Anticholinesterase ursane triterpenes from Eugenia catharinensis D. Legrand

Eugenia genus is one of the largest in the Myrtaceae family, and its common constituents are phenolic compounds and triterpenes. The species Eugenia catharinensis D. Legrand, popularly known as “guamirim-de-folha-miúda”, is an endemic tree grove in the coast of Santa Catarina and Paraná. Taking into consideration the scarce studies on this species in the literature, the objective of this work was to carry out the phytochemical study of Eugenia catharinensis and to determine the anticholinesterase activity of crude extracts and isolated compounds of this species. Leaves of Eugenia catharinensis were collected in Blumenau-SC, dried, ground and macerated for seven days in dichloromethane (EDCM), ethyl acetate (EAE) and 70% ethanol (EBH). To evaluate the anticholinesterase activity, 325 µL of tamponed Tris-HCl (pH 8), 100 µL of the essential oil solution and 25 µL of a solution containing enzyme (0.28 UI mL-1, from E. eletricus) were incubated for 15 min. Subsequently, 75 µL of acetylchonine iodide solution (0.023 mg mL-1) and 475 µL of DTNB were added and the final mixture incubated for 30 min at room temperature. The absorbance of the mixture was measured at 405 nm. A negative control was prepared using 100 µL of methanol and a positive control with 100 µL of neostigmine (0.1 mg mL-1). The inhibition was calculated, and the results expressed as IC50 (µg mL-1). All extracts showed interesting activity on the acetylcholinesterase, being the best results obtained in EBH (53.35 ± 8.3 µg mL-1) and EAE (57.08 ± 12.1 µg mL-1). EAE extract was chosen for fractionation study by chromatographic methods. Three triterpenes with ursane skeleton could be isolated: ursolic acid, pomolic acid and rotungenic acid.The anticholinesterase activity of the compounds was tested, and pomolic acid showed the best result (65.33 ± 17.96 µg mL-1), followed by rotungenic acid (680.01 ± 50 µg mL-1) and ursolic acid (530.0 ± 10 µg mL-1). The evaluation of the structure-activity relationship of the isolated triterpenes of Eugenia catharinensis, showed that ursane skeleton with -OH at positions C-3 and C-19, as presented in pomolic acid, showed the best inhibition value. In ursolic acid, where does not have a methyl moiety at position C-19, the activity decreased significantly. In rotungenic acid, in addition to the -OH groups at positions C-3 and C-19, this molecule also has a -OH group at C-24, which may have been the determining factor to reduce the inhibition effect, since this compound presented the highest IC50 value among the triterpenes. No studies were found in the literature on the structure-activity relationship of pentacyclic triterpenes in relation to activity against acetylcholinesterase. Further studies need to be performed to elucidate the chemical features of ursane triterpenes on the acethylcholinesterase inhibition, however, Eugenia catharinensis showed potential anticholinesterasic activity.