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, 2013, 279726

Eugenia Uniflora L. Essential Oil as a Potential Anti-Leishmania Agent: Effects on Leishmania Amazonensis and Possible Mechanisms of Action

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Eugenia Uniflora L. Essential Oil as a Potential Anti-Leishmania Agent: Effects on Leishmania Amazonensis and Possible Mechanisms of Action

Klinger Antonio da Franca Rodrigues et al. Evid Based Complement Alternat Med.

Abstract

Eugenia uniflora L. is a member of the Myrtaceae family and is commonly known as Brazilian cherry tree. In this study, we evaluated the chemical composition of Eugenia uniflora L. essential oil (EuEO) by using gas chromatography-mass spectrometry (GC-MS) and assessed its anti-Leishmania activity. We also explored the potential mechanisms of action and cytotoxicity of EuEO. Thirty-two compounds were identified, which constituted 92.65% of the total oil composition. The most abundant components were sesquiterpenes (91.92%), with curzerene (47.3%), γ -elemene (14.25%), and trans- β -elemenone (10.4%) being the major constituents. The bioactivity shown by EuEO against promastigotes (IC50, 3.04 μ g·mL(-1)) and amastigotes (IC50, 1.92 μ g·mL(-1)) suggested significant anti-Leishmania activity. In the cytotoxicity determination, EuEO was 20 times more toxic to amastigotes than to macrophages. Hemolytic activity was 63.22% at the highest concentration tested (400 μ g·mL(-1)); however, there appeared to be no toxicity at 50 μ g·mL(-1). While the data show that EuEO activity is not mediated by nitric oxide production, they do suggest that macrophage activation may be involved in EuEO anti-Leishmania activity, as evidenced by increases in both the phagocytic capacity and the lysosomal activity. More studies are needed to determine in vivo activity as well as additional mechanisms of the anti-Leishmania activity.

Figures

Figure 1
Figure 1
Effect of Eugenia uniflora essential oil (400, 200, 100, 50, 25, 12.5, 6.25, and 3.12 μg·mL−1) or amphotericin B (Amph) (2 μg·mL−1) on Leishmania amazonensis promastigotes. Cultures of log-phase promastigotes (1 × 106) were incubated at 26°C for 24, 48, and 72 h in different essential oil concentrations. Data represent the mean percentage of growth inhibition ± standard error of 3 experiments carried out in triplicate. *P < 0.05.
Figure 2
Figure 2
Cytotoxicity of Eugenia uniflora essential oil on the viability of murine peritoneal macrophages. Peritoneal macrophages were seeded at 1 × 105/well in 96-well microplates and incubated for 48 h in the presence of E. uniflora L. essential oil at concentrations of 100, 50, 25, 12.5, 6.25, and 3.12 μg·mL−1. Viability was determined with 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT), and the optical density was determined at 540 nm. Data represent the mean parasite density ± standard error of 3 experiments carried out in triplicate. *P < 0.05; **P < 0.01; and ***P < 0.001.
Figure 3
Figure 3
Hemolytic activity of Eugenia uniflora essential oil in a 4% suspension of human O+ red blood cells after 1 h of incubation.
Figure 4
Figure 4
Effect of Eugenia uniflora essential oil on macrophage infection after 48 h of exposure. Peritoneal macrophage cells were infected with promastigotes of Leishmania amazonensis and then treated with 3.12, 1.56, or 0.78 μg·mL−1 of Eugenia uniflora essential oil. Data represent the mean parasite density ± standard error of 3 experiments carried out in triplicate. ***P < 0.001.
Figure 5
Figure 5
Effect of Eugenia uniflora essential oil on the survival of Leishmania amazonensis amastigotes internalized in macrophages. Leishmania amazonensis-infected mouse peritoneal macrophages were treated with 3.12, 1.56, or 0.78 μg·mL−1 of Eugenia uniflora essential oil. After 48 h of incubation, amastigote survival was assessed. Data represent the mean parasite density ± standard error of 3 experiments carried out in triplicate. ***P < 0.001.
Figure 6
Figure 6
The influence of Eugenia uniflora essential oil on the lysosomal activity (a) and phagocytic activity (b) of peritoneal macrophages at concentrations of 100, 50, 25, 12.5, 6.25, and 3.12 μg·mL−1. Data represent the mean density ± standard error of 3 experiments carried out in triplicate. *P < 0.05 and ***P < 0.001. C: control. ABS: absorbance.
Figure 7
Figure 7
Production of nitric oxide (NO). Murine macrophages (2 × 105) were treated with Eugenia uniflora essential oil (100, 50, 25, 12.5, 6.25, and 3.12 μg·mL−1) in the absence (a) or presence of Leishmania amazonensis (b) over 24 h. Data represent the mean density ± standard error of 3 experiments carried out in triplicate. ***P < 0.001. C: control. ABS: absorbance.

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