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Brazilian Green Propolis: Effects In Vitro and In Vivo on Trypanosoma cruzi.

Salomão K, de Souza EM, Henriques-Pons A, Barbosa HS, de Castro SL - Evid Based Complement Alternat Med (2011)

Bottom Line: These effects were confirmed by flow cytometry analysis.The extract (25-300 mg kg(-1) body weight/day for 10 days) reduced the parasitemia, although not at significant levels; increased the survival of the animals and did not induce any hepatic, muscular lesion or renal toxicity.Et-Bra could be a potential metacyclogenesis blocker, considering its effect on reservosomes, which are an important energy source during parasite differentiation.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Biologia Celular, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Av. Brasil 4365, Manguinhos 21045-900, Rio de Janeiro, Brazil.

ABSTRACT
The composition of a Brazilian green propolis ethanolic extract (Et-Bra) and its effect on Trypanosoma cruzi trypomastigotes and other pathogenic microorganisms have already been reported. Here, we further investigated Et-Bra targets in T. cruzi and its effect on experimental infection of mice. The IC(50)/4 days for inhibition of amastigote proliferation was 8.5 ± 1.8 μg mL(-1), with no damage to the host cells. In epimastigotes Et-Bra induced alterations in reservosomes, Golgi complex and mitochondrion. These effects were confirmed by flow cytometry analysis. In trypomastigotes, Et-Bra led to the loss of plasma membrane integrity. The in vitro studies indicate that Et-Bra interferes in the functionality of the plasma membrane in trypomastigotes and of reservosomes and mitochondrion in epimastigotes. Acutely infected mice were treated orally with Et-Bra and the parasitemia, mortality and GPT, GOT, CK and urea levels were monitored. The extract (25-300 mg kg(-1) body weight/day for 10 days) reduced the parasitemia, although not at significant levels; increased the survival of the animals and did not induce any hepatic, muscular lesion or renal toxicity. Since Et-Bra was not toxic to the animals, it could be assayed in combination with other drugs. Et-Bra could be a potential metacyclogenesis blocker, considering its effect on reservosomes, which are an important energy source during parasite differentiation.

No MeSH data available.


Related in: MedlinePlus

Flow cytometry analysis of T. cruzi epimastigotes treated with Et-Bra for 24 h and incubated with Rh123 and PI or AO. (a–d) Rh123 fluorescence intensity in: (a) control, (b) 100 μg mL−1, (c) 150 μg mL−1, (d) 200 μg mL−1 Et-Bra. Rh123high parasites were delimited by a marker (M1) and the corresponding percent of the cells included. (e) Average fluorescence peak values for the AO fluorescence, from at least three independent assays, showing a dose-dependent decrease after treatment with Et-Bra. Asterisks indicate P < .05 when compared to untreated epimastigotes.
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fig5: Flow cytometry analysis of T. cruzi epimastigotes treated with Et-Bra for 24 h and incubated with Rh123 and PI or AO. (a–d) Rh123 fluorescence intensity in: (a) control, (b) 100 μg mL−1, (c) 150 μg mL−1, (d) 200 μg mL−1 Et-Bra. Rh123high parasites were delimited by a marker (M1) and the corresponding percent of the cells included. (e) Average fluorescence peak values for the AO fluorescence, from at least three independent assays, showing a dose-dependent decrease after treatment with Et-Bra. Asterisks indicate P < .05 when compared to untreated epimastigotes.

Mentions: Based on the ultrastructural data, treated parasites were incubated with fluorescent markers for flow cytometry analysis. FSCXSSC dot plots show that epimastigotes treated with Et-Bra suffered dramatic morphological alterations (data not shown). The extract induced a decrease in the percentage of Rh123+ cells from 87.7 (control) to 43.5 (300 μg mL−1 Et-Bra), indicating a loss of the mitochondrial membrane potential, although with membrane integrity, as indicated by PI analysis (Figures 5(b)–5(d)). AO labeling indicated, in comparison with untreated epimastigotes, a dose-dependent and statistically significant decrease in the average fluorescence peak at FL2 induced by Et-Bra (Figure 5(e)), a phenomenon also observed by epifluorescence microscopy (data not shown), indicating pH increase in the acidic compartments. In trypomastigotes, increasing doses of Et-Bra (15–150 μg mL−1) led to membrane damage, revealed by a dose-dependent increase in PI labeling and decreased fluorescence of Rh123high (Figure 6).


Brazilian Green Propolis: Effects In Vitro and In Vivo on Trypanosoma cruzi.

Salomão K, de Souza EM, Henriques-Pons A, Barbosa HS, de Castro SL - Evid Based Complement Alternat Med (2011)

Flow cytometry analysis of T. cruzi epimastigotes treated with Et-Bra for 24 h and incubated with Rh123 and PI or AO. (a–d) Rh123 fluorescence intensity in: (a) control, (b) 100 μg mL−1, (c) 150 μg mL−1, (d) 200 μg mL−1 Et-Bra. Rh123high parasites were delimited by a marker (M1) and the corresponding percent of the cells included. (e) Average fluorescence peak values for the AO fluorescence, from at least three independent assays, showing a dose-dependent decrease after treatment with Et-Bra. Asterisks indicate P < .05 when compared to untreated epimastigotes.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3094871&req=5

fig5: Flow cytometry analysis of T. cruzi epimastigotes treated with Et-Bra for 24 h and incubated with Rh123 and PI or AO. (a–d) Rh123 fluorescence intensity in: (a) control, (b) 100 μg mL−1, (c) 150 μg mL−1, (d) 200 μg mL−1 Et-Bra. Rh123high parasites were delimited by a marker (M1) and the corresponding percent of the cells included. (e) Average fluorescence peak values for the AO fluorescence, from at least three independent assays, showing a dose-dependent decrease after treatment with Et-Bra. Asterisks indicate P < .05 when compared to untreated epimastigotes.
Mentions: Based on the ultrastructural data, treated parasites were incubated with fluorescent markers for flow cytometry analysis. FSCXSSC dot plots show that epimastigotes treated with Et-Bra suffered dramatic morphological alterations (data not shown). The extract induced a decrease in the percentage of Rh123+ cells from 87.7 (control) to 43.5 (300 μg mL−1 Et-Bra), indicating a loss of the mitochondrial membrane potential, although with membrane integrity, as indicated by PI analysis (Figures 5(b)–5(d)). AO labeling indicated, in comparison with untreated epimastigotes, a dose-dependent and statistically significant decrease in the average fluorescence peak at FL2 induced by Et-Bra (Figure 5(e)), a phenomenon also observed by epifluorescence microscopy (data not shown), indicating pH increase in the acidic compartments. In trypomastigotes, increasing doses of Et-Bra (15–150 μg mL−1) led to membrane damage, revealed by a dose-dependent increase in PI labeling and decreased fluorescence of Rh123high (Figure 6).

Bottom Line: These effects were confirmed by flow cytometry analysis.The extract (25-300 mg kg(-1) body weight/day for 10 days) reduced the parasitemia, although not at significant levels; increased the survival of the animals and did not induce any hepatic, muscular lesion or renal toxicity.Et-Bra could be a potential metacyclogenesis blocker, considering its effect on reservosomes, which are an important energy source during parasite differentiation.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Biologia Celular, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Av. Brasil 4365, Manguinhos 21045-900, Rio de Janeiro, Brazil.

ABSTRACT
The composition of a Brazilian green propolis ethanolic extract (Et-Bra) and its effect on Trypanosoma cruzi trypomastigotes and other pathogenic microorganisms have already been reported. Here, we further investigated Et-Bra targets in T. cruzi and its effect on experimental infection of mice. The IC(50)/4 days for inhibition of amastigote proliferation was 8.5 ± 1.8 μg mL(-1), with no damage to the host cells. In epimastigotes Et-Bra induced alterations in reservosomes, Golgi complex and mitochondrion. These effects were confirmed by flow cytometry analysis. In trypomastigotes, Et-Bra led to the loss of plasma membrane integrity. The in vitro studies indicate that Et-Bra interferes in the functionality of the plasma membrane in trypomastigotes and of reservosomes and mitochondrion in epimastigotes. Acutely infected mice were treated orally with Et-Bra and the parasitemia, mortality and GPT, GOT, CK and urea levels were monitored. The extract (25-300 mg kg(-1) body weight/day for 10 days) reduced the parasitemia, although not at significant levels; increased the survival of the animals and did not induce any hepatic, muscular lesion or renal toxicity. Since Et-Bra was not toxic to the animals, it could be assayed in combination with other drugs. Et-Bra could be a potential metacyclogenesis blocker, considering its effect on reservosomes, which are an important energy source during parasite differentiation.

No MeSH data available.


Related in: MedlinePlus