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Research Article
Anthelmintic Activity of Crude Extract and Essential Oil of
Tanacetum vulgare (Asteraceae) against Adult Worms of
Schistosoma mansoni
Loyana Silva Godinho,1Lara Soares Aleixo de Carvalho,1
Clarissa Campos Barbosa de Castro,1Mirna Meana Dias,1Priscila de Faria Pinto,2
Antônio Eduardo Miller Crotti,3Pedro Luiz Silva Pinto,4Josué de Moraes,5
andAdemarA.DaSilvaFilho
1
1Faculdade de Farm´
acia, Departamento de Ciˆ
encias Farmacˆ
euticas, Universidade Federal de Juiz de Fora,
36036-900 Juiz de Fora, MG, Brazil
2Instituto de Ciˆ
encias Biol´
ogicas, Departamento de Bioqu´
ımica, Universidade Federal de Juiz de Fora,
36036-900 Juiz de Fora, MG, Brazil
3Departamento de Qu´
ımica, Faculdade de Filosoa, Ciˆ
encias e Letras de Ribeir˜
ao Preto, Universidade de S˜
ao Paulo,
14040-901 Ribeir˜
ao Preto, SP, Brazil
4N´
ucleo de Enteroparasitas, Instituto Adolfo Lutz, 01246-902 S˜
ao Paulo, SP, Brazil
5Faculdade de Ciˆ
encias de Guarulhos, FACIG/UNIESP, 07025-000 Guarulhos, SP, Brazil
Correspondence should be addressed to Ademar A. Da Silva Filho; ademar.alves@uf.edu.br
Received August ; Accepted December ; Published February
Academic Editors: Z.-G. Han and T. G. Moreels
Copyright © Loyana Silva Godinho et al. is is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
Schistosomiasis, a parasitic disease caused by trematode atworms of the genus Schistosoma,aectsmorethanmillionpeople
worldwide, and its control is dependent on a single drug, praziquantel. Tan a c etum vulg a r e (Asteraceae) is used in folk medicine
as a vermifuge. is study aimed to investigate the in vitro schistosomicidal activity of the crude extract (TV) and the essential oil
(TV-EO) from the aerial parts of T. vu l gare . TV-EO was obtained by hydrodistillation and analyzed by GC/MS, which allowed the
identication of 𝛽-thujone (.%) as the major constituent. TV and TV-EO, at 𝜇g/mL, decreased motor activity and caused
% mortality of all adult worms. At and 𝜇g/mL, only TV caused death of all adult worms, while TV-EO was inactive. TV
( 𝜇g/mL) was also able to reduce viability and decrease production of developed eggs. Confocal laser scanning microscopy
showed morphological alterations in the tegument of the S. mansoni surface aer incubation with TV ( and 𝜇g/mL).
Quantitative analysis on the schistosomes tegument showed that TV caused changes in the numbers of tubercles of S. mansoni
male worms in a dose-dependent manner. e ndings suggest that T. vul g are is a potential source of schistosomicidal compounds.
1. Introduction
Schistosomiasis, a parasitic disease caused by trematode
atworms of the genus Schistosoma,isthesecondmajor
neglected tropical disease, with major economic and public-
health consequences. e major etiological agent of human
schistosomiasis is Schistosoma mansoni,anditisestimated
that more than million people are infected and
million people are at risk of infection worldwide [,].
e treatment of schistosomiasis is based on the control
of adult worms in infected patients, with praziquantel (PZQ)
being the most widely used drug. However, PZQ does not
prevent reinfection, is inactive against juvenile schistosomes,
and has only a limited eect on already developed liver
and spleen lesions [,]. ese limitations, in combina-
tion with a considerable concern about the development of
PZQ resistance, have motivated the scientic community to
develop novel and inexpensive drugs against schistosomiasis
Hindawi Publishing Corporation
e Scientific World Journal
Volume 2014, Article ID 460342, 9 pages
http://dx.doi.org/10.1155/2014/460342
e Scientic World Journal
[,]. In this regard, there has been intensication in the
search for new schistosomicidal compounds from natural
sources, mainly from plants, which continue to be a major
source of biologically active metabolites that may provide
lead structures for the development of new drugs [,].
A number of recent studies have investigated the schisto-
somicidalactivityofplantsandtheirisolatedcompounds[–
]. A number of extracts, essential oils, and isolated natural
compounds displaying in vitro schistosomicidal activity have
been identied [–]. Among tested plants, several members
of the family Asteraceae have shown promising in vitro
schistosomicidal activity [,].
Plants of the genus Tanacetum (Asteraceae) are used
over the years in folk medicines all over the world for
many medicinal purposes, including anti-inammatory and
anthelmintic [,]. Tanacetum extracts and its isolated
compounds have been also reported to exhibit antiviral
activity []andtrypanocidal[] and leishmanicidal []
activities.
Tanacetum vulgare L., known as “Tansy” in Europe
and “catinga-de-mulata” in Brazil, is widely used in folk
medicine as a vermifuge and anti-inammatory []. Also,
the aerial parts of this plant are popularly used to treat
migraine, neuralgia, and rheumatism, and as an anthelmintic
and insect repellent []. Phytochemical studies have shown
that T. vulgar e contain several biologically active metabolites,
mainly sesquiterpene lactones [–].
On the basis of the folkloric uses of T. v u l g are as an
anthelmintic and a vermifuge, as well as in continuation of
our search for active natural sources against S. mansoni [–
], the present study evaluated the in vitro schistosomicidal
eects of the extract and the essential oil of T. vulg a re,which
have not yet been described.
2. Materials and Methods
2.1. Plant Material and Extraction. Aerial parts of T. v ulg a re
L. were collected from the Horto Medicinal da Faculdade de
F´
armacia, in Juiz de Fora, MG, Brazil. A voucher specimen
was deposited in the herbarium of the Botany Depart-
ment of the Universidade Federal de Juiz de Fora, MG,
Brazil. e plant material ( g) was dried, powdered, and
exhaustively extracted, by maceration at room temperature,
using EtOH/H2O : (v/v). Aer ltration, the solvent was
removed under reduced pressure to yield . g of the crude
hydroalcoholic extract of T. vu l g are (TV).
2.2. Essential Oil Extraction. e essential oil of T. vul g a re
(TV-EO) was obtained from aerial parts by hydrodistillation
using a Clevenger-type apparatus for h. Aer manual
collection of the essential oil, traces of water were removed
by freezing the sample below ∘C, followed by transfer of the
unfrozen essential oil to a new vial. e yield was calculated
as % (w/w) of the fresh aerial parts.
2.3. CG/MS Analysis. e TV-EO was analyzed by GC/MS
analysis using a Shimadzu QP Plus (Shimadzu Cor-
poration, Kyoto, Japan) system equipped with an AOC-
i autosampler and a Restek Rtx-MS fused-silica capil-
lary column (% phenyl-, % methylpolysiloxane; m ×
. mm i.d., lm thickness . 𝜇m). e oven temperature
was programmed to increase from to ∘Cat
∘C/min;
injector temperature, ∘C; ion-source temperature, ∘C;
carrier gas, He (. mL/min); split ratio, : ; injection
volume, . 𝜇L. e mass spectrometer was operated in the
electron ionization mode ( eV), and the spectra were taken
with a scan interval of . s over the mass range – Da.
e quantication of each TV-EO constituent was done by
internal normalization (%). e identication of the TV-EO
components was based on the comparison of their retention
indices (RI; determined in relation to the RT of n-alkanes
(C8–C26)) and mass spectra with those of the Wiley 7,NIST
08,andFFNSC 1.2 spectral libraries, as well as with those
reported in the literature [].
2.4. Parasite. S. mansoni. (BH strain Belo Horizonte, Brazil)
worms have been maintained in Biomphalaria glabrata snails
and Mesocricetus auratus hamsters hosts at the Adolfo Lutz
Institute (S˜
ao Paulo, Brazil). Female hamsters, weighting –
g, were infected by subcutaneous injection of cercariae.
Aer weeks, adults S. mansoni specimens were recovered
from the hamster by perfusion with RPMI medium
supplemented with heparin [].ewormswerewashedin
RPMI medium (Gibco) supplemented with 𝜇g/mL
of streptomycin, UI/mL of penicillin (Invitrogen), and
mMofHepes.Pairsofadultworms(maleandfemale)were
incubatedina-wellcultureplate(TechnoPlasticProducts,
TPP) containing mL of the same medium supplemented
with % heat-inactivated calf serum at ∘Cina%CO
2
atmosphere [,]. All experiments were authorized by the
CommitteeforEthicsinAnimalCareofAdolfoLutzInstitute,
in accordance with nationally and internationally accepted
principles for laboratory animal use and care.
2.5. In Vitro Studies with S. mansoni. For the in vitro test with
S. mansoni, TV and TV-EO were evaluated at concentrations
of , , , and 𝜇g/mL, according to works previously
described [,]. Samples were dissolved in .% DMSO
and added to RPMI medium containing one pair of
adult worms that had been allowed for h to adapt to
the culture medium. Worm motor activity, tegumental alter-
ations, changes in the pairing, and survival of the parasites
were monitored on daily basis for days using an inverted
microscope and a stereomicroscope (SMZ , Nikon). All
experiments were carried out in triplicate and repeated at
least three times, using 𝜇M praziquantel (PZQ) as positive
control group, and RPMI medium and RPMI with
.% DMSO as negative control groups.
2.6. Tegumental Changes. e quantication of the number
of tubercles was performed for TV (the most active sample in
tegument) using a confocal microscope. Aer the established
times or in the occurrence of death, the parasites were xed in
e Scientic World Journal
formalin-acetic-alcohol solution (FAA) and analyzed under a
confocal microscope (Laser Scanning Microscopy, LSM
META, Zeiss) at nm (exciting) and nm (emission)
as described by [,]. A minimum of three areas of the
tegument of each parasite were assessed. e numbers of
tubercles were counted in , 𝜇m2of area calculated with
theZeissLSMImageBrowsersoware.Ablindanalysis
was performed by observer with experience and training in
parasitology.
2.7. Viability Assay. For the viability assay against S. mansoni,
pairsofadultwormswereincubatedforhwithTV
(the most active sample) at concentration of 𝜇g/mL; the
viability assay was performed according to MTT assay [,
].Aerincubation,eachpairofadultwormswasplaced
individually into wells (-well plates) containing 𝜇L
of phosphate-buered saline (PBS) with mg MTT/mL
for min at ∘C. e solution was carefully removed
and replaced with 𝜇LofDMSOandthewormswere
allowed to stand in DMSO at room temperature for h. e
absorbance was read at nm using as negative control
groups RPMI medium and RPMI with .%
DMSO. Heat-killed worms at ∘Cwereusedaspositive
controls groups.
2.8. Statistical Analysis. e statistical tests were performed
with the GRAPHPAD PRISM (version .) soware. Sig-
nicant dierences were determined by one-way analysis of
variance (ANOVA) and applying Tukey’s test for multiple
comparisons with a level of signicance set at 𝑃 < 0.05.
3. Results and Discussion
Many plants have been used throughout the world in tra-
ditional medicine for the treatment of parasite diseases [].
In this scenario, several in vitro studies against Schistosoma
species have been performed with crude plant extracts and
essential oils, especially from species from the Asteraceae
family [,,]. T. vulgar e is popularly used as anthelmintic
and vermifuge, which encouraged us to evaluate the in vitro
eect of its crude extract and essential oil against adult worms
of S. mansoni, which have not been performed to date.
As shown in Table , positive control (PZQ, 𝜇M)
resulted in the death of all parasites within hours, whereas
no mortality was observed in the worms of the negative
(RPMI medium) and solvent control (RPMI medium plus
.% DMSO) groups. On the other hand, aer h of
incubation, both TV and TV-EO, at 𝜇g/mL, cause %
mortality of all adult worms. However, at and 𝜇g/mL,
only TV causes % mortality, while TV-EO was inactive
at concentrations of to 𝜇g/mL aer h of incubation
(Table ).
Also, all pairs of worms separated into individual male
and female when exposed to and 𝜇g/mL of both TV
and TV-EO aer h of incubation. However, at 𝜇g/mL,
% worm pairs were separated by TV-EO only aer h
of incubation. Moreover, male and female adults showed a
signicant decrease in motor activity aer h of incubation
with 𝜇g/mL of TV and TV-EO. However, motor activity
was signicantly decreased by the action of TV and TV-
EO (at concentrations ranging from to 𝜇g/mL) only
aer h of incubation. PZQ ( 𝜇M) caused % mortality
butnoseparationofwormpairs,whereasnoeectswere
observed in worms in the negative (RPMI medium)
and control (RPMI medium plus .% DMSO) groups. Addi-
tionally,onlyTV(,,and𝜇g/mL) caused signicant
tegumental alterations in adult worms aer h of incubation
(Table ).
In relation to the decrease in motor activity, studies
revealed that the musculature of S. mansoni can serve as a
therapeutictarget,becausethemotilityofwormsisassociated
with important neurotransmitters or neuromodulators [].
Nematode neuropeptide systems comprise an exceptionally
complex array of ∼ peptidic signaling molecules that
operate within a structurally simple nervous system of ∼
neurons. us, these signaling systems can provide tools for
the discovery of more amenable targets such as neuropeptide
receptors or neuropeptide processing enzymes [].
In recent years, some essential oils have been reported as
promising schistosomicidal agents [–,]. In this study,
the in vitro eects of dierent concentrations of the essential
oil from the T. v ulg a re (TV-EO) on S. mansoni adult worms
were assessed.
e essential oil of T. vulgare (TV-EO) was obtained by
hydrodistillation of the aerial parts, yielding .% (w/w).
e chemical composition of this oil is shown in Table and
Figure . A total of compounds were identied, being six
monoterpenes (.%) and one sesquiterpene (.%). 𝛽-
ujone (3, .%) was identied as the major constituent
(Figure )ofTV-EO.𝛽-ujone has als o been previously
described as major constituent of the essential oils obtained
from specimens of T. vulgare collected in Eurasian and North
America []. Considering the chemical composition of TV-
EO, its schistosomicidal in vitro activity may be related
to 𝛽-thujone, its major constituent. Studies have associated
the anthelmintic activity of the essential oils obtained from
specimens of T. v u l gare with 𝛽-thujone []. It has been
reported that thujone may act on GABAAreceptor, similarly
to some anthelmintic drugs, such as ivermectin [,].
Also, considering the mechanism by which TV-EO exerts
its in vitro schistosomicidal eect, essential oils may have
no specic cellular target, because lipophilic compounds,
typically present in essential oils, may pass through the cell
wall, tegument, and cytoplasmic membrane, damaging their
structures,whichmayleadtothecellularlysis[].
According to results observed in preliminary survival of
-day-old adult worms of S. mansoni test (Table ), TV was
moreactivethanTV-EO,duetoitsabilitytocause%
mortality of all parasites at lower concentrations, as well as
to cause signicant tegumental alterations. Because of that,
TV was further tested to assess viability, oviposition, and
morphological alterations in the parasite’s tegument.
e viability of adult S. mansoni worms was evaluated
during their in vitro incubation with TV ( 𝜇g/mL) aer a
period of hours (Figure ). A % reduction was observed
in the worms viability incubated with TV when compared
with the negative control groups. However, the viability of
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T : In v itro eects of crude hydroalcoholic extract (TV) and essential oil of T. vu l gare (TV-EO) against S. mansoni adult worms.
Groups Incubation
period (h)
Separated
worms (%)aDead worms
(%)a
Decrease in motor activity (%)aWorms with tegumental alteration (%)a
Slight Signicant Partial Extensive
Controlb
.% DMSO
PZQc
TV
𝜇g/mL
𝜇g/mL
𝜇g/mL
𝜇g/mL
TV-EO
𝜇g/mL
𝜇g/mL
𝜇g/mL
𝜇g/mL
aPercentages relative to the worms investigated. bRPMI . cTested at c oncentration of 𝜇M.
these worms was signicantly higher than that of the group
of worms dead by heating.
Regarding production of developed eggs by adult worms
of S. mansoni (Figure ), TV ( 𝜇g/mL) showed a signi-
cantdecreaseinthenumberofdevelopedeggs,by.%,aer
h of incubation, in comparison with the negative control
group treated with RPMI medium. However, the eect
of TV on egg production may be correlated with its ability to
separate adult worms into male and female.
e presence of S. mansoni eggs in the host tissues has
been reported to be closely related to the pathology of human
schistosomiasis [,]. e permanent pairing of the schis-
tosomes couples in the blood system of their hosts vertebrates
throughout their lifespan causes high rate of oviposition,
which is responsible for the resulting immunopathological
lesions, characterized by inammation and brosis in the
target [].
Moreover, light microscopic investigations (Table )
showed that TV caused morphological alterations in the
parasite’s tegument, with no distinction between male and
female worms (data not shown). No tegumental changes in
adult worms were observed for the negative control group,
O
OO
OH
(1) (2) (3) (4)
(5) (6) (7)
F : Chemical constituents identied by GC-MS in the
essential oil of T. vul g are (TV-EO).
e Scientic World Journal
T : Composition of the essential oil of T. vul g are (TV-EO) identied by CG/MS analysis.
Compounds RIexp
aRIlit
bContent (%)cIdenticationd
,-Cineol (1) . RI, MS
𝛼-ujone (2) . RI, MS
𝛽-ujone (3) . RI, MS
𝛼-Pinene (4) . RI, MS
𝑝-Cimene (5) . RI, MS
Terpinen--ol (6) . RI, MS
Germacrene-D (7) . RI, MS
Not identied — . RI, MS
Not identied — . RI, MS
Total 96.39
Monoterpenes: .%
Sesquiterpenes: .%
aRIexp: retention index determined relative to 𝑛-alkanes (C–C)ontheRtx-MScolumn.bRIlit : retention index from []. cCalculated from the peak area
relative to the total peak area. dCompound identication: RI, comparison of the RI with those of []; MS: comparison of the mass spectra with those of the
Wiley , NIST , and FFNSC . spectral libraries as well as with those of [].
0
0.2
0.4
0.6
Viability (550nm)
§
∗
∗
Control 0.5% DMSO TV Dead
F : In vitro eect of the crude hydroalcoholic extract of T.
vulgare (TV) on the viability of the S. mansoni adult worms. Pairs
of adult worms were treated with TV at 𝜇g/mL during h
and the viability was measured by MTT assay at nm. RPMI
mediumand.%DMSOinRPMImediumwereusedas
negative control group s. Heat-killed worms at ∘C (dead) were used
as positive control group. e viability was expressed as mean of the
absorbance values from three experiments. ∗Signicantly dierent
from control (𝑃 < 0.05); §signicantly dierent from dead group
(𝑃 < 0.05).
while the positive control (PZQ) had tegumental alteration
in % of the worms. In addition, the eect of TV on
the parasite’s tegument was monitored microscopically using
confocal laser scanning microscopy. As shown in Figure ,
morphological alterations of the tegument on the S. mansoni
surface were detected with TV at 𝜇g/mL (Figure (d))
and 𝜇g/mL (Figure (e)). Meanwhile, no abnormality
was seen in the worms maintained in the negative control
0
100
200
300
400
Number of developed eggs
∗
Control 0.5% DMSO TV
F : In vitro eect of the crude hydroalcoholic extract of
T. vulg a re (TV) at 𝜇g/mL on egg development (quantitative
analysis of the development phenotype). Aer treatment, the eggs
were microscopically examined and scored as developed or unde-
veloped based on the presence or absence of the miracidium. Data
are presented as the mean of developed eggs from two separate
experiments. ∗Signicantly dierent from control (𝑃 < 0.05).
group. us, a pattern consisting of a combination of changes
in the surface morphology was detected and correlated to
the death of the adult worms. ese pronounced changes in
the aspect of the tubercles, which oen appeared collapsed
and disrupted, were similar to those reported in studies
with some isolated natural compounds, such as piplartine,
epiisopiloturine, and (+)-limonene epoxide [,,].
Additionally, morphological alterations on S. mansoni
tegument were evaluated quantitatively aer exposure to
e Scientic World Journal
(a)
(b) (c)
(d) (e)
F : Confocal laser scanning microscopy investigation of the crude hydroalcoholic extract of T. vul g are (TV) in vitro schistosomicidal
eect. In these experiments, pairs of adult worms were incubated in -well culture plates containing RPMI medium and treated with
dierent concentrations of TV. Aer h of incubation or in the case of death, adult male worms were xed in FAA solution and uorescent
images were obtained using confocal microscopy (Carl Zeiss LSM META). General view of the anterior worm region showing, in red,
thelocationwheretegumentwasanalysed(a).ControlwormsinRPMIwith.%DMSO(b),and𝜇M praziquantel (c), compared to
worms t reated with 𝜇g/mL of TV (d), and 𝜇g/mL of TV (e). Scale bars, 𝜇m(a)and𝜇m (b–e).
dierent concentrations of TV. In this quantitative analysis,
areas of tegument of male worms were assessed, and the
numbers of tubercles on the dorsal surface of parasites were
counted [,–]. As shown in Figure ,TVcaused
changes on tubercles of S. mansoni male worms in a dose-
dependent manner. For example, the number of intact
tubercles in an area of 𝜇m2on male worms of the
negative control was , while in the groups exposed to ,
, , and 𝜇g/mL of TV the number was, respectively,
,,,and.Similarresults,withthepatternoftubercle
destruction in a dose-dependent manner, were obtained from
thepairedschistosomesexposedtosomenaturalproducts,
such as antimicrobial peptide dermaseptin and (+)-limonene
epoxide [,].
e tegument is extremely important to the infection
success and survival in the host, and it has been a major
target for the development of antischistosomal drugs [],
sincemostofthecurrentlyuseddrugsagainstschistosomes,
such as PZQ [], meoquine [], and artemether [], act
by damaging the schistosome tegument.
Several compounds, mainly sesquiterpene lactones
(STL), have been identied as active constituents in previous
phytochemical studies of the aerial parts of T. vu l g a re [–].
Some STL from T. vu lgare, mainly parthenolide, proved to
be active against some parasites, such as Tr y p ano s oma cr u z i
[]andLeishmania amazonensis []. Also, T. vulgare
shows remarkable antioxidant properties, mainly due to
its phenolic compounds content, especially avonoids and
caeoylquinic acid derivatives []. e wide spectrum of T.
vulgare activities can be mainly ascribed to the occurrence
of STL [].SomeSTLhavebeenreportedasmolluscicidal
compounds, showing activity against adult Biomphalaria
sp., a snail directly implicated in the transmission of schis-
tosomiasis [].
e mechanism by which TV exerts its in vitro schis-
tosom ici dal eect i s unc lear. Howev er, ad ult worms of
e Scientic World Journal
010 25 50 100 PZQ
0
10
20
30
40
50
Number of intact tubercles
TV (𝜇g/mL)
∗
∗∗
∗∗
∗∗∗
F : Quantitative analysis of morphological alterations on
the tegument of S. mansoni aer exposure to dierent concentra-
tions of the crude hydroalcoholic extract of T. vu lgar e (TV). e
quantication of the number of tubercles was performed using
three-dimensional images obtained from laser scanning confocal
microscopy. Indicated are numbers of intact tubercles, and these
numbers were measured in a 𝜇m2area in a dorsal region
of Schistosoma mansoni adult male worm (see Figure (a))and
calculated with the Zeiss LSM Image Browser soware. Praziquantel
(PZQ, 𝜇M) was used as a positive control. A minimum of three
tegument areas of each parasite were assessed. Values are means ±
SD (bars) of ten male adult worms. ∗𝑃 < 0.05,∗∗ 𝑃 < 0.01,and
∗∗∗𝑃 < 0.001 compared with untreated group.
S. mansoni died accompanied by destruction of the worm
body, and a relationship between tegumental damage and
the death of worms was observed in the in vitro assays.
Furthermore, in vivo investigations in mice infected by S.
mansoni are necessary to determine the clinical potential of
TV to treat schistosomiasis. In this case, mice may be treated
orally using single or multiple oral doses at dierent life-cycle
stages (e.g., schistosomula, juvenile, and adult worms). In
addition, toxicological studies (e.g., acute oral LD50 of TV)
should be examined.
4. Conclusion
In this study, we have reported an investigation on the in
vitro schistosomicidal eects of the crude hydroalcoholic
extract (TV) and the essential oil from T. vulgar e (TV-
EO) for the rst time. It was demonstrated that both TV
and TV-EO possess signicant activity against adult worms
of S. mansoni.eactivityofTV-EOmayberelated,at
least in part, to monoterpene thujones, which were detected
as major constituents in TV-EO. Our study reinforced the
traditional use of T. vulg a re as a vermifuge and anthelmintic.
Considering the obtained results, further biological studies,
as well as phytochemical investigations, are in progress with
TV in order to identify its major active compound and
elucidate its mechanism(s) of schistosomicidal action.
Conflict of Interests
e authors declare that there is no conict of interests.
Acknowledgments
e authors are grateful to FAPEMIG (Grant no.
/) and FAPESP for nancial support and CAPES,
PIBIC/CNPq/UFJF and CNPq for fellowships. ey are
grateful to Mr. Jeerson S. Rodrigues for excellent technical
assistance with S. mansoni life cycle maintenance at the
Adolfo Lutz Institute (S˜
ao Paulo, SP, Brazil). ey also thank
Dr. Henrique K. Roato and Dr. Ronaldo Z. Mendonc¸a
(Butantan Institute, S˜
ao Paulo, SP, Brazil) for expert help
with confocal microscope studies (FAPESP, Project /-
).
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