Potencial antiinflamatório do extrato aquoso de Echinodorus macrophyllus e de suas frações em modelo de inflamação aguda

Abstract

Echinodorus macrophyllus (Alismataceae), known as "chapéu de couro" in Brazil, is used popularly to treat rheumatic and inflammatory diseases. In this work we have evaluated the anti-inflammatory effects of the aqueous extract of E. macrophyllus (AEEm) and of its ethanolic fractions in mice air pouch model. Fractions were obtained by applying 7 g AEEm on a silica gel chromatography open column eluted with different ethanol concentrations. The fractions so obtained were evaporated under vacuum and lyophilized. Representatives chromatograms of EAEm/fractions were obtained using a HPLC system. We obtained four fractions, two with higher-yielding. Briefly, the air pouch was induced by 5 mL of sterile air injection (s.c.) on the back of male SW mice (25-35 g). After 3 days, 3 mL of sterile air has been injected again to keep it. After six days each group (n = 4) received intraperitoneal (i.p.) or oral (p.o.) treatment with AEEm (25 or 250 mg/kg), Fr20 or Fr40 (2.5, 25, 50 or 100 mg/kg) or controls indomethacin (10 mg/kg, p.o.) and vehicle (saline). One hour later 1 mL saline or carrageenan 1% sterile was injected into the pouch. After 4 h, the cavity was washed with NaCl 0.9%, EDTA 2 mM (1 mL), for determination of leukocyte numbers, final exudate volume and protein concentration. Cytospin preparations of exudates were stained with Panotic method for differential leukocyte count. Histological sections of tissue collected from different groups were fixed with 10% buffered formalin (pH 7.4) for 7 days and stained with HE and analyzed by MO. The iNOS and COX-2 expression analyses were performed on the exudate cells by RT-PCR. Accumulated nitrite (NO2−) in the media obtained from the RAW 264.6 cell cultures was determined using a colorimetric assay based on the Griess reaction. Results were expressed as mean ± SEM and compared using ANOVA and Dunnet's test. Experiments were performed in triplicate. In air pouch model, carrageenan 1% increased both the cell migration and the exudate protein level. However, while pretreatment with Fr40 increased inflammatory response, the pretreatment with AEEm and Fr20, mainly i.p. inhibited its when compared to the control group treated only the vehicle. So, the following rates of inhibition of cell migration were observed: AEEm, i.p. at 25 mg/kg (66.44%) and at 250 mg/kg (87.27%) and Fr20 at 2.5 mg/kg (26.89%), at 25 mg/kg (60.06%), at 50 mg/kg (63.13%) and at 100 mg/kg (77.47%). Regarding the differential count, the EAEm and Fr20 affected mainly the content of neutrophils, inhibiting the neutrophils migration in exudate. AEEm and Fr20 also reduced the total protein level in exudates mainly in the i.p. treatment. AEEm at 25 and 250 mg/kg showed 3.33 ± 0.55 and 2.05 ± 0.51 mg/mL, respectively, when compared to controls groups (Indomethacin 2.88 ± 0.64 mg/mL; vehicle 5.48 ± 0.88 mg/mL). Fr20 at 2.5, 25, 50 and 100 mg/kg showed 4.788 ± 0.444, 1.417 ± 0.519, 2.474 ± 0.529 and 2.215 ± 0.361 mg/mL. The histological analysis showed cellular infiltrate, mainly composed by polymorphonuclear leukocytes throughout the inflamed dermis of animals treated with vehicle. Treatment with AEEm or Fr20 reduced the leukocyte infiltrate on inflamed tissue. In addition, treatment with AEEm and Fr20 showed suppressive activity on iNOS and COX-2 expression, and showed inhibitory effects on LPS-induced nitric oxide production. In conclusion, all these findings support an anti-inflammatory potential suggested for this plant and provides a basis for understanding their action molecular mechanism. However, further studies should be undertaken to better elucidate the pathways by which AEEm and Fr20 exert their anti-inflammatory effects. In addition, phytochemical studies must be underway to identify active compounds in AEEm and Fr20.