Betulinic acid was extracted from the bark of Ziziphus joazeiro Mart., a native brazilian tree from the Rhamnaceae family, according to the methodology previously described (Barbosa-Filho et al., 1985). The semi-synthetic compound BA5 was prepared from betulinic acid as previously described (Barbosa-Filho et al., 1985) and used in antileishmanial assays (BA5; 94–98% purity by high performance liquid chromatography). Amphotericin B (Gibco Laboratories, Gaithersburg, MD) was used as positive control in antileishmanial assays. Gentian violet (Synth, São Paulo, SP, Brazil) was used as positive control in the cytotoxicity to mammalian cell assays. All compounds were dissolved in dimethyl sulfoxide (DMSO; PanReac, Barcelona, Spain) and diluted in cell culture medium for use in the assays. The final concentration of DMSO was less than 0.1% in all in vitro experiments.

Male 4–6-weeks old BALB/c were used. All mice were raised and maintained at the animal facilities of the Gonçalo Moniz Institute, Oswaldo Cruz Foundation, Salvador, Brazil in sterilized cages, under a controlled environment and receiving a balanced rodent diet and water ad libitum. All experiments were approved by the local Animal Ethics Committee (Approval number: 004/2019).

L. amazonensis (MHOM/BR88/BA-125 Leila strain), L. major (MHOM/RI/WR173), L. braziliensis (MHOM/BR88/BA-3456) promastigotes were cultivated in Schneider (Sigma, St. Louis, MO, United States) medium supplemented with 10% fetal bovine serum (FBS) (Gibco) and 50 μg ml⁻¹ Gentamicin (Sigma). L. infantum (MCAN/BR/89/BA262) promastigotes were cultivated in liver infusion tryptose (LIT) medium supplemented with 20% fetal bovine serum and 50 μg ml⁻¹ Gentamicin, pH 7.2, at 26°C until logarithmic phase. Log phase promastigotes were used to study the effects of the betulinic acid and BA5 derivative.

L. amazonensis, L. major, L. braziliensis and L. infantum chagasi promastigotes (1 × 10⁶ cells/well) were incubated into 96-well plates, cultivated in Schneider (Sigma) medium supplemented with 10% fetal bovine serum (FBS) (Gibco) and μg mL⁻¹ Gentamicin (Sigma). Drugs were added at six concentrations ranging from 1.56 to 50 µM in triplicate, and the plate was incubated for 72 h at 26°C. Amphotericin was added at eight concentrations ranging 0.0156–2.0 µM. Promastigotes viability was measured by twenty µL/well of AlamarBlue (Invitrogen, Carlsbad, CA, United States) during 2 h (L. amazonensis, L. major and L. braziliensis) or 24 h for L. infantum due to slower metabolism (Corral et al., 2013), after which colorimetric readings were performed at 570 and 600 nm.

Peritoneal exudate macrophages were obtained by washing of the peritoneal cavity of BALB/c mice with cold Dulbecco’s Modified Eagle’s Medium (DMEM Life Technologies, GIBCO-BRL), 5 days after injection of 3% thioglycolate in saline (1.5 ml per mice). Cells were added into 96-well plates at a density 1 × 10⁵ cells/well containing DMEM medium supplemented with 10% of fetal bovine serum (FBS; Gibco) and 50 μg ml⁻¹ Gentamicin (Novafarma, Anapolis, Brazil) and incubated for 24 h at 37°C and 5% CO₂. Drugs was added in triplicate at eight concentrations ranging from 0.04 to 100 µM and incubated for 72 h. Twenty µL/well of AlamarBlue (Invitrogen) was added to the plates during 10 h. Colorimetric readings were performed at 570 and 600 nm. CC₅₀ values were calculated using data-points gathered from three independent experiments. Gentian violet (Synth, Sao Paulo, Brazil) was used as positive control, at concentrations ranging from 0.04 to 10 µM.

Peritoneal exudate macrophages (5 × 10⁵ cells) were plated onto sterile coverslips in 24-well plates and kept for 24 h. The macrophages were infected with stationary growth phase promastigotes of L. amazonensis at a ratio of 10:1 macrophage at 35°C during 4 h and 5% CO₂. Infected macrophages were incubated with different atoxic concentrations with values below the IC₅₀ values of BA (9.4; 4.7; 2.3 µM) and BA5 (15.5; 7.7; 3.8 µM). After 24 h, the cells were fixed in methanol. The percentage of infected macrophages and the number of amastigotes/macrophages were determined by counting 100 cells per slides by counting the slides after Giemsa staining (Sigma) in an optical microscope (Olympus, Tokyo, Japan). Amphotericin B (Gibco) was used as a positive control in this assay.

Promastigotes of L. amazonensis (10⁶ cells/well) were incubated in 24-well plates and incubated with BA and BA5 in different concentrations (IC₅₀ or 2x IC₅₀) for 24 h at 26°C. Parasites were labeled with propidium iodide (PI) and annexin V using the annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (Sigma) according to the manufacturer’s instructions. The experiment was performed using a BD FACSCalibur flow cytometer (Becton Dickinson Biosciences, San Jose, CA, United States) by acquiring 10,000 events, and data were analyzed by BD software FlowJo v10 (Tree Star, Ashland, OR).

Promastigotes of L. amazonensis (1 × 10⁷/well) were incubated with BA5 (9.0 and 4.5 μM) for 48 h. Parasites were washed with saline, centrifuged for 10 min at 252.0 g and diluted in the lysis solution containing PI (0.1% Triton X-100 and 2 μg ml−1 propidium iodide in PBS) in the absence of light at 37°C. After 30 min, the samples were acquired on a LSRFortessa flow cytometer (Becton Dickinson Biosciences, San Jose, CA, United States) and analyzed by FlowJo v.10 software (Tree Star).

To determine the effect of the compound on mitochondrial membrane potential, L. amazonensis promastigotes were incubated with 9.0 and 4.5 μM of BA5 for 72 h. After the treatment, parasites were incubated with 10 μg/ml of rhodamine 123 (Sigma Aldrich, St. Louis, United States) for 15 min. Methanol was used as negative control. Data acquisition was performed using a LSRFortessa flow cytometer and the analysis was performed by FlowJo v.10 software.

L amazonensis promastigotes (1 × 10⁷) were incubated with three concentrations from the IC₅₀ values (2.25, 4.5 and 9.0 μM) of BA5 for 48 h at 26°C. The parasites were fixed in a 2% glutaraldehyde solution and 0.1 M sodium cacodylate buffer for 2 h at room temperature. After fixation, the cells were post-fixed in osmium tetroxide (1%) for 1 h at room temperature. The parasites were placed on glass cover slips with 0.01% poly-L-lysine, dehydrated in graded ethanol (30–100%) and submitted at critical point (replacement of ethanol by CO₂) LEICA CPD 030. Samples were metalized with gold and observed in the scanning electron microscope JEOL JSM-6390LV.

Isobolograms were constructed by the fixed ratio method. Serial double dilutions were performed in triplicate in ratios of 1:1 and 10:1, BA5 and amphotericin B, respectively, using L. amazonensis promastigotes. For each proportion, an IC₅₀ value was calculated for each drug and combination after 72 h of incubation. The fractional inhibitory concentrations (FIC) were calculated by (IC₅₀ when combined/IC₅₀ isolated drug). The FIC values of different ratios were used to construct the isobologram in Graph Pad Prism version 5.01 program (Graph Pad Software, San Diego, CA, United States). The analysis of the combined effects was performed by determining the combination index (CI) as described previously by Chou and Talalay, 2005. CI values were used as cutoff to determine synergism.

One-way analysis of variance and Newman-Keuls multiple comparison tests were employed by using Graph Pad Prism version 5.01 (Graph Pad Software, San Diego, CA, United States). Differences were considered significant when the values were of p < 0.05.

Betulinic acid and BA5 derivative (Figure 1) presented CC₅₀ values of 18.8 and 31.1 µM, respectively, to mammalian cells. Amphotericin B, the reference antileishmanial drug, presented a CC₅₀ value of 3.3 µM, and gentian violet, a known cytotoxic drug, had a CC₅₀ value of 0.5 μM (Table 1). The effect of BA5 on promastigote forms of different species of leishmania was evaluated at six different concentrations, ranging from 1.56 to 50 μM. As show in the Table 1, BA5 was effective against all tested species. After 72 h of incubation, BA5 inhibited L. amazonensis promastigote proliferation with an IC₅₀ of 4.5 ± 1.1 μM; L. major (IC₅₀ = 3.0 ± 0.8 μM), L. braziliensis (IC₅₀ = 0.9 ± 1.1 μM) and L. infantum (IC₅₀ 0.15 ± 0.05 μM). In addition, BA5 was 6.9 times more selective (IS) for L. amazonensis promastigotes, 10.4 times more selective for L. major, 34.5 more selective for L. braziliensis and 207 more selective for L. infantum when compared with mammalian cell. Furthermore, IS of BA5 was higher for L. braziliensis than amphotericin B. Betulinic acid exhibited little or no activity against promastigote forms of different species of leishmania. This prototype was not selective for L. amazonensis (IS = 0.66) and L. braziliensis (IS = 1.1) (Table 1).

BA and BA5 promoted a significant decrease in the number of L. amazonensis-infected macrophages after 24 h of treatment (Figure 2). BA5 decreased the percentage of infected cells and the number of intracellular parasites at all concentrations tested, in a concentration-dependent manner (Figures 2E,F). BA prototype reduced the number of intracellular forms per macrophage only in the highest concentration tested, presented IC₅₀ value greater than 200 and was not selective against the parasite (SI < 1) (Table 2). BA5 presented an IC₅₀ value of 4.1 ± 0.7 µM (SI = 7.5) and amphotericin B exhibited an IC₅₀ value of 0.05 ± 0.02 µM (SI = 66) (Table 2).

After determining the activity against promastigotes and amastigotes forms of Leishmania sp., assays were performed to elucidate a possible mechanism of action of the BA5. First, ultrastructural analysis by scanning electron microscopy (SEM) was used to evaluate the morphology of L. amazonensis promastigotes treated or not with BA5. Untreated promastigotes had the typical elongated shape of the parasite without visible alterations in the plasma membrane or in cell volume (Figure 3A). In contrast, parasites treated for 48 h with BA5 (2.2, 4.5 or 9.0 µM) presented several morphological alterations, such as membrane protrusions resembling surface blebs (Figure 3B), flagella damage, increase in size (Figure 3C), and body deformation (Figure 3D).

Because the formation of blebbing in the membrane, cell rounding, and flagella damage generally culminates in the formation of apoptotic bodies (Basmaciyan and Casanova, 2019), we evaluated the mechanism by which compound BA5 could cause parasite death. Promastigotes were double-stained with Annexin-V-FITC and propidium iodide (PI) for flow cytometry analysis. Untreated cells were Annexin-V and PI-negative, demonstrating cell viability. The percentage of promastigotes positive only for annexin-V was 27.3% after treatment with IC₅₀/2 value of BA5, 51.25% when cells were treated with the IC₅₀ value of BA5 and 54.45% when cells were treated with 2x IC₅₀ of BA5. This data suggests that these cells were in early stages of apoptosis-like death. No significant difference in the number of necrotic cells was observed after treatment when compared to untreated controls (Figure 4).

To better understand the pathways that lead to apoptosis-like death, the mitochondria potential of L. amazonensis was evaluated by flow cytometry, after BA5 treatment and incubation with rhodamine123. As shown in Figure 5, the intensity of rhodamine123 was not significantly altered by incubation with BA5 at IC₅₀ and 2xIC₅₀ values of the drug. Amphotericin B and methanol, two known drugs able to induce mitochondrial alterations, reduced the intensity of the rhodamine 123.

Next, flow cytometric analysis after cell permeabilization and labelling with PI was used for quantification of nuclear DNA of parasites. Promastigotes of L. amazonensis treated with BA5 and amphotericin B with IC₅₀ and 2xIC₅₀ values were marked with PI and analyzed by flow cytometry. Figure 6 shows the distribution of cellular DNA through the cell cycle of the parasites in the absence and presence of the tested compounds. A significant increase in population of cells in pre-phase G0 and a significant decrease in population of cells in G2/M were observed in cells treated with IC₅₀ value (Figure 6B), and 2x IC₅₀ value (Figure 6C) concentrations of BA5, compared to untreated control (Figure 6A), 24 h after incubation.

The antileishmanial effect of BA5 and amphotericin B combination was investigated on promastigote forms of L. amazonensis. The combination of the drugs reduced the IC₅₀ values of amphotericin B by seven times and decreased the IC₅₀ values of BA5 by 50 times compared to each drug separately. The combination index values (0.15 ± 0.09 µM) Table 3 associated with a concave isobologram revealed that BA5 and amphotericin B have synergistic effects (Figure 7).