Sustainable Use of Various Amaryllidaceae Plants against Alzheimer’s Disease

Alzheimer’s Disease (AD), a slowly progressive neuropsychiatric illness, principally characterized by memory deficits, has become the fourth leading cause of death in developed nations. Since the patients with AD suffer from marked reduction of cholinergic neuronal function resulting in a deficiency in acetylcholine (ACh) concentration in the brain, and these reductions are associated with impairments in memory, cholinergic enhancement strategies have been at the forefront of efforts to palliate pharmacologically the cognitive symptoms. Therefore, treatment approaches have been focused on the acetylcholinesterase inhibitors (AChEI) which are the most promising drugs demonstrating efficacy in the treatment of AD. Since, recently, galanthamine, an alkaloid isolated from different Galanthus species (Amaryllidaceae), has been found to be a potent and reversible acetylcholinesterase inhibitor, this development has prompted us to investigate the anticholinesterase activity in other plant species of Amaryllidaceae growing in Turkey, namely Galanthus elwesii, G. ikariae, Narcissus tazetta subsp. tazetta, Leucojum aestivum and Pancratium maritimum by Ellman method in comparison with galanthamine as the standard drug. Bioactivity-directed fractionation and isolation studies carried out on G. ikariae and N. tazetta subsp. tazetta extracts afforded 8 Amaryllidaceae-type alkaloids in total. We found that the activity of both of the plant extracts was due to the synergistic interaction of the alkaloids isolated. INTRODUCTION Alzheimer’s Disease (AD) is a chronic neurodegenerative disorder characterized by a progressive decline in cognitive function, including loss of memory and disturbances in function and behavior (Adams et al., 1984; Bachman et al., 1992; Arnold et al., 1993; Aisen and Davis, 1997). A deficit of central presynaptic cholinergic function has been demonstrated in AD by degeneration of cholinergic neurons in basal forebrain (Friede, 1965; Perry et al., 1978; lnan, 1999). This led to the suggestion that acetylcholinesterase inhibitors (AChEI) would preserve a putative deficit in acetylcholine levels associated with AD, and thus might reserve the memory impairments (Schneider, 2001; GuardadoSantervas, 2001; Gauthier, 2001). Therefore, a number of AChEI have been developed as candidates for the symptomatic treatment of AD, including natural compounds, such as physostigmine, huperzine A and galanthamine as well as various synthetic compounds such as tacrine, donepezile and rivastigmine (Davis and Mohs, 1982; Liu et al., 1986; Summers et al., 1986; Mutafova-Yambolieva et al., 1993; Rogers et al., 1998; Potkin et al., 2001). Among these, galanthamine, an Amaryllidaceae alkaloid isolated from the bulbs of Galanthus species. Amaryllidaceae alkaloids exhibit antitumor, antiviral and anticholinergic activities. Some of them have been used in the treatment of myastenia gravis, myopathy and diseases of the nervous system (Martin, 1987). The bulbs of some species of Amaryllidaceae cultivated in Turkey have been exported as ornamental plants. In the aforementioned context, we systematically study five uninvestigated Amaryllidaceae plants growing in Turkey for their potential anticholinesterase activity. Proc. WOCMAP III. Vol. 4: Targeted Screening of MAPs, Economics & Law Eds. C. Franz, Á. Máthé, L.E. Craker and Z.E. Gardner Acta Hort. 678, ISHS 2005 60 MATERIALS AND METHODS Plant Materials The bulbs of Galanthus elwesii Hooker fil., Narcissus tazetta subsp. tazetta L. and Pancratium maritimum L. from Antalya-Akseki, Antalya-Kumluca and Antalya-Belek located at the Mediterranean region of Turkey, respectively, G. ikariae L. and Leucojum aestivum L. from Trabzon-Sürmene and Samsun-Terme at the northeast of Turkey, respectively, were collected and identified by Prof. Dr. B. Şener. Voucher specimens are deposited in the Herbarium of Faculty of Pharmacy, Gazi University, Ankara, Turkey. Extraction Air-dried bulbs of Galanthus elwesii, G. ikariae, Narcissus tazetta subsp. tazetta, Leucojum aestivum and Pancratium maritimum (50 g for each) were powdered in a grinder mechanically and extracted three times with chloroform:methanol (1:1) at room temperature. After removal of the solvent in vacuo at 40°C until dryness, crude extract of each species was obtained. Anticholinesterase activity of the extracts was determined at 10 μg/ml concentration by Ellman method. Subsequently, the alkaloid extracts were prepared from the powdered bulbs of each species by the following procedure. The powdered materials were percolated with ethanol (96°) at room temperature, evaporated under vacuum until dryness. The crude extracts were acidified with HCl (5%), and left for 2 days in +4°. After filtration, hydro-acidic layers were made basic with NH4OH (25%) to adjust pH to 8, extracted with chloroform and evaporated in vacuo until dryness. Bioactivity-directed Isolation Alkaloid extracts of G. ikariae and N. tazetta subsp. tazetta were used for bioactivity-directed fractionation and isolation. Alkaloid extract of G. ikariae was subjected to column chromatography (Si 60) and eluted with chloroform:methanol mixtures in increasing polarity. 50 fractions (10 ml for each) were collected in total. By monitoring with TLC, similar fractions were combined and 5 subfractions were obtained. Anticholinesterase activity of the subfractions were determined at 10 μg/ml concentration. Third and fourth subfractions displayed activity and from them, lycorine (0.09 g), tazettine (0.008 g), crinine (0.06 g), galanthamine (0.003 g), 3-epihydroxybulbispermine (0.074 g) and 2-demethoxymontanine (0.047 g) were isolated by preparative TLC. Same isolation procedure was also applied for the alkaloid extract of N. tazetta subsp. tazetta and lycorine (0.3 g), tazettine (0.0018 g), 3-epihydroxybulbispermine (0.042 g), N-nor-galanthamine (0.007 g) and haemanthamine (0.005 g) were isolated by preparative TLC from the active subfractions. The characterization of the isolated alkaloids were determined by H-NMR and EIMS spectra in comparison with the published data (Grundon, 1985; Southon and Buckingham, 1989; Şener et al., 1993; Şener et al., 1998). Anticholinesterase activity of the pure compounds was determined by Ellman method at 10 μg/ml concentration. Chemicals For acetylcholinesterase inhibitory activity, electric eel acetylcholinesterase (AChE; True cholinesterase; EC 3.1.1.7., Type VI-S, EC no: 232-559, 2000 Units) was purchased from Sigma (St. Louis, MO). Sterile-apyrogen isotonic sodium chloride solution (0.9%) was obtained from Ibrahim Ethem Drug Company. The enzyme kit (Spinreact, Spain), based on the Ellman method, was used to evaluate the acetylcholinesterase inhibitory activity. The standard drug, galanthamine, was obtained from Johnson & Johnson drug company. Organic solvents and silica gel used in the isolation were from Merck Co. Determination of Anticholinesterase Activity Anticholinesterase activity was determined spectrophotometrically using acetylthiocholine as substrate by modifying the method of Ellman (Ellman et al., 1961).