Narcissus and Daffodil

Identification section
Separation section
HPLC
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injector
Bioactivity test section
DTNB
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ATCI
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HPLC column
Acetylcholinesterase inhibitors 373
50°C
AChE
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Figure 15.2 shows the scheme of the on-line HPLC-UV-MS-biochemical detection
system developed in our laboratory (Ingkaninan et al., 2000a). The separation
is performed in an HPLC column. The eluate is split into three flows: the major
flow travels to a UV detector, while two minor flows go to the MS and the
biochemical detection system. In the biochemical detection system, the eluate is
mixed with substrate (ATCI), enzyme (AChE) and DTNB for approximately 2 min
in the reaction coil. A spectrometer set at 405 nm detects the yellow product
obtained from the enzymatic reaction. Any inhibitory activity from the HPLC eluate
will result in a negative peak on the biochemical detector. After determination of
the delay times of the three detection lines, the results of these detections can be
related and thus UV spectra and molecular weight of the active compounds can
be determined. In this way, known inhibitors such as galanthamine can easily be
recognised.
The strongly AChE inhibiting extract from narcissus ‘Carlton’ (Figure 15.1) was
fractionated by means of centrifugal partition chromatography (CPC) and the
active fraction was injected into the on-line HPLC-UV-MS-biochemical detection
system (Ingkaninan et al., 2000a). The chromatogram from two detectors and the
mass spectra are shown in Figure 15.3. Although the chromatogram from the
HPLC is very complex, the biochemical detection system showed a high selectivity
for the AChE inhibitors. Two broad negative peaks from the biochemical detector
proved the presence of at least two inhibitors in the extract. The retention time of
the main active peak and the molecular weight derived from MS data ([M + H]
of 288) corresponded to those of galanthamine, a well-known AChE inhibitor.
MS
UV or PDA
Detector
Vis detector at
405nm
Figure 15.2 Scheme of the on-line HPLC-UV-MS-bioactivity detection for acetylcholinesterase
inhibitors.