Fig 1.
Structures of oleanolic (A) and ursolic acids (B).
Table 1.
EC50, CC50 and SI induced by OA, UA, amphotericin B and Miltefosine against L. (L.) amazonensis promastigotes and macrophages.
Fig 2.
Ultrastructural changes of L. (L.) amazonensis promastigote induced by in vitro treatment with UA.
Images of cultured promastigote forms before (A) and after treatment (B, C, D and E) with UA EC50 were captured by transmission electron microscopy. (N)–Nucleus, (K)–Complex kinetoplast-Mitochondria; red arrow: mitochondrion swelling; white arrow: membrane-containing vacuoles; white asterisk: blebs on nucleus and mitochondria.
Fig 3.
The mechanism of parasite death was investigated after in vitro treatment with UA.
By flow cytometry the pattern of Annexin V and PI staining was analyzed in non-treated promastigotes (A), UA-treated promastigotes (B) and H2O2-treated promastigotes (C); and it was characterized as “viable cells” in Q1 (Annexin V-/PI-), “early apoptosis stage” in Q2 (Annexin V+/PI-), “late apoptosis stage” in Q3 (Annexin V+/PI+) and “cellular death/necrosis” in Q4 (Annexin V-/PI+). D—Activity of caspase 3/7 was investigated in UA-treated parasites. E—Aspect of nuclear DNA from control parasites (lane 2), UA-treated parasites (lane 3) and H2O2-treated parasites, molecular marker is represented in lane 1; F—JC1 aggregates were analyzed in control, UA—and H2O2—treated parasites.
Fig 4.
Infection Index (II) of L. (L.) amazonensis-infected peritoneal macrophages submitted to different treatments (A). Peritoneal macrophages were infected with promastigote forms of L. (L.) amazonensis and treated for 24 and 72h with UA or amphotericin B (Amp B– 0.1 μg/mL). Illustration of control infected macrophages (B), infected cells treated with 0.1 μg/mL (C), 1.0 μg/mL (D) and 10.0 μg/mL (E) of UA as well as 0.1 μg/mL of amphotericin B (F) at 24 and 72h. Results are represented by mean and standard deviation of three independent experiments and three replicates per sample. * (p<0.05) indicate significant differences.
Fig 5.
Nitrite levels were evaluated in supernatant of L. (L.) amazonensis-infected peritoneal macrophages submitted to different treatments.
Peritoneal macrophages were infected with promastigote forms of L. (L.) amazonensis and treated for 24 or 72h with UA or amphotericin B (Amp B– 0.1 μg/mL). Results are represented by mean and standard error of three independent experiments and three replicates per sample. * (p<0.05) indicates significant differences.
Fig 6.
BALB/c mice were infected with promastigote forms of L. (L.) amazonensis and treated with 1.0 and 2.0 mg/kg of UA or 100.0 mg/kg of Glucantime (Glu).
The development of the disease was monitored for 5 weeks through weekly measurement of lesions. Results are represented by mean and standard error of three independent experiments, containing 5 animals per group. * (p<0.05) indicates significant differences when compared treated groups vs infected, non-treated group.
Fig 7.
Parasite inhibition after treatment. Footpad lesions of control mice and of mice treated with of UA (1.0 mg/kg or 2.0 mg/kg) or Glucantime (100.0 mg/kg).
Results are represented by mean and standard error of three independent experiments containing five animals per group. * and + (p<0.05) indicate significant differences when compared non-treated vs treated mice and, Glucantime vs UA treated mice, respectively.
Fig 8.
Histological sections of skin from L. (L.) amazonensis mice submitted or not with UA and amphotericin treatments.
A—Skin histological section of an infected control mice; B—Skin histological section of an infected BALB/c mice treated with UA (1.0mg/kg); C—Skin histological section of an infected BALB/c mice treated with UA (2.0mg/kg); D—Skin histological section of an infected BALB/c mice treated with amphotericin B (5.0mg/kg). Skin sections were staining with hematoxilin & eosin staining and observed under optical microscope at a magnification of 40x.