Caracterização química e estudo da atividade e do mecanismo de ação antiinflamatório do extrato hidroetanólico e compostos isolados da entrecasca da Dilodendron bipinnatum Radlk

Abstract

Ethnopharmacological relevance: Dilodendron bipinnatum Radlk., Sapindaceae, is a native plant, but not endemic in the Pantanal. The inner stem bark preparations are used in inflammatory disorders in the forms of decoction and maceration. We have recently shown that these preparations have anti-inflammatory properties, which are mediated by the inhibition of cell migration and the modulation of the Th1 and Th2 cytokines, although the mechanism of molecular action has not been elucidated. Objective: The objective of the present study was to investigate in detail the mechanisms responsible for the anti-inflammatory properties of the hydroethanolic extract of the stem bark of Dilodendron bipinnatum (EHDb). Materials and methods: The powder of inner sterm bark was macerated in 70% (w/v, 1:3) hydroethanolic solution and concentrated to obtain EHDb. Cell viability, intracellular redox status and changes in membrane potential mitochondrial (ΔΨm) were evaluated by the 1 h pretreatment with the HHDb in RAW 264.7 cells stimulated for 24 h with 1 μg/mL LPS, after labeling with Anexinna-V and 7-aminoactinomycin-D, DCFH-DA and JC-1, respectively, by flow cytometry. The effect of EHDb on cytokines levels (TNF-α, IL-1β and IL-10) and PGE2 were evaluated by ELISA and the expression of the cytoplasmic proteins MAPKs (p-p38, pJNK and p-ERK), MKP-1 and COX-2 by western blotting. The nuclear translocation of NFκB was evaluated by immunofluorescence microscopy and the results quantified nuclear nucleation: cytoplasm. Results: LPS, EHDb and NAC demonstrated to be non-cytotoxic, with 80% average viability. EHDb attenuated the production of pro-inflammatory cytokines (IL-1β and TNF-α) and PGE2 induced by LPS, but did not affect IL-10. ROS production was also inhibited by HEDb (1, 5 or 20 μg / mL), even at the lowest concentration; At 20 μg/mL, HEDb was more effective than NAC, which was used as a positive control (74.1% and 66.2% inhibition of LPS effect, respectively). LPS induced an increase of ΔΨm (19.2%, p<0.001), while HEDb inhibited the change in ΔΨm (7.7% at 20 μg/mL, p<0.001). Results from western blotting showed that HEDb inhibited the expression of MAPK p-p38, p-JNK and COX-2, whereas the expression of MKP-1 was increased. p-ERK was not affected. LPS promoted nuclear translocation of NF-қB p65 (67%, p<0.01) in RAW 264.7 cells, compared to the basal group (33%). Pretreatment with HEDb inhibited this translocation of NF-κBp65 (58% to 20 μg/ml, p<0.001). Conclusion: HEDb has a potent anti-inflammatory activity and acts on multiple targets and biological pathways of potential therapeutic relevance.