Investigação do potencial antiartrítico do ácido trans-aconítico: incorporação em microesferas mucoadesivas e síntese de ésteres

Abstract

Echinodorus grandiflorus (Cham. & Schltdl.) Micheli (Alismataceae), popularly known as “chapéu-de-couro”, is traditionally used to treat different inflammatory diseases, including arthritis. Previously, our research group has demonstrated the antiinflammatory and antinociceptive activity of different extracts, fractions and constituents from the species, including trans-aconitic acid (ATA), using in vitro and in vivo models. The present study aimed to improve the biopharmaceutical properties of ATA in order to explore its potential anti-arthritic activity based on two strategies: (i) production of mucoadhesive microspheres loaded with ATA (ATA-ME); and (ii) synthesis of esters from ATA. In the first case, seven batches of ME-ATA formulations were prepared employing different ratios of polymers and ATA. The preparations were characterized by their encapsulation rate, average particle size, and polydispersity index. The ATA content in the formulations was determined by a HPLC method developed and validated for this purpose. F3c formulation (composed by equal amounts (w/w) of HPMC, carbopol, and ATA) exhibited the highest encapsulation rate and was fully characterized by morphological (OM and SEM), physicochemical (FT-IR spectroscopy and zeta potential and thermal (DSC-TG,) analyses. The obtained data indicated stability of the formulation and little interaction between the matrix and ATA. The esters were synthesized by Fischer esterification reaction between ATA and three alcohols (ethanol, n-butanol and n-octanol) in the presence of H2SO4 (2.7 % v/v) at 75 ° C for 2 hours. Kinetic studies enabled the optimization of the reaction conditions. The overall yields (sum of mono-, di- and triesters) of the reactions with ethanol, n-butanol and n-octanol were found to be 85.4%, 37.7% and 71.3%, respectively. Monoesters and diesters were obtained as isomeric mixtures and were characterized by chromatographic (TLC, HPLC-DAD, and ULPC-ESI-MS) and spectroscopic (UV, IR and MS) techniques. The diesters mixture and the triesters were characterized by mono- and two-dimensional 1H and 13C and NMR analyses. F3c formulation and the obtained esters were evaluated in a model of LPS-induced acute arthritis. F3c formulation induced a prolonged antiinflammatory response by reducing neutrophilic accumulation in the joint cavity up to 30 hours of pretreatment, in comparison to free TAA. All esters showed similar or higher anti-inflammatory activity than TAA. The diesters were the most active derivatives, exhibiting higher potency than ATA in the tested dose range (1.73 to 17.3 µmol/Kg), promoting a decrease of cellular accumulation, in particular of neutrophils, and reduction of MPO and CXCL-1. The n-octyl diesters were evaluated on acute and chronic AIA models, inducing similar or higher reduction of inflammatory parameters than TAA. In conclusion, the obtained results indicate that F3c formulation is stable and induces a prolonged anti-inflammatory response than free ATA. In addition, the obtained data highlighted the promising anti-inflammatory activity for TAA esters, especially the dioctyl-esters, which were shown to be active in acute and chronic arthritis models at lower doses than ATA.