Echinodorus grandiflorus (Cham. & Schltdl.) Micheli (Alismataceae) is a medicinal plant popularly known as “chapéu-de-couro”, used in the Brazilian folk medicine to treat inflammatory conditions such as rheumatoid arthritis, among other uses. Our research group has previously reported the in vitro inhibition of TNF-α release and in vivo anti-arthritic activities of E. grandiflorus extract and fractions thereof, disclosing the participation of flavonoids and aconitic acid isomers in the biological response.1,2 Moreover, trans-aconitic acid (TAA) showed potent but short lasting anti-edematogenic activity3. Since TAA is a highly polar tricarboxylic acid, it is rapidly excreted. Hence, esterification was adopted as a strategy to obtain lipophilic TAA derivatives with improved biopharmaceutical properties. For this purpose, TAA ethylic esters were prepared via Fischer esterification and purified by column chromatography over silica-gel. The products were characterized by TLC, RP-HPLC-DAD, RP-UPLC-ESI-MS, and FTIR. Additionally, NMR experiments (1H and 13C, DEPT-135, COSY, HSQC, HMQC and HMBC) were performed to confirm the structure of the triester unequivocally, as well as of the major ester product in the diester isomeric mixture. The characterized esters had their anti-inflammatory activity evaluated in vivo by LPS-induced acute arthritis in Swiss mice. The evaluated parameters were total and differential leukocyte accumulation, CXCL-1 content and myeloperoxidase activity. Purification of the reactional mixture afforded two groups of compounds (mono- and diesters), each one containing three constitutional isomers, along with the isolated triester, yielding 12.2%, 43.0%, and 32.3%, respectively. The mixture of diesters was composed of nearly 70%, 25% and 5% of the isomers. HMBC correlations showed that the major diester was the one esterified at C1 and C6 (Figure 1). Only the diester mixture reduced the total cells and neutrophil accumulation in the femorotibial joint. When given orally to mice, at doses ranging from 0.017 to 172.3 μmol/Kg, the diester mixture reduced cellular infiltration into the knee joint, especially of neutrophils. The diester mixture showed to be more active than TAA at low doses, pointing out that an increase in lipophilicity enhances the anti-inflammatory activity. In conclusion, the study disclosed TAA diesters as potentially useful derivatives for management of inflammatory diseases like rheumatoid arthritis.

Figure 1: 1H-13C correlations (arrows) observed in the HMBC spectrum of the main constituent in the diethyl t-aconitates isomeric mixture.

References:
1E.d.F. Garcia, et al., Planta Medica, v. 82, p. 337-343, 2016.
2E.d.F. Garcia, et al., Planta Medica, v. 82, p. 407-413, 2016.
3E.d.F. Garcia, et al., Phytomedicine, v. 18, p. 80-86, 2010.