An Integration Method Based On Chromatographic-mass Spectrum Technology And Metabolomics To Explore The Pharmacodynamic Material Basis Of Lonicerae Japonicae Flos And Lonicerae Flos

Objective: In this study,we established a metabonomics based on GC-MS,UHPLC-Q-TOF-MS/MS technology to identify the volatile oil and flavonoids in Lonicerae Japonicae Flos(LJF)and Lonicerae Flos(LF),and screen out their differential components to provide reference for clinical alternative medicine.Network pharmacology was used to explore the mechanism of luteolin on non-small cell lung cancer.Then,metabonomics method was used to further study the changes of endogenous metabolites and elucidate the related metabolic pathways,so as to provide data support for luteolin in the treatment of NSCLC.Methods: The volatile oil of LJF and LF obtained by steam distillation was detected by GC-MS.The raw data were pretreated with n-alkanes for peak correction and peak alignment.The substance is retrieved in the NIST MS database according to the retention time,m/z and other parameters.Finally,the difference of volatile oil was analyzed;these samples obtained by ultrasound extraction were detected by UHPLC-Q-TOF-MS/MS platform.The obtained data were compared with the secondary fragments in the self built database by peakview,and qualitative analysis was carried out based on the cleavage rules of various types of flavonoids.After the raw data was imported into the Progenesis QI for normalization,it was imported into SIMCA-P 14.1for multivariate statistical analysis.The components with VIP>3 and P<0.05 were selected as the differential variables of the two herbs,then these differential variables were matched with the identified flavonoids to screen the differential flavonoids.In the research of network pharmacology,the target genes of luteolin and NSCLC were identified by multiple online databases,and the protein-protein interaction network was constructed to screen the core target.Go analysis and KEGG analysis of the core targets were performed using Metascape database.A549 cells were treated with different concentrations of luteolin and the cell survival rate was detected by cell counting kit-8(CCK-8).The drug concentration at 50% survival rate was used for further study.A549 cells were treated by luteolin for 48 h to obtain the analytical samples and the samples were detected by UHPLC-Q-TOF-MS/MS platform.The raw data was normalized by progenesis QI and imported into SIMCA-P 14.1 software for multivariate data statistical analysis to obtain VIP value.At the same time,the P value was obtained by t-test.Metabolites that satisfy both VIP>1 and P <0.05 were considered differential metabolites.Potential biomarkers were obtained after identified through the HMDB database.Finally,the online platform Metaboanalyst 4.0 and KEGG online database was used for thermographic analysis and metabolic pathway screening.The ROC curve analysis was performed to find potential diagnostic biomarkers.Results: According to the NIST MS database,40 kinds of volatile components were detected in LJF and LF,including hydrocarbons,esters,aldehydes,ketones,phenols,alcohols and anilines.Among them,24 kinds of components are same and 16 kinds are differential volatile oils.A total of 47 flavonoids were identified in LJF and LF by UHPLC-Q-TOF-MS/MS.By screening VIP and P value,77 differential variables were obtained.By matching with the fragment ions of identified flavonoids,seven differential flavonoids were selected.Network pharmacology method was used to screen the core target genes of luteolin on NSCLC,and 44 related pathways were obtained by enrichment analysis.The cell viability test showed that the concentration of luteolin was 38 μg/m L when the cell viability was 50%.With this concentration as treatment group,45 potential biomarkers were screened and identified through multivariate statistical analysis and HMDB database.The results involved 20 pathways,the most important of which were tyrosine metabolism,glutathione metabolism and pentose phosphate pathway.ROC curve analysis showed that D-ribose,glutathione,L-tyrosine and3-methoxytyramine had good accuracy in the prediction of luteolin treat for NSCLC.Conclusions: In this study,a metabonomics method based on GC-MS and UHPLC-Q-TOF-MS/MS was established to compare the differential components of volatile oil and flavonoids in LJF and LF,which provided an effective basis for the clinical use of the two herbs.Network pharmacology and metabonomics were used to analyze the mechanism of luteolin in the treatment of NSCLC.The related metabolic pathways and potential diagnostic biomarkers interfered by luteolin were identified.The research results provide a new idea for luteolin in the treatment of NSCLC.