Effects of sucrose and plant growth regulators on acetylcholinesterase inhibitory activity of alkaloids accumulated in shoot cultures of Amaryllidaceae

The influence of sucrose (30, 60, 90 and 120 g/L), activated charcoal (5 and 10 g/L), and various levels of several plant growth regulators (6-benzyladenine, naphthalene-1-acetic acid, 2,4-dichlorophenoxyacetic acid, and picloram) on organogenesis (bulb and root development) and the accumulation of alkaloid and galanthamine in shoot cultures of three Amaryllidaceae species (Narcissus pseudonarcissus, Galanthus elwesii, and Leucojum aestivum) was investigated in a full-factorial experiment. Alkaloid extracts were analyzed by gas chromatography-mass spectrometry, leading to the quantification of galanthamine and to the identification of other alkaloids. The different extracts were then subjected to an Ellman test to evaluate the inhibitory activity of acetylcholinesterase. The highest contents of galanthamine [0.02-0.1% dry weight (DW) depending on the plant species] were always accompanied with a high level of acetylcholinesterase inhibition (> 30%). However, some samples containing low amounts of galanthamine (0.005% DW) showed high inhibitory activities (40-80%). These findings demonstrate the presence of Amaryllidaceae alkaloids that have not yet been identified as having anti-acetylcholinesterase activity.