Characterization of an acetylcholinesterase inhibitor isolated from Ammocharis coranica (Amaryllidaceae)

Abstract

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder, with a mean duration of about 8.5 years between manifestation of clinical symptoms and death. The disorder is characterised by abnormal pathologic features, such as extracellular deposits of β-amyloid derived from amyloid precursor protein (APP) in senile plaques, intracellular formation of neurofibrillary tangles containing hyperphosphorylated form of a microtubule associated protein, tau, and the loss of neuronal synapses and pyramidal neurons, as well as a decrease in levels of the neurotransmitter acetylcholine (ACh) by nearly 90%. AD is the leading cause of dementia in elderly people; with the proportion of elderly people in the population increasing steadily, the burden of the disease, both to caregivers and national economies, is expected to become substantially greater over the next 2 to 3 decades. There are currently, 20 million people with the disorder world-wide. The “cholinergic hypothesis of AD”, which states that cognitive impairment in AD are mainly due to decline in the level of ACh forms the neurobiological incentive for treatment aiming at the improvement of cholinergic function in AD. Inhibition of acetylcholinesterase (AChE), the enzyme involved in the metabolic hydrolysis of acetylcholine (ACh) at the cholinergic synapses in central and peripheral nervous systems, promotes increase in the concentration, and duration of action of synaptic ACh, and serves as a strategy for the treatment of AD and discovery of active compounds. Drugs approved for the treatment of AD are mostly compounds with AChE inhibitory activity; these drugs have limitations to their therapeutic success, due to non selectivity, low efficacy, poor bioavailability, and adverse cholinergic side effects in the periphery, which include nausea, vomiting, diarrhoea, dizziness, and hepatotoxicity. This motivates the evaluation of more plants for AChE inhibitory compounds. Bulbs of Ammocharis coranica were selected for AChE inhibitory assay because the plant is used in traditional medicine to treat people with mental illnesses. It also belongs to the Amaryllidaceae family known to contain compounds with AChE inhibitory activity. A 96% ethanol crude extract of the bulbs of A. coranica had good AChE inhibitory activity with an IC50 of 14.3 ± 0.50 µg/ml in a quantitative assay. In a qualitative bioautographic assay of the extract based on Ellman’s method several white areas of inhibition against a yellow background were observed. No false positives were found. The minimum concentration of the ethanolic crude of the plant extract visualised as a white area of inhibition of AChE enzyme on the TLC plates was 15 µg. The crude extract was separated into several fraction of different polarity by solvent-solvent fractionation. The IC50 of the inhibition of the ethanolic crude extract of the bulbs of A. coranica was 14.3 ± 0.50 µg/ml. The intermediate polarity fractions (butanol and ethyl acetate) had IC50 of 0.05 ± 0.02 and 43.1 ± 1.22 µg/ml, respectively. Compound 1 was isolated from butanol fraction and compounds 1 and 2 were isolated from the ethyl acetate fraction using bioautographic assay guided fractionation. The structures of these compounds were elucidated using nuclear magnetic resonance (NMR) spectroscopy, and identified as lycorine (1) and 24-methylenecycloartan-3β-ol (2) respectively. Lycorine (1) had AChE inhibitory activity, with an IC50 of 102 ± 7.75 µM. This is equivalent to an IC50 of 29.4 µg/ml indicating that the butanol fraction was nearly 600 times more active. It is possible that the butanol fraction may have therapeutic uses if it is not toxic and the results imply that substantial synergism may have existed between different compounds in the butanol fraction. The 24-methylenecycloartan-3β-ol (2) was not active when tested using the microtitre plate assay although it did inhibit colour development in the bioautography assay. Copyright