Acetylcholinesterase inhibitory activity of Habranthus tubispathus

CAVALLARO, VALERIA; NATALIA P. ALZA; ANA PAULA MURRAY

Córdoba, Argentina

Congreso; Iº Reunión Internacional de Ciencias Farmacéuticas; 2010

FBIOyF-UNR

Habranthus tubispathus, (L`Hér.)Traub (Amaryllidaceae) is native to Argentina, Chile, Uruguay and Paraguay  (1). So far no phytochemical studies have been reported on this specie. Plants of the Amaryllidaceae family are known to contain isoquinoline alkaloids, commonly called Amaryllidaceae alkaloids. Some of these alkaloids are acetylcholinesterase (AChE) inhibitors (2). The main role of AChE is the termination of nerve impulse transmission at the cholinergic synapses by rapid hydrolysis of acetylcholine. Inhibition of this enzyme is currently used for the treatment of Alzheimer´s disease, senile dementia, ataxia and myasthenia gravis (3). We proposed the isolation and characterization of the bioactive alkaloids from the bulbs of H. tubispathus. Bulbs of H.  tubispathus were collected in November 2009 in Bahía Blanca, Argentina. Fresh bulbs (966 g) were chopped and macerated with EtOH at room temperature for two weeks, and then extracted with boiling EtOH for 2 hours. Both extracts were combined and after evaporation under reduced pressure, the residue was dissolved in 2% HCl and filtered 1 h later. The remaining solution was made basic (pH 9) with Na2CO3 3M and extracted with CH2Cl2 several times (Fraction A) and then with CH2Cl2:MeOH (3:1) (Fraction B). Both fractions, A and B, were subjected to Flash Column Chromatography, eluting initially with CH2Cl2 and increasing the polarity of the eluent with MeOH. Alkaloids were detected by Dragendorff´s reagent and the inhibitors were detected by the Ellman`s method. The AChE activity was quantified by a modification of Ellmann`s method (4). The IC50 value of the ethanolic extract of H. tubispathus was 976,6 mg/mL+/- . This value was obtained by probit analysis from four determinations, each one performed in triplicate. The Rf values of the white spots observed in the TLC bioautographic method for the detection of AChE inhibitors was coincident with those of the orange spots observed when we reveled with Dragendorff`s reagent. So, we proceeded to the isolation of the alkaloids, responsible of the AChE inhibition. We ’ll present the structural elucidation of these compounds as well as the AChE inhibition of these alkaloids. H. tubispathus shows an important inhibition of AChE in comparison with the IC50 value of other species of the Amaryllidaceae family previously reported (4). This activity can be explained by the presence of  active alkaloids