Study On The Chemical Constituents Of Dictamni Cortex And Its Anti-Rheumatoid Arthritis Activity Based On Network Pharmacology

Objective:As a traditional Chinese medicine to clear heat and dry dampness,dispel wind and detoxify toxins,Dictamni cortex mainly contains alkaloids,citrulline,sesquiterpenes and other major components,and studies have shown that its extract has various pharmacological activities such as antibacterial,anti-inflammatory,anti-tumor and arterial diastolic.Therefore,the research and development and utilization of Dictamni cortex has a broad prospect.Rheumatoid arthritis（RA）is a systemic autoimmune disease with pain,swelling and stiffness of peripheral joints as the main symptoms,which can cause joint damage or even disability in the late stage.Based on the previous experiments,this study was conducted to systematically investigate the chemical composition of ethyl acetate extracted parts of Dictamni cortex,to carry out the study on the mechanism of action of Dictamni cortex against RA using network pharmacology and molecular docking techniques,and to further explore the in vitro anti-inflammatory activity of monomeric components of Dictamni cortex,so as to lay the experimental foundation for the development of innovative drugs from Dictamni cortex.Methods:（A）The raw herbs of Dictamni cortex were crushed and the active ingredients were extracted by reflux extraction method with 70%ethanol-water solvent and concentrated to obtain the crude extract.The crude extracts were extracted with equal volumes of petroleum ether,methylene chloride,ethyl acetate and n-butanol in order to obtain four different polarities of the crude extracts.The ethyl acetate layer was separated into 12 streams by silica gel column chromatography with a gradient elution from small to large according to the polarity.A combination of silica gel column chromatography,Sephadex LH-20 gel column chromatography,and semi-preparative HPLC was used to separate the crude streams step by step to obtain the monomeric compounds.Structure identification were performed based on physicochemical properties and ¹H-NMR,¹³C-NMR and MS.（B）Network pharmacology:Screening the active ingredients of Dictamni cortex through TCMSP database and predicting their potential targets using Swiss Target Prediction database;obtaining RA-related gene targets through Gene Cards,OMIM,TTD,Dru GBank and Pharm Gkb databases;constructing target PPI networks through String platform,Cytoscape 3.9.0 software,and constructing"drug-ingredient-target-disease-pathway"networks using Cytoscape 3.9.0 software.Cytoscape 3.9.0 software was used to construct target PPI networks and screen key targets;"Drug-component-target-disease-pathway"network diagram constructed by Cytoscape 3.9.0 software;gene ontology（GO）function enrichment and KEGG pathway enrichment analysis were performed through DAVID database.The main potential active ingredients and protein receptors were molecularly docked using Autodock software and visualized using Py MOL software.（C）In this experiment,the potential anti-inflammatory activity of the monomeric components was initially investigated on the basis of the systematic isolation and identification of the chemical components of Dictamni cortex.The inhibition of NO release by the monomeric components isolated and identified from Dictamni cortex was initially investigated by MTT method using RAW264.7 cells induced by LPS stimulation as an in vitro activity screening model.Results:（A）A total of five compounds were isolated,containing four isoflavonoid glycosides,Puerarin（1）、Daidzin（2）、genistin（3）、Genistein 8-C-glucoside（4）,and dibutyl phthalate（5）.（B）16 potential active ingredients and 1304 RA targets were screened from Dictamni cortex,and 28 key targets were screened from 178 intersecting targets,acting on 20 major pathways.Five key targets and their corresponding compounds were selected for molecular docking validation.GO analysis and KEGG analysis showed that the chemical components of Dictamni cortex could exert therapeutic effects on rheumatoid arthritis through multiple biological pathways and multiple pathways;molecular docking results showed that the chemical components of Dictamni cortex could produce stable binding to the targets acting on the disease.（C）Compounds 2,3 and 4 all showed significant inhibitory effects on the growth of RAW264.7 cells and significantly reduced the amount of NO released from RAW264.7cells.Conclusion:Five compounds were isolated from the ethyl acetate layer of Dictamni cortex,of which compound 5 was obtained for the first time from Dictamnus L.The present study predicted the pharmacodynamic substance basis for the anti-RA activity of Dictamni cortex,indicating that Dictamni cortex may act in a synergistic manner with multiple components and targets,providing a scientific basis for further revealing its mechanism of action.Compounds 2,3 and 4 have potential anti-inflammatory activity...