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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.
(MC), a medium sized town in the northern region of groups of ve for each drug test and treatment was
MG. In BH, at one occasion, young and old plants were carried out by oral route, daily, for 4 consecutive days.
collected in the same area, at the same time, as judged Extracts were suspended in water plus 0.1% Tween-20
by their sizes. Thus, the smaller plants (average size immediately before use, then diluted with water so that
0.4 m, SD 0.1 m) were considered younger than the doses of 250, 500 or 1000 mg/kg were administered in a
bigger plants (1 m, SD 0.1 m). In MC, samples were 0.2 mL volume per animal, via gavage. In one experi-
collected on two occasions, once during the hot rainy ment, B. pilosa extracts were given at serial doses of
season (average temperature 34 C) and once during 500 mg/kg to determine the inhibitory drug concentra-
the cool dry season (average temperature 27 C), in tion. Two control groups were used in each test, one
February and August 1999, respectively. The wild-type receiving the standard antimalarial drug chloroquine in
samples were identied as Bidens pilosa L. (Asteraceae) subcurative doses while one group was not treated. In
by J. R. Stehmann, Department of Botany, Federal one experiment, the response of malaria-infected mice
University of Minas Gerais (BHCB), where a refer- to chloroquine was determined using daily doses be-
ence voucher is kept (BHCB 64919). The specimens tween 8 and 100 mg/kg for 4 consecutive days.
were dried at room temperature and immediately used Antimalarial activity was evaluated by counting
to prepare ethanol extracts, which were then kept re- parasitaemia in blood smears taken at days 5 and 7
frigerated until testing. after parasite inoculation, by microscopy, after metha-
nol xation and staining with Giemsa. Inhibition of
Cultivated B. pilosa plants. Plants selected from one parasite growth in the drug-treated groups was calcu-
strain of a wild sample were cultivated in a rigid lated in relation to the control (non-treated) group as
polyethylene pot (22.5 cm 15.2 cm) with 18 L of follows: average parasitaemia in the control (non-
substrate (75:25 of soil/sand), the standard soil, supple- treated) group minus parasitaemia in the test group,
mented with different concentrations per pot of either divided by parasitaemia in the control (non-treated)
limestone (3, 4, 5, 7 and 8 g per pot) or humus (80%, group. Results were expressed as a percentage of par-
65%, 50%, 35% and 30%), mixed until the soil was asitaemia reduction; extracts that reduced parasite growth
found to be homogeneous. The experimental units con- by 30%, were considered active (Carvalho et al., 1991).
tained two plants each, were kept in a shaded environ- Overall mortality was monitored daily for 4 weeks.
ment, covered with a roof of 75 m thick polyethylene
ultraviolet-resistant transparent lm, and harvested 71 P. falciparum in vitro culture and drug assays. B. pilosa
days after cultivation. plants cultivated under standard conditions were
tested for their antimalarial activity in vitro using
Crude extract preparation. Dried plant roots were three P. falciparum isolates with different susceptibi-
powdered (100 g each), extracted by percolation with lities to chloroquine: clone W2 (chloroquine-resistant,
80% ethanol at room temperature, and the solvent meoquine-sensitive); clone D6 (chloroquine-sensitive,
evaporated to dryness at 45 C maximum. Dried meoquine-resistant); and isolate BHz (partially resist-
extracts were kept under aluminium foil during the pro- ant to chloroquine), previously isolated at our labora-
cess of manipulation, stored in a refrigerator, and sub- tory in 1986, from an imported case of malaria from
mitted to in vivo and in vitro antimalarial assays or the Amazon.
chemical analysis. All parasites were maintained in continuous culture
on human erythrocytes (blood groups A+ or O+ ) using
Phytochemical analysis. The extracts were submitted RPMI medium supplemented with 10% human serum
to TLC with silica gel 60 F254 (ref. 105721; Merck, as described by Trager and Jensen (1976). The
Whitehouse Station, NJ, USA). The chloroform/acetone antiparasitic effect of the ethanol extracts from B. pilosa
system (9:1) was used for separation and identication cultivated under different soil conditions and of wild-
of acetylenes. The plates were revealed with vanillin type plants was measured at the same time by the
(1%) and sulphuric acid (10%) in ethanol as described [3H]-hypoxanthine incorporation assay as described by
(Brando et al., 1997). For separation and identica- Desjardins et al. (1979), modied by Zalis et al. (1998).
tion of avonoids, the ethyl acetate/acetic acid/formic Briey, trophozoite stages in sorbitol-synchronized
acid/water system was used (100:11:11:26) and revealed blood (Lambros and Vanderberg, 1979) were cultured
with NP/PEG reagents (Wagner and Bladt, 1996). at 1% to 2% parasitaemia and 2.5% haematocrit, then
incubated with the plant extracts (at various concentra-
Animal use and ethical approval. Swiss albino adult tions), diluted with 0.02% Tween-20 in culture medium
mice, weighing 2022 g, were used for the antimalarial (RPMI 1640) without hypoxanthine; a chloroquine con-
and toxicity tests. The animals received water and trol (as a reference antimalarial drug) was used in
food ad libitum. Their use was approved by the Ethical each experiment. The stock solutions of extracts and
Committee for Using Animals (CEUA-P0094-01) at chloroquine were further diluted in a complete medium
Fundao Instituto Oswaldo Cruz (FIOCRUZ). (RPMI 1640 plus 10% human serum) without hypo-
xanthine, to reach the concentrations shown in the
Antimalarial tests in vivo. The antimalarial suppres- results. Inhibition of parasite growth was evaluated
sive test described by Peters (1985) and modied by through the levels of [3H]-hypoxanthine incorporation
Carvalho et al. (1991) was performed in mice infected plotted to generate dose-response curves. The half-
with P. berghei, strain NK-65, originally received from maximal inhibitory response (IC50) compared with para-
New York University Medical School. Parasites were site growth in the drug-free controls was estimated
maintained by weekly blood passage in mice by the by curve-tting using a software program [Microcal,
intraperitoneal route, 10 5 infected red blood cells Origin Software, Inc. (Northampton, MA, USA) ]. All
per mouse. The animals were randomly separated into assays were performed in triplicate.
Copyright 2004 John Wiley & Sons, Ltd. Phytother. Res. 18, 634639 (2004)
636 V. F. ANDRADE-NETO ET AL.
RESULTS
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
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
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
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 activity (IC50 = 10.417.0 g/mL) than did the cultivated
still alive (Table 1). Mortality was higher among the samples.
malaria-infected mice treated with 1000 mg/kg. The The in vitro sensitivities of all P. falciparum isolates
extract alone caused no mortality to normal mice at to the plant extracts were approximately similar and
doses up to 3000 mg/kg; 4000 mg/kg caused 20% mor- reproducible in assays in triplicate on separate
tality (not shown). occasions for each plant (Table 3). The growth of
The in vitro antimalarial activity of cultivated B. pilosa chloroquine-resistant parasites (clone W2 and iso-
tested against three P. falciparum isolates with various late BHz) or P. falciparum chloroquine-sensitive para-
susceptibilities to chloroquine was similar, based on sites (clone D6) was similarly inhibited by B. pilosa
each inhibitory concentration dose ( ICs) (Table 3). extracts. The wild-type plants were more active than
The IC50 of plants cultivated in standard soil ranged the cultivated samples, and the humus-cultivated plants
from 25 to 36 g/mL; for plants cultivated in soil with were more active than the limestone plant samples
65% to 80% humus, it ranged from 16.4 to 22 g/mL. (Table 3).
Plants cultivated with 3 g/pot of limestone were less To investigate whether the cultivated plants were less
active, with IC50 from 36.7 to 49.8 g/mL (Table 3). active because they were younger (71 days of culture)
Plants cultivated in 4 to 8 g/pot of limestone or 30% to and smaller, young and old plants collected in BH at
50% humus were inactive (IC50 50 g/mL) (not the same time and place were tested in parallel. The
shown). The wild-type plants showed signicantly higher IC50 was similar, 9.6 and 12 g/mL, respectively.
Copyright 2004 John Wiley & Sons, Ltd. Phytother. Res. 18, 634639 (2004)
638 V. F. ANDRADE-NETO ET AL.
The IC of chloroquine, tested in parallel against the very effective against P. falciparum, whereas specimens
three P. falciparum isolates, conrmed that clone W2 is from other parts of the world showed little or no activ-
chloroquine resistant (IC50 = 0.22 g/mL), isolate BHz ity (Klayman, 1985; WHO, 2000). The antimalarial ac-
is partially resistant (IC50 = 0.09 g/mL), and clone D6 tivity of B. pilosa was less affected, even though plants
is chloroquine sensitive (IC50 = 0.04 g/mL). It also cultivated were not as active as the sylvatic plants,
shows that chloroquine efcacy was 50 to 100 times and humus soil produced better specimens than lime-
superior to the plant extracts in vitro. stone soil. Apparently, humus performs the appropri-
Chromatography analysis by TLC of the cultivated ate distribution of particles (sand, silt, clay) making up
or wild-type plant extracts showed the presence of acety- pores, and better storage of water and air, protecting
lenes and avonoids in all specimens, although with against rapid changes in soil pH and increased cation
different chromatographic proles (data not shown). exchange, important for root cell division and dry
matter production, resulting in healthier and stronger
plants (Swift, 1996). However, the ideal culture soil for
plants with a strong antimalarial activity is yet to be
DISCUSSION determined.
The ethanol extracts from the sylvatic and wild
Although the antimalarial activity of the B. pilosa B. pilosa roots contained avonoids shown by TLC,
ethanol extract was previously demonstrated (Brando although at different proles (data not shown). The
et al., 1997; Krettli et al., 2001), this work now shows HPLC analysis showed peaks for polyacetylene and
for the rst time that: (i) the human malaria parasite P. methoxylated avonoids, compounds corresponding to
falciparum, chloroquine-resistant or meoquine-resistant, quercetin-3,3-dimethoxy-7-0-rhamnoglucopyranose
displays similar in vitro antimalarial susceptibility to B. (Brando et al., 1998) and acetylene 1-phenyl-1,3-diyn-
pilosa, like that shown by the chloroquine-susceptible 5-en-7-ol-acetate (Brando et al., 1997) and were tested
parasites tested simultaneously; (ii) sylvatic B. pilosa in vivo. Next, fractions with or without avonoids were
specimens collected in different regions at different tested against malaria in vivo in parallel with the ethanol
times of the year and at various ages (as judged by extracts in malaria infected mice. The ether-methanol
plant size) were similarly active in vivo. fraction enriched with avonoid was more active than
Some differences observed with 250500 mg/kg of the ether fraction, (reactive in polyacetylenes, negative
crude extracts of B. pilosa may result from individual for avonoids) and as active as the ethanol fraction.
variations in the infected mice rather than from plant This corroborates our hypothesis that this avonoid is
toxicity. A dose of 1000 mg/kg of extracts was found to responsible for the strong antimalarial activity of B.
be toxic and less active, probably as a result of the pilosa (Krettli et al., 2001). Further studies should ex-
immunosuppressive activity of the plant in vivo (Pereira plore this compound as a prototype for an antimalarial
et al., 1999), down modulating the immune mechanisms aimed at the P. falciparum chloroquine-resistant para-
of natural resistance to malaria. Indeed, it has been sites which are rather frequent worldwide.
shown that the antimalarial effect of any given drug
depends on its synergism with the vertebrate immune
response (Peters, 1985; Targett, 1985; Melendez and Acknowledgements
Krettli, 1987).
The pharmacological action of plants is known to We thank the Brazilian agencies CNPq and FAPEMIG for nancial
support; Dr M. Wilcox for suggestions with the manuscript; Brenda
vary with the weather conditions, soil type and plant Rae Marshall for corrections to the English; Edmilson Cordeiro for
collection time, amongst other factors. In the case of kindly providing plants from Montes Claros; and Lindomar Reis for
A. annua (Asteraceae), plants collected in China were plant samples from Ibi.
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