Activity of essential oils on the growth of Leishmania infantum promastigotes†
This paper was published in Flavour and Fragrance Journal as part of the special issue based on the lectures given at the 40th International Symposium on Essential Oils, held at Savigliano, Italy, 6–9 September 2009, organized by Carlo Bicchi and Patrizia Rubiolo
In order to contribute to the search for new therapeutic agents for leishmaniasis, we report the effect of several essential oils on the growth of Leishmania infantum promastigotes. Eight of the tested essential oils revealed activity below 150 µg/ml, the most active being Cymbopogon citratus, Juniperus oxycedrus berries and Thymus capitelatus oils, with 50% effective concentration values in the range 16–51 µg/ml. The results support the concept that several essential oils or some of their constituents can become useful in the research of new therapeutic agents for leishmaniasis and in the clinical management of this parasitic disease. Copyright © 2010 John Wiley & Sons, Ltd.
Protozoan parasites of the genus Leishmania cause visceral, cutaneous and mucosal diseases in humans, which are collectively referred as leishmaniasis, affecting more than 12 million people worldwide and being responsible for high rates of mortality in tropical and subtropical countries.[1,2] At least 13 identified Leishmania species are able to infect humans, each one with its unique epidemic pattern and clinical manifestations of infection. Nevertheless, from a generic point of view, there are three main typical manifestations of leishmaniasis: visceral leishmaniasis (L. donovani, L. infantum); cutaneous leishmaniasis (L. major, L. tropica); and mucocutaneous leishmaniasis. In all cases, the leishmanial infection is transmitted to the vertebrate hosts by the bite of a sandfly.
The number and efficacy of drugs available for the treatment of human and animal leishmaniasis are limited. The treatment of choice, in spite of its toxicity, is still based on the use of pentavalent antimonial drugs or, alternatively, pentamidine and amphotericin B.[3,5] Considering the toxicity, side-effects, rate of relapse, cost, length of the treatment and the resistance that the parasites show to these drugs, more attention should be given to the search for new chemotherapeutics. One of the major obstacles to develop a good anti-Leishmania drug is the protection of parasites inside macrophages, requiring compounds effective on the parasite but preserving the host cell.[4,6,7]
Screening plant extracts is a valuable research option for the search of anti-Leishmania leads and drugs. Plant extracts offer a huge diversity of compositions and constituents, most of them commercially unavailable and structurally difficult to synthesize; several plant extracts have already been tested for anti-Leishmania activity, although most of them are aqueous or alcoholic extracts and so are exclusively composed of polar molecules. However, other kind of extracts, prepared by distillation (essential oils), composed of a huge diversity of small (<300 Da) hydrophobic molecules, most of them accomplishing theoretical criteria of drug-likeness prediction, offer peculiar advantages and expectations. Such molecules easily diffuse across cell membranes and consequently gain advantage with regard to interactions with intracellular targets. Our previous research showed the potential of essential oils as natural antimicrobial agents, particularly against yeasts, filamentous fungi and also flagellated protozoa, as the intestinal parasite Giardia lamblia.[12–25]
Rosa et al. and Ueda-Nakamura et al. reported the effects of the essential oils from Croton cajucara and Ocimum gratissimum on L. amazonensis. Monzote et al.[28–31] revealed the activity of Chenopodium ambrosioides against Leishmania amazonensis and L. donovani. The effects of essential oils from Cymbopogon citratus (DC) Stapf., Lippia sidoides Cham. and Ocimum gratissimum L. on Leishmania chagasi were investigated by Oliveira et al. As there are few reports on the activity of essential oils on endemic Old World Leishmania species, in the present work we focused on the screening of a set of 19 essential oils for their effects on L. infantum promastigotes, of one of those Old World species.
Materials and Methods
Essential oils from the aerial parts of Crithmum maritimum L., Cymbopogum citratus (DC) Stapf., Distichoselinum tenuifolium (Lag.) García Martín & Silvestre, Eryngium maritimum L., Lavandula viridis L'Hér., Lippia graveolens H.B.K., Mentha cervina L., M. × piperita L., Origanum virens Hoffmanns. & Link, Rosmarinus officinalis L., Seseli tortuosum L., Thymbra capitata (L.) Cav., Thymus capitelatus Hoffmanns. & Link, T. mastichina L. and T. zygis Loefl. Ex L. subsp. sylvestris (Hoffmanns & Link) Brot. ex Coutinho (two chemotypes), Lavandula viridis L'Hérand from the leaves, and from the berries of Juniperus oxycedrus L. were prepared at our laboratory (CEF, Coimbra) by water distillation, using a Clevenger-type apparatus and following the procedure described in the European Pharmacopoeia (1997). The oil from the buds of Syzygium aromaticum (L.) Merr. & Perry was acquired from Segredo da Planta (Portugal).
Essential oil analysis.
Analysis was carried out by gas chromatography (GC) and 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 (Bellefonte, PA, USA) fused silica capillary columns with different stationary phases: SPB-1 (polydimethylsiloxane 30 m × 0.20 mm i.d., film thickness 0.20 µm), and SupelcoWax-10 (polyethyleneglycol 30 m × 0.20 mm i.d., film thickness 0.20 µm); oven temperature programme, 70–220°C at 3°C/min, then held at 220°C for 15 min; injector temperature, 250°C; carrier gas, helium, adjusted to a linear velocity of 30 cm/s; split ratio, 1:40; detector temperature, 250°C. GC–MS was carried out in a Hewlett-Packard 6890 gas chromatograph fitted with a HP1 fused silica column (polydimethylsiloxane 30 m × 0.25 mm i.d., film thickness 0.25 µm), interfaced with an Hewlett-Packard mass selective detector 5973 (Agilent Technologies), operated by HP Enhanced ChemStation software, version A.03.00. GC parameters were as described above; interface temperature, 250°C; MS source temperature, 230°C; MS quadrupole temperature, 150°C; ionization energy, 70 eV; ionization current, 60 µA; scan range, 35–350 units; scans/s, 4.51.
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–C23n-alkanes, were compared with those of reference samples included in our laboratory database. Acquired mass spectra were compared with reference spectra from our own database, Wiley/NIST database and literature data.[35,36] Relative amounts of individual components were calculated based on GC raw data areas without FID response factor correction.
Parasites and Cultures
Promastigote forms of L. infantum Nicolle (zymodeme MON-1) were maintained at 26°C by weekly transfers in HEPES (25 mm)-buffered RPMI 1640 medium enriched with 10% inactivated fetal bovine serum (FBS).
Growth Inhibition Assays
Essential oils were dispersed in dimethyl sulphoxide (DMSO; Sigma Chemical) at 100 mg/ml and then diluted to 1 mg/ml with culture medium (RPMI 1640). Appropriated volumes of these solutions were incorporated afterwards to assays to give final oil concentrations in the range 10–400 µg/ml. The DMSO concentration never exceeded 0.4%, which was proved to be not toxic for the protozoa. Promastigotes of L. infantum (106 cells/ml) in the log phase of growth were incubated in HEPES (25 mm)-buffered RPMI 1640 medium enriched with 10% inactivated fetal bovine serum (FBS) in the absence or presence of different concentrations of essential oils and DMSO at 26°C.
The inhibitory effect on cell growth was then estimated by the tetrazolium-dye (MTT) colorimetric method. The concentration that inhibited culture growth by 50% (IC50) was determined after 24 h by dose–response regression analysis, plotted by GraphPad Prism 5.
The cytotoxic effects on mammalian cells of the most active essential oils were assessed with bovine aortic endothelial cells (primary culture), incubated under microaerophilic conditions in the presence of the essential oil at Leishmania's inhibitory concentration. The tetrazolium-dye colorimetric assay was used to detect living cells which have the ability to reduce yellow 3-(4,5-dimethylthiazole-2-yl)-2,5 (MTT) to a blue formazan product. Briefly, the cells were cultivated in 24-well tissue culture plates (500 µl, containing 106 cells/ml in RPMI 1640 medium/DMEM medium, supplemented with 10% FBS) at 37°C and 5% CO2 atmosphere. When the macrophages reached log phase (3–4 days), the medium was removed and the cells were then incubated with fresh medium added of essential oil, or with fresh medium as the control. After further incubation for 14 h, control and treated cells were washed three times with PBS, pH 7.2. 50 µl MTT solution (5 mg/ml in PBS) and 450 µl PBS were added to each well. After 1 h incubation at 37°C, the cells were then washed three times with PBS. 500 µl DMSO was added to the wells and the optical density was measured at 530 nm. Cell viability was determined using the following formula: [100 − (L2/L1) × 100], where L1 is the percentage of viable control cells and L2 is the percentage of viable treated cells, as previously described.
All experiments were performed in triplicate. The mean and standard deviation (SD) of at least six independent assays were determined. Statistical analysis between mean values obtained for the experimental groups was done by Student's t-test. p ≤ 0.05 was considered significant.
Results and Discussion
Chemical diversity and compositional data of the 19 tested essential oils are summarized in Table 1. The compositions were extensively elucidated, attaining identification rates > 90%, except for Eryngium maritimum oil, for which only 71.8% of the composition was elucidated. In general, compositions are analogous to those typical of the oils from each source species and type. In this set of 19 essential oils, >100 different compounds were identified, representing the major chemical families reported for this kind of plant extract – monoterpene hydrocarbons, sesquiterpene hydrocarbons, oxygen-containing mono- and sesquiterpenoids, diterpenoids, phenylpropanoids and volatile aliphatic compounds. Forty-nine of these compounds were found in concentrations >2.0% in at least one essential oil. Six monoterpene hydrocarbons (α-pinene, β-pinene, myrcene, limonene, p-cymene and γ-terpinene), 13 oxygen-containing monoterpenes (1,8-cineole, camphor, borneol, menthofuran, isomenthone, pulegone, neral, geranial, menthol, thymol, carvacrol, geraniol and geranyl acetate), the sesquiterpene germacrene D and the phenylpropanoids eugenol and dillapiole exceeded 10.0% of the total composition of at least one essential oil.
Table 1. Global composition and major constituents of the tested essential oils. (Information merely indicative)
|Crithmum maritimum||74.2||7.4||2.0||0.3||13.8||0.6||–||(31)/98.3||α-Pinene (2.1%), p-cymene (7.1%), β-phellandrene (8.0%), Z-β-ocimene (5.2%), γ-terpinene (48.7%), thymyl-methyl oxide (6.3%), dillapiole (13.8%)|
|Cymbopogon citratus||6.7||88.6||t||t||–||1.9||–||(16)/97.0||Myrcene (6.4%), neral (32.6%), geranial (48.4%)|
|Distichoselinum tenuifolium||91.2||0.5||0.5||0.5||0.1||–||–||(26)/92.7||Myrcene (84.6%), limonene (2.2%)|
|Eryngium maritimum||5.25||t||53.7||12.9||–||–||–||(21)/71.8||α-Pinene (3.6%), germacrene D (41.1%), bicyclogermacrene (2.1%), Germacrene B (2.4%), δ-cadinene (2.1%)|
|Juniperus oxycedrus (berries oil)||66.9||5.7||11.3||5.5||–||t||2.6||(44)/92.0||α-Pinene (56.4%), myrcene (4.0%), γ-cadinene (2.3%), manoyl oxide (2.2%)|
|Juniperus oxycedrus (leaves oil)||87.3||6.5||0.9||2.0||–||–||0.5||(40)/97.7||α-Pinene (76.4 %), δ-3-carene (2.7%)|
|Lavandula viridis||17.3||58.3||18.6||–||–||1.9||–||(38)/96.1||α-Pinene (9.2%), camphene (2.7%), 1,8-cineole (29.7%), linalool (9.0%), camphor (10.0%), borneol (2.7%), (Z)-α-bisabolene (6.3%), selina-3,7(11)-diene (6.6%)|
|Lippia graveolens||28.9||41.0||10.3||10.8||–||–||–||(46)/90.3||Myrcene (3.4%), δ-3-carene (4.3%), p-cymene (16.9%), 1,8-cineole (6.6%), trans-sabinene hydrate (2.3%), linalool (5.4%), α-terpineol (3.6%), thymol (19.8%), (E)-caryophyllene (2.4%), caryophyllene oxide (5.7%)|
|Mentha cervina||8.5||89.7||0.3||–||–||2.0||–||(16) 99.5||Limonene (5.4%), isomenthone (10.6%), pulegone (74.8%)|
|Mentha × piperita||5.6||90.5||1.9||0.3||–||0.3|| ||(29) 98.6||1,8-Cineole (5.8%), menthone (9.8%), menthofuran (10.9%), neo-menthol (4.0%), menthol (44.0%), neo-isomenthol (2.9%), pulegone (2.4%), menthyl acetate (7.8%)|
|Origanum virens||22.5||74.5||1.3||0.5||–||0.5||–||(28) 99.3||Myrcene (2.4%), p-cymene (7.4%), γ-terpinene (7.9%), thymol (2.1%), carvacrol (68.2%)|
|Rosmarinus officinalis||64.3||33.7||1.2||0.2||–||0.4||–||(31) 99.8||α-Pinene (11.1%), camphene (3.4%), myrcene (32.0%), p-cymene (3.8%), 1,8-cineole (13.7%), limonene (6.6%), linalool (2.1%), camphor (11.9%)|
|Seseli tortuosum||84.3||1.4||2.4||2.3||–||–||–||(27) 90.3||α-Pinene (27.4%), camphene (2.1%), sabinene (2.0%), β-pinene (16.0%), myrcene (3.0%), limonene (10.0%), (Z)-β-ocimene (8.0%), γ-terpinene (9.3%)|
|Syzygium aromaticum||0.8||1.6||9.5||0.4||85.3||–||–||(20) 97.3||Eugenol (85.3%), α-humulene (6.8%)|
|Thymbra capitata||12.5||79.7||4.7||0.8||–||0.3||–||(28) 98.1||p-Cymene (5.5%), γ-terpinene (3.6%), linalool (2.8%), carvacrol (74.6%), (E)-caryophyllene (3.9%)|
|Thymus capitelatus||18.2||78.8||0.5||0.5||–||–||–||(28) 97.9||α-Pinene (4.5%), camphene (6.5%), sabinene (3.0%), β-pinene (2.0%), 1,8-cineole (58.6%), borneol (10.0%)|
|Thymus mastichina||20.6||74.5||0.3||2.3||–||–||–||(24) 97.7||α-Pinene (3.6%), sabinene (3.7%), β-pinene (5.3%), myrcene (2.0%), 1,8-cineole (64.6%), limonene (5.0%), α-terpinyl acetate (5.0%)|
|Thymus zygis subsp. sylvestris (chemotype geraniol)||1.8||91.0||0.9||1.5||–||1.2||–||(27) 96.3||Camphor (3.9%), geraniol (33.1%), geranyl acetate (44.5%)|
|Thymus zygis subsp. sylvestris (chemotype thymol)||71.0||22.1||1.6||–||–||1.0|| ||(31) 95.7||α-Thujene (2.6%), myrcene (3.0%), α-terpinene (2.7%), p-cymene (36.6); γ-terpinene (21.0%), linalool (2.9%), thymol (15.2%)|
Table 2 summarizes the results of the inhibitory concentrations (IC50) of the essential oils on the growth of L. infantum. Cymbopogon citratus, Juniperus oxycedrus (berries oil), and Thymus capitelatus oils were generally the most effective to inhibit Leishmania growth (IC50 values of 16–51 µg/ml). The oils from Thymbra capitata, Crithmum maritimum, Juniperus oxycedrus (leaves oil), Seseli tortuosum and Thymus mastichina exhibited activity of <150 µg/ml, and Origanum virens, Lippia graveolens, Mentha cervina, Mentha × piperita, Thymus zygis subsp. sylvestris (chemotype geraniol) (IC50 values of 150–200 µg/ml), as well as Syzygium aromaticum, Thymus zygis subsp. sylvestris (chemotype thymol), Lavandula viridis, Distichoselinum tenuifolium and Eryngium maritimum (IC50 values > 200 µg/ml) inhibit Leishmania growing at higher concentrations. Only Rosmarinus officinalis essential oil did not show any activity.
Table 2. Inhibitory concentration (IC50) of the active essential oils against L. infantum
|Thymbra capitata||130 (119–142)|
|Origanum virens||196 (189–202)|
|Syzygium aromaticum||220 (204–238)|
|Thymus zygis subsp. sylvestris (chemotype thymol)||293 (261–334)|
|Lippia graveolens||171 (151–193)|
|Cymbopogon citratus||25 (20–31)|
|Mentha × piperita||198 (180–217)|
|Thymus zygis subsp. sylvestris (chemotype geraniol)||162 (151–174)|
|Lavandula viridis||263 (248–279)|
|Crithmum maritimum||122 (109–136)|
|Distichoselinum tenuifolium||295 (272–321)|
|Eryngium maritimum||205 (199–211)|
|Juniperus oxycedrus (berries oil)||51 (46–56)|
|Juniperus oxycedrus (leaves oil)||127 (107–150)|
|Mentha cervina||178 (152–209)|
|Seseli tortuosum||133 (115–153)|
|Thymus capitelatus||37 (30–46)|
|Thymus mastichina||133 (111–159)|
These in vitro results showed the potential of essential oils (C. citratus, T. capitelatus and J. oxycedrus berries) as hopeful sources for lead or active molecules against Leishmania. The ability of most of essential oil compounds, to easily diffuse through cell membranes and interact with intracellular targets, can minimize the problem on diffusing inside macrophages to inhibit intracellular promastigotes and differentiation into the amastigote form. Additionally, the most active essential oils tested did not show toxicity to mammalian endothelial cells at IC50 for Leishmania.
Considering that L. infantum may express visceral and cutaneous disease in humans and animals, we believe that the most active essential oils, particularly that of C. citratus, can be a hopeful alternative as an inhibitor of Leishmania growth to be used in human and animal disease.
The authors are grateful to Professor Jorge Paiva for help with plant taxonomy, 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 supplying Leishmania major BCN, and to Fundação para a Ciência e Tecnologia (FCT) and Programa Operacional Ciência e Inovacão 2010 (POCI)/FEDER for financial support.