63ª Reunião Anual da SBPC

C. Ciências Biológicas - 6. Farmacologia - 4. Farmacologia

BIOLOGICAL SCREENING OF Pimenta pseudocaryophyllus FOR ANTICHOLINESTERASE ACTIVITY IN VITRO

James Oluwagbamigbe Fajemiroye 1 Paulo Cesar Ghedini 2 Joelma Abadia Marciano de Paula 3 José Realino de Paula 4 Elson Alves Costa 5

1. Depto de Fisiologia, LFPN, ICB-UFG 2. Depto de Fisiologia,LFBM, ICB-UFG 3. Unidade Universitária de Ciências Exatas e Tecnológicas, Curso de Farmácia - UEG 4. Faculdade de Farmácia, LPPN - UFG 5. Prof. Dr./Orientador - Depto de Fisiologia, LFPN, ICB-UFG

INTRODUÇÃO:

One of the most debilitating and devastating of all neurological disorders is the dementia that occurs in the elderly population over 65 years of age. The most common cause of this illness is Alzheimer’s disease (AD). The degeneration of cholinergic cortical neurons, presence of extracellular plaques and intracellular neurofibrillary tangles among others are hallmarks of the cognitive deficit in Alzheimer-type of dementia. Cholinesterase inhibitors that can increase the cholinergic transmission by blocking the degradation of acetylcholine being used for alleviating the symptoms of patients are usual therapy for AD. Existing anticholinesterase drugs for the treatment of dementia are reported to have several dangerous adverse effects such as hepatotoxicity, short duration of biological action, low bioavailability, and adverse cholinergic side effects in the periphery. The history of drug discovery has shown that plants contain active compounds that have become new sources of investigation for the pharmaceutical industry. The Brazilian floral offers abundant biologically active compounds. In the present study, ethanol extract of P. pseudocaryophillus native to ``cerrado`` and its fractions were screened for possible anticolinesterase activity in search for innovative therapy.

METODOLOGIA:

EEPPM was obtained by soaking the dried leaf powder in ethanol (95%;1:5).The hexane (FHEEPPM), dichloromethane (FDDEEPPM), ethyl acetate (FEAEEPPM) and aqueous (FAQEEPPM) fractions were prepared through EEPPM fractionation with solvents of different polarities. Anticholinesterase assay were carried out using Ellman’s colorimetric method (1969). Various concentrations of the extracts in 1.2 ml of 0.1mM sodium phosphate buffer (pH 8.0) were added to 10 μl of DTNB (0.75 mM) and 3,3 μl mice brain homogenate (crude enzyme) and incubated for 5 min at 25°C. The reaction was then initiated by the addition of 8 μl of acetylthiocholine (0.01M). The hydrolysis of acetylthiocholine was monitored by the formation of yellow 2-nitro-5-sulfidobenzene-carboxylate anion as a result of the reaction of DTNB with thiocholine, released by the enzymatic hydrolysis of acetylthiocholine for 10 min, at a wavelength of 412 nm. Physostigmine was used as a positive control and all tests were carried out in triplicate.

RESULTADOS:

Being a viable therapeutic approach to the treatment of Alzheimer’s disease, screening of medicinal plants for acetylcholinesterase (AChE) inhibition remains a potent measure. The percentage inhibition data for ethanol and successive fractions obtained through solvent partitioning shows varying degree of anticholinesterase activity. Concentration producing 50% inhibition values (IC50) were obtained from the dose-effect curves by linear regression while maximum inhibition (%) was represented as (Mean ± S.E) obtained through Graphpad software (GraphPad software, San Diego, CA, USA). Among the fraction tested FDEEPPM and FAEEEPPM with IC50 values of 483(455-501) and 505(487-515) μg/ml, respectively inhibited AChE activity. These fractions had a maximum inhibition of 57,8±0,8% and 56,6±0,9 % respectively; FAQEEPPM and FHEEPPM with maximum inhibition of 18,5±0,9 % and 7,3±0,2 % respectively showed low activity against AChE. The inhibition observed with EEPPM was insignificant. It was interesting to observe a decrease in AChE activity at the highest concentration of EEPPM tested (1000 μg/ml). This effect may be as a result of enzyme saturation or solution turbidity. Physostigmine (standard drug used) showed an IC50 value of 0,052 (0, 01- 0, 23) μg/ml.

CONCLUSÃO:

Traditionally, plants have been used to enhance memory and alleviate other symptoms associated with AD. Some of the most prescribed herbal medicines and compounds like EGb 761 and galanthamine are sourced from Ginkgo biloba L and Galanthus woronowii respectively. The most effective treatments are therefore this type of the medications that attempt to increase the brain’s levels of acetylcholine, an important neurotransmitter whose levels decrease with onset of the disease. The inhibition of acetylcholinesterase activity observed by ethanol extract, aqueous and hexane fraction can be associated with nonspecific effect. However dichloromethane and ethyl acetate fractions of Pimenta pseudocaryophillus leaves with the lowest IC50 showed significant activity .This result suggest potent cognitive enhancing property and that the active(s) principle(s) responsible present low polarity. These results are promising regarding the use of this plant species in treating diseases involving cognitive deficit. Further studies are necessary to examine the ethyl acetate and dichloromethane fraction in memory model with mice and determine the compound(s) as well as the mechanisms of action involved in this effect.

Palavras-chave: anticholinesterase, Pimenta pseudocaryophyllus, Alzheimer´s disease.