Avaliação da atividade e do mecanismo de ação anti-infamatório do extrato da infusão das folhas de Copaifera malmei Harms

Abstract

Copaifera malmei Harms (Fabaceae), popularly known as óleo-mirim, is a native and endemic plant found in the states of Mato Grosso and Goiás of Brazil. The plant's leaves infusion is popularly used by riverine communities of the northern Araguaia microregion, Mato Grosso, Brazil, for the treatment of gastric ulcers and inflammatory diseases of the respiratory tract. Recently, our research team has shown that an infusion extract of the leaves of C. malmei has a strong antiulcer activity, anti-ulcerative colitis and its oral use gives no indications of toxicity. There is no information in the literature demonstrating the acute anti-inflammatory activity of C. malmei and its respective mechanism of action. This study aimed to evaluate the anti-inflammatory activity and mechanism of action of an infusion extract of the leaves of C. malmei (SIECm) using in vivo and in vitro experimental models. The leaves of C. malmei was prepared SIECm was prepared by infusion, by incubating the powdered dried leaves material in boiled water for 15 min, thus obtaining SIECm. The effect of SIECm on cell viability in murine macrophages (RAW 264.7 cells) was assessed by the Alamar blue assay. The in vivo anti-inflammatory activity and mechanism of action of SIECm (20, 100 or 400 mg/kg) was evaluated in lipopolysaccharide (LPS)-induced peritonitis model in Swiss mice. The peritoneal lavage was collected for the determination of TNF-α, IL-1β and IL-10, using commercial ELISA kits. The in vitro anti-inflammatory studies activity of SIECm was determined in RAW 264.7 cells stimulated with LPS. The supernatant of the stimulated cells was used for the determination of the aforementioned cytokines as well as oxide nitric (NO) by the Griess method. SIECm showed no cytotoxicity effect with IC50 > 400 μg/mL. In LPS-induced peritonitis model, SIECm reduced total leukocytes (59.7%, 51.8% and 56.9%, p < 0.001) and neutrophils migration (45.5%, 53.2%, 74.4%, p < 0.001), at all doses. SIECm also attenuated increase in the concentrations of the pro-inflammatory cytokines (IL-1β and TNF-α) and increased level of anti-inflammatory cytokine IL-10 in peritonitis. SIECm significant inhibition effect on the increased concentrations (1, 5 and 10 µg/mL) of TNF-α (60.9%, 60.6% and 60.2%, p< 0.001), IL-1β (73.7%, 67.4% and 53.1%, p< 0,001) and augmented IL-10 (130.5%, 136.3% and 131.1%, p < 0,001) in activated RAW 264.7 cells. Furthermore, SIECm reduced NO production by 51.1, 41.1 and 45.8% and IFN-γ (0.1 ng/mL) by 99.6%, 84.9% and 84.1% (p < 0.001) in RAW 264.7 cells with the treatment of LPS (0.1 µg/mL) at all concentrations. Similarly, in the LPS-induced peritonitis model, pre-treatment with SIECm reduced nitrite levels by 55.1%, 51.4% and 62.7% (p < 0.01) in the peritoneal lavage when stimulated by LPS. SIECm presented a new mechanism and multi-target anti-inflammatory action, due, at least in part, to the inhibition of cell migration, downregulation of TNF-α and IL-1β and upregulation of IL-10 and inhibition of NO. The results confirm the popular use of C. malmei leaves for inflammation. Based on the literature, it can be suggested that flavonoids, phenolic and phytosterol, are responsible, at least in part, for SIECm's anti-inflammatory activity.