Study On The Active Components Of Anti-rheumatoid Arthritis Synovitis In The Total Flavonoids From Artemisia Argyi And Their Mechanisms Based On Spectrum-effect Relationships And Network Pharmacology

Rheumatoid Arthritis（RA）is an autoimmune disease with high prevalence and disability.Inflammatory hyperplasia of RA synovial is the lesions center of RA and inhibition RA synovitis is an important strategy for the treatment of RA.Studies have found that a variety of flavonoids from plant have anti-RA effects.Artemisia argyi is the dry leaf of Artemisia argyi levl.et Vant.,which have the effects of warming menstruation,dispersing cold and relieving itching.A.argyi can be used to treat RA in various ways such as moxibustion,external application or internal administration,and flavonoids are the main non-volatile component for its efficacy.At present,the active components of total flavonoids from A.argyi（TFAA）in the treatment of RA synovitis are unclear.Therefore,the spectrum-effect relationships were adopted to screen the active components,and network pharmacology was used to explore their mechanism,so as to provide scientific basis for its subsequent clinical research.15 batches of A.argyi from Henan,Hubei,Zhejiang,Gansu and Shandong were collected.Total flavonoids were extracted by 70%ethanol and purified by D101 macroporous resin column chromatography.The flavonoids content was determined by ultraviolet-visible spectroscopy.The fingerprint of TFAA were established by HPLC method,and the common peaks were found and the similarity evaluation was carried out by using the"TCM chromatographic fingerprint similarity evaluation system".Fibroblast-like synoviocytes（MH7A）were induced by TNF-αto establish the synovitis model of RA.The toxic effect of15 batches of TFAA on MH7A cells was determined by MTT method.The content of NO in the supernate of cells was determined by Griess method,and the efficacy of 15 batches of TFAA against RA synovitis was evaluated by NO inhibition rate.The spectrum-effect relationships between the peak areas of common peak and the efficacy was analyzed by using gray correlation analysis and partial least squares regression analysis.In addition,LC-MS was used to identify the structure of the common peaks.The efficacy of the selected active components was further evaluated,and their content was determined by HPLC.The content of 15 batches of TFAA ranged from 24.98%to 97.59%.14 common peaks were identified on the fingerprint.The similarity of fingerprint ranged from 0.762 to 0.984.12 compounds were identified by LC-MS,which were X1:Schaftoside,X2:Isochlorogenic acid B,X3:Isochlorogenic acid A,X4:Isochlorogenic acid C,X6:Luteolin,X7:Nepetin,X9:3,4,5-Tricaffeoylquinic acid,X10:Hispidulin,X11:Jaceosidin,X12:centaureidin,X13:Eupatilin X14:Casticin.The 15 batches of TFAA had no obvious toxicity to cells when the concentration was no higher than 12.5μg/m L,and the inhibition rate of NO was ranged from56.71%±2.86%to 95.69%±3.30%.Four key active flavonoid components,including X10（Hispidulin）,X11（Jaceosidin）,X13（Eupatilin）,and X14（Casticin）,were screened by the spectrum-effect relationships.The efficacy test results showed that the four active components significantly inhibited the content of NO in the supernatant of MH7A cells induced by TNF-α,with the inhibition rates of 53.94%,81.56%,82.88%and 60.52%when the concentration at 1.56μM.After the content determination,the quality score of Hispidulin,Jaceosidin,Eupatilin,and Casticin were 0.456～3.980 mg·g^-1,1.431～18.701 mg·g^-1,2.162～18.763 mg·g^-1 and 0.448～1.9388 mg·g^-1,respectively.The highest content of all four components was found in Qichun origin of Hubei,which provides theoretical support for Qichun as a genuine drug and good efficacy.Network pharmacology was used to investigate the mechanism of 4 active components against RA synovitis.TCMSP and STP databases were used to search the targets of active components.Gene Cards,OMIM,TTD and Drug Bank databases were used to search the targets related to RA synovitis.And the potential targets of the active components for the treatment of RA synovitis were obtained by taking the intersection.The Potential targets were imported into the DAVID database for GO and KEGG analysis,and imported into the STRING database for protein interaction analysis to screen out the key targets.34 targets were screened.And the key targets were TP53,AKT1,CASP3,ESR1,PPARG,CCND1,MAPK14,NOS3,MMP2,MCL1,which involves sphingolipid signaling pathway,AGE-RAGE signaling pathway,PI3K-Akt signaling pathway,relaxin signaling pathway,prolactin signaling pathway,p53 signaling pathway,estrogen signaling pathway and other signaling pathways.In summary,4 key active flavonoid components against RA synovitis were screened by spectrum-effect relationships,including Hispidulin,Jaceosidin,Eupatilin,and Casticin,and their anti-RA synovitis effects were found by network pharmacology,probably through TP53,AKT1,CASP3,ESR1,PPARG and other targets,acting on sphingolipid signaling pathway,AGE-RAGE signaling pathway,PI3K-Akt signaling pathway,p53 signaling pathway and other pathways.This study provided a basis for the quality standard of TFAA against RA and drug discovery.And provided theoretical support for further clinical research.