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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
(i) it is hypotensive in rats with no adverse effects on
the cardiac system (Ritz, 1993) or the renal system
The global malaria situation is worsening: about 500 (Dimo et al., 1999); (ii) it is protective to the rat gastric
million new cases appear each year, 90% in Africa alone mucous layer, inhibiting the formation of ulcers after
(WHO, 1999) and half a million in the Brazilian Ama- depletion of neutral glycoproteins (Alvarez et al., 1999);
zon Region (FUNASA, 2002). Chloroquine resistance and (iii) it is antihyperglycaemic in mice (Ubillas et al.,
is the main problem affecting disease control, thus treat- 2000). The plant infusion is used to treat human dia-
ment of malaria relies mainly on quinine and artemisinin betes of the non-insulin dependent type (Oubr et al.,
derivatives, combined with other drugs. Both com- 1997), among other indications. This study tested the in
pounds, rst isolated from medicinal plants already vivo antimalarial activity of B. pilosa collected in vari-
used hundreds of years ago, are still active against P. ous parts of Brazil as well as of plants cultivated in soil
falciparum drug-resistant parasites (Ridley, 2002). supplemented with humus and limestone against drug-
The Amazon forest has many species of plants used resistant P. falciparum parasites in vitro. Thin-layer
in traditional medicine (Brando et al., 1992). Approxi- chromatography (TLC) studies detected the presence
mately 50 species cited to treat malaria and /or fever of avonoids in all B. pilosa plants tested, although in
were tested in our previous work, several being active varying proles.
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, MATERIAL AND METHODS
the latter rather unstable when isolated (Brando et al.,
Collection of wild-type B. pilosa plants. Plant samples
* Correspondence to: Dr A. U. Krettli, Laboratory of Malaria, Centro de were collected in three different regions of Minas Gerais
Pesquisas Ren Rachou/FIOCRUZ, Av. Augusto de Lima 1715, Belo (MG), Brazil: (i) in the metropolitan area of Belo
Horizonte, MG, CEP 30190-002 Brazil.
E-mail: akrettli@cpqrr.ocruz.br
Horizonte (BH), the state capital, on several occasions;
Contract/grant sponsor: CNPq. (ii) in the rural area of Ibi (IB), a village in the east-
Contract/grant sponsor: FAPEMIG. ern region; and (iii) at a farm close to Montes Claros
Copyright 2004 John Wiley & Sons, Ltd. Received
Phytother. Res. 21 July(2004)
18, 634639 2003
Accepted 21 April 2004
Copyright 2004 John Wiley & Sons, Ltd.
ANTIMALARIAL ACTIVITY OF BIDENS PILOSA 635

(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.

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 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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