Properties and limits of some essential oils: chemical characterisation, antimicrobial activity, interaction with antibiotics and cytotoxicity

Abstract Because of the emergence of multi-drug resistance bacteria and fungi, alternatives to conventional antimicrobial therapy are needed. This study aims to evaluate in vitro the antimicrobial activity of: Mirtus communis, Coriandrum sativum, Pelargonium capitatum, Cuminum cyminum, Ocimum basilicum, Citrus aurantium amara, Cymbopogon. winterianus, Cymbopogon martini, Salvia sclarea, Melaleuca alternifolia and Mentha suaveolens essential oils on bacteria and fungi, in relation to their chemical composition. The potential interaction of M. alternifolia (TTO), C. sativum (CDO) and M. suaveolens (EOMS) essential oils when used in combination with gentamicin and fluconazole has been evaluated. The results obtained showed a synergic effect on some bacteria and fungi, with FICI values ≤5. The cytotoxicity of TTO, CDO and EOMS was investigated towards HeLa cells. Only EOMS did not result cytotoxic at the active concentrations on micro-organisms. Further studies are necessary to obtain optimal ratios and dosing regimens for higher therapeutic efficacy and to decrease toxicological profiles.


Introduction
Plant-derived compounds are always a source of novel therapeutics. Plants are known to produce an enormous variety of small molecules (MW <500 kDa) known as phytoalexins such as terpenoids, glycosteroids, flavonoids and polyphenols. Most of these small molecules have weak antimicrobial activity, several orders of magnitude less than that of common antibiotics produced by bacteria and fungi. In spite of the fact that plant-derived antibacterials are less potent (Seow et al. 2014), plants fight infections successfully. Aromatic and medicinal plants also produce a wide variety of volatile aliphatic and cyclic hydrocarbons, corresponding oxygenated isoprenoid derivatives and analogues that form complex mixtures called essential oils (Hammer et al. 1999;Rios & Recio 2005). Essential oils are a rich source of biologically active compounds; they were shown to possess antibacterial, antifungal, antiviral, insecticidal and antioxidant properties (Solorzano-Santos & Mirandas-Novales 2012). Some oils have also been used in cancer treatment (Gautam et al. 2014) in food preservation, aromatherapy and fragrance industry. Because of all these properties, medicinal and aromatic plants which and constitute a major source of important organic compounds (Gurib-Fakim 2006) and World Health Organization noted that the majority of world's population depends on traditional medicine for primary healthcare. Commercially available essential oils from aromatic plants such as Melaleuca alternifolia (tea tree oil), Thymus vulgaris (Thyme), Mentha piperita (Peppermint), Rosmarinus officinalis (Rosemary), Citrus aurantium (Lime oil) and Cymbopogon martini (Palmarosa oil) have been extensively used for the treatment of topical bacterial (Prabuseenivasan et al. 2006) and fungal infections. Indeed, Coriandrum sativum has been recommended for dyspeptic complaints, loss of appetite, convulsion, insomnia and anxiety (Emamghoreishi et al. 2005) in addition to its antimicrobial activity (Silva et al. 2011). Also, essential oils from Cuminum cyminum (cumin) (Iacobellis et al. 2005), Ocimum basilicum (basil) (Hussain et al. 2008), Mirtus communis (myrtle) (Ragno et al. 2008) are known for antimicrobial and antioxidant properties. Gram positive and negative bacteria, in addition to fungi, are responsible for several human infections. Effective antimicrobials have been developed over the years; however, it has been observed a dramatic increase in resistance to antimicrobial drugs. Although antibiotics have been effective to fight infectious diseases for a long time, resistance to these drugs has also led to the reemergence of old infectious diseases. For this reason, the development of new antimicrobial compounds appears necessary. Actually, one strategy employed to overcome resistance mechanisms has been the use of combinations of drugs, such as beta-lactams together with beta-lactamase inhibitors or combinations of different classes of drugs (Rey-Jurado et al. 2013). Medicinal plants and herbs represent preferred sources of active molecules which could become lead compounds for new pharmaceutical products. Moreover, several plant extracts exhibited 'in vitro' synergic activity when utilised in combination with different drugs against several micro-organisms and could represent a new alternative approach to infectious diseases treatment. Further studies are necessary to better define cytotoxicity of EOs, since in literature, a data reported mainly the effect on tumoral cell lines. (Elsayed et al. 2015).This article reports the antimicrobial screening of eleven essential oils against some bacterial and fungal species, the interaction of C. sativum, M. alternifolia and Mentha suaveolens EOs with conventional antimicrobial drugs such as gentamicin or fluconazole and toxicity studies towards a cell line of human origin (HeLa).

Antimicrobial activity of essential oils by diffusion test
The antimicrobial activity of eleven EOs measured by diffusion test against selected species of bacteria and fungi is summarised in Table S1. The results reveal that EOs show antimicrobial activity with varying magnitudes and specie specificity. The zone of growth inhibition above 8 mm in diameter was taken as a positive result. Generally, most of the tested microorganisms were sensitive to many of the EOs. At a concentration of 20 mg/ml, the best activity has been expressed by M. alternifolia, M. communis, C. sativum, C. cyminum, Cymbopogon winteranius and M. suaveolens EOs, with inhibition halos between 33.7 and 10.4 mm diameter for gram-positive or gram-negative bacteria and between 70 and 12 mm for fungi. In particular, M. alternifolia, Coriandrum sativum and M. communis EOs produced inhibition halos between 17 and 33.7 mm, or between 18.4 and 25 mm against Staphylococcus epidermidis strains and Staphylococcus aureus ATCC 25923, respectively. In the case of gram-negative bacteria, inhibition halos ranged between 17 and 29.4 mm, 29-30 mm, 23.5 and 29.5 mm for the multi-resistant clinical isolate of Klebsiella pneumoniae, Acinetobacter baumanii and Escherichia coli ATCC 25922, respectively. Moreover, the same oils showed a strong activity towards Candida albicans strains with inhibition halos between 30 and 60 mm. M. suaveolens, C. cyminum and C. winteranius showed a lower antibacterial activity with inhibition halos ranging from 13.5 to 19.6 mm for all tested bacteria, while they resulted active towards fungi with inhibition halos between 20 and 40 mm. The observed variability in sensitivity might be due to the different chemical composition of each essential oil and to the kind of micro-organism tested. These differences could also be referred to a different rate of essential oils constituent's penetration through the cell wall and cell membrane structures. For example, the antibacterial properties of tea tree oil (TTO) have been attributed to the monoterpenoid, terpinen-4-ol. Because of its lipophilic nature, it is thought to diffuse into and damage cell membrane structures, or to inhibit membrane-bound enzymes (zengin & Baysal 2014). Linalool, which is the main constituent in C. sativum EO, is known to bind to membrane ergosterol, increasing ionic permeability and causing membrane damage leading to cell death. Linalool is inactive (Freires et al. 2014) on cell wall biosynthesis-related pathways. The ability of essential oils to disrupt the permeability barrier of the cell membrane structures and the accompanying loss of chemiosmotic control are the most likely reasons for their lethal action. The active concentrations of oils were generally about 100 times higher than those of gentamicin or fluconazole. This difference could be explained taking into account that essential oils are complex mixtures of different compounds, where only some molecules could have antimicrobial properties. Identification and purification of the different components could highlight the most active antimicrobial molecules.

Chemical composition of essential oils
Taking into account the chemical composition of the EOs reported in Table 1, their activity towards gram-positive or gram-negative bacteria and fungi could be related to the functional group that characterises the main component. Nevertheless, it has to be considered that the presence of minor components might also play a role to determine such an effect. The antibacterial and antifungal activity of C. aurantium amara essential oil could be attributed to the presence of limonene, a monoterpene that represents 90.7% of the total extract. The  antimicrobial activity described is in agreement with literature data (Viuda-Martos et al. 2008;ulukanli et al. 2014). C. winteranius and C. cyminum essential oils are both active against fungi. The antifungal activity of these extracts can be ascribed to the two aldehydes representing the main components: cuminal (49.6%) for C. cyminum and β-citronellal (41.8%) for C. winteranius. Cuminal, an uncommon aldehyde, is present in Eucalyptus camaldulensis essential oils from Sardinia, which is known for antifungal activity towards common phytopathogenic fungi (Barra et al. 2010). A main component with alcoholic function characterises C. sativum, C. martini, P. capitatum and M. alternifolia essential oils; in particular, β-citronellol, the main component of P. capitatum (58.8%), may be responsible for the antifungal activity of this essential oil, whereas terpinen-4-ol, geraniol and linalool which are the main components of M. alternifolia (53.7%), C. martini, (83.9%) and C. sativum (48.4%), respectively, could account for the antibacterial activity showed by these extracts. O. basilicum essential oil is the only extract characterised by an ether as a main component: estragole, representing 91.1% of this oil. It is partially active against gram-positive bacteria and fungi. Regard to this component, literature data show different biological activities, in particular, it is known to increase phagocytic activity of macrophages (Silva-Comar et al. 2014). The observed antifungal and antibacterial activity of M. suaveolens essential oil utilised in this study could be attributed, at least partially to piperitenone, a ketone oxide that represents 38.1% of extract. It must be observed that in other extracts of M. suaveolens, EOs piperitenone oxide was about 90% (Angiolella et al. 2010). This fact can be attributed to the different plant cultivation zone in agreement with Khaoukha et al. (2014) and to the seasonal variations of the active constituents (Settanni et al. 2014). In the tested extract, other components are limonene 9.1%, linalool 7.8%, and other minor chemical constituents. M. communis essential oil is active versus fungi and gram-negative bacteria. This oil contains as main component eucalyptol, a monoterpene oxide representing 36.4% of the total extract, in agreement with Akin et al. (2010), and also other constituents such as myrtenyl acetate, a terpene ester 31.8%, limonene and alphapinene about 10%. As to, S. sclarea essential oil tested in this work, it is not characterised by a main component but contains more than ten different molecules with a percentage that never exceeds 20%, except for caryophyllene 25.8% and α-caryophyllene 22.4%. In our study, this oil demonstrated only poor antibacterial and antifungal activity (Table S1). On the contrary, low percentages of borneol and α-pinene in Ampelopsis megalophylla as reported by Xie et al. (2014) and caryophyllene (Joycharat et al. 2014) show high microbiological activity. TTO which exhibited MIC values of 3.12 mg/ml. Altogether these results are in agreement with the high variability of the antimicrobial activity of EOs observed with the disc diffusion method. All EOs MIC values were in the order of magnitude of milligrams and therefore significantly higher than those obtained with the reference antimicrobial drugs. The combination of antimicrobials with EOs could represent a possible alternative strategy to increase the efficacy of antibacterial and antifungal drugs. A clear synergic effect of essential oils with fluconazole has been reported against C. albicans (Stringaro et al. 2014) and with different antibacterial substances towards gram-negative bacteria (Toroglu 2007(Toroglu , 2011. FICI values reported in Table 2 indicate a positive interaction between gentamicin and fluconazole with TTO, CDO and EOMS against all bacteria and fungi tested, respectively. In particular, a synergic effect was observed for TTO plus gentamicin towards S. aureus ATCC 25923, S. epidermidis, A. baumanii and for TTO plus fluconazole for C. albicans with FICI values = 0.5. For C. albicans ATCC 24433 and other bacterial strains, the combination TTO-antimicrobial drugs was found to be additive with FICI value >0.5. An additive effect was also reported for CDO plus gentamicin against all bacteria obtained strains tested, except for A. baumanii with FICI value = 0.25, in agreement with data of Duarte et al. (2012). Interestingly, the combination of fluconazole and CDO showed a synergic effect towards fungi with FICI values = 0.5. Also, FICI values of 0.13 and 0.37 for C. albicans CO23RFLu and CO23 strains respectively, obtained with EOMS plus fluconazole, indicated a strong synergic effect. In the case of bacteria, EOMS plus gentamicin produced a prevalent additive effect; only for K. pneumoniae, the action was synergic with FICI value = 0.5. Therefore, in the case of fungi, all tested combinations of EOs plus fluconazole produced a synergic effect, except for C. albicans ATCC 24433. On the contrary, in the case of tested bacterial strains, the effect of EOs plus gentamicin was mainly additive, although it has been observed even towards multi-drug resistant strains. However, it must be observed that EOs concentrations necessary to decrease MIC values of gentamicin or fluconazole appear very high and therefore probably toxic for therapeutic purposes. To address this issue, TTO, CDO and EOMS were evaluated for their cytotoxicity towards an epithelial cell line of human origin (HeLa), utilised as a model. Cell monolayers were exposed to EOs diluted in cell culture medium for the same time length as for antimicrobial testing. The values of the maximum non-cytotoxic concentration (MNCC) were 1.6 mg/ml for both CDO and EOMS and 0.4 mg/ml for TTO. Comparison of data obtained between EOs cytotoxicity studies and MIC values of antimicrobial activity for bacteria and fungi, point out that TTO and CDO result cytotoxic in this cell model. Only EOMS was found not to be toxic for HeLa cell cultures at the MIC values for fungi and gram-positive bacteria. Interestingly, MICs values obtained for EOMS in combination with gentamicin or fluconazole (Table 2) were comparable to those of MNCC for eukaryotic cells. Also, the combination of reference antimicrobials with CDO produced similar results on gram-positive bacteria and fungi while in the case of gram-negative bacteria a major variability has been observed. For TTO, all MIC values were always higher than MNCC values for eukaryotic cells. As reported by Reichlinga et al. (2009), essential oils may exert cytotoxic effects to tissue cells at concentrations which do not yet show an antimicrobial effect. In addition, their use could also be limited by the concentrations that they can achieve at the site of action. Correlations studies of in vitro and in vivo toxicity data are necessary in order to develop models that allow a prediction of systemic toxicity in vivo from cell culture data.

Conclusions
Data reported in this study suggest that the antifungal and antibacterial activity of EOs could be related to their main chemical components. Some EOs can show an additive or synergic antimicrobial effect when tested in combination with sub-inhibitory concentrations of gentamicin or fluconazole, even against clinical multi-drug resistant isolates.
Although results are encouraging, further studies are necessary to define the main active constituents of each oil and the optimal ratio between EOs and reference antimicrobial drugs to increase their efficacy. Finally, in a hypothetical antimicrobial therapy with EOs in combination with known antimicrobials, maximum benefit could be achieved using isolated active components from each oil. Also dosing regimens of EOs, when in combination with reference antimicrobials, should be explored for higher therapeutic efficacy and to decrease toxicological profiles.