Antifungal and Cytotoxic Activities of Sixty Commercially-Available Essential Oils

There is an urgent and unmet need for new antifungal therapies. Global fungal infection rates continue to rise and fungal infections pose increasing burdens on global healthcare systems. Exacerbating the situation, the available antifungal therapeutic arsenal is limited and development of new antifungals has been slow. Current antifungals are known for unwanted side effects including nephrotoxicity and hepatotoxicity. Thus, the need for new antifungals and new antifungal targets is urgent and growing. A collection of 60 commercially-available essential oils has been screened for antifungal activity against Aspergillus niger, Candida albicans, and Cryptococcus neoformans, as well as for cytotoxic activity against MCF-7 and MDA-MB-231 human breast tumor cell lines; the chemical compositions of the essential oils have been determined by gas chromatography-mass spectrometry (GC-MS). Ten essential oils showed remarkable antifungal and cytotoxic activities: Indian, Australian, and Hawaiian sandalwoods; melissa; lemongrass; cilantro; cassia; cinnamon; patchouli; and vetiver.


Introduction
Fungi are ubiquitous in nature. Of the estimated 1.5 million species of fungi [1], there are approximately 100 species that cause human infection [2]. These infections include aspergillosis, candidiasis, and cryptococcosis, among others [3]. Invasive fungal infections from these opportunistic pathogens have been increasing in recent decades, causing substantial morbidity and mortality [2,4]. The most common Aspergillus species causing pulmonary aspergillosis is A. fumigatus, but A. flavus, A. terreus, and A. niger can also cause Aspergillus lung disease, particularly in immunosuppressed individuals [5]. Likewise, the principle agents of candidiasis are Candida albicans, C. glabrata, C. tropicalis, C. parapsilosis, and C. krusei [6]. Cryptococcus neoformans is the main fungal species responsible for cryptococcosis, but Cryptococcus taxonomy has undergone several revisions [7]. Treatment options for invasive fungal infections include amphotericin B, as well as several azole compounds, such as fluconazole and itraconazole [8]. However, there have been severe side effects associated with these antifungal agents [8], and antifungal resistance continues to increase [9].
Essential oils are complex mixtures of volatile compounds derived principally from higher plants [10]. These materials have been used to treat human infections and other maladies for centuries. The biological activities associated with essential oils depend on the compositions, both the concentrations of the major components and the possible synergistic interactions with minor components. In this report, we present the antifungal screening of a collection of 60 essential oils obtained from commercial sources against Aspergillus niger, Candida albicans, and Cryptococcus neoformans. In addition, the essential oils were also screened against two human breast tumor cell lines, MCF-7 (estrogen receptor positive breast adenocarcinoma) and MDA-MB-231 (estrogen receptor negative breast adenocarcinoma).

Results
The antifungal and cytotoxicity screening results are summarized in Table 1. The most active essential oils, both in terms of antifungal activity and cytotoxic activity, were the sandalwood species (Santalum album, S. austrocaledonicum, and S. paniculatum), rich in santalols; cassia (Cinnamomum cassia) and cinnamon (C. zeylanicum), both dominated by cinnamaldehyde; lemongrass (Cymbopogon flexuosus), melissa (Melissa officinalis), and cilantro (Coriandrum sativum leaf oil), which were dominated by aldehydes; patchouli (Pogostemon cablin), rich in patchouli alcohol; and vetiver (Vetiveria zizanoides), with isovalencenol and khusimol as major components. Of the fungal species tested, Cryptococcus neoformans was the most susceptible and Candida albicans was the least sensitive. Both breast tumor cells lines showed similar activities and correlated well with C. neoformans antifungal activity.
A hierarchical cluster analysis ( Figure 1) revealed four apparent clusters based on compositions and bioactivities: (1) a largely inactive cluster that is dominated by oxygenated monoterpenoids; (2) an inactive cluster with aromatics as the predominant chemical class; (3) a largely inactive cluster, dominated by monoterpene and sesquiterpene hydrocarbons; and (4) the biologically active cluster, which is rich in oxygenated sesquiterpenoids and aldehydes.
Cluster 3 can be subdivided into a sub-cluster rich in monoterpene hydrocarbons (3a) and a sub-cluster with both monoterpene hydrocarbons and sesquiterpene hydrocarbons (3b). Sub-cluster 3a is made up of gymnosperm essential oils and the Citrus essential oils and are, by and large, inactive.
Sub-cluster 3b, on the other hand, has significant concentrations of sesquiterpenoids and generally showed moderate cytotoxic activity. Thus, for example, Cistus ladanifer essential oil had IC 50 values of 36.6 and 46.3 µg/mL against MCF-7 and Hs578T cell lines; copaiba oils, rich in β-caryophyllene, showed moderate cytotoxic activities on both MCF-7 and MDA-MB-231 cells (IC 50 values range from 22.7 to 67.2 µg/mL). Frankincense (Boswellia carteri) essential oil is also rich in β-caryophyllene and showed comparable cytotoxic activity. Sesquiterpene hydrocarbons, such as β-caryophyllene and α-humulene, have shown moderate cytotoxic activity against several human tumor cell lines [11,13,24]; the relatively high concentrations of sesquiterpene hydrocarbons in the essential oils of sub-cluster 3b may account for the observed moderate cytotoxicities.
Sandalwood oil (species not reported) had shown antifungal activity against C. neoformans with MIC of 100 µg/mL [26]. Indian sandalwood (S. album) had previously shown only marginal activity against C. albicans [27] with MIC values of around 600 µg/mL [28,29], consistent with this current investigation. Santalum album essential oil had previously demonstrated in vitro cytotoxic activity on both MCF-7 and MDA-MB-231 cells [25,30], as well as several other tumor cell lines [31]. The antifungal and cytotoxic activities of sandalwood oils can be attributed to the high concentrations of αand β-santalols [32,33].
Both patchouli (Pogostemon cablin) and vetiver (Vetiveria zizanoides) essential oils showed notable antifungal activity against C. neoformans (MIC = 20 µg/mL), as well as cytotoxic activity against MCF-7 cells (IC 50 = 25.0 and 23.9 µg/mL, respectively). Both of these essential oils are rich in sesquiterpene alcohols, patchouli alcohol in P. cablin, and (E)-isovalencenol and khusimol in V. zizanoides. Previous studies on the antifungal activity of patchouli oil showed no activity against Aspergillus spp. [21,47], whereas in this work, patchouli oil showed inhibition against A. niger with MIC of 156 µg/mL. Likewise, vetiver oil inhibited the growth of A. niger and C. albicans (MIC = 78 and 313 µg/mL), but previous reports in the literature showed no activity against these two organisms [21,22].

Gas Chromatography-Mass Spectrometry
Essential oils obtained from dōTERRA International were analyzed by gas chromatography-mass spectrometry (GC-MS) using a Shimadzu GCMS-QP2010 Ultra operated in the electron impact (EI) mode (electron energy = 70 eV), scan range = 40-400 atomic mass units, scan rate = 3.0 scans/s, and GC-MS solution software version. 4.20 (Shimadzu Scientific Instruments, Columbia, MD, USA). The GC column was a ZB-5 fused silica capillary column (Phenomenex, Torrance, CA, USA) with a (5% phenyl)-polymethylsiloxane stationary phase and a film thickness of 0.25 µm. The carrier gas was helium with a column head pressure of 552 kPa and flow rate of 1.37 mL/min. The injector temperature was 250 • C and the ion source temperature was 200 • C. The GC oven temperature program was programmed for 50 • C initial temperature, temperature increased at a rate of 2 • C/min to 260 • C. A 5% w/v solution of the sample in CH 2 Cl 2 was prepared and 0.1 µL was injected with a splitting mode (30:1). The remaining essential oils (Ameo, Mountain Rose Herbals, Albert Vielle) were analyzed with an Agilent 6890 GC, Agilent 5973 MSD, EI (70 eV); range of 40-400 amu, scan rate of 3.99 scans/s, HP-5ms column (Agilent Technologies, Santa Clara, CA, USA), He carrier gas, head pressure of 92.4 kPa, flow rate of 1.5 mL/min, GC oven temperature program of 60 • C initial temperature, held for 5 min, then increased at 3 • C/min up to 280 • C, 1% solutions of essential oils in CH 2 Cl 2 , splitless injection. Identification of the oil components was based on their retention indices determined by reference to a homologous series of n-alkanes, and by comparison of their mass spectral fragmentation patterns with those reported in the literature [48], and stored in our in-house library [49].

Antifungal Screening
Candida albicans (ATCC 18804) and Cryptococcus neoformans (ATCC 24607) were grown on potato dextrose agar (PDA) for 48 or 72 h, respectively. Three milliliters of potato dextrose broth (PDB) was inoculated with a single colony. These liquid cultures were grown at 37 • C for another 48 or 72 h for microdilution assays. Minimum inhibitory concentrations (MICs) were determined by microdilution in 96-well round bottom plates from triplicates. Briefly, 100-µL aliquots of MOPS (3-(N-morpholino)propanesulfonic acid) buffered RPMI (Roswell Park Memorial Institute) medium pH 7.0 were added to each well. In addition, aliquots of 100 µL of essential oil (1% in DMSO) or the positive and negative controls of 100% DMSO and 100 µM amphotericin B (AMB) and 100% DMSO, respectively, were added to the first row. Each well was serially diluted two-fold down the column excluding the negative control (medium alone). Subsequently, 100 µL of 4 × 10 3 cells/mL of inoculum in MOPS buffered RPMI were added to each well, resulting in a final concentration of 2 × 10 3 cells/mL. The microplates were incubated at 37 • C without agitation for 48 or 72 h for C. albicans or C. neoformans, respectively. MIC values were determined visually as the last well with no turbidity by comparison with the positive and negative controls.
Aspergillus niger (ATCC 16888) was grown for seven days at room temperature on potato dextrose agar (PDA) plates. Using an inoculum loop, the spores were gently gathered from the top of the PDA plate and suspended in 1 mL of potato dextrose broth (PDB). Before further use, the spore solution was filtered using sterile cheesecloth. The OD 625 was adjusted to 0.15 by dilution with fresh PDB. For screening, 100 µL of MOPS buffered RPMI was added to each well of a 96-well plate. A sample or a control of 100 µL was then added to the first well in each row and serially diluted down the column. Lastly, 100 uL of the adjusted spore solution was added to each well of the plate. The plates were incubated at room temperature for seven days. Amphotericin B was used as a positive control, while DMSO and RPMI media alone were used as negative controls. Inhibition was determined visually by comparing the growth of the positive and negative controls with the samples.

Cytotoxicity Screening
Cell culturing and cytotoxicity screening were carried out as previously reported [50]. Briefly, MCF-7 and MDA-MB-231 cells were each grown in sterile RPMI 1640 media with L-glutamine, 26 mL of 7.5% sodium bicarbonate per liter medium; 10,000 units penicillin and 10,000 µg/mL streptomycin per liter of medium; and 15 mL of 1 M HEPES per liter medium, buffered with 5.0 N NaOH to pH 7.35. MCF-7 and MDA-MB-231 cells were plated at concentrations of 1.44 × 10 6 and 1.44 × 10 4 cells per well, respectively, in 96-well plates in volumes of 100 µL/well. Test samples were diluted in growth medium to a concentration of 0.01% (w/v). Tingenone was used as a positive control; growth medium and DMSO were used as negative controls for each plate. The final concentrations of test samples and tingenone controls were 100 µg/mL. The cells were incubated with the test samples at 37 • C and 5% CO 2 for 48 h. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to determine cell viability. The supernatant medium was removed from each well using suction and a solution of MTT solution (1:10 dilution of 5 mg/mL of stock MTT in growth medium) was added to each well, and the plates were incubated for an additional 4 h at 37 • C and 5% CO 2 . After the incubation period, the medium was carefully aspirated from the wells, and 100 µL of ISO PBS, containing 100 mL isopropyl alcohol, 4.0 µL 5.0 N HCl, and 50.0 mL phosphate-buffered saline, was added to the wells, and the plate was gently shaken to dissolve the crystals. Absorbance was measured using a SpectraMax plate reader at 570 nm and percent viability was determined. For essential oils showing <50% viability, dilutions of the samples (50,40,30,20, and 10 µg/mL) were further assayed. Median inhibitory concentrations (IC 50 ) were determined using the Reed-Muench method [51].

Hierarchical Cluster Analysis
The chemical classes of the commercial essential oils, along with the antifungal and cytotoxic activities, were used in the cluster analysis. The 60 essential oils were treated as operational taxonomic units (OTUs) and the 12 chemical classes (monoterpene hydrocarbons, oxygenated monoterpenoids, sesquiterpene hydrocarbons, oxygenated sesquiterpenoids, diterpenoids, aromatic compounds, fatty-acid derivatives, aliphatic esters, aldehydes, phenolics, sulfur-containing compounds, and others) and five bioactivities (Aspergillus niger, Candida albicans, Cryptococcus neoformans, MCF-7, and MDA-MB-231) were used to determine the associations between the essential oils using agglomerative hierarchical cluster (AHC) analysis using XLSTAT Premium, version 19.5.47159 (Addinsoft, Paris, France). Dissimilarity was determined using Euclidean distance, and clustering was defined using Ward's method.

Conclusions
Several essential oils have shown notable antifungal activities against opportunistic fungal pathogens. These readily-available materials may add to our treatment options, as agents themselves or as adjuvant therapies, to combat fungal infections. In addition to the antifungal and cytotoxic activities of the essential oils in this study, those essential oil that do not show appreciable cytotoxic activity to human cells may be considered relatively safe for other uses such as cosmetics, flavoring, and aromatherapy.

Conflicts of Interest:
The authors declare no conflict of interest.