Avaliação in vitro e in vivo dos mecanismos imunomoduladores envolvidos na atividade antiinflamatória de subfrações de Echinodorus macrophyllus

Abstract

Echinodorus macrophyllus (Alismataceae), known as "chapéu-de-couro" in Brazil, is a popularly specie used to treat inflammatory diseases. Since previous studies have shown an anti-inflammatory effect of aqueous extract of E. macrophyllus (AEEm) and its ethanolic fraction (Fr20), the aim of this study was to evaluate this response and the mechanisms of action in Fr20 subfractions (SFs). The Fr20 was fractionated, and four subfractions (SF1 to SF4) had a sufficient yield for monitoring in vitro and in vivo. Nitrite accumulation (NO2) supernatant was determined by Griess reaction and cell viability by reducing mitochondrial activity (MTT) in RAW 264.7 cells. The subfractions SF1, SF3, and mainly the SF4 decreased (p <0.05) NO levels, without affecting cell viability. The air pouch model was used to monitoring in vivo. The carrageenan stimulus for 4h increased cell migration and protein content in the exudate. The SF2 and SF3 subfractions were not effectives. SF1 inhibited the cell migration 90.5% (100 mg/kg) and SF4 up to 54.0% (50 mg/kg), mainly affecting the number of neutrophils. The protein level in the exudate of the control (12.08 ± 4.58 mg) has been reduced to 2.25 ± 1.24 mg, by SF1 and 3.90 ± 0.79 mg, by SF4 at the highest dose (100 mg/kg). SF1 was more effective to inhibit neutrophil migration in transwell assay (46,3%) and reduced the LTB4 (86,1%) level in the exudate. The SF1 and SF4 subfractions were more effectives, exercising greater activities on neutrophils and macrophages, respectively. The SF4 increased IL10 (19,3%); reduced NO (100%), TNF-α (54,2%) and IL-1β (65,8%) through of inhibition of mRNA for iNOS, TNF-α (73,6%) and IL-1β (73,3%) and showed antioxidant effect, in macrophages stimulated with LPS; and reduced migration of macrophages in vitro (wound healing) and in vivo (peritonitis, 66,1%). We also evaluated the anti-inflammatory effect (air pouch model) after induction of inflammation with carrageenan for 24 h, and SF1 toxicological effects after the subacute treatment (5 days). Despite the high level of inflammation of the control group, in this condition, the SF1 continued to show significant anti-inflammatory action (reduced vasodilation, redness, fibrin deposition and migration of neutrophils to the pouch), and increased the level of IL-10 (74,1%), and decreased TNF-α (87%), IL-1β (77%) and CKCL1/KC (71,3%) and PGE2 (97,8%) on exudates. The weigh and histopathology of organs; hematological, biochemical, genotoxicity of liver and GST and EROD activity s analysis showed low toxicity to SF1. Chromatographic analysis (HPLC-DAD and UPLC-ESI-TOF-MS) indicated the presence of monoglicosilates and diglicosilates flavonoids in SF4, and monoglicosilate in SF1. These results confirm the anti-inflammatory potential of this plant and show important mechanisms of action.