IN VIVO AND IN VITRO ANTILEISHMANIAL EFFECTS OF METHANOLIC EXTRACT FROM BARK OF BURSERA APTERA

Abstract

Background: Cutaneous leishmaniasis lacks effective and well-tolerated treatments. The current therapies mainly rely on antimonial drugs that are inadequate because of their poor efficacy. Traditional medicine offers a complementary alternative for the treatment of various diseases. Additionally, several plants have shown success as anti-leishmanial agents. Therefore, we sought to evaluate the in vitro and in vivo activity of MEBA against Leishmania mexicana.

Materials and methods: Methanolic extract of B. aptera was obtained by macetration, after we determined in vitro anti-leishmanial activity of MEBA by MTT assay and the induced apoptosis in promastigotes by flow cytometry. To analyze the in vivo anti-leishmanial activity, we used infected mice that were treated and not treated with MEBA and we determined the levels of cytokines using ELISA. The phytochemical properties were determined by CG-MS and DPPH assay.

Results: We determined of LC₅₀ of 0.408 mg/mL of MEBA for in vitro anti-leishmanial activity. MEBA induced apoptosis in promastigotes (15.3% ± 0.86). Treated mice exhibited smaller lesions and contained significantly fewer parasites than did untreated mice; in addition, we found that IFN-γ and TNF-α increased in the sera of MEBA-treated mice. GC-MS analysis showed that podophyllotoxin was the most abundant compound. Evaluation of the activity by DPPH assay demonstrated an SC₅₀ of 11.72 μg/mL.

Conclusion: Based on the above data, it was concluded that MEBA is a good candidate in the search for new anti-leishmanial agents.

Keywords: Leishmania; cutaneous leishmaniasis; traditional medicine.

Figure 1

Analysis of externalization of phosphatidylserine in promastigotes treated with MEBA. L. Mexicana promastigotes were incubated with MEBA (0.408 mg/mL) for 1 h, stained with PI and annexin V–FITC and analyzed by flow cytometry. As observed, MEBA promotes the externalization of phosphatidylserine in treated promastigotes, which indicated that the leishmanicidal activity of MEBA occurs primarily via apoptosis. Dotplots shown are from representative experiments. Numbers represent the mean percentage values of expression ± SE of five independent experiments. P < 0.05, unpaired t test with Welch’s correction.

Figure 2

Analysis of changes in the mitochondrial membrane potential of L. mexicana promastigotes treated with MEBA for 1 h, followed by staining with JC-1 and flow cytometry analysis. Histogram showing a reduction in JC-1 red staining in the parasite population treated with MEBA compared to that in untreated parasites. Treatment with MEBA caused a loss of the membrane potential, which reduced the red staining of the parasite. Histograms shown are from representative experiments.

Figure 3

Evaluation of the effect of MEBA on lesion size. Untreated mice developed large lesions (●), while MEBAtreated mice showed a significant absence of the increase in the size of the lesions (○). From the fourth week, the size of lesions in MEBA-treated mice decreased significantly in relation to the control. The significance of differences was determined with multiple t tests (one per row). * P < 0.05 was considered statistically significant.

Figure 4

Parasite burden was determined at 8 weeks following infection with L. mexicana by limiting dilution. Data are expressed as the mean log dilution (n = 6). Significant differences in the number of parasites in the lesions of treated and untreated mice (6.8 ± 0.247 and 9.6 ± 0.175, respectively) were observed. As shown, these findings indicate that MEBA has anti-leishmanial activity against L. mexicana. Untreated mice developed large ulcerative lesions full of parasites, whereas the mice treated with MEBA developed small lesions with few parasites. The significance of differences was determined with unpaired t tests (two-tailed) with Welch’s correction. *P < 0.05 was considered statistically significant.

Figure 5

Cytokine levels in sera of mice infected with L. mexicana by ELISA sandwich assay. In treated mice, a significant increase in IFN-γ and TNF-α was observed compared with the untreated group, whereas IL-4 and IL-10 showed a significant decrease in relation to the untreated group. The significance of differences was determined with multiple t tests (one per row). * P < 0.05 was considered statistically significant.