Narcissus and Daffodil

Acetylcholinesterase inhibitors 371
[ 14C]sodium bicarbonate to generate [ 14C]carbon dioxide, which is measured using
an ionisation chamber system. Compared with Ellman’s colorimetric method, these
radiometric techniques are more sensitive. However, the radiometric method is an
endpoint measurement and cannot be used for kinetic studies. Furthermore, the
limited linear range of the method demands the proper calibration of the activity
prior to incubation.
A fluorometric method has also been developed based on the reaction of
thiocholine produced by the enzyme from acetylthiocholine with the fluorogenic
compound N-(4-(7-diethylamino-4-methylcoumarin-3-yl)phenyl) maleimide (CPM)
(Parvari et al., 1983). This assay combines the specificity and the technical advantages
of the Ellman technique with a wide linear range of accuracy and a limit of
detection which is 100-fold lower than that of the radiometric method. Roger et al.
(1991) developed the optical sensor for anti-AChE using immobilised fluorescein
isothiocyanate (FITC)-tagged eel electric organ AChE on quartz fibres for monitoring
enzyme activity. This biosensor is reusable, sensitive and easy to operate.
It shows potential adaptability to field use as the instrument is portable.
Recently, Pasini et al. (1998) reported a chemiluminescent (CL) method for a
384-well microtiter format assay for high throughput screening of AChE inhibitors.
The CL detection of AChE was based on coupled enzymatic reactions involving
choline oxidase and horseradish peroxidase as the indicator enzymes. The reaction
leads to light emission and luminol is used for the detection of the peroxide
formed. This method had a detection limit 1000-fold lower than that of the colorimetric
method.
THE SEARCH FOR AChE INHIBITORS FROM NATURAL SOURCES
Nature is a rich source of biological and chemical diversity. It has been shown
many times that natural products could be developed as drug candidates. The
unique and complex structures of natural products such as paclitaxel cannot be
obtained easily by chemical synthesis. The well-known AChE inhibitors, galanthamine
and physostigmine, are also obtained from plants. The clinical studies of
AChE inhibitors for Alzheimer’s disease are still ongoing. Until now, no drug of
choice has been decided upon. Therefore, the search for new AChE inhibitors
from natural sources is of great interest. However, in searching for new leads from
crude natural products extracts, one might encounter the problem of isolation of
known active compounds or compounds that give aspecific effects on the bioassays
used. A strategy of ‘dereplication’ of active compounds, or rapid identification of
known active compounds at an early stage, is important in the lead finding
process. One of the approaches for dereplication is to couple a separation technique
with a bioactivity detection method. The known active compounds can be
rapidly identified by the aid of data analysis and the unknown active fraction can
be further investigated.
Narcissus plants are a potential source for AChE inhibitors as they contain a
variety of compounds, especially alkaloids, the group of compounds that shows
many biological activities including AChE inhibitory effect. Since narcissus are
cultivated plants, there could be a large variation of chemical constituents among
different cultivars. Figure 15.1 shows the inhibitory effect of the methanol extracts