Narcissus and Daffodil

376 K. Ingkaninan, H. Irth and R. Verpoorte
FUTURE PERSPECTIVES
It is to be noted that in the on-line HPLC-UV-MS-biochemical detection, only a
small amount, i.e., one-thirtieth of the eluate from the HPLC, was split to the
biochemical detector. Therefore, it is possible to apply the on-line system for a
preparative separation technique such as preparative HPLC or CPC and on-line
fraction collection can be performed. In both separation techniques, gradient
elution might be useful for broad range separation. Furthermore, to gain more
information for the identification of active peaks, a PDA and an NMR can be coupled
to this on-line system.
Although the Ellman colorimetric method used in this on-line biochemical
detection system is not the most sensitive, it is the simplest and the most
inexpensive screening method for AChE inhibitors. In natural productsbased
research, where milligrams of new leads are still needed for the structure
elucidation, selectivity for the bioactive compounds is more important
than sensitivity. However, the sensitivity of the on-line biochemical detection
could be increased by using a fluorometric method. Some of the fluorescent
probes such as N-[4-[7-(diethylamino)-4-metylcoumarin-3-yl]phenyl]maleimide
(CPM), which forms a fluorescent product when it reacts with thiocholine
(Berman and Leonard, 1990) or the substrate, 7-acetoxy-N-metylquinolinium
iodide (Menger and Johnston, 1991), have been used in an ultrasensitive
assay for acetylcholinesterase and are commercially available. These fluorescent
probes could be further developed for the on-line biochemical detection
system.
Another method that could be useful for the screening of AChE inhibitors from
natural products is thin-layer chromatography (TLC). Some TLC methods using
an enzyme, a substrate and a dye as the spraying reagent have been reported
(Bunyan, 1964; Menn and McBain, 1966; Kiely et al., 1991). The inhibition zone
from the TLC indicates the inhibitory activity of the test compound to the enzyme.
When complex mixtures such as crude plant extracts are tested, the presence of
known inhibitors in the extract will be rapidly identified. This technique can be
helpful in the selection of extracts to be further investigated for new AChE
inhibitors.
In conclusion, narcissus cultivars are potential sources for AChE inhibitors. As
these plants commonly contain the known active compound galanthamine, the
dereplication step of galanthamine is crucial. This step can be done by using the
on-line HPLC-UV-MS-biochemical detection that has already been developed, or
by other techniques that could lead to the rapid separation and identification of
the active compounds from complex mixtures. From our studies, it is clear that
screening on the level of cultivars using such methodology may result in new compounds
with AChE inhibitory activity.
ACKNOWLEDGEMENTS
The authors would like to acknowledge the van Leersum Fund for the financial
support for an HPLC pump.