Trypanocidal activity of Brazilian plants against epimastigote forms from Y and Bolivia strains of Trypanosoma cruzi

Trypanocidal activity of Brazilian plants against epimastigote forms from Y and Bolivia strains of Trypanosoma cruzi

Renata Tomé Alves^*, I; Luis Octávio Regasini^II; Cristiano Soleo Funari^II; Maria Cláudia Marx Young^III; Aline Rimoldi^IV; Vanderlan da Silva Bolzani^II; Dulce Helena Siqueira Silva^II; Sérgio de Albuquerque^V; João Aristeu da Rosa^I

^ILaboratório de Parasitologia, Faculdade de Ciências Farmacêuticas de Araraquara-SP, Universidade Estadual Paulista, Brazil

^IIDepartamento de Química Orgânica, Instituto de Química de Araraquara-SP, Universidade Estadual Paulista, Brazil

^IIIInstituto de Botânica de São Paulo, Brazil

^IVInstituto de Biologia, Universidade Estadual de Campinas-SP, Brazil

^VLaboratório de Parasitologia, Faculdade de Ciências Farmacêuticas de Ribeirão Preto-SP, Universidade de São Paulo, Brazil

ABSTRACT

Chagas disease is one of the main public health problems in Latin America. Since the available treatments for this disease are not effective in providing cure, the screening of potential antiprotozoal agents is essential, mainly of those obtained from natural sources. This study aimed to provide an evaluation of the trypanocidal activity of 92 ethanol extracts from species belonging to the families Annonaceae, Apiaceae, Cucurbitaceae, Lamiaceae, Lauraceae, Moraceae, Nyctaginaceae, and Verbenaceae against the Y and Bolivia strains of Trypanosoma cruzi. Additionally, cytotoxic activity on LLCMK2 fibroblasts was evaluated. Both the trypanocidal activity and cytotoxicity were evaluated using the MTT method, in the following concentrations: 500, 350, 250, and 100 µg/mL. Benznidazole was used for positive control. The best results among the 92 samples evaluated were obtained with ethanol extracts of Ocotea paranapiacabensis (Am93) and Aegiphila lhotzkiana (Am160). Am93 showed trypanocidal activity against epimastigote forms of the Bolivia strain and was moderately toxic to LLCMK2 cells, its Selectivity Index (SI) being 14.56, while Am160 showed moderate trypanocidal activity against the Bolivia strain and moderate toxicicity, its SI being equal to 1.15. The screening of Brazilian plants has indicated the potential effect of ethanol extracts obtained from Ocotea paranapiacabensis and Aegiphila lhotzkiana against Chagas disease.

Keywords: biodiversity, Chagas, cytotoxicity, disease, strains, trypanocidal activity,Trypanosoma cruzi

Introduction

In Latin America, Chagas disease is an important cause of morbidity, affecting around 10 million people and representing a risk for 25 million from the South of the United States to the South of Argentina (WHO, 2010).

Since this disease affects mostly poor populations, the development of new therapeutic solutions is not an attractive business for the large pharmaceutical companies, and currently it can be said that this initiative is being extremely neglected, which is a very concerning fact on account of the needs of those people (Nwaka & Ridley, 2003). The two drugs available for the treatment of Chagas disease, nifurtimox and benznidazole, have potential toxic side effects and variable efficiency, both of them being ineffective in eradicating the infection during its chronic phase, which contributes to its low use rates (Coura, 2009). For this reason, the screening of potential new compounds is essential (Coura & Castro, 2002).

The difficulty to find a substance capable of fighting the parasite can be directly related to the morphological characteristics of the strain, mainly considering the presence of different populations, which present distinct tissue tropism. Therefore, different strain groups of T. cruzi should be considered in the evaluation of new drugs (Macedo et al., 2002).

This scenario clearly shows that it is necessary to develop therapies that stop the multiplication of T. cruzi without causing any severe side effect (Coura & Castro, 2002). Medicinal plants have been used in the treatment for parasitic diseases for a long time, and many works sustain the therapeutic value of products from plant origin, also describing the trypanocidal activity of natural active compounds (Bastos et al., 1999; Saraiva et al., 2007; Batista Jr. et al., 2008).

Continuing our studies on the discovery of trypanocidal agents obtained from plants from both the Cerrado and the Atlantic Forest (Cotinguiba et al. 2009; Lopes et al., 2008; Regasini et al. 2009), 92 ethanol extracts of species belonging to the families Annonaceae, Apiaceae, Cucurbitaceae, Lamiaceae, Lauraceae, Moraceae, Nyctaginaceae, and Verbenaceae were tested against epimastigote forms of Trypanosoma cruzi (Y and Bolivia strains), and their cytotoxic activity on LLCMK₂ fibroblasts was evaluated. The emergency to find new antiprotozoal agents with trypanocidal activity and the evidence that some species of the aforementioned families have trypanocidal activity against parasitic forms of T. cruzi provided the motivation to carry out the screening of such extracts (Buainain et al., 1992; Fournet et al., 2007; Osorio et al., 2007; Cabral et al., 2010).

Material and methods

Parasites

In the assays both the Y and Bolivia strains were used, the former belonging to lineage I and the latter, to lineage II. The strains were kept in BALB/c mices and in LIT (Liver Infusion Tryptose) culture medium, in BOD incubator at 28 ºC, at the Laboratory of Parasitology of the Faculty of Pharmaceutical Sciences of Araraquara-SP, Unesp.

Plant material and extraction

The plant material was collected by Maria Cláudia Marx Young in remaining areas of Atlantic Forest and Cerrado in the State of São Paulo, and it was identified by Inês Cordeiro, Institute of Botany, State Department of the Environment, São Paulo-SP. The voucher specimens were then deposited in the herbarium "Maria Eneyda P. Kaufmann" at the IBT-SMA. The codes of the extracts and voucher specimens can be found in Table 1.

After the collection, the botanical material was dried in the absence of light and then powdered using a cutting mill. A 30 g portion of the powder was extracted with ethanol (5x100 mL) during three weeks, at room temperature. After the filtration, the solvent was evaporated under reduced pressure, which resulted in the crude extracts.

Twenty-eight of the 92 ethanol extracts evaluated belong to the genera Rollinia, Xylopia, Anaxagorea, Annona, Guatteria and Duguetia, family Annonaceae; one to the genus Hydrocotyle, family Apiaceae; two to the genus Cayaponia, family Cucurbitaceae; two to the genera Aegiphila, family Lameaceae 36 to the genera Nectandra and Ocotea, family Lauraceae; one to the genus Dorstenia, family Moraceae; eight to the genera Bougainvilleae, Pisonia and Guapira, family Nyctaginaceae; and fourteen to the genera Lantana, Starchytarpheta, and Lippia, family Verbenaceae (Table 1).

In vitro assay for trypanocidal activity

Trypanocidal activity was evaluated by means of the MTT method, with changes (Muelas-Serrano et al., 2000).

The epimastigote forms (1.10⁷ parasites/mL), obtained from culture in stationary phase, were cultured in plates with 96 wells in BOD incubator at 28 ºC for 24 h, concentrations for the ethanol extracts being 500, 350, 250 and 100 µg/mL. After this period, the MTT (2.5 mg/mL) and PMS (0.22 mg/mL) solutions were added to each well, and the plate was incubated for 1 h. Then 100 µL of HCl (1M) and SDS (10%) were added to it. The plate was kept at room temperature for 30 min, and the reading was performed on a spectrophotometer at 595 nm. Benznidazole was used in the same concentrations for positive control.

The assays were in triplicate, and the results were expressed as IC50, calculated by the statistical method of sigmoid concentration-response curve using the GraphPad Prisma 4.0 software.

Cytotoxicity assay

Extracts with trypanocidal activity against epimastigote forms of T. cruzi were evaluated regarding their cytotoxicity on LLCMK2 fibroblasts by means of the MTT method, with changes (Muelas-Serrano et al., 2000).

LLCMK₂ cells (1.10⁶ /mL) were cultured in plates with 96 wells and ethanol extracts in the following concentrations: 500, 350, 250 and 100 µg/mL. The plates were incubated in a CO₂ incubator at 5% and 37 ºC for 24 h. After that period, 10 µL of MTT solution (5mg/mL) were added to each well, and the plates were incubated for 4 h. Then 100 µL of acid isopropyl were added, and the plate was kept at room temperature for 1 h. The reading was performed on a spectrophotometer at 595 nm. RPMI culture medium was used for positive control, whereas LLCMK₂ cells were used for negative control.

The assays were carried out in triplicate, and the results were expressed as CC50, calculated by the statistical method of sigmoid concentration-response curve using the GraphPad Prisma 4.0 software.

The cytotoxic activity (CC50) was related to the trypanocidal activity (IC50) in order to determine the correspondent Selectivity Index (IS=CC50/IC50).

Results and Discussion

Ninety-two ethanol extracts of different species of Brazilian flora were tested. The trypanocidal activity of the samples was classified according to criteria set by Osorio et al. (2007). The extracts were classified as highly active (IC50<10 µg/mL), active (IC50>10<50 µg/mL), moderately active (IC50>50<100 µg/mL) and inactive (IC50>100 µg/mL). With regard to their cytotoxicity, the samples were classified as highly toxic (CC50<10 µg/mL), toxic (CC50>10<100 µg/mL), moderately toxic (CC50>100<1000 µg/mL) and potentially non-toxic (CC50>1000 µg/mL).

According to this classification, all the 92 ethanol extracts tested against epimastigote forms of the Y strain of T. cruzi are inactive (Table 2).

Regarding the Bolivia strain, the fruit extract of Ocotea paranapiacabensis (Lauraceae) (Am93) is considered active, whereas the fruit extract of Aegiphila lhotzkiana (Lamiaceae) (Am160) and the leaf extract of Ocotea elegans (Am73) were respectively classified as moderately active and inactive against the same parasitic forms (Table 3).

The IC50 values for benznidazole against epimastigote forms of the Y and Bolivia strains were 0.99 and 11.77, respectively (Tables 2 and 3).

Regarding the cytotoxicity analysis, the extracts of Duguetia lanceolata (Am379) and Xylopia aromatica (M1103) were classified as toxic to LLCMK₂ cells, whereas the extracts of Ocotea paranapiacabensis (Am93), Ocotea elegans (Am73), Ocotea indecora (R60), Aegiphila lhotzkiana (Am160), Guapira oppositta (Am116), Guatteria elliptica (Am03), and Duguetia lanceolata (Am352) were classified as moderately toxic (Tables 2 and 3).

The most promising samples were those that proved to be more active against epimastigote forms of T. cruzi and less toxic to LLCMK₂ cells.

According to this classification, the most promising extracts for chemical and pharmacological investment were the fruit of Ocotea paranapiacabensis, Lauraceae (Am93), which proved to be active against epimastigote forms of the Bolivia strain and moderately toxic to LLCMK2 cells, its SI being equal to 14.56, and the fruit extract of Aegiphila lhotzkiana, Lamiaceae (Am160), which was also tested against the Bolivia strain and showed moderate activity regarding the parasites and the LLCMK2 cells.

By comparing the trypanocidal activity of the extracts against the Y strain and the Bolivia strain, a clear difference could be noted. The material tested against the Y strain did not show a satisfactory activity. On the other hand, two extracts (Am93 and Am160), which were tested against the Bolivia strain, were found to be, respectively, active and moderately active against such parasitic forms.

This difference in sensitivity between the strains can be explained by the fact that T. cruzi populations show large intraspecific variability, as it can be noted by differences in their morphology, virulence, pathogenicity, evasion ability in case of an immune response from the host, antigenic composition and biochemical properties (Fernandes et al., 1998; Tibayrenc & Ayala, 2002).

The trypanocidal activity of the ethanol extract of Ocotea paranapiacabensis (Lauraceae) against epimastigote forms of the Bolivia strain is reported for the first time in this work. Data from the literature report the activity of isolated alkaloids of Ocotea odorifera against promastigote forms of Leishmania braziliensis, L. donovan and L. amazonensis and trypomastigote forms of T. cruzi (Fournet et al., 2007). Extracts of branches and roots of the same species were found to be active against Plasmodium falciparum. Popular medicine recommends the use of these plants in the treatment for dermatoses, rheumatism, fever and syphilis (Botsaris, 2007).

Aegiphila lhotzkiana, which showed trypanocidal activity against the Bolivia strain, is widely distributed in Northeastern Brazil, where it is popularly known as pau-de-sebo. The oil obtained from its fruit is used in popular medicine for treating pediculosis and scabies, and its extract is used as an antidote to snakebite (Costa-Lotufo et al., 2004). The activity of this crude extract was unknown until this research was carried out, because there are no reports in the literature on the trypanocidal activity of this species, not even on the genus it belongs to.

The screening of Brazilian plants has indicated the potential effect of ethanol extracts obtained from fruits of Ocotea paranapiacabensis (Lauraceae) and Aegiphila lhotzkiana (Lamiaceae) against Chagas disease, considering the epimastigote forms of the Bolivia strain of T. cruzi.

These data reinforce the importance of the efforts to promote the sustainable use of Brazilian biodiversity, focusing on the search for new therapeutic agents for the treatment of some neglected diseases that affect millions of people in Brazil and other countries.

Acknowledgments

The authors want to thank Miriam P. A. Toldo, Mariana Rosa, Mariana Bryan Augusto and Isabel Martinez for helping in the laboratory procedures, and the BIOTA-FAPESP (03/02176-7) and BIOPROSPECTA-FAPESP (04/07932) programs and the CNPq for the scholarships and resources granted.

Received 19 Feb 2011

Accepted 22 Oct 2011

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Publication Dates

• Publication in this collection
  24 Jan 2012
• Date of issue
  June 2012

History

• Received
  19 Feb 2011
• Accepted
  22 Oct 2011