Essential Oil of Common Sage (Salvia officinalis L.) from Jordan: Assessment of Safety in Mammalian Cells and Its Antifungal and Anti-Inflammatory Potential

Salvia officinalis L. (Lamiaceae) is a Mediterranean species, naturalized in many countries. In Jordan, it is used in traditional medicine as antiseptic, antiscabies, antisyphilitic, and anti-inflammatory, being frequently used against skin diseases. This study aimed the assessment of the antifungal and anti-inflammatory potential of its essential oils, and their cytotoxicity on macrophages and keratinocytes. The oils were investigated by gas chromatography and gas chromatography-mass spectrometry and the antifungal activity was evaluated against yeasts, dermatophyte and Aspergillus strains. Assessment of cell viability was made by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and the in vitro anti-inflammatory potential was evaluated by measuring nitric oxide production using lipopolysaccharide-stimulated mouse macrophages. The main compounds of S. officinalis oils were 1,8-cineole (39.5–50.3%) and camphor (8.8–25.0%). The oils revealed antifungal activity against dermatophyte strains and significantly inhibited NO production stimulated by LPS in macrophages, without affecting cell viability, in concentrations up to 0.64 μL/mL. This is the first report addressing the in vitro anti-inflammatory potential of S. officinalis oil. These findings demonstrated that bioactive concentrations of S. officinalis oils do not affect mammalian macrophages and keratinocytes viability making them suitable to be incorporated in skin care formulations for cosmetic and pharmaceutical purposes.


Introduction
The genus Salvia L. (Lamiaceae) comprises about 900 species, spread throughout the world, some of which with great economic value since they are used as spices and flavoring agents by perfumery and cosmetic industries [1,2].
Salvia sp. has also been used for a long time in folk medicine as medication against fever, rheumatism, perspiration, sexual debility, and in the treatment of chronic bronchitis, as well as mental and nervous diseases [3].
S. officinalis (sage, garden sage, or common sage) is a perennial, evergreen subshrub, with woody stems, grayish leaves, and blue to purplish flowers. It is native to the Mediterranean region, being currently cultivated in various countries [4,5]. Sage is one of the oldest medicinal plants, and the etymology of the Latin name suggests its healing properties, 2 BioMed Research International with Salvia deriving from the Latin verb salvare = to save or to cure, and officinalis also means medicinal.
Essential oil of sage and their preparations are externally used for inflammations and infections of the mucous membranes of throat and mouth (stomatitis, gingivitis, and pharyngitis). Internally, the essential oil is used for dyspeptic symptoms and excessive perspiration [4].
Environmental conditions such as temperature, day length, and light influence quantitative compositions of essential oils [16]. These conditions change significantly and predictably during the vegetation period, leading to a pattern of seasonal variation in plant metabolites, that is, generally repeated every year. In Salvia, the major monoterpenes 1,8cineole, camphor, and the two thujones show pronounced dynamics during a vegetative cycle that has been confirmed under different geographical conditions [17][18][19][20]. According to Grausgruber-Gröger et al. 2012 [21], 1,8-cineole steadily decreases during a vegetative period (approx. May to October), camphor peaks in the middle of the vegetative period, and -and -thujone increase gradually during the vegetative period.
The antibacterial properties of sage oils have been attributed to the presence of 1,8-cineole, thujone, and camphor [2]. However in 2007, Pinto et al. [22] stated that thujones do not play an important role against yeasts and filamentous fungi. This work also suggests 1,8-cineole and camphor as the main responsible for the antifungal activity in the tested strains.
Common sage is well known in Palestitian traditional medicine by possessing antimicrobial properties [23]. In 2008, a taxonomical and pharmacological survey of therapeutic plants in Jordan indicates the main traditional uses S. officinalis as antiseptic, antiscabies, antisyphilitic, and antiinflammatory, being frequently used against skin and eye diseases and also in pleurisy [24,25]. In Jordan and the Middle East, it is also being reported the use of common sage for fever, digestive disorders, and stomach ache [26].
Thus, taking into account these traditional uses of S. officinalis, in the present work we aim to characterize the chemical composition of the essential oil obtained from the aerial parts of S. officinalis collectedin different regions of Jordan, as well as to evaluate the antifungal and the antiinflammatory activities of the oil at concentrations safe to mammalian cells. All the six samples were used for essential oil isolation and the oils were characterized. Only the oils chemically more distinct (S1 and S2) were chosen for biological assays.

Essential Oil Isolation.
The essential oil of the six samples was isolated by hydrodistillation for 3 h using a Clevengertype apparatus, according to the procedure described in the European Pharmacopoeia [27].

Qualitative and Quantitative Analyses.
Components of the volatile oils were identified by their retention indices on both SPB-1 and SupelcoWax-10 columns, calculated by linear interpolation relative to retention times of C 8 -C 24 of nalkanes and compared with those of reference compounds included in CEF laboratory database or literature data [28], and by their mass spectra by matching with reference spectra from the CEF laboratory own spectral database, Wiley/NIST database or literature data [28][29][30]. Relative amounts of individual components were calculated based on GC raw data areas without FID response factor correction.
2.6. Antifungal Activity Evaluation 2.6.1. Fungal Strains. The antifungal activity of the oils obtained from the aerial parts of S. officinalis collected in Shoubak, South Jordan (S1) and Ma'an, South Jordan (S2), was evaluated against yeasts and filamentous fungi strains (Aspergillus spp. and dermatophytes).   Candida parapsilosis ATCC 90018 was used as control.
The fungal isolates were identified by standard microbiology methods and stored on Sabouraud broth with glycerol at -70 ∘ C. Prior to antifungal susceptibility testing, each isolate was inoculated on Sabouraud agar to ensure optimal growth characteristics and purity.

Antifungal Activity Methods.
All chemicals were supplied by Sigma Aldrich, except fluconazole, supplied by Pfizer, UK under the name Diflucan.
Broth macrodilution methods based on the Clinical and Laboratory Standards Institute (CLSI) reference protocols M27-A3 [31] and M38-A2 [32], for yeasts and filamentous fungi, respectively, were used to determine minimum inhibitory concentrations (MICs) and minimum lethal concentrations (MLCs) of the essential oil.
The serial doubling dilution of the essential oil was prepared in dimethyl sulfoxide (DMSO), with concentrations ranging from 0.32 to 20 L/mL. Final concentration of DMSO never exceeded 2%. Recent cultures of each strain were used to prepare the cell suspension adjusted to 1-2 × 10 3 cells per mL for yeasts and 1-2 × 10 4 cells per mL for filamentous fungi. The concentration of cells was confirmed by viable count on Sabouraud agar. The test tubes were incubated aerobically at 35 ∘ C for 48 h/72 h (Candida spp. and Aspergillus spp./Cryptococcus neoformans) and at 30 ∘ C for 7 days (dermatophytes) and MICs were determined. To evaluate MLCs, aliquots (20 L) of broth were taken from each negative tube after MIC reading and cultured in Sabouraud dextrose agar plates. Plates were then incubated at 35 ∘ C for 48 h (Candida spp. and Aspergillus spp.) and 72 h for Cryptococcus neoformans and 30 ∘ C for 7 days (dermatophytes). In addition, two reference antifungal compounds, amphotericin B (Sigma Aldrich) and fluconazole (Pfizer), were used to control the sensitivity of the tested microorganisms. All tests were performed in Roswell Park Memorial Institute medium (RPMI 1640 medium). For each strain tested, both growth conditions and sterility of the medium were checked in two control tubes. The innocuity of DMSO was also checked at the highest tested concentration. All experiments were performed in triplicate and repeated if the results differed.

Nitric Oxide (NO) Measurement.
The anti-inflammatory activity of the oils from samples S1 and S2 was evaluated in the mouse macrophage cell line Raw 264.7.
The production of NO was measured by the accumulation of nitrite in the culture supernatants, using a colorimetric reaction with the Griess reagent [33]. The cells were plated at 0.6 × 10 6 cells/well in 48-well culture plates, allowed to stabilize for 12 h, and then incubated with culture medium (control), or stimulated with 1 g/mL LPS, or with 1 g/mL LPS in the presence of different concentrations of the essential oil during 24 h. Briefly, 170 L of culture supernatants was collected and diluted with equal volume of the Griess reagent (0.1% (w/v) N-(1-naphthyl) ethylenediamine dihydrochloride and 1% (w/v) sulphanilamide containing 5% (w/v) H 3 PO 4 ) during 30 min, in the dark. The absorbance at 550 nm was measured using an automatic plate reader (SLT, Austria). Nitrite concentration was determined using a sodium nitrite standard curve. serum, 100 U/mL penicillin, and 100 g/mL streptomycin, at 37 ∘ C in a humidified atmosphere of 95% air and 5% CO 2 .

Evaluation of
In the experiments, cells were monitored by microscope observation in order to detect any morphological change.

MTT Assay for Cell Viability.
Cell viability was assessed for the oils from samples S1 and S2 using the mouse macrophage cell line Raw 264.7 and the human keratinocyte cell line HaCaT.
Assessment of cell viability was made through a colorimetric assay, using MTT [34]. The HaCaT cells (0.2 × 10 6 cells/well, cultured in 48-well microplates) were incubated in a final volume of 600 L, allowed to stabilize for 12 h, and then incubated for 24 h with different concentrations of the essential oil. After adding 60 L of MTT solution (5 mg/mL in PBS) to each well, the cells were further incubated at 37 ∘ C for 15 min, in a humidified atmosphere of 95% air/5% CO 2 . Supernatants were then discarded and 300 L of acidified isopropanol (0.04 N HCl in isopropanol) was added to the cultures and mixed thoroughly to dissolve the dark blue crystals of formazan. Formazan quantification was performed using an ELISA automatic microplate reader (SLT, Austria) at 570 nm, with a reference wavelength of 620 nm.
In the case of the cell line Raw 264.7, after collection of 170 L of culture supernatants for NO measurement, 43 L of MTT solution (5 mg/mL in PBS) was added and cells were incubated at 37 ∘ C for 15 min, in a humidified atmosphere of 95% air and 5% CO 2 . Supernatants were then discarded, and 300 L of acidified isopropanol (0.04 N HCl in isopropanol) was added to the cultures and mixed thoroughly to dissolve the dark blue crystals of formazan. Formazan quantification was performed as described above.

Data Analysis.
All the experiments were performed in duplicate, being the results expressed as mean ± SEM of three independent experiments. The means were statistically compared using one-way ANOVA, with a Dunnett's multiple comparison test. The differences between the means were considered significant for values of < 0.01. The statistical tests were applied using GraphPad Prism, version 5.02 (GraphPad Software, San Diego, CA, USA).

Essential Oil Composition.
The essential oil of the six samples was obtained in yields of 1.2-2.8% (w/w), based on the dry weight of the plant.
The qualitative and quantitative composition of the oils is presented in Table 1, where the compounds are listed by order of their elution on a polydimethylsiloxane column.

Antifungal Activity.
Evaluation of MIC and MLC of the essential oil showed a variability of inhibition among all the fungal strains tested. The results of the antifungal tests are summarized in Table 2.
The dermatophyte strains showed more sensibility to these oils when compared with Candida and Aspergillus strains, particularly for Trichophyton rubrum and Epidermophyton floccosum with MIC of 0.64 L/mL. Among the tested yeasts, Cryptococcus neoformans was the strain that showed more sensibility with MIC of 0.64 L/mL.

Effect of the Essential Oil on Keratinocytes Viability. As shown in
A cell-free control was performed in order to exclude nonspecific effects of the oils on MTT (data not shown).

Discussion and Conclusions
The essential oil composition of S. officinalis is highly influenced by genetic and environmental factors, organ age, t: traces (<0.05%). RI a : Retention indices on the SPB-1 column relative to C 8 to C 24 -alkanes. RI p : Retention indices on the SupelcoWax-10 column relative to C 8 to C 24 -alkanes.
These results are in agreement with the revision of Grausgruber-Gröger et al. 2012 [21], where they stated that 1,8cineole steadily decreases during a vegetative period (approx. May to October), camphor peaks in the middle of the vegetative period, and -and -thujones increase gradually during the vegetative period. Our samples were collected in April, and they possess very low amount of thujones and a quite high amount of 1,8-cineole.
In 1990, Tucker et al. [37] categorized sage commercial oils in five chemotypes according to the amount of the major compounds: (i) camphor > -thujone   Thujones have been reported to be toxic to both brain and liver cells, presenting -thujone higher toxicity thanthujone [38,39]. The samples from Jordan are very promising as they possess very low amount of thujones, thereby evidencing its safety profile. Accordingly, the oils did not affect macrophages and keratinocytes viability at concentrations up to 0.64 L/mL. The absence of keratinocyte toxicity is crucial to make an essential oil suitable for inclusion in topical formulations. Indeed, there is a lack of knowledge addressing keratinocytes cytotoxicity induced by essential oils and a current screening in our lab points that keratinocytes are very sensitive to several essential oils, which highlights the use of safe bioactive concentrations of S. officinalis essential oil for topical applications.
Macrophages produced a variety of proinflammatory mediators, such as nitric oxide, upon activation with the pro-inflammatory stimulus LPS. Since NO is a wellestablished inflammatory marker, the inhibition of its production might be a useful strategy to treat inflammatoryrelated diseases. Therefore, we also evaluated, for the first time, the in vitro anti-inflammatory activity of S. officinalis essential oil. The oils significantly inhibited NO production elicited by LPS in macrophages which demonstrates its strong antiinflammatory potential. However, sample 2 (S2) has a slightly higher activity than sample 1 (S1), what can be related to the chemical composition. Although the samples possess similar chemical composition, they are distinguished by the content of camphor. Moreover, the higher amount of camphor in S2 did not affect the cell viability.
Concerning the antifungal properties of the essential oil, the results presented here demonstrate that the dermatophyte strains showed more susceptibility to these oils when compared with Candida and Aspergillus strains, particularly for Trichophyton rubrum and Epidermophyton floccosum with MIC of 0.64 L/mL. Among the tested yeasts, Cryptococcus neoformans was the strain that showed more susceptibility with MIC of 0.64 L/mL.
Although the MIC and MLC results varied among tested organisms, in various cases the MIC was equivalent to the MLC, indicating fungicidal activity of the oils (Table 2).
Taking together, our results demonstrated that bioactive concentrations of S. officinalis oils do not affect mammalian macrophages and keratinocytes viability making them suitable to be incorporated in skin care formulations for cosmetic and pharmaceutical purposes.