Chemical Composition and Cytotoxic and Antibacterial Activities of the Essential Oil of Aloysia citriodora Palau Grown in Morocco

The aim of this work is to investigate the in vitro cytotoxic and antibacterial effects of the essential oils of Aloysia citriodora Palau, harvested in different regions of Morocco. The chemical profile was established using gas chromatography-mass spectrometry analysis. The cytotoxic activity against P815, MCF7, and VERO cell lines as well as the normal human peripheral blood mononuclear cells (PBMCs) was evaluated using the MTT assay. Standard, ATCC, strains of bacteria (Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa) were cultivated in Muller Hinton media. Then, agar disc diffusion, minimum inhibitory concentrations (MICs), and minimal bactericidal concentrations (MBCs) were determined using microdilution method. The essential oils obtained were predominantly composed of β-spathulenol (15.61%), Ar-curcumene (14.15%), trans-caryophyllene oxide (14.14%), and neral (10.02%). The results of the assays showed that the cytotoxic effect of the essential oil of A. citriodora was high on P815 and moderate on MCF7 and on VERO cell lines. However, no cytotoxic effect was observed on PBMCs. On the other hand, essential oils showed a significant antimicrobial activity against both Gram-negative and Gram-positive bacteria. MICs ranged between 2.84 and 8.37 mg/ml. Essential oil of A. citriodora leaves possesses significant antibacterial effect and cytotoxic activity against tumor cell lines.


Introduction
The genus Aloysia belongs to the Verbenaceae family and consists of approximately 200 species of herbs, shrubs, and small trees which are often aromatic [1]. The species Aloysia citriodora Palau or Lippia citriodora (H.B. & K.) is commonly known as Lemon Verbena, Verbena grass Louise, Arabic tea, and lemongrass. This plant is growing spontaneously in South America, especially in Argentina and Chile. In Morocco, this species is cultivated for more than a century and has been used in folk medicine as herbal tea preparations, for its antispasmodic, digestive, stomachic, sedative, and antipyretic properties. The essential oil extracted from the dried leaves of A. citriodora is indicated for anxiety, stress, insomnia, some depressions, nervous fatigue, multiple sclerosis, psoriasis, tachycardia, rheumatism, enterocolitis, Crohn's disease, anorexia, dyspepsia, intestinal parasites (amebiasis and amebic cysts), and prevention of asthma attacks [2][3][4]. The broad range of biological activities of essential oils could be generally correlated to the chemical composition. Therefore, this biological difference can be partly explained by the variation in their chemical composition [5]. It is well established that sesquiterpenoids and their derivatives are credited with many biological activities such as anti-inflammatory, antibacterial, antiasthmatic, and antifungal properties [6]. Thus, structure-activity relationships describe broad classes of activities for the different chemical groups of molecules found in essential oils.
In the present study, we analyzed the chemical composition analysis; the cytotoxic and the antibacterial activities of essential oil of A. citriodora distilled from the shade-dried 2 Advances in Pharmacological Sciences leaves harvested in the summer season (July-August 2015) in different Moroccan regions.

Plant Material.
The leaves of A. citriodora Palau were collected during July-August 2015 (the period of maximum essential oils production) [7], from the following Moroccan regions: Marrakech (Ait Imour region), Beni Mellal (Laâyayta locality), Agadir (Oulad Tayma), and Berkane and Demnate (mountain region). The plants were grown in organic farming without pesticide treatments. The botanical determination was performed by Pr. A. Boulli (Laboratory of Natural Resources Valorization, Faculty of Science and Technology, Beni Mellal), and a voucher specimen was deposited in the Herbarium at the Faculty of Science and Technology, Beni Mellal, Morocco, under reference: FSTBMCOLL72015.

Extraction of the Essential Oil.
The collected leaves of A. citriodora Palau were shade-dried during two weeks. Then the essential oil was obtained by hydrodistillation using a Clevenger-type device (samples were 100 g of crushed leaves in 1.5 l distilled water). At the end of the distillation process, the organic phase which contains the essential oil is separated from the hydrosol (aqueous phase) with ether followed by gentle removal of the solvent by evaporation. The yield of essential oil ranged from 0.1% to 0.2%.

GC-MS.
The obtained essential oil samples were analyzed with a gas chromatograph Trace GC Ultra equipped with a FID detector and a capillary column (30 m × 0.25 mm, 0.25 m film thickness) DB-5 (methyl polysiloxane with 5% phenyl); the injection volume was 1 L. The gas chromatograph was coupled to a mass spectrometer Q Polaris MS that performs the analysis of the mass spectrometry (70 eV with an ion trap). The temperature program was 40 ∘ C for 2 min and then heated to 180 ∘ C at a rate of 4 ∘ C/min. The carrier gas was helium (1.4 ml/min).

Cytotoxicity Assay.
The cytotoxic effect of essential oils was evaluated against P815 (Murine Mastocytoma, cell line), MCF7 (human breast adenocarcinoma), and VERO (kidney carcinoma cell line of monkey). The used cell lines were obtained as a gift of Professor G. Lemaire, Institute of Biochemistry, University of Paris XI, France. PBMCs were isolated using the standard Ficoll-Hypaque density gradient from samples of healthy volunteers under medical supervision. These cells were maintained and grown in special culture medium (RPMI-1640, Sigma-Aldrich, France) supplemented with 10% heat inactivated fetal calf serum (FCS) and 1% penicillin-streptomycin (Sigma-Aldrich). Cultures were maintained at 37 ∘ C in humidified 5% CO 2 incubator. The cytotoxicity assays were performed in triplicate on each cell line using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay (Sigma-Aldrich), as described and modified by Mosmann [8] and Mouse et al. [9].
All experiments were performed with twice PBS washed cells at a density of about 1.5 × 10 4 cells per well (1.5 × 10 5 cells/ml), in flat-bottomed 96-well microtiter plates (Thermo Fisher Scientific) in 100 l complete medium per well. Then 100 l of complete culture medium containing different concentrations of the tested essential oils (dissolved in DMSO) was added to each well. After 48 h exposure of cells to these concentrations of tested essential oils at 37 ∘ C and 5% CO 2 , 100 l of medium was carefully aspirated from each well and replaced with 20 l of MTT solution (5 mg/ml of PBS). After incubation in the same conditions for 4 hours, the plates were treated with 80 l of an HCl/isopropanol (24 : 1) solution to dissolve the blue intracellular formazan produced by living cells. One hour later, the absorbance (optical density OD) of the plates was determined by a Multiskan EX spectrophotometer reader at two wavelengths (540 and 630 nm). DMSO (0.5%) was used as the negative control and methotrexate (MTX) as the positive one.
The viability was evaluated by the following formula: OD is the optical density of the solution in wells containing cells treated with essential oils and OD 0 is the optical density of the solution in wells containing DMSO treated cells (negative control  [10]. The Mueller-Hinton agar media in Petri dishes was swabbed with 100 l of the bacterial suspension 10 8 CFU/ml (0.5 NTU McFarland) and kept for 30 min at 4 ∘ C. Sterile filter paper discs (6 mm in diameter) [11] were soaked with 6 l essential oil and placed aseptically on the surface of the inoculated Muller-Hinton agar plates. Plates were then incubated at 37 ∘ C for 24 h and the diameters of bacterial growth inhibition zones (in millimeters) were recorded. Standard antibiotic discs (6 mm) were used as positive controls: ceftriaxone (30 g), ofloxacin (5 g), and blank discs were used as negative control. the essential oil concentrations to be incubated at 37 ∘ C for 24 h with the bacterial suspensions adjusted to 10 5 CFU/ml in wells of 96-well microplates (Thermo Fisher Scientific). Untreated control (wells containing bacteria with DMSO at a concentration of 1%) was included in the assay. After incubation, the wells were optical density examined (OD 600 nm) with a spectrophotometer (Multiskan EX) for bacterial growth.

MICs and MBCs
The MIC was determined as follows: OD essential oils is the optical density of the suspension in wells containing essential oils treated bacteria and OD Untreated control is the optical density of the suspension in wells containing DMSO (without essential oils) treated bacteria (negative control). The MIC is defined as the lowest concentration of the essential oil at which the bacteria does not show visible growth.
MBC is defined as the lowest concentration of the essential oil at which incubated microorganisms were completely killed.

Statistical
Analysis. Data were expressed as mean values ± SEM of three different experiments; each one was performed in duplicate. Statistical significance was determined with the one-way analysis of variance followed by a post hoc Scheffe's test. The differences were considered statistically significant at < 0.05.

Extraction and Chemical Composition of the Essential Oils.
Distilled essential oil of A. citriodora features: appearance (mobile liquid, clear), color (yellow, more or less dark), characteristic odor (lemony, fresh), and density at 20 ∘ C (0.879).
Among 72 peaks revealed by chromatography ( Figure 1), 64 peaks (96%) were identified (according to National Institute of Standards and Technology database). Twenty-four among 64 peaks represent 80% of the essential oil compounds (Table 1). Eight peaks were for unknown compounds and probably represent 4% of the essential oils.

Essential Oils Mediated Cell Cytotoxicity.
The assays showed that the essential oil of A. citriodora exerted a dose dependent cytotoxic effect on P815, MCF7, and VERO tumor cell lines with IC 50 ranging from 6.60 to 79.63 g/ml. (Figures 2, 3, 4, and 5). However, no cytotoxicity was observed against PBMCs. In fact, the viability was over 80% at the concentration used to induce tumor cell lysis ( Figure 6).

Disc Diffusion Assay.
Inhibition zone diameters of the disc diffusion assays were recorded in

Chemical Composition Analysis.
The chromatographic spectra of different essential oils from different Moroccan regions revealed the same qualitative chemical composition with some quantitative differences. -spathulenol is the most abundant compound and represents 15% of the essential oil from Berkane region. trans-Caryophyllene oxide prevails in essential oils from all regions with an average rate of 14%. Arcurcumene is found to represent 14% of the essential oils from Beni Mellal and Agadir regions and 11% of those from other regions. Neral represents 10% of the essential oil from Agadir region and 6% to 8% of those from other regions. cis-Verbenol represents nearly 8% of the essential oil from Agadir region and 4% to 6% of other essential oils. Paracymene was only present in the essential oil from Agadir region at 0.02%.muurolene was only found at 0.04% in the essential oils from Beni Mellal and Demnate regions. 8-Cedren-13-ol was not found in the essential oil from Agadir and represented 0.01 to 0.05% of the essential oils from other regions.
The major molecules dominating the chemical composition of essential oils are caryophyllene oxide, -spathulenol, Ar-curcumene, neral, and cis-verbenol (Table 2). Other products such as -caryophyllene, isoledene, elemene, 1.8-cineole, copaene, and nerol are also present with variable ratios. Unlikely, limonene and 6-methyl-5-hepten-2-one, which are compounds constantly found in A. citriodora essential oils distilled in Morocco at proportions of 14% for the first and 3% for the second, are present in the studied essential oils in the present work at very low averaging rates of 0.02% to 0.5%. In fact, the compositions of the essential oils depend on the operating (drying, extraction methods) and on the harvest and the storage conditions [12,13].
The main compounds found in the present investigation were different quantitatively and qualitatively from those         reported in the literature. The average rate of limonene and geranial was 17% and 9%, respectively, in different essential oils distilled from A. citriodora grown in Morocco (leaves of A. citriodora harvested in May period), whereas the amount of the monoterpene (limonene) is only 0.52% and the terpene aldehyde (geranial) is not detected in the present study. However, the rates of sesquiterpenes ( -caryophyllene, Ar-curcumene, -elemene, etc.), terpene oxides (caryophyllene oxide), some monoterpene alcohols (cis-verbenol), and sesquiterpene alcohols (spathulenol) were higher in the present studied essential oils [16]. It is interesting to note that limonene, -caryophyllene, p-cymene, linalool, citral,pinene, and 1.8-cineole are common compounds of essential oils of Lippia sp. and thought to be responsible for specific

Cytotoxicity against P815, MCF7, VERO, and PBMCs Cell
Lines. Based on the essential oils cytotoxicity ranking [17], the five essential oils studied marked very high cytotoxic effect against P815 (BE essential oil is the most cytotoxic with IC 50 = 6.60 g/ml) and high to moderate activity against MCF7 and VERO cell lines (DE essential oil is the most cytotoxic with IC 50 = 34.72 g/ml and 32.90 g/ml, resp.). To the best of our knowledge, there is no previous report about the cytotoxic activity of A. citriodora essential oil. However, the cytotoxic effect of Lippia alba, with its main constituent citral on HeLa and VERO cell lines was reported [18]. The interesting cytotoxicity toward cell lines could be due to  the main compounds in the essential oil. Literature reports the induction of apoptosis by citrals (neral and geranial) in chronic lymphoid leukemia by activation of caspase-3 [19,20]. Moreover, it has also been demonstrated thatcaryophyllene and caryophyllene oxide were also reported to induce apoptosis in tumor cells and exert analgesic, antiinflammatory, antibacterial, antifungal, and sedative activities [21,22]. Furthermore, -elemene and spathulenol were reported as having anticancer activities on human glioblastoma and on MCF7 cancer cell lines [23][24][25]. Interestingly,   [26]. Interestingly, it was observable that cytotoxic and antibacterial activities of essential oils were different; for example, MA presented the moderate cytotoxicity but a very strong antibacterial activity. This may be explained by qualitative and quantitative variability of chemical compounds identified in these essential oils and their differential molecular mechanisms.
Generally, the first site of action of essential oils on the bacterial cells is the membranes. This is directly related to the hydrophobicity of the essential oils components. This property ensures and facilitates permeability of membrane phospholipids bilayer to these molecules. The result is a destabilization of the plasma membrane structure and a change in its permeability to ions, protons, and other cellular components [27][28][29][30]. In addition to the induced membrane alterations, such molecules can cross the lipid bilayer and interact with intracytoplasmic targets [31]. Given the molecular diversity of the essential oils, it appears more likely that their antibacterial activity results from the combination of several mechanisms, acting synergistically on different cellular targets [32]. In fact, among the dominant molecules in the five studied essential oils, caryophyllene and -caryophyllene oxide have antibacterial and antifungal activities [33]. Oxygenates (caryophyllene oxide, 1.8-cineole, etc.) have in vitro antibacterial activity [34]; citral (neral and geranial) has a strong antibacterial effect [35]. The interaction between p-cymene, -terpinene, and the phenolic compounds could exhibt an antibacterial activity [36]. Furthermore, the essential oils showing high antibacterial activity are phenol and cinnamic aldehyde rich oils (thymol, carvacrol, and eugenol) as in the essential oils of Thymus vulgaris, Origanum compactum, Satureja montana, Eugenia caryophyllata, and Cinnamomum zeylanicum [37]. However, antimicrobial effectiveness of an essential oil is due to the nature and content of these various constituents that may act synergistically; the effect of minor compounds is not always negligible. Synergism between constituents can cause a much more pronounced effect than the expected activity of the major compounds [38].

Conclusion
The results obtained in this study showed that the molecular profile of the essential oil of A. citriodora grown in Morocco is slightly variable depending on the region where the plant was harvested. In vitro tested essential oils of A. citriodora have a strong cytotoxic activity against P815 (IC 50 = 6.60 g/ml), compared to MCF7 (IC 50 = 34.72 g/ml) and VERO (IC 50 = 32.90 g/ml). A moderate antibacterial activity on E. coli ATCC 25922 and on S. aureus ATCC 25923 was recorded. However, P. aeruginosa ATCC 27853 strain was resistant to these essential oils. Cytotoxic and antibacterial activities depend on the chemical nature and interactions of A. citriodora essential oil's compounds. Further studies need to be conducted on apoptosis induction, genes expression (Bcl2, p53, Bax, Jun/Fos, etc.), and antitumor activity of the main compounds of A. citriodora essential oil to understand the involved molecular pathways.

Conflicts of Interest
The authors do not have any conflicts of interest regarding the content of the present work.