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Antimalarial activity of Bidens pilosa L.

(Asteraceae) ethanol extracts from wild plants
collected in various localities or plants cultivated
in humus soil

ARTICLE in PHYTOTHERAPY RESEARCH SEPTEMBER 2004

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 PHYTOTHERAPY RESEARCH
Phytother. Res. 18, 634639 (2004)
634 Published online in Wiley InterScience (www.interscience.wiley.com).
V. F. ANDRADE-NETO DOI: 10.1002/ptr.1510
ET AL.

Antimalarial Activity of Bidens pilosa L.

(Asteraceae) Ethanol Extracts From Wild
Plants Collected in Various Localities or Plants
Cultivated in Humus Soil

Valter F. Andrade-Neto1, 2, Maria G. L. Brando3, Francielda Q. Oliveira3,

Vicente W. D. Casali4, Brian Njaine4, Mariano G. Zalis5, Luciana A. Oliveira1 and
Antoniana U. Krettli1*
1
Laboratory of Malaria, Centro de Pesquisas Ren Rachou/FIOCRUZ, Belo Horizonte, Brazil
2
Department of Parasitology, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, Brazil
3
Laboratory of Pharmacognosy, Faculty of Pharmacy, UFMG, Belo Horizonte, MG, Brazil
4
Department of Agronomy, Universidade Federal de Viosa, Viosa, MG, Brazil
5
Institute of Biophysics, Universidade Federal do Rio de Janeiro, RJ, Brazil

Bidens pilosa (Asteraceae), a medicinal plant used worldwide, has antimalarial activity as shown in previous
work. This study tested ethanol extracts from wild plants collected in three different regions of Brazil and from
plants cultivated in various soil conditions. The extracts were active in mice infected with P. berghei: doses of
500 mg/kg administered by oral route reduced malaria parasitaemia and mouse mortality; higher doses were
found to be less effective. Tested in vitro against three P. falciparum isolates, two chloroquine resistant and
one meoquine resistant, the plants cultivated under standard conditions, and in humus enriched soil, were
active; but the wild plants were the most active. Analysis using thin layer chromatography demonstrated the
presence of avonoids (compounds considered responsible for the antimalarial activity) in all plants tested,
even though at different proles. Because B. pilosa is proven to be active against P. falciparum drug-resistant
parasites in vitro, and in rodent malaria in vivo, it is a good candidate for pre-clinical tests as a phytotherapeutic
agent or for chemical isolation of the active compounds with the aim of nding new antimalarial drugs.
Copyright 2004 John Wiley & Sons, Ltd.

Keywords: Bidens pilosa; avonoids; antimalarial activity.

1997). B. pilosa has other important biological effects:

INTRODUCTION
The global malaria situation is worsening: about 500
million new cases appear each year, 90% in Africa alone
(WHO, 1999) and half a million in the Brazilian Ama-
zon Region (FUNASA, 2002). Chloroquine resistance
is the main problem affecting disease control, thus treat-
ment of malaria relies mainly on quinine and artemisinin
derivatives, combined with other drugs. Both com-
pounds, rst isolated from medicinal plants already
used hundreds of years ago, are still active against P.
falciparum drug-resistant parasites (Ridley, 2002).
The Amazon forest has many species of plants used
in traditional medicine (Brando et al., 1992). Approxi-
mately 50 species cited to treat malaria and /or fever
were tested in our previous work, several being active
against P. falciparum in culture and P. berghei in mice
(Carvalho et al., 1991). In the case of B. pilosa and
other Bidens species (Asteraceae), antimalarial activity
was attributed to avonoid compounds and acetylenes,
the latter rather unstable when isolated (Brando et al.,
* Correspondence to: Dr A. U. Krettli, Laboratory of Malaria, Centro de
Pesquisas Ren Rachou/FIOCRUZ, Av. Augusto de Lima 1715, Belo
Horizonte, MG, CEP 30190-002 Brazil.
E-mail: akrettli@cpqrr.ocruz.br
Contract/grant sponsor: CNPq.
Contract/grant sponsor: FAPEMIG.
Copyright 2004 John Wiley & Sons, Ltd.
Copyright 2004 John Wiley & Sons, Ltd.
(i) it is hypotensive in rats with no adverse effects on
the cardiac system (Ritz, 1993) or the renal system
(Dimo et al., 1999); (ii) it is protective to the rat gastric
mucous layer, inhibiting the formation of ulcers after
depletion of neutral glycoproteins (Alvarez et al., 1999);
and (iii) it is antihyperglycaemic in mice (Ubillas et al.,
2000). The plant infusion is used to treat human dia-
betes of the non-insulin dependent type (Oubr et al.,
1997), among other indications. This study tested the in
vivo antimalarial activity of B. pilosa collected in vari-
ous parts of Brazil as well as of plants cultivated in soil
supplemented with humus and limestone against drug-
resistant P. falciparum parasites in vitro. Thin-layer
chromatography (TLC) studies detected the presence
of avonoids in all B. pilosa plants tested, although in
varying proles.
MATERIAL AND METHODS
Collection of wild-type B. pilosa plants. Plant samples
were collected in three different regions of Minas Gerais
(MG), Brazil: (i) in the metropolitan area of Belo
Horizonte (BH), the state capital, on several occasions;
(ii) in the rural area of Ibi (IB), a village in the east-
ern region; and (iii) at a farm close to Montes Claros
Received
Phytother. Res. 21 July(2004)
18, 634639 2003
Accepted 21 April 2004
 ANTIMALARIAL ACTIVITY OF BIDENS PILOSA
(MC), a medium sized town in the northern region of
MG. In BH, at one occasion, young and old plants were
collected in the same area, at the same time, as judged
by their sizes. Thus, the smaller plants (average size
0.4 m, SD 0.1 m) were considered younger than the
bigger plants (1 m, SD 0.1 m). In MC, samples were
collected on two occasions, once during the hot rainy
season (average temperature 34 C) and once during
the cool dry season (average temperature 27 C), in
February and August 1999, respectively. The wild-type
samples were identied as Bidens pilosa L. (Asteraceae)
by J. R. Stehmann, Department of Botany, Federal
University of Minas Gerais (BHCB), where a refer-
ence voucher is kept (BHCB 64919). The specimens
were dried at room temperature and immediately used
to prepare ethanol extracts, which were then kept re-
frigerated until testing.
Cultivated B. pilosa plants. Plants selected from one
strain of a wild sample were cultivated in a rigid
polyethylene pot (22.5 cm 15.2 cm) with 18 L of
substrate (75:25 of soil/sand), the standard soil, supple-
mented with different concentrations per pot of either
limestone (3, 4, 5, 7 and 8 g per pot) or humus (80%,
65%, 50%, 35% and 30%), mixed until the soil was
found to be homogeneous. The experimental units con-
tained two plants each, were kept in a shaded environ-
ment, covered with a roof of 75 m thick polyethylene
ultraviolet-resistant transparent lm, and harvested 71
days after cultivation.
Crude extract preparation. Dried plant roots were
powdered (100 g each), extracted by percolation with
80% ethanol at room temperature, and the solvent
evaporated to dryness at 45 C maximum. Dried
extracts were kept under aluminium foil during the pro-
cess of manipulation, stored in a refrigerator, and sub-
mitted to in vivo and in vitro antimalarial assays or
chemical analysis.
Phytochemical analysis. The extracts were submitted
to TLC with silica gel 60 F254 (ref. 105721; Merck,
Whitehouse Station, NJ, USA). The chloroform/acetone
system (9:1) was used for separation and identication
of acetylenes. The plates were revealed with vanillin
(1%) and sulphuric acid (10%) in ethanol as described
(Brando et al., 1997). For separation and identica-
tion of avonoids, the ethyl acetate/acetic acid/formic
acid/water system was used (100:11:11:26) and revealed
with NP/PEG reagents (Wagner and Bladt, 1996).
Animal use and ethical approval. Swiss albino adult
mice, weighing 2022 g, were used for the antimalarial
and toxicity tests. The animals received water and
food ad libitum. Their use was approved by the Ethical
Committee for Using Animals (CEUA-P0094-01) at
Fundao Instituto Oswaldo Cruz (FIOCRUZ).
Antimalarial tests in vivo. The antimalarial suppres-
sive test described by Peters (1985) and modied by
Carvalho et al. (1991) was performed in mice infected
with P. berghei, strain NK-65, originally received from
New York University Medical School. Parasites were
maintained by weekly blood passage in mice by the
intraperitoneal route, 10 5 infected red blood cells
per mouse. The animals were randomly separated into
Copyright 2004 John Wiley & Sons, Ltd.
635
groups of ve for each drug test and treatment was
carried out by oral route, daily, for 4 consecutive days.
Extracts were suspended in water plus 0.1% Tween-20
immediately before use, then diluted with water so that
doses of 250, 500 or 1000 mg/kg were administered in a
0.2 mL volume per animal, via gavage. In one experi-
ment, B. pilosa extracts were given at serial doses of
500 mg/kg to determine the inhibitory drug concentra-
tion. Two control groups were used in each test, one
receiving the standard antimalarial drug chloroquine in
subcurative doses while one group was not treated. In
one experiment, the response of malaria-infected mice
to chloroquine was determined using daily doses be-
tween 8 and 100 mg/kg for 4 consecutive days.
Antimalarial activity was evaluated by counting
parasitaemia in blood smears taken at days 5 and 7
after parasite inoculation, by microscopy, after metha-
nol xation and staining with Giemsa. Inhibition of
parasite growth in the drug-treated groups was calcu-
lated in relation to the control (non-treated) group as
follows: average parasitaemia in the control (non-
treated) group minus parasitaemia in the test group,
divided by parasitaemia in the control (non-treated)
group. Results were expressed as a percentage of par-
asitaemia reduction; extracts that reduced parasite growth
by 30%, were considered active (Carvalho et al., 1991).
Overall mortality was monitored daily for 4 weeks.
P. falciparum in vitro culture and drug assays. B. pilosa
plants cultivated under standard conditions were
tested for their antimalarial activity in vitro using
three P. falciparum isolates with different susceptibi-
lities to chloroquine: clone W2 (chloroquine-resistant,
meoquine-sensitive); clone D6 (chloroquine-sensitive,
meoquine-resistant); and isolate BHz (partially resist-
ant to chloroquine), previously isolated at our labora-
tory in 1986, from an imported case of malaria from
the Amazon.
All parasites were maintained in continuous culture
on human erythrocytes (blood groups A+ or O+ ) using
RPMI medium supplemented with 10% human serum
as described by Trager and Jensen (1976). The
antiparasitic effect of the ethanol extracts from B. pilosa
cultivated under different soil conditions and of wild-
type plants was measured at the same time by the
[3H]-hypoxanthine incorporation assay as described by
Desjardins et al. (1979), modied by Zalis et al. (1998).
Briey, trophozoite stages in sorbitol-synchronized
blood (Lambros and Vanderberg, 1979) were cultured
at 1% to 2% parasitaemia and 2.5% haematocrit, then
incubated with the plant extracts (at various concentra-
tions), diluted with 0.02% Tween-20 in culture medium
(RPMI 1640) without hypoxanthine; a chloroquine con-
trol (as a reference antimalarial drug) was used in
each experiment. The stock solutions of extracts and
chloroquine were further diluted in a complete medium
(RPMI 1640 plus 10% human serum) without hypo-
xanthine, to reach the concentrations shown in the
results. Inhibition of parasite growth was evaluated
through the levels of [3H]-hypoxanthine incorporation
plotted to generate dose-response curves. The half-
maximal inhibitory response (IC50) compared with para-
site growth in the drug-free controls was estimated
by curve-tting using a software program [Microcal,
Origin Software, Inc. (Northampton, MA, USA) ]. All
assays were performed in triplicate.
Phytother. Res. 18, 634639 (2004)
636 V. F. ANDRADE-NETO ET AL.

Statistical analysis. The data of in vivo antimalarial tests

were double-entered using the EPI Info 2000 (CDC,
Atlanta GA, USA) and analysed using the Stata Soft-
ware package (Stata Corporation, College Station, TX,
USA). Students t-test was used to compare the inhibi-
tion of parasite growth. The cumulative mouse mortal-
ity was analysed by the KruskalWallis test method
[Biostat 1.0 (MCT-CNPq, Manaus, AM, Brazil) ].
Parameters that showed signicant differences were
subsequently incorporated into a multiple comparison
analysis using Anova/Post Hoc test. Statistical differ-
ences between the geometric average of IC50 values were
calculated by the Mann-Whitney U test method per-
formed with Biostat 1.0 MCT-CNPq because these data
were not normally distributed.

RESULTS

Ethanol crude extracts from B. pilosa wild-type plants,

collected at three different locations in the State of
MG-Brazil, given to mice inoculated with P. berghei
blood parasites, reduced parasitaemia up to 60% at
doses of 250 and 500 mg/kg; however, at a dose of
1000 mg/kg, only the BH samples caused a reduction
in parasitaemia. Table 1 summarizes the results of one
experiment of the three performed. Plants collected Figure 1. (A) Mean reduction of P. berghei parasitaemia growth
under different weather conditions, the cool-dry and in mice treated with ethanol extract from B. pilosa wild-type
hot-rainy seasons, at the same location (MC), were plants collected at BH in relation to controls (n = 5 per group)
found to have similar activities (not shown). A dose- and the inhibitory concentration (IC50) dose. (B) Flavonoid com-
response curve using extracts from BH showed that the pounds isolated first from the wild plant roots (Brando et al.,
1998) responsible for antimalarial activity.
drug-inhibitory concentration (IC50) was 485 mg/kg; the
reduction of parasitaemia was statistically signicant
( p = 0.0027) for the higher dose tested (Figure 1A).
The reduction of parasitaemia with 250 mg/kg B. B. pilosa extracts (250 mg/kg) increased the survival of
pilosa extracts was similar to that caused by 12 mg/kg infected mice in relation to non-treated control mice,
of chloroquine in mice, although the latter caused a but not at a signicant level. By day 15, all controls had
better reduction in mortality (Table 2). Treatment with died, and half of the B. pilosa- treated mice were alive;

Table 1. Antimalarial activity of Bidens pilosa ethanol extracts from roots collected in different localities of Minas Gerais against
Plasmodium berghei in mice infected with blood parasites

Inhibition of
parasite growth Cumulative mortality of 5 mice at days of
Plant Dose
(%) at days infection (%)
collection (mg/kg daily by
site oral route, 4x) 5 7 9 15 21 24

BH 0 2 (40%) 5 (100%) 5 (100%) 5 (100%)

250 38a 44b 0 2 (40%) 2 (40%) 5 (100%)
500 55b 40a 1 (20%) 3 (60%) 3 (60%) 5 (100%)
1000 54b 60c 2 (40%) 4 (80%) 5 (100%) 5 (100%)
IB 0 1 (20%) 5 (100%) 5 (100%) 5 (100%)
250 30 48b 2 (40%) 2 (40%) 3 (60%) 5 (100%)
500 43b 38a 3 (60%) 3 (60%) 4 (80%) 5 (100%)
1000 0 10 0 4 (80%) 5 (100%) 5 (100%)
MC 0 2 (40%) 5 (100%) 5 (100%) 5 (100%)
250 55c 60c 0 2 (40%) 3 (60%) 5 (100%)
500 32 36a 0 3 (60%) 5 (100%) 5 (100%)
1000 17 24 0 4 (80%) 5 (100%) 5 (100%)

O, Control non-treated groups.

BH, Belo Horizonte; IB, Ibi; MC, Montes Claros.
Significant differences in relation to non-treated controls by multiple comparison test (Anova/Post Hoc test): a p = 0.04; b
p = 0.0077;
c
p = 0.00137.

Copyright 2004 John Wiley & Sons, Ltd. Phytother. Res. 18, 634639 (2004)
 ANTIMALARIAL ACTIVITY OF BIDENS PILOSA 637

Table 2. Inhibition of parasite growth and mortality by Plasmodium berghei in mice treated with
different doses of chloroquine in one representative experiment

Inhibition of
parasite growth Cumulative mortality of 5 mice at days of
(%) at days infection (%)
Chloroquine
orally (mg/kg) 5 7 12 18 24

0 2 (40%) 5 (100%) 5 (100%)

8 20 5 1 (20%) 3 (60%) 5 (100%)
12 51a 30 1 (20%) 2 (40%) 5 (100%)
15 75a 54a 0 1 (20%) 2 (40%)
30 99b 93b 0 0 1 (20%)
50 99b 95b 0 0 1 (20%)
100 100b 100b 0 0 0

Significant differences in relation to non-treated controls by multiple comparison test (Anova/

Post Hoc test) with a p < 0.001; b p < 0.0001.

Table 3. Comparison of the drug inhibitory concentration dose (IC) to P. falciparum (Pf) growth (clones W2, D6 and strain BHz) in
the presence of B. pilosa extracts using plant roots cultivated under different soil conditions

ICs (g/mL)
Strain of Pf
Plants cultivated in or drug reference used in vitro IC25 IC50 IC75

Wild-type soil W2 3.8 0.01 12.6 0.01b 34.0 0.20

D6 3.1 0.01 10.4 0.01b 45.0 0.09
BHz 5.4 0.02 17.0 0.03a 43.0 0.04
Humus (65%) W2 3.4 0.02 22.0 0.03a >50
D6 3.0 0.01 16.4 0.02a >50
BHz 4.2 0.02 16.6 0.02a >50
Limestone (3 g/pot) W2 5.0 0.08 49.8 0.55 >50
D6 3.7 0.03 47.8 0.46 >50
BHz 3.5 0.03 36.7 0.10 >50
Cultivated controls (standard soil) W2 8.8 0.80 25.1 0.20 >50
D6 11.2 0.54 36.0 0.10 >50
BHz 9.0 0.33 27.6 0.30 >50
Chloroquine (antimalarial drug reference) W2 0.11 0.03 0.22 0.05 0.43 0.02
D6 0.02 0.03 0.04 0.03 0.09 0.05
BHz 0.04 0.05 0.09 0.03 0.18 0.04

Values are mean standard deviation in one representative experiment in triplicate. Significant differences in relation to controls by
Mann-Whitney U-test were seen with a p = 0.04; b p = 0.01.
The P. falciparum isolates used for each test were: chloroquine sensitive and mefloquine resistant ( D6); chroloquine resistant and
mefloquine sensitive (W2) and chloroquine partially resistant (BHz).

by day 21, approximately 40% of the treated mice were
still alive (Table 1). Mortality was higher among the
malaria-infected mice treated with 1000 mg/kg. The
extract alone caused no mortality to normal mice at
doses up to 3000 mg/kg; 4000 mg/kg caused 20% mor-
tality (not shown).
The in vitro antimalarial activity of cultivated B. pilosa
tested against three P. falciparum isolates with various
susceptibilities to chloroquine was similar, based on
each inhibitory concentration dose ( ICs) (Table 3).
The IC50 of plants cultivated in standard soil ranged
from 25 to 36 g/mL; for plants cultivated in soil with
65% to 80% humus, it ranged from 16.4 to 22 g/mL.
Plants cultivated with 3 g/pot of limestone were less
active, with IC50 from 36.7 to 49.8 g/mL (Table 3).
Plants cultivated in 4 to 8 g/pot of limestone or 30% to
50% humus were inactive (IC50 50 g/mL) (not
shown). The wild-type plants showed signicantly higher
Copyright 2004 John Wiley & Sons, Ltd.
activity (IC50 = 10.417.0 g/mL) than did the cultivated
samples.
The in vitro sensitivities of all P. falciparum isolates
to the plant extracts were approximately similar and
reproducible in assays in triplicate on separate
occasions for each plant (Table 3). The growth of
chloroquine-resistant parasites (clone W2 and iso-
late BHz) or P. falciparum chloroquine-sensitive para-
sites (clone D6) was similarly inhibited by B. pilosa
extracts. The wild-type plants were more active than
the cultivated samples, and the humus-cultivated plants
were more active than the limestone plant samples
(Table 3).
To investigate whether the cultivated plants were less
active because they were younger (71 days of culture)
and smaller, young and old plants collected in BH at
the same time and place were tested in parallel. The
IC50 was similar, 9.6 and 12 g/mL, respectively.
Phytother. Res. 18, 634639 (2004)
638 V. F. ANDRADE-NETO ET AL.
The IC of chloroquine, tested in parallel against the
three P. falciparum isolates, conrmed that clone W2 is
chloroquine resistant (IC50 = 0.22 g/mL), isolate BHz
is partially resistant (IC50 = 0.09 g/mL), and clone D6
is chloroquine sensitive (IC50 = 0.04 g/mL). It also
shows that chloroquine efcacy was 50 to 100 times
superior to the plant extracts in vitro.
Chromatography analysis by TLC of the cultivated
or wild-type plant extracts showed the presence of acety-
lenes and avonoids in all specimens, although with
different chromatographic proles (data not shown).
DISCUSSION
Although the antimalarial activity of the B. pilosa
ethanol extract was previously demonstrated (Brando
et al., 1997; Krettli et al., 2001), this work now shows
for the rst time that: (i) the human malaria parasite P.
falciparum, chloroquine-resistant or meoquine-resistant,
displays similar in vitro antimalarial susceptibility to B.
pilosa, like that shown by the chloroquine-susceptible
parasites tested simultaneously; (ii) sylvatic B. pilosa
specimens collected in different regions at different
times of the year and at various ages (as judged by
plant size) were similarly active in vivo.
Some differences observed with 250500 mg/kg of
crude extracts of B. pilosa may result from individual
variations in the infected mice rather than from plant
toxicity. A dose of 1000 mg/kg of extracts was found to
be toxic and less active, probably as a result of the
immunosuppressive activity of the plant in vivo (Pereira
et al., 1999), down modulating the immune mechanisms
of natural resistance to malaria. Indeed, it has been
shown that the antimalarial effect of any given drug
depends on its synergism with the vertebrate immune
response (Peters, 1985; Targett, 1985; Melendez and
Krettli, 1987).
The pharmacological action of plants is known to
vary with the weather conditions, soil type and plant
collection time, amongst other factors. In the case of
A. annua (Asteraceae), plants collected in China were
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