Aldheides monoterpenes as potential anti-Leishmania … c... · 2 Aldheides monoterpenes as...

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Experimental Parasitology xxx (2012) xxx–xxx

YEXPR 6347 No. of Pages 9, Model 5G

5 January 2012

Contents lists available at SciVerse ScienceDirect

Experimental Parasitology

journal homepage: www.elsevier .com/locate /yexpr

Aldheides monoterpenes as potential anti-Leishmania agents: Activity ofCymbopongon 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, Portugalb Departamento Farmácia, Escola Superior de Saúde do Vale do Ave/Centro de Investigação em Tecnologias da Saúde IPSN-CESPU, 4760 Vila Nova de Famalicão, Portugalc Laboratório de Microscopia Electrónica, Departamento das Ciências da Vida, Faculdade de Ciências e Tecnologia da Universidade de Coimbra, Portugald Instituto de Imunologia da Faculdade de Medicina da Universidade de Coimbra, Portugal

a r t i c l e i n f o a b s t r a c t

33343536373839404142434445

Article history:Received 1 August 2011Accepted 20 December 2011Available online xxxx

Keywords:Leishmania infantumLeishmania tropicaLeishmania majorEssential oilAntiprotozoalsIn vitro activityDrug actionInfectious diseases (ID)UltrastructureFlow cytometryDrug developmentMedicinal plants

0014-4894/$ - see front matter � 2011 Elsevier Inc. Adoi:10.1016/j.exppara.2011.12.012

⇑ Corresponding author. Address: Faculdade de FCoimbra, Pólo das Ciências da Saúde, Azinhaga de SantPortugal.

E-mail address: [email protected] (M.C. Sousa).

Please cite this article in press as: Machado, M.,citral on L. infantum, L. tropica and L. major. Exp

In order to contribute for the search of new drugs for leishmaniasis, we study the susceptibility of Leish-mania infantum, Leishmania tropica and Leishmania major to Cymbopogon citratus essential oil and majorcompounds, mrycene and citral. C. citratus and citral were the most active inhibiting L. infantum, L. tropicaand L. major growth at IC50 concentrations ranging from 25 to 52 lg/ml and from 34 to 42 lg/ml, respec-tively. L. infantum promastigotes exposed to essential oil and citral underwent considerable ultrastruc-tural alterations, namely mitochondrial and kinetoplast swelling, autophagosomal structures,disruption of nuclear membrane and nuclear chromatin condensation. C. citratus essential oil and citralpromoted the leishmanicidal effect by triggering a programmed cell death. In fact, the leishmanicidalactivity was mediated via apoptosis as evidenced by externalization of phosphatidylserine, loss of mito-chondrial membrane potential, and cell-cycle arrest at the G(0)/G(1) phase. Taken together, ours findingslead us to propose that citral was responsible for anti-Leishmania activity of the C. citratus and both mayrepresent a valuable source for therapeutic control of leishmaniasis.

� 2011 Elsevier Inc. All rights reserved.

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1. Introduction

Leishmania, a unicellular trypanosomatid protozoan parasite, isthe causative organism of leishmaniasis, which comprises a widedisease spectrum ranging from localized, self-healing, cutaneouslesions to disfiguring mucocutaneous leishmaniasis and the vis-ceral form, which can be fatal if neglected (Murray et al., 2005;WHO, 2009; Postigo, 2010). In the past decade, unresponsivenessto antimonials, the first line of treatment, has increased substan-tially in visceral leishmaniasis, mainly at endemic areas like India(Croft et al., 2006; Natera et al., 2007). Amphotericin B, pentami-dine and miltefosine have been used as alternative drugs. Currenttreatments are limited, have the potential to develop resistance,

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ll rights reserved.

armácia da Universidade dea Comba, 3000-548 Coimbra,

et al. Aldheides monoterpenes. Parasitol. (2012), doi:10.1016

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 distilla-tion, are composed by a huge diversity of small hydrophobic mol-ecules, most of them accomplishing theoretical criteria’s ofdruglikeness prediction (Lipinski et al., 1997). Such molecules eas-ily diffuse across cell membranes and consequently gain advantagein what concerns to interactions with intracellular targets, being avaluable research option for the search of anti-Leishmania leadsand drugs (Edris, 2007).

C. citratus (DC) Stapf, Family Poaceae, is a widely used herb intropical countries namely on Southeast Asia, African and SouthAmerica countries and is also known as a source of ethnomedi-cines. C. citratus is commonly used in folk medicine in Angola forthe treatment of gastrointestinal disturbances, and as an antispas-modic, anti-inflammatory, anti-pyretic, and diuretic. Some studieshave demonstrated its antimicrobial activity, namely antibacterial,antifungal, and antiprotozoa properties (Santoro et al., 2007a,b;

as potential anti-Leishmania agents: Activity of Cymbopongon citratus and/j.exppara.2011.12.012

http://dx.doi.org/10.1016/j.exppara.2011.12.012

mailto:[email protected]


http://www.sciencedirect.com/science/journal/00144894

http://www.elsevier.com/locate/yexpr


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2 M. Machado et al. / Experimental Parasitology xxx (2012) xxx–xxx


5 January 2012

Mayaud et al., 2008; Irkin and Korukluoglu, 2009; Oliveira et al.,2009).

However, there are few reports on the activity of essential oilson endemic Leishmania species responsible for cutaneous and vis-ceral leishmaniasis of the old world. So, the present work we fo-cused on the leishmanicidal activity of C. citratus and majorcompounds, myrcene and citral, on three old world Leishmania spe-cies, namelyLeishmania infantum, Leishmania tropica and Leishmaniamajor. Additionally, we undertake other essays to demonstrate thesafety of the essential oil and compounds and elucidate the mech-anisms that contribute to leishmanicidal activity.

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2. Material and methods

2.1. Plant material

2.1.1. OriginPlant 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 (CaboS. Vicente COI00033066; Arrifana COI00033067) were depositedat the Herbarium of the Department of Botany of the Universityof Coimbra (COI).

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2.1.2. Essential oilThe essential oil from the aerial parts C. citratus (DC) Stapf was

isolated by water distillation for 3 h from air dried material, using aClevenger-type apparatus, following the procedure described inthe European Pharmacopoeia (Council of Europe, 1997).

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2.1.3. Essentials oils analysisAnalysis was carried out by gas chromatography (GC) and by

gas chromatography-mass spectroscopy (GC/MS). Analytical GCwas carried out in a Hewlett–Packard 6890 (Agilent Technologies,Palo Alto, CA, USA) gas chromatograph with a HP GC ChemStationRev. A.05.04 data handling system, equipped with a single injectorand two flame ionization detection (FID) systems. A graphpak divi-der (Agilent Technologies, part No. 5021-7148) was used for simul-taneous 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 silicacolumn, interfaced with an Hewlett–Packard mass selective detec-tor 5973 (Agilent Technologies) operated by HP Enhanced Chem-Station software, version A.03.00. Components of each essentialoil were identified by their retention indices on both SPB-1 andSupelcoWax-10 columns and from their mass spectra. Retentionindices, calculated by linear interpolation relative to retentiontimes of C8–C23 of n-alkanes, were compared with those of authen-tic samples included in our own laboratory database. Acquiredmass spectra were compared with reference spectra from ourown database; Wiley/NIST database (Wiley, 2007) and literaturedata (Joulain and Konig, 1998; Adams, 2004). Relative amounts ofindividual components were calculated based on GC peak areaswithout FID response factor correction.

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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 �Cby weekly transfers in HEPES (25 mM)-buffered RPMI 1640 med-ium enriched with 10% inactivated fetal bovine serum (FBS). Thesecells were used to study the effects of essential oils on Leishmaniapromastigotes growth.

Please cite this article in press as: Machado, M., et al. Aldheides monoterpenescitral on L. infantum, L. tropica and L. major. Exp. Parasitol. (2012), doi:10.1016

2.3. Viability assays

Essential oil and major compounds (citral and myrcene) wereinitially diluted in dimethyl sulfoxide (DMSO; Sigma Chemical) at100 mgmL�1 and then in culture medium in order to get a rangeof concentrations from 10 to 400 lgmL�1. Log phase promastigotesof L. infantum, L. tropica and L. major (106 cells/ml) were incubatedin HEPES (25 mM)-buffered RPMI 1640 medium enriched with 10%inactivated FBS in the presence of different concentrations ofessential oil and compounds or DMSO (vehicle control) at 26 �C. Ef-fects on viability were estimated by tetrazolium-dye (MTT) color-imetric method (Monzote et al., 2007).The concentration thatinhibited 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 cit-ral at concentrations that inhibit viability by 50% (IC50) and themorphological alterations were investigated by electronic micros-copy. For ultrastructural studies with transmission electronicmicroscopy, the samples were treated as reported previously (Sou-sa et al., 2001). Briefly, cell were fixed with glutaraldehyde in so-dium cacodylate buffer, post fixed in osmium tetroxide anduranyl acetate, dehydrated in ethanol and in propylene oxide andembedded in Epon 812 (TAAB 812 resin). Ultrathin sections werestained with lead citrate and uranyl acetate. For Scanning elec-tronic microscopy, the samples were fixed and postfixed as de-scribed for transmission, dehydrated in ethanol, critical pointdried using CO2 and sputter-coat with gold. The specimens wereexamined 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 analysisFor flow cytometry analysis of DNA content, exponentially

grown L. infantum promastigote cells (106) were treated with C. cit-ratus essential oil and citral at IC50 concentrations for 3, 5, 7, and24 h at 26 �C. Promastigote suspension was then fixed in 200 llof 70% ethanol for 30 min. at 4 �C. Next, cells were washed inPBS, and resuspended in 500 ll of PI solution (PI/Rnase, Immuno-step) 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 2.0programme.

2.5.2. Analysis of phosphatidylserine externalizationDouble 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 toessential oil and citral at IC50 concentrations for 3 h, 5 h, 7 h, and24 h at 26 �C. Cells were then washed with PBS and ressuspendedin binding buffer (10 mM HEPES–NaOH, pH 7.4, 140 NaC1, 2.5 mMCaCI2). To 100 ll of this suspension were added 5 ll of Annexin VFITC and 5 ll of PI (AnnexinV-FITC Apoptosis detection Kit, Imm-munostep). After 15 min incubation in the dark at room tempera-ture, it was added 400 ll binding buffer and cells were thenanalyzed 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 agiven marker, relatively to the number of cells analyzed.



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Table 1Global composition of Cymbopogon citratus essential oil.

RIa RIb Compound %

959 1338 6-Methyl-5-hepten-2-one

0.6

980 1161 b-Myrcene [1] 11.51020 1206 Limonene t1025 1235 Z-b-Ocimene 0.41035 1250 E-b-Ocimene 0.31073 n.d. Z-Epoxyocimene 0.21082 1543 Linalool 0.81085 1295 Perillene 0.11115 n.d. Photocitral B 0.11124 n.d. Photocitral A 0.21129 1480 Citronellal 0.11150 n.d. Rosefurane epoxide 0.11197 n.d. 2.3-Epoxyneral 0.11207 n.d. 2.3-Epoxygeranial 0.11214 1679 Neral [2] 32.51233 1842 Geraniol 1.31242 1730 Geranial [3] 45.71272 1594 2-Undecanone 0.11335 1660 Citronellyl acetate t1357 1756 Geranyl acetate 0.81410 1590 E-b-Caryophyllene 0.11427 1580 E-a-Bergamotene t1474 1801 2-Tridecanone tMonoterpene hydrocarbons 12.3Oxygen containing monoterpenes 82.0Sesquiterpene hydrocarbons 0.1Other compounds 0.7Total identified 95.1

[1]

CHO

[2]

CHO

[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.

M. Machado et al. / Experimental Parasitology xxx (2012) xxx–xxx 3


5 January 2012

2.5.3. Measurement of mitochondrial membrane potentialTo assess the mitochondrial membrane potential (Dwm), a cell-

permeable cationic and lipophilic dye, JC-1 (5,50,6,60-tetrachloro-1,10,3,30-tetraethylbenzimidazolcarbocyanine iodide), was used aspreviously described (Cossarizza et al., 1993). This probe aggre-gates within mitochondria and fluoresces red (590 nm) at higherDwm. However, at lower Dwm, JC-1 cannot accumulate withinthe mitochondria and instead remains in the cytosol as monomers,which fluoresce green (490 nm). Therefore, the ratio of red to greenfluorescence gives a measure of the transmembrane electrochem-ical gradient. L. infantum promastigotes (106 cells) promastigoteswere exposed to essential oil and citral at IC50 concentrations for3 h, 5 h, 7 h, and 24 h at 26 �C. Promastigotes were then incubatedJC-1 (5 lg/ml) (Molecular Probes, Invitrogen) in the dark for15 min at room temperature. Then, cells were washed in PBS, sus-pended in 400 ll of PBS and analyzed by flow cytometry. Dataanalysis was carried out using the program Paint-a-gate.

2.6. Mammalian cell cytotoxicity assay

For cytotoxicity assays, log phase of macrophages (ATCC, RAW264.7 cell line) and bovine aortic endothelial cells were trypsinizedand incubated at 37 �C in 24-well tissue culture plates in RPMI1640 medium (macrophages) and DMEM medium (endothelialcells) supplemented with 10% FBS under microaerophilic condi-tion. When the monolayers reached confluence, the medium wasremoved and the cells were incubated with fresh medium plusessential oil and citral at IC50 concentrations for 14 h. The cells via-bility was evaluated by MTT test and by morphological observationby optical microscopy.

2.7. Statistical analysis

All experiments were performed in triplicate and in three inde-pendent assays (n = 6). Values were expressed as mean ± SEM andthe means were statistically compared using student t and ANOVAtest, with a Dunnett’s post-test. The significance level was⁄p < 0.05, ⁄⁄p < 0.01 and ⁄⁄⁄p < 0.001.

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Fig. 1. Effect of Cymbopogon citratus essential oil on Leishmania promastigotesviability. 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. Results

3.1. Essential oils analysis

The compounds identified in the essential oil, their retentiontime, and their relative proportions are listed in Table 1. The maincomponents 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 onthe 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% ofthe L. infantum and L. major promastigotes and 80% of L. tropicapromastigotas (Fig. 1), and citral, at the same concentration, killedabout 45% of L. infantum and L. tropica promastigotes, and about60% of L. major promastigotas (Fig. 2). All three strains of Leish-mania were susceptible to C. citratus essential oil (Tabela 2), show-ing 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) prom-astigotes viability. The citral was also very effective against Leish-mania promastigotas, presenting IC50 values of 42 lg/ml for L.infantum, 34 lg/ml for L. tropica and 36 lg/ml for L. major (Table2). Myrcene revealed to be the less active component of the essen-tial oil with an IC50 value of 164 lg/ml (Table 2).


3.3. Ultrastructural effects

In order to investigate the ultrastructural effects, L. infantumpromastigotes were incubated for 7 h in the presence or absence



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Fig. 2. Effect of citral on Leishmania promastigotes viability. Cultures of log-phasepromastigotes (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 andSEM.

Table 2Inhibitory concentration (IC50) of Cymbopogon citratus essential oil and majorconstituents on Leishmania strains.

IC50 (lgmL�1)

L. infantum L. tropica L. major

Cymbopogon citratus 25 (20–31) 52 (49–55) 38 (31–44)Citral (neral 40% + geranial 60%) 42 (31–58) 34 (25–48) 36 (27–47)Myrcene 164 (158–170) n.d. n.d.

⁄95% Confidence intervals.



5 January 2012

of the C. citratus essential oil and citral, and then observed by scan-ning (Fig. 3) and transmission (Fig. 4) electron microscopy. Un-treated promastigotes (control) observed by scanning electronmicroscopy presented a typical elongated body shape and anteriorflagella (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-septationof the cell body (Fig. 3E). Note the irregular surface with blebs for-mation, of all treated parasites, and membrane disruption with lostof intracellular content (Fig. 3D, F, G, H and J). Control parasites, ob-served by transmission electron microscopy, presented normalnucleus, kinetoplast, mitochondria and flagellar pocket (Fig. 4Aand B). The most prominent ultrastructural effect observed inpromastigotes treated with C. citratus and citral, were the appear-ance of aberrant-shaped cells (Fig. 4E and G) with cytoplasmaticdisorganization (Fig. 4C, D, E, G and I), besides an increase in cyto-plasmatic clearing (Fig. 4C, E,G and I). There was an increase in thenumber of autophagosomal structures, characterized by intensecytoplasmic vacuolization (Fig. 4C and H). Treated parasites alsopresented swelling of cell body (Fig. 4E, H and I), mitochondria(Fig. 4C, E and G–I) and kinetoplast (Fig. 4E). The swelling of theunique and highly branched mitochondria, resulted in a innermitochondrial membrane disorganization, displaying several andcomplex invaginations and forming concentric membranous struc-tures (Fig. 4E, H and I). It was also noted an increase on acidocalci-somes (Fig. 4F, G and H) and the presence of myelin-like figures asmultilamelar bodies (Fig. 4E and H). Other alteration frequently ob-served was on the nuclear chromatin organization, resembling thenucleus of apoptotic cells (Fig. 4C, D and H), and disruption ofnuclear membrane (Fig. 4F and I). Large amounts of membrane


vesicles could be seen within the flagellar pocket of many treatedcells (Fig. 4H). Although the degree of cell damage varied, citralwhere slightest drastic, the observed ultrastructural effects foundfor 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 afterPI staining of the parasites incubated with essential and citral for24 h at IC50 concentrations (Table 3). Fig. 5 shows the distributionof cell DNA trough cell cycle of parasites in the absence and pres-ence of the essential oil and the compound. After 24 h of incuba-tion, the majority of treated parasite cells were arrested on G0/G1 phase of cell cycle (essential oil, 84%; citral, 92.3%), oppositeto what occurs in not treated cells (35.9%).

3.5. Phosphatidylserine externalization

During early apoptosis, the plasma membrane loses asymmetrycausing PS to be translocated from the cytoplasmic face of the plas-ma membrane to the external face which can be detected usingAnnexin V. To distinguish apoptotic cell death from necrotic celldeath, cells were counterstained with PI, a non-permeable stainwith an affinity for nucleic acids, as it selectively enters necroticcells. Therefore, co-staining of annexin V and PI can differentiatebetween cells undergoing early apoptosis (annexin V-positive,PI-negative), necrosis (PI-positive, annexin V-negative) and livecells (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 was1.3%, 1.6%, 1.3% and 3.3%, respectively. Following treatment ofpromastigotes with C. citratus essential oil at its IC50 values, thepercentage 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, positivecells to annexin V increased with time, 2.3% at 3 h, 3.2% at 5–7 hand 21.6% at 24 h. The percentage of PI-stained cells ranged from0.4% 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 isessential for parasite survival, as Leishmania has a single mitochon-drion. The 585/530 nm ratio, i.e. J-aggregates, within the mito-chondria vs monomers in the cytosol represents the Dwm. Inuntreated cells, the red/green fluorescence ratio was 1.56; 2.2and 2.68 at 3 h, 5 h, 7 h and 24 h, respectively. However, the addi-tion of C. citratus essential oil caused a loss of mitochondrial mem-brane potential, blocking JC-1 entry to the mitochondria, leavingthe JC-1 monomers to fluoresce green within the cytoplasm. Thiswas reflected in the red/green fluorescence ratio, which decreasedto 1.1; 1.6; 1.68 and 2.38 following drug treatment for 3, 5, 7 and24 h, respectively. Moreover, treatment with citral revealed tohave a pronounced effect on mitochondrial membrane potentialwith a red/green fluorescence ratio of 1.2 at 3 h; 1.56 at 5 h; 1.47at 7 h and 1.87 at 24 h (Table 5). C. citratus essential oil and citralinduced an immediate decrease on Dwm. Data indicate that essen-tial oil up to the 3rd hour caused a higher number of cells with lowDwm (34.2%), which was followed by a sustained decrease in theDwm thereafter (Fig. 6). Therefore, a more prolonged incubationtime (24 h) showed that C. citratus (18.8%) and citral (27.6%) main-tain the profile characterized by a higher number of cells with lowDwm, 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 typicalelongated 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, Iand 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.



5 January 2012

3.7. Mammalian cell cytotoxicity assay

The cytotoxicity of C. citratus essential oil and citral were eval-uated in cultures of bovine aortic endothelial cells (primaryculture) and macrophages cell line using the MTT test. The resultsshowed that they did not induced toxicity against these mamma-lian cells (not shown).

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4. Discussion

Despite of the last years advances, in the knowledge of the biol-ogy of several forms of leishmaniasis, pentavalent antimonials re-main the first-line treatment for this infection in most endemicareas despite the limitations imposed by the need of parenteraladministration, high toxicity and increasing drug resistance, asfor many other drugs used (Leandro and Campino, 2003; Croftet al., 2006; Natera et al., 2007).

In recent years, several screenings of medicinal plants used forthe 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 essen-tial oils as potential sources for the isolation of novel compoundswith leishmanicidal effect. So, in the present study, we evaluatedthe activity of C. citratus essencial oil and their major compoundson three Leishmania species. C. citratus oil contains two isomericaldehydes, neral (cis-citral) and geranial (trans-citral) which to-gether represents around 81% of the oil, and myrcene that appearwith 6.4%. It has been pointed out that the anti-Leishmania activityof 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 com-pounds usually point at cell membranes as the primary target(Bakkali et al., 2008). In our study we have observed an increasein cell and organelle volume and cytoplasm vacuolization in trea-ted cells. In this sense, the essential oil and citral may have a pas-sive entry and may accumulate in parasite cell membranes leadingto an increase of membrane permeability, as observed by propidi-um iodide assay. Similar ultrastructural alterations were also ob-served in Trypanosoma cruzi and Leishmania amazonensis treatedwith other essential oils and/or their main constituents (Pedrosoet al., 2006; Santoro et al., 2007a,b).

The presence of autophagosomal structures was very promi-nent. Other authors also observed this alteration in T. cruzi and inL. amazonensis treated with drugs like ketoconazole, terbinafine,among others (Lazardi et al., 1990; Lorente et al.,2004). Anotherimportant alteration is the presence of membrane elaboratedstructures in the cytoplasm, appearing as myelin-like figures.The presence of these structures suggests the occurrence of anautophagic process, with the formation of structures known asautophagosomes (Rodrigues et al., 2002). These structures areprobably involved in the breakdown and recycling of abnormalmembrane structures, suggesting an intense process of remodelingof intracellular organelles irreversibly damaged by the essential oiland citral.

The presence of membrane vesicles in the flagellar pocket of cit-ral treated parasites is characteristic of an exocytose process, andwe cannot exclude the possibility that they result from the secre-tion into this region of abnormal lipids, which accumulate as a con-sequence 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, parasitestreated 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 3Effect 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 Phase S Phase G2/M

3 h 5 h 7 h 24 h 3 h 5 h 7 h 24 h 3 h 5 h 7 h 24 h

C. citratus 88.6 91 77.6 84 3.8 6 22.4 11.3 2.7 2.9 0 4.7Citral 96.4 90.3 85.1 92.3 5.5 3.9 11.5 6.7 5.9 5.7 3.9 0.9Control 88.7 90.4 73.2 35.9 3.8 4.8 26.8 44.5 7.6 4.9 0 19.3



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


characterized by their acidic content, high electron density, andhigh concentration of polyphosphates, calcium, magnesium, andother 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. citratusessential oil (B) and citral (C) at IC50 concentrations. Propidium iodide staining was performed and samples were analyzed by flow cytometry.

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


Anexine PI Anexine/PI

3 h 5 h 7 h 24 h 3 h 5 h 7 h 24 h 3 h 5 h 7 h 24 h

C. citratus 2.2 2.9 1.7 4.1 0.4 0.7 0.8 2.5 0.5 2.2 1.7 2.5Citral 2.3 3.2 3.2 21.6 0.3 0.3 0.6 6.7 1.9 1.9 0.7 6.5Control 1.3 1.6 1.3 3.3 0.4 0.2 0.3 1.1 0.5 0.3 0.3 1.6

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


JC1Mon JC1Agreg MIFAgreg / MIFMon membrane Pot

3 h 5 h 7 h 24 h 3 h 5 h 7 h 24 h 3 h 5 h 7 h 24 h

C. citratus 34.2 12 10.8 18.8 65 87.2 88.1 80.4 1.1 1.6 1.68 2.38Citral 20.9 14.8 14.3 27.6 77.9 83.5 84.5 71.5 1.2 1.56 1.47 1.87Control 13.3 10.3 9.5 3.6 82.6 89 88.8 95.8 1.56 2 2 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) orpresence 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, thereader is referred to the web version of this article.)



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terpenoids constituents from some essential oils have toxic effectson many biological systems by interacting at the cellular level withcytosolic Ca2+, through an intracellular calcium store release andcalcium channel blockage (Interaminense et al., 2007). Since terpe-noids are found in the essential oil tested, the increase in numberand volume of acidocalcisomes in treated cells could be due todirect or indirect action of citral on membrane ion flow in theseorganelles.

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 mito-chondrial matrix, forming complex and elaborate structures. Sub-sequently, the mitochondrial matrix became less electrondense,and a typical swelling of the mitochondrion was observed. Thosealterations have been also seeing in previous studies on L. brasilien-sis (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 asustained depolarization of mitochondrial membrane. This is acharacteristic feature of metazoan apoptosis and has been



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observed to play a key role in drug-induced death in protists suchas Leishmania (Sen et al., 2004). Moreover, we also find condensa-tion and fragmentation of DNA on treated cells. These nuclear fea-tures, characteristic of apoptosis, are being related with alterationsin the mitochondrial membrane potential (Green and Kroemer,2004). Data indicate that C. citratus oil and citral exerts its anti-leishmanial activity primarily via apoptosis and secondary tonecrosis.

Apoptosis is a programmed cell death process, which is charac-terized by a series of events involving morphological and biochem-ical changes. In parasites, apoptosis appears to be the predominantform of cell death, as has been observed in kinetoplastids (Arnoultet al., 2002) in response to chemotherapeutic agents such asamphotericin B and plant extracts such as Aloe vera leaf exudates(Dutta et al., 2007a,b).

Taken together, results demonstrated that essential oil and cit-ral induced Leishmania promastigotes death sharing several pheno-typic features observed with metazoan apoptosis, which includedphosphatidylserine exposure, depolarization of mitochondrial po-tential, arrested G0/G1 cell cycle phase and nuclear disorganiza-tion, with chromatin condensation.

C. citratus essential oil and citral besides being active againstpromastigotes are expected to exhibit a stronger activity onamastigotes forms as generally occurs among natural extractsand synthetic drugs (Dutta et al., 2007a,b; Santin et al., 2009;Nakayama et al., 2007; Lakshmi et al., 2007). Furthermore, the abil-ity of essential oil compounds to easily diffuse cell membranes andinteract with intracellular targets will be extremely important toinduce the inhibitory activity on intracellular parasites.

The main reasons for which a number of plant metabolites withleishmanicidal activity have not made into clinical evaluation istheir high toxicity on mammalian cells. This lack of selectivity isnot observed on C. citratus oil and citral, once they did not showtoxicity against mammalian cells tested.

In conclusion, C. citratus essential oil and citral may representvaluable sources for lead or active molecules against Leishmaniainfections.

533534535536537538539540

Source of funding

This work was supported by ‘‘Programa Operacional Ciência eInovacão 2010 (POCI)/FEDER’’ da Fundação para a Ciência e Tecno-logia (FCT).

541542543544545546547548549550551552553554

Acknowledgments

Authors are grateful to Prof. Jorge Paiva for help in plant taxo-nomic, to José Correia da Costa from Centro de Imunologia e Biolo-gia Parasitária, Instituto Nacional. Dr. Ricardo Jorge, Porto forsupplying the Leishmania infantum Nicolle (zymodeme MON-1),to António Osuna, Departamento de Parasitología, Facultad deCiencias, Instituto de Biotecnología, Universidad de Granada, forsuplly Leishmania major BCN.

555556557558559560561562563564565566567568569

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