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2010 Antiprotozoal Markhamia tomentosa

publicité
Annals of Tropical Medicine & Parasitology, Vol. 104, No. 5, 391–398 (2010)
Antiprotozoal activities of some constituents of
Markhamia tomentosa (Bignoniaceae)
F. TANTANGMO*, B. N. LENTA{, F. F. BOYOM{, S. NGOUELA*, M. KAISER1,
E. TSAMO*, B. WENIGER", P. J. ROSENTHAL** and
C. VONTHRON-SÉNÉCHEAU",{{
*
Department of Organic Chemistry, Faculty of Science, TWAS Research Unit of the University of
Yaoundé I, P.O. Box 812, Yaoundé, Cameroon
{
Department of Chemistry, Higher Teachers’ Training College, University of Yaoundé I, P.O. Box
47, Yaoundé, Cameroon
{
Department of Biochemistry, Faculty of Science, University of Yaoundé I, P.O. Box 812,
Yaoundé, Cameroon
1
Swiss Tropical and Public Health Institution, Socinstrasse 57, CH-4002 Basel, Switzerland
"
Laboratoire de Pharmacognosie et Molécules Naturelles Bioactives, UMR 7200, Faculté de
Pharmacie, Université de Strasbourg, B.P. 60024, 67401, Illkirch Cedex, France
**
Division of Infectious Diseases, Department of Medicine, University of California, San
Francisco, 1001 Portero Avenue, San Francisco, CA 94943, U.S.A
{{
Laboratoire de Biologie et de Biotechnologies Marines, UMR M IFREMER 100, Université de
Caen Basse-Normandie, Esplanade de la Paix, 14032 Caen Cedex, France
Received 10 May 2010, Accepted 17 May 2010
Phytochemical investigation of an ethyl-acetate extract of the stem bark of Markhamia tomentosa (Bignoniaceae),
which had good antimalarial activity in vitro, resulted in the isolation of eight known compounds: 2acetylnaphtho[2,3-b]furan-4,9-dione (1), 2-acetyl-6-methoxynaphtho[2,3-b]furan-4,9-dione (2), oleanolic acid
(3), pomolic acid (4), 3-acetylpomolic acid (5), tormentic acid (6), b-sitosterol (7) and b-sitosterol-3-O-b-Dglucopyranoside (8). The structures of these compounds were established by spectroscopic methods. Each of
compounds 1, 2, 4 and 5 was evaluated in vitro for its antiprotozoal activities against the ring stages of two
chloroquine-resistant strains of Plasmodium falciparum (K1 and W2), the amastigotes of Leishmania donovani, and
the bloodstream trypomastigotes of Trypanosoma brucei rhodesiense (the species responsible for human malaria,
visceral leishmaniasis and African trypanosomiasis, respectively). Although compounds 1 and 2 exhibited potent
antiprotozoal activities, they also showed high toxicity against a mammalian (L-6) cell line.
Plants of the genus Markhamia are widely
distributed in Africa, where they are used
for the treatment of several human diseases
(Kerharo, 1978; Letouzey, 1982; Adjanouhoun
et al., 1996). In Tanzania, for example,
aqueous extracts of the root bark of M. lutea
are used to treat anaemia and diarrhoea
(Kerharo, 1978; Adjanouhoun et al., 1996).
Reprint requests to: C. Vonthron-Sénécheau.
E-mail: [email protected]
# W. S. Maney & Son Ltd 2010
DOI: 10.1179/136485910X12743554760180
In Cameroon, M. lutea and M. tomentosa
are both used to cure various microbial
and parasitic diseases (Adjanouhoun et al.,
1996). In previous phytochemical studies of
Markhamia spp, bio-active lignans, phenyl
glycosides, anthraquinones, alkaloids, phenyl
propanoids and terpenoids have been isolated
(Adesanya and Nia, 1997; Kernan et al., 1998;
Khan and Mlungwana, 1999; Kanchanapoom
et al., 2002; Lacroix et al., 2009). As part of
an ongoing investigation of antiprotozoal
agents in Cameroonian medicinal plants, an
392
TANTANGMO ET AL.
ethyl-acetate extract of M. tomentosa was
prepared and found to have good antimalarial activity in vitro against two strains (K1
and W2) of Plasmodium falciparum (see
below). Chromatographic fractionation of
this extract led to the isolation of eight
known compounds: 2-acetylnaphtho[2,3-b]
furan-4,9-dione (1), 2-acetyl-6-methoxynaphtho[2,3-b]furan-4,9-dione(2), oleanolic
acid (3), pomolic acid (4), 3-acetylpomolic
acid (5), tormentic acid (6), b-sitosterol (7)
and b-sitosterol-3-O-b-D-glucopyranoside (8).
The preparation of three extracts of M.
tomentosa stem bark, the isolation of compounds 1–8 from the ethyl-acetate extract,
and evaluation of the in-vitro activities of the
extracts and some of the isolated compounds, against strains of Plasmodium,
Leishmania and Trypanosoma and a mammalian cell line, are described below.
MATERIALS AND METHODS
General
Melting points were determined on an M-540
melting-point unit (Büchi, Flawil, Switzerland).
Optical rotations were measured, in chloroform solution, on a DIP-3600 digital polarimeter (JASCO, Tokyo). Infra-red (IR) spectra
were determined on a Fourier transform
IR spectrometer (Jasco) while ultra-violet
(UV) spectra were determined on a Unicam
spectrophotometer (Spectronic Analytical
Instruments, Leeds, U.K.). 1H and 13C
nuclear-magnetic-resonance (NMR) spectra
were investigated on a Bruker spectrometer
(Bruker Optik, Ettlingen, Germany) equipped
with 5-mm 1H and 13C probes operating at
500 and 125 MHz, respectively, with tetramethylsilane as the internal standard. Silica
gels of 230- to 400-mesh and 70- to 230-mesh
(Merck, Darmstadt, Germany) were used for
flash and column chromatography, respectively, while aluminium sheets precoated
with silica gel 60 F254 (Merck) were used
for thin-layer chromatography (TLC), with
various mixtures of petroleum ether, nhexane, ethyl acetate, and acetone as eluents.
Spots were visualized with UV light (at 254
and 365 nm) or using methanol–H2SO4
reagent.
Plant Material
The stem bark of M. tomentosa was collected
in June 2007 at Mont Eloundem (Yaoundé)
in the Central province of Cameroon. The
plants were identified by N. Victor, a
botanist at the National Herbarium of
Cameroon, where a voucher specimen
(6475/SRF/Cam) was deposited.
Extraction and Isolation
The dried stem bark (4 kg) of M. tomentosa
was extracted successively with hexane,
ethyl acetate and methanol, by maceration.
In each extraction, two 10-litre volumes of
solvent were used over a period of 48 h.
After concentration under vacuum at room
temperature, a green hexane (9 g), a brown
ethyl-acetate (50 g) and a brown methanolic
extract (100 g) were produced. When each
of these extracts was screened in vitro for its
activities against the W2 and K1 strains of
Plasmodium falciparum, the ethyl-acetate
extract was found to have the highest
antimalarial activity against both strains
(see below). This extract was therefore
fractionated by column chromatography on
silica gel (230- to 400-mesh), with nhexane–ethyl-acetate mixtures of increasing
polarity being used as the eluents. Ninety
fractions, each of 400 ml, were collected
and combined, on the basis of the results of
TLC, to yield four main fractions that were
labelled F1 (4.0 g), F2 (7.1 g), F3 (9.3 g)
and F4 (11.7 g).
Fraction F1 (4.0 g) was essentially an oil
that was not further investigated. Fraction
F2 (7.1 g) was subjected to column chromatography over silica gel (70- to 230mesh), eluting with n-hexane–ethyl-acetate
gradient mixtures. This resulted in the
collection of 76 sub-fractions, each of
150 ml, which were combined on the basis
of the results of TLC analysis. Further
purification of sub-fractions 40–45 afforded
ANTIPROTOZOAL ACTIVITIES OF Markhamia
b-sitosterol 7 (300 mg) and 2-acetylnaphtho[2,3-b]furan-4,9-dione 1 (30 mg).
Sub-fractions 53–55 yielded 2-acetyl-6methoxynaphtho[2,3-b] furan-4,9-dione 2
(35 mg). Successive chromatography of fractions 68–70 afforded oleanolic acid 3
(625 mg). Fraction F3 (9.3 g) was also subjected to column chromatography over silica
gel (70- to 230-mesh), eluting with n-hexane–
ethyl-acetate mixtures (80 : 20–75 : 25) to
yield pomolic acid 4 (200 mg) and a
powder of a mixture of compounds
(400 mg). The powder was rechromatographed, using silica gel (70- to 230-mesh)
and eluting with dichloromethane–methanol mixtures (99 : 1–98.5 : 1.5), to yield 3acetylpomolic acid 5 (300 mg), tormentic
acid 6 (30 mg) and b-sitosterol-3-O-b-Dglucopyranoside 8 (600 mg). Fraction F4
(11.7 g) was another complex mixture
that was not studied further.
Assays of Biological Activity
ANTIMALARIAL ACTIVITY (W2 STRAIN)
Antimalarial activity was first determined,
in vitro, using the W2 strain of P. falciparum,
which is resistant to chloroquine and
some other antimalarial drugs (Singh and
Rosenthal, 2001). The parasites were cultured in sealed flasks at 37uC, in an atmosphere containing 3% (v/v) O2, 5% (v/v)
CO2 and 91% (v/v) N2, in RPMI 1640
medium with 25 mM HEPES (pH 7.4),
10% (v/v) heat-inactivated human serum,
and sufficient human erythrocytes to achieve
a 2% haematocrit. Parasites were synchronized at the ring stage, by serial treatment
with 5% (v/v) sorbitol (Sigma; Lambros
and Vanderberg, 1979) and studied at 1%
parasitaemia.
Each of compounds 1, 2, 4 and 5 was
prepared as a 10-mM stock solution in dimethyl
sulphoxide (DMSO) and diluted as needed
for the individual experiments, with each
test dilution tested in triplicate. The stock
solutions were diluted with HEPES- and
serum-supplemented RPMI 1640 medium
to give (0.2% (v/v) DMSO. Each test
393
dilution was gently mixed with an equal
volume of parasite culture (showing 1%
parasitaemia and at a 4% haematocrit).
Negative controls contained the same concentrations of DMSO but no test compound whereas the cultures used as positive
controls contained 1 mM chloroquine phosphate (Sigma) and no test compound. Once
set up, the test cultures were incubated at
37uC for 48 h (representing one cycle of
erythrocytic invasion and intra-erythrocytic
multiplication) before being fixed by replacing the medium with an equal volume of
1% (w/v) formaldehyde in 0.1 M phosphatebuffered saline at pH 7.2 (PBS). Aliquots
(50 ml) of each fixed culture were then added
to 5-ml round-bottomed polystyrene tubes,
each of which contained 0.5 ml PBS holding
0.1% (v/v) Triton X-100 and 1.0 nM YOYO
nuclear dye (Molecular Probes, Eugene,
OR). Parasitaemias in the test and control
cultures were then compared using a flow
cytometer (FACSortTM; BD, Franklin Lakes,
NJ) to count the nucleated (i.e. parasitised)
erythrocytes in each sample. The counts were
recorded using the CellQuestTM software
package (BD), with the test-culture counts
normalized to percentages of the corresponding counts for the positive-control cultures.
(K1 STRAIN)
The antimalarial activity of each crude
extract and each of compounds 1, 2, 4 and
5 was also assessed quantitatively, in vitro,
using the microculture radio-isotope technique described by Desjardins et al. (1979),
as modified by Ridley et al. (1996). The
assay uses the uptake of [3H]hypoxanthine
by parasites as an indicator of viability.
Continuous in-vitro cultures of the asexual
erythrocytic stages of the pyrimethamineand chloroquine-resistant K1 strain of P.
falciparum (Thaithong and Beale, 1981)
were maintained following the methods of
Trager and Jensen (1976). Each extract or
compound was tested after two-fold serial
dilution, at seven concentrations between
20 and 0.31 mg/ml. After incubation of the
ANTIMALARIAL ACTIVITY
394
TANTANGMO ET AL.
parasites with the extract/compound for
48 h at 37uC, [3H]hypoxanthine (Amersham
International, Little Chalfont, U.K.) was
added to each well and the incubation was
continued for another 24 h at the same
temperature. Chloroquine (Sigma) was again
used as a positive reference.
LEISHMANICIDAL ACTIVITY
For the in-vitro assays of leishmanicidal
activity, 50 ml of culture medium — a 1 : 1
mixture of SM medium (Cunningham, 1977)
and
SDM-79
medium
(Brun
and
Schönenberger, 1979) at pH 5.4, supplemented with 10% (v/v) heat-inactivated foetal calf
serum (FCS) — was added to each well of a
96-well microtitre plate (Costar, Cambridge,
MA). Serial dilutions of a crude extract,
compounds 1, 2, 4 or 5 or the reference drug
(miltefosine; Zentaris, Frankfurt, Germany)
were prepared in duplicate, in the wells, to
give 50 ml/well and concentrations between
30 and 0.041 mg/ml. Then 105 axenicallygrown amastigotes (Bates, 1993) of
Leishmania donovani (MHOM/ET/67/L82)
in 50 ml medium were added to each well,
before the plate was incubated at 37uC,
under a 5%-CO2 atmosphere, for 72 h. A
12.5% (w/v) aqueous solution of resazurin
was then added, at 10 ml/well, and incubation continued for a further 2–4 h. The plate
was then read in a microplate fluorometer
(Spectramax Gemini XS; Molecular Devices,
Sunnyvale, CA), using an excitation wavelength of 536 nm and an emission wavelength
of 588 nm (Raz et al., 1997). Fluorescence
development was measured and expressed as
a percentage of the corresponding positivecontrol (miltefosine) value.
ANTITRYPANOSOMAL ACTIVITY
The procedures described by Freiburghaus
et al. (1996) were used to test the in-vitro
activity of each crude extract and isolated
compounds 1, 2, 4 and 5. Working stock
solutions (of 180 mg/ml) were prepared in
the rabbit-serum-containing culture medium described by Baltz et al. (1985) and
dispensed, at 100-ml/well, into the first row
of wells of a 96-well microtitre plate
(Costar). Complete culture medium was
then added to the other wells, so that threefold serial dilutions of each extract/compound could be prepared. After the addition
to each well of 26103 of the bloodstream
forms of Trypanosoma brucei rhodesiense,
from axenic culture (Baltz et al., 1985), the
concentration of the extract/compound in
each 100-ml culture ranged from 90 to
0.13 mg/ml. After incubation for 72 h in
a humidified atmosphere at 37uC, with
5% (v/v) CO2, parasite development was
assessed with resazurin — as for L. donovani
but incubating with the resazurin for 24 h
and using melarsoprol (ArsobalH; Rhône
Poulenc Rorer, Paris), not miltefosine, as
the reference drug.
CYTOTOXICITY
The cytotoxicity of each crude extract and
compounds 1, 2, 4 and 5 was assessed using
the L-6 cell line (of rat skeletal-muscle
myoblasts) and the method of Pagé et al.
(1993) as modified by Ahmed et al. (1994).
The rat cells were seeded in 96-well microtitre plates (Costar) to give 103 cells in
50 ml complete medium [MEM supplemented with 10% (v/v) heat-inactivated FCS]
in each well. A three-fold serial dilution of
an extract or isolated compound, prepared
in the complete culture medium, was
then added, at 50 ml/well, to give final
concentrations of 90 to 0.13 mg extract or
compound/ml. Plates were then incubated
at 37uC for 72 h in a humidified incubator,
with 5% (v/v) CO2. Resazurin was again
added as a viability indicator (Ahmed et al.,
1994), incubating with the resazurin for 2 h
before each plate was ‘read’ on the fluorescence scanner. Podophyllotoxin (Polysciences
Inc., Warrington, PA) was used as the
reference drug.
DATA ANALYSIS
Median inhibitory concentrations (IC50)
were calculated, for each extract and iso-
ANTIPROTOZOAL ACTIVITIES OF Markhamia
lated compound, using the Prism 3.0 software package (GraphPad Software, La Jolla,
CA). For this, the assay data were fitted, by
non-linear regression, to the variable-slope
sigmoidal dose–response formula y5100/
(1z10(logIC502x)H), where H is the Hill
coefficient or slope factor (Singh and
Rosenthal, 2001).
Selectivity indexes (SI) were calculated,
from the results of the assays of antimalarial
and cytotoxic activities, as (IC50 for the L-6
cells)/(IC50 for a strain of P. falciparum).
The values given in the Table are mean
results (and S.D.) for either two independent
assays (extracts) or three (pure compounds),
with each assay run in duplicate.
395
ethyl-acetate extract had the best antimalarial
activity, with similarly low IC50 and similarly
high SI (.50) recorded for the two strains of
P. falciparum that were tested (see Table).
Fractionation of the ethyl-acetate extract
by flash and successive column chromatography yielded eight compounds. Their
structures were established, by 1H- and,
13
C-NMR spectroscopy (including one- and
two-dimensional techniques) and mass spectrometry, as 2-acetylnaphtho[2,3-b]furan4,9-dione (1; Rao and Kingston, 1982),
2-acetyl-6-methoxynaphtho[2,3-b]furan-4,9dione (2; Zani et al., 1991), oleanolic acid (3;
Mahato and Kundu, 1994), pomolic acid (4;
Mahato and Kundu, 1994), 3-acetylpomolic
acid (5; Mahato and Kundu, 1994), tormentic acid (6; Delgado et al., 1989; Mahato and
Kundu, 1994; Li et al., 2009), b-sitosterol
(7; Kovganko et al., 1999) and b-sitosterol-3O-b-D-glucopyranose (8; Moghaddam et al.,
2007) (Fig. 1).
Compounds 1, 2, 4 and 5 were then
evaluated for their activities against P.
falciparum, L. donovani and T. b. rhodesiense,
to check whether or not each could contribute to the revealed antiprotozoal activity
of the crude ethyl-acetate extract [the anti-
RESULTS AND DISCUSSION
In the present study, three extracts of the
stem bark of M. tomentosa were evaluated
in vitro for their activities against (the
chloroquine-resistant K1 and W2 strains
of) P. falciparum, L. donovani, and T. brucei
rhodesiense — parasites that can cause
human malaria, visceral leishmaniasis and
African trypanosomiasis, respectively. The
TABLE. In-vitro antiprotozoal and cytotoxic activities of the three crude extracts and isolated compounds
Mean (S.D.) median inhibitory concentration (mg/ml):
Plasmodium
falciparum
(K1)
Sample
P.
falciparum
(W2)
Leishmania
donovani
Trypanosoma
brucei
rhodesiense
Selectivity index
L-6
cell
line
P.
P.
falciparum falciparum
(K1)
(W2)
CRUDE EXTRACT
Hexane
Ethyl-acetate
Methanolic
.5
4.93 (0.39) .5
2.81 (0.06)
1.46 (0.12) .5
.5
ND
.5
.5
1.5
.5
83
.90
.90
18
.50
–
17
.50
–
ISOLATED COMPOUND
1
2
4
5
0.11
0.44
3.47
2.10
(0.02)
0.16 (0.02)
(0.04)
0.93 (0.04)
(0.90) .5
(0.7) .5
0.10 (0.02) 0.016 (0.004)
0.77 (0.04) 0.05 (0.01)
0.31 (0.03) .5
3.40 (0.7) .5
0.1
0.1
4.2
16.3
REFERENCE DRUG
Chloroquine
Miltefosine
Melarsoprol
Podophyllotoxin
ND, Not determined.
0.094 (0.042)
0.039 (0.002)
0.12 (0.05)
0.003 (0.002)
0.007
0.9
0.2
1.2
7.8
0.6
0.1
13.7
–
396
TANTANGMO ET AL.
FIG. 1. Structures of the isolated compounds 1–6.
protozoal and cytotoxic activities of compounds 3 and 6 had already been investigated
by Ngangoue (2008)]. Compounds 1 and 2
exhibited strong activities not only against the
two strains of P. falciparum but also against
the leishmanial amastigotes and trypanosomes tested (0.01 mg/ml,IC50,0.9 mg/ml;
see Table). Unfortunately, both compounds
also showed strong cytotoxic activity when
tested against the L-6 cell line (IC5050.1 mg/
ml). Compounds 4 and 5 showed weak
antiprotozoal activities and relatively low SI
(see Table).
Both active compounds, 1 and 2, possess an
anthraquinone skeleton and are derived from
lapachol (Fig. 2), a compound that is known
to have antiparasitic activities (Hussain et al.,
2007). Compared with 1, 2 bears an additional
methoxy group at C-6, which may explain the
differences in the activities of the two compounds. Several naphthoquinones and anthraquinones from plant sources, and synthetic
derivatives, have been reported to possess
good antimalarial and other antiprotozoal
activities in vitro (Ribeiro-Rodrigues et al.,
1995; Sittie et al., 1999; Onegi et al., 2002;
Mbwambo et al., 2004; Noungoue et al.,
2009). Furthermore, the naphthoquinone
sterekunthal A (Fig. 2), isolated from the
root bark of Stereospermum kunthiamum
(which, like M. tomentosa, is a member of
the Bignoniaceae), was found to exhibit good
activity in vitro against P. falciparum, with an
IC50 of 1.3 mg/ml (Onegi et al., 2002). Thus,
the good antimalarial activity of the ethylacetate extract of M. tomentosa could be
ANTIPROTOZOAL ACTIVITIES OF Markhamia
397
FIG. 2. Structures of lapachol and sterokunthal A.
explained, at least partially, by the occurrence of the two naphthoquinones 1 and 2.
Unfortunately, the antimalarial activity of
these two compounds may reflect general
toxicity, as shown by the low IC50 recorded
against the mammalian cell line. The crude
ethyl-acetate extract, which gave a much
better SI for its antimalarial activity than any
of the isolated compounds, must contain
other compounds that moderate the antimammalian cytotoxicity of these two
naphthoquinones. The extract may, of course,
also contain antimalarial compounds that
were not isolated in the present study.
The present results highlight the antimalarial
potency of naphthoquinones derivatives and
partially validate the use of M. tomentosa, in
Cameroonian folk medicine, to cure malaria.
The authors thank
the European Commission for a Marie Curie
Post-doctoral Fellowship (awarded to
B.N.L.) and the Academy of Sciences for
the Developing World (TWAS) for awarding
a research grant (07-141 LDC/CHE/AF/
AC-UNESCO FR: 3240171776) to the
University of Yaoundé I’s TWAS Research
Unit. The assistance of M. Nana (of the
National Herbarium of Cameroon), in the
collection and the identification of plant
material, is gratefully acknowledged.
ACKNOWLEDGEMENTS.
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