In vitro cytotoxic activity of Cymbopogon citratus L. and Cymbopogon nardus L. essential oils from Togo

The leaf essential oils of Cymbopogon citratus L. and  Cymbopogon nardus L. (Poaceae) from Togo were steam-distilled, analyzed for percentage composition and investigated in vitro for their potential cytotoxic activity on human epidermic cell line HaCat. The percentage composition showed that the main constituents of essential oils samples were respectively geranial (45.2%), neral (32.4%) and myrc¨ne (10.2%) for C. citratus essential oil and citronellal (35.5%), geraniol (27.9%) and citronellol (10.7%) for that of C. nardus . The in vitro cytotoxicity bioassays on human epidermic cell line HaCaT revealed that the toxicity of the essential oil from C. citratus (IC 50 : 150 µL.mL -1 ) was higher than that of the essential oil from C. nardus (IC 50 : 450 µL.mL -1 ). Pure commercial neral, geranial, and citronellal standards showed respectively the following IC 50 values: 100, 250 and 300 µL.mL -1 ). Conversely, pure citronellol standard appeared almost non-toxic (IC 50 >1000 µL.mL -1 ), proving the major role played in synergy by neral and geranial in the overall toxicity showed by the citratus oil sample tested in this work.


Introduction
During the last decades, chemists and biologists have been intensively investigating tropical and subtropical plants species with potential medicinal properties in order to assess the feasibility of developing natural, sustainable, and affordable drugs and cosmetics (Aké Assi and Guinko, 1991;Iwu, 2000;Orafidiya et al., 2001).
Cymbopogon citratus L. and Cymbopogon nardus L. essential oils, apart from their broad use in food or drinks, are widely involved in perfumery, body care products and soap manufacture.Also important is their pharmaceutical usage, which still remains under exploited especially in West Africa where these aromatic herbs are widely distributed.
Recently, particular attention has been given by researchers to the use of essential oils from tropical origin as active ingredients in various pharmaceutical formulations against some skin troubles (Orafidiya et al., 2001;Orafidiya et al., 2002) like human mycosis.Indeed, naturally occurring molecules of essential oils are more and more considered as valid additives to conventional antibiotherapies (Chaumont et al., 2001;Chaumont, 2003;Koba, 2003).
While intensive work is done to record the antimicrobial potential of plant essential oils and their other biological applications, some important works have been reported in the relevant literature regarding their possible cytotoxicity on human cutaneous cell lines (Foray et al., 1999;Hayes et al., 1999;Koba et al., 2007) and leukemia cells (Dubey et al., 1997a).
The objective of this paper was to investigate chemical composition and the in vitro cytotoxic potential of C. citratus and C. nardus essential oils.The assumption was made that these essential oils, commonly used or proposed for use in soap and body care and in different formulations to treat skin troubles like acnes and tinea in West Africa (Orafidiya et al., 2001;Orafidiya et al., 2002) may be toxic to the human epidermic cell line HaCaT.Hence the cytotoxicity evaluation of these volatile essential oils undertaken in this work constitutes one of the important steps prior to their possible exhaustive assessment for pharmaceutical use against superficial skin mycosis.

Plant material and volatile oils isolation
Aerial parts (leaves) of the tested plants were harvested in April 2005, from the experimental field of the Unité de Recherche sur les Agroressources et la Santé Environnementale at the Université de Lomé, Togo.
Plant material was identified by Prof. Akpagana at the Université de Lomé, where voucher specimens were stored in the herbarium respectively under references N'247 for C. citratus and 268 (K) for C. nardus.
A sample (50 g) of air-dried plant material was extracted by the hydrodistillation technique during 2 hours in a modified Clevenger-type apparatus (Craveiro et al., 1976).The extracted essential oils were stored in hermetically sealed dark glass flasks with rubber lids, covered with aluminium foil to protect the contents from light and kept under refrigeration at 4°C until use without any prior purification.

Essential oils analyses
Gas chromatographic analysis was carried out on a Varian 3300 type gas chromatograph equipped with FID detector.An apolar capillary column DB-5 (30 m x 0.25 mm i.d.; film thickness 0.25 µm) and on a polar column supelcowax 10 with the same characteristics as above mentioned were used.DB-5 column operating conditions were as follows: from 50°C (5 min), 50°C to 250°C at the rate of 2°C/min and Supelcowax 10 from 50°C (5 min), 50 to 200°C at 2°C/min.The injector and detector temperatures were 250 and 300°C respectively.The carrier gas was helium at a flow rate of 1.50 mL/ min.Samples (0.2 µL) of undiluted essential oil were injected manually.
The GC/MS analysis was carried out on a Hewlett Packard 5890 SERIES II chromatograph, coupled with a mass spectrometer of the Hewlett Packard 5971 SERIES type operating in the EI mode at 70 eV.The capillary column type was DB5-MS (30 m x 0.25 mm i.d.; film thickness 0.25 µm).The amount of sample injected and GC/MS parameters were the same as above.

Identification of components
The components of oils samples were identified by their retention time, retention indices relative to C8-C24 nalkanes, computer matching with Willet 275.L library and as well as by comparison of their mass spectra with the authentic samples or with data already available in the literature (Kondjoyan and Berdagué, 1996;Adams, 2001).
The percentage of composition of the identified compounds was computed from the GC peak area without any correction factor and was calculated relatively.

HaCaT cell culture
HaCaT, an immortalized human keratinocyte line was a generous gift from Nathalie Gault (Commissariat à l'Énergie Atomique, Bruyère Le Châtel, France) (Boukamp et al., 1988;Gault et al., 2002).Cells were routinely grown in Costar plastic flasks in monolayer cultures in DMEM medium supplemented with 10% (v/v) FCS, and 5 M of HEPES + 80 mg.L -1 of gentamicin.They were grown in a humidified atmosphere of 5% CO2 in air.The medium was routinely renewed 2, 4 and 6 days after passage and when confluence was reached; cells were trypsinized and split for subcultures (seeding density 3,500 cells/ cm 2 in a 75 cm 2 flask) or used for cytotoxicity assays.Cells were used for experiments within 10 passages to ensure cell line stability.All the experiments were carried out at 37°C.

Cytotoxicity assay
HaCaT cells were seeded at a density of 6 x 10 4 cells per well in 100 µL culture medium containing 10% FCS on 96 multi well culture plates and incubated overnight for adherence.The next day, the medium was removed and cells were incubated in FCS-free medium containing increasing concentrations (from 50 to 1,000 µL.mL -1 ) of C. citratus, C. nardus essential oils or their major components from commercial origin (Pure neral, geranial, citronellal and geraniol) solubilized in 1/10 Tween 80 ® in culture medium (with <0.1% Tween 80 ® an ethanol vehicle).Each experiment was carried out in triplicate.

Determination of cell viability
After the exposure period, the reaction medium was removed and the adhering cells washed with PBS.One hundred microliter of MTT solution (0.5 g.L -1 in medium) was added to each culture well.After incubating for 4 hours at 37°C, the MTT reaction medium was removed and formazan-blue was solubilized in 100 µL DMSO.This assay is based on the reduction of yellow tetrazolium salt MTT by the mitochondrial succinate dehydrogenase to form an insoluble formazan-blue product.Only viable cells with active mitochondria reduce significant amounts of MTT (Mosmann, 1983) and formazan-blue formation absorbance was recorded in an ELX800 UV, universal microplate reader spectrophotometer at 570 nm.Values of absorbance were converted into percentage of residual viability.Usually, inhibition concentration 50% (IC50) is chosen as the best biological marker of cytotoxicity.
The inhibition (I) of the essential oils dilutions in percent was calculated as follows: I = (A0-A1/A0) x 100 Ao is the absorbance of the control reaction (containing all reagents except the tested compounds), and A1 the absorbance with the tested substances (crude essential oils or pure commercial component).
The IC50 value represented the concentration of the tested compounds or essential oils that caused 50% cells inhibition.

Results and Discussion
The light yellow essential oil of C. citratus and C. nardus were obtained in yields of 1.6 and 1.3% respectively, based on dried extracted material.The percentage composition of the studied oil samples are listed in Table I.
The sample of C. citratus essential oil was notably richer in citral (neral and geranial upper than 77%) than that of C. nardus (with only a total of 1.1% in citral).
The percentage of HaCaT cell viability and the IC50 values recorded for both tested essential oil samples and their four major constituents from commercial origin are shown in Table II.
At lower concentrations in the range from 25 to 100 µL.mL -1 , none of the six tested substances showed any cytotoxicity.Conversely, both tested essential oils induced significantly increased cell cytotoxicity, at higher concentrations ranging from 100 to 1,000 µL.mL - 1 .Two types of profiles were observed: (i) tested citratus and nardus oils and pure neral, geranial and citronellal standards showed cytotoxicity towards HaCaT with following respective IC50 values (150, 450, 100, 250 and 300 µL.mL -1 ) shown in Table II; (ii) pure geraniol standard did not show any cytotoxicity up to 1,000 µL.mL -1 with cell viability up to 154%.
These experimental results demonstrated that, of the four major constituents of both tested essential oils, neral, geranial and citronellal were the most toxic for HaCaT cell line.Consequently, the cytotoxic effect of the essential oils of C. citratus and C. nardus found in this study was undoubtedly due to citral (neral and geranial), for the citratus oil, and to citronellal for that of nardus.Previous works only assumed what we experimentally established to a large extent in this investigation, but unfortunately authors did not provide any numerical data on the toxicity of the essential oils rich in citral which they used in high concentrations on human skin (Franchomme et al., 1996;Baudoux, 2002;Nakamura et al., 2003).Citral in vitro cytotoxicity was established on leukemia cells (Dubey et al., 1997b).Apart from its inherent toxicity, some authors have reported that citral, a mixture of both geranial and neral stereoisomers, had a significant ability to suppress oxidative stress possibly through induction of the endogenous anti-oxidant glutathione system, providing a new insight into skin cancer (Nakamura et al., 2003).Besides, geraniol, as one of the main constituents of the nardus sample tested here, did not show any cytotoxicity.But it could probably expressed its potential as a cytoprotector or as an antioxidant in subsequent oxidative stress assays.

Conclusion
Our findings clearly showed that essential oils of C. citratus and C. nardus, with a percentage composition identical to our chemotypes, when used in appropriate doses, could be quite suitable as active components in pharmaceutical formulations for skin treatment and its damages repairing.