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Cardamonin, a schistosomicidal chalcone from Piper aduncum L. (Piperaceae) that inhibits Schistosoma mansoni ATP diphosphohydrolase.

ABSTRACT

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 diphosphohydrolase inhibitory activity of cardamonin, as well as, its molecular docking with S. mansoni ATPDasel, 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 ATPDasel 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 [micro]M) 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 5. mansoni ATP diphosphohydrolase ([IC.sub.50] of 23.54 [micro]M). 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 demonstrated, for the first time, that this chalcone is highly effective in inhibiting S. mansoni ATP diphosphohydrolase, opening the route to further studies of chalcones as prototypes for new S. mansoni ATP diphosphohydrolase inhibitors.

Keywords:

Cardamom

Chalcone

SmATPDases

Piper aduncum

Apyrase

Schistosomicidal activity

Introduction

Schistosomiasis, caused by trematode flatworms of the genus Schistosoma, is one of the most significant neglected tropical diseases, causing serious public health problems in more than 70 tropical and subtropical countries (Gryseels et al. 2006). It is estimated that more than 200 million people are infected and that 779 million are at risk of infection (Gaba et al. 2014; Veras et al. 2012). Schistosoma mansoni is the major etiological agent of human schistosomiasis, for which the treatment is dependent on a single drug, praziquantel (PZQ).

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 appearance 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 parasite 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 localized on the external tegument surface of S. mansoni that hydrolyze a variety of nucleoside tri- and diphosphates to corresponding nucleoside monophosphates (Vasconcelos et al. 1993,1996; DeMarco et al. 2003). Also, the literature suggests thatS. mansoni ATP diphosphohydrolases are involved in the evasion of the host defense, due to the location of these isoforms in the parasite and the importance of nucleoside di- and triphosphates in activating cells of the host immune system (de Souza et al. 2014; Da'dara et al. 2014). Besides, S. mansoni 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 parasite within blood vessels and its survival over a long period in the human host (Da'dara et al. 2014; Faria-Pinto et al. 2004). Two S. mansoni ATP diphosphohydrolase isoforms (SmATPDasel 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 SmATPDasel 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-ruao", is widely used in folk medicine as antiinflammatory and antiseptic (Carrara et al. 2013; Morandim et al. 2009). Previous phytochemical studies of the aerial parts of P. aduncum reported the isolation of chalcones, flavanones, and dihydrochalcones (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 schistosomicidal 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 schistosomicidal 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 ATPDasel 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 C[H.sub.2] [Cl.sub.2] as solvent. After extraction, the solvent was removed under vacuum to yield 24 g of the crude dichloromethane extract (PaI), which was able to cause 100% mortality in schistosomes. PaI (24 g) was suspended in methanol:[H.sub.2]0 (7:3 v/v), and submitted to sequential partition, with equal volume of solvents of increasing polarities, namely nhexane (PaI-H, 8 g), chloroform (PaI-C, 6 g) and ethyl acetate (PaI-A, 7 g). PaI-C was selected for chromatographic fractionation based on its schistosomicidal activity. PaI-C (5 g) was chromatographed over silica gel using a vacuum liquid chromatography system and hexane: ethyl acetate mixtures in increasing proportions as eluents, furnishing 12 fractions. Of them, fractions IV (150 mg) and V (341 mg) were submitted to column chromatography over silica gel using CHCI3 :Me2CO in increasing proportions as eluent, affording the following compounds: 5,7-dihydroxyflavanone (pinocembrin, 30 mg) from 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 of all isolated compounds were established by by [sup.1]H and [sup.13]C NMR data analysis in comparison to literature (Krishna and Chaganty 1973; Posso et al. 1994; Avila et al. 2011; Gonealves et al. 2014).

Maintenance of the S. mansoni life cycle

S. mansoni (BH strain Belo Horizonte, Brazil) worms were maintained in Biomphalaria glabrata snails as intermediate hosts and Mesocricetus auratus hamsters as definitive host at the Adolfo Lutz Institute (Sao Paulo, Brazil), according to standard procedures previously described (de Moraes et al. 2012). At 49 days post-infection, adult S. mansoni specimens were recovered from each hamster by perfusion in Roswell Park Memorial Institute (RPMI) 1640 medium (Invitrogen, Sao Paulo, Brazil) supplemented with heparin. All experiments were authorized by the Committee for Ethics in Animal Care of Adolfo Lutz Institute (Sao Paulo, Brazil), in accordance with nationally and internationally accepted principles for laboratory animal use and care (CEUA, 11.794/08). The study was conducted in adherence to the institution's guidelines for animal husbandry.

In vitro studies of adult schistosomes

Adult schistosomes were washed in RPMI-1640 medium supplemented with 200 p.g/ml streptomycin, 200 1U [ml.sup.-1] penicillin (Invitrogen), and 25 mM HEPES. Adult worm pairs (male and female) were incubated in a 24-well culture plate (Techno Plastic Products, TPP, St. Louis, MO, USA), containing the same medium supplemented with 10% heat-inactivated calf serum (Gibco BRL) at 37[degrees]C in a 5% C02 atmosphere. A preliminary screening of crude extract (PaI) and fractions (PaI-H, PaI-C, and PaI-A) was performed at 200 [micro]g/ml, and isolated compounds at 100 [micro]M, as described by Ramalhete et al. (2012). The most active compound (cardamonin) was also evaluated at 50, 25, 10 and 5 [micro]M. Samples were added to the culture from a 4000 [micro]g/ml stock solution in RPMI-1640 containing dimethyl sulfoxide (DMSO). The final volume in each well was 2 ml. The control worms were assayed in RPMI-1640 medium and RPMI1640 with 0.5% DMSO as negative control groups and PZQ (5 [micro]M) as positive control group. All experiments were repeated at least three times independently for each treatment, and plating was carried out in triplicate for each concentration. Parasites were maintained for 48 h and monitored every 24 h using a light microscope in order to evaluate their general condition, with emphasis on changes in motor activity, mortality rate, and tegumental alterations. Worm mortality was assessed by lack of movement (de Moraes et al. 2012).

Assessment of the reproductive fitness of adult worms

In order to evaluate the sexual fitness of worms exposure to non-lethal concentrations of cardamonin, parasites were continually monitored and schistosome egg output in vitro was determined by counting the number of eggs, as previously described (de Moraes et al. 2012). In this case, adult worm pairs were incubated with cardamonin (2.5,5, and 10 [micro]M) and in vitro parasite egg output was determined, on daily basis for five days, by counting the number of eggs using an inverted microscope and a stereomicroscope (SMZ 1000, Nikon, Melville, NY, USA).

Confocal laser scanning microscopy

Tegumental alteration and quantification of the number of tubercles were performed for cardamonin (10,25,50 and 100 [micro]M) using a confocal laser scanning microscope (de Moraes et al. 2012). After the established times or in the occurrence of death, the parasites were fixed in Formalin-acetic-alcohol solution (FAA) and analyzed under a confocal microscope (Laser Scanning Microscopy, LSM 510 META, Zeiss) at 488 nm (exciting) and 505 nm (emission). A minimum of three areas of the tegument of each parasite were assessed. The number of tubercles was counted in 20,000 [micro][m.sup.2] of area calculated with the Zeiss LSM Image Browser software.

Cytotoxicity assay

Vero mammalian cells (African green monkey kidney fibroblast) used in this study were obtained from the American Type Culture Collection (ATCC CCL-81; Manassas, VA) and provided by Dr. Ronaldo Z. Mendonea (Laboratorio de Parasitologia, Instituto Butantan, Sao Paulo, Brazil). Cytotoxicity was determined as previously described (de Moraes et al. 2012) using different concentrations of cardamonin (25, 50,100, and 200 [micro]M).

Determination of S. mansoni ATP diphosphohydrolase activity

The total homogenized of adults worms of S. mansoni (0.05 mg protein/ml), containing ATP diphosphohydrolases, was pre-incubated for 30 min at 37[degrees]C with cardamonin (5-40 [micro]M) in a standard reaction medium, adapted according to method previously described (Faria-Pinto et al. 2004). The hydrolytic assay was initiated by the addition of sufficient ATP or ADP to give a final concentration of 3 mM, and stopped by the addition of 0.1 N HC1. Inorganic phosphate (Pi) liberated was determined spectrophotometrically (Penido et al. 2007). Control samples were incubated in the same medium without the addition of cardamonin and in the presence and absence of 10% DMSO (used to dissolve cardamonin). In the absence of 10% DMSO, the adults worms homogenized presented an ADPase activity of 76.49 [+ or -] 11.47 nmol Pi [mg.sup.-1] [min.sup.-1] and an ATPase activity of 55.80 [+ or -] 8.36 nmol Pi [mg.sup.-1] [min.sup.-1]. The half-inhibitory concentration ([IC.sub.50]) was calculated from dose-response curves by non-linear regression analysis using Graphpad Prism version 5.0 (Graphpad software Inc., La Jolla, CA, USA).

Statistical analysis

Statistical tests were performed with Graphpad Prism software. Significant differences were determined by a one-way analysis of variance (ANOVA) and by applying Turkey's test for multiple comparisons with the level of significance set at P < 0.05.

Molecular modeling

The amino acid sequence of S. mansoni ATPDase 1 was retrieved from PUBMED database (code: Gl: 33114187) (DeMarco et al. 2003). The initial homology models of S. mansoni ATPDase 1 were built using Swiss-MODEL and Swiss-PDB viewer programs (http://swissmodel.expasy.org/ and http://www.expasy.org/spdbv/) as described by Wermelinger et al. (2009). We used the ATPDase 1 of Rattus norvegicus (PDB 3ZX3) as template with the homology degree of S. mansoni ATPDase 1 (39%). The structure derived from homology modeling was submitted to the validation process, using QMEAN scoring function (Benkert et al. 2011) and the PROCHECK program (Laskowski et al. 1993).

[FIGURE 1 OMITTED]

Molecular docking was performed for cardamonin using Autodock 4.2 along with AutodockTools 1.5.6 (ADT) (Morris et al. 1998). The cardamonin was built using SPARTAN'14 (Wavefunction Inc., Irvine, CA, USA) and energy minimization was performed with convergence criterion of the lowest energy. Non-polar hydrogen atoms and lone pairs were merged, and each atom was assigned with Gasteiger partial charges. The grid size was set to 40 [Angstrom] x 40 [Angstrom] x 40 [Angstrom] with a grid spacing of 0.508 [Angstrom]. The grid box was positioned at the center of Nucleotide-Binding domain of the enzyme. One hundred independent dockings were carried out for each docking experiment. The lowest docked energy of each conformation in the most populated cluster was selected. Analysis and visualization of the docking results were done using Visual molecular dynamics (VMD) and SPDBviewer.

Results and discussion

The development of new schistosomicidal drugs and the identification of new schistosomicidal molecules have been highly encouraged, mainly because the treatment of schistosomiasis relies on a single drug, PZQ (Gaba et al. 2014). In this regard, the Brazilian flora is rich in several medicinal plants with high potential for providing biologically active compounds against Schistosoma species (Barbosa de Castro et al. 2013). Among then, several species of Piper have been reported as promising sources of antiparasitic compounds (de Moraes et al. 2012; Hermoso et al. 2003; Ruiz et al. 2011). Recently, our previous study showed that extracts from P. aduncum were active against adult worms of S. mansoni (Carrara et al. 2013).

Now, as a part of our program devoted to the search for schistosomicidal molecules from Brazilian plants, we performed a bioassayguided fractionation of the crude extract of P. aduncum L. (PaI). As shown in Table 1, the crude extract (PaI, 200 [micro]g/ml) caused 100% mortality in all adult parasites after 24 h of incubation. Then, PaI was further partitioned into three organic fractions. Among then, the chloroform fraction (PaI-C, 200 [micro]g/ml) was found to be the most active, causing 100% mortality, significant decrease in motor activity, and tegumental alterations on adult worms (Table 1). On the other hand, n-hexane (PaI-H) and ethyl acetate (PaI-A) fractions were inactive. PZQ (5 [micro]M) caused 100% mortality, whereas no effect was observed 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 compounds, which were chemically identified by [sup.1]H and [sup.13]C NMR data analysis in comparison to literature as: pinocembrin (Posso et al. 1994), uvangoletin (Avila et al. 2011), and cardamonin (Krishna and Chaganty 1973; Gonealves et al. 2014) (Fig. 1). Purity of all the isolated compounds was estimated to be higher than 95% by both [sup.13]C NMR and HPLC analysis using different solvent systems (MeOH/MeCN/[H.sub.2]0,65:5:30; MeOH/H20 50 to 100% in 20 min).

In a preliminary survival of adult worms of S. mansoni test, all isolated compounds were tested at 100 [micro]M. 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 [micro]M, while no activity was found at concentrations of 5 and 10 [micro]M, even after 48 h of incubation. Because 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 reproductive fitness of worms treated with non-lethal concentrations of cardamonin (2.5, 5 and 10 [micro]M) (Fig. 2). It was observed that cardamonin also reduced the total number of eggs laid at sub-lethal doses (5 and 10 |iM). Based on three independent experiments, performed in triplicate, cardamonin (5 and 10 [micro]M) shows the same inhibition percentage of egg laying throughout the incubation period, compared with the control group. These results suggest that the inhibition of oviposition by cardamonin is irreversible. Reproductive fitness 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 schistosomiasis, which is characterized by immunopathological lesions, including inflammation and fibrosis in the target (Gryseels et al. 2006). Moreover, changes in the reproductive ability of S. mansoni may be associated with alterations in the reproductive system of worms when exposed to drugs with schistosomicidal properties (Barth et al. 1996). According to de Moraes et al. (2012), compounds with schistosomicidal activity can be also effective suppressive, inhibiting oviposition by schistosomes.

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

The effects of cardamonin on the tegument of schistosomes were analyzed by confocal microscopy analysis in order to examine morphological alterations at the surface of male worms. Morphological studies revealed progressive damages and prominent alterations of the tegumental surfaces, in which tubercles appeared collapsed and disrupted (Fig. 3A-E). While in the negative control group, adult worms of S. mansoni showed intact surface structure and topography (Fig. 3A), after incubation with 25 and 50 [micro]M of cardamonin, teguments showed massive disintegration of tubercles (Fig. 3D and E). PZQ(5 [micro]M) and cardamonin (10 [micro]M) also caused damages in tubercles (Fig. 3B and C).

Morphological alterations on the Schistosoma tegument were also quantitatively analyzed by counting the tubercles on the dorsal surface of male schistosomes (Fig. 4). Cardamonin caused disintegration of the tegumental surface in a dose-dependent manner (Fig. 4). No intact tubercles were seen at 100 [micro]M, while in the group exposed to 50 [micro]M of cardamonin the number of intact tubercles was 2 [+ or -] 1, differing significantly from the negative control group (P < 0.001). Thus, a pattern consisting of a combination of changes in the surface morphology were detected and correlated to the death of the adult worms.

[FIGURE 4 OMITTED]

[FIGURE 5 OMITTED]

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). Also, in case of severe damages on the tegument, the host's immune response can affect the reparation process (Veras et al. 2012). The tegument destruction induced by cardamonin will probably not allow reparation, since the damages lead to extensive peeling of the tegument surface, as well as formation and collapsing of tubercles, indicating straight damage to the cells in a direct dose-dependent effect. Furthermore, our results showed that cardamonin is nontoxic to the mammalian Vero cells at concentrations that effectively kill the adult worms of S. mansoni (25, 50,100, and 200 [micro]M) (data not shown).

[FIGURE 6 OMITTED]

According to obtained results, we speculated if cardamonin may act on a specific target in the S. mansoni tegument. Then, the in vitro ATP diphosphohydrolase activity of cardamonin was determined. Cardamonin (40 [micro]M) inhibited S. mansoni ATPase activity by approximately 82%, showing an [IC.sub.50] of 23.54 [micro]M (Fig. 5), whereas the ADPase activity remained unaffected (ca. < 10%) up to the highest concentration (data not shown).

ATP diphosphohydrolases are ecto-enzymes present in the tegument of S. mansoni. Its purine recovery activity has been suggested as a mechanism for Schistosoma protection against host organism (Faria-Pinto et al. 2004; Vasconcelos et al. 1993, 1996). S. mansoni ATP diphosphohydrolases specifically counteract ATP damage-associated molecular patterns (DAMPS)-mediated inflammatory signaling and limit the host's attempts to focus inflammatory mediators around the worms (de Souza et al. 2014; Da'dara et al. 2014). In this manner, these tegumental enzymes help impair host immune defenses and promote parasite survival. Therefore, targeting S. mansoni ATP diphosphohydrolase has been considered a promising approach to the study of novel drug candidates to treat schistosomiasis (Da'dara et al. 2014; Penido et al. 2007). However, no study has been reported on the ATP diphosphohydrolase activity of chalcones.

Considering the importance of studying novel S. mansoni ATP diphosphohydrolases inhibitors, cardamonin presents better results for ATPase activity compared to other compounds described in literature, such as thapsigargin (Martins et al. 2000) and alkylaminoalkanethiosulfuric acids (Penido et al. 2007). In addition, because there is broad expression of ATP diphosphohydrolase during all stages of the S. mansoni life cycle (Vasconcelos et al. 1993, 1996; DeMarco et al. 2003; Faria-Pinto et al. 2004), new schistosomicidal S. mansoni ATP diphosphohydrolase inhibitors could be also active against juvenile schistosomes, unlike PZQ, which is inactive. Moreover, since ATP diphosphohydrolases have also been characterized in other parasites, such as Trypanosoma cruzi and Leishmania amazonensis, their inhibitors could also be used to treat other parasitic diseases (Maia et al. 2011).

Regarding the high ATPase inhibitory activity, we performed a molecular modeling approach of cardamonin and SmATPDase 1. It is important to point out that this is the first report of docking using the SmATPDase 1. Cardamonin was docked into the extracellular region of the 3D SmATPDase 1 structure, which was constructed based on the best homology crystal structure present in the RCSB protein data bank. Also, the Nucleotide-Binding site was proposed as the docking area, since cardamonin was highly active in the ATPase inhibitory assay. The obtained negative free energy values (-7.27 kcal/mol to -4.73 kcal/mol) from docking analyses support the assumption that binding of cardamonin to Nucleotide-Binding of SmATPDase 1 is a spontaneous process. Herein in Fig. 6A and B, it is possible to observe nine different docks positions of cardamonin interacting with SmATPDase 1. Most of them possess a negative free energy values, and only one (colored in pink) was located outside of the Nucleotide-Binding domain, showing the highest Estimated Free Energy of Binding (-4.73 kcal/mol) (Fig. 6B). On the other hand, the lowest energy docked conformation (-7.27 kcal/mol) is represented in Fig. 6C and D. Results revealed that cardamonin is located in the hydrophobic binding cleft (gray spheres in Fig. 6D), lined with residues at Nucleotide-Binding, represented by Asp78, Ala79, Gly80, Ser81, Ser83, Lys85, Glu201, Asp232, Leu233, Phe234 and Gly235. The analysis of the higher energy interactions, such as ionic and H-bonds, reveals that in cardamonin polar groups containing oxygen interact with polar groups of the SmATPDase 1 residues Glu201, Asp232, and Tyr397, performing H-bonds. In addition, it is observed an ionic interaction between Trp483 and the oxygen of the acetophenone ring (blue spheres in Fig. 6D). All docking results suggest that cardamonin interacts with the Nucleotide-Binding of SmATPase 1, which corroborates with its high inhibitory ATPase activity. Also, the nature of SmATPase 1-cardamonin interactions is mainly hydrophobic and hydrogen bonding.

Cardamonin is the main chalcone found in large amounts of cardamom spice (fruits oiAmomum subulatum Roxb.) and other medicinal plants of Zingiberaceae family, such as Alpinia katsumadae Hayata, Alpinia speciosa (Blume) D. Dietr., and Elettaria cardamomum (L.) Maton (Bheemasankara Rao et al. 1976; Yadav et al. 2012; Goncalves et al. 2014), showing a number of biological activities, such as anti-inflammatory, antimutagenic, and antioxidant (Goncalves et al. 2014).

Previous studies also showed that cardamonin is highly active against promastigote and amastigote forms of L amazonensis (Ruiz et al. 2011; Goncalves et al. 2014). Our results demonstrated that cardamonin not only kills adult schistosomes but also inhibits egg laying and damaging the worm's tegument without affecting mammalian cells. The obtained results indicated that cardamonin seems to cause death and damage of the S. mansoni tegument by inhibiting S. mansoni ATP diphosphohydrolase. Also, to date, this is the first time that the in vitro schistosomicidal activity and the S. mansoni ATP diphosphohydrolase inhibition were reported for a chalcone.

Conclusion

This report provides evidence for the in vitro schistosomicidal activity of cardamonin and demonstrated that this chalcone is highly effective in killing adult worms and inhibiting S. mansoni ATP diphosphohydrolase. Also, taken together, all experimental and theoretical data suggest that cardamonin is a promising compound that could be evaluated in additional in vivo schistosomicidal investigations. Finally, the findings of this study open the route to further studies of chalcones as prototypes for the development of new S. mansoni ATPDase 1 inhibitors.

Conflict of interest

The authors declare that there are no conflicts of interest.

ARTICLE INFO

Article history:

Received 24 April 2015

Revised 11 June 2015

Accepted 18 June 2015

Abbreviations: PZQ, praziquantel; PaI, crude dichloromethane extract from inflorescences of P. aduncum; PaI-H, hexane fraction of the crude extract of P. aduncum; PaI-C, chloroform fraction of the crude extract of P. aduncum; PaI-A, ethyl acetate fraction of the crude extract of P. aduncum.

Acknowledgments

The authors are grateful to FAPEMIG (Grant numbers # APQ 0171/11; BPD 00284-14; APQ 02015/14), CNPq (Grant number # 487221/2012-5), and FAPESP for financial support, as well as to CAPES, PIBIC/CNPq/UFJF and CNPq (Grant number # 307729/2012-5) for fellowships. We are also grateful to Mr. Jefferson S. Rodrigues for excellent technical assistance with S. mansoni life cycle maintenance at the Adolfo Lutz Institute (Sao Paulo, SP, Brazil). We also thank Dr. Henrique K. Roffato and Dr. Ronaldo Z. Mendonca (Butantan Institute, Sao Paulo, SP, Brazil) for expert help with confocal microscope studies (FAPESP Grant number # 00/11624-5). The author JM received no public or private funding.

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Clarissa C.B. de Castro (a), Poliana S. Cost (a), Gisele T. Laktin (a), Paulo H.D. de Carvalho (a), Reinaldo B. Geraldo (a), Josue 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 Farmacia Departamento de Ciencias Farmaceuticas, Universidade Federal de Juiz de Fora 36036-900Juiz de Fora, MC, Brazil

(b) Nucleo de Pesquisa em Doeneas Negligenciadas (FACIC), 07025-000 Guarulhos, SP, Brazil

(c) Nucleo de Enteroparasitas, Instituto Adolfo Lutz, 01246-902 Sao Paulo, SP, Brazil

(e) Departamento de Quimica, Universidade Federal de Juiz de Fora, 36036-330Juiz de Fora, MC, Brazil

(e) Instituto de Ciencias Biologicas, Departamento de Bioquimica Universidade Federal de Juiz de Fora, 36036-900Juiz de Fora, MG, Brazil

* 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 Filho).

http://dx.doi.Org/10.1016/j.phymed.2015.06.009
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    Dead    Motor activity
                             incubation   worms   reduction (%) (a)
                             (h)          (%)
                                          (a)     Slight   Significant

Control (b)                  24           --      --       --
                             48           --      --       --
DMSO 0.5%                    24           --      --       --
                             48           --      --       --
PZQ(5 [micro]M)              24           100     --       100
                             48           100     --       100
Extract and fractions (c)
Pal                          24           100     --       100
                             48           100     --       100
Pal-C                        24           100     --       50
                             48           100     --       100
Pal-H                        24           --      --       --
                             48           --      --       --
Pal-A                        24           --      --       --
                             48           --      --       --
Compounds
Uvangoletin (100 [micro]M)
                             24           --      --       --
                             48           --      --       --
Pinocembrin (100 [micro]M)
                             24           --      --       --
                             48           --      --       --
Cardamonin
100 [micro]M                 24           100     --       100
                             48           100     --       100
50 [micro]M                  24           o o     --       100
                             48           100     --       100
25 [micro]M                  24           100     --       100
                             48           100     --       100
10 [micro]M                  24           --      --       --
                             48           --      --       --
5 [micro]M                   24           --      --       --
                             48           --      --       --

Groups                       Period of    Worms with tegumental
                             incubation   alterations (%) (a)
                             (h)
                                          Partial   Extensive

Control (b)                  24           --        --
                             48           --        --
DMSO 0.5%                    24           --        --
                             48           --        --
PZQ(5 [micro]M)              24           --        100
                             48           --        100
Extract and fractions (c)
Pal                          24           --        100
                             48           --        100
Pal-C                        24           --        50
                             48           --        100
Pal-H                        24           --        --
                             48           --        --
Pal-A                        24           --        --
                             48           --        --
Compounds
Uvangoletin (100 [micro]M)
                             24           --        --
                             48           --        --
Pinocembrin (100 [micro]M)
                             24           --        --
                             48           --        --
Cardamonin
100 [micro]M                 24           --        100
                             48           --        100
50 [micro]M                  24           --        100
                             48           --        100
25 [micro]M                  24           --        100
                             48           --        100
10 [micro]M                  24           --        --
                             48           --        --
5 [micro]M                   24           --        --
                             48           --        --

(a) Percentages relative to the 20 worms investigated.

(b) RPMI 1640.

(c) Crude extract and fractions were tested at 200 [micro]g/ml.
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Author:de Castro, Clarissa C.B.; Costa, Poliana S.; Laktin, Gisele T.; de Carvalho, Paulo H.D.; Geraldo, Re
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Date:Sep 15, 2015
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