Journal of Ethnopharmacology 57 (1997) 131 – 138

Antimalarial activity of extracts and fractions from Bidens

pilosa and other Bidens species (Asteraceae) correlated with the
presence of acetylene and flavonoid compounds

M.G.L. Brandão a, A.U. Krettli b,*, L.S.R. Soares b, C.G.C. Nery a, H.C. Marinuzzi b
a
Laboratório de Farmacognosia, Faculdade de Farmácia, Uni6ersidade Federal de Minas Gerais (UFMG),
A6. Olegário Maciel 2360, 30180 -112 Belo Horizonte, Brasil
b
Centro de Pesquisas René Rachou, FIOCRUZ/MG, A6. Augusto de Lima, 1715, 30190 -002 Belo Horizonte MG, Brasil

Received 21 January 1997; received in revised form 1 May 1997; accepted 13 May 1997

Abstract

After interviewing natives and migrants from the Amazon region of Brazil about plants traditionally used for
treatment of malaria fever and/or liver disorders, we selected and identified 41 different species, including the native
Bidens (Asteraceae). We have undertaken an antimalarial study of Bidens pilosa and other species of Bidens from
abroad. The crude ethanol extracts (whole plant, leaves and roots) and the chloroform and butanol fractions from B.
pilosa at concentrations of 50 mg/ml caused up to 90% inhibition of Plasmodium falciparum growth in vitro. In vivo
the fractions caused partial reduction of Plasmodium berghei parasitemia in mice. The ethanol extracts from nine
different Bidens species collected outside Brazil were tested, and seven inhibited parasite growth in vitro by 65 – 91%.
As B. pilosa appears to be a promising antimalarial agent, we further characterized the substances responsible for
such activity. HPLC analysis using a photo diode-array detector showed phenyl acetylene and flavonoids in the
ethanol extract from the leaves and roots. The chloroform fractions from the roots, which caused 86% inhibition of
parasite growth in vitro, contained a major component identified as 1-phenyl-1,3-diyn-5-en-7-ol-acetate. The
association of antimalarial activity and the presence of acetylene compounds is discussed. In summary, all species of
Bidens which had aliphatic acetylenes (6–14 each) were also very active, whereas extracts of B. par6iflora and of B.
bitternata with none or the three acetylenes, respectively as reported in literature, were inactive or had a borderline
activity in vitro. © 1997 Elsevier Science Ireland Ltd.

Keywords: Malaria; Chemotherapy; Plant extracts; Bidens pilosa; Acetylenes

1. Introduction

The malaria problem has worsened in the

* Correponding author. world. There are increasing numbers of cases and

0378-8741/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved.

PII S 0 3 8 - 8 7 4 1 ( 9 7 ) 0 0 0 6 0 - 3
132 M.G.L. Brandão et al. / Journal of Ethnopharmacology 57 (1997) 131–138
new areas of transmission. New antimalarials are
needed because most P. falciparum parasites are
now resistant to those currently used. Interest in
plants as new antimalarials has been stimulated
by the isolation of artemisinin from Artemisia
annua a compound very active against drug resis-
tant malaria parasites (Klaymann, 1985). This
plant, used to treat malaria in China for thou-
sands of years, does not exist in Brazil where one
native species of the genus, A. 6erlotorum, exists
but has proven inactive experimentally (our un-
published data). Other plants traditionally used
for treatment of malaria in Brazil have been de-
scribed (Brandão et al., 1985) by interviewing
natives and migrants in the Amazon region, where
the disease has always been prevalent. Of the 41
plants discovered (Brandão et al., 1992) several
were active as crude extracts against experimental
malaria in vitro and in vivo (Carvalho et al.,
1991).
Bidens pilosa, a plant also used in traditional
medicine to treat malaria (N’Dounga et al., 1983),
is found in tropical and subtropical regions of the
world. It is used in traditional medicine as an
anti-inflammatory, diuretic, anti-rheumatic, an-
tibiotic agent to treat nematodes and against dia-
betes (Wat et al., 1980; Geissberger and Sequin,
1991). Nevertheless, the 1-phenyl-3,5,7-heptatriyn,
isolated from the plant extract had a low in vitro
activity against P. falciparum and was inactive in
rodent malaria and other parasitic infections
(N’Dounga et al., 1983). In Brazil, B. pilosa and
B. bipinnatus are considered liver protective agents
the latter being used to treat malaria in the Ama-
zon region (Brandão et al., 1992). We here report
on the antimalarial activity and chemical compo-
sition of B. pilosa, a plant which is indigenous to
Brazil, as well as of nine other Bidens species
collected in the Old World.
2. Methodology
2.1. Plant samples
B. pilosa L. was collected around Pampulha
Lake, Belo Horizonte (Minas Gerais), Brazil and
identified by T.S.M. Grandi. A voucher specimen
(GR101) is deposited at the Pharmacognosy Lab-
oratory, Faculty of Pharmacy, Belo Horizonte.
Other species of Bidens collected outside Brazil—
B. frondosa, B. tripartitus, B. pilosa, B. ferulaefo-
lia, B. bipinnatus, B. maximo6icziana, B.
campylotheca, B. bitternata and B. par6iflora—
were tested. They were kindly provided as dried
whole plants in 1992 by Professor H. Wagner,
from the Institute of Pharmaceutical Biology,
University of Munich, Germany, where voucher
specimens are deposited.
2.2. Extract and fraction preparation
Two methods were used to prepare extracts
and fractions from the air-dried plant samples:
(i) 300 g samples (whole plant) of B. pilosa and 5
g of the other species of Bidens were percolated
exhaustively with 90% ethanol; after evaporation
at 40–50°C, a crude residue was obtained; (ii)
300 g of air-dried leaves, stems and roots of B.
pilosa were percolated sequentially with ether
and ethanol; the dried ethanolic extracts were
dissolved in water and extracted with chloro-
form. The chloroform fraction was separated and
the aqueous solution (fraction H2OA) was ex-
tracted with n-butanol to provide fraction H2OB.
The ether/ethanol extracts and the chloroform/
n-butanol fractions were vacuum-dried; the
aqueous fraction was then lyophilized and used
for further tests.
2.3. Chromatographic and spectroscopic analysis
Analytical TLC was carried out on silica gel
60 F254, 0.25 mm plates (Merck, Darmstadt).
For separation and identification of compounds
the following solvent systems (a–b) and spray
reagent (c) were used. (a) Chloroform-acetone
(9:1); (b) hexane-ethylacetate (8:2); and (c)
vanillin/sulfuric acid, followed by heating. The
HPLC analysis was performed on an HP 1090
liquid chromatography unit equipped with an
HP 1040A pump and a UV diode-array detector,
using a Hibar LiChroCART Lichrospher 100
RP-18, 5 mm, 125-4 column (Merck). The ex-
tracts and fractions B. pilosa were evaporated
 M.G.L. Brandão et al. / Journal of Ethnopharmacology 57 (1997) 131–138 133

Fig. 1. (A) HPLC chromatogram of the chloroform fraction from Bidens pilosa roots shows four peaks identified by the diode array
method through their UV spectrum as either acetylene (I, II, IV) or chalcone (III). The predominant compound, (II), showed a
retention time of 19.82 min and a UV spectrum with typical bands of phenylacetylene (B). The structure being illustrated in (C) is
1-phenyl-1,3-diyn-5-en-7-ol-acetate.

to dryness and solubilized in methanol. Chloro-
phyll was removed by chromatography on Sep-
Pak C-18 cartridges with methanol as eluent.
Separation of compounds was performed with
acetonitrile-H2O mixtures as mobile phase, with
the following gradient systems: (d) Gradient of
acetonitrile: water from 10:90 to 90:10 in 20 min,
for chloroform fractions and ethanol extracts and
(e) gradient of acetonitrile 10:90 to 25:75 in 30
min, for butanol and aqueous fractions. Diode-ar-
ray detection allowed a rapid characterization of
the separated compounds by their Ultraviolet
spectra from 210 to 365 nm. IR spectrum was
recorded in Kbr pellets. 1H NMR was recorded in
CDCl3 at 250 MHz, d relative to TMS (0 ppm)
and J in Hz.
2.4. Isolation and identification of compound II
(Fig. 1c)
1-phenylhepta-1,3-diyne-5-en-7-ol-acetate was
described previously (Bohlmann et al., 1964; So-
erensen and Soerensen, 1958). It is a yellow oil at
room temperature (around 28°C) when isolated
from the ethanol extract by flash chromatography
on silica gel, using mixtures of hexane:ethyl ac-
etate (90:10–10:90). The column yielded a total of
68 fractions which were monitored by TLC with
solvent systems (a) and spray reagent (c). Purifica-
tion of compound II was performed by rechro-
matography of fractions 22–28 on silica gel with
the mixture hexane:ethyl acetate (70:30). The pu-
rity of the compound was confirmed by HPLC
which showed the presence of a single peak at
17.82 min and the same characteristic UV spec-
trum. All procedures were performed in darkness
and at temperatures not exceeding 20°C. It be-
came a yellow pale powder when stored at 4°C
and suffered gradual darkening on exposure to air
and light. By TLC this compound showed on
Rf = 0.64 in solvent system (a), Rf = 0.35 in (b)
and developed a brown colour with (c). It also
showed strong blue fluorescence at 254 nm and
yellowish fluorescence at 365 nm. The spectro-
scopic data are UV l max 202, 239, 250, 264, 278,
296 and 317 (Lam and Hansen, 1990). The
ence of acetate groups in the molecule was confi-
rmed by IR spectrum (Kbr), which showed a
134 M.G.L. Brandão et al. / Journal of Ethnopharmacology 57 (1997) 131–138
strong absorption at 1730 cm − 1. Other absorp-
tions were at 2460, 2410, 2380 (conjugated tryin
system), 1450, 1370, 1280, 1270, 1120, 1080, 1030
and 740 cm − 1. 1HNMR (250 MHz, ppm, CDCl3):
d 7,5 (5H, m, aromatic-H), d 2,4 (3H, s, CH3
acetate), d 4,9 (2H, dd, CH2 −0).
2.5. Antimalarial tests in 6itro
For the in vitro tests we used Plasmodium falci-
parum parasites, strain BHz 26/86, isolated from a
patient from Rondônia, Brazil in our laboratory
(Rocha et al., 1987). Cultured blood samples with
high parasitemia have been maintained in liquid
nitrogen and are defrosted as needed. Cultivation
was performed according to Trager and Jensen,
1976. Complete medium was added to a final
volume of 8 ml in culture Petri dishes (Falcon,
NJ) with human blood at 1% parasitemia and a
5% hematocrit. The experiments were set up as
described below, with 8 – 12 drug-free wells ran-
domly spread in each plate containing only com-
plete medium (RPMI supplemented with 10%
human serum) and control wells with standard
doses of the antimalarial chloroquine or with
complete medium plus 0.01% Tween-80.
The in vitro tests of extracts and fractions were
performed in cultures for a total of 72 h, in
96-well plates (Falcon, NJ) as described (Carvalho
et al., 1991). Briefly, the drugs were dissolved in
0.01% of Tween-80 in water, used at final concen-
trations of 50 and 25 mg/ml and incubated with
the parasite suspension (1% parasitemia). In some
experiments lower doses of plant extracts were
used as specified in the results. At 24 and 48 h of
culture, the medium was aspirated and replaced
with fresh medium, with or without drugs. Blood
samples were taken at 72 h, smeared, methanol
fixed, Giemsa-stained and examined microscopi-
cally to determine the percent parasitemia. Each
extract dose was tested in duplicate. A total of
3000 erythrocytes were counted and all smears
were read coded.
2.6. Antimalarial tests in 6i6o
The rodent Plasmodium berghei, strain NK, is
maintained in the laboratory through weekly pas-
sages of erythrocytic parasites into mice by in-
traperitoneal injection (ip) (Carvalho et al., 1991).
The experimental mice were inoculated with 106
infected red blood cells (ip) and the next day were
randomly divided into the following groups of
four to six mice each: (a) Untreated controls; (b)
chloroquine-treated; (c) treated with the test drugs
dissolved in Tween-80 in water; and (d) controls
treated with Tween-80 (final dilution of 0.01% in
(c) and (d)). Treatment was given orally (1000
mg/kg for the test drugs, and 100 mg/kg for
chloroquine) for four consecutive days. On the
5th day after inoculation, blood smears were pre-
pared, fixed with methanol and stained with
Giemsa. Parasitemia was determined in coded
smears by counting 3000 erythrocytes randomly.
By comparison with the untreated controls, the
inhibition of parasite growth was then calculated.
A drug was considered partially active when it
reduced the parasitemia by more than 30%.
3. Results
In vitro parasitemia of the P. falciparum cul-
tures by day 4 in one representative experiment is
shown in Table 1. Without drug (medium only) it
was on average, 2.5%. In the presence of 0.01%
Tween-80, parasitemias were similar to controls
whereas chloroquine, added at concentrations of
6.5–102 ng/ml, significantly inhibited parasite
Table 1
Percent parasitemia and reduction of parasite growth at day 4
of P.falciparum cultures in control wells with only complete
culture medium, in the presence of either 0.01% Tween-80 or
chloroquine used at different concentrations
Culture wells Mean para- Reduction of parasite
sitemiaa ( 9 growth (%)
S.D.)
Medium 2.53 ( 90.78) 0
Tween-80 2.47 ( 90.62) 0
Chloroquine (ng/ml)
102 0.02 ( 90.03) 99.2
25.5 0.22 ( 90.25) 91.3
6 0.65 ( 90.77) 74.3
a
Average of eight wells (medium) or four wells (Tween and
chloroquine) expressed as a percentage.
 M.G.L. Brandão et al. / Journal of Ethnopharmacology 57 (1997) 131–138
Table 2
In vitro inhibition of Plasmodium falciparum growth (%) by
extracts and fractions from different parts of Bidens pilosa
Part used Ether Ethanol Chloroform Butanol
Whole Plant NT 90a NT NT
Stems 33 NT 47 NT
Leaves 0 90 94a 79
Roots 0 90 86 68
B. pilosa used at a concentration of 50 mg/ml in the culture
medium evaluated in comparison to control cultures without
antimalarial drugs (no inhibition). NT, not tested.
a
At the dose of 25mg/ml parasite growth inhibition was about
80%.
growth (Table 1). In each of the experiments
with Bidens, these controls were included (data
not shown).
Crude extracts and fractions of Bidens pilosa
were initially tested in vitro against P. falci-
parum in cultures (Table 2). The crude ethanol
extract of the whole plant, leaves and roots at
concentrations of 25 – 50 mg/ml caused signifi-
cant in vitro inhibition of P. falciparum growth,
in relation to control cultures with no drugs
added to the medium. The chloroform fractions
obtained from stems, leaves and roots, caused
47, 94 and 86% inhibition, respectively of para-
site growth, whereas the butanol fractions of the
leaves and roots inhibited growth 79 and 68%.
The results of the antimalarial tests with
crude ethanol extracts from nine other species of
Bidens show that seven of them were very active
at concentrations of 25 and 50 mg/ml or even
lower (Table 3). The total number of acetylene
and thiophene compounds, previously identified
by others in such plants (Table 3), are also
shown. They varied from six to 14 among the
more active species. No acetylene compounds
have yet been isolated from B. par6iflora, shown
to be inactive in our experiments, whereas B.
bitternata, with a borderline activity, had only
three isolated acetylenes. The water extracts
from all species were inactive when tested in
vitro (data not shown).
HPLC analysis of the active ethanol extract
from the leaves of B. pilosa showed the presence
of several constituents, identified as acetylene
135
and phenolic compounds based on their UV
spectra, generated by diode-array detection. The
ethanol extract from the roots had two main
components with gradient d, at tR 19.82 and
10.42 min, identified as a phenyl acetylene and
flavonoid, respectively. The major compound de-
tected in the chloroform fraction was phenyl
acetylene at tR 19.82 min. The HPLC chro-
matogram (A) and the UV spectra (B) of this
compound (compound II), is shown (Fig. 1).
The other constituents were identified as
aliphatic acetylenes (I and III) and a chalcone
(IV) by their characteristic UV spectra. HPLC
analysis of the butanol fractions with gradient e
showed the absence of acetylene and the pres-
ence of several polar compounds, especially
flavonoids, chalcones and chlorogenic acids.
The aqueous fraction A from the roots, with
low activity (34% parasitemia inhibition, data
not shown), showed the presence of a flavonoid
at tR 10.42 min with gradient e. The aqueous
root fraction B, and the ether extracts obtained
from leaves, stems and roots were inactive (not
shown). By TLC and HPLC, we verified the ab-
sence of acetylenes and flavonoids in these ex-
tracts.
In the in vivo tests in mice the antimalarial
chloroquine control inhibited parasite growth by
100% whereas 0.01% Tween-80, used to dissolve
the extracts, had no effect on malaria para-
sitemia. The crude ethanol extract and the chlo-
roform fractions of B. pilosa were tested in mice
infected with P. berghei. The whole plant, but
not the stems, caused reduction of parasitemia
by around 40% (data not shown).
The dried ethanol extract was fractionated by
column chromatography purifying the phenyl
acetylene, 1-phenylhepta-1,3-diyne-5-en-7-ol-ac-
etate, the principal constituent of the chloroform
fraction (compound II, Fig. 1C), identified by
UV, IR and NMR. This compound had been
previously isolated from the roots of B. pilosa
(Bohlmann et al., 1964) and from Coreopsis spe-
cies (Soerensen and Soerensen, 1958), both from
Asteraceae family. It was not tested as a
purified component due to its quick decomposi-
tion, as explained above.
 136

Table 3
In vitro inhibition of Plasmodium falciparum growth (%) by crude ethanol extracts of several species of Bidens (whole plant) collected outside Brazil and the presence
of acetylene and related compounds (thiophene, phenyl and aliphatic acetylene) previously identified by other authors

Bidens species tested Reduction of parasite growth Author reference Number of com-
at doses (mg/ml) pounds identified

50 25 20 10 5

B. frondosa, L. 91 84 77 19 0 Christensen and Lam, 1991 6

B. tripartitus, L. 87 63 70 36 20 Marchant, 1988; Christensen et al., 1990; Christensen and Lam, 1991 13
B. pilosa, L. 83 72 62 8 0 Bohlmann et al., 1964; Wat et al., 1979; Hoffman and Hoelzl, 1988; 11
N’Dounga et al., 1983; Christensen and Lam, 1991
B. ferulaefolia, DC. 82 71 98 75 Christensen and Lam, 1991 12
B. bipinnatus, L. 71 46 ND ND ND Christensen and Lam, 1991 9
B. maximo6icziana, Willd. 70 71 ND ND ND Christensen and Lam, 1991 6
B. campylotheca, Schultz Bip. spp. 65 56 ND ND ND Christensen and Lam, 1991; Bauer et al., 1992; Redl et al., 1994 14
campylotheca
B. bitternata, DC. 38 37 ND ND ND Christensen and Lam, 1991 3
B. par6iflora, Willd. 0 0 ND ND ND No acetylene reported in the literature 0

Experiment 1 (50, 25 mg/ml) and experiment 2 (20, 10 and 5 mg/ml) were performed at different times.
ND, not done.
M.G.L. Brandão et al. / Journal of Ethnopharmacology 57 (1997) 131–138
 M.G.L. Brandão et al. / Journal of Ethnopharmacology 57 (1997) 131–138
4. Discussion
The first evaluation of B. pilosa as an anti-
malarial was reported long ago by Spencer et al.
(1947), who observed that the methanol and
aqueous extracts of the whole plants were inactive
against Plasmodium cathemerium, a species that
causes malaria in birds. More recently, a low in
vitro activity against P. falciparum and in vivo
against P. berghei was reported (N’Dounga et al.,
1983). Bidens species are largely used in tradi-
tional medicine as a source of antibiotics, liver
protection, antimalarials and their activities are
explained by the abundant production of phenyl
acetylenes. Biological evaluations have been per-
formed with 1-phenyl-3,5,7-heptatriyn, present in
the leaves of Bidens plants (N’Dounga et al.,
1983) as well as other phenyl acetylene com-
pounds, some of which require ultraviolet light
for expression of activity. The phenyl acetylene
compounds and derivatives (thiophene), for in-
stance, present potent phototoxic activity against
a range of organisms, including fungi, bacteria,
nematodes, insects and protozoa (Arnason et al.,
1980; Wat et al., 1979).
Our data show that ethanol crude extracts ob-
tained from leaves and roots of B. pilosa had
strong antimalarial activity against the human
parasite P. falciparum. The active chloroform
fraction from the roots has the phenyl acetylene,
1-phenyl-1,3-dyne-5-en-7-ol-acetate as a major
component, plus two other acetylenes and one
chalcone (compound III in Fig. 1). We suggest
therefore, that acetylenes may be responsible for
the observed antimalarial activity. This hypothesis
is strengthened by our results with other species of
Bidens active in vitro, from which several acetyle-
nes have been previously described (Table 3).
Furthermore, the crude ethanol extract of B.
par6iflora, which has no acetylene compounds, as
yet described, was inactive. In addition, 1-phenyl-
3,5,7-heptatriyn has been found in B. tripartitus,
B. ferulaefolia and B.campylotheca (Bauer et al.,
1992; Redl et al., 1994) and is here shown to be
active, while 1-phenylhepta-1,3-diyne-5-en-7-ol-
acetate has been found in B. bitternata (Chris-
tensen and Lam, 1991) with only borderline
antimalarial activity. Data showing that the iso-
137
lated 1-phenyl-3,5,7-heptatriyn has low or no ac-
tivity against P. falciparum and P. berghei
(N’Dounga et al., 1983) may be attributed to its
instability under the assay conditions. Our present
experience shows that the isolated compound II,
an yellow oil, went dark and solid when exposed
to air for 24–48h, at room temperature, or after 1
week at 4°C. Decomposition by air and heat is
likely to explain the negative antimalarial results
with the acetylene isolated from this plant, as
described by N’Dounga et al. (1983) as well as in
our present results. In the crude ethanol extract,
the presence of chlorophyll and other phenolic
constituents seems to increase its stability. By
HPLC analysis we have observed the presence of
compound II, in crude ethanol extracts stored at
4°C, for about 6 months (data not shown).
Several extracts and fractions of B. pilosa were
inactive in vivo against P. berghei when tested
weeks after isolation (data not shown). Only the
ethanol extract and the chloroform fraction of the
whole plant caused a consistent, although low,
reduction of parasitemia, a result attributed to the
possible protective effect of chlorophyll. The ther-
apeutic usefulness of acetylene compounds seems
limited since they are easily oxidized by air and
light, a problem we are now trying to overcome.
Another possibility is that the antimalarial effects
of Bidens result from association of acetylenes
with other phenolic compounds and with the
flavonoids since in the chloroform fraction from
the roots we showed one chalcone and two other
acetylenes. The flavonoids are substances with
anti-oxidative properties which could increase the
stability of the acetylenes (Marchant, 1988).
The solubility of acetylene compounds in
aqueous solutions is low and these substances are
easily decomposed by heating. The ‘garrafadas’
(maceration of the plant in alcohol) of B. bipinna-
tus, not teas or tisanes, are used in the Amazon to
treat malaria. They may cure the disease since
ethanol increases the solubility of acetylene and
flavonoids. Indeed, the ethanol extract of B. pilosa
was shown here to be active. The involvement of
phenolic compounds, as in the case of
artemisinine activity against malaria has been
shown (Elford et al., 1987). Therefore, it is also
possible that in theB. pilosa extracts, the lipid
138 M.G.L. Brandão et al. / Journal of Ethnopharmacology 57 (1997) 131–138
soluble compounds (acetylenes) have their activity
potentiated by hydrophilic substances (flavo-
noids).
In conclusion, we consider B. pilosa a promis-
ing antimalarial because of its natural occurrence,
its wide distribution in the country and the strong
activity of its extracts and fractions shown here.
Several other Bidens species from outside Brazil
also have antimalarial action and they should be
further investigated.
Acknowledgements
We thank Prof. H. Wagner (Munich) for HPLC
diode-array facilities and for supplying the other
Bidens species tested, to Jessica Kissinger, Márcio
M. Coelho and Dr Paul Williams for English
corrections. Research supported by CNPq
(Process No. 521279/93 – 3 and 523231/94),
FAPEMIG (CBS 860/90) and IFS/Stockholm
(F2191-1).
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