Pharmacology & Pharmacy, 2011, 2, 47-55
doi:10.4236/pp.2011.22006 Published Online April 2011 (http://www.SciRP.org/journal/pp)
47
Antitrypanosomal Activity of a Semi-Purified
Subfraction Rich in Labdane Sesquiterpenes,
Obtained from Flowers of Anthemis tinctoria,
against Trypanosoma cruzi
Nilza de Lucas Rodrigues Bittencourt1, Tânia Ueda-Nakamura1,2, Benedito Prado Dias Filho1,2,
Celso Vataru Nakamura1,2
1Programa de Pós-graduação em Ciências Farmacêuticas; 2Departamento de Ciências Básicas da Saúde, Laboratório de Inovação
Tecnológica no Desenvolvimento de Fármacos e Cosméticos, Universidade Estadual de Maringá, Maringá, Paraná, Brazil.
Received September 27th, 2010; revised December 15th, 2010; accepted December 29th, 2010.
ABSTRACT
In Brazil and several other Latin American countries, Chagas’ disease still constitutes a serious medical and social
problem, and there is a need to develop new, more-potent drugs with fewer side effects to effectively treat this disease.
We investigated the antitrypanosomal effect of a crude extract, fractions, and a semi-purified subfraction rich in a mix-
ture of isomeric labdane sesquiterpenes, obtained from flowers of Anthemis tinctoria, against Trypanosoma cruzi. In
epimastigote forms, the aqueous crude extract, dichloromethane fraction, and semi-purified subfraction showed a
dose-dependent inhibitory activity, with IC50 of 2.3 μg/ml, 1.8 μg/ml, and 0.2 μg/ml, respectively. In the interaction in-
dex, the semi-purified subfraction showed a reduction in both the percentage of infected LLCMK2 cells and the mean
number of amastigotes per infected cell. The cytotoxicity evaluation demonstrated that the cytotoxic concentrations of
the semi-purified subfraction were higher for LLCMK2 cells than for the protozoans, with a selectivity index of 35.0.
Epimastigote forms treated with the semi-purified subfraction showed ultrastructural and morphological alterations
such as rounding of the cells and bleb formation in the flagellum and cytoplasmic membrane. These results show that
the flowers from A. tinctoria may be a source of new drugs with antiprotozoal activity. However, additional in vitro and
in vivo studies are needed to validate the use of A. tinctoria in the treatment of Chagas’ disease.
Keywords: Antiprotozoan Activity, Medicinal Plants, Trypanosoma cruzi, Ultrastructure Alterations
1. Introduction
About 65% - 80% of the population in developing
countries essentially depends on plants for primary health
care. Some 25% of all modern medicines are derived
directly or indirectly from plants [1]. Many plants from
Brazilian ecosystems such as the savanna, and the
Atlantic and Amazon forests are used in traditional medi-
cine [2]. Also, many exotic plants that were introduced
into Brazil and incorporated into traditional medicine
display curative properties [3]. Various studies have
demonstrated a strong correlation between popular use
and experimenttally demonstrated pharmacological acti-
vity. Many plant extracts and essential oils have been
shown to exert in vitro and in vivo activity, which jus-
tifies research on plants used in traditional medicine [4].
Plants produce a variety of compounds with antimi-
crobial properties, which have led to the development of
new drugs for treatment of infectious diseases [5].
The family Compositae is one of the most species-rich
among the flowering plants. Anthemis L. is the second-
largest genus in this family with approximately 25 000
species, widely distributed in subtropical and temperate
areas. Species of Anthemis are widely used in the phar-
maceutical, cosmetic and food industries. Anthemis tinc-
toria L. is a perennial herb cultivated in Mediterranean
countries, and several secondary metabolites have been
identified in this species, such as volatile oils, triterpenes,
polyacetylenes, and flavonoids [6]. In traditional medi-
cine, this plant is used to treat liver problems and jaun-
dice [7]. Its flowers have well-known antiseptic and me-
dicinal properties, derived from flavonoids as well as es-

Antitrypanosomal Activity of a Semi-Purified Subfraction Rich in Labdane Sesquiterpenes, Obtained 48
from Flowers of Anthemis tinctoria, against Trypanosoma cruzi
sential oils [8]. In Europe, extracts, dyes, teas, and oint-
ments are used as anti-inflammatory, antibacterial, anti-
spasmodic, and sedative agents [9]. The antimicrobial ac-
tivity of essential oils and extracts from different species
of Anthemis has been studied previously [10,11].
Trypanosoma cruzi is the etiologic agent of Chagas’
disease, which in Brazil and several other Latin Ameri-
can countries constitutes, a serious social and medical
problem [12]. Transmission to vertebrates occurs through
feces of hemipteran insects contaminated with metacyclic
trypomastigotes, the infective stage of the parasite [13].
The acute phase of Chagas’ disease is frequently asymp-
tomatic, and the chronic phase usually develops 10 to 20
years after the infection, affecting about 10% to 30% of
infected individuals [14].
In spite of the impressive progress in the understand-
ing of the biology of T. cruzi, the drugs available (nifur-
timox and benzonidazole) are active on the acute stage of
Chagas’ disease, with about 80% effectiveness; but have
limited utility against the established chronic disease [15].
The side effects of both compounds can be quite severe
[16]. Therefore, trypanocidal drugs with less-serious side
effects are necessary. In this context, plants are a reser-
voir of chemical and biological diversity that has led to
the development of hundreds of pharmaceutical drugs
[17]. The objective of the present study was to investi-
gate the activity of the extracts, fractions, and a sequiter-
pene-rich semi-purified subfraction, obtained from flow-
ers of A. tinctoria, against T. cruzi.
2. Materials and Methods
2.1. General Experimental Procedures
The NMR spectra were obtained in VARIAN GEMINI
300 (7.05 T) spectrometers, using deuterated solvent,
TMS as the internal standard and a constant temperature
of 298 K. Sephadex LH-20; Silica gel 60 (70 - 230 and
230 - 400 mesh); TLC: silica gel plates F254 (0.25 mm
thickness).
2.2. Collections of the Plant
Flowers of Anthemis tinctoria were collected in Novem-
ber 2004 at the “Profa. Irenice Silva” Garden of Medici-
nal Plants of the State University of Maringá. The plant
was identified through authentic comparison by Dr. Cir-
ino Correia Júnior, and a voucher specimen (No. HUM
1133) is deposited at the Herbarium of the State Univer-
sity of Maringá, Paraná, Brazil.
2.3. Separation of the Components
Dried flowers of A. tinctoria (130 g) were extracted with
ethanol:water (9:1 v/v) by maceration for 8 days at room
temperature. The solvent was removed in a rotating
evaporator, to give an aqueous extract and a dark-green
residue. The aqueous extract was lyophilised (21.0 g) and
the water-insoluble residue was diluted with ethyl-acetate,
yielding the ethyl-acetate extract (4.71 g). The aqueous
and ethyl-acetate extracts were assayed against the epi-
mastigote form of T. cruzi. The aqueous extract (13 g) -
was submitted to vacuum-column chromatography (32 g
silica gel) and eluted with hexane, dichloromethane, ethyl
acetate, methanol, and methanol/water (9:1 v/v). Each
fraction (hexane, F1; dichloromethane, F2; ethyl acetate,
F3; methanol, F4; and methanol/water 9:1, F5) was as-
sayed for antitrypanosomal activity. The dichloromethane
fraction (500 g), which showed the highest inhibitory
activity, was chromatographed by column chromatography
in Sephadex-LH-20 and eluted with chloroform/methanol
(1:1 v/v). This process yielded 8 subfractions, denomi-
nated F2a, F2b, F2c, F2d, F2e, F2f, F2g, and F2h. Sub-
fraction F2 g (140 g) was chromatographed in Sephadex
phase with movable phase, chloroform/methanol (1:1
v/v), yielding the subfraction (11 g). The subfraction
was identified as a mixture of isomeric labdane ses-
quiterpenes by analyses of spectral data of 1H and 13C
(Chart 1).
2.4. Parasites
Epimastigote forms of T. cruzi Y strain were cultured in
LIT (Liver Infusion Triptone broth) [18] with 10% fetal
bovine serum (SFB) (Gibco Invitrogen Corporation, New
York, USA) at 28˚C for 96 h. Amastigote and trypomas-
tigote forms were grown in monolayers of LLCMK2 cells
in DMEM medium (Gibco Invitrogen) with 10% SFB in
5% CO2 at 37˚C.
2.5. Antiproliferative Activity against
Epimastigote Forms
The aqueous and ethyl-acetate crude extracts, fractions
(hexane, dichloromethane, ethyl acetate, methanol, and
methanol/water ) and the semi-purified subfraction were
dissolved in dimethyl sulphoxide (DMSO, Sigma Chemi-
cal Co., St. Louis, Missouri, USA) at a final concentra-
tion not exceeding 1% [19] and assayed against the epi-
mastigote form of T. cruzi. The experiments were per-
formed on 24-well polystyrene plates containing 1 ml of
diluted compound at different concentrations (from 1.0 to
1,000 μg/ml). The starting inoculum consisted of 106
parasites in logarithmic growth phase per well. The cells
were incubated at 28˚C and the growth was determined
by counting the parasites with a Neubauer hemocytome-
ter (Improved Double Neubauer Ruling) every 24 h over
a 7-day period. Benznidazole (N-benzyl-2-nitro-1-imida-
zolacetamide, Roche Pharmaceuticals, Rio de Janeiro,

Antitrypanosomal Activity of a Semi-Purified Subfraction Rich in Labdane Sesquiterpenes, Obtained
from Flowers of Anthemis tinctoria, against Trypanosoma cruzi
49
Chart 1. Fractionation scheme of sequiterpene from aqueous extract of flowers of Anthe m is t inctoria.
Brazil) was used as a reference drug. The assays were
performed in duplicate on separate occasions.
2.6. Growth Inhibition Assay of
Mammalian-Stage Amastigote
Form of T. cruzi
LLCMK2 cells (kidney cells from Mulatto monkey) were
plated on glass coverslips (diameter 13 mm) into
24-multiwell tissue culture plates and maintained in a
humidified 5% CO2 atmosphere at 37˚C. The cells were
infected with trypomastigote forms at a parasite/cell ratio
of 10:1. After 24 h, non-adhered parasites were washed
out, and fresh DMEM medium, and the semi-purified
subfraction containing sesquiterpene in various concen-
trations, was added. The cells were incubated in a hu-
midified 5% CO2 atmosphere at 37˚C for 96 h. The cov-
erslips were washed 3 times with PBS, fixed with
methanol and stained with Giemsa. Next, the coverslips
were mounted on glass slides using Entellan (Merck),
and the percentage of LLCMK2 cells with internalised
parasites (1) and the number of internalised parasites per
LLCMK2 cell (2) were determined by counting at least
200 cells per sample under a microscope (Olympus CX
31). The product of 1 and 2 determined the survival index.
2.7. Cytotoxicity Activity
LLCMK2 cells were seeded onto 24-well microtitre
plates at a concentration of 2.0 × 105 cells/ml and al-
lowed to proliferate for 48 h in DMEM containing 5%
FBS at 37˚C with 5% CO2 to form a cell monolayer.
Different concentrations of the semi-purified subfraction
containing sesquiterpene were applied to the monolayer
and incubated at 37˚C with 5% CO2 for 72 h. Next, the
cells were treated with 10% trichloroacetic acid at 4˚C
for one hour, gently washed in tap water, and allowed to
dry at room temperature. A solution of 0.4% sulforho-
damine B (in 1% acetic acid) was added to each well,
and the plate was kept protected from light for 30 min at
4˚C. The wells were washed four times with 1% acetic
acid, and 150 µl of 10 mM Tris-base was added and ho-
mogenised for 15 min. The absorbance was read at 530
nm in a microplate spectrophotometer (Bio Tek-Power
Wave XS). The CC50 (concentration that lysed 50% of
cells) of the subfraction was calculated.

Antitrypanosomal Activity of a Semi-Purified Subfraction Rich in Labdane Sesquiterpenes, Obtained 50
from Flowers of Anthemis tinctoria, against Trypanosoma cruzi
2.8. Evaluation of Morphological Alterations by
Scanning Electron Microscopy
Epimastigote forms treated with IC50 (0.2 µg/ml) or IC90
(1.0 µg/ml) of the semi-purified sesquiterpene subfrac-
tion for 96 h were collected by centrifugation, washed in
PBS and fixed with 2.5% glutaraldehyde in 0.1 M so-
dium cacodylate buffer, pH 7.2, containing 1.0 mM
CaCl2
at 4˚C. After fixation, small drops of the sample
were placed on a specimen support with poly-L-lysine.
Subsequently, the samples were dehydrated in graded
ethanol, critical-point dried in CO2, sputter-coated with
gold and observed in a SHIMADZU SS-550 Scanning
electron microscope.
2.9. Evaluation of the Ultrastructural Alterations
in T. cruzi by Transmission Electron
Microscopy
After treatment with IC50 or IC90 of the semi-purified
sesquiterpene subfraction for 96 h, epimastigote forms
were washed in 0.01 M phosphate-buffered saline and
fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate
buffer, pH 7.2. The cells were postfixed in a solution
containing 1% OsO4 and 0.8% potassium ferrocyanide in
0.1 M cacodylate buffer, dehydrated in acetone, and em-
bedded in Epon. Thin sections were collected on a cop-
per grid (300 mesh), stained with uranyl acetate and lead
citrate, and observed in a Zeiss 900 transmission electron
microscope.
2.10. Statistical Analysis
Statistical analysis was performed with the program
GraphPad Prism 4 (GraphPad Software, San Diego,
California, USA). One-Way Anova was applied, and a p-
value less than 0.05 was regarded as significant.
3. Results and Discussion
In the attempt to develop new drugs for treatment of in-
fectious diseases, studies are carried out with compounds
of both natural and synthetic origin. Many studies have
demonstrated that crude extracts, fractions, and com-
pounds isolated from medicinal plants exhibit antiproton-
zoal activity [20-26]. In this study, we evaluated the anti-
trypanosomal activity of the crude extract, fractions, and
a subfraction rich in a mixture of isomeric labdane ses-
quiterpenes, obtained from flowers of A. tinctoria, against
epimastigote, and amastigote forms of T. cruzi.
The analyses of the 1H and 13C NMR spectrum of the
semi-purified subfraction showed a signal of a mixture of
isomers of cross-conjugated terpenoid ketones derived
from labdane sesquiterpenes that are frequently found in
the family Asteraceae [27]. Three types of classes of
secondary metabolites have been detected in Anthemis:
polyacetylenes [28], flavonoids [29], and sesquiterpene
lactones [30]. Previous chemical studies of species of
Anthemis have shown the presence of sesquiterpene lac-
tones [31-37]. The three major types of sesquiterpene
lactones are germacrolides, eudesmanolides, and guaia-
nolides [38]. Recently it was demonstrated that anthecu-
larin, a sesquiterpene lactone isolated from Anthemis
auriculata, shows antimalarial activity, and also antitry-
panosomal activity against Trypanosoma brucei [39].
The hydroalcoholic crude extracts, fractions, and semi-
purified subfraction obtained from A. tinctoria flowers
were used in order to investigate the antiprotozoal activ-
ity of this plant against T. cruzi. A progressive increase
in the antitrypanosomal effect was observed in the course
of the purification process. Figure 1(a) shows the per-
centage of growth inhibition of the epimastigote form
treated with the aqueous phase of the crude extract for 96
h of incubation at 28˚C. This extract showed a dose-de-
pendent inhibitory activity of 77.4% and 91.9% at 5 and
10 μg/ml, respectively. In the same concentrations, the
ethyl-acetate extract showed an inhibitory activity of
31.7% and 76.8%. The 50% inhibitory concentration (IC50)
of the crude extract aqueous phase was 2.3 g/ml. On the
basis of this finding, the crude aqueous extract was
fractionated on silica gel into five fractions: hexane (F1),
dichloromethane (F2), ethyl-acetate (F3), methanol (F4),
and methanol:water (F5). The F2 fraction showed an IC50
of 1.8 g/ml (Figure 1(b)), and the F3 fraction an IC50 of
5.0 g/ml (data not shown). The other fractions (F1, F4,
and F5) showed lower inhibitory activity, with IC50 of
26.8, 23.6, and 145 g/ml, respectively. Subsequently,
the F2 fraction was submitted to the Sephadex column,
yielding 8 subfractions: F2a, F2b, F2c, F2d, F2e, F2f,
F2g, and F2h. Figure 1(c) illustrates the inhibitory ac-
tiveity of the F2g subfraction, with a percentage of inhi-
bition of growth of the epimastigote form above 90% for
all concentrations used (1 - 1000 µg/ml). The F2g sub-
fraction, which was rich in a mixture of isomeric labdane
sesquiterpenes, showed an IC50 at 0.2 g/ml. In com-
parison, Luize et al. [40] investigated the in vitro anti-
proliferative effect of eupomatenoid-5, a sesquiterpene
isolated from leaves of Piper regnellii var. pallecens, a
plant of the same family (Compositae) as A. tinctoria,
against T. cruzi. Our results obtained with a mixture of
sesquiterpenes also showed that this mixture was more
active than Benzonidazole.
The effect of the semi-purified subfraction obtained
from flowers of A. tinctoria on the interaction of T. cruzi
with LLCMK2 cells was evaluated. The treatment of
LLCMK2 cells infected with the amastigote form showed
that the semi-purified subfraction had a dose-dependent

Antitrypanosomal Activity of a Semi-Purified Subfraction Rich in Labdane Sesquiterpenes, Obtained 51
from Flowers of Anthemis tinctoria, against Trypanosoma cruzi
(a)
(b)
(c)
Figure 1. Effects of crude extract aqueous phase (a), di-
chloromethane fraction (b) and semi-purified subfraction (c)
obtained from flowers of Anthemis tinctoria, on the growth
of Trypanosoma cruzi epimastigote form.  Control;  1000
µg/ml; ▲ 100 µg/ml;  50 µg/ml;  10 µg/ml;  5 µg/ml;
 1 µg/ml.
antitrypanosomal activity (Figure 2), leading to consid-
erable reduction in both the percentage of infected cells
and the mean number of parasites per infected cell. After
96 h of incubation, the percentage of LLCMK2 cells with
internalised parasites was higher for the control than for
cells treated with the semi-purified subfraction. At that
time, the control showed a mean of 34.1 amastigotes per
cell, with 40.6% of cells infected. Cells treated with 1.0
g/ml showed a mean of 18.1 internalised amastigotes
per cell, with 22.7% of cells infected. Treatment of the
cells with 5.0 g/ml resulted in only 4.7% infected cells
and 4.2 parasites per cell.
Important criteria for the investigation of compounds
with activity against T. cruzi are both their therapeutic
potential, and the lack of a cytotoxic effect on mam-
malian cells. We evaluated the cytotoxicity in LLCMK2
cells treated with the crude extract aqueous phase, di-
chloromethane fraction, and the semi-purified subfraction
(Figure 3). The 50% cytotoxicity concentration (CC50) in
LLCMK2 cells treated with the crude extract aqueous
phase, dichloromethane fraction, and semi-purified sub-
fraction were 17.3 μg/ml, 4.0 μg/ml, and 7.0 μg/ml,
respectively. The best selectivity index (SI) ratio (CC50
for LLCMK2 cells/IC50 for antiprotozoa) was obtained by
the semi-purified subfraction, with SI of 35.0. The di-
chloromethane fraction and crude extract showed SI of
2.2 and 7.5, respectively (Table 1).
Morphological alterations in epimastigote forms treated
Figure 2. Growth inhibition assay of mammalian-stage
amastigote form of Trypanosoma cruzi treated with a semi-
purified subfraction obtained from Anthemis tinctoria.
Figure 3. Cytotoxicity activity on LLCMK2 cells. () aqueous
extract, (▲) dichloromethane fraction; () semi-purified
subfraction obtained from flowers of Anthemis tinctoria.
Table 1. Citotoxicity, growth inhibition and selectivity index
of the crude extract aqueous phase, dichlorometane fraction,
and semi-purified subfraction obtained of Anthemis tinctoria.
CC50 IC50
Drugs
(µg/ml) (µg/ml)
IS
Crude extract aqueous phase 17.3 2.3 7.5
Dichlorometane fraction 4.0 1.8 2.2
Semi-purified subfraction 7.0 0.2 35.0
IS = CC50/IC50

Antitrypanosomal Activity of a Semi-Purified Subfraction Rich in Labdane Sesquiterpenes, Obtained 52
from Flowers of Anthemis tinctoria, against Trypanosoma cruzi
with the semi-purified subfraction were observed by
scanning electron microscopy. Epimastigote forms in the
presence of the semi-purified subfraction at IC50 (0.2
g/ml) showed distortions of the parasite cell body, and
the cell shape was severely affected. Increase of the cell
volume and rounding of the cell body were observed
(Figures 4(b), (c), and (d)). At IC90 (1.0 g/ml), altera-
tions were more evident (Figures 4 (e) and (f)). Figure
4(a) shows the characteristic elongated shape of an un-
treated protozoan, or treated only with 1% DMSO (con-
trol), with a terminal flagellum that emerges from the
flagellar pocket and remains tightly attached to the cell
body along its length, typical of the epimastigote form.
Ultrastructural alterations of the epimastigote form
treated with the semi-purified subfraction were observed
by transmission electron microscopy. Untreated T. cruzi
or epimastigotes treated with 1% DMSO showed the
typical ultrastructure (Figure 5(a)). Epimastigotes treated
with the semi-purified sesquiterpene-rich subfraction at
concentrations of IC50 (Figures 5(b), (c), and (d)) and IC90
(Figures 5(e) and (f)) showed ultrastructural alterations.
One important change took place in the membrane lining
the cell body and flagellum, with portions of the mem-
brane detached from the body, forming blebs at the mem-
brane flagellar (Figures 5(b) and (d)). The formation of
blebs was also observed in T. cruzi treated with com-
(a) (b)
(c) (d)
(e) (f)
Figure 4. Morphological alterations of Trypanosoma cruzi
epimastigote form cultured at 28˚C for 96 h, observed by
scanning electron microscopy, in the absence (a) and pres-
ence of the semi-purified subfraction at concentrations of
IC50 (b), (c), and (d) and IC90 (e) and (f). Bar = 1 μm.
(a) (b)
(c) (d)
(e) (f)
Figure 5. Ultrastructural alterations of Trypanosoma cruzi
epimastigote forms cultured at 28˚C for 96 h, observed by
transmission electron microscopy, in the absence (a) and
presence of semi-purified subfraction at concentrations of
IC50 (b), (c) and (d) and IC90 (e) and (f) er-endoplasmic
reticulum; f-flagellum; m-mitochondrion; n-nucleus;
v-vacuole. Bar = 1 μm.
pounds ER27856 and BPQ-OH (3-biphenyl-4yl)-3-hy-
droxy quinuclidine BPQ-OH) [41]. These compounds are
inhibitors of scalene synthase, the enzyme involved in the
biosynthesis of ergosterol. The change in the chemical
composition of the membrane alters the ratio of phos-
pholipids and sterols, affecting the stability of the mem-
branes, and especially the integrity of the cell body [42].
This may explain the morphological changes observed
in epimastigotes treated with the sesquiterpene-rich sub-
fraction of the flower of A. tinctoria. Pedroso et al. [43]
also showed the presence of blebs in cell and flagel-
lar-pocket membranes in Crithidia deanei treated with
essential oil from Cymbopogons citratus.
4. Conclusions
In conclusion, this study showed that extracts, fractions,
and a semi-purified subfraction containing sesquiterpene
obtained from flowers of A. tinctoria have potential anti-

Antitrypanosomal Activity of a Semi-Purified Subfraction Rich in Labdane Sesquiterpenes, Obtained 53
from Flowers of Anthemis tinctoria, against Trypanosoma cruzi
proliferative activity in vitro against T. cruzi. Therefore,
natural products may be a source of new drugs with anti-
protozoal activity that could be used to treat parasitic
infectious diseases such as Chagas’ disease.
5. Acknowledgements
This study was supported through grants from DECIT
/SCTIE/MS and MCT by Conselho Nacional de Desen-
volvimento Científico e Tecnológico (CNPq), Financia-
dora de Estudos e Projetos (FINEP), Programa de Núcleos
de Excelência (PRONEX/Fundação Araucária), and Pro-
grama de Pós-graduação em Ciências Farmacêuticas da
Universidade Estadual de Maringá.
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