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Search for Bioactive Alkaloids in Hymenocallis

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Nury Rivero, Matilde Gómez & J. D. Medina

To cite this article: Nury Rivero, Matilde Gómez & J. D. Medina (2004) Search for
Bioactive Alkaloids in Hymenocallis Species, Pharmaceutical Biology, 42:4-5, 280-285, DOI:
10.1080/13880200490511774

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Pharmaceutical Biology
2004, Vol. 42, Nos. 4–5, pp. 280–285
Search for Bioactive Alkaloids in Hymenocallis Species
Nury Rivero1,2, Matilde Gómez2 and J. D. Medina2
1
Facultad de Farmacia, Universidad Central de Venezuela, Caracas Venezuela; 2Centro de Química, Instituto Venezolano de
Investigaciones Científicas, Caracas Venezuela
Abstract
Extracts from five species of Hymenocallis Salisbury
(Amaryllidaceae) were examined by means of gas chro-
matography-mass spectrometry (GC-EIMS), for sources of
bioactive alkaloids; especially of the isocarbostyril type such
as the antitumor compound narciclasine. Ten different alka-
loids were identified, all of them exhibiting skeletons of
the lycorine, galanthamine, and pyrrolophenanthridine
types considered typical for the family. The detection of
1-O-acetyl-pseudolycorine and 4,5-dehydro-anhydrolycorine
constitutes the first report of these alkaloids for the genus. In
addition, the identification of anhydropseudolycorine and
4,5-dehydro-anhydropseudolycorine from several species
constitutes the first report of these alkaloids as naturally
occurring compounds. Neither of the species contains iso-
carbostyrils and, due to the low alkaloid concentrations
observed, they show no promise as a source of
Amaryllidaceae alkaloids.
Keywords: Amaryllidaceae alkaloids, antitumor,
Hymenocallis species, isocarbostyrils.
Introduction
Hymenocallis Salisbury (Amaryllidaceae) has been chemi-
cally studied as a genus since 1920, when the isolation
of lycorine (1; Fig. 1) from H. littoralis Salisb.
(Amaryllidaceae) was first reported by Gorter (1920). This
alkaloid is widely distributed within the Amaryllidaceae. It
was first isolated from bulbs of Narcissus pseudonarcissus
L. (Amaryllidaceae) in 1887, and its biological activity was
demonstrated as early as 1898, when it was shown that
lycorine produces an emetic effect (Morishima, 1898). This
finding stimulated the pharmacological study of extracts and
pure compounds isolated from plants of this family. Since
Accepted: June 28, 2004
Address correspondence to: José D. Medina, Centro de Química, Instituto Venezolano de Investigaciones Científicas, Apartado 21827,
Caracas 1020-A, Venezuela. E-mail: jmedina@quimica.ivic.ve
DOI: 10.1080/13880200490511774
then, and only to mention a few of the activities reported for
lycorine, this alkaloid has been shown active as an antifun-
gal (Cook & Loudon, 1952), antiviral (Dabire & Murav’eva,
1982), and as an antifeedant (Singh & Pant, 1980). It has also
been shown to have activity against tumors with P388-PS
system and protein synthesis inhibitor related to ADN and
ARN (Suffnees & Cordell, 1985) and sarcomas (Pettit et al.,
1986).
Despite the variety of activities reported for
Hymenocallis alkaloids, they generated little scientific inter-
est until 1993 when Pancratium littorale Jacq. (Amarylli-
daceae), from which an antineoplastic alkaloid with an
isocarbostyril-type structure (pancratistatin, 2) had been
isolated, was reclassified as Hymenocallis littoralis Salisb.
(Amaryllidaceae) (Pettit et al., 1993). Pancratistatin has
proven active against the P388-PS system (Suffnees &
Cordell, 1985; Pettit et al., 1986; 1993), sarcomas
M5076 (Martin, 1987; Pettit et al., 1995), melanomas and
viruses (Lewis, 1997; Pettit et al., 1995). In addition,
narciclasin (3) and 7-deoxy-narciclasin (4) were isolated
from the same plant, showing various interesting activities
(Suffnees & Cordell, 1985; Martin, 1987; Pettit et al., 1993).
These events induced a reinvestigation of many
Hymenocallis species because these alkaloids are neutral
(lactams), and they might have been overlooked in the search
for basic compounds.
Capillary gas chromatography coupled with a mass detec-
tor (GC-MS) has been successfully used to determine
Amaryllidaceae alkaloids as trimethylsilyl ethers (Onyiriuka
& Jackson, 1977), N-oxide derivatives (Kobayashi et al.,
1991), and, recently, extracts without derivatization (Kreh et
al., 1995; Bastos et al., 1996). This tool provides a very rapid
method of examining the alkaloid content of the plants of this
family with very accurate identification through the frag-
mentation of the alkaloids. This paper reports the search for
© 2004 Taylor & Francis Ltd.
 Bioactive alkaloids in Hymenocallis 281

OH OH

HO HO OH
2
1
H
H
O O
OH
H H
N NH
O O
O
1 2

OH

OH OH

H
H
O B
OH O
H3C O
NH
O
O
R N
CH3
3 R = OH
4 R=H 5

OH

H
B
O
H3C O
O

N N
H
O

6 7
Figure 1. Alkaloids discussed in the text.

new bioactive alkaloids, especially the isocarbostyril type,
through the analysis of the extracts from five Hymenocallis
species: (1) H. bolivariana Traub., (2) H. guianensis (Ker
Gawler) Herb., (3) H. lobata Klotzsch, (4) H. tubiflora Salisb.
[syn. H. guianensis var. tubiflora (Salisb.) Herb. (two popu-
lations)] and (5) H. venezuelensis Traub.
Materials and Methods
General
Samples were dried in a 6-l Labconco Lyph-Lock Benchtop
Freeze Dry System (model 77520). GC-MS was performed
in a Hewlett-Packard model HP5973 instrument, with an HP-
5MS [film 0.25 mm, poly(5%-diphenyl-95%-dimethylsi-
loxane)], 30 m capillary column, using software G1034C,
version C03.00 (HP 1989–1994). Temperature programming:
200–250 °C (4 °/min), hold 5 min, 250–280 °C (4 °/min),
hold 1 min. Injector temperature: 250 °C, split mode 50 : 1,
pressure 23 psi, flow 75 ml/min. Helium as carrier gas,
1.5 ml/min, at constant pressure (23 psi). Transfer line tem-
perature: 280 °C. Source temperature: 250 °C. Analyzer
temperature: 150 °C. Lycorine, isolated from H. caribaea,
was used as standard at a concentration of 1 mg/ml
and its spectrum compared with the databases of the
instrument (Wiley 275.L. library and NIST, version 1.1a).
Samples were injected as methanol and/or n-butanol
solutions.
282 N. Rivero et al.
HO
N N
H3C O
8 9
HO
N H3C O
H3C O
10
Figure 2. Alkaloids discussed in the text.
Plant material
The bulbs of the five species studied were collected in 1992
in different regions of Venezuela. They were taxonomically
identified by Dr. M. Reymúndez (Universidad Central de
Venezuela, Caracas, Venezuela). Bulbs were kept frozen until
used. Names were checked with the International Plant Name
Index (IPNI) and W3Tropicos.
H. bolivariana was collected in a shaded site along the
Orituco River near the town of Calabozo in the state of
Guárico. A voucher specimen was filed with the National
Herbarium (no. 113 1992, M. Reymundez).
H. venezuelensis was collected to the south of the Orituco
River in a flooded savanna exposed to direct sunlight.
Voucher specimens were filed with the National Herbarium
(nos. 106–112 1992, M. Reymúndez).
H. guianensis was collected from the understory of the
gallery forest alongside the rivers in the vicinity of the road
that connects the towns of Tumeremo and Bochinche in
the state of Bolívar. Voucher specimens were filed with
the National Herbarium (nos. 132–33, 276 1992, M.
Reymúndez).
H. lobata was collected in a partially flooded sandy
savanna, south of the Orinoco River, along the road con-
necting Caicara with Maripa in the state of Bolívar. Voucher
specimens were filed with the National Herbarium (nos. 127,
228–232 1992, M. Reymúndez).
H. tubiflora was collected from two populations: one from
Caripe del Guácharo (H. tubiflora Caripe), in the state of
Monagas (no. 144 1992, M. Reymúndez), and the other, H.
tubiflora Guaraunos (no. 142 1992, M. Reymúndez), from
the understory of the gallery forest alongside the rivers near
O
O
OH
R O
2
1
H
HO
H
N
11 R = acetyl
12 R = H
the road that connects Guaraunos with Ajíes in the state of
Sucre. These, too, were filed with the National Herbarium.
Extraction of alkaloidal fractions
Alkaloids were extracted from the five species using the
method reported by Pettit et al. (1993), with some minor
modifications. The plant material, freeze-dried and pow-
dered, was macerated with ethanol 95% for 15 days at room
temperature. The extract was filtered through a cheesecloth
and concentrated under vacuum. The dried extract was par-
titioned with water : chloroform (1 : 1). The CHCl3 extract
was dried under vacuum and then extracted with 10%
aqueous HCl. The acidic solution was back-washed with
CHCl3, then basified with sodium bicarbonate and extracted
with chloroform and, successively, with n-butanol to obtain
fractions B and C (basic alkaloids). The aqueous phase
obtained from the partition water : chloroform was extracted
with n-butanol, and the organic phase was dried and evapo-
rated under vacuum. The dry extract was dissolved in
methanol. Acetone was then added until a 1 : 5 relation was
obtained, and the solution was then placed in a refrigerator
overnight. A precipitate resulted, and the supernatant was
completely evaporated to give a fraction D (neutral alka-
loids). During the GC-MS runs, it became evident that the
alkaloid content of the fractions was very small, with yields
of little significance.
Anhydro-pseudolycorine (8)
The observed retention time for this alkaloid under the
experimental conditions was 11.90–11.94 min (anhydroly-
 Bioactive alkaloids in Hymenocallis
corine’s retention time under the same conditions was
10.41–10.48 min), which, together with the major peaks
shifted by 2 atomic mass units from those of anhydroly-
corine, is indicative of the polar sustituent (OH) generated
by the opening of the dioxymethylene ring. Mass spectrum
(% relative abundance): m/z 253 (53), 252 (100), 237 (19),
209 (15).
4,5-dehydro-anhydro-pseudolycorine (10)
Under experimental conditions, the retention time observed
for this alkaloid was 13.78–13.80 min (for 4,5-dehydro-
anhydro-lycorine it was 11.99–12.04 min). An argument
similar to the one mentioned above supports the structure
assigned. Mass spectrum (% relative abundance): m/z 251
(75), 250 (100), 235 (15), 222 (4), 207 (21), 191 (8), 178
(15).
Results
A total of 17 extracts obtained from the plant material were
examined by the GC-MS technique. H. bolivariana did
not yield a fraction D. Due to the nature of the extraction,
fraction D cannot be considered exclusively as alkaloids.
Table 1 shows the alkaloids identified from the extracts of
the five species. The alkaloid content in all fractions was very
small. Ten different alkaloids were identified by means of
their fragmentation patterns, exhibiting skeletons typical
for the family, of the type lycorine, galanthamine, and
pyrrolophenanthridine.
Discussion
The use of the combined GC-MS technique for the exami-
nation of the five species of Hymenocallis allowed for the
determination of the structures of the main constituents of
the alkaloidal fractions. There are ample studies in the liter-
Table 1. Alkaloids identified for each species, listed in elution order.
Alkaloid
Lycoramine (5)
N-Demethyl-galanthamine (6)
Galanthamine isomer
Anhydrolycorine (7)
Anhydro-pseudolycorine (8)
4,5-Dehydro-anhydrolycorine (9)
4,5-Dehydro-anhydro-pseudolycorine (10)
Lycorine (1)
1-O-Acetyl-pseudolycorine (11)
Pseudolycorine (12)
a
1, H. bolivariana; 2, H. guianensis; 3. H. lobata; 4. H. tubiflora (Caripe); 5. H. tubiflora
(Guaraunos); 6, H. venezuelensis.
283
ature that demonstrate the power of this technique for the
determination of structures of Amaryllidaceae alkaloids,
especially for those for which skeletal features have been
determined. For the alkaloids detected with the skeleton of
the lycorine type (lycorine, 1, 1-O-acetyl-pseudolycorine, 11,
and pseudolycorine, 12), the findings by Kinstle et al. (1966)
and Llabrés et al. (1986) establish the principal fragments
one should expect from this type of alkaloid. The loss of the
C1–C2 bridge with their substituents gives, in general, the
base peak of the spectrum. The spectra obtained for these
three alkaloids were in close agreement with those reported
in the literature (The Wiley Registry of Mass Spectral Data,
6th ed.; Llabrés et al., 1986).
Those alkaloids with a galanthamine-type skeleton (lyco-
ramine, 5, N-demethyl-galanthamine, 6, and an isomer of
galanthamine) are without doubt defined by the presence of
a very intense molecular ion, the (M–1) fragment as the base
peak (or vice versa), and a major peak that represents the loss
of ring B from the (M–1) fragment (Razakov et al., 1969;
Kreh et al., 1995; Bastidas et al., 1987). The alkaloid men-
tioned as an isomer of galanthamine gives all the fragments
typical of that alkaloid reported in the literature, but their
relative abundances are much smaller. For that reason, it was
assumed that it is not galanthamine itself.
Finally, the alkaloids with a pyrrolophenanthridine-type
skeleton (anhydro-lycorine, 7, anhydro-pseudolycorine, 8,
4,5-dehydro-anhydro-lycorine, 9, and 4,5-dehydro-anhydro-
pseudolycorine, 10) due to their extended aromaticity present
the base peak at (M–1), with a few peaks in their spectra
shifted according to the sustituents present. In these cases,
comparison of the spectra and retention times of the new
alkaloids with the ones of their reported homologs (anhydro-
lycorine, The Wiley Registry of Mass Spectral Data, 6th
ed., and 4,5-dehydro-anhydro-lycorine, Ghosal et al., 1986)
allowed for a full identification.
Furthermore, considering the characteristics of the chro-
matography column [nonpolar, poly(5%-diphenyl-95%-
Speciesa
1 2 3 4 5 6
+ + + +
+
+ + + +
+ + + + +
+ + +
+ + +
+ + +
+ + + + +
+
+ + + + +
284
Table 2. Elution order of the alkaloids related to their melting points and structural features.
Alkaloid
Lycoramine (5)
N-Demethyl-galanthamine (6)
Anhydrolycorine (7)
Anhydro-pseudolycorine (8)
4,5-Dehydro-anhydrolycorine (9)
4,5-Dehydro-anhydro-pseudolycorine (10)
Lycorine (1)
1-O-Acetyl-pseudolycorine (11)
Pseudolycorine (12)
a
Kihara et al. (1987); b Wildman (1960); c Ghosal et al. (1986).
dimethylsiloxane)], one should expect the compounds to
elute from the column in an order that follows their boiling
(melting) points, with allowance for structural features that
confer more or less polarity to the compounds. Table 2 shows
a very good correlation in this respect, giving additional
support to the proposed structures.
In conclusion, research of the five Hymenocallis species
revealed the presence of two new natural compounds,
namely anhydro-pseudolycorine (8) from H. guianensis,
H. lobata, and H. tubiflora (Guaraunos), and 4,5-dehydro-
anhydro-pseudolycorine (10) from H. lobata. In addition,
we obtained two alkaloids that had not been previously
reported for species of Hymenocallis, 4,5-dehydro-anhy-
drolycorine (9) (Ghosal et al., 1986) from H. tubiflora (both
populations) and H. venezuelensis and 1-O-acetyl-pseudoly-
corine (11) (Llabrés et al., 1986) from H. tubiflora
(Guaraunos). Despite a careful search, none of the species
showed the presence of isocarbostyril-type alkaloids. Taking
into account the low concentrations of alkaloids observed,
these species show no promise as a source of Amaryllidaceae
alkaloids.
Acknowledgments
We wish to thank the Instituto Venezolano de Investigaciones
Científicas and the Universidad Central de Venezuela for
financial support as well as Dr. M. Reymundez for her help
in collecting and identifying the plant material. We are
grateful to Dr. Werner Wilbert, of the Department of
Anthropology of IVIC, for his kind review of the manuscript.
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