Journal of Ethnopharmacology 137 (2011) 998–1002

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm

In vitro antitrypanosomal and antileishmanial activity of plants used in Benin in

traditional medicine and bio-guided fractionation of the most active extract
Joanne Bero a,∗ , Véronique Hannaert b , Gabrielle Chataigné a , Marie-France Hérent a ,
Joëlle Quetin-Leclercq a
a
Université catholique de Louvain, Louvain Drug Research Institute, Pharmacognosy Research Group, Avenue E. Mounier, 72, B-1200 Brussels, Belgium
b
Université catholique de Louvain, de Duve Institute, Research Unit for Tropical Diseases Trop74-39, Avenue Hippocrate 74, B-1200 Brussels, Belgium

a r t i c l e i n f o a b s t r a c t

Article history: Ethnopharmacological relevance: The aim of the study was to evaluate the in vitro antitrypanosomal and
Received 22 April 2011 antileishmanial activity of crude extracts of 10 plant species traditionally used in Benin to treat parasitic
Received in revised form 16 June 2011 infections.
Accepted 6 July 2011
Materials and methods: For each species, dichloromethane, methanol and aqueous extracts were tested.
Available online 18 July 2011
Their antitrypanosomal and antileishmanial activities were evaluated in vitro on Trypanosoma brucei
brucei (strain 427) (Tbb) and on promastigotes of Leishmania mexicana mexicana (MHOM/BZ/84/BEL46)
Keywords:
(Lmm).
Antitrypanosomal activity
Antileishmanial activity
Results: The best growth inhibition was observed with the dichloromethane extracts of aerial parts of
Benin Acanthospermum hispidum DC. (Asteraceae) (IC50 = 14.5 ␮g/ml on Tbb and 11.1 ␮g/ml on Lmm), twigs of
Acanthospermum hispidum Keetia leucantha (K. Krause) Bridson (syn. Plectronia leucantha Krause) (IC50 = 5.8 ␮g/ml on Tbb), aerial
Keetia leucantha parts of Byrsocarpus coccineus Schumach. & Thonn (syn. Rourea coccinea (Schumach. & Thonn.) Hook.f.)
Byrsocarpus coccineus (IC50 = 14.7 ␮g/ml on Tbb) and aerial parts of Carpolobia lutea G.Don. (IC50 = 18.3 ␮g/ml on Tbb). All these
extracts had a low cytotoxicity. It is not the case for the methanolic and water extracts of roots of
Anchomanes difformis (Blume) Engl. (IC50 = 14.7 and 13.8 ␮g/ml on Tbb) which were toxic at the same
concentration range on WI38, human cells. A bio-guided fractionation of the most active extract of Keetia
leucantha allowed to identify oleanolic acid and ursolic acid as responsible for the observed activities.
Conclusion: Our study gives some justification for antiparasitic activity of some investigated plants.
© 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction fer from cutaneous leishmaniasis today (World Health Organisation

Trypanosoma brucei is the parasite responsible for human
African trypanosomiasis or sleeping sickness, an illness affect-
ing 300,000–500,000 people, while up to 60 million people in
36 countries are at risk of contracting the disease (World Health
Organisation (WHO), 2002). This parasite is transmitted by the bite
of infected tsetse flies of the genus Glossina.
Leishmaniasis is also a disease caused by protist parasites of
the genus Leishmania and is transmitted by the bite of a female
phlebotomine sand fly. Leishmaniasis can occur as three different
forms: cutaneous, mucocutaneous and visceral leishmaniasis. In
the world, one person becomes infected by cutaneous leishmani-
asis every 20 s. The disease is endemic in 82 countries of tropical
and subtropical areas around the world, and 10 million people suf-
∗ Corresponding author. Tel.: +32 2 764 7292; fax: +32 2 764 7253.
E-mail address: joanne.bero@uclouvain.be (J. Bero).
(WHO), 2007).
These two parasitic diseases are the cause of considerable
mortality and morbidity throughout the world (World Health
Organisation (WHO), 2007).
Resistance, toxicity and variable efficacy between strains or
species of most of drugs used to treat these diseases as well as,
for some of them, the need for long course parenteral administra-
tion led to the search for new antitrypanosomal and antileishmanial
compounds, particularly in plants used in traditional medicine, as
a source of new leads with new mechanism of action (Hoet et al.,
2004).
The present study investigates the in vitro antitrypanosomal and
antileishmanial activity of crude extracts from ten plants used as
antiparasitic in traditional medicine in Benin. These plants were
already evaluated for their antimalarial activity (Bero et al., 2009)
but as many plants or compounds active on a parasite often also
show an activity on another one we evaluated them for their
antitrypanosomal and antileishmanial activities. Each plant pow-
der was macerated with dichloromethane, methanol and water.
Extracts were evaluated for their antitrypanosomal and antileish-

0378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2011.07.022
 J. Bero et al. / Journal of Ethnopharmacology 137 (2011) 998–1002
manial activities while cytotoxicity was previously determined on
a mammalian cell line (Bero et al., 2009).
The best antitrypanosomal extract was fractionated to obtain
two known triterpenoids (oleanolic and ursolic acids) possessing
antiparasitic activity (Torres-Santos et al., 2004; Hoet et al., 2007).
The presence of these compounds in the dichloromethane extracts
of Keetia leucantha could justify a part of the activity observed.
2. Materials and methods
2.1. Plant material
Plant materials (leaves, twigs, aerial parts and roots) were col-
lected from the South of Benin, especially from Abomey-Calavi
(South-West) to the border area with Nigeria (South-East) between
July 2006 and September 2006. Voucher specimens were identified
and deposited at the Herbier National of Abomey-Calavi Univer-
sity in Benin and at the Herbarium of National Botanic Garden of
Belgium, at Meise. The voucher specimen, the family and traditional
uses are given in Bero and Ganfon (Bero et al., 2009).
2.2. Preparation of crude plant extracts
Plant extracts of leaves, twigs, roots or aerial parts were pre-
pared by macerating 10–30 g (see Table 1) of dried and powdered
plant material at room temperature for about 24 h under shak-
ing. The material was extracted sequentially with dichloromethane
and methanol. A total aqueous extract was also prepared from
another sample. The quantity of solvent used for each extraction
was 10 times the quantity of plant material. Thus, three extracts
were obtained for each plant part. The filtrates were evaporated
to dryness under reduced pressure with a rotary evaporator at a
temperature of 30 ◦ C while the water filtrates were freeze-dried to
powders. Yields for each extraction are indicated in Table 1.
2.3. Parasites, cells and media
Trypanosoma brucei brucei (strain427) bloodstream forms were
cultured in vitro in HMI9 medium containing 10% heat-inactivated
foetal bovine serum (Hirumi and Hirumi, 1994).
Leishmania mexicana mexicana promastigotes (MHOM/BZ/
84/BEL46) were cultivated in vitro in a semi-defined medium (SDM-
79) (Brun and Lun, 1994) supplemented with 15% heat-inactivated
foetal bovine serum.
The human normal fibroblast cell line, WI-38, was culti-
vated in vitro in DMEM medium containing 4 mM l-glutamine,
1 mM sodium pyruvate supplemented with 10% heat-inactivated
foetal bovine serum and penicillin–streptomycin (100 UI/ml to
100 ␮g/ml).
All cells were incubated in a humidified atmosphere with 5% CO2
at 37 ◦ C except the Leishmania promastigotes which were incubated
at 28 ◦ C.
2.4. In vitro test for antitrypanosomal and antileishmanial
activity
The in vitro test was performed as described by Hoet et al.
(2004). Amphotericin B (a commercial antileishmaniasis drug) and
Suramine (a commercial antitrypanosomal drug) were used as
positive controls in all experiments with an initial concentration
of 1 ␮g/ml. First stock solutions of crude extracts and compounds
were prepared in DMSO or in ethanol/water (2:1) for water extracts
at 20 mg/ml. The solutions were further diluted in medium to
give 0.2 mg/ml stock solutions. Extracts and compounds were
tested in eight serial three-fold dilutions (final concentration
999
range: 100–0.05 ␮g/ml) in 96-well microtiter plates. All tests were
performed in duplicate.
2.5. Cytotoxicity assay
The cytotoxicity of the extracts on WI-38 cells was evaluated as
previously described (Bero et al., 2009).
2.6. Bio-guided fractionation of twigs dichloromethane extract of
Keetia leucantha
A total of 2.620 kg of powder of twigs of Keetia leucantha
was extracted by maceration under shaking at room tempera-
ture with 2620 ml of dichloromethane for 24 h followed by 48 h
with 1310 ml of new fresh dicholoromethane, yielding 12.40 g of
extract. 12.11 g of the dichloromethane extract was solubilised in
the two phases of a mixture of hexane–methanol–water (10:8:2)
and a liquid–liquid extraction was realised. The lower phase was
evaporated and subjected to elution through sephadex LH-20 col-
umn (4.5 cm x 56 cm) with dichloromethane–methanol–petroleum
ether 40–60◦ (1:1:1) and gave 8 fractions which were pooled
following their TLC profiles. F6 (441.4 mg) was submitted to
a silica (Si 70–200 ␮m) gel column (2.4 cm × 27.2 cm) with
dichloromethane-petroleum ether 40–60◦ -diethyl ether (1:1:1)
to give 14 fractions. F6-3 and F6-4 (22.7 mg) were submit-
ted to another sephadex LH-20 column (2.1 cm × 30.0 cm) with
dichloromethane–methanol–petroleum ether 40–60◦ (1:1:1) and
afforded a mixture of ursolic and oleanolic acids.
The identification of these compounds was realised with a LC
MS/MS system consisting in a Thermo Accela pump, autosampler
and photodiode array detector. The column used was a Phe-
nomenex Licrospher C18 column, 4 mm × 250 mm packed with
5 ␮m particles. The flow rate was 800 ␮l/min using a binary solvent
system: solvent A, HPLC grade water–acetonitrile (9:1) 0.1% formic
acid and solvent B, acetonitrile with 0.1% formic acid (0–5 min:
100% A, 70–85 min: 0% A, 86–96 min: 100% A). High-resolution
MS was measured on a Thermo Scientific LTQ orbitrap XL mass
spectrometer with APCI source in positive mode. Data acquisition
and processing were performed with Xcalibur software. Injected
standard compounds were ursolic acid (Sigma) and oleanolic acid
(Sigma). The same analytical procedure was used to identify these
two compounds in the crude leaves dichloromethane extract of the
plant.
3. Results
In vitro antitrypanosomal, antileishmanial and cytotoxic activi-
ties are summarized in Table 1.
Concerning the antitrypanosomal activity, we observed that 6
extracts could be considered as having good activity with IC50
values ≤20 ␮g/ml, 14 had a moderate activity with IC50 values
between 21 and 60 ␮g/ml, 4 showed a low activity with IC50 values
between 60 and 100 ␮g/ml and 12 may be considered as inactive
with IC50 > 100 ␮g/ml. The dichloromethane extracts were gener-
ally more active than the methanol and water extracts. The best
growth inhibitions of Trypanosoma brucei brucei were observed
with the dichloromethane extracts of Acanthospermum hispidum,
Byrsocarpus coccineus, Carpolobia lutea, Keetia leucantha twigs and
the methanol and water extracts of Anchomanes difformis. All these
extracts had a low or no cytotoxicity except the two last ones.
Concerning the antileishmanial activity, we observed that one
extract could be considered as having promising activity with an
IC50 values ≤ 20 ␮g/ml, 3 had a moderate activity with IC50 values
between 21 and 50 ␮g/ml, 2 showed a low activity with an IC50
value between 50 and 100 ␮g/ml and 30 may be considered as inac-
tive with IC50 > 100 ␮g/ml. The best growth inhibition of Leishmania
 1000
Table 1
In vitro antitrypanosomal, antileishmanial and antiplasmodialb activity, cytotoxicityb and selectivity index of the selected plant extracts.

Plant species Part studieda Extract Amount of Cytotoxicity Antitrypanosomal Selectivity Antileishmanial Selectivity Antiplasmodial Selectivity
plant WI38b (IC50 , activity Tbb indexc activity Lmm indexc activity Pf indexb,c
extracted (g) ␮g/ml) (IC50 , ␮g/ml) WI38/Tbb (IC50 , ␮g/ml) WI38/Lmm (IC50 , ␮g/ml) WI38/3D7
and yield of Average ± Average ± Average ± Average ±
extract (%) standard standard standard standard
deviation deviation deviation deviationb

3D7 W2
Acanthospermum hispidum AP CH2 Cl2 10.92 (3.2) 34.9 ± 6.4 14.5 ± 5.5 2.4 11.1 ± 5.7 3.1 7.5 ± 1.2 4.8 ± 1.6 4.7
CH3 OH 10.92 (3.8) >100 47.5 ± 0.6 >2.1 >100 nd 47.1 ± 3.5 nd >2.1
H2 O 10.33 (9.6) >100 54.8 ± 0.5 >1.8 >100 nd 55.6 ± 28.7 nd >1.8
Anchomanes difformis R CH2 Cl2 9.95 (0.2) 26.0 ± 4.2 50.7 ± 7.3 0.5 >100 <0.3 >100 nd <0.3
CH3 OH 9.95 (4.9) 14.6 ± 3.5 14.7 ± 3.8 1.0 >100 <0.1 >100 nd <0.1
H2 O 10.57 (3.0) 12.7 ± 1.0 13.8 ± 2.3 0.9 >100 <0.1 >100 nd <0.1

J. Bero et al. / Journal of Ethnopharmacology 137 (2011) 998–1002

Byrsocarpus coccineus AP CH2 Cl2 10.55 (2.0) >100 14.7 ± 1.2 >6.8 >100 nd 41.6 ± 22.1 nd >2.4
CH3 OH 10.55 (16.4) >100 21.1 ± 1.9 >4.7 >100 nd 54.7 ± 21.9 nd >1.8
H2 O 10.18 (11.3) >100 49.5 ± 4.9 >2.0 >100 nd >100 nd nd
Carpolobia lutea AP CH2 Cl2 10.51 (2.1) 65.4 ± 15.1 18.3 ± 2.5 3.6 31.1 ± 9.0 2.1 19.4 ± 6.5 8.1 ± 4.5 3.4
CH3 OH 10.51 (12.0) >100 >100 nd >100 nd 85.4 ± 2.2 nd >1.2
H2 O 10.18 (12.6) >100 >100 nd >100 nd >100 nd nd
Dialium guineense AP CH2 Cl2 10.39 (2.1) 77.3 ± 0.2 >100 <0.8 >100 <0.8 42.1 ± 17.7 nd 2.3
CH3 OH 10.39 (9.1) >100 42.3 ± 3.1 >2.4 >100 nd >100 nd nd
H2 O 10.43 (14.9) >100 52.0 ± 5.2 >1.9 >100 nd 65.5 ± 3.9 nd >1.5
Heliotropium indicum AP CH2 Cl2 10.38 (1.2) >100 58.3 ± 1.5 >1.7 >100 nd >100 nd nd
CH3 OH 10.38 (6.4) >100 80.2 ± 8.0 >1.2 >100 nd >100 nd nd
H2 O 10.51 (15.9) >100 >100 nd >100 nd >100 nd nd
Keetia leucantha LF CH2 Cl2 30.17 (5.6) 65.6 ± 1.3 24.4 ± 1.8 2.7 21.2 ± 1.1 3.1 13.8 ± 8.3 26.5 ± 9.5 4.8
CH3 OH 30.17 (9.7) >100 47.6 ± 5.2 >2.1 >100 nd >100 nd nd
H2 O 22.36 (9.0) >100 70.2 ± 10.8 >1.4 >100 nd >100 nd nd
TW CH2 Cl2 10.18 (0.5) >100 5.8 ± 1.3 >17.2 23.5 ± 0.2 >4.3 11.3 ± 3.8 15.8 ± 2.3 >8.8
CH3 OH 10.18 (4.0) >100 53.1 ± 2.8 >1.9 >100 nd >100 nd nd
H2 O 22.25 (2.9) >100 86.9 ± 5.3 >1.2 >100 nd >100 nd nd
Pupalia lappacea AP CH2 Cl2 9.90 (0.7) 68.3 ± 4.9 52.0 ± 2.8 1.3 >100 <0.7 50.29 ± 16.1 nd 1.4
CH3 OH 9.90 (4.3) >100 >100 nd >100 nd >100 nd nd
H2 O 10.14 (10.2) >100 >100 nd >100 nd >100 nd nd
Sansevieria liberica AP CH2 Cl2 10.09 (1.7) >100 60.1 ± 1.5 >1.7 61.6 ± 4.7 >1.7 44.5 ± 3.7 nd >1.3
CH3 OH 10.09 (7.1) >100 >100 nd >100 nd >100 nd nd
H2 O 8.83 (11.6) >100 >100 nd >100 nd >100 nd nd
Schrankia leptocarpa LF CH2 Cl2 10.83 (0.9) >100 49.2 ± 2.9 >2.0 >100 nd 34.3 ± 13.5 nd >2.9
CH3 OH 10.83 (6.5) >100 >100 nd >100 nd >100 nd nd
H2 O 10.07 (10.3) >100 >100 nd >100 nd >100 nd nd
TW CH2 Cl2 30.29 (1.5) >100 55.9 ± 3.5 >1.8 58.8 ± 3.9 >1.7 29.6 ± 5.1 nd >3.4
CH3 OH 30.29 (7.7) >100 >100 nd >100 nd >100 nd nd
H2 O 20.52 (5.5) >100 >100 nd >100 nd >100 nd nd
Campthotecinb 0.4 ± 0.2 >100 nd >250
Chloroquineb 0.02 ± 0.01 0.49 ± 0.15
Artemisininb 0.01 ± 0.001 0.005 ± 0.001
Amphotericin B 0.10 ± 0.01
Suramine 0.11 ± 0.02
WI38 = human normal fibroblast cells.
a
Plant part used: LF = leaves, TW = twigs, R = roots, AP = aerial parts.
b
Bero et al., 2009.
c
Selectivity index = IC50(WI38) /IC50(Tbb or Lmm or 3D7) .
 J. Bero et al. / Journal of Ethnopharmacology 137 (2011) 998–1002
mexicana mexicana was observed with the dichloromethane extract
of Acanthospermum hispidum.
Considering the selective activity of the dichloromethane
extract of twigs of Keetia leucantha, a bio-guided fractionation
was realised and led to the identification of oleanolic and urso-
lic acids, as major compounds of one active fraction. These two
triterpenic acids were tested for their antitrypanosomal activity
(IC50 = 1.0 ± 0.2 ␮g/ml and 2.8 ± 0.5 ␮g/ml respectively for ursolic
and oleanolic acids).
4. Discussion and conclusions
The present study showed the antiparasitic activity of the
lipophilic extract of Acanthospermum hispidum on Tbb strain
(IC50 = 14.5 ␮g/ml) and on Lmm strain (IC50 = 11.1 ␮g/ml) with
some selectivity (IC50 on WI38 >30 ␮g/ml). This extract was previ-
ously shown to possess a higher antiplasmodial activity (IC50 = 7.5
and 4.8 ␮g/ml respectively on 3D7 and W2) (Bero et al., 2009), sug-
gesting some interesting selectivity on parasites and particularly
plasmodium.
The methanolic and water extracts of Anchomanes difformis
showed a selective antitrypanosomal activity with IC50 = 14.7 and
13.8 ␮g/ml, respectively, but also cytotoxicity at the same concen-
tration range while no antileishmanial nor antiplasmodial activities
were observed (Bero et al., 2009). Because of its toxicity, this plant
should not be recommended for use as traditional medicine.
The dichloromethane and methanol extracts of Byrsocarpus coc-
cineus have no cytotoxicity but a good antitrypanosomal activity
(14.7 and 21.1 ␮g/ml, respectively), giving a more interesting selec-
tivity.
The dichloromethane extract of Carpolobia lutea displayed a
comparable activity against Tbb (IC50 = 18.3 ␮g/ml) and Plasmod-
ium (IC50 of 19.4 and 8.1 ␮g/ml, respectively on 3D7 and W2 strains)
with a selectivity index of more than 3 (Bero et al., 2009).
Lipophilic extracts of leaves and twigs of Keetia leucantha
showed interesting antitrypanosomal and antileishmanial activ-
ities in addition to the already observed antiplasmodial activity
(Bero et al., 2009). The best activity was observed for twigs with IC50
of 5.8 ␮g/ml on Trypanosoma and a selectivity index higher than
17.2. Their antileishmanial activities were 21.2 and 23.5 ␮g/ml,
respectively.
Bio-guided fractionation of the twigs extract led to the isolation,
as major components of an active fraction, of ursolic and oleanolic
acids which may be considered as the antiparasitic active compo-
nents of this fraction. The antitrypanosomal activities of the two
standard compounds were similar to those reported in the lit-
erature. These compounds have previously shown an activity on
Tbb with IC50 = 2.9 ␮g/ml with SI of 20.6 for oleanolic acid and
IC50 = 1.1 ␮g/ml with SI of 6.0 for ursolic acid (Hoet et al., 2007). Oth-
ers studies showed antitrypanosomal activity with IC50 = 8.8 ␮g/ml
on Tbb and Trypanosoma brucei congolense for ursolic acid (Taketa
et al., 2004) and an in vivo significant reduction of parasites of
75.7% at 2 mg/kg/d intraperitoneally during 13 days (Cunha et al.,
2006). At a concentration of 20 mg/kg/d intraperitoneally during 20
days, reductions of parasitaemia of 60% for ursolic acid and 40% for
oleanolic acid were observed (Ferreira et al., 2010). Ursolic acid also
stopped all the movements of Trypanosoma cruzi epimastigotes at
the minimum concentration of 40 ␮g/ml after 48 h of incubation
while the same effect was observed at 250 ␮g/ml for oleanolic acid
(Abe et al., 2002). These two compounds also possess toxicity with
50% lysis at 21 and 80.4 ␮M, respectively, on Trypanosoma cruzi
trypomastigotes (Cunha et al., 2003).
Antileishmanial activity of ursolic acid was also evaluated
against promastigotes of Leishmania amazonensis (IC50 = 43.8 and
5 ␮g/ml, depending on the test) (Torres-Santos et al., 2004; Gnoatto
1001
et al., 2008) and against promastigotes of Leishmania donovani
(IC50 = 3.5 ␮g/ml) (Moulisha et al., 2010). The activity of oleanolic
acid against promastigotes of Leishmania amazonensis gave an IC50
of 10 ␮g/ml (Torres-Santos et al., 2004).
Antiplasmodial activity of these two triterpenic acids were
also observed on Plasmodium falciparum with IC50 of 15.2 and
3.1 ␮g/ml (Cimanga et al., 2006) and IC50 of 9.3 and 4.9 ␮g/ml
respectively for oleanolic and ursolic acids (van Baren et al., 2006).
Furthermore, they also possess among other activities, interesting
anti-inflammatory properties (Recio et al., 1995).
HPLC–MS analysis showed that these two acids were major
compounds in the crude leaves and twigs extracts. Nevertheless,
other fractions obtained at lower quantities during the bio-guided
fractionation also displayed interesting activities and the com-
pounds they contain may also account for the overall activity of
the crude extracts and would be worth being further isolated and
identified.
Extracts of Dialium guineense, Heliotropium indicum, Pupalia lap-
pacea, Sansevieria liberica, Schrankia leptocarpa did not show any
strong antiparasitic activity.
These investigations showed that various extracts active against
malaria and used traditionally also possess activity against other
parasites as Trypanosoma and Leishmania and for some of them, a
certain selectivity for one parasite is found. Moreover, this is the
first report on the analysis of these plant extracts for their antit-
rypanosomal and antileishmanial activities. The presence of two
triterpenoid acids (ursolic and oleanolic acids) was also demon-
strated for the first time in Keetia leucantha and could explain a
part of the activity of the dichloromethane extracts of leaves and
twigs. As they are major active compounds, they could be used as
active markers for the standardization of treatments with this crude
plant or extracts. According to the results obtained, the properties
of Acanthospermum hispidum, Keetia leucantha, Byrsocarpus coccinea
and Carpolobia lutea need to be further investigated by isolation and
identification of pure bioactive compounds by bio-guided fraction-
ation.
Acknowledgements
The authors are grateful to Mr. Agabani (botanist of Univer-
sity of Abomey-Calavi, Cotonou, Benin), Dr Gbaguidi Fernand, Dr
Gbenou Joachim and Mr. Ganfon Habib (Centre Béninois de la
Recherche Scientifique et Technique, Cotonou, Benin and Labo-
ratoire de pharmacognosie et huiles essentelles UFR pharmacie,
Faculté des Sciences de la Santé, Université d’Abomey Calavi,
Cotonou, Benin) for plant collections as well as Professor Elmar Rob-
brecht and Olivier Lachenaud (botanists of National Botanic Garden
of Belgium, Meise, Belgium) for clarifying botanical information.
We wish to thank Marie-Christine Fayt for her skillful technical
assistance.
The authors gratefully thank the Belgian National Fund for Sci-
entific Research (FNRS) (FRFC 2.4555.08), the Special Fund for
Research (FSR) and the faculty of medicine of UCL.
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