Study On The Pharmacological Activity Of Aconitum Soongaricum Stapf.and Hydrolysis Products In Xinjiang Province Based On The Cell Membrane Chromatography

Objective:An EGFR/CMC-online-HPLC system developed to screen anti-EGFR compounds from Aconitum soongaricum Stapf.and its hydrolysis products,then in vestigate their inhibitory effect by cell assay.RT-PCR and WB to investigate different components intervention,HIF-1? VEGF?TLR4 and protin expression level,to explore the mechanism of the treatment of RA.Method:1 A two-dimensional online system(EGFR/CMC-HPLC)was built to screen active compounds of anti-EGFR from Aconitum soongaricum Stapf.and its hydrolysis products,then the components retained on CMC were collected while used offline MS to identify the anti-EGFR active components;Molecular docking assay was performed to determine the binding affinity of the screened components to EGFR;MTT was used to confirm the inhibition effect of screening compounds on HEK293 cells and EGFR/HEK293 high expression cell lines.2 CCK8 was used to evaluate the inhibition activty of aconitine,songorine and benzoylaconite on Human fibroblast-Like Synoviocyte Rheumatoid Arthritis(HFLS-RA);ELISA method to detect the cell culture supernatant of IL-1,IL-6,TNF alpha factor expression;WB and RT-PCR method to detect HIF-1?,TLR4,VE GFA mRNA and protein expression.Result:1 The screening results showed that aconitine,12-epi-napellin,songorine and benzoylaconite were to be the active components acted on EGFR like gefitinib;molecular docking assay showed target components act on EGFR in similar manner with gefitinib as a positive control drug;MTT assay comfirmed screened components have inhibitory effects on HEK293 cells and EGFR/HEK293 high expression cell lines.2 CCK8 experiment show that different concentrations of aconitine,songorine and benzoylaconite have inhibitory effects on HFLS-RA,of which aconitine 500?g/mL,songorine 350?g/mL and benzoylaconite 1000?g/mL after 24h.Conclusion:1 The online EGFR/CMC-HPLC-MS method has a good prospect for the rapid screening of the active compounds in the complex system of traditional Chinese medicine,and fully demonstrates the advantages of cell me mbrane chromatography simple,rapid and high efficiency.2 Aconitine,songorine and benzoylaconite have certain antirheumatoid arthritis activity in vitro,speculated that the mechanism of anti rheumatoid arthritis control effect might be through the in hibition of inflammatory cytokine production and the regulation of HIF-1?,TLR4,VEGFA pathway.