Esterification of trans-aconitic acid improves its anti-inflammatory activity in LPS-induced acute arthritis.

trans-Aconitic acid (TAA) is an abundant constituent in the leaves of Echinodorus grandiflorus, a medicinal plant used to treat rheumatoid arthritis in Brazil. Esterification was explored as a strategy to increase lipophilicity and biopharmaceutical properties of TAA, a highly polar tricarboxylic acid. We herein report the synthesis of TAA esters via Fischer esterification with ethanol, n-butanol and n-octanol. The reaction kinetics was investigated to produce mono-, di- and tri- derivatives. Mono- and diesters of TAA were obtained as a mixture of positional isomers, whereas the triesters were recovered as pure compounds. The obtained esters were screened in a model of acute arthritis induced by the injection of LPS in the knee joint of Swiss mice. The diesters were the most active compounds, regardless of the alcohol employed in the reaction, whereas bioactivity of the derivatives improved by increasing the length of the aliphatic chain of the alcohol employed in esterification. In general, the esters showed higher potency than TAA. When administered orally to mice at doses of 0.017-172.3 µmol/Kg, the diethyl, di-n-butyl and di-n-octyl esters of TAA reduced the cellular infiltration into the knee joint, especially of neutrophils. The study identified diesters of TAA as potential useful derivatives for the management of rheumatoid arthritis and other inflammatory diseases.

Encapsulation of trans-aconitic acid in mucoadhesive microspheres prolongs the anti-inflammatory effect in LPS-induced acute arthritis.

trans-Aconitic acid (TAA) is the main constituent of the leaves from the medicinal plant Echinodorus grandiflorus, used to treat different inflammatory diseases. TAA induces a potent but short-lasting biological response, credited to its high polarity and unfavorable pharmacokinetics. Here we developed, characterized and evaluated the anti-inflammatory activity of mucoadhesive microspheres loaded with TAA. Seven batches of mucoadhesive microspheres were prepared by the emulsification/solvent evaporation method, employing different proportions of TAA and Carbopol 934 or/and hydroxypropylmethylcellulose. All batches were characterized for their particle medium size, polydispersity index and entrapment percentage. The batch coded F3c showed highest entrapment percentage and was characterized by infrared spectroscopy (ATR-FTIR), scanning electron microscopy (SEM), differential scanning calorimetry (DSC), thermogravimetric analyses (TGA) and zeta potential. The anti-inflammatory activity of F3c was assessed in a model of acute arthritis induced by injection of LPS in the knee joint of Swiss mice. The granulometric analyses indicated heterogeneous size distribution for F3c. SEM characterization indicated microspheres with slightly irregular shape and rough surface. Results from ATR-FTIR and thermal analyses (DSC and TGA) pointed out absence of incompatibility between the components of the formulation; thermal events related to the constituents were isolated and randomly located, suggesting amorphous distribution of TAA in the formulation matrix. The zeta potential of the formulations varied from -30 to -34 mV, which may contribute to good stability. When given orally to mice, F3c induced a prolonged anti-inflammatory response by reducing total cell count and neutrophilic accumulation in the joint cavity even when given 48 and 36 h before the stimulus, respectively, in comparison to free TAA (up to 24 and 6 h, respectively). Therefore, the encapsulation of TAA in mucoadhesive microspheres provided its sustained release, indicating that this drug delivery system is a potential agent to treat inflammatory diseases by regulating cell influx.

Antiedematogenic activity and phytochemical composition of preparations from Echinodorus grandiflorus leaves.

The leaves of Echinodorus grandiflorus (Alismataceae) are traditionally used in Brazil to treat inflammatory conditions. The aim of the present study was to evaluate the antidematogenic activity of crude aqueous, dichloromethane and hydroethanolic extracts from E. grandiflorus leaves using the carrageenan-induced paw edema model in mice, along with of fractions enriched in diterpenes, flavonoids and hydroxycinnamoyltartaric acids (HCTA). Significant inhibitions of paw edema were elicited by the 50% and 70% EtOH extracts (1000 mg/kg, p.o.), as well as by the fractions enriched in diterpenes (70-420 mg/kg, p.o.) and flavonoids (7.2-36 mg/kg, p.o.). Isovitexin, isoorientin, trans-aconitic and chicoric acids were identified in all extracts by HPLC analysis. Trans-aconitic acid itself exhibited significant antiedematogenic effect (270 mg/kg, p.o.). The biological activity correlated positively with the contents of flavonoids and diterpenes, but negatively with HCTA concentrations, demonstrating the participation of the two classes of compounds in the antiedematogenic activity of E. grandiflorus.

Evaluation of antileishmanial activity of trans-aconitic acid.

TAA, an inhibitor of the enzyme aconitase, inhibits the growth of L. donovani promastigotes. Morphogenic transformation of the amastigote to the promastigote (table; see text) form in vitro was also inhibited by 2 mM TAA. TAA also reduced multiplication of the parasite in macrophage culture. In the hamster model of leishmania, TAA significantly reduced the parasitic burden of liver. In acute toxicity tests with BALB/c mice no deaths were recorded even at a dose level of 2 g/kg body wt/day.

Experimental visceral leishmaniasis: role of trans-aconitic acid in combined chemotherapy.

We previously reported the effectiveness of trans-aconitic acid (TAA) as an antileishmanial compound. Inhibitory effects of TAA along with other antileishmanial compounds on transformation and in vitro multiplication in macrophage cultures of Leishmania donovani have been assessed. The efficacy of TAA in combined chemotherapy of experimental visceral leishmaniasis has also been evaluated along with those of commonly used antileishmanial compounds such as sodium stibogluconate, pentamidine, and allopurinol. TAA (2 mM) inhibited transformation of L. donovani amastigotes to promastigotes by 95.2%, whereas in combination with pentamidine (5 micrograms/ml), allopurinol (10 micrograms/ml), and sodium stibogluconate (50 micrograms of Sb per ml), it inhibited transformation by about 100, 99, and 98.5%, respectively. Sodium stibogluconate (20 micrograms of Sb per ml), pentamidine (2 micrograms/ml), and allopurinol (5 micrograms/ml) suppressed the amastigote burden in peritoneal macrophage cultures from BALB/c mice by 32.6, 56.1, and 46.3%, respectively. When these three drugs were used along with TAA (5 mM), the parasite loads were reduced by 100, 100, and 88.1%, respectively. TAA (5 mM) alone suppressed the amastigote burden by 59.5%. In experimental visceral leishmaniasis in hamsters (1-month model), TAA at a dose of 200 mg/kg of body weight per day suppressed the spleen parasite load by 73.5%, and TAA in combination with sodium stibogluconate (50 mg of Sb per kg per day), pentamidine (8 mg/kg/day), and allopurinol (15 mg/kg/day) inhibited the spleen parasite load by 98, 98.9, and 97%, respectively. Individually, these three drugs inhibited the parasite load by 35, 20, and 22%, respectively. TAA (400 mg/kg/day) inhibited the spleen parasite load by 99.8%, but an inhibitory effect of approximately 100% was noted when TAA was supplemented with an antileishmanial drug. TAA was administered in experimental animals through oral, intraperitoneal, and intramuscular routes; the intramuscular route was most effective.