Isolamento de substâncias do extrato e frações de Echinodorus macrophyllus (Kunth) Mich. com atividade antinociceptiva e estudo de seus mecanismos de ação

Abstract

In this study we evaluated the antinociceptive potential of the hexane extract (HEEm), aqueous extract (AEEm) and fractions (Fr) from the leafs of Echinodorus macrophyllus (Kunth) Mich., known in Brazil as " chapéu de couro ", in different nociceptive responses generated by a chemical or thermal noxious stimulus and perform phytochemical analysis. The AEEm was obtained by infusion in distilled water (50 g d.w./L, 100°C), followed by filtration and lyophilization. The AEEm was fractionated on Sephadex LH 20, generating Fr20 and Fr40 fractions. The chromatographic profiles of fractions were analyzed by HPLC-DAD in a reverse phase C18 column. The chromatographic profile of HEEm was analyzed by GC-MS using a DB 5 (30 m x 0.25 mm x 0.25 µm) column. We used Swiss Webster mice (2- 4 months of age (n= 5/group)) for biological tests (CEUA 05/2009 e 07/2013). The antinociceptive effects of HEEm, AEEm, Fr20 and Fr40 were evaluated using hyperalgesia models induced by intraperitoneal (i.p.) injection of acetic acid, formalin test and tail immersion in a bath at 50°C. Treatment (p.o.) with AEEm and HEEm showed higher inhibition of the contortions number of 75% and 52% respectively at a dose of 25 mg/kg. The Fr20 (5 mg/kg) inhibited 39% of the contortions, and Fr40 treatment (25 mg/kg) showed 96% inhibition when compared to control. Treatment with dipyrone inhibited contortions induced by acetic acid by 51% on average in relation to the control group. In the tail immersion model in a bath at 50°C, treatment with AEEm showed no antinociceptive activity. In this model, the EHEm showed antinociceptive activity by treatment with 50 mg/kg, at 60min and 90 min and the Fr20 increased the latency time, 90 min after treatment with the three doses (25, 50 and 100 mgkg), when compared with the control. Morphine, used as standard drug showed antinociceptive effect after treatment (i.p.) in all time analyzed (30-180 min). In the MTT assay with NEURO-2A cells, AEEm exhibited the higher inhibition of the mitochondrial reducing activity (MRA) in a concentration dependent manner (IC50 of 36.15 mg/mL). HEEm showed MRA reduction reaching 49% at 100 mg/mL concentration and morphine exhibited maximum reduction of 23%. Fr20 fraction showed inhibition of 38.1% of MRA, and FR40 did not interfere with MRA in most concentrations tested. The precise mechanisms involved in the antinociceptive response of the Fr40 fraction from E. macrophyllus are not completely understood, but the presence of flavonoids and other aromatic compounds, suggests the possible contribution of NO adrenergic activation. By the analysis of the Fr40 compounds by HPLC-DAD and standards co-injection, and TLC evaluation isovitexin and isoorientin were identified in this fraction.