In vitro antiprotozoal activity of alkaloids from Phaedranassa dubia

(Amaryllidaceae)
Edison J. Osorio
a,b
, Strahil Berkov
b
, Reto Brun
c
, Carles Codina
b
, Francesc Viladomat
b
,
Fabio Cabezas
d
, Jaume Bastida
b,
*
a
Grupo de Investigacion en Sustancias Bioactivas, Facultad de Qumica Farmaceutica, Universidad de Antioquia, A.A. 1226, Medelln, Colombia
b
Departament de Productes Naturals, Facultat de Farma`cia, Universitat de Barcelona, Avn. Joan XXIII s/n, 08028 Barcelona, Spain
c
Parasite Chemotherapy, Swiss Tropical Institute, Socinstrasse 57, CH-4051 Basel, Switzerland
d
Departamento de Qumica, Grupo de Qumica de Compuestos Bioactivos, Universidad del Cauca, A.A. 304, Popayan, Colombia
1. Introduction
Protozoal diseases like leishmaniasis, trypanosomiasis and
malaria are among the most common chronic infections that occur
primarily in rural and poor urban areas of tropical and subtropical
regions around the world. They are considered as an important
health and socioeconomic problem wherever these diseases are
endemic. Their chemotherapy is not satisfactory due to a lack of
effectiveness and also the toxicity associated with long-term
treatments with empirically discovered drugs. The rapidly
increasing problem of drug resistance and a lack of suitable
vaccines, make the development of efcient, non-toxic, and
inexpensive new drugs an urgent task (Edwards and Biagini,
2006; Osorio et al., 2007, 2008; Singh, 2006).
Plants of the Amaryllidaceae family are known to produce
structurally unique alkaloids with a wide range of interesting
physiological effects, including antitumor, antiviral, cytotoxic,
acetylcholinesterase inhibitory, immunostimulatory, antiinam-
matory, analgesic, and DNA-binding activities and some of them
have also been used in the treatment of Alzheimers disease
(Bastida et al., 2006). Some of these alkaloids are of particular
interest because of their potential antiprotozoal activity. Thus,
lycorine, augustine and crinamine were found to be the principal
antimalarial constituents from the bulbs of Crinum amabile and
among them, augustine appeared to be the most active alkaloid
(Likhitwitayawuid et al., 1993). Haemanthamine and 6-hydro-
xyhaemanthamine exhibited antimalarial activity against both
chloroquine-sensitive and chloroquine-resistant strains of Plas-
modium falciparum (Herrera et al., 2001a; Sener et al., 2003).
Haemanthidine, pancracine, 3-O-acetylsanguinine and 1,2-O-
diacetyllycorine showed biological activity in in vitro assays
against Trypanosoma brucei rhodesiense and/or T. cruzi strains
(Herrera et al., 2001a,b; Labrana et al., 2002; Machocho et al.,
2004). These results prompted us to investigate the alkaloids of
Phaedranassa dubia J.F. Macbr. and to screen the in vitro
antiprotozoal activity. In this paper, we report the isolation and
structural characterization of eight compounds and the results of
their in vitro evaluation against T. brucei rhodesiense, T. cruzi,
Leishmania donovani and P. falciparum. Phaedranamine (1) is
reported here for the rst time as a natural product.
2. Results and discussion
The structures of the isolated compounds (Fig. 1) were assigned
by NMR, MS, UV, and CDspectroscopy. The IR spectra of compound
1 displayed a broad absorption band centred at 3200 cm
1
as well
as a band at 936 cm
1
associated with hydroxyl and methylene-
dioxy groups, respectively. Its HR-ESI-MS suggested a molecular
formula of C
17
H
19
NO
4
with a [M+H]
+
peak at m/z 302.1390 [M]
+
(calcd 301.1392). In the
1
H NMR spectrum (400 MHz, CD
3
OD), the
Phytochemistry Letters 3 (2010) 161163
A R T I C L E I N F O
Article history:
Received 5 May 2010
Received in revised form 25 June 2010
Accepted 28 June 2010
Keywords:
Phaedranassa dubia
Amaryllidaceae
Antiprotozoal activity
Phaedranamine
A B S T R A C T
The bulbs of Phaedranassa dubia (Amaryllidaceae) were found to contain the novel compound
phaedranamine, together with seven known alkaloids. The structure and stereochemistry of the
alkaloids were determined by physical and spectroscopic methods. An in vitro screening against four
different parasitic protozoa was carried out using the isolated compounds. The alkaloids ungeremine,
pseudolycorine and haemanthamine showed good activity in in vitro assays against Trypanosoma brucei
rhodesiense, T. cruzi and Plasmodium falciparum with IC
50
values in the range of 3.66 mM or lower.
2010 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
* Corresponding author. Tel.: +34 934020268; fax: +34 934029043.
E-mail address: jaumebastida@ub.edu (J. Bastida).
Contents lists available at ScienceDirect
Phytochemistry Letters
j our nal homepage: www. el sevi er . com/ l ocat e/ phyt ol
1874-3900/$ see front matter 2010 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.phytol.2010.06.004
coupling constants between H-1 and H-2 (J = 5.6 Hz), H-2 and H-3
(J = 10.0 Hz), H-3 and H-4b (J = 3.4 Hz), together with the geminal
coupling of around 19.2 Hz between H-4aand H-4b, allowed us to
assign the hydroxyl group at the C-1 position and the unsaturation
between C-2 and C-3. The nuclear Overhauser effect (NOE) contour
between H-1 and the two H-11 conrmed the suggested b-
orientation of the OH group. The multiplicity of H-4a conrms the
assignment of the double bond, with the notable difference of the
axial H-4a in haemanthamine type alkaloids (Bastida et al., 2006).
The two H-6 protons were clearly differentiated as an AB system
with geminal coupling of 16.6 Hz. The singlet aromatic proton
resonance was ascribable to H-10 due to three-bond HMBC
correlations to C6a and C-10b, in addition to a NOESY correlation to
H-1, allowing the aromatic methoxyl group to be placed at C-7. The
H-6a was assigned at high elds as a consequence of the NOE
contour correlation with H-12endo. Furthermore, H-4a showed a
NOESY correlation with H-12exo and H-6b with H-4a, all of which
conrmed the proposed assignments. The quaternary carbons C-6a
and C-10a were ascribed by means of their correlations with the
methine protons H-10 and H-1, respectively. Finally, the singlet at
d = 48.96 was assigned to C-10b taking into account their three-
bond connectivities with H-10, H-4a and H-4b. The absolute
conguration of this alkaloid was determined from the CD
spectrum, where the curve was qualitatively similar to those of
the known haemanthamine type alkaloids with the 5,10b-ethano
bridge in an a orientation (Pham et al., 1998; Wagner et al., 1996),
having a maximum of around 280 nm and a minimum of 259 nm.
Thus, the structure of 1 was determined as phaedranamine, a new
natural product. From a chemotaxonomic point of view, this is the
rst report of a 7-substituted haemanthamine type alkaloid in the
Amaryllidaceae family.
The biological activity tests against the parasitic protozoa and
for cytotoxicity were performed as described by Labrana et al.
(2002). Antiprotozoal activity of the individual P. dubia alkaloids is
summarized in Table 1. Ungeremine showed higher activity than
the other alkaloids against three of the four protozoan parasites
tested. It was active against T. brucei rhodesiense (IC
50
= 3.66 mM),
T. cruzi (IC
50
= 3.20 mM) and P. falciparum (IC
50
= 0.33 mM), but not
against L. donovani. In spite of showing a degree of cytotoxicity
against L6 cells (rat skeletal myoblasts, IC
50
= 65.28 mM), their
selectivity index (IC
50 L6
/IC
50 K1
) for P. falciparum was around 197,
which conrms the selective activity of the compound for this
protozoan. Pseudolycorine showed good activity in in vitro assays
against P. falciparum (IC
50
= 0.83 mM) and haemanthamine pre-
sented biological activity against T. brucei rhodesiense
(IC
50
= 1.62 mM) and P. falciparum (IC
50
= 2.29 mM), the latter
agreeing with previous reports (Herrera et al., 2001a; Sener et al.,
2003). For the last two alkaloids, no cytotoxic activity for L6 cells
was found, which conrms their selective antiprotozoal activity for
T. brucei rhodesiense and P. falciparum.
Structureantiprotozoal activity relationships have not been
studied in Amaryllidaceae alkaloids. Some results suggest that the
methylenedioxy group and a tertiary non-methylated nitrogen
contribute to a higher activity in these alkaloids (Sener et al., 2003).
Nevertheless, we have observed strong activity in alkaloids with
quaternary nitrogen. We have also found that the mechanism of
antiplasmodial action of Amaryllidaceae alkaloids does not depend
on interactions with heme, at least in the case of alkaloids such as
ungeremine, pseudolycorine and haemanthamine (data not
published). Additional investigation is required to specify the
necessary structural requirements for antiparasitic activity, and, in
particular, the possible mode of action.

Fig. 1. Structures of the isolated alkaloids from Phaedranassa dubia.

Table 1
In vitro antiprotozoal and cytotoxic activities of P. dubia alkaloids.
Parasite: T. b. rhodesiense T. cruzi L. donovani P. falciparum Cytotoxicity
Stage: Trypomastigote Amastigote in L6 cells Amastigote, axenic Intraerythrocytic
Strain: STIB 900 (IC
50
a
) Tulahuen C4 (IC
50
a
) MHOM-ET-67/L82 (IC
50
a
) K1 (IC
50
a
) L6 (IC
50
a
)
Standard/compound
Phaedranamine (1) 95.51 >100 >100 14.31 >300
Haemanthamine 1.62 >100 >100 2.29 >300
Pseudolycorine Ne >100 >100 0.83 >300
Ungeremine 3.66 3.20 >100 0.33 65.28
Zefbetaine 132.94 >100 >100 >15 >300
Sanguinine 82.38 >100 >100 >15 >300
Galanthamine 132.12 >100 >100 >15 >300
Epinorgalanthamine Ne >100 >100 >15 >300
Melarsoprol 0.007
Benznidazole 0.84
Pentostam 0.25
Chloroquine 0.14
Podophyllotoxin 0.02
Ne, evaluation not possible.
a
All values as: mM.
E.J. Osorio et al. / Phytochemistry Letters 3 (2010) 161163 162
3. Experimental
3.1. General experimental procedures
1
H,
13
C NMR, COSY, HMQC, and HMBC spectra were recorded on
a Mercury 400F (400 MHz/100 MHz) spectrometer in CD
3
OD or
CDCl
3
with TMS as internal standard. Chemical shifts were
reported in d units (ppm) and coupling constants (J) are expressed
in Hz. Optical rotations were carried out on a Perkin-Elmer 241
Polarimeter. UV spectra were recorded on a Hitachi U-2000
spectrophotometer. HR-ESI-MS spectra were obtained on an
LC/MSD-TOF (2006) Mass spectrometer (Agilent technologies). A
Jasco-J-810 Spectropolarimeter was utilized to run the CD spectra,
all recorded in MeOH. Silica gel SDS chromagel 60 A CC (635 mm)
was used for VLC, and silica gel 60 F254 Macherey-Nagel for
analytic and prep. TLC. Sephadex LH20 (Amersham Biocience) was
used for gel ltration. Spots on chromatograms were detected
under UV light (254 nm) and by Dragendorffs reagent.
3.2. Plant material
Bulbs of P. dubia were collected in the Sierra municipality, in the
department of Cauca (Colombia) in September of 2003. The
samples were authenticated by Dr. Bernardo Ramrez, University
of Cauca (Colombia). A specimen voucher (BR 17-169) has been
deposited in the Museum of Natural History, Section Herbarium,
University of Cauca.
3.3. Extraction and isolation
The dry vegetable material (771.3 g of bulbs) was macerated
thoroughly with EtOHat roomtemperature for 48 h (31.5 L). The
solution was evaporated under reduced pressure and the residue
dissolved in H
2
O (100 mL) and acidied with 5% H
2
SO
4
to pH 34.
After removing the neutral material with Et
2
O (3 50 mL), the
acidic solution was basied with NH
4
OH solution to pH 89 and
then extracted with EtOAc several times. The extracts were
combined to give extract A (2.03 g), which was subjected to VLC on
silica gel 60, eluting initially with n-Hexane, increasing the polarity
with EtOAc and later up to EtOAcMeOH (1:1). Three major
fractions containing alkaloids were obtained. Fr. I (0.34 g), which
was rst treated by CC on Sephadex LH20 eluting with MeOH and
then rechromatographed by prep. TLC, eluting with EtOAcMeOH
(7:3), yielded pseudolycorine (28 mg), phaedranamine (1) (12 mg)
and haemanthamine (15 mg). Prep. TLC separation of fr. II (0.22 g)
with Me
2
COMeOH(7:3), and nal purication on Sephadex LH20
using MeOH as an eluent, afforded sanginine (5 mg), epinorga-
lanthamine (4 mg), and galanthamine (5 mg). Fr. III (0.14 g) was
rechromatographed by CC on silica gel using Me
2
CO as a solvent
and then, by prep. TLC, eluting with BuOHAcOHH
2
O in NH
3
atmosphere, gave betaine-type alkaloids, zefbetaine (8 mg) and
ungeremine (6 mg).
3.4. Phedranamine (1)
White needles (EtOAc); mp 176178 8C; [a]
D
20
+1728 (c 0.82,
MeOH); CD [Q]
l
20
: [u]259 12,400, [u]280 +9399 (MeOH); IR
n
max
cm
1
: 3200, 2886, 1735, 1619, 1476, 1079, 1045, 977, 936,
753;
1
HNMR spectral data (400 MHz, CD
3
OD): d 6.67 (1H, s, H-10),
5.99 (1H, dd, J = 10.0, 5.6 Hz, H-2), 5.90 (2H, 2d, J = 1.2 Hz, OCH
2
O),
5.78 (1H, ddd, J = 10.0, 4.4, 3.4 Hz, H-3), 4.65 (1H, d, J = 5.6 Hz, H-1),
4.33 (1H, d, J = 16.6 Hz, H-6b), 4.05 (1H, d, J = 16.6 Hz, H-6a),
4.00 (3H, s, OCH
3
), 3.75 (1H, t, J = 7.6 Hz, H-4a), 3.64 (1H, ddd,
J = 12.5, 9.5, 4.5 Hz, H-12exo), 3.05 (1H, ddd, J = 12.5, 8.4, 8.0 Hz,
H-12endo), 2.64 (1H, ddd, J = 19.2, 7.4, 3.4 Hz, H-4b), 2.10 (1H, m,
H-11exo), 2.04 (1H, ddd, J = 19.2, 7.8, 4.4 Hz, H-4a), 1.97 (1H, m,
H-11endo);
13
C NMR (100 MHz, CD
3
OD) d 148.47 (C, C-9), 140.22
(C, C-7), 137.92 (C, C-10a), 133.81 (C, C-8), 126.32 (CH, C-2), 126.32
(CH, C-3), 116.24 (C, C-6a), 101.16 (CH
2
, OCH
2
O), 100.01 (CH, C-10),
63.89 (CH, C-1), 59.25 (CH, C-4a), 58.26 (CH
3
, OCH
3
), 56.38 (CH
2
,
C-6), 51.12 (CH
2
, C-12), 48.96 (C, C-10b), 38.93 (CH
2
, C-11), 28.00
(CH
2
, C-4); EIMS (70 eV): m/z (rel. int.): 301 [M]
+
(100), 300 [MH]
+
(19), 284 (16), 282 (18), 272 (13), 256 (20), 231 (20), 218 (16),
204 (20), 77 (21); HR-ESI-MS m/z 302.1390 [M+H]
+
(calcd for
C
17
H
20
NO
4
302.1392).
Acknowledgements
This work was partially nanced by the Generalitat de
Catalunya (2005SGR-00020). EJO is grateful to the Agreement
Foundation Carolina, University of Antioquia for provision of a
doctoral fellowship. This investigation received nancial support
from the UNICEF/World Bank/WHO Special Program for Research
and Training in Tropical Diseases (RB).
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