ACETYLCHOLINESTERASE ASSAY OF THE LEAVES AND BARK FROM TRICHILIA SILVATICA (MELIACEAE)

JACENIR VIEIRA DA SILVA, ANELISE SAMARA NAZARI FORMAGIO, RAQUEL O. NASCIMENTO, KAMILLA FELIPE DO NASCIMENTO, DIEGO CEGÓBIA FERREIRA, CARLA ROBERTA FERREIRA VOLOBUFF, MATHEUS SANTIAGO SILVA

Resumo


Introduction: Neurodegenerative disorders, like Alzheimer’s disease (AD) are frequent in elderly people,
as a result of malfunctioning of different biochemical pathways. The drugs approved for the AD therapy
act by counteracting the acetylcholine (Ach) deficit, that is, they try to enhance the acetylcholine level in
the brain. The drugs used so far, take advantage of their action as acetylcholinesterase inhibitors
(AChEIs) but these drugs have been reported to have their adverse effects including gastrointestinal
disturbances, hepatotoxicity, and problems associated with bioavailability which increases the interest
in finding better acetylcholinesterase inhibitors from natural resources.1,2 Trichilia silvatica (Meliceae) is
known as "coffee-to-kill" or "catiguá-white". Studies of the leaves reveal the presence of
sesquiterpenes, triterpenes, lignan glycosides, steroids and limonoids, as well anti-inflammatory and
antioxidant activities.3,4 Objectives: Evaluation the acetylcholinesterase activity of the extract obtained
of the bark and leaves from T. silvatica. Material and Methods: T. silvatica leaves and bark were
collected in Dourados-MS, Brazil. Air-dried leaves (TSL) and bark (TSB) were exhaustively extracted by
maceration with methanol. The rats were deprived of food overnight and then euthanized by heart
puncture. After that, the brain were carefully removed and the structures were gently removed and
separated into cerebellum (CE) cerebral cortex (CC), striatum (ST) and hippocampus (HP). The four brain
structures selected for the experiment (CE, CC, ST and HP) were maintained refrigerated in a solution of
10 mM Tris-HCl, pH 7.4, on ice. AChE activity was determined by a modification of the
spectrophotometric method of Ellman et al. (1961)5. After pre-incubation, the reaction speed was
measured by increasing absorbance to 412 nm. The enzyme activity was expressed in mmol AcSCh/h/mg
protein. In all the enzyme preparations, protein was measured according to Bradford (1976)6 using
bovine serum albumin as standard. For brain tissues, the protein was determined previously and
adjusted for each structure: CE (0.5 mg/ml), CC (0.7mg/ml), HP (0.8 mg/ml) and ST (0.4 mg/ml). Results:
The TSL samples not showed alteration in AChE activity in brain structures. AChE activity was decreased
with TSB in 0.5mg/ml (CC; 2.86 µmol Ach.h-1.mg; HP:0.93 µmol Ach.h-1.mg; CE; 0.33 µmol Ach.h-1.mg;);
in 2.5mg/ml (CC: 10,25 µmol Ach.h-1.mg; HP:2,59 µmol Ach.h-1.mg; CE: 6.27 µmol Ach.h-1.mg;); 4.0
mg/ml (CC: 8,27 µmol Ach.h-1.mg; HP:12,4 µmol Ach.h-1.mg; CE: 7.81 µmol Ach.h-1.mg;); samples
compared to the control. Discussion and Conclusion: The methanol extracts obtained from T. silvatica
of the bark anticholinesterase effect in the analyzed structures, however, it is necessary to continue the
study in ex vivo models in order to characterize its inhibitory action of acetylcholinesterase.

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