Narcissus and Daffodil

372 K. Ingkaninan, H. Irth and R. Verpoorte
% Inhibition of acetylcholinesterase
100
90
80
70
60
50
40
30
20
10
0
Avalanche Carlton Grand
Soleil d'Or
Sir Winston
Churchill
Figure 15.1 Percentage of inhibition effect of methanol extracts from some narcissus
cultivars at the concentration of 0.1 mg/ml measured with the microplate
assay.
from some narcissus cultivars tested in our laboratory. The microtiter plate assay
used was according to the method of Ellman et al. (1961), as described by Ingkaninan
et al. (2000a). All extracts showed an inhibitory activity for AChE. However, the
activity might be due to the known active compound, galanthamine. A dereplication
step for the rapid identification of galanthamine in crude extracts is necessary.
The extracts also contain other unknown, active compounds that will be selected
for further studies.
IDENTIFICATION OF AChE INHIBITORS BY HPLC WITH ON-LINE
COUPLED UV, MS AND BIOCHEMICAL DETECTION
Recently, strategies for the on-line coupling of biochemical detection to separation
methods such as high-performance liquid chromatography (HPLC) have been
developed (Irth et al., 1995). This technique allows the simultaneous separation
and detection of bioactive compounds from complex mixtures such as natural
products. To obtain additional information on the compounds separated, the
on-line system can also be coupled with other detection methods such as mass
spectrometry (MS) and ultraviolet (UV) or photodiode array (PDA) detection. In a
natural products-based drug discovery programme, this technique can be useful
for the detection of new active compounds in the presence of the already known
active compounds. It opens the possibility for the rapid screening of galanthaminecontaining
plants for other AChE inhibitors.
The colorimetric assay for AChE as reported by Ellman et al. (1961) is suitable
for development into a continuous-flow biochemical detection system. AChE from
electric eels is inexpensive and has satisfactory stability at room temperature. The
assay reagents, DTNB and ATCI, are also commercially available. The short reaction
time of the assay, approximately 2 min, made it possible to develop it for continuousflow
biochemical detection system without causing much band-broadening.