehisandra chinensis,including schisandrin, schisandrol B,schisantherin A,deoxyschisandrin,γ-schisandrin,schisandrin C was developed by HPLC-DAD,which reflect contents of lignans in WWZ,control and evaluate the quality of Chinese medicinal herbs.Six lignans of thirty-three batches of Sehisandra chinensis from different regions and five Chinese medicinal remedies including Sehisandra chinensis were determined.The results indicated that the contents of six lignans in Sehisandra chinensis varied greatly,which showed regulary distribution by regions.Six lignans in plasma of SD rats were determined by LC-MS,which was applied in the pharmaceutics of six lignans in extract of Sehisandra chinensis.After administration of deoxyschisandrin suspl and the Sehisandra chinensis extract,the parameters of pharmacokinetics were different greatly.The C_max and T_max of six lignans in SD plasma increased after administration of the Sehisandra chinensis extract.The results of characterization and determination of chemical components in vivo and in vitro by modern combinative analytical techniques indicated that HPLC-MS＞HPLC-TOF/MS＞HPLC-DAD in the aspect of detective sensitivity.In terms of identification,HPLC-DAD,HPLC-TOF/MS and HPLC-QIT/MS were compared.The compounds were identified according to characteristic ultraviolet absorption and retention time,elementarily identified according to accurate mass determination and possible molecule matching by HPLC-TOF/MS,and elucidated by HPLC-QIT/MS.It can provide help for the selection of optimum analytical techniques and utilization rate of modern analytical techniques in Chinese medicinal herbal analysis.