Narcissus and Daffodil

374 K. Ingkaninan, H. Irth and R. Verpoorte
Figure 15.3 The spectra and the chromatograms obtained after injection of the fraction
from narcissus ‘Carlton’ extract into the on-line HPLC-UV-MS-bioactivity
detection system. Left: ESI-MS. Right: Chromatograms from the biochemical
detection set at 405 nm (upper line) and from the UV set at 215 nm
(lower line). The delay time of the peaks between the biochemical detection
and the UV was 2.3 ± 0.1 min and the delay time of the peaks between
the biochemical detector and the MS was 2.1 ± 0.1 min.
However, MS showed that there was another molecule present in the same peak at
[M + H] of 290. It would be interesting to identify this compound further and test
it for inhibitory effect. The minor peak in the chromatogram detected by the biochemical
detector was caused by an unknown AChE inhibitor. Further studies
should, therefore, focus on the isolation and identification of this active compound.
A second example is a study of the extract from narcissus ‘Sir Winston Churchill’
(Ingkaninan et al., 2000b). From the microplate assay, this extract showed much
less activity than that of narcissus ‘Carlton’ (Figure 15.1). The extract was fractionated
by the same CPC procedure as that of narcissus ‘Carlton’. The active fraction
was also obtained at the same retention. However, when the active fraction was
injected into the on-line system, no activity corresponding to galanthamine was
found. The activity was from another compound with a different molecular weight
and retention time. As this extract possibly contains new AChE inhibitors, it was
chosen for further investigation. The active fraction was further separated by
another CPC run and the fractions obtained were injected into the on-line system
(Figure 15.4).
The active peak from the biochemical detector at 15.2 min corresponded to the
UV peak at 12.9 min and the MS peak at 13.1 min. However, MS spectra showed
that there were two compounds present at 13.1 min having molecular weights of
265 ([M + H] of 266) and 317 ([M + H] of 318). These two compounds were
isolated by preparative HPLC and tested for AChE inhibitory activity by the