Studies On The Anti-inflammation Mechanisms And Quality Control Methods Of Aconitum Vilmorinianum Kom

Objective:This paper aimed to study the anti-inflammatory effect and mechanisms of Aconitum vilmorinianum Kom by in vitro and in vivo inflammatory models for improving and controlling the quality of Aconitum vilmorinianum Kom.Methods:Five fractions were obtained from A. vilmorinianum, i.e.Fr. water fractions (Fr.H2O), ethanol fractions (Fr. EtOH), chloroform fractions (Fr. CHCl3), chloroform-methanol fractions (Fr. CHCl3-MeOH) and methanol-water fractions (Fr. MeOH-H2O). The anti-inflammatory roles for the fractions were evaluated by testing their effects on the NO and PGE2 production in LPS-stimulated RAW264.7 macrophages. The fraction(s) that had been tested to have anti-inflammatory activities were subsequently studied further for their anti-inflammatory effects utilizing peritoneal capillary permeability model induced by acetic acid in mice and rat paw edema model induced by carrageenan.ELISA method was used for understanding the action of the effective components on the inflammatory cytokines.Furthermore, the related inflammatory cytokines gene expression and regulation of NF-κ B, MAPKs signaling pathway related proteins were also investigated by real-time fluorescence quantitative PCR and Western Blotting Technology.In addition,we also evaluated the anti-inflammatory activity of the compounds which were isolated from the effective fraction. Therefore, we can choose the active ingredient which had higher purity and content as the reference compound to control the quality of medicinal extract.At last,we also detected medicinal material moisture content, total ash, acid insoluble ash, heavy metals and harmful elements and organochlorine pesticide residues and other related items of different batches.Results:In addition to Fr. H2O, the other fractions were able to inhibit the releasing of NO and TNF-α on LPS-stimulated RAW 264.7 cells and CHCl3-MeOH Fr.showed the best roles in inhibiting NO and TNF-a release(53.76+1.08,59.84+2.12,**P<0.01):CHCl3-MeOH Fr. could reduce peritoneal exudate capillaries and rat paw edema and also inhibit releasing inflammatory mediators including PGE2 and TNF-α (P<0.01) in inflammatory toes. Similarly, Fr. CHCl3-MeOH inhibited the production of cytokines IL-6, TNF-α, IL-1β and inflammatory mediators,NO, PGE2 in a dose-dependent manner in RAW264.7 cell (P<0.05, P<0.01). Fr. CHCl3-MeOH also significantly inhibited phosphorylation of p38、JNK/SAP、Erkl/2 kinase in the inflammatory signaling pathway MAPKs (P<0.05, P<0.01) and up-regulated IκBα protein and down regulated the expression of p-IκBα protein (P<0.01) in the NF-κB pathway.The anti-inflammatory activity evaluation experiments for various components showed these components had anti-inflammatory activities except for Hemsleyaconitine A and 8-deacetyl-yunaconitine had the strongest inhibition effect on the releasing of NO and TNF-a in LPS-stimulated RAW264.7 cell.8-deacetyl-yunaconitine as the marker compound in different batches of medicinal materials were determined, and the results showed that the content should not be less than 0.018%. Heavy metals and material moisture content were within the acceptable criteria. Conclusions:Fr. CHC13-MeOH is the main effective fraction of anti-inflammatory roles in Aconitum vilmorinianum Kom. This fraction might inhibit the exudation of the NO, PGE2, TNF-a, IL-6, IL-1β by regulating and controlling the MAPKs and NF-κB pathway. The assay method for 8-deacetyl-yunaconitine was accurate, sensitive and reliable. The results of this study provided scientific basis for further and deeper study of Aconitum vilmorinianum Kom in the future.