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Synergistic effects of parthenolide and benznidazole on Trypanosoma cruzi.

ABSTRACT

Parthenolide previously isolated from Tanacetum vulgare was tested for its in vitro combinatory effect with benznidazole against Trypanosoma cruzi. Parthenolide showed a strong synergistic activity against epimastigote forms, reducing 23-fold the concentration of benznidazole necessary to inhibit 50% of cell growth ([IC.sub.50] of 1.6 to 0.07 [mu]g/ml) when in combination with parthenolide. In addition, an additive effect against trypomastigote forms (FIC 1.06), followed by an antagonistic effect on the cytotoxicity (FIC 2.36), was observed for the combination of both drugs. Parthenolide induced morphological alterations in the body shape of trypomastigote forms, causing rounding and shortening of the parasite and loss of integrity of the plasma membrane, as previously described by other workers.

ARTICLE INFO

Keywords: Tanacetum vulgare Parthenolide Trypanosoma cruzi Trypanocidal activity Synergism

[C] 2010 Elsevier GmbH. All rights reserved.

Introduction

The importance of natural products that have been traditionally used by native cultures to treat infectious diseases is clearly enormous (Rates 2001). Natural products are valuable sources of chemotherapeutic agents, and because of the diversity of their molecular structures, they became targets for drug discovery (Shu 1998). Parthenolide, a sesquiterpene lactone isolated from many members of the genus Tanacetum, exhibits anti-inflammatory properties and is used clinically for treatment of migraine (Murphy et al. 1998; Pfarrenrath et al. 2002). Parthenolide has also been shown to inhibit the growth of Leishmania amazonensis (Tiuman et al. 2005) and Trypanosoma cruzi (Izumi et al. 2007).

American trypanosomiasis (Chagas' disease) is a zoonotic disease caused by the flagellate protozoan T. cruzi, transmitted to humans by triatomine bugs, through blood transfusion (Viotti et al. 1994), or by the oral route (Yoshida 2009). Chagas' disease is endemic in Latin America, where it affects 15 million people. Each year 41,200 new cases are recorded, and 12,500 deaths occur (WHO 2006). The infection is characterized by a rapid acute phase followed by a chronic phase, in which most patients remain asymptomatic, and 30-40% of cases develop cardiac symptoms or digestive lesions.

At present, curative treatments for Chagas' disease do not exist. Nifurtimox and benznidazole are the only antichagasic drugs with clinical efficacy, but they are far from optimal because of collateral effects. The improvement of the efficacy of the usual drugs by combination with natural compounds has been evaluated (reviewed by Wagner and Ulrich-Merzenich 2009). In this study, we evaluated the effect of parthenolide isolated from T. vulgare on the morphology of T. cruzi, as well as its combinatory effect with benznidazole.

Materials and methods

Isolation of compounds

Leaves of Tanacetum vulgare were collected at the Irenice Silva Medicinal Plants Garden of the State University of Maringa, in Maringa city, Brazil. A voucher of this species was deposited at the herbarium of the same university (register number HUM 8425), and identified by comparison with an authenticated specimen. Plant material was dried, pulverized, and macerated. The crude extract was filtered, its solvent was evaporated, and it was lyophilized. Then, the crude extract was chromatographed on a silica-gel 60 column (70-230 mesh - Merck[R]), and the dichloromethane fraction of the ethyl-acetate crude extract (ECE-DF) was subfractioned with several solvent combinations. This resulted in Isolation of a pure compound that was identified by nuclear magnetic resonance (NMR; Gemini 2000 BB; Varian), (1) H NMR (300 MHz), and (13) C NMR (75.5MHz) analyses in [CDCl.sub.3]; infrared analysis (Bomem-MV 100; Hartmann & Braun-Michelson); and UV analysis (CARY 1E UVVis; Varian).

Synergistic effect

Epimastigotes (1 x [10.sup.6] cells/ml) from exponential phase culture were resuspended in LIT medium supplemented with 10% FBS. The drugs were added to the cell suspension, alone or in combination [parthenolide and benznidazole), at the desired final concentrations in 24-well plates, and incubated at 28 [degrees] C. The number of parasites was determined by counting with a Neubauer hemocytometer after 96 h. Trypomastigotes (1 x [10.sup.7] cells/ml) were resuspended in DMEM with 10% Balb/c mice blood. This suspension was added to the drugs, alone or in combinations, previously prepared in 96-well plates, followed by incubation at 37 [degrees]C. The surviving trypomastigotes were counted as described by Brener (1962) after 24 h. The cytotoxicity was analyzed after treatment of monolayers of [LLCMK.sub.2] cells with the drugs alone or in combinations for 96 h. This assay was evaluated by the sulforhodamine B technique (Skehan et al. 1990). Classical isobolograms were constructed by plotting concentrations of drugs that alone or in combination induced 50% inhibition of growth ([IC.sub.50]) of epimastigote forms, 50% effective concentration ([EC.sub.50]) of trypomastigote forms, or a 50% cytotoxic effect ([CC.sub.50]) in [LLCMK.sub.2] cells. Fractional inhibitory concentrations (FIC) were calculated according to Hallander et al. (1982).

Scanning electron microscopy

Trypomastigote forms were treated with 1 [mu]g/ml (4.0 [mu]M) of parthenolide for 24 h. After treatment, the parasites were washed with 0.01 M PBS and fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer for 2 h. The parasites were washed three times in 0.1 M sodium cacodylate buffer, placed on the poly-L-lysine-coated coverslip, dehydrated in different concentrations of ethanol, critical point-dried in [CO.sub.2], sputter-coated with gold, and observed with a Shimadzu SS-550 scanning electron microscope.

Results and discussion

There is a general lack of effective and inexpensive chemotherapeutic agents for the treatment of Chagas' disease. Natural products have potential in the search for new and selective agents for the treatment of important tropical diseases caused by protozoa. Plants belonging to the genus Tanacetum are reputed to have excellent medicinal value, and several sesquiterpenoids and sesquiterpene lactones, which are typical constituents of these plants, have been isolated from Tanacetum species (Tiuman et al. 2005).

Here we report the synergistic effect of a compound obtained from T. vulgare in combination with benznidazole. The compound isolated from ECE-DF obtained from T. vulgare was identified as the sesquiterpene lactone, 4[alpha],5[beta]-epoxy-germacra-l-(10),11(13)-dien-12,6[alpha]-olide (parthenolide), as previously described (Tiuman etal. 2005).

Parthenolide has been reported to have many biological activities, including antiviral (Onozato et al. 2009) and antiprotozoal (Tiuman et al. 2005; Izumi et al. 2007). Izumi et al. (2007) described the effect of parthenolide isolated from Tanacetum parthenium, and in the present study we confirmed this activity (data not shown) and showed the synergistic effect of parthenolide isolated from T. vulgare in combination with benznidazole. The chemical structures of parthenolide and benznidazole are given in Fig. 1.

[FIGURE 1 OMITTED]

Synergistic effect

Synergy is defined as an effect produced by a combination of components that is greater than the sum of the effects produced by the components alone (Wagner and Ulrich-Merzenich 2009):

E([d.sub.benz],[d.sub.part])> [Ed.sub.benz] + [Ed.sub.part]

where E is the observed effect, and [d.sub.benz] and [d.sub.part] are the doses of benznidazole and parthenolide.

The combination effect can be demonstrated by an isobologram, which uses the fractional inhibitory concentration (FIC) (Urbina et al. 1995, 1996). When the sum of these fractions is 1, the combination is additive; when the sum is <1, the combination is synergistic; when the sum is >1, the combination is antagonistic (Chou 2006). Parthenolide displayed a strong synergistic effect when used in combination with benznidazole against epimastigotes (FIC = 0.35), reducing the [IC.sub.50] of the latter drug almost 23-fold. The isobologram of this combination shows the synergistic effect (Fig. 2A).

[FIGURE 2 OMITTED]

In the present study, the [IC.sub.50] of benznidazole against epimastigote forms varied from 1.6 to 1.8 [mu]g/ml when this medicine was tested separately. This concentration could be reduced to 0.1 [mu]g/ml in synergistic action with 0.19 [mu]g/ml of parthenolide, which gave the same inhibition results. The treatment of the protozoa with parthenolide alone resulted in an [IC.sub.50] of 0.63 [mu]g/ml.

When tested against trypomastigote forms, benznidazole alone showed [EC.sub.50] of 1.3 [mu]g/ml, and in association with parthenolide showed an FIC of 1.06, i.e., having only an additive effect. Contrary to the effect of combinations on epimastigotes, in trypomastigotes the association of the drugs was more efficient for parthenolide, for which the [EC.sub.50] was reduced almost 15 times in the presence of benznidazole.

The cytotoxic effect was also evaluated, with the drugs alone or in combination. The [CC.sub.50] of benznidazole was 200 [mu]g/ml, and that of parthenolide was 9.5 [mu]g/ml. The FIC value of both drugs combined was 2.36, indicating strong antagonism (Fig. 2B), which resulted in greater protection of the cells from drug damage.

A few researchers have studied antibiotic synergism against T. cruzi. A systematic study found that mevinolin (hypocholesterolemic agent) combined with azoles (ergosterol biosynthesis inhibitors) such as ketoconazole can be used in the treatment of Chagas' disease (Urbina et al. 1993). The same group observed the antiproliferative synergism of allylamine-SF86-327 (terbinafine) and [[DELTA].sup.24(25)] sterol methyltransferase inhibitors with ketoconazole on T. cruzi (Urbina et al. 1988, 1995).

Recently, a synergistic effect of aspirin with nifurtimox and benznidazole was observed on T. cruzi infection of RAW cells, with FICs of 0.71 and 0.61, respectively. These synergistic effects result from the capacity of aspirin to increase the antiparasitic activity of macrophages (Lopez-Munoz et al. 2010).

Benaim et al. (2006) showed that amiodarone, a drug used to treat arrhythmias, has direct activity against T. cruzi, both in vitro and in vivo, and that it acts synergistically with posaconazole to block ergosterol biosynthesis. A synergistic effect was also observed for the combination of benznidazole and ketoconazole in mice infected with CL and Y isolates of T. cruzi, but not with the Colombiana strain. Therefore, the synergistic effects of these drugs may differ according to genetic attributes of the parasite (Araujo et al. 2000).

Electron microscopy

Several studies have also reported alterations in T. cruzi treated with synthetic or natural compounds (Salas et al. 2008; Valdez et al. 2009). Izumi et al. (2007) demonstrated the morphological activity of parthenolide on epimastigotes. Here we observed that the treatment of trypomastigotes with 1 [mu]g/ml (4.0 [mu]M) of parthenolide induced alterations in the body shape, causing rounding and shortening of the parasite and loss of integrity of the plasma membrane (Fig. 3). The changes in the structure and shape of the plasma membrane may be caused by the action of the drug on submembrane microtubules, since parthenolide exerts in vitro stimulatory activity on tubulin assembly (Miglietta et al. 2004).

[FIGURE 3 OMITTED]

Conclusion

Searching for new drugs with high activity is essential, especially considering that in Brazil parasitic diseases constitute a serious public-health problem. The present study, unlike most, evaluated the synergy potential of natural compound. Our results showed that parthenolide changed the structure of trypomastigote forms and showed a synergistic effect with benznidazole when tested against epimastigotes, and an additive effect against trypomastigotes. The antagonistic cytotoxicity shown by the combination of parthenolide and benznidazole demonstrated that combinations of drugs can not only control the development of the parasite infection but also reduce the damage in host cells. Further studies are necessary to increase understanding of the mode of action of parthenolide, and the possibility that it might in the future be used, alone or in combination with other drugs, for treatment of Chagas' disease.

Acknowledgements

This study was supported through grants from DECIT/SCTIE/MS and MCT by the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq), Financiadora de Estudos e Projetos (FINEP), and Programa de Nucleo de Excelencia (PRONEX/Fundacao Araucaria).

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Karin Juliane Pelizzaro-Rocha (a), Tatiana Shioji Tiuman (b), Erika Izumi (a), Tania Ueda-Nakamura (a), (b), Benedito Prado Dias Filho (a), (b), Celso Vataru Nakamura (a), (b), *

(a) Laboratorio de Inovacao Tecnologica no Desenvolvimento de Farmacos e Cosmeticos, Departamento de Ciencias Basicas da Saude, Universidale Estadual de Maringa, Maringa, Parana, Brazil

(b) Programa de Pos Graduacao em Ciencias Farmaceuticas, Universidade Estadual de Maringa, Maringa, Parana, Brazil

* Corresponding author at: Universidade Estadual de Maringa, Departamento de Ciencias Basicas da Saude, Laboratorio de Inovacao Tecnologica no Desenvolvimento de Farmacos e Cosmeticos, Bloco B-08 Sala 006 CCS, Avenida Colombo, 5790. BR-87020-900 Maringa, Parana, Brazil. Tel.: +55 44 3041 5012; fax: +55 44 3011 4860.

E-mail address: cvnakamura@gmail.com (C.V. Nakamura).
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Title Annotation:Short communication
Author:Pelizzaro-Rocha, Karin Juliane; Tiuman, Tatiana Shioji; Izumi, Erika; Ueda-Nakamura, Tania; Filho, B
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:3BRAZ
Date:Dec 15, 2010
Words:2865
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