In vitro and in vivo anti-inflammatory effects of different extracts from Epigynum auritum through down-regulation of NF-κB and MAPK signaling pathways

Epigynum auritum has been historically used as a “dai” or traditional medicine for the treatment of inflammation, swelling and severe pain during injury; these may reduce risk of disease and lead to healthier aging. Apart from this, Epigynum auritum extract was also used in arhritis treatment which is also a type of inflammation. Previous phytochemical studies of E. auritum revealed that steroids are main characteristic components with a number of biological activities (especially immunosuppressive and anti-inflammatory activity) Nevertheless, the underlying mechanism of the E. auritum on inflammatory diseases is still unresolved. This study aimed to comparatively investigate the anti-inflammatory potential of different fractions from the extract of E. auritum (EAE), with their possible active ingredients to reveal the underlying mechanism. The EAE was fractionated by column chromatography with macroporous resin D101 which yielded six fractions. The potential anti-inflammatory properties of different fractions of EAE were evaluated in in vitro and in vivo model. The lipopolysaccharide (LPS)-induced RAW264.7 macrophages cells were used for in vitro studies however two typical acute inflammation murine models (xylene-induced ear edema and carrageenan-induced paw edema) were used for anti-inflammatory studies. The important molecular mechanisms related to inflammation were also analyzed by ELISA, western blotting and immunofluorescence. UHPLC-MS/MS was used to analyze the chemical composition of 100% EAE fraction. Different EAE fractions (especially the Fr. 100% of MeOH:H2O) significantly reduced the productions of NO, ROS, TNF-α, and IL-6 by LPS-induced RAW264.7 macrophages and increased the expression of IL-10. The expression levels of iNOS and COX-2 enzymes were significantly down-regulated by 100% EAE fraction. Furthermore, 100% EAE fraction inhibited the phosphorylation of the ERK1/2, JNK, and p38 MAPK, and reduced the nuclear translocation of NF-κB which prevents its activation by blocking the phosphorylation and degradation of inhibitor protein of IκBα. In addition two inflammatory animal models; xylene-induced ear edema and carrageenan-stimulated paw edema were also developed with significantly ameliorated inflammatory cytokines. The treatment of these inflammatory models with 100% EAE fraction (Fr. 100%) suppressed the expressions of elevated inflammatory cytokines. Besides the UHPLC-HRMS/MS analysis was also carried out in which the androstane analo