Food and Chemical Toxicology 68 (2014) 71–77

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Food and Chemical Toxicology

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Evaluation of toxicity of essential oils palmarosa, citronella, lemongrassand vetiver in human lymphocytes

http://dx.doi.org/10.1016/j.fct.2014.02.0360278-6915/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author. Tel.: +91 9831061998; fax: +91 033 24614849.E-mail address: anitamukherjee28@gmail.com (A. Mukherjee).

Sonali Sinha, Manivannan Jothiramajayam, Manosij Ghosh, Anita Mukherjee ⇑Department of Botany, Centre of Advanced Study, Cell Biology and Genetic Toxicology Laboratory, University of Calcutta, 35, Ballygunge Circular Road, Kolkata 700019, India

a r t i c l e i n f o

Article history:Received 8 January 2014Accepted 26 February 2014Available online 17 March 2014

Keywords:Essential oilsHuman lymphocytesCytotoxicityGenotoxicityReactive oxygen speciesApoptosis

a b s t r a c t

The present investigation was undertaken to study the cytotoxic and genotoxic potential of the essentialoils (palmarosa, citronella, lemongrass and vetiver) and monoterpenoids (citral and geraniol) in humanlymphocytes. Trypan blue dye exclusion and MTT test was used to evaluate cytotoxicity. The genotoxicitystudies were carried out by comet and DNA diffusion assays. Apoptosis was confirmed by Annexin/PIdouble staining. In addition, generation of reactive oxygen species was evaluated by DCFH-DA stainingusing flow cytometry. The results demonstrated that the four essential oils and citral induced cytotoxicityand genotoxicity at higher concentrations. The essential oils were found to induce oxidative stress evi-denced by the generation of reactive oxygen species. With the exception of geraniol, induction of apop-tosis was confirmed at higher concentrations of the test substances. Based on the results, the fouressential oils are considered safe for human consumption at low concentrations.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction intended use. The essential oils from Cymbopogon and vetiver are

Essential oils extracted from the aromatic grasses like Cymbopo-gon martini, Cymbopogon winterianus, Cymbopogon citratus andVetiveria zizanioides are of enormous commercial value for environ-mental, agricultural, food and medical applications as well as inperfumery and aromatherapy. Essential oils are complex naturalmixtures containing about 20–60 components of different concen-trations. They are characterized by one or two major componentsat high concentrations (20–70%) compared to the others compo-nents present in trace amounts (Bakkali et al., 2008). Phytochemi-cal analysis of the essential oil of C. martini (palmarosa) indicatedthe presence of geraniol (65–85%) and geranyl acetate (5–20%) asmajor components (Raina et al., 2003). Geraniol (20–40%) is alsothe major constituent of C. winterianus Jowitt (java citronella)along with citronellal (20–30%) and citronellol (10–15%). Essentialoil from C. citratus (lemongrass) is rich in citral (50–88%) withother components such as linalool, myrcene, geraniol, geranyl ace-tate and camphene (Anonymous, 2001; Ganjewala, 2009).

The greatest use of these essential oils and their major compo-nents (monoterpenoids) is in food as flavouring and preservativeagents (Burt, 2004). The recent interest in ‘green’ consumerism haslead to the renewal of scientific interest in these substances. Palma-rosa, citronella, lemongrass, vetiver, citral and geraniol are classifiedas GRAS (Generally recognized as safe) by the US FDA for their

used at levels of 5–40 ppm to impart flavour in alcoholic and non-alcoholic beverages, chewing gum, candies, dairy and baked foodproducts (Council of Europe, 2000). In addition, they are used as pre-servatives for their antimicrobial (Hammer et al., 1999; Kim et al.,1995a,b; Onawunmi, 1989; Prashar et al., 2003), antifungal (Liet al., 2013) and antiprotozoal (Monzote et al., 2012) properties.

In vitro studies have demonstrated antibacterial activity of theessential oils at levels between 0.2 and 10 ll/ml and a number oftheir components has been identified having minimum inhibitoryconcentrations (MICs) of 0.05–5.0 ll/ml (Burt, 2004). To achievethe same antibacterial activity in foods, it has generally been foundthat a greater concentration of the essential oils or their compo-nent is used (Shelef, 1983; Smid and Gorris, 1999). The ratio hasbeen approximately 50-fold in soup (Ultee and Smid, 2001) and25- to 100-fold in soft cheese (Mendoza-Yepes et al., 1997). Theseconcentrations are much higher than the Admissible Daily Intake(ADI) values. The ADI values of citral and geraniol is 0.5 mg/kgbody weight/day (Council of Europe, 2000).

The random and inappropriate use of the essential oils may en-tail risks to human health due to mutational events, carcinogeniceffects and genetic damages (Sousa et al., 2010). Moreover, thereis a demand for their comprehensive safety evaluation and this is-sue is becoming more complicated as the use of the essential oils isnot under any regulatory control in many countries.

The present study was undertaken to investigate the toxiceffects of essential oils (palmarosa, citronella, lemongrass, vetiveracetate) and monoterpenoids (citral and geraniol) on human

72 S. Sinha et al. / Food and Chemical Toxicology 68 (2014) 71–77

lymphocytes. Cytotoxicity and genotoxicity of test substances wereevaluated by trypan blue dye exclusion test, MTT assay, single cellgel electrophoresis and DNA diffusion assay respectively. The reac-tive oxygen species generation and detection of apoptosis/necrosiswas determined using flow cytometry.

2. Materials and methods

2.1. Chemicals

Methyl methanesulphonate (MMS), normal melting point agarose (NMA), lowmelting point agarose (LMPA), ethylenediaminetetraacetic acid (EDTA) disodiumsalt, ethidium bromide (EtBr), 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazoliumbromide (MTT), Trizma base, Spermine, Triton X-100, DMSO and 20 ,70-dichlorfluo-rescein-diacetate (DCFH-DA), trypan blue and Histopaque were also purchasedfrom Sigma–Aldrich Chemical Co., Bengaluru, India. Phosphate-buffered saline(PBS; Ca2+, Mg2+ free), and Roswell Park Memorial Institute media (RPMI-1640)were purchased from Hi Media, Mumbai, India.

2.2. Test substance

Palmarosa oil (CAS No. 8014-19-5; source-C. martini), citronella oil (CAS No.8000-29-1; source-C. winterianus), lemongrass oil (CAS No. 8007-02-1; source-C.citratus Stapf), vetiver acetate (CAS No. 62563-80-8; source-Vetiver java), citral(CAS No. 5392-40-58) and geraniol (CAS No. 106-24-1), were purchased fromSigma–Aldrich Fine Chemicals, St. Louis, MO, USA.

2.3. Test system

Human peripheral blood was collected into heparinized vacutainers by veinpuncture from healthy male volunteers (n = 5; 20–25 year old, non-smokers, non-alcohol consuming and not undergoing any medication) after obtaining written in-formed consent. The lymphocytes were isolated from fresh blood according to themethod of Boyum (1976) with minor modifications. Fresh blood was diluted with

Fig. 1. Effect of the test substances on cell viability of human lymphocytes evaluated bgeraniol, (d) lemongrass, (e) citral, and (f) vetiver acetate. (For interpretation of the referarticle.)

equal volume of PBS (Ca2+, Mg2+ free; pH 7.4) and layered over 2 ml of Histopaqueand centrifuged at 1000 rpm for 40 min. The buffy coat was aspirated into 3–5 ml ofPBS and centrifuged at 1000 rpm for 10 min. The pellet was resuspended in RPMI-1640 media (1 � 106 cells/ml) and viability was checked. Lymphocytes with 98%and above viability were used in the experiments (Tennant, 1964). All the experi-ments were done in triplicate and in accordance with the Institutional guidelines(Institutional Ethical Committee, University of Calcutta, India).

2.4. Cytotoxicity assays

2.4.1. Dose selectionThe dose selection for the present study was based on initial cytotoxicity

screening of the test substances using trypan blue dye exclusion method andMTT assay. Stock solutions of the test substances were prepared in DMSO and fur-ther dilutions were made with PBS. The cytotoxicity assays were carried out over awide range of concentrations of 0–5000 lg/ml and the cut-off point was considered70% cell viability as suggested by Henderson et al., 1997. Following the initialscreening, the final treatment concentrations were selected. Palmarosa, citronellaoils and geraniol were tested at different concentrations of 100, 200, 500, 1000,1500 and 2000 lg/ml. Whereas lemongrass oil, citral and vetiver acetate oil havebeen tested at different concentrations of 25, 50, 100, 200, 400 and 800 lg/ml. Neg-ative control in all the experiments was maintained as lymphocytes suspended inRPMI-1640 medium added with 0.01% DMSO. The detailed protocols of above men-tioned cytotoxicity assays are described in the following sections.

2.4.2. Trypan blue dye exclusion methodThe lymphocytes (1 � 106/ml) were incubated with different concentrations of

the test substances. After treatment for 3 h at 37 �C, the lymphocytes were washedby centrifugation and fresh media was added (Tennant, 1964). The lymphocyteswere stained with trypan blue dye (0.4% w/v) and the number of viable and deadcells were scored under the light microscope with Neubauer’ hemocytometer.

2.4.3. MTT assayCytotoxicity of the test substances at concentrations of the test substances were

also evaluated using MTT assay. The MTT assay is based on reduction of the yellowtetrazolium salt (MTT), to form a soluble blue formazan product by mitochondrial

y trypan blue dye exclusion test and MTT assay; (a) palmarosa, (b) citronella, (c)ences to colour in this figure legend, the reader is referred to the web version of this

Fig. 2. Comet data (% tail DNA) of human lymphocytes treated with different concentrations of the test substances; (a) palmarosa, (b) citronella, (c) geraniol, (d) lemongrass,(e) citral, and (f) vetiver acetate.

S. Sinha et al. / Food and Chemical Toxicology 68 (2014) 71–77 73

enzymes. The amount of formazan produced is directly proportional to the numberof living cells. Briefly, after treatment with the test substances for 3 h, 10 ll 0.5 mg/ml of MTT solution was added and maintained at 37 �C till purple precipitate wasvisible. Then 100 ll of DMSO was added and mixed thoroughly to dissolve the darkblue crystals of formazan (Mosmann, 1983). Formation of formazan was quantifiedon iMark™ Microplate Absorbance Reader (BIO-RAD, CA, USA) at 570 nm, with630 nm as a reference wavelength.

2.5. Genotoxicity assays

2.5.1. Single cell gel electrophoresis (comet assay)Genotoxicity of the test substances was evaluated using comet assay according

to the method of Singh et al. (1988) with slight modifications (Tice et al., 2000). Thelymphocytes (1 � 106/ml) after incubation for 3 h at 37 �C were centrifuged andresuspended in 1% low melting point agarose. Simultaneously negative (0.01%DMSO) and positive (Methyl methanesulphonate, 50 lM) controls were main-tained. Then, 100 ll of cell suspension was layered on slides coated with 1% normalmelting point agarose and immersed in cold lysis buffer (2.5 M NaCl, 100 mM Na2-

EDTA, 10 mM Tris–HCl, 1% (v/v) Triton-X-100, 10% (v/v) DMSO; pH 10). Followed bylysis, the DNA was allowed to unwind in electrophoresis buffer for 20 min and elec-trophoreses at a constant voltage of 25 V and 300 mA at 4 �C. Slides were neutral-ized in 0.4 M Tris (pH 7.5) for 15 min and finally rinsed with water. Slides werescored using image analysis system (Kinetic imaging; Andor technology, Notting-ham, UK) attached to a fluorescence microscope (Leica, Wetzlar, Germany)equipped with appropriate filters (N2.1). The microscope was connected to a com-puter through a charge-coupled device (CCD) camera to transport images to soft-ware (Komet 5.5) for analysis. One hundred and fifty nuclei were scored pertreatment and expressed as percentage of tail DNA.

2.5.2. DNA diffusion assayDNA diffusion assay was done at similar concentrations to determine the

amount of DNA fragmentation associated with apoptosis and necrosis (Gichneret al., 2005; Singh, 2005). Apoptotic cells show nuclear DNA with a dense centralzone and a lighter hazy outer zone with halo like appearance due to nucleosome-sized DNA whereas nuclei due to necrosis has well defined outer boundary with rel-atively clear appearance. For this assay, cells (1 � 106/ml) were incubated for 3 h at37 �C and processed in a manner similar to the comet assay except the nuclei were

not subjected to electrophoresis. Followed by lysing, the slides were placed for 1 hin a solution of 50% ethanol and 50% Tris buffer (400 mM, pH 7.4), with a final con-centration of 1 mg spermine/ml to remove the precipitated salts and detergentswhile retaining DNA in the agarose. Simultaneously negative and positive (Methylmethanesulphonate, 50 lM) controls were maintained. The slides were stainedwith EtBr and nuclei were analyzed under fluorescent microscope. Slides werescored using image analysis system (Kinetic imaging; Andor technology, Notting-ham, UK) attached to a fluorescence microscope (Leica, Wetzlar, Germany)equipped with appropriate filters (N2.1). The microscope was connected to a com-puter through a charge-coupled device (CCD) camera to transport images to soft-ware (Komet 5.5) for analysis. From each replicate experiment, the percentage ofdiffused nuclei and the average nuclear area on each slide were used to expressthe nuclear DNA diffusion.

2.6. Detection of apoptosis/necrosis using Annexin V-FITC/PI staining

Annexin V-FITC/PI staining was performed for quantification of apoptosis andnecrotic cell death using FITC Annexin V apoptosis detection kit I. Lymphocytes(1 � 106 cells/ml) were incubated with the test substances at concentrations(palmarosa, citronella and geraniol-1000, 1500 and 2000 lg/ml; lemongrass, citraland vetiver acetate-200, 400 and 800 lg/ml) as cytotoxicity and genotoxicity in-creased at higher concentrations. After treatment for 3 h at 37 �C, the lymphocyteswere washed in cold PBS and resuspended in 1� 100 ll calcium containing bindingbuffer (10 mM HEPES, 140 mM NaCl, 5 mM CaCl2; pH 7.4) at a concentration of1 � 106 cells/ml and stained for 15 min, with 5 ll Annexin V-FITC (BD Pharmingen,USA) and 5 ll PI at 1 lg/ml. 20,000 cells were analysed at an excitation wavelengthof 488 nm and emission wavelengths of 530 nm for FITC fluorescence and 610 nmfor PI fluorescence. The percentages of viable (Annexin V�PI�), early apoptotic(Annexin V+PI�), late apoptotic/necrotic (Annexin V+PI+) and necrotic cells (AnnexinV�PI+) were evaluated with the CellQuestPro� software (Becton, Dickinson,Heidelberg, Germany).

2.7. Reactive oxygen species level in human peripheral lymphocytes

Intracellular ROS generation was evaluated using the fluorescent probe20 ,70-dichlorofluorescein diacetate (DCFH-DA) in human lymphocytes. The cells(1 � 106 cells/ml) exposed to the test substances (palmarosa, citronella and

Fig. 3. DNA diffusion assay in human lymphocytes treated with different concentrations of the test substances; (a) palmarosa, (b) citronella, (c) geraniol, (d) lemongrass, (e)citral, and (f) vetiver acetate.

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geraniol-1000, 1500 and 2000 lg/ml; lemongrass, citral and vetiver acetate-200,400 and 800 lg/ml) for 3 h at 37 �C were collected by centrifugation and washedin PBS at 1000 rpm for 5 min. Thereafter, cells were resuspended in PBS andDCFH-DA was added to a final concentration of 25 lM. The suspension was incu-bated in dark for 30 min at 37 �C. The samples were analyzed by a flow cytometer(FACS Aria III with cell Sorter, BD, San Jose, CA, USA). Approximately 20,000 eventswere recorded from each sample and processing was performed with FACS Divasoftware (BD, San Jose, CA, USA). Results were expressed as the fold change overthe control.

2.8. Statistical analyses

The data are represented as Mean ± SEM. The data were analyzed using the Sta-tistical Programme – SigmaStat 3.0 (SPSS Inc., Chicago, IL, USA) and the level of sig-nificance was established at P 6 0.05. One-way analysis of variance (ANOVA) testwas done for trypan blue dye exclusion test, MTT assay, single cell gel electropho-resis, DNA diffusion assay, Annexin V/PI and DCFH-DA staining. Dunnett’s multiplecomparison tests were performed to compare the data of individual concentrationswith that of the negative control.

3. Results

3.1. Cytotoxicity assays in human lymphocytes

The results of the cytotoxicity assays revealed reduction in via-bility of human lymphocytes treated with the essential oils, citraland geraniol (Fig. 1a–f). Trypan blue dye exclusion test showed de-crease in cell viability in lymphocytes but was not found signifi-cant. Relative to the control, all the test samples except geraniolexhibited statistically significant cytotoxicity at the higher concen-trations as demonstrated by the MTT assay. Citral exhibited maxi-mum cytotoxic potential reducing cell viability to 75.69%. The cell

viability curve of trypan blue dye exclusion method showed simi-lar pattern like MTT assay but with lesser toxicity.

3.2. DNA damage induced by the essential oils, citral and geraniol

Palmarosa and citronella oils could induce significant DNA dam-age at concentrations of 1000 lg/ml and above (Fig. 2a and b) andtheir major component geraniol did not reveal any genotoxicity(Fig. 2c). DNA strand breaks induced by the lemongrass oil and itsmajor component citral exhibited significant DNA damaging poten-tial on human lymphocytes at 100 and 25 lg/ml and above respec-tively (Fig. 2d and e). Vetiver acetate oil was found significantlygenotoxic at concentrations 100 lg/ml and above. Methyl metha-nesulphonate used as positive control induced significant increaseof DNA strand-breaks (65–70% tail DNA). Results of the DNA diffu-sion assay showed significant DNA damaging potential similar tothe comet assay as demonstrated by increased nuclear area andthe number of diffused nuclei (Fig. 3a–f). Methyl methanesulpho-nate resulted in an increase in the nuclear area (5056 lm2) andthe number of diffused nuclei (34%; data not shown).

3.3. Apoptosis induced by the essential oils, citral and geraniol

Annexin V-FITC/PI staining was performed to assess the extentand mode of cell death. Experimental results indicated that celldeath was primarily due to apoptosis in all the test substances incomparison with the proportion of necrosis (Fig. 4a–f). A smallfraction of cells were found to undergo apoptosis in lymphocytestreated with the palmarosa oil (10.77%) and geraniol (7.16%) as

Fig. 4. Flow cytometric analysis of Annexin V-FITC/PI stained human lymphocytes treated with different concentrations of the test substances; (a) palmarosa, (b) citronella,(c) geraniol, (d) lemongrass, (e) citral, and (f) vetiver acetate.

S. Sinha et al. / Food and Chemical Toxicology 68 (2014) 71–77 75

interpreted in Fig. 4a and c. Citronella and lemongrass oils inducedconsiderable apoptosis in lymphocytes (39.62% and 30.12% respec-tively) whereas citral resulted in maximum number of cell deathby apoptosis (43.94% increase with respect to the control) as repre-sented in Fig. 4e.

3.4. Estimation of DCFH-DA oxidation in human lymphocytes by flowcytometry

Upon oxidation by ROS, the nonfluorescent DCFH-DA is con-verted to the highly fluorescent 20,70-dichlorofluorescein (DCF)which was quantified by flow cytometry. Results indicated signifi-cant increase in ROS production at all the treatment concentrationstested for citronella, lemongrass and citral (Fig. 5b, d and e).Vetiver acetate oil generated ROS significantly at concentrationsof 400 lg/ml and above (Fig. 5f). Palmarosa oil and geraniolshowed non-significant amount of ROS generation in human lym-phocytes in comparison with the control set (Fig. 5a and c).

4. Discussion

There is an increasing demand for screening of new synthetic andnatural active constituents with applications in pharmaceutical andfood industry. A number of essential oils and their components havebeen registered and classified as GRAS (Generally recognized assafe) by the US FDA and approved for use as food additives. Never-theless, report by Naik et al., 2003 mentioned there is an inverserelationship between the dietary intake of anti-oxidant rich foodsand incidence of human disease. Tripathi et al., 2006 showed oral

administration of vetiver oil in rats for 45–90 days elicited mildhematotoxic effect. Hepatotoxic and nephrotoxic effects in micetreated with the extracts of C. citratus (30% and 80%) were observedby Guerra et al., 2000. Recently Sousa et al., 2010 reported cytogeno-toxicity of the extract from C. citratus (DC) Stapf inducing chromo-some aberration and cell death in roots of Lactuca sativa. Inaddition, estragole and methyl eugenol were found genotoxic andwere delisted from GRAS (Commission Decision of 23 January,2002). Considering this, the present study was focused on the safetyevaluation of the four essential oils per se and the two major compo-nents citral and geraniol. The present investigation suggest that hu-man lymphocytes exposed to the test substances except geraniolsignificantly modified mitochondrial activity, induced DNA damage,generated ROS and come up against death primarily by apoptosis.

The four essential oils and citral were found to be significantlycytotoxic at high concentrations whereas geraniol did not showany cytotoxicity as revealed by the MTT assay. Results of trypanblue dye exclusion test differed from the MTT assay which didnot reflect significant reduction in cell viability. As trypan bluedye reflects only a loss of plasma membrane integrity associatedwith necrosis (Bonfoco et al., 1995), the results could be correlatedto the effect on metabolic activity or apoptosis as evidenced by theMTT assay. The genotoxicity assays confirmed DNA damages inlymphocytes at concentrations reflecting significant cytotoxicity.Similar to the results of alkaline comet assay, the DNA diffusion as-say demonstrated formation of DNA fragments at the high concen-trations which might be due to apoptosis/necrosis. Therefore, inorder to establish the mode of death in lymphocytes, we conductedAnnexin/PI double staining method using flow cytometry thatshowed the percentages of cells undergoing apoptotic and necrotic

Fig. 5. Flow cytometric estimation of reactive oxygen species (ROS) generation in human lymphocytes, at different concentrations of the test substances; (a) palmarosa, (b)citronella, (c) geraniol, (d) lemongrass, (e) citral, and (f) vetiver acetate.

76 S. Sinha et al. / Food and Chemical Toxicology 68 (2014) 71–77

cell death. Flow cytometry analysis of lymphocytes exhibited pre-dominant apoptosis in citral, lemongrass and vetiver acetate oils incomparison with the other test substances.

To elucidate the possible cause of apoptosis we paid muchattention to the intracellular redox status among the various othermechanisms. In recent years, antioxidants and prooxidants havebeen extensively studied and it seems that most of the antioxi-dants can behave as prooxidants which depends on their concen-tration and the nature of the neighbouring molecules (Villanuevaand Kross, 2012). Therefore, we determined the ability of the testsubstances to generate reactive oxygen species. ROS generationand cell death was low in human lymphocytes treated with gera-niol and palmarosa oil (less genotoxic) whereas it was found tobe highest in citral. The degree of ROS generation was in thedecreasing order of citral > lemongrass > vetiver acetate > citro-nella > palmarosa > geraniol. The toxic effects of the lemongrassoil may be due to the presence of citral as the major componentwhereas considerable less toxic potentials of palmarosa and citro-nella oils could be attributed to the presence of geraniol. The resultof DCFH-DA staining may lead to the speculation that the test sub-stances can behave as a prooxidant at certain concentrations. Sim-ilarly, well known antioxidants such as vitamin C, a-Tocopherol,carotenoids, flavonoids and phenols were found to become proox-idants at high concentrations (Cillard et al., 1980; Duarte and Lu-nec, 2005; Galati and O’Brien, 2004; Yordi et al., 2012; Youngand Lowe, 2001).

In conclusion, based on the results of the present study the fouressential oils can be considered safe at low concentration for hu-man consumptions.

Conflict of Interest

The authors declare that there is no conflict of interest.

Transparency Document

The Transparency document associated with this article can befound in the online version.

Acknowledgment

This work was supported by the DST–PURSE program, Depart-ment of Botany, University of Calcutta. The authors (SS, MJ andMG) would like to thank CSIR and UGC-RFSMS, Government of In-dia for providing financial support [Sanction number: 09/028(0728)/2008-EMR-I and 09/028(0860)/2012-EMR-I].

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