1. Introduction
The laurel,
Laurus nobilis L., an evergreen tree or shrub belonging to the family Lauraceae, is native to the south parts of Europe and the Mediterranean area; this plant is widely cultivated in many countries of this region. Its dried leaves and the essential oil (EO) deriving from leaves are used as a valuable spice and flavoring agent in culinary and food industry. The leaves have been used, in Iranian folk medicine, to treat epilepsy, neuralgia, and parkinsonism [
1,
2]. Leaves and fruits have been reported to possess aromatic, stimulant, and narcotic properties [
3]. Several studies reported the antimicrobial and the antioxidant properties of laurel essential oil and/or extracts [
4,
5,
6]. The leaves of
L. nobilis are traditionally used orally to treat the symptoms of gastrointestinal problems, such as epigastric bloating and flatulence [
7]. The essential oil of laurel leaves is widely used in the perfume and soap industries [
8]. Moreover, it has been used for relieving hemorrhoid and rheumatic pains [
2]. It also has diuretic and antifungal activities [
1,
2,
9].
The present study describes the composition of the essential of leaves of L. nobilis and some of its biological activities. In particular, we evaluated the possible antimicrobial effects against different strains of bacteria and fungi, its cytotoxicity on SH-SY5Y cells and its possible subcellular effects in SH-SY5Y cells are used to evaluate the possible effects on Central Nervous System (CNS).
3. Discussion
In the composition of the essential oil of L. nobilis, 1,8-cineole, sabinene, and linalool are the main components, with other compounds being present in low percentages or even in traces.
The comparison with most recent literature concerning the chemical composition of the essential oil of
L. nobilis from other Mediterranean areas, showed substantial differences: 1,8-cineole percentage found (31.9%) is lower than the values recorded in other studies: Turkey 44.97% [
10], Tunisia 56.0% [
11], Cyprus 58.59% [
12], Morocco 52.43% [
5], but similar to Algerian essential oil (34.62%) [
13]. In our sample, the amount of sabinene was 12.2%, higher than in the essential oils analyzed by Derwich and coworkers [
5], Snuossi and co-workers [
11], and Yalcin [
12] who found percentages of sabinene of 6.13, 3.5, and 6.13%, respectively. Linalool was found in concentrations comparable to previous studies [
10,
13] but in essential oil analyzed by Derwick and coworkers [
5] linalool was not found.
The inhibition halo technique is often used to assess antibacterial activity of vegetal extracts and essential oils [
14]. Our results on the antimicrobial activity confirm that the behavior of an antimicrobial agent can be not only species-specific but also strain-specific [
15,
16]. The available literature reports several studies ascertaining the antibacterial activity of the essential oil of
L. nobilis against different pathogens [
6,
17,
18,
19]. The Algerian laurel essential oil versus
E. coli and
Ps. aeruginosa produced an inhibition halo of 13.73 mm and 10.73, respectively. The oil produced an inhibition halo of 13.03 mm when assayed versus
S. aureus [
19]. In our experiments, the antimicrobial activity resulted higher than that of the essential oil of
L. nobilis collected in Turkey, if tested against
E. coli O157:H7 [
20]. Considering the chemical composition of the essential oil, we could hypothesize a synergistic action at least of the main compounds present in, specifically monoterpenes and oxygenate monoterpenes, which have per se low antimicrobial activity when used as single compounds [
21]. Other terpenes—such as α-pinene, β-pinene, γ-terpinene δ-3-carene, (+)-sabinene, and α-terpinene—showed a very low or no antimicrobial activity against 25 genera of bacteria [
22]. These in vitro tests confirm that terpenes show ineffective antimicrobial activity when used as single compounds [
23]. The
L. nobilis essential oil showed different antifungal activity (
Table 4) respect to previous studies: in fact, our results indicate a marked antifungal activity, superior, for example, respect to that observed by Simic and co-workers both against
Aspergillus and
Penicillium spp., in spite of its high content of 1,8-cineole [
24]. Therefore, the diameter of the halo produced by 1,8-cineole against the fungi was less evident. For this reason, we hypothesize a synergistic effect among the different components of the
L. nobilis EO, which, in a certain sense, cancel out the weakness demonstrated by the individual components. The remarkable effectiveness exhibited by the studied essential oil may be interesting for example in the manufacturing and food processing, which, as is known, is increasingly directed to the use of green technologies able to safeguard the quality food, the environment, and especially the health of the consumer [
25]. Therefore, the comforting results obtained using the essential oil of
L. nobilis indicate that such oil could be used as an antimicrobial agent, also for health and food purposes. The use of this essential oil prolonged the shelf life of modified atmosphere-packed (MAP) fish fillets [
26]. The laurel essential oil was also used to safeguard the quality and safety of fresh sausages, prolonging their shelf life too and also avoiding the problems related to fat oxidation [
27].
The cytotoxic activity of the 1,8-cineole and the essential oil were evaluated in human neuroblastoma cell line (SH-SY5Y). The IC
50 values were > 400 µg/mL, indicating that the substances were not cytotoxic, as judged by the criterion set by the National Cancer Institute, that stated that only natural substances with IC
50 < 20 µg/mL were considered to be cytotoxic against the treated cells [
28]. However, comparing the IC
50 values, our findings indicated that
L. nobilis EO is more cytotoxic than 1,8-cineole. This cytotoxicity can be probably attributed to a synergistic activity of this and other components.
Specific induction of apoptosis by 1,8-cineole was observed in human leukemia Molt 4B and HL-60 cells, but not in human stomach cancer KATO III cells [
29]. However, this monoterpene was not cytotoxic against the human cancer in vitro models in the present study. Our results showed that the essential oil showed less cytotoxicity than the one tested on ACHN and C32 cell lines (IC50 202.62 and 209.69 µg/mL for ACHN and C32, respectively) [
30] and the leaf extract tested on human neuroblastoma cell lines SK-N-BE(2)-C and SH-SY5Y [
31].
Essential oils have been used for centuries as a traditional medicine, and currently used worldwide in the management of depression, anxiety, and stress-related disorders but there is very little verified science behind their use [
32]. To clarify the traditional belief in the antiepileptic effects, and the use in neuralgia and parkinsonism of
L. nobilis in folk medicine [
1], we investigated the possible influence of the essential oil and its major components on ADCY1 expression. Our results showed that the treatment with 200 and 100 µg/mL of
L. nobilis essential oil reduced ADCY1 expression in SH-SY5Y cell and, consequently, the intracellular production of cAMP. 1,8-Cineole had no effect on ADCY1 expression but we hypothesized that the essential oil effect is due to the presence of linalool (10.2%). Elisabetsky and coworkers showed that linalool possesses dose-dependent sedative effects in the Central Nervous System [
33]. In our previous study, we reported that linalool is a component of many essential oils that can influence the expression of ADCY1 and ERK [
34].
Moreover, in the complex phytochemical composition of the essential oil, one or more components might act synergistically to influence ADCY1 expression in SH-SY5Y cells.
4. Materials and Methods
4.1. Plant Material
L. nobilis leaves were collected in February 2016, in Montecorice, Cilento area (Campania, Southern Italy, 40°14′9″24 N, 14°59′13″20 E), 90 m above sea level. Representative homogeneous samples of population were collected during the balsamic time. The plant was identified by Prof. V. De Feo, on the basis of
Flora d’Italia [
35] and a voucher specimen has been deposited in the Herbarium of the Medical Botany Chair of the University of Salerno.
4.2. Isolation of Volatile Oil
One hundred grams of dried leaves of
L. nobilis were ground in a Waring blender and then subjected to hydrodistillation for 3 h according to the standard procedure described in the European Pharmacopoeia [
36]. The yellow essential oil was solubilized in
n-hexane, filtered over anhydrous sodium sulphate, and stored under N
2 at +4 °C in the dark, until tested and analyzed.
4.3. GC-FID Analysis
Analytical gas chromatography was carried out on a Perkin-Elmer Sigma-115 gas chromatograph (Pelkin-Elmer, Waltham, MA, USA) equipped with a FID and a data handling processor. The separation was achieved using a HP-5MS fused-silica capillary column (30 m × 0.25 mm i.d., 0.25 μm film thickness). Column temperature: 40 °C, with 5 min initial hold, and then to 270 °C at 2 °C/min, 270 °C (20 min); injection mode splitless (1 μL of a 1:1000 n-hexane solution). Injector and detector temperatures were 250 °C and 290 °C, respectively. Analysis was also run by using a fused silica HP Innowax polyethylenglycol capillary column (50 m × 0.20 mm i.d., 0.25 µm film thickness). In both cases, helium was used as carrier gas (1.0 mL/min).
4.4. GC/MS Analysis
Analysis was performed on an Agilent 6850 Ser. II apparatus (Agilent Technologies, Inc., Santa Clara, CA, USA), fitted with a fused silica DB-5 capillary column (30 m × 0.25 mm i.d., 0.33 μm film thickness), coupled to an Agilent Mass Selective Detector MSD 5973; ionization energy voltage 70 eV; electron multiplier voltage energy 2000 V. Mass spectra were scanned in the range 40–500 amu, with scan time of 5 scans/s. Gas chromatographic conditions were as reported in the previous paragraph; transfer line temperature, 295 °C.
4.5. Identification of Essential Oil Components
The identification of the essential oil constituents was based on the comparison of their Kovats retention indices (RIs), determined relative to the tR values of
n-alkanes (C
10–C
35) with either those of the literature [
37,
38,
39] and mass spectra on both columns with those of authentic compounds available in our laboratories by means NIST 02 and Wiley 275 mass spectral libraries [
40]. The components’ relative concentrations were obtained by peak area normalization. No response factors were calculated.
4.6. Antimicrobial Activity
The inhibition halo test was performed in order to evaluate the potential antimicrobial activity of the
L. nobilis essential oil and 1,8-cineole [
15]. The Gram-negative
Escherichia coli DMS 8579 and
Pseudomonas aeruginosa ATCC 50071, and the Gram positive
Bacillus cereus DSM 4313,
Bacillus cereus 4384 and
Staphylococcus aureus DSM 25693 were the bacterial strains tested in this study. Bacteria were purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ). Nutrient broth (Sigma Aldrich, Milano, Italy) was used as medium of bacterial growth at 37 °C for 18 h. The optical densities of all cultures were adjusted to match a 0.5 McFarland standard of 1 × 10
8 colony-forming units (CFU)/mL and spread onto Nutrient agar plates. The essential oil of
L. nobilis as well as the pure component 1,8-cineole were re-suspended in dimethyl sulfoxide (DMSO), and then diluted to be subjected to biological analyses. Sterile filter paper discs (5 mm) were impregnated with volumes corresponding to 0.4, 1 and 2 µL of the essential oil and placed on the plates. These last were left for 30 min at room temperature under sterile conditions before their incubation at 37 °C for 24 h; the diameter of the clear zone shown on plates (inhibition halo zone) was accurately measured (“Extra steel Caliper mod 0289”, mm/inch reading scale, precision 0.05 mm, Mario De Maio, Milan, Italy). A disc treated with DMSO alone served as the negative control. Tetracycline (7 µg/disc; Sigma Aldrich, Milano, Italy) was used as reference drugs. The experiments were performed in triplicate and averaged.
4.7. Minimum Inhibitory Concentration (MIC)
A modified version of the resazurin microtitre-plate assay [
41] was used to evaluate the minimal inhibitory concentration of the
L. nobilis essential oil and 1,8-cineole. Briefly, different volumes of the test material—prepared as described above—were pipetted in a multi-well with different volumes of Muller-Hinton broth (Sigma Aldrich, Milano, Italy). Two fold serial dilutions were performed such that each well had 50 μL of the test material in serially descending concentrations. 30 μL of 3.3 × strength isosensitised broth (Sigma Aldrich) and 5 μL of resazurin indicator solution (previously prepared by dissolving 270 mg tablet purchased from Sigma Aldrich, in 40 mL of sterile distilled water) were added in each well, to reach a final volume/well of 240 µL. Finally, 10 μL of bacterial suspension was added to each well to achieve a concentration of approx. 5 × 10
5 cfu/mL. Ciprofloxacin (3 µM in DMSO, Sigma Aldrich) and DMSO were used as positive and negative controls, respectively. Plates were prepared in triplicate, wrapped with cling film to avoid the bacterial dehydration, and incubated at 37 °C for 24 h. The lowest concentration at which a colour change (visually assessed from blu/dark purple to pink, or colorless) occurred indicated the MIC value.
4.8. Antifungal Activity
The possible antifungal activity of the essential oil and 1,8-cineole was tested against four fungal tester strains of agro-food interest, Aspergillus versicolor DSM 1943, Penicillium expansum DSM 1994, Penicillium citrinum DSMZ 1179, and Aspergillus niger DSM 1957 following the method of Marrufo and co-workers [
42]. The strains were purchased from DSMZ. They were grown in potato dextrose broth (Sigma Aldrich, Milano, Italy) at 28 °C. A cell suspension of fungi was prepared in sterile distilled water, adjusted to contain approximately 10
6 CFU/mL, and plated onto potato dextrose agar. After 30 min under sterile conditions, the inoculated plates were spotted with different amount of the samples, previously diluted 1:10 (
v/
v) in DMSO. After 20 min under sterile conditions at room temperature, plates were incubated at 28 °C for 48–72 h. When the mycelium of fungi reached the edges of the control plate (negative control without the samples added), the diameter of the clear zone shown on plates (inhibition halo zone) was accurately measured (“Extra steel Caliper mod 0289”, mm/inch reading scale, precision 0.05 mm, Mario De Maio, Milano, Italy); DMSO was used as negative control (10 μL/paper disc). The antifungal activity was expressed in mm. Samples were tested in triplicate and the results are expressed as mean ± standard deviation.
4.9. Cell Cultures
Human neuroblastoma (SH-SY5Y) cancer cells were cultured in Roswell Park Memorial Institute Medium (RPMI) supplemented with 1% l-glutamine, 10% heat-inactivated fetal bovine serum (FBS), 1% penicillin/streptomycin (all from Sigma Aldrich) at 37 °C in an atmosphere of 95% O2 and 5% CO2.
4.10. MTT Assay
Cells were plated (7 × 103) in 96-well culture plates in 150 µL of culture medium and incubated at 37 °C in humidified atmosphere of 95% O
2 and 5% CO
2. The day after, a 150 µL aliquot of serial dilutions of 1,8- cineole and the essential oil (1600–50 µg/mL) was added to the cells and incubated for 24 h. DMSO alone was used as control. Cell viability was assessed through MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. Briefly, 30 µL of MTT (5 mg/mL) was added and the cells incubated for additional 3 h. Thereafter, cells were lysed and the dark blue crystals solubilized with 30 µL of a solution containing 50%,
v/
v,
N,
N-dimethylformamide, 20%,
w/
v, SDS with an adjusted pH of 4.5. The optical density (OD) of each well was measured with a microplate spectrophotometer (Thermo Scientific Multiskan GO) equipped with a 520 nm filter. Cell viability in response to treatment was calculated as a percentage of control cells treated with DMSO at the final concentration 0.1% viable cells = (100 × OD treated cells)/OD control cells [
43].
4.11. Extraction Proteins and Western Blotting
Cells were treated with different concentrations (400–50 µg/mL) of the 1,8-cineole and essential oil, and after 24 h, they were collected and lysed using Laemmli buffer to extract total proteins. For Western blot analysis, an aliquot of total protein was run on 8% SDS-PAGE gels and transferred to nitrocellulose. Nitrocellulose blots were blocked with 10% non-fat dry milk in Tris buffer saline 0.1% Tween-20 over night at 4 °C. After blocking, blots were incubated with antibodies raised against ADCY1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), phosphorylated p44/42 MAP kinase (ERK), or total phosphorylated p44/42 MAP kinase (ERK) for 3 h at room temperature. Immunoreactivity was detected by sequential incubation with horseradish peroxidase-conjugated secondary antibody (Amersham Biosciences, Pittsburgh, PA, USA) and enhanced chemiluminescence reagents (ImmunoCruz, Santa Cruz Biotechnology, Santa Cruz, CA, USA) [
44].
4.12. Statistical Analysis
All experiments were carried out in triplicate. Data of each experiment were statistically analyzed using GraphPad Prism 6.0 software (GraphPad Software Inc., San Diego, CA, USA) followed by comparison of means (two-way ANOVA) using Dunnett’s multiple comparisons test, at the significance level of p < 0.05.
