Ethanolic extract of Croton blanchetianus Ball induces mitochondrial defects in Leishmania amazonensis promastigotes

Abstract

Leishmaniasis is a neglected disease caused by Leishmania. Chemotherapy remains the mainstay for leishmaniasis control; however, available drugs fail to provide a parasitological cure, and are associated with high toxicity. Natural products are promising leads for the development of novel chemotherapeutics against leishmaniasis. This work investigated the leishmanicidal properties of ethanolic extract of Croton blanchetianus (EECb) on Leishmania infantum and Leishmania amazonensis, and found that EECb, rich in terpenic compounds, was active against promastigote and amastigote forms of both Leishmania species. Leishmania infantum promastigotes and amastigotes presented IC₅₀ values of 208.6 and 8.8 μg/mL, respectively, whereas Leishmania amazonensis promastigotes and amastigotes presented IC₅₀ values of 73.6 and 3.1 μg/mL, respectively. Promastigotes exposed to EECb (100 µg/mL) had their body cellular volume reduced and altered to a round shape, and the flagellum was duplicated, suggesting that EECb may interfere with the process of cytokinesis, which could be the cause of the decline in the parasite multiplication rate. Regarding possible EECb targets, a marked depolarization of the mitochondrial membrane potential was observed. No cytotoxic effects of EECb were observed in murine macrophages at concentrations below 60 µg/mL, and the CC₅₀ obtained was 83.8 µg/mL. Thus, the present results indicated that EECb had effective and selective effects against Leishmania infantum and Leishmania amazonensis, and that these effects appeared to be mediated by mitochondrial dysfunction.

Key words
Leishmania amazonensi; Leishmania infantum; mitochondrial metabolism; natural products

MATERIALS AND METHODS

Plant material

The bark of C. blanchetianus was collected in the village Fazenda Serrote, in the Municipality of Piranhas, state of Alagoas, Brazil. Botanical identification was performed by Dr. Ana Paula Silveira, botanist of the Department of Biology, Universidade Federal de Sergipe, Brazil (DB-UFS). A voucher specimen was deposited in the DB-UFS herbarium under the registration number ASE 24664.

Preparation of the C. blanchetianus extract and phytochemical analysis

A total of 1.2 kg of C. blanchetianus bark was dried at room temperature and pulverized in a blade mill to obtain the powder, which was extracted by maceration in 90% ethanol for five days. Afterwards, the material was filtered and concentrated in a rotatory evaporator under reduced pressure at 45°C, to give 114.35 g of the ethanolic extract (HEE, yield of 9.53%).

To obtain a chromatographic profile of the extract, High Performance Liquid Chromatography (HPLC) analyses were performed on a series of liquid chromatograph Shimadzu Prominence LC comprised of two pumps 6AD, photodiode array detector (DAD) and a SPD M20A analytical C18 column (25.0 × 0.46 cm, 5 μm particles). The solvents used were HPLC grade H₂O and CH₃OH. From hydroethanolic extract was made a solution with final concentration of 1 mg/ml subjected to elution through gradient 5 to 100% CH₃OH exploration, exploratory gradient: 20 to 100% CH₃OH up to 40 min followed by isocratic gradient of 40 to 60 min 100% CH₃OH to investigate the chromatographic profile. The substances were detected using a photodiode array detector (DAD - UV/Vis) using a 254 nm wavelength (Queiroz et al. 2002). For the acquisition and processing of chromatographic data was used LC Solution software.

Parasites

For the assays, L. amazonensis (MHOM/BR/77/LTB0016) and L. infantum (MHOM/MA67ITNAB263) were maintained as promastigotes at 26°C in Schneider’s insect medium (Sigma-Aldrich), supplemented with 10% and 20% heat-inactivated fetal calf serum (HIFCS) for L. amazonensis and L. infantum, respectively, in addition to 100 μg/mL streptomycin and 100 U/mL penicillin.

Antipromastigote activity

L. amazonensis and L. infantum promastigotes (5 x 10⁶ cells/mL) were cultivated in Schneider’s insect medium supplemented with HIFCS, as previously described. Parasites were incubated at 26°C for 24 h, in either the absence or presence of C. blanchetianus extract dissolved in DMSO (50 to 500 µg/mL). The final concentration of DMSO never exceeded 0.4%, a concentration which is not toxic for the protozoa (Oliveira et al. 2009). The viability of promastigotes was assessed by the AlamarBlue® assay (Fonseca-Silva et al. 2011). Pentamidine was used as reference drug. The vehicle of extract (DMSO 0.4%) was added to the control group.

Antiamastigote activity

Peritoneal macrophages were collected from BALB/c mice (6–8 weeks old) and plated in cold RPMI medium supplemented with 10% HIFCS, 100 μg/mL streptomycin, and 100 U/mL penicillin, at a concentration of 2 x 10⁶ cells/mL (0.4 mL/well), in Lab-Tek eight-chamber slides. Cells were then incubated for 3 h at 37^oC in a 5% CO₂ atmosphere, for cell attachment. Non-adherent cells were removed by phosphate buffered saline (PBS) washing, and L. amazonensis or L. infantum promastigotes were added to the peritoneal macrophages at a parasite ratio of 3:1 and 5:1, respectively. After 5 h incubation, free parasites were removed by successive washes with warmed RPMI 1640, and Leishmania-infected macrophages were then incubated in either the absence or presence of C. blanchetianus extract (3.0 to 30.0 µg/mL) for 72 h. The percentage of infected macrophages was determined by light microscopy and random counts of a minimum of 300 cells in each treatment, in duplicate. Results were expressed as an infection index (% of infected macrophages X number of amastigotes/total number of macrophages). Pentamidine was used as a reference drug.

Macrophage viability assay and NO production

Peritoneal macrophages (2 x 10⁶ cell/mL) collected from BALB/c mice (6–8 weeks old) were allowed to adhere in 96-well tissue culture plates for 1 h, at 37^oC, in a 5% CO₂ atmosphere, for cell attachment. Nonadherent cells were removed by washes with RPMI 1640 medium. Serial dilutions of C. blachetianus extract in concentrations from 120 to 1.875 μg/mL were added to the wells containing adherent macrophages. After 72 h incubation, the medium was discharged, and the macrophages were washed with RPMI 1640 medium; cell viability was assessed after 12 h incubation with AlamarBlue® (Fonseca-Silva et al. 2013).

The selectivity index (SI) was determined using the following equation: macrophage CC₅₀/intracellular amastigote IC₅₀ (Weniger et al. 2001). Peritoneal macrophages were lysed with 0.1% Triton X-100 and used as positive controls. For the estimation of nitric oxide (NO) in EECb treated macrophages, the cell supernatants were collected and distributed (50 μL/well) in 96-well plates, and equal volume of Griess reagent was added to each well; these were then incubated for 10 min at 25°C, and the absorbance was taken at the wavelength 540 nm. The concentration of NO was given in μM.

Analysis of promastigote cellular morphology

Morphological changes in parasites as a result of C. blanchetianus extract treatment were identified microscopically. Briefly, L. amazonensis and L. infantum promastigotes in exponential growth phase were incubated in either the absence or presence of C. blanchetianus extract (100 μg/mL) for 24 h. Cytospin smears from cell suspensions were stained with 10% Giemsa staining solution and examined under an optical microscope (Olympus). At least 20 microscope fields were observed for each sample.

Determination of promastigotes growth rate and doubling time

L. amazonensis promastigotes were cultivated in Schneider’s insect medium in the absence or presence of 70.0 µg/mL or 140.0 µg/mL EECb (starting from 5 x 10⁵ parasites/mL). The parasites growing were determined by direct counting with a Neubauer chamber either every 24 h for five days for growth curve determination, or every 6 h for 48 h for doubling time determination. The doubling time was calculated by the Least Squares Fitting-Exponential method accessed in the online platform Doubling Time, available at http://www.doubling-time.com/compute_more.php.

Cellular membrane integrity assessment

L. amazonensis promastigotes (5 x 10⁵ cells/mL) were cultivated in Schneider’s insect medium supplemented with HIFCS, as previously described, in either the absence or presence of 70.0 µg/mL and 140.0 µg/mL of C. blanchetianus extract, at 26°C for 24 h. Promastigotes were then harvested, washed twice with PBS (pH 7.2), and stained with propidium iodide (PI) (40 µg/mL) for 15 min, in the dark, at room temperature. PI-positive cells were evaluated by flow cytometry using an Attune® Acoustic Focusing (Life Technologies) cytometer. A total of 10,000 events were obtained and analyzed using Attune® Cytometer Software.

Analysis of the cell cycle

L. amazonensis promastigotes (1 x 10⁵ cells/mL) were incubated in either the absence or presence of C. blanchetianus extract (70.0 µg/mL or 140.0 µg/mL), for 24 h, and then washed twice with PBS (pH 7.2). Cells were fixed in chilled 70% ethanol and incubated for 1 h on ice. Parasites were again washed twice with PBS and resuspended in 0.5 mL PBS containing PI (40 µg/mL) and RNase A (Sigma: R464; 200 μg/mL). The mixture was incubated for 1 h at room temperature in the dark. Data was obtained using an Attune® Acoustic Focusing (Life Technologies) cytometer and analyzed using Attune® Cytometer Software.

Determination of mitochondrial membrane potential (Δψm)

The variation in mitochondrial membrane potential (Δψ_m) was monitored using JC-1 dye. L. amazonensis promastigotes (0.5 to 1 x 10⁶ cells/mL) were incubated in either the absence or presence of C. blanchetianus extract (70.0 µg/mL and 140.0 µg/mL) for 24 h, and then washed twice with PBS (pH 7.2). Afterwards, promastigotes were incubated with JC-1 (10 µg/mL in HBBS) for 20 min, at room temperature, in the dark. JC-1 stained cells were washed twice with HBSS, and fluorescence was measured spectrofluorometrically at both 530 nm and 590 nm, using an excitation wavelength of 480 nm. The ratio of values obtained at 590 nm and 530 nm was plotted as the relative ΔΨ_m. The mitochondrial uncoupling agent carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP; 20 μM; 20 min) was used as positive control (Fonseca-Silva et al. 2016).

Statistical analysis

All experiments were performed in triplicate, except the antiamastigote activity, which was performed in duplicate. The minimum EECb concentration that caused 50% reduction in the survival of the amastigote and promastigote Leishmania forms (IC₅₀), and in macrophages (CC₅₀), was obtained by regression analysis using GraphPad Prism 5.0 software (GraphPad Software, La Jolla, CA, USA). Data were analyzed statistically using two-way analysis of variance (ANOVA) and Student’s t-test.

Ethical Approval

Macrophages from BALB/c mice were obtained following protocols approved by the Ethics Committee for Animal Use of the Instituto Oswaldo Cruz (L026/2015).

RESULTS AND DISCUSSION

During the last few decades, studies have demonstrated leishmanicidal effects of some Croton species. Dey et al. (2015) showed that the hexanic extract of C. caudatus is an effective growth inhibitor of L. donovani promastigotes and amastigotes. Also, extracts significantly altered the biochemical parameters (protein, lipids, and carbohydrates) in promastigotes, and caused morphological changes, DNA condensation, and subsequent apoptosis. Previously, Rosa et al. (2003) showed that at the nanogram level, the essential oil of C. cajucara caused gross morphological changes in L. amazonensis promastigotes. Moreover, after 30 min in the presence of the essential oil, the parasites were completely destroyed. These data show that the genus Croton is a source of promising leads for novel leishmaniasis chemotherapeutics. These findings encouraged our study of the antileishmanial activity of ethanolic extract of C. blanchetianus (EECb) on L. amazonensis and L. infantum.

Phytochemical characterization

The chemistry of the Croton genus is very diverse. Terpenoids are the predominant metabolites, especially diterpenes. Triterpenoids, pentacyclic or steroids, are also often reported. These compounds may be associated with the antibacterial, antifungal, antiviral, antimalarial, anti-inflammatory and anticancer activities, corroborating with the traditional therapeutic uses of plants of this genus (Salatino et al. 2007).

The chromatographic profile of ethanolic bark extract of C. blanchetianus was focused from 22 to 55 min of chromatographic running in which the most prevalent compounds or groups of compounds were eluted between 37 and 55 min, that is characteristic of low polarity compounds (Fig. 1a). Also, UV scan for the selected chromatographic peaks with retention time of 37.63, 41.08, 50.81 and 54.33 min are showed on Fig. 1b, which complies with a chemical profile of terpenic compounds, whose absorption spectra typically range between 200 and 270 nm. Besides, a plain I Band, characteristic of terpenes (Kong et al. 2001) could be observed. Based on the compound absorption profile, we can observe that the main groups of terpenes found in the ethanolic bark extract of C. blanchetianus are unsaturated, whose absorption ranges between 215 and 250 nm and aromatic terpenes, whose absorption ranges between 250 and 270 (Ugaz 1994).

Figure 1
Phytochemical characterization of ethanolic bark extract of C. blanchetianus. a: Chromatographic profile at 60 min of chromatographic running; b: UV scan (190–800 nm) for the chromatographic peaks with retention time of 37.63 min (B1); 41.08 min (B2); 50.81 min (B3) and 54.33 min (B4) complying with compounds of terpene nature. (*) Chromatographic peaks analyzed in B1; B2; B3 and B4.

One of the mechanisms of action of terpenes is the attack on the plasma membrane. Mendanha & Alonso (2015) demonstrated that terpenes act as lipid spacers, increasing the amount of the most mobile component. Thus, the insertion of terpenes in the lipid bilayer, increase the membrane fluidity. This is consistent with the reported broad spectrum of terpene activity against protozoa, pathogenic fungi and tumor cells (Camargos et al. 2014).

Plant extracts have been traditionally used in the treatment of protozoan diseases (Cock et al. 2018, Da Silva et al. 2018) and several scientific studies with the chemical characterization of the extracts and/or terpenoids compounds and their leishmanicidal potential have been developed in the last years (Teles et al. 2015, de Almeida et al. 2016, Garcia et al. 2017).

The trypanocide activity of the methanolic extract of the stem bark of Croton cajucara and of the isolated terpenes, transdehydrocrotonin and acetyl aleuritolic acid, were investigated on Trypanosoma cruzi. The extract and the terpenes isolated inhibited T. cruzi for cause inhibition of the trypanothione reductase pathway, alterations of the plasma membrane and mitochondrial damage (Campos et al. 2010). Diterpenes from Croton cajucara displayed in vitro antileishmanial effects against promastigotes and amastigotes of L. amazonensis. The possible mechanism of action of C. cajucara diterpenes was the inhibition of the trypanothione reductase enzyme (Lima et al. 2015).

Effect on parasite and macrophage survival

The in vitro leishmanicidal activity of EECb was evaluated in both axenic promastigote and intracellular amastigote forms of L. amazonensis and L. infantum. The effect of different concentrations of EECb on the parasite survival and the 50% inhibitory concentrations (IC₅₀) are shown in Figure 2 and Table I respectively. Besides the IC₅₀ value for L. amazonensis promastigotes being nearly three times lower than that for L. infantum, at 100 μg/mL EECb, a reduction of 70% in the L. amazonensis promastigote survival was observed (Figure 2a). This level of reduction was only observed in promastigotes of L. infantum at an EECb concentration of 300.0 µg/mL. Likewise, an 80% reduction in L. amazonensis amastigote survival was detected in macrophages treated with EECb concentrations between 5.0 and 10.0 µg/mL, whereas in L. infantum amastigotes, this level of survival reduction was detected in concentrations between 20 and 30 µg/mL (Figure 2b). Based on these results, we can notice that promastigote and amastigote forms of L. amazonensis are more sensitive to EECb, when compared to L. infantum.

Table I
In vitro antileishmanial activity, cytotoxicity, specificity, and selectivity index for EECb.

Treatment	Murine Macrophage	L. amazonensis					L. infantum
		Promastigote	Amastigote	SIa	SPIb		Promastigote	Amastigote	SI a	SPI b
	CC50 (µg/mL)	IC50 (µg/mL)					IC50 ( µg/mL)
EECb	83.8 (64.0-101.4)	73.6 (63.0–86.0)	3.1 (2.4–3.8)	27.0	23.7		208.7 (176.7-246.5)	8.8 (7.4–10.2)	9.5	23.6
Pentamidine	2.8 (2.5-3.3)	1.63 (1.60-1.66)	0.64 (0.61-0.67)	4.3	2.5		1.94 (1.90-1.98)	0.14 (0.007-0.21)	20.0	13.8

• ^a SI: selectivity index (macrophage CC₅₀/amastigote IC₅₀ ratio).
  ^b SPI: specificity index (promastigote/amastigote IC₅₀ ratio).
  95% confidence intervals are shown in brackets.

Figure 2
Effect of EECb on L. amazonensis and L. infantum survival. a: Effect of EECb on L. amazonensis and L. infantum promastigotes survival after 24 h of treatment, and b: effect of EECb on L. amazonensis and L. infantum amastigotes survival after 72 h of treatment. Results from three experiments in duplicate (a), and from two experiments in duplicate (b), are shown as percentages and standard errors of survival inhibition, compared to the untreated control.

Rosa et al. (Rosa et al. 2003) showed that the linalool-rich essential oil from the leaves of C. cajucara has a strong effect on the viability of L. amazonensis promastigotes (LD₅₀ = 8.3 ng/mL) and amastigotes (LD₅₀ = 22.0 ng/mL). The effect of C. cajucara essential oil included gross morphological changes in L. amazonensis promastigotes, such as disruption of flagellar membranes, mitochondrial swelling, and nuclear and kinetoplast chromatins destruction. More recently, Rodrigues et al. (2013) showed similar effects of C. cajucara essential oil on the morphology of L. infantum promastigotes. However, the IC₅₀ obtained was 67.0 µg/mL, considerably higher than that previously observed for L. amazonensis. These observations corroborate the findings in the present study, since L. amazonensis promastigotes are more sensitive to the action of Croton products than L. infantum.

Even though antileishmanial drugs currently in use have strong in vitro activity against amastigote forms, they usually present high toxicity to mammalian host cells. On the other hand, several studies have shown that, at effective concentrations, essential oils of Croton, and their major compounds, present low toxicity to host cells, when compared with parasites (Guimaraes et al. 2010, Lima et al. 2015, Rodrigues et al. 2013, Rosa et al. 2003). Thus, to test the selectivity of EECb for the parasite, murine macrophages were treated with increasing concentrations of EECb; after 72 hours treatment, no cytotoxic effects were observed at concentrations below 60 µg/mL, and the CC₅₀ obtained was 83.8 µg/mL.

Despite the difference in the sensitivity to EECb observed between L. amazonensis and L. infantum, the effect of the extract on parasite survival of both species was higher on amastigotes, when compared with promastigotes. The specificity index (SPI), corresponding to the ratio between the IC₅₀ for promastigote and the IC₅₀ for intracellular amastigotes, indicates the specificity of antileishmanial activity of a given compound. De Muylder et al. (2011) considered an SPI value greater than 2.0 as the cut-off point to define a compound as more active against intracellular amastigotes. The SPI obtained for EECb was higher than 20.0 for both L. amazonensis and L. infantun (Table I).

This is a relevant finding, as the targets for new chemotherapy are the intracellular amastigotes, i.e., the parasite form that survives and multiplies inside human macrophages (Croft & Coombs 2003). According to Guimarães et al. (2010) one of the possible explanations for the higher activity of a compound on intracellular amastigotes is that this compound may be responsible for increasing the effectiveness of the macrophage’s microbicide response by producing NO. However, this seems not to be the case, since EECb did not cause macrophages to increase their NO production (data not shown). Alternatively, macrophages might accumulate higher levels of EECb, and this could be the possible explanation for the distinct action of EECb on promastigotes alone, and on amastigotes in an intracellular environment (Inacio et al. 2014).

The ratio between the CC₅₀ of EECb for macrophages, and the IC₅₀ of EECb for amastigotes, which corresponds to the SI, was 27.0 for L. amazonensis and 9.5 for L. infantum. Therefore, at concentrations lower than 10.0 µg/mL, EECb effectively reduced the survival of both L. infantum and L. amazonensis intracellular amastigotes. Besides, the extract appeared to be acting directly on the parasite, with no cytotoxic or immunomodulatory effect on mammalian macrophages.

Effect on parasite morphology and growth rate

Although C. blanchetianus extract showed leishmanicidal activity, its mechanism of action on the parasites remains unknown. Optical microscopic analysis of promastigotes treated with EECb at 100 µg/mL revealed a volume reduction and rounding of the cellular body, as well as flagellum duplication, as shown in Figure 3. Similar morphological changes were observed by da Silva et al. (da Silva et al. 2015b) in L. amazonensis promastigotes exposed to 100 µg/mL of Physalis angulata aqueous extract. Atypical flagella duplication has already been shown in L. amazonensis promastigotes treated with essential oil from Cymbopogon citratus (Santin et al. 2009). Although P. angulata and C. citratus are representative of genera different from that studied in this work, and certainly contain chemical compounds different from that present in EECb composition, morphological alterations such as flagella duplication seem to be a recurrent result of interference of plant extracts in the process of cell division, as suggested by Adade & Souto-Padrón (2010). Therefore, the present results suggest that EECb may be interfering with the process of cytokinesis, which could be causing a decline in the parasite multiplication rate.

Figure 3
Effect of EECb on Leishmania promastigotes morphology as observed by microscopy. Untreated L. amazonensis and L. infantum promastigotes showing the typical elongated shape. L. amazonensis and L. infantum promastigotes treated with 100 µg/mL of EECb showed body rounding, cellular volume reduction, and flagellum duplication (arrowed). Magnification: 1000X.

In order to verify this hypothesis, the effect of EECb on promastigotes growth was monitored over five days. Considering that the same morphological changes were detected in both L. amazonensis and L. infantum, but since the results obtained so far have shown that L. amazonensis survival is more strongly affected by EECb action, L. amazonensis promastigotes were chosen to investigate the mechanism of action on Leishmania. The growth curves presented in Figure 4a show that, compared to the control, treatment with 70.0 and 140.0 µg/mL (concentration corresponding to nearly one and two times the IC₅₀) produced reductions of around 60% and 70%, respectively, in parasite count from 24 to 72 hours of growth. This caused the treated parasites to reach the stationary phase after only 96 hours, whereas the untreated reached this phase after 72 hours of cultivation. Indeed, a 35% reduction in the growth rate of the parasites was observed, as evidenced by the comparison of parasite doubling time, which increased from 6.9 hours in untreated parasites, to around 10.0 hours for parasites exposed to EECb (Figure 4b).

Figure 4
Effect of EECb on L. amazonensis promastigotes proliferation. L. amazonensis was cultivated in Schneider’s insect medium in the absence (circle) or presence of 70.0 µg/mL (square) or 140.0µg/mL EECb (triangle), and the number of parasites was determined by direct counting with a Neubauer chamber, every 24 h for 5 days (a), and every 6 h for 48 h (b). The doubling time was calculated by the Least Squares Fitting-Exponential method accessed in the online platform Doubling Time, available at http://www.doubling-time.com/compute_more.php. Values are presented as the mean ± standard error of three different experiments in triplicate.

A possible explanation for the growth rate decrease could be the delay in the cell cycle caused by EECb. However, no significant changes in the cell cycle progression were observed in L. amazonensis promatigotes exposed to EECb, when compared with the control cells (data not shown). These results indicate that the effect of the extract on the growth kinetics is not due to an arrest on the cell cycle of the parasites. A similar scenario was observed by Scher et al. (2012) in response to overexpression of the gene anti-silencing factor 1 (ASF1) in L. major. The authors suggest that the delay in the growth rate could be a strategy used by the parasite to recover the cellular homeostasis compromised by adverse conditions. Therefore, it can be considered that, at the evaluated conditions, EECb affects the metabolism of L amazonensis promastigotes, so that it reduces parasite proliferation.

Effect on cell membrane integrity and mitochondrial membrane potential

Mitochondrial metabolism could be a relevant target for EECb action. Mitochondria are essential organelles for any cell survival, since they play an important role in energy metabolism. Maintaining the proper mitochondrial membrane potential is vital to the ATP production process, and consequently, to energy production by cells (Joshi & Bakowska 2011, Machado et al. 2014). In kinetoplastids parasites, such as Trypanosoma and Leishmania, mitochondrion is a single organelle essential for survival, and its proper functioning is extremely important, when compared with organisms that have multiple mitochondria, in which viable organelles can compensate for malfunctioning of the damaged ones (Shaha 2006). Thus, the mitochondria of trypanosomatides are considered one of the most fascinating target for drugs within protozoan organisms (Fidalgo & Gille 2011). Indeed, several studies have shown an association between trypanocidal action of some compounds and disorders in mitochondrial membrane potential, in Leishmania (Antinarelli et al. 2016, Britta et al. 2014, Corral et al. 2016, Fonseca-Silva et al. 2015, Kathuria et al. 2014), Trypanosoma cruzi (Martins et al. 2016, Menna-Barreto et al. 2009, Volpato et al. 2015), and T. brucei (Alkhaldi et al. 2016).

Thus, to verify if the reduction detected in the growth rate was related to the effect of EECb on mitochondrial metabolism, the mitochondrial membrane potential (ΔѰm) was assessed using JC-1. This dye is lipophilic and concentrates in mitochondria in proportion to the membrane potential; increased JC-1 aggregate (red fluorescent) accumulation is observed inside mitochondria with greater ΔΨm, whereas JC-1 monomers (green fluorescent) accumulate in the cytoplasm when ΔΨm decreases. The histograms of the fluorescence (Figure 5) demonstrated that the treatment with 70.0 and 140.0 μg/mL of EECb decreased the parasite mitochondrial membrane potential by 30.0% and 43.0%, respectively. In this case, the level of mitochondrial membrane depolarization of L. amazonensis promastigotes treated with 140 μg/mL of EECb did not differ from that observed following treatment with the standard drug Carbonylcyanide-4(trifluoromethoxy)phenylhydrazone (FCCP) (20µM).

Figure 5
The effect of EECb on mitochondrial membrane potential (ΔΨm) in Leishmania amazonensis. L. amazonensis promastigotes were cultivated in Schneider’s insect medium in the absence or presence of 70.0 µg/mL or 140.0µg/mL EECb for 24 h. Positive control was treated with FCCP (20 µM) for 20 min, and in the negative control (absence of EECb), the same volume of vehicle (DMSO) was added to the growth medium. Dose-dependent alterations in relative ΔΨm values are expressed as the ratio of the JC-1 fluorescence measurements at 590 nm (for J-aggregate) versus 530 nm (for J-monomer). Data are expressed as the means ± standard errors of three different experiments. Significant differences, relative to the control group, are indicated by * (p<0.05), ** (p<0.01), and *** (p<0.001).

These results suggest that the leishmanicidal activity of EECb is related to a marked depolarization of the mitochondrial membrane of parasites. Similar results were obtained by Da Silva et al. (2015) concernig the antipromastigote activity of phytol rich hexane fraction (PRF) from leaves of Lacistema pubescens on L. amazonensis. Sifaoui et al. (Sifaoui et al. 2014) demonstrated that treatment with maslinic acid extracted from Limouni olive leaf decreased the L. amazonensis and L. infantum mitochondrial membrane potential by 22.0% and 9.7%, respectively. Also, 81.7% reduction in ΔѰm of L. amazonensis promastigotes was induced by Neem (Azadirachta indica) leaf extract (Dayakar et al. 2015). Further, mitochondrial dysfunction associated with ultrastructural changes on the mitochondrial matrix, with the appearance of complex structures and swelling of this organelle, was detected in L. amazonensis (Rosa et al. 2003) and L. chagasi promastigotes (Rodrigues et al. 2013) treated with linalool-rich essential oil from Croton cajucara. The fact that previous studies have shown that secondary products from plants from a range of genera, including Croton, can induce mitochondrial damage, supports our finding that the antiproliferative effect of EECb on Leishmania promastigotes is due to the generation of a defect in mitochondrial metabolism.

To better understand the effects of EECb on parasite viability, the membrane integrity was evaluated with PI staining. PI is a DNA and RNA intercalating dye that diffuses selectively through plasma membranes whose integrity has been disrupted (Britta et al. 2014). No changes in the PI fluorescence intensity were detected in parasites exposed to 70.0 and 140.0 μg/mL EECb after 24 hours (data not shown).

Taken together, the results showed that EECb presents activity against the promastigote and intracellular amastigote forms of L. amazonensis and L. infantum, with no effect upon mammalian cells. Moreover, mitochondrial dysfunction with no change in plasma membrane permeability appear to be the main effects involved in the delay in the growth rate caused by EECb on L. amazonensis promastigotes.

ACKNOWLEGMENTS

The authors thank Conselho Nacional de Desenvolvimento Científico e Tecnológico/Programa Estratégico de Apoio à Pesquisa em Saúde – Brazil (CNPq/PAPES), Fundação de Apoio à Pesquisa e à Inovação Tecnológica do Estado de Sergipe – Brazil (Fapitec/SE), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Grant number 2342/2012 PROEF), Financiadora de Estudos e Projetos – Brazil (FINEP), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Instituto Oswaldo Cruz/Fundação Oswaldo Cruz (IOC/Fiocruz) that financed this study.

REFERENCES

• ADADE CM & SOUTO-PADRÓN T. 2010. Contributions of ultrastructural studies to the cell biology of Ttypanosmatids: targets for anti-parasitic drugs. Open Parasitol J 4: 178-187.
• ALKHALDI AA, MARTINEK J, PANICUCCI B, DARDONVILLE C, ZIKOVA A & DE KONING HP. 2016. Trypanocidal action of bisphosphonium salts through a mitochondrial target in bloodstream form Trypanosoma brucei. Int J Parasitol-Drug 6: 23-34.
• ALVAR J, VELEZ ID, BERN C, HERRERO M, DESJEUX P, CANO J, JANNIN J, DEN BOER M & TEAM WHOLC. 2012. Leishmaniasis worldwide and global estimates of its incidence. PloS ONE 7: e35671.
• ANGÉLICO EC, RODRIGUES EC, DA COSTA JGM, LUCENA MFA, QUEIROGA NETO V & DE MEDEIROS SR. 2014. Chemical characterization and antimicrobial activity of essential oils and Croton’s varieties modulator in the Brazilian’s Northeast semiarid. Afr J Plant Sci 87: 392-397.
• ANTINARELLI LM, SOUZA IO, GLANZMANN N, ALMEIDA AD, PORCINO GN, VASCONCELOS EG, DA SILVA AD & COIMBRA ES. 2016. Aminoquinoline compounds: Effect of 7-chloro-4-quinolinylhydrazone derivatives against Leishmania amazonensis. Exp Parasitol 171: 10-16.
• BRITTA EA, SCARIOT DB, FALZIROLLI H, UEDA-NAKAMURA T, SILVA CC, FILHO BP, BORSALI R & NAKAMURA CV. 2014. Cell death and ultrastructural alterations in Leishmania amazonensis caused by new compound 4-Nitrobenzaldehyde thiosemicarbazone derived from S-limonene. BMC Microbiol 14.
• CAMARGOS HS, R. MOREIRA A, MENDANHA SA, FERNANDES KS, DORTA ML & ALONSO A. 2014. Terpenes increase the lipid dynamics in the Leishmania plasma membrane at concentrations similar to their IC50 values. PLoS ONE 9: e104429.
• CAMPOS MC, SALOMAO K, CASTRO-PINTO DB, LEON LL, BARBOSA HS, MACIEL MA & CASTRO SL. 2010. Croton cajucara crude extract and isolated terpenes: activity on Trypanosoma cruzi. Parasitol Res 107: 1193-1204.
• COCK IE, SELESHO MI & VAN VUUREN SF. 2018. A review of the traditional use of southern African medicinal plants for the treatment of selected parasite infections affecting humans. J Ethnopharmacol 220: 250-264.
• CORRAL MJ, BENITO-PENA E, JIMENEZ-ANTON MD, CUEVAS L, MORENO-BONDI MC & ALUNDA JM. 2016. Allicin induces calcium and mitochondrial dysregulation causing necrotic death in Leishmania. PLoS Neglect Trop D 10: e0004525.
• CROFT SL & COOMBS GH. 2003. Leishmaniasis-current chemotherapy and recent advances in the search for novel drugs. Trends Parasitol 19: 502-508.
• CROFT SL & OLLIARO P. 2011. Leishmaniasis chemotherapy--challenges and opportunities. Clin Microbiol Infec 17: 1478-1483.
• DA SILVA BJM, HAGE AAP, SILVA EO & RODRIGUES APD. 2018. Medicinal plants from the Brazilian Amazonian region and their antileishmanial activity: a review. J Integr Med 16: 211-222.
• DA SILVA JM, ANTINARELLI LM, RIBEIRO A, COIMBRA ES & SCIO E. 2015a. The effect of the phytol-rich fraction from Lacistema pubescens against Leishmania amazonensis is mediated by mitochondrial dysfunction. Exp Parasitol 159: 143-150.
• DA SILVA RR, DA SILVA BJ, RODRIGUES AP, FARIAS LH, DA SILVA MN, ALVES DT, BASTOS GN, DO NASCIMENTO JL & SILVA EO. 2015b. In vitro biological action of aqueous extract from roots of Physalis angulata against Leishmania (Leishmania) amazonensis. BMC Complem Altern M 15: 249.
• DAYAKAR A, CHANDRASEKARAN S, VERONICA J, SUNDAR S & MAURYA R. 2015. In vitro and in vivo evaluation of anti-leishmanial and immunomodulatory activity of Neem leaf extract in Leishmania donovani infection. Exp Parasitol 153: 45-54.
• DE ALMEIDA BC ET AL. 2016. Antiprotozoal activity of extracts and isolated triterpenoids of ‘carnauba’ (Copernicia prunifera) wax from Brazil. Pharm Biol 54: 3280-3284.
• DE MUYLDER G, ANG KK, CHEN S, ARKIN MR, ENGEL JC & MCKERROW JH. 2011. A screen against Leishmania intracellular amastigotes: comparison to a promastigote screen and identification of a host cell-specific hit. PLoS Neglect Trop D 5: e1253.
• DEY S ET AL. 2015. Protective effect of Croton caudatus Geisel leaf extract against experimental visceral leishmaniasis induces proinflammatory cytokines in vitro and in vivo. Exp Parasitol 151-152: 84-95.
• DORLO TP, BALASEGARAM M, BEIJNEN JH & DE VRIES PJ. 2012. Miltefosine: a review of its pharmacology and therapeutic efficacy in the treatment of leishmaniasis. J Antimicrob Chemother 67: 2576-2597.
• FIDALGO LM & GILLE L. 2011. Mitochondria and trypanosomatids: targets and drugs. Pharm Res 28: 2758-2770.
• FONSECA-SILVA F, CANTO-CAVALHEIRO MM, MENNA-BARRETO RF & ALMEIDA-AMARAL EE. 2015. Effect of apigenin on Leishmania amazonensis is associated with reactive oxygen species production followed by mitochondrial dysfunction. J Nat Prod 78: 880-884.
• FONSECA-SILVA F, INACIO JD, CANTO-CAVALHEIRO MM & ALMEIDA-AMARAL EE. 2011. Reactive oxygen species production and mitochondrial dysfunction contribute to quercetin induced death in Leishmania amazonensis. PloS ONE 6: e14666.
• FONSECA-SILVA F, INACIO JD, CANTO-CAVALHEIRO MM & ALMEIDA-AMARAL EE. 2013. Reactive oxygen species production by quercetin causes the death of Leishmania amazonensis intracellular amastigotes. J Nat Prod 76: 1505-1508.
• FONSECA-SILVA F, INACIO JD, CANTO-CAVALHEIRO MM, MENNA-BARRETO RF & ALMEIDA-AMARAL EE. 2016. Oral efficacy of apigenin against cutaneous leishmaniasis: involvement of reactive oxygen species and autophagy as a mechanism of action. PLoS Neglect Trop D 10: e0004442.
• GARCIA AR, AMARAL ACF, AZEVEDO MMB, CORTE-REAL S, LOPES RC, ALVIANO CS, PINHEIRO AS, VERMELHO AB & RODRIGUES IA. 2017. Cytotoxicity and anti-Leishmania amazonensis activity of Citrus sinensis leaf extracts. Pharm Biol 55: 1780-1786.
• GUIMARAES LR, RODRIGUES AP, MARINHO PS, MULLER AH, GUILHON GM, SANTOS LS, DO NASCIMENTO JL & SILVA EO. 2010. Activity of the julocrotine, a glutarimide alkaloid from Croton pullei var. glabrior, on Leishmania (L.) amazonensis. Parasitol Res 107: 1075-1081.
• INACIO JD, GERVAZONI L, CANTO-CAVALHEIRO MM & ALMEIDA-AMARAL EE. 2014. The effect of (-)-epigallocatechin 3-O--gallate in vitro and in vivo in Leishmania braziliensis: involvement of reactive oxygen species as a mechanism of action. PLoS Neglect Trop D 8: e3093.
• JOSHI DC & BAKOWSKA JC. 2011. Determination of mitochondrial membrane potential and reactive oxygen species in live rat cortical neurons. JoVE-J Vis Exp: e2704.
• KATHURIA M, BHATTACHARJEE A, SASHIDHARA KV, SINGH SP & MITRA K. 2014. Induction of mitochondrial dysfunction and oxidative stress in Leishmania donovani by orally active clerodane diterpene. Antimicrob Agents Ch 58: 5916-5928.
• KONG L, LI X, ZOU H, WANG H, MAO X, ZHANG Q & NI J. 2001. Analysis of terpene compounds in Cimicifuga foetida L. by reversed-phase high-performance liquid chromatography with evaporative light scattering detection. J Chromatogr A 936: 111-118.
• LIMA GS, CASTRO-PINTO DB, MACHADO GC, MACIEL MA & ECHEVARRIA A. 2015. Antileishmanial activity and trypanothione reductase effects of terpenes from the Amazonian species Croton cajucara Benth (Euphorbiaceae). Phytomedicine 22: 1133-1137.
• LIRA R, SUNDAR S, MAKHARIA A, KENNEY R, GAM A, SARAIVA E & SACKS D. 1999. Evidence that the high incidence of treatment failures in Indian kala-azar is due to the emergence of antimony-resistant strains of Leishmania donovani. J Infect Dis 180: 564-567.
• MACHADO M, DINIS AM, SANTOS-ROSA M, ALVES V, SALGUEIRO L, CAVALEIRO C & SOUSA MC. 2014. Activity of Thymus capitellatus volatile extract, 1,8-cineole and borneol against Leishmania species. Vet Parasitol 200: 39-49.
• MARTINS SC, LAZARIN-BIDOIA D, DESOTI VC, FALZIROLLI H, DA SILVA CC, UEDA-NAKAMURA T, SILVA SO & NAKAMURA CV. 2016. 1,3,4-Thiadiazole derivatives of R-(+)-limonene benzaldehyde-thiosemicarbazones cause death in Trypanosoma cruzi through oxidative stress. Microbes Infect 18: 787-797.
• MELO GFA, COSTA ACV, GARINO JUNIOR F, MEDEIROS RS, MADRUGA MS & QUEIROGA NETO V. 2013. The sensitivity of bacterial foodborne pathogens to Croton blanchetianus Baill essential oil. Braz J Microbiol 44: 1189-1194.
• MENDANHA SA & ALONSO A. 2015. Effects of terpenes on fluidity and lipid extraction in phospholipid membranes. Biophys Chem 198: 45-54.
• MENNA-BARRETO RF, GONCALVES RL, COSTA EM, SILVA RS, PINTO AV, OLIVEIRA MF & DE CASTRO SL. 2009. The effects on Trypanosoma cruzi of novel synthetic naphthoquinones are mediated by mitochondrial dysfunction. Free Radical Bio Med 47: 644-653.
• MITTAL MK, RAI S, ASHUTOSH, RAVINDER, GUPTA S, SUNDAR S & GOYAL N. 2007. Characterization of natural antimony resistance in Leishmania donovani isolates. Am J Trop Med Hyg 76: 681-688.
• MURRAY HW, BERMAN JD, DAVIES CR & SARAVIA NG. 2005. Advances in leishmaniasis. Lancet 366: 1561-1577.
• OLIVEIRA VC, MOURA DM, LOPES JA, DE ANDRADE PP, DA SILVA NH & FIGUEIREDO RC. 2009. Effects of essential oils from Cymbopogon citratus (DC) Stapf., Lippia sidoides Cham., and Ocimum gratissimum L. on growth and ultrastructure of Leishmania chagasi promastigotes. Parasitol Res 104: 1053-1059.
• PACE D. 2014. Leishmaniasis. J Infect 69 Suppl 1: S10-18.
• RODRIGUES IA, AZEVEDO MM, CHAVES FC, BIZZO HR, CORTE-REAL S, ALVIANO DS, ALVIANO CS, ROSA MS & VERMELHO AB. 2013. In vitro cytocidal effects of the essential oil from Croton cajucara (red sacaca) and its major constituent 7- hydroxycalamenene against Leishmania chagasi. BMC Complem Altern M 13: 249.
• ROSA MDSS, MENDONCA-FILHO RR, BIZZO HR, DE ALMEIDA RODRIGUES I, SOARES RM, SOUTO-PADRON T, ALVIANO CS & LOPES AH. 2003. Antileishmanial activity of a linalool-rich essential oil from Croton cajucara. Antimicrob Agents Ch 47: 1895-1901.
• SALATINO A, SALATINO MLF & NEGRI G. 2007. Traditional uses, chemistry and pharmacology of Croton species (Euphorbiaceae). J Braz Chem Soc 18: 11-33.
• SANTIN MR, DOS SANTOS AO, NAKAMURA CV, DIAS FILHO BP, FERREIRA IC & UEDA-NAKAMURA T. 2009. In vitro activity of the essential oil of Cymbopogon citratus and its major component (citral) on Leishmania amazonensis. Parasitol Res 105: 1489-1496.
• SCHER R, GARCIA JB, PASCOALINO B, SCHENKMAN S & CRUZ AK. 2012. Characterization of anti-silencing factor 1 in Leishmania major. Mem I Oswaldo Cruz 107: 377-386.
• SEN R & CHATTERJEE M. 2011. Plant derived therapeutics for the treatment of Leishmaniasis. Phytomedicine 18: 1056-1569.
• SHAHA C. 2006. Apoptosis in Leishmania species & its relevance to disease pathogenesis. Indian J Med Res 123: 233-244.
• SIFAOUI I ET AL. 2014. In vitro effects of triterpenic acids from olive leaf extracts on the mitochondrial membrane potential of promastigote stage of Leishmania spp. Phytomedicine 21: 1689-1694.
• SILVA F DOS S, ALBUQUERQUE UP, COSTA JUNIOR LM, LIMA ADA S, DO NASCIMENTO AL & MONTEIRO JM. 2014. An ethnopharmacological assessment of the use of plants against parasitic diseases in humans and animals. J Ethnopharmacol 155: 1332-1341.
• SINGH B & SUNDAR S. 2012. Leishmaniasis: vaccine candidates and perspectives. Vaccine 30: 3834-3842.
• SOTO J ET AL. 2007. Treatment of Bolivian mucosal leishmaniasis with miltefosine. Clin Infect Dis 44: 350-356.
• SOTO J, REA J, BALDERRAMA M, TOLEDO J, SOTO P, VALDA L & BERMAN JD. 2008. Efficacy of miltefosine for Bolivian cutaneous leishmaniasis. Am J Trop Med Hyg 78: 210-211.
• SUNDAR S, JHA TK, THAKUR CP, ENGEL J, SINDERMANN H, FISCHER C, JUNGE K, BRYCESON A & BERMAN J. 2002. Oral miltefosine for Indian visceral leishmaniasis. N Engl J Med 347: 1739-1746.
• TARIKU Y, HYMETE A, HAILU A & ROHLOFF J. 2010. Constituents, antileishmanial activity and toxicity profile of volatile oil from berries of Croton macrostachyus. Nat Prod Commun 5: 975-980.
• TELES CB, MOREIRA-DILL LS, SILVA AA, FACUNDO VA, AZEVEDOJ WF, SILVA LH, MOTTA MC, STABELI RG & SILVA-JARDIM I. 2015. A lupane-triterpene isolated from Combretum leprosum Mart. fruit extracts that interferes with the intracellular development of Leishmania (L.) amazonensis in vitro. BMC Complement Altern Med 15: 165.
• VOLPATO H, DESOTI VC, VALDEZ RH, UEDA-NAKAMURA T, SILVA SDE O, SARRAGIOTTO MH & NAKAMURA CV. 2015. Mitochondrial dysfunction induced by n-butyl-1-(4-dimethylamino)phenyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxamide is required for cell death of Trypanosoma cruzi. PloS ONE 10: e0130652.
• WENIGER B, ROBLEDO S, ARANGO GJ, DEHARO E, ARAGON R, MUNOZ V, CALLAPA J, LOBSTEIN A & ANTON R. 2001. Antiprotozoal activities of Colombian plants. J Ethnopharmacol 78: 193-200.
• WHO. 2016. Leishmaniasis in high-burden countries: an epidemiological update based on data reported in 2014. Weekly epidemiological record, Genève: World Health Organization: 285-296.

Publication Dates

• Publication in this collection
  28 Oct 2020
• Date of issue
  2020

History

• Received
  14 Sept 2018
• Accepted
  06 Jan 2019