Padronização químico-biológica de extrato de Echinodorus macroplyllus e desenvolvimento de formulação anti-inflamatória com potencial antiartrítico.

Abstract

The species Echinodorus macrophyllus and Echinodorus grandiflorus are commonly referred to as "chapéu-de-couro" (leather hat) and have a long-standing traditional use in Brazil for treating rheumatism and arthritis. This study aimed to conduct both chemical and biological analyses of E. macrophyllus leaves to establish a standardized extract and a formulation with antiarthritic activity. The leaves of E. macrophyllus were obtained from commercial sources (lots DV1 and DV2) and also from a reference material (DVMus). Additionally, a commercial batch of E. grandiflorus (EG) leaves was included in the study. Molecular and phylogenetic analyses of DVMus were performed using the markers matK MG1/MG15, leafy (Flint2 F1/R1), 5S-NTS (PI/PII), ITS (5S/ER), and psbA-trnH (trnH2/psbA). However, it was not possible to unequivocally identify DVMus at the species level using the aforementioned markers and the sequences deposited in GenBank. A comparison of DV1, DV2, DVMus, and EG was carried out through the analysis of chemical markers contents (cis- and trans-aconitic acids, homoorientin, chicoric acid, swertisin, caffeoyl-feruloyl-tartaric acid, and diferuloyl-tartaric acid) and in vitro biological activities (anti-inflammatory and antioxidant). The obtained data were subjected to principal component analysis (PCA) and the results did not allow differentiation between the analyzed samples of E. macrophyllus and E. grandiflorus. To develop the standardized extract of E. macrophyllus, extractive tests were carried out with DV2 using the methods of percolation, dynamic maceration, and static maceration, employing the solvents 96°GL EtOH, 90% EtOH, 70% EtOH and 50% EtOH. The extracts obtained by percolation showed higher yields, especially the 70% EtOH and 50% EtOH extracts, in addition to higher levels of chemical markers. The activity of the above mentioned extracts on the release of pro-inflammatory cytokines in THP-1 cells stimulated by LPS was evaluated. The 70% EtOH and 50% EtOH extracts, obtained by percolation, induced a greater reduction in TNF and IL-1β release. All extracts prepared by percolation were evaluated in an acute murine model of antigen-induced arthritis (AIA). The extracts, administered orally, reduced the migration of total inflammatory cells, neutrophils, and monocytes to the intra-articular cavity, with the highest activity observed for the 96°GL EtOH, 70% EtOH, and 50% EtOH extracts. In the sequence, the 96°GL EtOH and 70% EtOH extracts were evaluated in a murine model of chronic AIA. Both extracts reduced the migration of total inflammatory cells, neutrophils, and monocytes to the intra-articular cavity, in addition to the concentrations of IL-1β and CXCL1 in the periarticular tissue. Additionally, the histopathological analysis of the articular cartilage indicated a decrease in the loss of proteoglycans. The mechanism of anti-inflammatory action of the 70% EtOH and 50% EtOH extracts was evaluated in vitro in primary culture of murine chondrocytes stimulated with LPS. The extracts reduced the release of IL-6 and the expression of type 3 metalloproteinases, justifying the reduction in proteoglycan degradation and in the inflammatory process observed in the chronic AIA model. Based on these results, the 70% EtOH and 50% EtOH extracts were selected for pharmacotechnical development. The extracts were prepared by percolation and the ethanol was evaporated under reduced pressure in a rotary evaporator. The remaining water was eliminated by drying under optimized conditions in a spray dryer, with the addition of 25% w/w of Aerosil®. The 50% EtOH extract showed better solubility in water and fluidity of the powder and, therefore, it was selected to develop the formulation. The formulation was obtained by adding 10% w/w of microcrystalline cellulose to the standardized dried 50% EtOH extract. The resulting formulation exhibited suitable moisture content, particle size, particle uniformity, and powder flow for encapsulation. Additionally, the formulation's chemical markers contents were equivalent to those found in the original extract. The obtained formulation was evaluated in an acute AIA model and induced an anti-inflammatory response similar to the original extract. In conclusion, different plant drugs from E. macrophyllus showed similarities in the chemical composition and in vitro anti-inflammatory activity. The 70% EtOH and 50% EtOH extracts were the most active in murine models of antigen-induced arthritis and in chondrocyte culture. They demonstrated a significant reduction in inflammatory response and a decrease in proteoglycan loss. The formulation of the 50% EtOH extract with the addition of 25% w/w Aerosil® and 10% w/w microcrystalline cellulose showed appropriate pharmacotechnical parameters for encapsulation.