Treatment of Trypanosoma cruzi-infected mice with propolis promotes changes in the immune response
Introduction
The flagellate protozoan Trypanosoma cruzi is the etiological agent of Chagas disease, an endemic Latin America parasitosis which has infected 16–18 million infected people (WHO, 2002). This disease is characterized by an acute phase with detectable parasitemia and a long-lasting asymptomatic phase (Laguens et al., 1994). The immune system is also involved in the parasite persistence (Zhang and Tarleton, 1999), the lymphoid system being a possible target and an active component of the Trypanosoma cruzi infection. The development of alternative drugs to replace nifurtimox and benznidazole, currently used for the treatment of chagasic patients, is urgent (Coura and De Castro, 2002). In this context, our laboratory is involved in the investigation of new natural and synthetic agents for the treatment of experimental Trypanosoma cruzi-infected animals (De Castro and Higashi, 1995, Pereira et al., 1998, Olivieri et al., 2002, Garzoni et al., 2004).
Propolis is a bee product, made from plant exudates, used for the construction and repair of the hive, as well as protection against micro-organisms. It is a complex mixture, with more than 200 compounds already identified in different samples (Bankova et al., 2000). Propolis presents a variety of biological activities, and its effect as anti-inflammatory, analgesic, and tissue regenerative agent has been demonstrated by different groups in in vivo models (De Castro, 2001). Such effects have been associated with the presence of phenolic compounds, such as flavonoids, and aromatic acids, and to their anti-oxidative properties (Heim et al., 2002, Ichikawa et al., 2002). Propolis extracts present low toxicity to experimental animals, with LD50 higher than 7 g/kg for mice (Arvouet-Grand et al., 1993). In relation to pathogenic protozoa, ethanol extracts showed activity against Toxoplasma gondii, Trichomonas spp., Giardia lamblia, and Trypanosoma cruzi (Starzyk et al., 1977, Torres et al., 1990, Higashi and De Castro, 1994, De Castro and Higashi, 1995, Marcucci et al., 2001, Cunha et al., 2004).
We have already reported the in vitro activity of the ethanol extract of Bulgarian propolis (Et-Blg), with a known chemical composition, against Trypanosoma cruzi and different species of fungi and bacteria of medical importance (Prytzyk et al., 2003, Salomão et al., 2004). In different mouse models, this extract also exhibited analgesic and anti-inflammatory effects which were associated with its high content of flavonoids (Paulino et al., 2003). These results encouraged us to assay the effect of Et-Blg on mice experimentally infected with Trypanosoma cruzi, and to verify the potential toxicity and immunomodulatory properties of this standardized propolis extract.
Section snippets
Propolis extract
A propolis sample was collected in 1999 at Burgas (Southeast Bulgaria), cut into small pieces, and extracted with 70% ethanol (1:10, w/v) at room temperature. After 1 day, the extract was filtered, evaporated to dryness under vacuum and the residue, named as Et-Blg (yield 62%, w/w), was stored at 4 °C, as previously described (Prytzyk et al., 2003).
Mice infection and treatment animals
Swiss mice weighing 18–20 g were maintained in our animal facility in stable conditions of temperature and light with 12-h light/12-h dark cycles.
Results
Swiss mice infected with the Y strain of Trypanosoma cruzi exhibited the classical pattern of parasitemia and survival rate, as previously described (Brener, 1962). Treatment with 50 mg of Et-Blg/kg body weight led to a statistically significant decrease in parasitemia, as compared with the control group (Table 1; Fig. 1a). No important differences were detected when comparing the survival of the animals (Table 1; Fig. 1b). The decrease in body weight of mice treated with all doses of the
Discussion and conclusions
We have previously determined by high temperature high resolution gas chromatography coupled to mass spectrometry that Et-Blg contains 42% flavonoids (pinostrobin, pinocembrin, chrysin, and a series of pinobanksins) and 12% phenolic acids (ferulic, isoferulic, caffeic acids, and esters) and was active in vitro against Trypanosoma cruzi, bacteria, and fungi (Prytzyk et al., 2003, Salomão et al., 2004).
In continuity, in the present work, we analyzed the effect of Et-Blg on Trypanosoma cruzi
Acknowledgements
We are grateful to Drs. Tania Araujo-Jorge and Maurilio J. Soares for a critical reading of this manuscript, to Marcos Meuser Batista for his excellent technical assistance and Marcos Antônio Pereira Marques for the support given in the biochemical analysis (Cecal/Fiocruz).
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