Study On The Material Foundation Of Qixianqingming Granule By UPLC-ESI-Q-TOF-MS

Qixianqingming granule was come from the clinical practice of Putuo Hospital,affiliated Hospital of Shanghai University of Chinese Medicine,and has been used to treat bronchial asthma more than 6 years.It is 8-herb formula composed of yinyanghuo(Epimedii Folium),huangqi(Astragali Radix),huzhang(Polygoni Cuspidati Rhizoma et Radix),bajitian(Morindae Officinalis Radix),pipaye(Eriobotryae Folium),dihuang(Rehmanniae Radix),chuanxiong(Chuanxiong Rhizoma)and xuanfuhua(Inulae Flos).For investigation and analysis effective chemical substance of Qixianqingming granule,the chemical components of the original and metabolite were analyzed by using ultra performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry(UPLC-ESI-Q-TOF-MS).Combination network pharmacology,the mechanism of Qixianqingming granule prescription was explored.The quality standard of Qixianqingming granule was established.1.Identification of chemical compounds of Qixianqingming granuleThe UPLC-ESI-Q-TOF-MS analytical method of the Qixianqingming granule was established for the first time.The results showed that the main chromatographic peaks were analyzed and 72 compounds were deduced based on the molecular weight,major MS~2 fragments and retention time,and combined with the fragmentation pathways summarized from the literature.Among them,there are 31 flavonoids,15 terpenoids,4anthraquinones,9 phenylethanoid glycosides,4 stilbenes,1 torachryson,1 alkaloid,2organic acids,1 sugar and 4 others.We found that 32 compounds from yinyanghuo,10compounds come from huangqi,10 compounds derived from huzhang,1 compound from bajitian,17 compounds from dihuang,3 compounds from pipaye and 3 compounds from xuanfuhua,nothing detected in chuanxiong.2.Quantitative analysis of compounds in Qixianqingming granuleThe HPLC analysis method of the Qixianqingming granule was established for the first time.According to UV absorption and retention time of reference substances and compounds identified based on LC-MS technology,we identified 29 compounds in the HPLC chromatogram of Qixianqingming granule.Among them,there are 2 organic acids,7 phenylethanoid glycosides,2 stilbenes,4 anthraquinones and 14 flavonoids.The content of sugar and protein were 70~90%and 2~4%respectively in Qixianqingming granule which were determined by UV spectrophotometry.The total content of material foundation more than 80%was clear in qixianqingming granule.3.Study of the absorbed constituents after oral administration of Qixianqingming granule and mechanism of action analysisUPLC-ESI-Q-TOF-MS method and UNIFI software were be used to analyze the absorbed constituents of Qixianqingming granule in rat serum and urine.13 original components and 30 metabolites were identified in rat serum,and 22 original components and 21 metabolites were identified in rat urine.These metabolites are many pairs of isomers,indicating that diversity of metabolism of Qixianqingming granule in vivo.Metabolic reactions included hydroxylation,demethylation,methylation,formate reaction,hydrogenation,deglycosylation,glucuronide conjugation and sulfation reaction.Asthma-related targets and absorbed constituents related targets were be summarized by using multi-database combination with the plugins in Cytoscape software.The core targets were obtained by setting the parameters after core analysis of intersection of the absorbed constituents target-target interaction(PPI)map with the asthma target PPI map.The“disease-compound-metabolism pathway”correlation network was established by metabolic pathway illumination on the core target.Finally,the results showed that Qixianqingming granule treatment of asthma may be achieved through 14 metabolism pathways,including MAPK signaling pathways,Wnt signaling pathways,Cell adhesion molecules,Toll-like receptor signaling pathway and T cell receptor signaling pathway.Results revealed Qixianqingming granule may play a synergistic role from multiple components,multiple targets,multiple paths,in the treatment of asthma.4.Quality standard for Qixanqingming granuleThe HPLC analytical methods has been established,which polydatin and icariin were selected as components of quantitative determination.TLC was used to identify the huangqi in Qixianqingming granule,the spot of Astragaloside IV were clear and negative sample without interference.HPLC was used to identified bajitian,the resolution of chromatographic peak nystose was well and negative sample without interference.This method is accurate,stable and feasible,and can be used for the quality control of Qixianqingming Granule.In conclusion,the main material foundation of Qixianqingming granule were identified through the UPLC-ESI-Q-TOF-MS technology.The content of various components was determined by HPLC and UV spectrophotometry in Qixianqingming granule,the total content of material foundation more than 80%is clear in the Qixianqingming granule.Using network pharmacology method,analysis the metabolic pathways of Qixianqingming granule in vivo,and their anti-asthmatic mechanisms were initially elucidated.The quality standard for Qixianqingming granule have been established.