| Chemical analysis is basis to appreciate the pharmacodynamic material and overall curative effects of TCM formulas.Due to the structure diversity of constituent in vivo,complexity of biological matrix and the low concentration of compounds in vivo,it is a significant challenge in their chemical analysis with a rapid and effect analysis strategy.Qi-Jing-Sheng-Bai granule(QJSB)is an effective traditional Chinese medicine formula that has been widely used for the treatment of leukopenia post radiotherapy or chemotherapy,it is composed of ten medicinal materials,including Astragalus membranaceus(Huangqi),Cervil Cornus Colla(Lujiaojiao),Panax quinquefolium(Xiyangshen),Epimedium brevicornum(Yinyanghuo),Angelica sinensis(Danggui),Polygonatum sibiricum(Huangjing),Eclipta pwstmta(Mohanlian),Lycium barbarum(Gouqi),Psoralea corylifolia(Buguzhi)and Spatholobus suberectus(Jixueteng)with a ratio of 6:1:2:3:2:2:3:2:2:6.With ten herb medicines above,QJSB shows significant clinical effects on the treatment of leukopenia post radiotherapy or chemotherapy,and improving the body immunity.In order to understand the underlying mechanism for its pharmacological activities and improve the moderation of QJSB,systematic characterization of chemical compositions is necessary.In this study,QJSB was chosen as the research target.Under the guide of various innovation theory,taking advantage of advanced analysis technique,the main chemical constituents in vitro and the multiple absorbed prototype compounds and metabolites in rat plasma after treated QJSB were screened out.In addition,the potential bioactive components was quantified by HPLC-QqQ-TOF-MS in QJSB extract.The specific contents are as follows:Chemical profiling of QJSB extract by ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry combined with numerical platform.The first step was to set up an in-house library including the information of chemical components from the target herb based on the literature,a total of 1501 compounds were summarized.All the compounds were listed on an excel spread-sheet with specific template,which included information such as compound name,molecular formula.The second step was to screen and verify the known ingredients from raw data.Screening known compounds was automatically performed by UNIFI platform.After importing the in-house library into the analysis method and setting the appropriate acquisition parameters,automated detection and data filtering were performed by the UNIFI and the peaks detected evaluated against a compounds library of TCMs,and then analyzing MS fragmentation behaviors of interests.As a result,138 known constituents of QJSB were tentatively characterized structurally belonging to flavonoids,saponins,alkaloids,phenol acids,glycosides,coumarins,anthraquinone,lignans and amino acids,and then summarized and propose fragmentation patterns for different types of known components individually.The characteristic fragment ions,neutral loss,were selected for screening out the components with similar MS fragment behaviors as known compounds.The last step was to identify or characterize the chemical structures of the potential novel components with similar MS fragmentation pattern of the identified ingredients.As the MS fragmentation patterns of known compounds had already been summarized and proposed in the second step,we used several tools provided by UNIFI platforms,including common fragments search and neutral loss to screen out and then characterize MS fragmentation patterns for compounds of interests.As a result,five unknown cycloartane-type saponins were characterized.Based on the chemical constituents in vitro,under the guide of serum pharmacochemistry,a method for screening and analysis of the multiple absorbed prototype compounds and metabolites of serum was established.The first step was to characterize the prototype constituents in the serum.Based on in-house library,deducted the matrix in the blank serum,the common perks between QJSB extract and drug serum were prototype constituents.As a result,24 prototype constituents were characterized in the drug serum for the first time including flavonoids(13),saponins(six),amino acid(two),phenolic acid(two)and coumarin(one).The second step was to set up another database in vivo by MetaboLynx.This software contained most of the possible metabolites from I phase and/or II phase,liver metabolic and other pathways.A total of 382 proposed metabolites were obtained.The last step was to characterize the metabolites in the serum,based on metabolite database,deducted the common perks in the QJSB extract,the different perks between drug serum and blank serum are metabolites.Taking advantage of data mining technique including neutral search and EIC,18 metabolites were characterized in the drug serum including isoflavones(six),isoprenylated flavones(six),dihydroflavone(two),phenolic acids(two),saponins(two).The main biotransformation reactions were hydrolysis(phase I),sulfation and glucuronidation(phase II).In conclusion,this work serves the first reference for Qi-Jing-Sheng-Bai chemical components and metabolites.Moreover,it provided a rapid and valid analytical strategy for characterization of the chemical compounds and metabolites of traditional Chinese medicine formula. |
PDF Full Text Request