Evaluating the Potential of Ursolic Acid as Bioproduct for Cutaneous and Visceral Leishmaniasis

Leishmaniasis affects around 12 million people worldwide and is estimated to cause the ninth-largest disease burden. There are three main forms of the disease, visceral (VL), cutaneous (CL), and mucocutaneous (MCL), leading to more than one million new cases every year and several thousand deaths. Current treatments based on chemically synthesized molecules are far from ideal. In this study, we have tested the in vitro and in vivo efficacy of ursolic acid (UA), a multifunctional triterpenoid with well-known antitumoral, antioxidant, and antimicrobial effects on different Leishmania strains. The in vitro antileishmanial activity against the intracellular forms was six and three-fold higher compared to extracellular forms of L. amazonensis and L. infantum, respectively. UA also showed to be a potent antileishmanial drug against both VL and CL manifestations of the disease in experimental models. UA parenterally administered at 5 mg/kg for seven days significantly reduced the parasite burden in liver and spleen not only in murine acute infection but also in a chronic-infection model against L. infantum. In addition, UA ointment (0.2%) topically administered for four weeks diminished (50%) lesion size progression in a chronic infection model of CL caused by L. amazonensis, which was much greater than the effect of UA formulated as an O/W emulsion. UA played a key role in the immunological response modulating the Th1 response. The exposure of Leishmania-infected macrophages to UA led to a significant different production in the cytokine levels depending on the Leishmania strain causing the infection. In conclusion, UA can be a promising therapy against both CL and VL.


Introduction
Leishmaniasis affects around 12 million people worldwide and is estimated to cause the ninth-largest disease burden [1]. There are three main forms of the disease, visceral (VL), cutaneous (CL) and mucocutaneous (MCL) [2]. Approximately 0.7 to 1.2 million new cases of CL, 0.2 to 0.4 million new VL cases, and 20,000 to 30,000 deaths occur annually. Around 310 million people are at risk of L. infantum [36]. UA extracted from Petiveria alliaceae showed similar activity against L. amazonensis associated with programmed cell death of parasites and an increase in NO [37].
The aim of this study was to evaluate the in vitro activity of UA on promastigotes and intracellular amastigotes of different Leishmania species in order to select an adequate dose for in vivo efficacy studies. Moreover, topical formulations containing UA were developed, and its efficacy was tested in a chronic model of CL. The activity of parenterally administered UA was assessed on both an acute and chronic model of VL.

Assessment of Topical Formulations and Quantification of UA
UA is a triterpenoid with negligible aqueous solubility. For this reason, it was compounded in semisolid formulations to facilitate its solubility and its topical administration. UA was readily dispersed using a mixture of glycerin: propylene glycol (1:1, w:w) that avoided drug agglomeration after mixing with the preformed emulsion or Orabase in the case of the cream or ointment, respectively. Both formulations exhibited a pH of 5.5, which is suitable for skin administration. UA was chemically stable over one month at 25 ± 2 • C. Consistency, color, and pH were kept unchanged over this period of time. UA concentration was determined using a linear regression calibration curve between 0.78 to 200µg/mL (y = 3.2423x + 0.4212; R 2 = 0.9999).

In Vitro Activity against Leishmania spp. and Cytotoxicity against J774 Macrophages
Miltefosine showed greater in vitro activity on promastigotes than UA. However, UA exhibited an SI ranging from two to four, depending on the Leishmania spp., further in vitro investigations were performed against Leishmania amastigotes (Table 1). a IC50, concentration of the compound that produced a 50% reduction in parasites; SD: standard deviation. b Selectivity index, SI = CC50/IC50. c CC50, concentration of the compound that produced a 50% reduction of cell viability in treated culture cells with respect to untreated ones.
The anti-leishmanial activity of UA against amastigotes showed a six and three-fold increase compared to extracellular forms of L. amazonensis and L. infantum, respectively (Table 2). Also, the selectivity index of UA was much greater (three and eight-fold higher depending on the strain) than miltefosine. The greater activity of UA observed in amastigotes rather than in promastigotes can be explained by the dual mode of action of this compound not only on the parasite cell but also stimulating the immunological response of the cell host, mainly increasing NO production in macrophages. Supported by the in vitro leishmanicidal efficacy in both intracellular and extracellular forms, UA was also evaluated in vivo in a murine model of acute infection by L. infantum. The compound was administered by the intraperitoneal route at 5 mg/kg daily for seven consecutive days. The results are summarized in Table 3. UA showed a high parasite growth inhibition corresponding to 99.83% and 99.78% reduction in the number of parasites in spleen and liver, respectively, compared to the untreated group (p < 0.05). These results suggest a good UA bioavailability from the site of injection to the target organs. No evident signs of toxicity, like loss in weight or hair loss, were observed at the end of the experiment in any of the animals, which suggests that UA was well-tolerated by the infected mice at the administered dose.  Table 4. In spite of the lack of evident signs of toxicity at the end of the treatment in any of the animals, UA was less effective against chronic than acute infection. At the end of the experiment, the mean weight in the treated group was higher (145.5 ± 13.4) than in the control group (126.6 ± 10.52), but there were no statistically significant differences. The natural compound showed a 58% and 79% reduction in the number of parasites in spleen and liver, respectively, compared to the untreated group.  of UA ointment (0.2%) for 28 days resulted in a significant (p < 0.05) 42% and 50.3% reduction in the lesion size compared to the untreated group at weeks 10 and 15. However, parasites were not completely eradicated after UA topical administration as inflammation increased at week 15.
2.3.3. In Vivo Activity Against L. amazonensis in An Experiment Model of CL Prior to topical treatment, disease progression was similar in all animals, and no statistically significant differences were observed after 35 days post-infection ( Figure 1). Daily topical administration of UA ointment (0.2%) for 28 days resulted in a significant (p < 0.05) 42% and 50.3% reduction in the lesion size compared to the untreated group at weeks 10 and 15. However, parasites were not completely eradicated after UA topical administration as inflammation increased at week 15. In a subsequent study, a higher dose of UA (0.5%) was tested ( Figure 2). Daily topical administration of either UA ointment (0.5%) or UA cream (0.5%) for 28 days did not result in a significant reduction in the lesion size compared to the untreated group at week 10, although a significant reduction (47.54%) in inflammation size was achieved at week 15 after the administration of UA ointment. In a subsequent study, a higher dose of UA (0.5%) was tested ( Figure 2). Daily topical administration of either UA ointment (0.5%) or UA cream (0.5%) for 28 days did not result in a significant reduction in the lesion size compared to the untreated group at week 10, although a significant reduction (47.54%) in inflammation size was achieved at week 15 after the administration of UA ointment. Molecules 2020, 25, x FOR PEER REVIEW 6 of 15

Production of Cytokines
The efficacy of UA was higher on the intracellular amastigotes than in the promastigotes. This can be linked with an immunological reaction of the macrophages. For this reason, the effect of UA on the production of cytokines in different types of cell cultures was evaluated: i) naïve splenocytes from mouse spleen and ii) infected macrophages with L. infantum and L. amazonensis. On the splenocytes, out of the 17 studied cytokines, the following ones showed a significant increase in the presence of UA: GM-CSF, IFN-γ, IL-4, IL-6, IL-9, IL-10, and decrease of RANTES (Table 5). Nevertheless, IL4, IL6, and IL-10 decreased at higher concentrations of UA. In contrast, higher UA concentrations led to an increase in IFN-γ, which can make the eradication of intracellular forms of Leishmania favorable, opposite to the decrease in IL-10.

Production of Cytokines
The efficacy of UA was higher on the intracellular amastigotes than in the promastigotes. This can be linked with an immunological reaction of the macrophages. For this reason, the effect of UA on the production of cytokines in different types of cell cultures was evaluated: (i) naïve splenocytes from mouse spleen and (ii) infected macrophages with L. infantum and L. amazonensis. On the splenocytes, out of the 17 studied cytokines, the following ones showed a significant increase in the presence of UA: GM-CSF, IFN-γ, IL-4, IL-6, IL-9, IL-10, and decrease of RANTES (Table 5). Nevertheless, IL4, IL6, and IL-10 decreased at higher concentrations of UA. In contrast, higher UA concentrations led to an increase in IFN-γ, which can make the eradication of intracellular forms of Leishmania favorable, opposite to the decrease in IL-10. It is also key to investigate the production of cytokines on infected macrophages and not only just on lymphocytes. The exposure of Leishmania-infected macrophages to UA led to a significant different production in the cytokines levels depending on the Leishmania strain causing the infection. Except for the production of TNF-α that was exacerbated in both cases, several differences were observed in the levels of: GM-CSF, IFN-γ, IL-1b, IL-2, IL-6, IL-10, and MCP-1.
In L. infantum-infected macrophages, the production of GM-CSF, IL-6, and IL-10 was augmented compared to the untreated control while the levels of IFN-γ, MCP-1, and IL-1b were diminished significantly (p < 0.05) when L. amazonensis infected macrophages were exposed to UA (Table 6).

Discussion
In vitro antileishmanial activity of UA has been previously reported by other authors [35][36][37]. However, this is one of the first works that reports the in vivo leishmanicidal activity of UA against both VL and CL in different states of the disease. There is a wide variability in the reported in vitro efficacy against Leishmania spp. Many factors can contribute to this variability, but the natural source from which UA is extracted plays a key role in the final activity of this metabolite.
Tan et al. extracted UA from Salvia cilicica roots, which exhibited a very high in vitro activity in the nanomolar range against L. donovani and L. major promastigotes (IC 50  On the contrary, UA extracted from Miconialang sdorffii exhibited a very low antileishmanial activity against L. amazonensis promastigotes (IC 50 de 350 µM) [41]. Chemically synthesized UA showed an IC 50 of 20 and > 25 µM against L. amazonensis and L. infantum promastigotes, respectively [30].
This variable efficacy is one of the major problems of natural compounds. Extraction and purification processes should also be standardized in order to obtain a compound with reproducible characteristics, making its further use in clinical studies easier. For this reason, chemical synthesized UA (purchased by Sigma-Aldrich) was used to perform the in vitro and in vivo assays.
The higher leishmanicidal activity of UA against intracellular compared to extracellular forms has also been reported by other authors [39]. This fact may be related to its ability to modulate the macrophage nitric oxide production, which would enhance the death of the intracellular parasites [42].
Parenteral administration of UA at 5 mg/kg for seven consecutive days has shown to significantly reduce parasite growth in liver and spleen in both acute and chronic infection models of VL. In the acute-infection mouse model, although there is parasite growth in the liver and spleen, the infection is usually resolved by a Th 1 dependent granulomatous response after multiple weeks [43]. However, VL developed by hamsters closely resembles human VL. It is characterized by a relentless growth of parasites in the spleen, liver, and bone marrow, being more complicated to eradicate and usually ends up killing the animal [43]. It has been reported that UA can improve the Th1 immune response in infected mice, which could explain its potent antileishmanial activity [20]. In this sense, the reduction in parasite burden not only in the acute-infection model but also in the chronic model makes UA a promising drug for VL therapy.
Results obtained after UA topical administration against CL were also encouraging suggesting that: (i) higher doses than 0.5% topically administered can lead to an altered immune-reaction in the cutaneous infected lesion, (ii) UA ointment formulation has a better permeability across the skin than UA cream, and (iii) a successful therapy against CL could be achieved if prolonged topical treatments using UA ointment (0.2%) are applied. Despite the fact that creams combine properties, such as lubrication, occlusion, and hydration, that lead to higher patient compliance and better suitability for cutaneous lesions (being especially useful in intertriginous areas where ointments might not be used because they may cause maceration or folliculitis [44,45]), ointments can be more convenient when an occlusive effect is required to enhance drug permeation [45].
The immune response associated with the protection against a Leishmania infection is attributed to the development of a Th1-type immunity characterized by the production of cytokines, such as IFN-γ, TNF-α, IL-12, and GM-CSF, which can lead to the activation of macrophages and production of nitric oxide (NO).
Passero et al. demonstrated that the production of cytokines in an in vivo model of L. infantum-infected hamsters was characterized by an increase in the IL-10 levels at all the doses tested of UA and a decrease in the IFN-γ at higher doses [35]. Our in vitro results correlate with those observed by Passero et al. In the latter study, it was also remarked that even though the parasite load were reduced by 96%, the complete eradication of the parasites was not achieved, which can be related to an immunoregulator effect of the parasite on the macrophages [36].
Other studies have shown that the antileishmanial activity of UA depends on two factors: the parasite strain as well as the interaction with the immune system. The production of IFN-γ in infected macrophages seems to be more regulated by the presence of the parasites rather than the concentration of UA. This can explain the lack of efficacy of UA in the chronic models of VL [46].
Other studies have shown that GM-CSF has a beneficial output in the disease progression, especially in CL, because it triggers the activation of macrophages to kill Leishmania [47,48]. GM-CSF can improve healing and scarring of cutaneous lesions caused by CL combined con different treatments, such as glucantime or miltefosine [48,49]. In our study, an increase in the GM-CSF levels was observed on lymphocytes independently of the dose of the UA tested. However, this increase was only noticeable in those macrophages infected by L. infantum along with higher levels of TNF-α. This can explain the higher activity of UA observed against amastigotes compared to promastigotes.

Chemical Reagents
All chemicals used, unless otherwise stated, were obtained from Sigma-Aldrich (Madrid, Spain) or Panreac S.A. (Barcelona, Spain) and used without further purification.

Preparation of UA Topical Formulations
UA cream consisted of an aqueous phase with tween 80 (6%) dispersed in water and an oil phase constituted by white wax (1%) and cetyl alcohol (15%) melted at 60 0 . The aqueous phase was heated at 60 0 and then was added onto the oil phase at the same temperature. Continuous stirring was required until the cream reached the desired consistency at room temperature. Finally, UA (0.5%) dispersed in a mixture (1:1) of glycerin:propylene glycol (12%) was added. UA ointment was prepared by dispersing the UA (0.2 or 0.5%) in a mixture (2:1) of glycerin:propylene glycol (5%), which, in a second step, was mixed with Orabase ® (Fagron, Madrid, Spain), a plasticized hydrocarbon gel composed of gelatin, pectin, and sodium carboxymethyl cellulose.

Quantification of UA in the Topical Formulations
UA concentration was assessed by HPLC. Formulations (50 mg) were weighted and dissolved with methanol (100 mL). Samples were centrifuged at 3000× g for 10 min, and supernatant was collected and injected in the HPLC. HPLC was equipped with a Jasco PU-1580 pump, a Jasco AS-2050 Plus autosampler, and a Jasco UV-1575UV-visible detector (Jasco, Madrid, Spain). Integration of the peaks was performed with the program Borwin 1.5 for PC (JMBS Developments, Tokyo, Japan). The samples (100 µL) were chromatographed over a ThermoHypersil BDS C18 reverse-phase column (Thermo Fisher Scientific, Madrid, Spain) (200 × 4.6 mm, 5 µm) at a flow rate of 1 mL/min. Elution was carried out isocratically with a mobile phase that consisted of a methanol:water (95:5, v/v) mixture filtered through a 0.45 µm hydrophilic polypropylene filter membrane (GH polypro, Pall Corp., DeLand, FL, USA). UA was detected at a wavelength of 210 nm, and its retention time was 5 min.

Parasites and Culture Procedure
The following species of Leishmania were used: an autochthonous isolate of L. infantum

In Vitro Promastigote Susceptibility Assay
The assay was performed as previously described [50]. Briefly, log-phase promastigotes (2.5 × 10 5 parasites/well) were cultured in 96-well plastic plates. Compounds (UA and miltefosine) were dissolved in dimethylsulfoxide (DMSO) and different concentrations of each (100, 50, 25, 12.5, 6.25 3.12, 1.56, and 0.78 µg/mL) were carried out up to 200 µL final volume. Growth control and signal-to-noise were also included. The final solvent (DMSO) concentrations never exceeded 0.5% (v/v) warranting no effect on parasite proliferation or morphology. After 48 h at 26 • C, 20 µL of a 2.5 mM resazurin (Sigma-Aldrich, Madrid, Spain) solution was added to each well, and the plates were returned to the incubator for another 3 h. The Relative Fluorescence Units (RFU) (535-590 nm excitation-emission wavelength) was determined in a fluorometer (Infinite 200Tecan i-Control, Barcelona, Spain). Growth inhibition (GI) was calculated by the following equation: All tests were carried out in triplicate. Miltefosine was used as the reference drug and was evaluated under the same conditions. The efficacy of each compound was estimated by calculating the IC 50 (concentration of the compound that produced a 50% reduction in parasites) using a multinomial probit analysis incorporated in SPSS software v21.0 (IBM, Madrid, Spain).

In Vitro Intracellular Amastigote Susceptibility Assay
The assay was carried out as previously described [51]. Briefly, 5 × 10 4 J774 macrophages and stationary promastigotes in a 1:10 ratio were seeded in each well of a microtiter plate, suspended in 200 µL of culture medium and incubated for 24 h at 33 • C in 5% CO 2 chamber. After this first incubation, the temperature was increased up to 37 • C for another 24 h. Thereafter, cells were washed several times in culture medium by centrifugation at 1500 rpm for 5 min in order to remove free non-internalized promastigotes. Finally, the supernatant was replaced by 200 µL/well of culture medium containing 2-fold serial dilutions of the test compounds (50, 25, 12.5, 6.25 3.12, 1.56, 0.78, and 0.39 µg/mL). Growth control and signal-to-noise were also included. All tests were carried out in a triplicate assay. Following incubation for 48 h at 37 • C, 5% CO 2 , the culture medium was replaced by 200 µL/well of the lysis solution (RPMI-1640 with 0.048% HEPES and 0.006% SDS) and incubated at room temperature for 20 min. Thereafter, the plates were centrifuged at 3500 rpm for 5 min, and the lysis solution was replaced by 200 µL/well of Schneider´s insect medium. The culture plates were then incubated at 26 • C for another 4 days to allow the transformation of viable amastigotes into promastigotes and proliferation. Afterward, 20 µL/well of 2.5 mM resazurin was added and incubated for another 3 h. Finally, fluorescence emission was measured, and IC 50 was estimated as described above. All tests were carried out in triplicate. Miltefosine was used as the reference drug and was evaluated at the same conditions.

Cytotoxicity Assay on Macrophages
The assay was carried out as previously described [52]. J774 macrophages cell lines were seeded (5 × 10 4 cells/well) in 96-well flat-bottom microplates with 100 µL of RPMI 1640 medium. The cells were allowed to attach for 24 h at 37 • C, 5% CO 2 , and the medium was replaced by different concentrations of the compounds in 200 µL of medium and exposed for another 24 h. Growth controls and signal-to-noise were also included. Afterward, a volume of 20 µL of the 2.5 mM resazurin solution was added, and plates were returned to the incubator for another 3 h to evaluate cell viability. The reduction of resazurin was determined by fluorometry as in the promastigote assay. Each concentration was assayed three times. Cytotoxicity effect of compounds was defined as the 50% reduction of cell viability of treated culture cells with respect to untreated culture (CC 50 ). Each mouse was infected by an intracardiac route with 1 × 10 7 promastigotes of L. infantum according to a model previously described by Dea-Ayuela et al. [53]. Mice were randomly split into two groups each of six animals. Treatment started on day thirty-five post-infection and lasted for seven consecutive days. One group received UA at 5 mg/kg once daily intraperitoneally administered in 0.1 mL of saline solution. The other group was left untreated (control group). One week after the last treatment, all mice were sacrificed, and parasite burden in liver and spleen was estimated by the limit dilution assay describe elsewhere [54]. Briefly, spleen (0.15 g) and liver (0.5 g) were homogenized in a 5 mL of PBS-50 mM glucose-2 mM ethylene diamine tetraacetic acid (EDTA) solution at 4 • C using a sterilized steel stainless tissue grinder. Cell debris was removed by passage through a glass wool column. The suspension obtained was centrifuged at 1500 rpm for 15 min at 4 • C. Afterward, the supernatants were discarded, and the pellets were collected and resuspended in Schneider medium supplemented as described above, and then, 200 µL of this suspension were transferred to the first well of a 96-well microtiter plate containing medium supplemented with antibiotics, as described above. Serial dilutions were repeated, transferring 100 µL from the previous well to the next one and adding 100 µL of Schneider medium. After incubation at 26 • C for 7 days, microplates were examined using an inverted microscope (Olympus, model IM) at a magnification of 40X, and the presence or absence of promastigotes in each well was recorded. The final titer was the last dilution for which the well contained at least one parasite [54].

Chronic Infection Model of VL in Golden Hamster
Chronic infection in Golden hamster was developed, as previously described by Dea-Ayuela et al. [55]. Each hamster was infected by an intracardiac route with 1 × 10 7 promastigotes of L. infantum. Hamsters were randomly split into two groups of six animals. Treatment started on day thirty-five post-infection and lasted for seven consecutive days. One group received UA at 5 mg/kg once daily intraperitoneally administered in 0.1 mL of saline solution. The other group was left untreated (control group). Parasite burden in liver and spleen was estimated by the limit dilution assay described above [54].

Chronic Infection Model of CL in Golden Hamster
This experiment was carried out as described by Morais-Teixeira et al., with some modifications [56]. Hamsters were randomly split into two groups of six animals. A suspension (25 µL) containing 1 × 10 7 stationary promastigotes of L. amazonensis in Schneider medium was injected subcutaneously in the footpad of the left hind paw at day 0. Right hind paw was used as a negative control (no infection). After 5 weeks post-infection, chronic CL was developed, and topical treatment with UA started. In a first experiment, UA ointment (0.2%) was administered once daily for 4 consecutive weeks. The other group received no treatment. Disease progression was monitored at weeks 0, 5, 10, and 15 using a Vernier caliper to measure footpad size. In a second experiment, hamsters were randomly split into three groups of six animals. One group was treated with UA ointment (0.5%), and the other group was tread with UA cream (0.5%). As in the first experiment, the third group received no treatment (control group).

Effect of UA in the Cytokines Production in Macrophages Infected with Leishmania In Vitro
In vitro Leishmania infection was carried out as described previously. After the incubation with 200 µL/well of culture medium containing UA at the IC 50 concentration previously determined for each of the species (L. infantum and L. amazonensis) for 48 h the supernatants were collected and stored at −80 • C for cytokine quantification.

Splenocytes Culture and Evaluation of Cytokines Production
Cell suspension of splenocytes was prepared from the spleen of non-infected BALB/c mice and then was rinsed with RPMI 1640 medium three times. Then, 5 mL of the cell suspension was added to 1 mL of ammonium chloride 0.8% to isolate the splenocytes, and the suspension was again washed three times with RPMI 1640 medium. The splenocytes, 2.5 × 10 9 cells/L, were cultured in RPMI 1640 medium containing 10% fetal bovine serum and incubated at 37 • C for 24 h. After incubation for 24 h, the supernatants were collected and stored at −80 • C for cytokine quantification.

Analysis of Cytokines
The analyses were carried out by using the Quantibody ® array (RayBiotech, Madrid, Spain), a multiplexed sandwich ELISA-based quantitative array platform [57]. Nineteen different markers were analyzed: GM-CSF, IFNγ, IL-1a, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12, IL-13, IL-17, KC, MCP-1, M-CSF, RANTES, VEGF. A pair of cytokine specific antibodies was used for detection. A capture antibody was first bound to the glass surface. After incubation with the sample, the target cytokine was trapped on the solid surface. A second biotin-labeled detection antibody was added, which recognized a different epitope of the target cytokine. The cytokine-antibody-biotin complex was visualized through the addition of the streptavidin-conjugated Cy3 equivalent dye, using a laser scanner (GenePix ® 4000B Scanner, UCM Facilities, Madrid, Spain).
A sandwich ELISA kit was used to determine the concentrations of TNF-α according the manufacturer's instructions. Briefly, standards and samples (50 µL) were diluted with 50 µL of commercial diluting solution in triplicate and incubated for 2 h at 37 • C. Plates were washed five times, and 100 µL of conjugated anti-TNF-α was added and incubated for 1 h at 37 • C. Finally, 100 µL of TMB was added as substrate and incubated for 30 min in dark at room temperature. The reaction was stopped with 3M H 2 SO 4, and the optical densities (OD) were read at 450 nm.

Statistical and Data Analysis for In Vitro Assays
The efficacy against the parasite (IC 50 ) and cytotoxicity effect (CC 50 ) of compounds were calculated from Probit analysis using SPSS v21.0 software (IBM, Madrid, Spain) Finally, selective index (SI) was calculated defined as the ratio between CC 50 /IC 50. The analysis of cytokines in vitro was performed by Tukey's HSD test using the statistical program IBM ® SPSS v21.0 (IBM, Madrid, Spain) .

Statistical and Data Analysis for in Vivo Assays
The group mean, standard deviation, standard error, and the differences were compared by the Mann-Whitney U nonparametric test, using the statistical programs of Microsoft Excel 2011 ® (Microsoft, Washington, DC, USA) and IBM ® SPSS v21.0. Statistical significance was set at a p-value < 0.05.

Conclusions
Currently, plant-based therapies have shown great potential in the treatment of leishmaniasis. However, it is challenging to find a compound that possesses efficacy not only against cutaneous but also against visceral leishmaniasis. In this work, we have demonstrated that UA, a ubiquitous triterpenoid in nature, has a potent antileishmanial efficacy against both VL and CL, having a direct effect on the immunological response of the host. UA parenterally administered at 5 mg/kg significantly reduced parasite growth in liver and spleen not only in acute-infection but also in a chronic infection model. In addition, UA ointment (0.2%) topically administered could constitute a promising therapy against CL being able to penetrate across the skin and to diminish disease progression. In conclusion, formulating UA with suitable excipient can lead to promising treatments for cutaneous and visceral leishmaniasis.