Narcissus and Daffodil

370 K. Ingkaninan, H. Irth and R. Verpoorte
postulated, that the application of galanthamine in Alzheimer’s disease was
studied. Based on the cholinergic hypothesis (Perry, 1986), memory impairments
in patients suffering from this disease result from a defect in the cholinergic system.
One approach to the treatment for this disease is to enhance the acetylcholine
level in the brain by AChE inhibitors (Winblad et al., 1993). Galanthamine hydrobromide
is now being developed for Alzheimer’s disease. It has shown clear evidence
for being suitable for the treatment of mild and moderate Alzheimer’s disease. At
least three corporate pharmaceutical companies are involved in developing galanthamine
for the Alzheimer’s disease market: Waldheim Pharmazeutika (Austria)
for developing a method of synthesis, Shire Pharmaceuticals (UK) for clinical studies,
and Janssen Pharmaceutica (Belgium) for final registration and marketing.
The preclinical and clinical studies of galanthamine were reviewed by Mucke
(1997a,b) and Rainer (1997), respectively.
Galanthamine is commercially isolated from Leucojum aestivum (Amaryllidaceae)
(Paskov, 1986). Although the chemical synthesis of galanthamine was accomplished
in 1960 (Barton and Kirby, 1960), it was only very recently that an industrial
scale synthesis of galanthamine hydrobromide was established (Czollner et al.,
1996). Several analytical techniques have been described to quantify galanthamine
in natural sources. Kreh et al. (1995) and Bastos et al. (1996) reported capillary
column gas chromatographic methods for quantification and identification of
galanthamine and other Amaryllidaceae alkaloids. A radioimmunoassay developed
by Tanahashi et al. (1990) provided specific and precise quantitation of galanthamine
in unpurified plant extracts. Three years later, Poulev et al. (1993) established a
more sensitive enzyme immunoassay for the quantitation of fmol amounts of
galanthamine.
SCREENING ASSAYS FOR ACETYLCHOLINESTERASE INHIBITORS
Several assays have been established for the detection of AChE activity and its
inhibitors. The colorimetric method developed by Ellman et al. (1961) is the most
widely used. This assay is based on the enzymatic hydrolysis of acetylthiocholine
iodide (ATCI) to yield thiocholine. When thiocholine reacts with 5,5′-dithiobis-2nitrobenzoate
(DTNB), it will produce the yellow product of 5-thio-2-nitrobenzoic
acid which can be detected at 405 nm by a spectrometer.
acetylthiocholine + H2O AChE
acetate + thiocholine
thiocholine + DTNB 5-thio-2-nitrobenzoic acid
+ 2-nitrobenzoate-5-mercaptothiocholine
This spectrometric assay has been used for the screening of plant extracts for anti-
AChE activity (Park et al., 1996; Kim et al., 1999). The use of a 96-well plate reader
for measuring the AChE activity, reported by Ashour et al. (1987), allowed the
rapid screening of series of samples.
A radiometric technique for the detection of AChE activity based on hydrolysis
of [ 3 H] labelled acetylcholine was reported by Johnson and Russell (1975). Guilarte
et al. (1983) developed a simpler method using [ 14 C]sodium bicarbonate. The
acetic acid formed from the enzymatic hydrolysis of acetylcholine reacts with