Phytomedicine 22 (2015) 921–928

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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.

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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: ademar.alves@ufjf.edu.br, silvafilhoaa@gmail.com (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 2

fter 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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OH

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

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200

250

300

24 h48 h72 h96 h120 h

0 2.5 5 10

******

******

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*

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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.

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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,

h

ncluding 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.

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0 10 25 50 100 PZQ

0

10

20

30

40

50

**

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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.

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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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