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Phytomedicine 22 (2015) 921–928
Contents lists available at ScienceDirect
Phytomedicine
journal homepage: www.elsevier.com/locate/phymed
Cardamonin, a schistosomicidal chalcone from Piper aduncum L.
(Piperaceae) that inhibits Schistosoma mansoni ATP diphosphohydrolase
Clarissa C.B. de Castro a, Poliana S. Costa a, Gisele T. Laktin a, Paulo H.D. de Carvalho a,Reinaldo B. Geraldo a, Josué de Moraes b, Pedro L.S. Pinto c, Mara R.C. Couri d,Priscila de F. Pinto e, Ademar A. Da Silva Filho a,∗
a Faculdade de Farmácia, Departamento de Ciências Farmacêuticas, Universidade Federal de Juiz de Fora, 36036-900 Juiz de Fora, MG, Brazilb Núcleo de Pesquisa em Doenças Negligenciadas (FACIG), 07025-000 Guarulhos, SP, Brazilc Núcleo de Enteroparasitas, Instituto Adolfo Lutz, 01246-902 São Paulo, SP, Brazild Departamento de Química, Universidade Federal de Juiz de Fora, 36036-330 Juiz de Fora, MG, Brazile Instituto de Ciências Biológicas, Departamento de Bioquímica, Universidade Federal de Juiz de Fora, 36036-900 Juiz de Fora, MG, Brazil
a r t i c l e i n f o
Article history:
Received 24 April 2015
Revised 11 June 2015
Accepted 18 June 2015
Keywords:
Cardamom
Chalcone
SmATPDases
Piper aduncum
Apyrase
Schistosomicidal activity
a b s t r a c t
Background: Schistosomiasis is one of the world’s major public health problems, and praziquantel (PZQ) is
the only available drug to treat this neglected disease with an urgent demand for new drugs. Recent studies
indicated that extracts from Piper aduncum L. (Piperaceae) are active against adult worms of Schistosoma
mansoni, the major etiological agent of human schistosomiasis.
Purpose: We investigated the in vitro schistosomicidal activity of cardamonin, a chalcone isolated from the
crude extract of P. aduncum. Also, this present work describes, for the first time, the S. mansoni ATP diphos-
phohydrolase inhibitory activity of cardamonin, as well as, its molecular docking with S. mansoni ATPDase1,
in order to investigate its mode of inhibition.
Methods: In vitro schistosomicidal assays and confocal laser scanning microscopy were used to evaluate the
effects of cardamonin on adult schistosomes. Cell viability was measured by MTT assay, and the S. mansoni
ATPase activity was determined spectrophotometrically. Identification of the cardamonin binding site and its
interactions on S. mansoni ATPDase1 were made by molecular docking experiments.
Results: A bioguided fractionation of the crude extract of P. aduncum was carried out, leading to identification
of cardamonin as the active compound, along with pinocembrin and uvangoletin. Cardamonin (25, 50, and
100 μM) caused 100% mortality, tegumental alterations, and reduction of oviposition and motor activity of all
adult worms of S. mansoni, without affecting mammalian cells. Confocal laser scanning microscopy showed
tegumental morphological alterations and changes on the numbers of tubercles of S. mansoni worms in a
dose-dependent manner. Cardamonin also inhibited S. mansoni ATP diphosphohydrolase (IC50 of 23.54 μM).
Molecular docking studies revealed that cardamonin interacts with the Nucleotide-Binding of SmATPDase 1.
The nature of SmATPDase 1–cardamonin interactions is mainly hydrophobic and hydrogen bonding.
Conclusion: This report provides evidence for the in vitro schistosomicidal activity of cardamonin and demon-
strated, for the first time, that this chalcone is highly effective in inhibiting S. mansoni ATP diphosphohydro-
lase, opening the route to further studies of chalcones as prototypes for new S. mansoni ATP diphosphohydro-
lase inhibitors.
© 2015 Elsevier GmbH. All rights reserved.
c
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Abbreviations: PZQ, praziquantel; PaI, crude dichloromethane extract from inflores-
ences of P. aduncum; PaI-H, hexane fraction of the crude extract of P. aduncum; PaI-C,
hloroform fraction of the crude extract of P. aduncum; PaI-A, ethyl acetate fraction of
he crude extract of P. aduncum.∗ Corresponding author. Tel.: +55 32 21023893; fax: +55 32 21023801.
E-mail address: [email protected], [email protected] (A.A. Da Silva
ilho).
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ttp://dx.doi.org/10.1016/j.phymed.2015.06.009
944-7113/© 2015 Elsevier GmbH. All rights reserved.
ntroduction
Schistosomiasis, caused by trematode flatworms of the genus
chistosoma, is one of the most significant neglected tropical diseases,
ausing serious public health problems in more than 70 tropical and
ubtropical countries (Gryseels et al. 2006). It is estimated that more
han 200 million people are infected and that 779 million are at risk
f infection (Gaba et al. 2014; Veras et al. 2012). Schistosoma mansoni
s the major etiological agent of human schistosomiasis, for which
he treatment is dependent on a single drug, praziquantel (PZQ).
922 C.C.B. de Castro et al. / Phytomedicine 22 (2015) 921–928
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However, PZQ does not prevent re-infections, has a limited effect on
developed liver and spleen lesions, and is inactive against juvenile
schistosomes (Ramalhete et al. 2012; Gryseels et al. 2006). Also, the
exclusive dependency on PZQ is alarming, raising concerns about the
reliance on a single drug that might lead to the potential appear-
ance of massive resistance to the drug (Barbosa de Castro et al. 2013;
de Moraes et al. 2012). In the light of the increasing incidences of
drug resistant schistosomiasis, there is an urgent and unmet need to
discover novel therapeutic agents against this pathogen (Gaba et al.
2014; Barbosa de Castro et al. 2013).
The major target for the development of new schistosomicidal
drugs is the tegument of Schistosoma, which is crucial for the par-
asite survival and its host immune defense (de Moraes et al. 2012;
Van Hellemond et al. 2006). S. mansoni ATP diphosphohydrolases (EC
3.6.1.5), also known as apyrases or SmATPDases, are ecto-enzymes lo-
calized on the external tegument surface of S. mansoni that hydrolyze
a variety of nucleoside tri- and diphosphates to corresponding nucle-
oside monophosphates (Vasconcelos et al. 1993, 1996; DeMarco et al.
2003). Also, the literature suggests that S. mansoni ATP diphosphohy-
drolases are involved in the evasion of the host defense, due to the
location of these isoforms in the parasite and the importance of nu-
cleoside di- and triphosphates in activating cells of the host immune
system (de Souza et al. 2014; Da’dara et al. 2014). Besides, S. man-
soni ATP diphosphohydrolases are directly involved in the parasite’s
ability to decrease the immune response, as well as the thrombotic
effect around adult worms and eggs, ensuring the mobility of para-
site within blood vessels and its survival over a long period in the hu-
man host (Da’dara et al. 2014; Faria-Pinto et al. 2004). Two S. mansoni
ATP diphosphohydrolase isoforms (SmATPDase1 and SmATPDase2)
of approximately 63 kDa, differing in their catalytic properties, were
observed in adult worm tegument (de Souza et al. 2014). Recently, it
has been shown that only SmATPDase1 is actually capable of cleaving
exogenous ATP (Da’dara et al. 2014). The inhibition of S. mansoni ATP
diphosphohydrolases is therefore considered a novel and important
therapeutic option in the treatment of schistosomiasis.
Piper aduncum L. (Piperaceae), known as “pimenta-de-macaco”
and “aperta-ruão”, is widely used in folk medicine as anti-
inflammatory and antiseptic (Carrara et al. 2013; Morandim et al.
2009). Previous phytochemical studies of the aerial parts of P. adun-
cum reported the isolation of chalcones, flavanones, and dihydrochal-
cones (Morandim et al. 2009). Recently, our previous work has
demonstrated that some crude extracts, obtained from Piper species
(Piperaceae), including P. aduncum, exhibit in vitro schistosomici-
dal activity against adult worms of S. mansoni (Carrara et al. 2013).
However, no active schistosomicidal compounds have been identified
from P. aduncum.
Thus, this present work describes, for the first time, the schis-
tosomicidal activity of cardamonin, a chalcone identified as a S.
mansoni ATP diphosphohydrolase inhibitor, isolated from P. aduncum
using bioguided fractionation procedures. Additionally, we have
performed molecular docking of cardamonin to investigate its mode
of S. mansoni ATPDase1 inhibition.
Material and methods
Plant material
Inflorescences of P. aduncum L. were colleted at the Faculty of
Pharmacy’s Medicinal Herb Garden, Juiz de Fora city, MG, Brazil, in
February, 2012. A voucher specimen (CES J59018) was stored at the
Herbarium of the Botany Department of the Federal University of Juiz
de Fora, MG, Brazil.
Extraction and bioactivity-guided isolation
Inflorescences of P. aduncum (160 g) were dried, powdered, and
exhaustively extracted, by Soxhlet, for 5 h, using CH Cl as solvent.
2 2fter extraction, the solvent was removed under vacuum to yield
4 g of the crude dichloromethane extract (PaI), which was able
o cause 100% mortality in schistosomes. PaI (24 g) was suspended
n methanol:H2O (7:3 v/v), and submitted to sequential partition,
ith equal volume of solvents of increasing polarities, namely n-
exane (PaI-H, 8 g), chloroform (PaI-C, 6 g) and ethyl acetate (PaI-A,
g). PaI-C was selected for chromatographic fractionation based
n its schistosomicidal activity. PaI-C (5 g) was chromatographed
ver silica gel using a vacuum liquid chromatography system and
exane:ethyl acetate mixtures in increasing proportions as elu-
nts, furnishing 12 fractions. Of them, fractions IV (150 mg) and V
341 mg) were submitted to column chromatography over silica gel
sing CHCl3:Me2CO in increasing proportions as eluent, affording the
ollowing compounds: 5,7-dihydroxyflavanone (pinocembrin, 30 mg)
rom subfraction IV; 2′,4′-dihydroxy-6′-methoxydihydrochalcone
uvangoletin, 15 mg), and 2′,4′-dihydroxy-6′-methoxychalcone
cardamonin, 76 mg) from subfraction V. The chemical structures
f all isolated compounds were established by by 1H and 13C
MR data analysis in comparison to literature (Krishna and Cha-
anty 1973; Posso et al. 1994; Avila et al. 2011; Gonçalves et al.
014).
aintenance of the S. mansoni life cycle
S. mansoni (BH strain Belo Horizonte, Brazil) worms were main-
ained in Biomphalaria glabrata snails as intermediate hosts and
esocricetus auratus hamsters as definitive host at the Adolfo Lutz
nstitute (São Paulo, Brazil), according to standard procedures previ-
usly described (de Moraes et al. 2012). At 49 days post-infection,
dult S. mansoni specimens were recovered from each hamster by
erfusion in Roswell Park Memorial Institute (RPMI) 1640 medium
Invitrogen, São Paulo, Brazil) supplemented with heparin. All ex-
eriments were authorized by the Committee for Ethics in An-
mal Care of Adolfo Lutz Institute (São Paulo, Brazil), in accor-
ance with nationally and internationally accepted principles for
aboratory animal use and care (CEUA, 11.794/08). The study was
onducted in adherence to the institution’s guidelines for animal
usbandry.
n vitro studies of adult schistosomes
Adult schistosomes were washed in RPMI-1640 medium supple-
ented with 200 μg/ml streptomycin, 200 IU ml−1 penicillin (In-
itrogen), and 25 mM HEPES. Adult worm pairs (male and female)
ere incubated in a 24-well culture plate (Techno Plastic Products,
PP, St. Louis, MO, USA), containing the same medium supplemented
ith 10% heat-inactivated calf serum (Gibco BRL) at 37 °C in a 5%
O2 atmosphere. A preliminary screening of crude extract (PaI) and
ractions (PaI-H, PaI-C, and PaI-A) was performed at 200 μg/ml,
nd isolated compounds at 100 μM, as described by Ramalhete et
l. (2012). The most active compound (cardamonin) was also eval-
ated at 50, 25, 10 and 5 μM. Samples were added to the cul-
ure from a 4000 μg/ml stock solution in RPMI-1640 containing
imethyl sulfoxide (DMSO). The final volume in each well was 2 ml.
he control worms were assayed in RPMI-1640 medium and RPMI-
640 with 0.5% DMSO as negative control groups and PZQ (5 μM)
s positive control group. All experiments were repeated at least
hree times independently for each treatment, and plating was car-
ied out in triplicate for each concentration. Parasites were main-
ained for 48 h and monitored every 24 h using a light micro-
cope in order to evaluate their general condition, with emphasis on
hanges in motor activity, mortality rate, and tegumental alterations.
orm mortality was assessed by lack of movement (de Moraes et al.
012).
C.C.B. de Castro et al. / Phytomedicine 22 (2015) 921–928 923
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Uvangoletin CardamoninPinocembrin
Fig. 1. Chemical structures of compounds isolated from inflorescences of
Piper aduncum L.
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ssessment of the reproductive fitness of adult worms
In order to evaluate the sexual fitness of worms exposure to
on-lethal concentrations of cardamonin, parasites were continually
onitored and schistosome egg output in vitro was determined by
ounting the number of eggs, as previously described (de Moraes et
l. 2012). In this case, adult worm pairs were incubated with carda-
onin (2.5, 5, and 10 μM) and in vitro parasite egg output was deter-
ined, on daily basis for five days, by counting the number of eggs
sing an inverted microscope and a stereomicroscope (SMZ 1000,
ikon, Melville, NY, USA).
onfocal laser scanning microscopy
Tegumental alteration and quantification of the number of tuber-
les were performed for cardamonin (10, 25, 50 and 100 μM) using a
onfocal laser scanning microscope (de Moraes et al. 2012). After the
stablished times or in the occurrence of death, the parasites were
xed in Formalin–acetic–alcohol solution (FAA) and analyzed under
confocal microscope (Laser Scanning Microscopy, LSM 510 META,
eiss) at 488 nm (exciting) and 505 nm (emission). A minimum of
hree areas of the tegument of each parasite were assessed. The num-
er of tubercles was counted in 20,000 μm2 of area calculated with
he Zeiss LSM Image Browser software.
ytotoxicity assay
Vero mammalian cells (African green monkey kidney fibroblast)
sed in this study were obtained from the American Type Culture
ollection (ATCC CCL-81; Manassas, VA) and provided by Dr. Ronaldo
. Mendonça (Laboratório de Parasitologia, Instituto Butantan, São
aulo, Brazil). Cytotoxicity was determined as previously described
de Moraes et al. 2012) using different concentrations of cardamonin
25, 50, 100, and 200 μM).
etermination of S. mansoni ATP diphosphohydrolase activity
The total homogenized of adults worms of S. mansoni (0.05 mg
rotein/ml), containing ATP diphosphohydrolases, was pre-incubated
or 30 min at 37 °C with cardamonin (5–40 μM) in a standard re-
ction medium, adapted according to method previously described
Faria-Pinto et al. 2004). The hydrolytic assay was initiated by the
ddition of sufficient ATP or ADP to give a final concentration of
mM, and stopped by the addition of 0.1 N HCl. Inorganic phos-
hate (Pi) liberated was determined spectrophotometrically (Penido
t al. 2007). Control samples were incubated in the same medium
ithout the addition of cardamonin and in the presence and absence
f 10% DMSO (used to dissolve cardamonin). In the absence of 10%
MSO, the adults worms homogenized presented an ADPase activ-
ty of 76.49 ± 11.47 nmol Pi mg−1 min−1 and an ATPase activity of
5.80 ± 8.36 nmol Pi mg−1 min−1. The half-inhibitory concentration
IC50) was calculated from dose–response curves by non-linear re-
ression analysis using Graphpad Prism version 5.0 (Graphpad soft-
are Inc., La Jolla, CA, USA).
tatistical analysis
Statistical tests were performed with Graphpad Prism software.
ignificant differences were determined by a one-way analysis of
ariance (ANOVA) and by applying Turkey’s test for multiple com-
arisons with the level of significance set at P < 0.05.
olecular modeling
The amino acid sequence of S. mansoni ATPDase 1 was re-
rieved from PUBMED database (code: GI: 33114187) (DeMarco
t al. 2003). The initial homology models of S. mansoni ATPDase
were built using Swiss-MODEL and Swiss-PDB viewer programs
http://swissmodel.expasy.org/ and http://www.expasy.org/spdbv/)
s described by Wermelinger et al. (2009). We used the ATPDase 1 of
attus norvegicus (PDB 3ZX3) as template with the homology degree
f S. mansoni ATPDase 1 (39%). The structure derived from homol-
gy modeling was submitted to the validation process, using QMEAN
coring function (Benkert et al. 2011) and the PROCHECK program
Laskowski et al. 1993).
Molecular docking was performed for cardamonin using Autodock
.2 along with AutodockTools 1.5.6 (ADT) (Morris et al. 1998). The
ardamonin was built using SPARTAN’14 (Wavefunction Inc., Irvine,
A, USA) and energy minimization was performed with convergence
riterion of the lowest energy. Non-polar hydrogen atoms and lone
airs were merged, and each atom was assigned with Gasteiger par-
ial charges. The grid size was set to 40 A × 40 A × 40 A with a
rid spacing of 0.508 A. The grid box was positioned at the center
f Nucleotide-Binding domain of the enzyme. One hundred indepen-
ent dockings were carried out for each docking experiment. The
owest docked energy of each conformation in the most populated
luster was selected. Analysis and visualization of the docking results
ere done using Visual molecular dynamics (VMD) and SPDBviewer.
esults and discussion
The development of new schistosomicidal drugs and the iden-
ification of new schistosomicidal molecules have been highly
ncouraged, mainly because the treatment of schistosomiasis relies
n a single drug, PZQ (Gaba et al. 2014). In this regard, the Brazilian
ora is rich in several medicinal plants with high potential for
roviding biologically active compounds against Schistosoma species
Barbosa de Castro et al. 2013). Among then, several species of Piper
ave been reported as promising sources of antiparasitic compounds
de Moraes et al. 2012; Hermoso et al. 2003; Ruiz et al. 2011).
ecently, our previous study showed that extracts from P. aduncum
ere active against adult worms of S. mansoni (Carrara et al. 2013).
Now, as a part of our program devoted to the search for schisto-
omicidal molecules from Brazilian plants, we performed a bioassay-
uided fractionation of the crude extract of P. aduncum L. (PaI). As
hown in Table 1, the crude extract (PaI, 200 μg/ml) caused 100%
ortality in all adult parasites after 24 h of incubation. Then, PaI
as further partitioned into three organic fractions. Among then, the
hloroform fraction (PaI-C, 200 μg/ml) was found to be the most ac-
ive, causing 100% mortality, significant decrease in motor activity,
nd tegumental alterations on adult worms (Table 1). On the other
and, n-hexane (PaI-H) and ethyl acetate (PaI-A) fractions were inac-
ive. PZQ (5 μM) caused 100% mortality, whereas no effect was ob-
erved in worms in the negative (RPMI 1640 medium) and control
RPMI medium plus 0.5% DMSO) groups.
Chromatographic fractionation of PaI-C yielded three pure com-
ounds, which were chemically identified by 1H and 13C NMR data
nalysis in comparison to literature as: pinocembrin (Posso et al.
994), uvangoletin (Avila et al. 2011), and cardamonin (Krishna
nd Chaganty 1973; Gonçalves et al. 2014) (Fig. 1). Purity of all
he isolated compounds was estimated to be higher than 95% by
924 C.C.B. de Castro et al. / Phytomedicine 22 (2015) 921–928
Table 1
In vitro effects of crude extract of P. aduncum (PaI), organic fractions (n-hexane PaI-H, chloroform PaI-C, and ethyl acetate PaI-A) and compounds
against adult worms of S. mansoni.
Groups Period of incubation (h) Dead worms (%) a Motor activity reduction (%)a Worms with tegumental alterations (%)a
Slight Significant Partial Extensive
Controlb 24 – – – – –
48 – – – – –
DMSO 0.5% 24 – – – – –
48 – – – – –
PZQ (5 μM) 24 100 – 100 – 100
48 100 – 100 – 100
Extract and fractionsc
PaI 24 100 – 100 – 100
48 100 – 100 – 100
PaI-C 24 100 – 50 – 50
48 100 – 100 – 100
PaI-H 24 – – – – –
48 – – – – –
PaI-A 24 – – – – –
48 – – – – –
Compounds
Uvangoletin (100 μM)
24 – – – – –
48 – – – – –
Pinocembrin (100 μM)
24 – – – – –
48 – – – – –
Cardamonin
100 μM 24 100 – 100 – 100
48 100 – 100 – 100
50 μM 24 100 – 100 – 100
48 100 – 100 – 100
25 μM 24 100 – 100 – 100
48 100 – 100 – 100
10 μM 24 – – – – –
48 – – – – –
5 μM 24 – – – – –
48 – – – – –
a Percentages relative to the 20 worms investigated.b RPMI 1640.c Crude extract and fractions were tested at 200 μg/ml.
0
50
100
150
200
250
300
24 h48 h72 h96 h120 h
0 2.5 5 10
******
******
***
*
***
Cardamonin (μM)
Num
ber
of
eggs
Fig. 2. In vitro effects of cardamonin on S. mansoni oviposition. Adult worm couples
were incubated with cardamonin, and at the indicated time periods, the cumulative
number of eggs per worm couple was assessed and scored using an inverted micro-
scope. Values are means ± SD (bars) of 10 worm couples. ∗P < 0.05, ∗∗P < 0.01, and∗∗∗P < 0.001 compared with untreated groups.
i
2
s
o
(
w
both 13C NMR and HPLC analysis using different solvent systems
(MeOH/MeCN/H2O, 65:5:30; MeOH/H2O 50 to 100% in 20 min).
In a preliminary survival of adult worms of S. mansoni test, all
isolated compounds were tested at 100 μM. Cardamonin exhibited
the most pronounced activity, causing 100% mortality, tegumental
alterations, and reduction in motor activity of all adult worms of S.
mansoni, after 24 h of in vitro drug exposure (Table 1). In contrast,
pinocembrin and uvangoletin were inactive. When analyzed at lower
concentrations, investigations revealed that all adult worms were
killed by cardamonin at 25 and 50 μM, while no activity was found
at concentrations of 5 and 10 μM, even after 48 h of incubation. Be-
cause cardamonin was active against adult schistosomes, we further
analyzed its effects on oviposition and on S. mansoni tegument.
We monitored the in vitro oviposition to assess the sexual repro-
ductive fitness of worms treated with non-lethal concentrations of
cardamonin (2.5, 5 and 10 μM) (Fig. 2). It was observed that car-
damonin also reduced the total number of eggs laid at sub-lethal
doses (5 and 10 μM). Based on three independent experiments, per-
formed in triplicate, cardamonin (5 and 10 μM) shows the same in-
hibition percentage of egg laying throughout the incubation period,
compared with the control group. These results suggest that the inhi-
bition of oviposition by cardamonin is irreversible. Reproductive fit-
ness of S. mansoni has been an important strategy used to evaluate
new schistosomicidal drugs, because egg production is responsible
for the transmission of the schistosome and the maintenance of its
life cycle (Godinho et al. 2014). Also, the presence of S. mansoni eggs
in the host tissues is closely related to the pathology of human schis-
tosomiasis, which is characterized by immunopathological lesions,
hncluding inflammation and fibrosis in the target (Gryseels et al.
006). Moreover, changes in the reproductive ability of S. man-
oni may be associated with alterations in the reproductive system
f worms when exposed to drugs with schistosomicidal properties
Barth et al. 1996). According to de Moraes et al. (2012), compounds
ith schistosomicidal activity can be also effective suppressive, in-
ibiting oviposition by schistosomes.
C.C.B. de Castro et al. / Phytomedicine 22 (2015) 921–928 925
Fig. 3. Confocal laser scanning microscopy observations of S. mansoni male worms after in vitro incubation with cardamonin. Pairs of adult worms were incubated in 24-well
culture plates containing RPMI-1640 medium with 0.5% DMSO and treated with cardamonin at different concentrations. (A) Control containing RPMI-1640 with 0.5% DMSO,
showing tubercles (T), (B) PZQ (5 μM), (C) 10 μM cardamonin, (D) 25 μM cardamonin, showing tubercles shrunken (sh), and disintegrate (di), and (E) 50 μM cardamonin, showing
tubercles disintegrate and dorsal tegumental surface sloughing (sl). Images: bars = 200 μm.
a
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E
b
q
f
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d
T
m
w
0 10 25 50 100 PZQ
0
10
20
30
40
50
**
***
******
**
Cardamonin (μM)
Num
ber
of in
tact
tub
ercl
es
Fig. 4. Effect of cardamonin on tubercles of S. mansoni male worms. The quantifica-
tion of the number of tubercles was performed using confocal microscopy. Numbers of
intact tubercles were measured in a 20,000 μm2 of area calculated with the Zeiss LSM
Image Browser software. Praziquantel (PZQ, 5 μM) was used as positive control. A min-
imum of three tegument areas of each parasite were assessed. Values are means ± SD
(bars) of ten male adult worms. ∗∗P < 0.01 and ∗∗∗P < 0.001 compared with untreated
groups.
The effects of cardamonin on the tegument of schistosomes were
nalyzed by confocal microscopy analysis in order to examine mor-
hological alterations at the surface of male worms. Morphologi-
al studies revealed progressive damages and prominent alterations
f the tegumental surfaces, in which tubercles appeared collapsed
nd disrupted (Fig. 3A–E). While in the negative control group, adult
orms of S. mansoni showed intact surface structure and topogra-
hy (Fig. 3A), after incubation with 25 and 50 μM of cardamonin,
eguments showed massive disintegration of tubercles (Fig. 3D and
). PZQ (5 μM) and cardamonin (10 μM) also caused damages in tu-
ercles (Fig. 3B and C).
Morphological alterations on the Schistosoma tegument were also
uantitatively analyzed by counting the tubercles on the dorsal sur-
ace of male schistosomes (Fig. 4). Cardamonin caused disintegration
f the tegumental surface in a dose-dependent manner (Fig. 4). No
ntact tubercles were seen at 100 μM, while in the group exposed
o 50 μM of cardamonin the number of intact tubercles was 2 ± 1,
iffering significantly from the negative control group (P < 0.001).
hus, a pattern consisting of a combination of changes in the surface
orphology were detected and correlated to the death of the adult
orms.
926 C.C.B. de Castro et al. / Phytomedicine 22 (2015) 921–928
0 5 1 0 1 5 2 0 2 5 3 0 3 5 4 0 4 5 5 0
0
5 0
1 0 0
1 5 0
C a rd am o n in [μM ]
%ATPaseactivity
IC 5 0 = 23 .54 μM
Fig. 5. ATPase activity of cardamonin. IC50 of cardamonin was calculated from dose-
response curves by a non-linear regression analysis. This assay was performed in trip-
licate. The results are expressed as the average of three experiments in duplicate and
data are shown as mean ± SD. In presence of 2.5% DMSO, the homogenized of adults
worms presented an ATPase activity of 80 ± 8,36 nmol Pi mg−1 min−1.
A
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d
m
The tegument of schistosomes is usually considered for its key
role in nutrient uptake, secretory functions and parasite protection
against the host immune system, so it has been a major target for the
development of schistosomicidal drugs (Van Hellemond et al. 2006).
Fig. 6. Molecular docking analysis of cardamonin with SmATPDase 1. (A) Nine different doc
docking position of cardamonin inside the Nucleotide-Binding domain of SmATPDase 1, show
energy docked conformation of cardamonin interacting with the SmATPase 1. (D) Binding o
SmATPDase 1. Cardamonin surface are represented as follow: apolar surface of cardamonin
interaction with the cardamonin: apolar interaction (gray spheres); a negative polar interact
colored using the CPK pattern. (For interpretation of the references to color in this figure lege
lso, in case of severe damages on the tegument, the host’s immune
esponse can affect the reparation process (Veras et al. 2012). The
egument destruction induced by cardamonin will probably not al-
ow reparation, since the damages lead to extensive peeling of the
egument surface, as well as formation and collapsing of tubercles,
ndicating straight damage to the cells in a direct dose-dependent ef-
ect. Furthermore, our results showed that cardamonin is nontoxic
o the mammalian Vero cells at concentrations that effectively kill
he adult worms of S. mansoni (25, 50, 100, and 200 μM) (data not
hown).
According to obtained results, we speculated if cardamonin may
ct on a specific target in the S. mansoni tegument. Then, the in vitro
TP diphosphohydrolase activity of cardamonin was determined.
ardamonin (40 μM) inhibited S. mansoni ATPase activity by approxi-
ately 82%, showing an IC50 of 23.54 μM (Fig. 5), whereas the ADPase
ctivity remained unaffected (ca. < 10%) up to the highest concentra-
ion (data not shown).
ATP diphosphohydrolases are ecto-enzymes present in the tegu-
ent of S. mansoni. Its purine recovery activity has been suggested
s a mechanism for Schistosoma protection against host organism
Faria-Pinto et al. 2004; Vasconcelos et al. 1993, 1996). S. mansoni ATP
iphosphohydrolases specifically counteract ATP damage-associated
olecular patterns (DAMPs)-mediated inflammatory signaling and
ks positions of cardamonin interacting with the SmATPDase 1. (B) Zoom view of the
ing only one mode outside of the Nucleotide-Binding domain (in pink). (C) The lowest
rientation and interaction of cardamonin with proteins residues at the active site of
surface (gray); polar and negative surfaces of cardamonin (red). Spheres indicate the
ion (red spheres); a positive polar interaction (blue spheres). Amino acids residues are
nd, the reader is referred to the web version of this article.)
C.C.B. de Castro et al. / Phytomedicine 22 (2015) 921–928 927
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imit the host’s attempts to focus inflammatory mediators around
he worms (de Souza et al. 2014; Da’dara et al. 2014). In this man-
er, these tegumental enzymes help impair host immune defenses
nd promote parasite survival. Therefore, targeting S. mansoni ATP
iphosphohydrolase has been considered a promising approach to
he study of novel drug candidates to treat schistosomiasis (Da’dara
t al. 2014; Penido et al. 2007). However, no study has been reported
n the ATP diphosphohydrolase activity of chalcones.
Considering the importance of studying novel S. mansoni ATP
iphosphohydrolases inhibitors, cardamonin presents better results
or ATPase activity compared to other compounds described in liter-
ture, such as thapsigargin (Martins et al. 2000) and alkylaminoalka-
ethiosulfuric acids (Penido et al. 2007). In addition, because there
s broad expression of ATP diphosphohydrolase during all stages of
he S. mansoni life cycle (Vasconcelos et al. 1993, 1996; DeMarco et
l. 2003; Faria-Pinto et al. 2004), new schistosomicidal S. mansoni
TP diphosphohydrolase inhibitors could be also active against ju-
enile schistosomes, unlike PZQ, which is inactive. Moreover, since
TP diphosphohydrolases have also been characterized in other par-
sites, such as Trypanosoma cruzi and Leishmania amazonensis, their
nhibitors could also be used to treat other parasitic diseases (Maia et
l. 2011).
Regarding the high ATPase inhibitory activity, we performed a
olecular modeling approach of cardamonin and SmATPDase 1. It is
mportant to point out that this is the first report of docking using
he SmATPDase 1. Cardamonin was docked into the extracellular
egion of the 3D SmATPDase 1 structure, which was constructed
ased on the best homology crystal structure present in the RCSB
rotein data bank. Also, the Nucleotide-Binding site was proposed
s the docking area, since cardamonin was highly active in the
TPase inhibitory assay. The obtained negative free energy values
−7.27 kcal/mol to −4.73 kcal/mol) from docking analyses support
he assumption that binding of cardamonin to Nucleotide-Binding
f SmATPDase 1 is a spontaneous process. Herein in Fig. 6A and B, it
s possible to observe nine different docks positions of cardamonin
nteracting with SmATPDase 1. Most of them possess a negative free
nergy values, and only one (colored in pink) was located outside of
he Nucleotide-Binding domain, showing the highest Estimated Free
nergy of Binding (−4.73 kcal/mol) (Fig. 6B). On the other hand, the
owest energy docked conformation (−7.27 kcal/mol) is represented
n Fig. 6C and D. Results revealed that cardamonin is located in
he hydrophobic binding cleft (gray spheres in Fig. 6D), lined with
esidues at Nucleotide-Binding, represented by Asp78, Ala79, Gly80,
er81, Ser83, Lys85, Glu201, Asp232, Leu233, Phe234 and Gly235. The
nalysis of the higher energy interactions, such as ionic and H-bonds,
eveals that in cardamonin polar groups containing oxygen interact
ith polar groups of the SmATPDase 1 residues Glu201, Asp232,
nd Tyr397, performing H-bonds. In addition, it is observed an ionic
nteraction between Trp483 and the oxygen of the acetophenone
ing (blue spheres in Fig. 6D). All docking results suggest that carda-
onin interacts with the Nucleotide-Binding of SmATPase 1, which
orroborates with its high inhibitory ATPase activity. Also, the nature
f SmATPase 1-cardamonin interactions is mainly hydrophobic and
ydrogen bonding.
Cardamonin is the main chalcone found in large amounts of car-
amom spice (fruits of Amomum subulatum Roxb.) and other medic-
nal plants of Zingiberaceae family, such as Alpinia katsumadae Hay-
ta, Alpinia speciosa (Blume) D. Dietr., and Elettaria cardamomum (L.)
aton (Bheemasankara Rao et al. 1976; Yadav et al. 2012; Gonçalves
t al. 2014), showing a number of biological activities, such as anti-
nflammatory, antimutagenic, and antioxidant (Gonçalves et al. 2014).
Previous studies also showed that cardamonin is highly active
gainst promastigote and amastigote forms of L. amazonensis (Ruiz et
l. 2011; Gonçalves et al. 2014). Our results demonstrated that carda-
onin not only kills adult schistosomes but also inhibits egg laying
nd damaging the worm’s tegument without affecting mammalian
ells. The obtained results indicated that cardamonin seems to cause
eath and damage of the S. mansoni tegument by inhibiting S. man-
oni ATP diphosphohydrolase. Also, to date, this is the first time that
he in vitro schistosomicidal activity and the S. mansoni ATP diphos-
hohydrolase inhibition were reported for a chalcone.
onclusion
This report provides evidence for the in vitro schistosomicidal ac-
ivity of cardamonin and demonstrated that this chalcone is highly
ffective in killing adult worms and inhibiting S. mansoni ATP diphos-
hohydrolase. Also, taken together, all experimental and theoretical
ata suggest that cardamonin is a promising compound that could
e evaluated in additional in vivo schistosomicidal investigations. Fi-
ally, the findings of this study open the route to further studies of
halcones as prototypes for the development of new S. mansoni ATP-
ase 1 inhibitors.
onflict of interest
The authors declare that there are no conflicts of interest.
cknowledgments
The authors are grateful to FAPEMIG (Grant numbers # APQ
171/11; BPD 00284-14; APQ 02015/14), CNPq (Grant number #
87221/2012-5), and FAPESP for financial support, as well as to
APES, PIBIC/CNPq/UFJF and CNPq (Grant number # 307729/2012-5)
or fellowships. We are also grateful to Mr. Jefferson S. Rodrigues for
xcellent technical assistance with S. mansoni life cycle maintenance
t the Adolfo Lutz Institute (São Paulo, SP, Brazil). We also thank Dr.
enrique K. Roffato and Dr. Ronaldo Z. Mendonça (Butantan Institute,
ão Paulo, SP, Brazil) for expert help with confocal microscope stud-
es (FAPESP Grant number # 00/11624-5). The author JM received no
ublic or private funding.
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