Cardamonin, a schistosomicidal chalcone from … that extracts from Piper aduncum L. (Piperaceae) are active against adult worms of Schistosoma mansoni,themajoretiologicalagentofhumanschistosomiasis.
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
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-
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,
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,
during in vitro cultivation. Rev. Inst. Med. Trop. São Paulo 38, 423–426.enkert, P., Biasini, M., Schwede, T., 2011. Toward the estimation of the absolute quality
of individual protein structure models. Bioinformatics 27, 343–350.heemasankara Rao, Ch., Namosiva Rao, T., Suryaprakasam, S., 1976. Cardamonin and
alpinetin from the seeds of Amomum subulatum. Planta Med. 29, 391–392.arrara, V.S., Vieira, S.C.H., de, P.R.G., Rodrigues, V., Magalhaes, L.G., Cortez, D.A.G.,
da Silva Filho, A.A., 2013. In vitro schistosomicidal effects of aqueous and
dichloromethane fractions from leaves and stems of Piper species and the isola-tion of an active amide from P. amalago L. (Piperaceae). J. Helminthol. 1–6.
a’dara, A.A., Bhardwaj, R., Skelly, P.J., 2014. Schistosome apyrase SmATPDase1, but notSmATPDase2, hydrolyses exogenous ATP and ADP. Purinergic Signal. 10, 573–580.
eMarco, R., Kowaltowski, A.T., Mortara, R.A., Verjovski-Almeida, S., 2003. Molec-ular characterization and immunolocalization of Schistosoma mansoni ATP-
e Moraes, J., Nascimento, C., Yamaguchi, L.F., Kato, M.J., Nakano, E., 2012. Schistosomamansoni: In vitro schistosomicidal activity and tegumental alterations induced by
Structural comparative analysis of secreted NTPDase models of Schistosoma man-soni and Homo sapiens. In: Campos, S. (Ed.), Advances in Bioinformatics and Com-
putational Biology. Springer International Publishing, pp. 91–98.aria-Pinto, P., Meirelles, M.N., Lenzi, H.L., Mota, E.M., Penido, M.L., Coelho, P.M.,
Vasconcelos, E.G., 2004. ATP diphosphohydrolase from Schistosoma mansoni egg:
characterization and immunocytochemical localization of a new antigen. Parasitol-ogy 129, 51–57.
aba, S., Jamal, S., Drug Discovery Consortium, O.S., Scaria, V., 2014. Cheminformaticsmodels for inhibitors of Schistosoma mansoni thioredoxin glutathione reductase.
Valladares, B., 2003. Antileishmanial activities of dihydrochalcones from Piperelongatum and synthetic related compounds. Structural requirements for activity.
Bioorg. Med. Chem. 11, 3975–3980.Krishna, B.M., Chaganty, R.B., 1973. Cardamonin and alpinetin from the seeds of Alpinia
Fessel, M.R., Marques, M.J., Souza, M.A., Coelho, P.M., Estanislau, J.A., da CostaRocha, M.O., de Oliveira Santos, M., Faria-Pinto, P., Vasconcelos, E.G., 2011. Occur-
rence of a conserved domain in ATP diphosphohydrolases from pathogenic organ-isms associated to antigenicity in human parasitic diseases. Dev. Comp. Immunol.
35, 1059–1067.
Martins, S.M., Torres, C.R., Ferreira, S.T., 2000. Inhibition of the ecto-ATPdiphosphohydrolase of Schistosoma mansoni by thapsigargin. Biosci. Rep.
series of schistosomicide drugs, the alkylaminoalkanethiosulfuric acids, partiallyinhibit the activity of Schistosoma mansoni ATP diphosphohydrolase. Eur. J. Phar-
macol. 570, 10–17.
osso, O.R., Diaz, P.P., de Diaz, A.M.P., 1994. Chemical study of Piper steerni. Rev. Colomb.Quim. 23, 53–62.
amalhete, C., Magalhaes, L.G., Rodrigues, V., Mulhovo, S., Da Silva Filho, A.A.,Ferreira, M.J., 2012. In vitro schistosomicidal activity of balsaminol F and karavi-
lagenin C. Planta Med. 78, 1912–1917.uiz, C., Haddad, M., Alban, J., Bourdy, G., Reategui, R., Castillo, D., Sauvain, M.,
Deharo, E., Estevez, Y., Arevalo, J., Rojas, R., 2011. Activity-guided isola-tion of antileishmanial compounds from Piper hispidum. Phytochem. Lett. 4,
363–366.
an Hellemond, J.J., Retra, K., Brouwers, J.F.H.M., van Balkom, B.W.M.,Yazdanbakhsh, M., Shoemaker, C.B., Tielens, A.G.M., 2006. Functions of the
tegument of schistosomes: clues from the proteome and lipidome. Int. J. Parasitol.36, 691–699.
asconcelos, E.G., Nascimento, P.S., Meirelles, M.N.L., Verjovski-Almeida, S.,Ferreira, S.T., 1993. Characterization and localization of an ATP-
diphosphohydrolase on the external surface of the tegument of Schistosoma
mansoni. Mol. Biochem. Parasitol. 58, 205–214.asconcelos, E.G., Ferreira, S.T., Carvalho, T.M., Souza, W., Kettlun, A.M., Mancilla, M.,
Valenzuela, M.A., Verjovski-Almeida, S., 1996. Partial purification andimmunohistochemical localization of ATP diphosphohydrolase from Schis-
tosoma mansoni. Immunological cross-reactivities with potato apyrase andToxoplasma gondii nucleoside triphosphate hydrolase. J. Biol. Chem. 271, 22139–