Monoterpenic aldehydes as potential anti-Leishmania agents: Activity of Cymbopogon citratus and citral on L. infantum, L. tropica and L. major

Experimental Parasitology 130 (2012) 223–231

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Monoterpenic aldehydes as potential anti-Leishmania agents: Activity of Cymbopogon citratus and citral on L. infantum, L. tropica and L. major M. Machado a,b, P. Pires a,b, A.M. Dinis c, M. Santos-Rosa d, V. Alves d, L. Salgueiro a, C. Cavaleiro a, M.C. Sousa a,⇑ a

Faculdade de Farmácia/CEF, Universidade de Coimbra, Azinhaga de Santa Comba, 3030-548 Coimbra, Portugal Departamento Farmácia, Escola Superior de Saúde do Vale do Ave/Centro de Investigação em Tecnologias da Saúde IPSN-CESPU, 4760 Vila Nova de Famalicão, Portugal Laboratório de Microscopia Electrónica, Departamento das Ciências da Vida, Faculdade de Ciências e Tecnologia da Universidade de Coimbra, Portugal d Instituto de Imunologia da Faculdade de Medicina da Universidade de Coimbra, Portugal b c

a r t i c l e

i n f o

Article history: Received 1 August 2011 Accepted 20 December 2011 Available online 2 January 2012 Keywords: Leishmania infantum Leishmania tropica Leishmania major Essential oil Antiprotozoals In vitro activity Drug action Infectious diseases (ID) Ultrastructure Flow cytometry Drug development Medicinal plants

a b s t r a c t In order to contribute for the search of new drugs for leishmaniasis, we study the susceptibility of Leishmania infantum, Leishmania tropica and Leishmania major to Cymbopogon citratus essential oil and major compounds, mrycene and citral. C. citratus and citral were the most active inhibiting L. infantum, L. tropica and L. major growth at IC50 concentrations ranging from 25 to 52 lg/ml and from 34 to 42 lg/ml, respectively. L. infantum promastigotes exposed to essential oil and citral underwent considerable ultrastructural alterations, namely mitochondrial and kinetoplast swelling, autophagosomal structures, disruption of nuclear membrane and nuclear chromatin condensation. C. citratus essential oil and citral promoted the leishmanicidal effect by triggering a programmed cell death. In fact, the leishmanicidal activity was mediated via apoptosis as evidenced by externalization of phosphatidylserine, loss of mitochondrial membrane potential, and cell-cycle arrest at the G(0)/G(1) phase. Taken together, ours findings lead us to propose that citral was responsible for anti-Leishmania activity of the C. citratus and both may represent a valuable source for therapeutic control of leishmaniasis. Ó 2011 Elsevier Inc. All rights reserved.

1. Introduction Leishmania, a unicellular trypanosomatid protozoan parasite, is the causative organism of leishmaniasis, which comprises a wide disease spectrum ranging from localized, self-healing, cutaneous lesions to disfiguring mucocutaneous leishmaniasis and the visceral form, which can be fatal if neglected (Murray et al., 2005; WHO, 2009; Postigo, 2010). In the past decade, unresponsiveness to antimonials, the first line of treatment, has increased substantially in visceral leishmaniasis, mainly at endemic areas like India (Croft et al., 2006; Natera et al., 2007). Amphotericin B, pentamidine and miltefosine have been used as alternative drugs. Current treatments are limited, have the potential to develop resistance,

⇑ Corresponding author. Address: Faculdade de Farmácia da Universidade de Coimbra, Pólo das Ciências da Saúde, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal. E-mail address: [email protected] (M.C. Sousa). 0014-4894/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2011.12.012

are expensive, are long length and possess unacceptable toxicity (Leandro and Campino, 2003). In the ongoing search for better leishmanicidal compounds, plant-derived products are gaining ground (Anthony et al., 2005; Sen et al., 2010). Essential oils, plant extracts prepared by distillation, are composed by a huge diversity of small hydrophobic molecules, most of them accomplishing theoretical criteria’s of druglikeness prediction (Lipinski et al., 1997). Such molecules easily diffuse across cell membranes and consequently gain advantage in what concerns to interactions with intracellular targets, being a valuable research option for the search of anti-Leishmania leads and drugs (Edris, 2007). C. citratus (DC) Stapf, Family Poaceae, is a widely used herb in tropical countries namely on Southeast Asia, African and South America countries and is also known as a source of ethnomedicines. C. citratus is commonly used in folk medicine in Angola for the treatment of gastrointestinal disturbances, and as an antispasmodic, anti-inflammatory, anti-pyretic, and diuretic. Some studies have demonstrated its antimicrobial activity, namely antibacterial, antifungal, and antiprotozoa properties (Santoro et al., 2007a,b;

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Mayaud et al., 2008; Irkin and Korukluoglu, 2009; Oliveira et al., 2009). However, there are few reports on the activity of essential oils on endemic Leishmania species responsible for cutaneous and visceral leishmaniasis of the old world. So, the present work we focused on the leishmanicidal activity of C. citratus and major compounds, myrcene and citral, on three old world Leishmania species, namelyLeishmania infantum, Leishmania tropica and Leishmania major. Additionally, we undertake other essays to demonstrate the safety of the essential oil and compounds and elucidate the mechanisms that contribute to leishmanicidal activity. 2. Material and methods 2.1. Plant material 2.1.1. Origin Plant material from C. citratus was obtained from a local market in Luanda, Angola. The plants were identified by a taxonomist (Dr. Jorge Paiva, University of Coimbra), and voucher specimens (Cabo S. Vicente COI00033066; Arrifana COI00033067) were deposited at the Herbarium of the Department of Botany of the University of Coimbra (COI). 2.1.2. Essential oil The essential oil from the aerial parts C. citratus (DC) Stapf was isolated by water distillation for 3 h from air dried material, using a Clevenger-type apparatus, following the procedure described in the European Pharmacopoeia (Council of Europe, 1997). 2.1.3. Essentials oils analysis Analysis was carried out by gas chromatography (GC) and by gas chromatography-mass spectroscopy (GC/MS). Analytical GC was carried out in a Hewlett–Packard 6890 (Agilent Technologies, Palo Alto, CA, USA) gas chromatograph with a HP GC ChemStation Rev. A.05.04 data handling system, equipped with a single injector and two flame ionization detection (FID) systems. A graphpak divider (Agilent Technologies, part No. 5021-7148) was used for simultaneous sampling to two Supelco (Supelco, Bellefonte, PA, USA) fused silica capillary columns with different stationary phases: SPB-1 and SupelcoWax-10. GC–MS was carried out in a Hewlett– Packard 6890 gas chromatograph fitted with a HP1 fused silica column, interfaced with an Hewlett–Packard mass selective detector 5973 (Agilent Technologies) operated by HP Enhanced ChemStation software, version A.03.00. Components of each essential oil were identified by their retention indices on both SPB-1 and SupelcoWax-10 columns and from their mass spectra. Retention indices, calculated by linear interpolation relative to retention times of C8–C23 of n-alkanes, were compared with those of authentic samples included in our own laboratory database. Acquired mass spectra were compared with reference spectra from our own database; Wiley/NIST database (Wiley, 2007) and literature data (Joulain and Konig, 1998; Adams, 2004). Relative amounts of individual components were calculated based on GC peak areas without FID response factor correction. 2.2. Parasites and cultures Promastigote forms of L. infantum Nicolle (zymodeme MON-1), L. tropica (ATCC 50129) and L. major BCN were maintained at 26 °C by weekly transfers in HEPES (25 mM)-buffered RPMI 1640 medium enriched with 10% inactivated fetal bovine serum (FBS). These cells were used to study the effects of essential oils on Leishmania promastigotes growth.

2.3. Viability assays Essential oil and major compounds (citral and myrcene) were initially diluted in dimethyl sulfoxide (DMSO; Sigma Chemical) at 100 mg mL1 and then in culture medium in order to get a range of concentrations from 10 to 400 lg mL1. Log phase promastigotes of L. infantum, L. tropica and L. major (106 cells/ml) were incubated in HEPES (25 mM)-buffered RPMI 1640 medium enriched with 10% inactivated FBS in the presence of different concentrations of essential oil and compounds or DMSO (vehicle control) at 26 °C. Effects on viability were estimated by tetrazolium-dye (MTT) colorimetric method (Monzote et al., 2007).The concentration that inhibited viability by 50% (IC50) was determined after 24 h for L. infantum and L. tropica and after 48 h for L. major, through dose– response regression analysis, plotted by GraphPad Prism 5.

2.4. Transmission and scanning electron microscopy L. infantum promastigotes were exposed to essential oil and citral at concentrations that inhibit viability by 50% (IC50) and the morphological alterations were investigated by electronic microscopy. For ultrastructural studies with transmission electronic microscopy, the samples were treated as reported previously (Sousa et al., 2001). Briefly, cell were fixed with glutaraldehyde in sodium cacodylate buffer, post fixed in osmium tetroxide and uranyl acetate, dehydrated in ethanol and in propylene oxide and embedded in Epon 812 (TAAB 812 resin). Ultrathin sections were stained with lead citrate and uranyl acetate. For Scanning electronic microscopy, the samples were fixed and postfixed as described for transmission, dehydrated in ethanol, critical point dried using CO2 and sputter-coat with gold. The specimens were examined in JEOL JEM-100 SX transmission electron microscopy (TEM) at 80 kV and in JEOL JSM-5400 scanning electron microscope (SEM) at 15 kV.

2.5. Flow cytometry 2.5.1. Cell cycle analysis For flow cytometry analysis of DNA content, exponentially grown L. infantum promastigote cells (106) were treated with C. citratus essential oil and citral at IC50 concentrations for 3h, 5h, 7h, and 24 h at 26 °C. Promastigote suspension was then fixed in 200 ll of 70% ethanol for 30 min. at 4 °C. Next, cells were washed in PBS, and resuspended in 500 ll of PI solution (PI/Rnase, Immunostep) for 15 min. at room temperature (Darzynkiewicz et al., 2001). Cells were then analyzed by flow cytometry (Facs Calibur– Beckton–Dickinson). Results were treated using ModFit LT V 2.0 programme.

2.5.2. Analysis of phosphatidylserine externalization Double staining for annexin V-FITC and propidium iodide (PI) was performed as described previously (Vermes et al., 1995). Briefly, L. infantum promastigotes (106 cells) were exposed to essential oil and citral at IC50 concentrations for 3 h, 5 h, 7 h, and 24 h at 26 °C. Cells were then washed with PBS and ressuspended in binding buffer (10 mM HEPES–NaOH, pH 7.4, 140 NaC1, 2.5 mM CaCI2). To 100 ll of this suspension were added 5 ll of Annexin V FITC and 5 ll of PI (AnnexinV-FITC Apoptosis detection Kit, Immmunostep). After 15 min incubation in the dark at room temperature, it was added 400 ll binding buffer and cells were then analyzed by flow cytometry (Facs Calibur–Beckton–Dickinson). Data analysis was carried out using the program Paint-a-gate, and values are expressed as a percentage of positive cells for a given marker, relatively to the number of cells analyzed.

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2.5.3. Measurement of mitochondrial membrane potential To assess the mitochondrial membrane potential (Dwm), a cellpermeable cationic and lipophilic dye, JC-1 (5,50 ,6,60 -tetrachloro1,10 ,3,30 -tetraethylbenzimidazolcarbocyanine iodide), was used as previously described (Cossarizza et al., 1993). This probe aggregates within mitochondria and fluoresces red (590 nm) at higher Dwm. However, at lower Dwm, JC-1 cannot accumulate within the mitochondria and instead remains in the cytosol as monomers, which fluoresce green (490 nm). Therefore, the ratio of red to green fluorescence gives a measure of the transmembrane electrochemical gradient. L. infantum promastigotes (106 cells) promastigotes were exposed to essential oil and citral at IC50 concentrations for 3 h, 5 h, 7 h, and 24 h at 26 °C. Promastigotes were then incubated JC-1 (5 lg/ml) (Molecular Probes, Invitrogen) in the dark for 15 min at room temperature. Then, cells were washed in PBS, suspended in 400 ll of PBS and analyzed by flow cytometry. Data analysis was carried out using the program Paint-a-gate. 2.6. Mammalian cell cytotoxicity assay For cytotoxicity assays, log phase of macrophages (ATCC, RAW 264.7 cell line) and bovine aortic endothelial cells were trypsinized and incubated at 37 °C in 24-well tissue culture plates in RPMI 1640 medium (macrophages) and DMEM medium (endothelial cells) supplemented with 10% FBS under microaerophilic condition. When the monolayers reached confluence, the medium was removed and the cells were incubated with fresh medium plus essential oil and citral at IC50 concentrations for 24 h. The cells viability was evaluated by MTT test and by morphological observation by optical microscopy. 2.7. Statistical analysis All experiments were performed in triplicate and in three independent assays (n = 6). Values were expressed as mean ± SEM and the means were statistically compared using student t and ANOVA test, with a Dunnett’s post-test. The significance level was ⁄ p < 0.05, ⁄⁄p < 0.01 and ⁄⁄⁄p < 0.001.

Table 1 Global composition of Cymbopogon citratus essential oil. RIa

RIb

Compound

%

959

1338

0.6

980 1020 1025 1035 1073 1082 1085 1115 1124 1129 1150 1197 1207 1214 1233 1242 1272 1335 1357 1410 1427 1474 Monoterpene hydrocarbons Oxygen containing monoterpenes Sesquiterpene hydrocarbons Other compounds Total identified

1161 1206 1235 1250 n.d. 1543 1295 n.d. n.d. 1480 n.d. n.d. n.d. 1679 1842 1730 1594 1660 1756 1590 1580 1801

6-Methyl-5-hepten-2one b-Myrcene [1] Limonene Z-b-Ocimene E-b-Ocimene Z-Epoxyocimene Linalool Perillene Photocitral B Photocitral A Citronellal Rosefurane epoxide 2,3-Epoxyneral 2,3-Epoxygeranial Neral [2] Geraniol Geranial [3] 2-Undecanone Citronellyl acetate Geranyl acetate E-b-Caryophyllene E-a-Bergamotene 2-Tridecanone

11.5 t 0.4 0.3 0.2 0.8 0.1 0.1 0.2 0.1 0.1 0.1 0.1 32.5 1.3 45.7 0.1 t 0.8 0.1 t t 12.3 82.0 0.1 0.7 95.1

CHO CHO

[1]

[2]

[3]

Compounds listed in order to their elution on the SPB-1 column; t: traces (<0.05%); n.d.: not determined. a Retention indices on the SPB-1 column relative to C8–C22 n-alkanes. b Retention indices on the SupelcoWax-10 column relative to C8–C22 n-alkanes.

3. Results 3.1. Essential oils analysis The compounds identified in the essential oil, their retention time, and their relative proportions are listed in Table 1. The main components from C. citratus were geranial (48.4%), neral (32.6%) and myrcene (6.4%). 3.2. Anti-Leishmania activity The effects of C. citratus essential oil and major compounds on the viability of L. infantum, L. tropica and L. major were studied (Figs. 1 and 2). Essential oil at 50 lg/ml was able to kill 65% of the L. infantum and L. major promastigotes and 80% of L. tropica promastigotas (Fig. 1), and citral, at the same concentration, killed about 45% of L. infantum and L. tropica promastigotes, and about 60% of L. major promastigotas (Fig. 2). All three strains of Leishmania were susceptible to C. citratus essential oil (Tabela 2), showing a marked effect on L. infantum (IC50/ 24 h = 25 lg/ml), L. tropica (IC50/ 24 h = 52 lg/ml) and L. major (IC50/ 48 h = 38 lg/ml) promastigotes viability. The citral was also very effective against Leishmania promastigotas, presenting IC50 values of 42 lg/ml for L. infantum, 34 lg/ml for L. tropica and 36 lg/ml for L. major (Table 2). Myrcene revealed to be the less active component of the essential oil with an IC50 value of 164 lg/ml (Table 2).

Fig. 1. Effect of Cymbopogon citratus essential oil on Leishmania promastigotes viability. Cultures of log-phase promastigotes (106) were incubated at 26 °C for 24 h (L. infantum, L. tropica) or 48 h (L. major), as function of essential oil concentration. Values are expressed as means and SEM.

3.3. Ultrastructural effects In order to investigate the ultrastructural effects, L. infantum promastigotes were incubated for 7 h in the presence or absence

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vesicles could be seen within the flagellar pocket of many treated cells (Fig. 4H). Although the degree of cell damage varied, citral where slightest drastic, the observed ultrastructural effects found for C. citratus treatment were similar for citral.

3.4. Cell-cycle arrest at the G(0)/G(1) phase The cell cycle analysis were performed by flow cytometry after PI staining of the parasites incubated with essential and citral for 24 h at IC50 concentrations (Table 3). Fig. 5 shows the distribution of cell DNA trough cell cycle of parasites in the absence and presence of the essential oil and the compound. After 24 h of incubation, the majority of treated parasite cells were arrested on G0/ G1 phase of cell cycle (essential oil, 84%; citral, 92.3%), opposite to what occurs in not treated cells (35.9%).

3.5. Phosphatidylserine externalization Fig. 2. Effect of citral on Leishmania promastigotes viability. Cultures of log-phase promastigotes (106) were incubated at 26 °C for 24 h (L. infantum, L. tropica) or 48 h (L. major), as function of citral concentration. Values are expressed as means and SEM.

Table 2 Inhibitory concentration (IC50) of Cymbopogon citratus essential oil and major constituents on Leishmania strains. IC50 (lg mL1)

Cymbopogon citratus Citral (neral 40% + geranial 60%) Myrcene ⁄

L. infantum

L. tropica

L. major

25 (20–31) 42 (31–58) 164 (158–170)

52 (49–55) 34 (25–48) n.d.

38 (31–44) 36 (27–47) n.d.

95% Confidence intervals.

of the C. citratus essential oil and citral, and then observed by scanning (Fig. 3) and transmission (Fig. 4) electron microscopy. Untreated promastigotes (control) observed by scanning electron microscopy presented a typical elongated body shape and anterior flagella (Fig. 3A). Essential oil and citral treated promastigotas (Fig. 3B–F and G–J, respectively) shown round (Fig. 3C, D, E, I, and J) and aberrant forms (Fig. 3B, G and H) with multi-septation of the cell body (Fig. 3E). Note the irregular surface with blebs formation, of all treated parasites, and membrane disruption with lost of intracellular content (Fig. 3D, F, G, H and J). Control parasites, observed by transmission electron microscopy, presented normal nucleus, kinetoplast, mitochondria and flagellar pocket (Fig. 4A and B). The most prominent ultrastructural effect observed in promastigotes treated with C. citratus and citral, were the appearance of aberrant-shaped cells (Fig. 4E and G) with cytoplasmatic disorganization (Fig. 4C, D, E, G and I), besides an increase in cytoplasmatic clearing (Fig. 4C, E,G and I). There was an increase in the number of autophagosomal structures, characterized by intense cytoplasmic vacuolization (Fig. 4C and H). Treated parasites also presented swelling of cell body (Fig. 4E, H and I), mitochondria (Fig. 4C, E and G–I) and kinetoplast (Fig. 4E). The swelling of the unique and highly branched mitochondria, resulted in a inner mitochondrial membrane disorganization, displaying several and complex invaginations and forming concentric membranous structures (Fig. 4E, H and I). It was also noted an increase on acidocalcisomes (Fig. 4F, G and H) and the presence of myelin-like figures as multilamelar bodies (Fig. 4E and H). Other alteration frequently observed was on the nuclear chromatin organization, resembling the nucleus of apoptotic cells (Fig. 4C, D and H), and disruption of nuclear membrane (Fig. 4F and I). Large amounts of membrane

During early apoptosis, the plasma membrane loses asymmetry causing PS to be translocated from the cytoplasmic face of the plasma membrane to the external face which can be detected using Annexin V. To distinguish apoptotic cell death from necrotic cell death, cells were counterstained with PI, a non-permeable stain with an affinity for nucleic acids, as it selectively enters necrotic cells. Therefore, co-staining of annexin V and PI can differentiate between cells undergoing early apoptosis (annexin V-positive, PI-negative), necrosis (PI-positive, annexin V-negative) and live cells (PI- and annexin V negative). In untreated promastigotes, the degree of binding of annexin V at 3 h, 5 h, 7 h and 24 h was 1.3%, 1.6%, 1.3% and 3.3%, respectively. Following treatment of promastigotes with C. citratus essential oil at its IC50 values, the percentage of annexin V-positive cells slightly increased to 2.2% at 3 h, 2.9% at 5 h, 1.7% at 7 h and 4.1% at 24 h. With citral, positive cells to annexin V increased with time, 2.3% at 3 h, 3.2% at 5–7 h and 21.6% at 24 h. The percentage of PI-stained cells ranged from 0.4% to 2.5% on the presence of C. citratus and from 0.3% to 6.7% with citral (Table 4).

3.6. Depolarization of mitochondrial membrane potential Maintenance of the mitochondrial transmembrane potential is essential for parasite survival, as Leishmania has a single mitochondrion. The 585/530 nm ratio, i.e. J-aggregates, within the mitochondria vs monomers in the cytosol represents the Dwm. In untreated cells, the red/green fluorescence ratio was 1.56; 2.2 and 2.68 at 3 h, 5 h, 7 h and 24 h, respectively. However, the addition of C. citratus essential oil caused a loss of mitochondrial membrane potential, blocking JC-1 entry to the mitochondria, leaving the JC-1 monomers to fluoresce green within the cytoplasm. This was reflected in the red/green fluorescence ratio, which decreased to 1.1; 1.6; 1.68 and 2.38 following drug treatment for 3, 5, 7 and 24 h, respectively. Moreover, treatment with citral revealed to have a pronounced effect on mitochondrial membrane potential with a red/green fluorescence ratio of 1.2 at 3 h; 1.56 at 5 h; 1.47 at 7 h and 1.87 at 24 h (Table 5). C. citratus essential oil and citral induced an immediate decrease on Dwm. Data indicate that essential oil up to the 3rd hour caused a higher number of cells with low Dwm (34.2%), which was followed by a sustained decrease in the Dwm thereafter (Fig. 6). Therefore, a more prolonged incubation time (24 h) showed that C. citratus (18.8%) and citral (27.6%) maintain the profile characterized by a higher number of cells with low Dwm, compared to control (3.6%), being more pronounced for citral (Table 5).

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Fig. 3. Scanning electron micrographs of Leishmania infantum promastigotes exposed to Cymbopogon citratus essential oil and citral. Untreated cells showing the typical elongated shape (A), parasite body (B), anterior flagella (F); B–F, treated promastigotas with essential oil; G–J, treated promastigotas with citral. Note round forms (C, D, E, I and J) and aberrant forms (B, G and H) with multi-septation of the cell body (E). Note the irregular surface (asterisks) and membrane disruption (arrows). A–H, J Bars = 5 lm; I, bar = 1 lm.

3.7. Mammalian cell cytotoxicity assay The cytotoxicity of C. citratus essential oil and citral were evaluated in cultures of bovine aortic endothelial cells (primary culture) and macrophages cell line using the MTT test. The results showed that they did not induced toxicity against these mammalian cells (not shown).

4. Discussion Despite of the last years advances, in the knowledge of the biology of several forms of leishmaniasis, pentavalent antimonials remain the first-line treatment for this infection in most endemic areas despite the limitations imposed by the need of parenteral administration, high toxicity and increasing drug resistance, as for many other drugs used (Leandro and Campino, 2003; Croft et al., 2006; Natera et al., 2007). In recent years, several screenings of medicinal plants used for the treatment of leishmaniasis have been carried (Anthony et al., 2005; Sen et al., 2010) namely by our group (Machado et al., 2010), confirming the importance of many plant species and essential oils as potential sources for the isolation of novel compounds with leishmanicidal effect. So, in the present study, we evaluated the activity of C. citratus essencial oil and their major compounds on three Leishmania species. C. citratus oil contains two isomeric aldehydes, neral (cis-citral) and geranial (trans-citral) which together represents around 81% of the oil, and myrcene that appear with 6.4%. It has been pointed out that the anti-Leishmania activity of C. citratus essential oil was mainly due to citral.

A common feature of plant volatiles is their hydrophobic nature, and several studies addressing the mode of action of such compounds usually point at cell membranes as the primary target (Bakkali et al., 2008). In our study we have observed an increase in cell and organelle volume and cytoplasm vacuolization in treated cells. In this sense, the essential oil and citral may have a passive entry and may accumulate in parasite cell membranes leading to an increase of membrane permeability, as observed by propidium iodide assay. Similar ultrastructural alterations were also observed in Trypanosoma cruzi and Leishmania amazonensis treated with other essential oils and/or their main constituents (Pedroso et al., 2006; Santoro et al., 2007a,b). The presence of autophagosomal structures was very prominent. Other authors also observed this alteration in T. cruzi and in L. amazonensis treated with drugs like ketoconazole, terbinafine, among others (Lazardi et al., 1990; Lorente et al.,2004). Another important alteration is the presence of membrane elaborated structures in the cytoplasm, appearing as myelin-like figures. The presence of these structures suggests the occurrence of an autophagic process, with the formation of structures known as autophagosomes (Rodrigues et al., 2002). These structures are probably involved in the breakdown and recycling of abnormal membrane structures, suggesting an intense process of remodeling of intracellular organelles irreversibly damaged by the essential oil and citral. The presence of membrane vesicles in the flagellar pocket of citral treated parasites is characteristic of an exocytose process, and we cannot exclude the possibility that they result from the secretion into this region of abnormal lipids, which accumulate as a consequence of citral effect.

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Fig. 4. Transmission electron micrographs of Leishmania infantum promastigotas exposed to Cymbopogon citratus essential oil and citral. A–B, Control parasites; C–I, parasites treated with essential oil and citral, showing promastigotas with different degrees of damage. Note the disruption of nuclear membranes (arrowheads in panels F and I), mitochondrial swelling (MS) (C, E, G–I), gross alterations in the organization of cytoplasm (D, E and G), nuclear chromatins (D) and kinetoplast swelling (KS) (E). N, nucleus; K, kinetoplast, F, flagellum, FP, flagelar pocket, MB, multilamelar bodies. Bars, 2 lm.

Table 3 Effect of Cymbopogon citratus essential oil and citral on cellular cycle of Leishmania infantum promastigotes by flow cytometry analysis. Leishmania intracellular entities (% of cells) Phase G0/G1

C. citratus Citral Control

Phase S

Phase G2/M

3h

5h

7h

24 h

3h

5h

7h

24 h

3h

5h

7h

24 h

88.6 96.4 88.7

91 90.3 90.4

77.6 85.1 73.2

84 92.3 35.9

3.8 5.5 3.8

6 3.9 4.8

22.4 11.5 26.8

11.3 6.7 44.5

2.7 5.9 7.6

2.9 5.7 4.9

0 3.9 0

4.7 0.9 19.3

The presence of acidocalcisome in several microorganisms and their apparent absence in mammalian cells make them promising targets for chemotherapy (Docampo and Moreno, 2008). They are

characterized by their acidic content, high electron density, and high concentration of polyphosphates, calcium, magnesium, and other elements. Previous studies have demonstrated that

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Fig. 5. Cell cycle histograms of Leishmania infantum promastigotes. L. infantum promastigotas were incubated at 26 °C for 24 h in the absence (A) or presence of C. citratus essential oil (B) and citral (C) at IC50 concentrations. Propidium iodide staining was performed and samples were analyzed by flow cytometry.

Table 4 Flow cytometry analysis of Leishmania infantum promastigotes treated with Cymbopogon citratus essential oil and citral showing the percentage of propidium iodide (PI) and annexin-V positive cells. Leishmania intracellular entities (% of cells) Anexine

C. citratus Citral Control

PI

Anexine/PI

3h

5h

7h

24 h

3h

5h

7h

24 h

3h

5h

7h

24 h

2.2 2.3 1.3

2.9 3.2 1.6

1.7 3.2 1.3

4.1 21.6 3.3

0.4 0.3 0.4

0.7 0.3 0.2

0.8 0.6 0.3

2.5 6.7 1.1

0.5 1.9 0.5

2.2 1.9 0.3

1.7 0.7 0.3

2.5 6.5 1.6

Table 5 Flow cytometry analysis of Leishmania infantum promastigotes treated with Cymbopogon citratus essential oil and citral showing the percentage of JC-1 positive cells. Leishmania intracellular entities (% of cells) JC1Mon

C. citratus Citral Control

JC1Agreg

MIFAgreg / MIFMon membrane Pot

3h

5h

7h

24 h

3h

5h

7h

24 h

3h

5h

7h

24 h

34.2 20.9 13.3

12 14.8 10.3

10.8 14.3 9.5

18.8 27.6 3.6

65 77.9 82.6

87.2 83.5 89

88.1 84.5 88.8

80.4 71.5 95.8

1.1 1.2 1.56

1.6 1.56 2

1.68 1.47 2

2.38 1.87 2.68

Fig. 6. Representative dot plots showing JC-1 staining of Leishmania infantum promastigotes. L. infantum promastigotas were incubated at 26 °C for 24 h in the absence (A) or presence of C. citratus essential oil (B) and citral (C) at IC50 concentrations. JC-1 staining was performed and samples were analyzed by flow cytometry. J-aggregates (blue) reflect functioning mitochondria and monomers are indicative of compromised mitochondria (pink). (For interpretation of the references in color in this figure legend, the reader is referred to the web version of this article.)

terpenoids constituents from some essential oils have toxic effects on many biological systems by interacting at the cellular level with cytosolic Ca2+, through an intracellular calcium store release and calcium channel blockage (Interaminense et al., 2007). Since terpenoids are found in the essential oil tested, the increase in number and volume of acidocalcisomes in treated cells could be due to direct or indirect action of citral on membrane ion flow in these organelles. Another change observed, especially in citral-treated parasites, takes place in the mitochondria. This modification started at the

inner mitochondrial membrane, which folded toward the mitochondrial matrix, forming complex and elaborate structures. Subsequently, the mitochondrial matrix became less electrondense, and a typical swelling of the mitochondrion was observed. Those alterations have been also seeing in previous studies on L. brasiliensis (Brenzan et al., 2007) and L. amazonensis (Rosa et al., 2003; Ueda-Nakamura et al., 2006). Along with above findings, C. citratus oil and citral promoted a sustained depolarization of mitochondrial membrane. This is a characteristic feature of metazoan apoptosis and has been

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observed to play a key role in drug-induced death in protists such as Leishmania (Sen et al., 2004). Moreover, we also find condensation and fragmentation of DNA on treated cells. These nuclear features, characteristic of apoptosis, are being related with alterations in the mitochondrial membrane potential (Green and Kroemer, 2004). Data indicate that C. citratus oil and citral exerts its antileishmanial activity primarily via apoptosis and secondary to necrosis. Apoptosis is a programmed cell death process, which is characterized by a series of events involving morphological and biochemical changes. In parasites, apoptosis appears to be the predominant form of cell death, as has been observed in kinetoplastids (Arnoult et al., 2002) in response to chemotherapeutic agents such as amphotericin B and plant extracts such as Aloe vera leaf exudates (Dutta et al., 2007a,b). Taken together, results demonstrated that essential oil and citral induced Leishmania promastigotes death sharing several phenotypic features observed with metazoan apoptosis, which included phosphatidylserine exposure, depolarization of mitochondrial potential, arrested G0/G1 cell cycle phase and nuclear disorganization, with chromatin condensation. C. citratus essential oil and citral besides being active against promastigotes are expected to exhibit a stronger activity on amastigotes forms as generally occurs among natural extracts and synthetic drugs (Dutta et al., 2007a,b; Santin et al., 2009; Nakayama et al., 2007; Lakshmi et al., 2007). Furthermore, the ability of essential oil compounds to easily diffuse cell membranes and interact with intracellular targets will be extremely important to induce the inhibitory activity on intracellular parasites. The main reasons for which a number of plant metabolites with leishmanicidal activity have not made into clinical evaluation is their high toxicity on mammalian cells. This lack of selectivity is not observed on C. citratus oil and citral, once they did not show toxicity against mammalian cells tested. In conclusion, C. citratus essential oil and citral may represent valuable sources for lead or active molecules against Leishmania infections.

Source of funding This work was supported by ‘‘Programa Operacional Ciência e Inovacão 2010 (POCI)/FEDER’’ da Fundação para a Ciência e Tecnologia (FCT). Acknowledgments Authors are grateful to Prof. Jorge Paiva for help in plant taxonomic, to José Correia da Costa from Centro de Imunologia e Biologia Parasitária, Instituto Nacional. Dr. Ricardo Jorge, Porto for supplying the Leishmania infantum Nicolle (zymodeme MON-1), to António Osuna, Departamento de Parasitología, Facultad de Ciencias, Instituto de Biotecnología, Universidad de Granada, for suplly Leishmania major BCN. References Adams, R.P., 2004. Identification of Essential Oil Components by Gas Chromatography Quadrupole Mass Spectroscopy. Allured Publishing, Carol Stream, Illinois. Anthony, J.P., Fyfe, L., Smith, H., 2005. Plant active components, a resource for antiparasitic agents? Trends Parasitol. 21, 462–468. Arnoult, D., Akarid, K., Grodet, A., Petit, P.X., Estaquier, J., Ameisen, J.C., 2002. On the evolution of programmed cell death: apoptosis of the unicellular eukaryote Leishmania major involves cysteine proteinase activation and mitochondrion permeabilization. Cell Death Differ. 9, 65–81. Bakkali, F., Averbeck, S., Averbeck, D., Idaomar, M., 2008. Biological effects of essential oils—a review. Food Chem. Toxicol. 46, 446–475.

Brenzan, M.A., Nakamura, C.V., Prado Dias Filho, B., Ueda-Nakamura, T., Young, M.C., Aparício Garcia Cortez, D., 2007. Antileishmanial activity of crude extract and coumarin from Calophyllum brasiliense leaves against Leishmania amazonensis. Parasitol. Res. 101, 715–722. Cossarizza, A., Baccaranicontri, M., Kalashnikova, G., Franceschi, C.A., 1993. New method for the cytofluorometric analysis of mitochondrial membrane potential using the J-aggregate forming lipophilic cation 5,50 ,6,60 -tetrachloro-1,10 ,3,30 tetraethylbenzimidazolcarbocyanine iodide (JC-1). Biochem. Biophys. Res. Commun. 197, 40–45. Council of Europe, 1997. Methods of pharmacognosy. In: European Pharmacopoeia. European Department for the Quality of Medicines, Strasbourg. Croft, S.L., Sundar, S., Fairlambm, A.H., 2006. Drug resistance in leishmaniasis. Clin. Microbiol. Rev. 19, 111–126. Darzynkiewicz, Z., Juan, G., Bedner, E., 2001. Determining cell cycle stages by flow cytometry. Curr. Protoc. Cell Biol. (New York). Docampo, R., Moreno, S.N., 2008. The acidocalcisome as a target for chemotherapeutic agents in protozoan parasites. Curr. Pharm. Des. 14, 882– 888. Dutta, A., Mandal, G., Mandal, C., Chatterjee, M., 2007a. In vitro antileishmanial activity of Aloe vera leaf exudate: a potential herbal therapy in leishmaniasis. Glycoconjugate J. 24, 81–86. Dutta, A., Mandal, G., Mandal, C., Chatterjee, M., 2007b. Racemoside A, an antileishmanial, water-soluble, natural steroidal saponin, induces programmed cell death in Leishmania donovani. J. Med. Microbiol. 56, 1196–1204. Edris, A.E., 2007. Pharmaceutical and therapeutic potentials of essential oils and their individual volatile constituents: a review. Phytother. Res. 21, 308–323. Green, D.R., Kroemer, G., 2004. The pathophysiology of mitochondrial cell death. Science 305, 626–629. Interaminense, L.F., Jucá, D.M., Magalhães, P.J., Leal-Cardoso, J.H., Duarte, G.P., Lahlou, S., 2007. Pharmacological evidence of calcium channel blockade by essential oil of Ocimum gratissimum and its main constituent, eugenol, in isolated aortic rings from DOCA salt hypertensive rats. Fundam. Clin. Pharmacol. 21, 497–506. Irkin, R., Korukluoglu, M., 2009. Effectiveness of Cymbopogon citratus L. essential oil to inhibit the growth of some filamentous fungi and yeasts. J. Med. Food 12, 193–197. Joulain, D., Konig, W.A., 1998. The Atlas of Spectral Data of Sesquiterpene Hydrocarbons. EB–Verlag, Hamburg. Lakshmi, V., Pandey, K., Kapil, A., Singh, N., Samant, M., Dube, A., 2007. In vitro and in vivo leishmanicidal activity of Dysoxylum binectariferum and its fractions against Leishmania donovani. Phytomedicine 14, 36–42. Lazardi, K., Urbina, J.A., de Souza, W., 1990. Ultrastructural alterations induced by two ergosterol biosynthesis inhibitors, ketoconazole and terbinafine, on epimastigotes and amastigotes of Trypanosoma (Schizotrypanum) cruzi. Antimicrob. Agents Chemother. 34, 2097–2105. Leandro, C., Campino, L., 2003. Leishmaniasis: efflux pumps and chemoresistance. Int. J. Antimicrob. Agents 22, 352–357. Lipinski, C.A., Lombardo, F., Dominy, B.W., Feeney, P.J., 1997. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv. Drug Deliv. Rev. 23, 3–25. Lorente, S.O., Rodrigues, J.C., Jiménez, C., Joyce-Menekse, M., Rodrigues, C., Croft, S.L., 2004. Novel azasterols as potential agents for treatment of leishmaniasis and trypanosomiasis. Antimicrob. Agents Chemother. 48, 2937–2950. Machado, M., Santoro, G., Sousa, M.C., Salgueiro, L., Cavaleiro, C., 2010. Activity of essential oils on the growth of Leishmania infantum. Flavour Fragr. J. 25, 156–160. Mayaud, L., Carricajo, A., Zhiri, A., Aubert, G., 2008. Comparison of bacteriostatic and bactericidal activity of 13 essential oils against strains with varying sensitivity to antibiotics. Lett. Appl. Microbiol. 47, 167–173. Monzote, L., Montalvo, A.M., Scull, R., Miranda, M., Abreu, J., 2007. Combined effect of the essential oil from Chenopodium ambrosioides and antileishmanial drugs on Promastigotes of Leishmania amazonensis. Rev. Inst. Med. Trop. (Sao Paulo) 49, 257–260. Murray, H.W., Berman, J., Davies, C., Saravia, N., 2005. Advances in leishmaniasis. Lancet 366, 1561–1577. Nakayama, H., Desrivot, J., Bories, C., Franck, X., Figadère, B., Hocquemiller, R., 2007. In vitro and in vivo antileishmanial efficacy of a new nitrilquinoline against Leishmania donovani. Biomed. Pharmacother. 61, 186–188. Natera, S., Machuca, C., Padrón-Nieves, M., Romero, A., Díaz, E., Ponte-Sucre, A., 2007. Leishmania spp.: proficiency of drug-resistant parasites. Int. J. Antimicrob. Agents 29, 637–642. Oliveira, V.C., Moura, D.M., Lopes, J.A., de Andrade, P.P., da Silva, N.H., Figueiredo, R.C., 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. Pedroso, R.B., Ueda-Nakamura, T., Dias Filho, B.P., Cortez, D.A.G., Cortez, L.E.R., Morgado-Díaz, J.A., 2006. Biological activities of essential oil obtained from Cymbopogon citratus on Crithidia deanei. Acta Protozool. 45, 231–240. Postigo, J.A., 2010. Leishmaniasis in the World Health Organization Eastern Mediterranean Region. Int. J. Antimicrob. Agents 36, S62–S65. Rodrigues, J.C., Attias, M., Rodriguez, C., Urbina, J.A., Souza, W., 2002. Ultrastructural and biochemical alterations induced by 22,26-azasterol, a delta(24(25))-sterol methyltransferase inhibitor, on promastigote and amastigote forms of Leishmania amazonensis. Antimicrob. Agents Chemother. 46, 487–499. Rosa, M.D.S., Mendonça-Filho, R.R., Bizzo, H.R., Rodrigues, I.A.R., Soares, R.M., SoutoPadron, T., 2003. Antileishmanial activity of a linalool-rich essential oil from Croton cajucara. Antimicrob. Agents Chemother. 47, 1895–1901.

M. Machado et al. / Experimental Parasitology 130 (2012) 223–231 Santin, M.R., Dos Santos, A.O., Nakamura, C.V., Dias Filho, B.P., Ferreira, I.C., UedaNakamura, 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. Santoro, G.F., Cardoso, M.G., Guimarães, L.G., Freire, J.M., Soares, M.J., 2007a. Antiproliferative effect of the essential oil of Cymbopogon citratus (DC) Stapf (lemongrass) on intracellular amastigotes, bloodstream trypomastigotes and culture epimastigotes of Trypanosoma cruzi (Protozoa: Kinetoplastida). Parasitology 134, 1649–1656. Santoro, G.F., Cardoso, M.G., Guimarães, L.G., Mendonça, L.Z., Soares, M.J., 2007b. Trypanosoma cruzi: activity of essential oils from Achillea millefolium L., Syzygium aromaticum L. and Ocimum basilicum L. on epimastigotes and trypomastigotes. Exp. Parasitol. 116, 283–290. Sen, N., Bandyopadhyay, S., Dutta, A., Mandal, G., Ganguly, S., Saha, P., 2004. Camptothecin induced mitochondrial dysfunction leading to programmed cell death in unicellular hemoflagellate Leishmania donovani. Cell Death Differ. 11, 924–936.

231

Sen, R., Ganguly, S., Saha, P., Chatterjee, M., 2010. Efficacy of artemisinin in experimental visceral leishmaniasis. Int. J. Antimicrob. Agents 36, 43–49. Sousa, M.C., Goncalves, C.A., Bairos, V.A., Poiares-da-Silva, J., 2001. Adherence of Giardia lamblia trophozoites to Int-407 human intestinal cells. Clin. Diagn. Lab. Immunol. 8, 258–265. Ueda-Nakamura, T., Mendonca-Filho, R.R., Morgado-Diaz, J.Á., Korehisa Maza, P., Prado Dias Filho, B., Garcia-Cortez, A.D., 2006. Antileishmanial activity of Eugenol-rich essential oil from Ocimum gratissimum. Parasitol. Int. 55, 99–105. Vermes, I., Haanen, C., Steffens-Nakken, H., Reutelingsperger, C.A., 1995. Novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J. Immunol. Methods 184, 39–51. WHO (World Health Organization), 2009. Leishmaniasis: Background Information. A Brief History of the Disease. World Health Organization. www.who.int/ leishmaniasis/en/. Wiley, 2007. Registry, eighth ed. with NIST 05 MS Spectra, Revision 2005 D.06.00. s.l. Agilent Technologies.