Narcissus and Daffodil

Analysis of Amaryllidaceae alkaloids 287
EXTRACTION OF AMARYLLIDACEAE ALKALOIDS
In quantitative analytical studies, alkaloids are usually extracted using the traditional
method. Plant material is typically dried and ground prior to extraction.
However, fresh plant materials are often used for this purpose, the water
content of which fluctuates with season, age, tissue type and storage conditions.
For this reason, quantitative data generated for fresh plant samples collected
and processed under different conditions may not be reliable for comparative
purposes. Traditionally, dried ground plant material, or fresh plant material
after maceration, is extracted with ethanol or a dilute acid solution. To remove
lipophilic non-alkaloids, the dilute acid extract is directly partitioned with an
organic solvent, whereas the ethanol extract must be evaporated and reconstituted
in dilute aqueous acid prior to partitioning. The use of fresh plant material
can present problems in the evaporation process, as large amounts of water and
partially water-soluble polysaccharides, especially in bulbs, make evaporation of
the ethanol extract difficult. After the removal of ethanol, the concentrated
extract is diluted with an acid solution and partitioned with an organic solvent.
The polysaccharides can also interfere with solvent extraction processes, tending
to make thick emulsions and causing incomplete extraction of non-alkaloids
and alkaloids in the subsequent steps. After the removal of lipophilic constituents,
the aqueous layer is basified and alkaloids are extracted into an organic
solvent.
In quantitative studies, where small samples are involved, problems due to
emulsions in the solvent extraction process can be overcome by centrifugation
(Davey et al., 1998; Bastos et al., 1996). However, a thick interface between organic
and aqueous layers may cause incomplete removal of the target compounds. The
error caused by this can be corrected by the addition of an internal standard at the
beginning of extraction, and by carrying out recovery studies for the substrates
and the internal standard (Claessens et al., 1983; Tencheva et al., 1987; Bastos
et al., 1996).
Queckenberg and Frahm (1994) developed a supercritical fluid extraction
procedure to remove the alkaloid fraction selectively from methanol extracts. The
re-extraction of dried methanol extract of Amaryllidaceae plants with supercritical
nitrous oxide (N 2 O) in the presence of a modifier (methanol containing ammonia
gas) efficiently removed the alkaloids, which were subsequently analysed by chromatographic
methods (Queckenberg and Frahm, 1993; Queckenberg et al., 1996).
Zhu et al. (1993) developed a supercritical fluid extraction method to isolate
lycorine from bulbs of Amaryllidaceae, and optimum conditions for the separation
were determined.
The procedure for the extraction of alkaloids (and their metabolites) from biological
fluids or tissues is somewhat similar to that used for plant materials. However,
due to the complex nature of the biological samples, more steps are involved
(Bickel et al., 1991a,b; Tencheva et al., 1987). Recently, Bores et al. (1996) used a
solid phase extraction procedure to separate galanthamine and its metabolite,
6-O-demethylgalanthamine, from biological samples. This procedure involves
the application of biological fluids containing test compounds to reversed-phase
cartridges and selective elution of the compounds of interest with appropriate
solvents.