Rapid And Complete Analysis The Components Of Abelmoschus Manihot And Screening Its Potential Anti-inflammatory Active Substance

As a kind of medicinal plant that was once on the verge of extinction,Abelmoschus manihot has therapeutic effects on liver disease/kidney disease,promoting vascular activity,anti-convulsion,anti-depression,anti-oxidation,anti-inflammation and so on.The anti-inflammatory effect of Abelmoschus manihot has been verified continuously by experiments,but the pharmacodynamic substance basis of its anti-inflammatory has not been explicitly clarified.In the study,a two-dimensional microscale carbon fiber/active carbon fiber system combined with a quadrupole time of flight high-resolution mass spectrometry（2DμCFs-QTOF-HRMS）system was proposed to achieve comprehensive analysis of the compositions of Abelmoschus manihot.The potential anti-inflammatory targets were predicted by network pharmacology,theoretical verified by molecular docking.And potential anti-inflammatory active ingredients were screened by Multi-compartment electrophoresis（MCESS）experiment.The purpose of this study was to explore the substance basis of anti-inflammatory efficacy of Abelmoschus manihot and to provide reference for molecular mechanism research.The research contents were as follows:Part one:Comprehensive analysis the components of Abelmoschus manihot flower,bud and stem extractsFirst,2DμCFs-QTOF-HRMS and HPLC-MS were used to analyze the extracts of flower.The feasibility and accuracy of 2DμCFs-QTOF-HRMS for the analysis of Abelmoschus manihot were verified by comparing with the results of HPLC-MS.Compared to HPLC-MS,2DμCFs-QTOF-HRMS had the advantages of rapid（70min to 5 min）,solvent saving（35 m L to 1.5 m L）and more components detection.Based on the above,2DμCFs-QTOF-HRMS was used to comprehensively analyze the components of bud/stem extracts.By comparison of the compounds of identified:stem did not contain typically flavonoids such as Hyperin found in flower/bud.The results of principal component analysis（PCA）showed that flower,bud and stem were divided into three categories,indicating that there were differences in their components.But on the whole,bud was much more similar to flower than stem.Through the comprehensive analysis of the three samples,it providesd the basis for the study of the pharmacodynamic substances of Abelmoschus manihot.In consideration of the compounds identified in flower/bud/stem,PCA results,and related reports on anti-inflammatory studies of Abelmoschus manihot,flower were selected for the second part of the screening study on potential anti-inflammatory active ingredients.Part two:Screening of potential anti-inflammatory active ingredients from Abelmoschus manihot flower extractsFor flower,the core potential anti-inflammatory target was predicted to be TNF-α（Tumor necrosis factor-α）through network pharmacology.Furthermore,the binding energies between compounds identified in flower and the active site of TNF-αwere theoretically calculated using molecular docking,and four small molecules with strong binding energies to TNF-α(binding energies<-4.50 kcal mol^-1)were obtained:Quercetin,Myricetin,Scopoletin,Quercetin-7-O-glucoside.Based on the above theoretical analysis of network pharmacology and molecular docking,TNF-αwas selected as the target of MCESS to screening the potential anti-inflammatory active ingredients from flower extracts,and the compounds that specifically bound to TNF-αwere screened out:Scopoletin,Glucosamine,and N-Lauryldiethanolamine.Among them,Scopoletin was one of the small molecules predicted by molecular docking(Binding energy-4.72 kcal mol^-1;hydrogen bonds with TYR151 of chain B,hydrophobic interactions with TYR59 and TYR119 of chain B,respectively),suggesting that Scopoletin may indeed bind specifically to the reported active site of TNF-α.Scopoletin and Glucosamine with clear anti-inflammatory activity by referring to relevant literatures.Combined with this study,it was speculated that them may play anti-inflammatory role as potential inhibitors of TNF-α.The anti-inflammatory activity of N-Lauryldiethanolamine has not been reported so far,whether it has anti-inflammatory activity and plays anti-inflammatory role as a potential inhibitor of TNF-αneeds further experimental verification.This study comprehensively analyzed the components of Abelmoschus manihot,and explored the potential anti-inflammatory active ingredients and targets,providing a reference for the research of its anti-inflammatory pharmacodynamic substance basis and molecular mechanism.In addition,network pharmacology prediction target,molecular docking theoretical verification target,provided a scientific basis for target selection of MCESS,avoided the blindness of target selection,effectively improved the success rate of screening experiment,reduced the cost of time and economic.This method provided a new idea for the target selection of affinity screening technology similar to MCESS,and provided a new direction for the study of screening active ingredients from medicinal plants such as traditional Chinese medicine.