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Laurus nobilis: Composition of Essential Oil and Its Biological Activities

Article  in  Molecules · June 2017

DOI: 10.3390/molecules22060930

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Article
Laurus nobilis: Composition of Essential Oil and Its
Biological Activities
Lucia Caputo 1, Filomena Nazzaro 2, Lucéia Fatima Souza 1,3, Luigi Aliberti 1, Laura De Martino 1,
Florinda Fratianni 2, Raffaele Coppola 4, and Vincenzo De Feo 1,*
1 Department of Pharmacy, University of Salerno, Via Giovanni Paolo II, 132, 84084 Fisciano (Salerno), Italy;
lcaputo@unisa.it (L.C.); luceia.souza@ufrgs.br (L.F.S.); luigialiberti83@libero.it (L.A.);
ldemartino@unisa.it (L.D.M.)
2 Istituto di Scienze dell’Alimentazione, Consiglio Nazionale delle Ricerche (ISA-CNR), Via Roma 64,

83100 Avellino, Italy; mena@isa.cnr.it (F.N.); fratianni@isa.cnr.it (F.F.)

3 Post-Doctoral by National Counsel of Technological and Scientific Development (CNPq/Brazil), 70000-000

Brasília, Brazil
4 Dipartimento di Agricoltura, Ambiente e Alimenti-Università del Molise, Via de Sanctis snc, 86100

Campobasso, Italy; coppola@unimol.it

* Correspondence: defeo@unisa.it; Tel.: +39-089-969-751; Fax: +39-089-969-602

Academic Editor: Olga Tzakou

Received: 10 May 2017; Accepted: 1 June 2017; Published: 3 June 2017

Abstract: Laurus nobilis is native to the southern Mediterranean region and cultivated mainly in
Europe and the USA as an ornamental and medicinal plant. The chemical composition of the
essential oil (EO) from leaves of L. nobilis, collected in Southern Italy, was studied by GC and GC-
MS. In all, 55 compounds were identified, accounting for 91.6% of the total oil. 1,8-Cineole (31.9%),
sabinene (12.2%), and linalool (10.2%) were the main components. Antimicrobial and antifungal
activities of EO and 1,8-cineole were determined in vitro. The cytotoxicity of the EO was evaluated
against SH-SY5Y cell line, as well as the influence of the EO on the expression of adenylate cyclase
1 (ADCY1), suggesting possible oil effects on the Central Nervous System.

Keywords: Laurus nobilis L.; essential oil; antibacterial activity; antifungal activity; cytotoxicity;
adenylate cyclase 1 (ADCY1); Central Nervous System (CNS)

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 [4–6]. 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).

Molecules 2017, 22, 930; doi: 10.3390/molecules22060930 www.mdpi.com/journal/molecules

Molecules 2017, 22, 930 2 of 11

2. Results

2.1. Essential Oil Yield and Composition

The hydrodistillation of the leaves of L. nobilis, harvested in Montecorice (Campania, Southern
Italy) provided an essential oil characterized by a typical odor, in a yield of 0.57% of the fresh weight.
Table 1 reports the chemical composition of the oil; the compounds are listed according to their
elution on an HP-5 MS capillary column.

Table 1. Chemical composition of the essential oil (EO) isolated from the leaves of L. nobilis.

No. Compound % Ri a Ri b Identification c

1 Methyl pentanoate 0.1 850 828 1,2
2 Ethyl isovalerate 0.1 853 858 1,2
3 α-Thujene 0.7 916 930 1,2
4 α-Pinene 5.8 922 939 1,2,3
5 Camphene 0.8 935 954 1,2
6 Sabinene 12.2 962 975 1,2
7 β-Pinene 1.4 980 979 1,2,3
8 α-Phellandrene 0.5 991 1002 1,2,3
9 δ-2-Carene 0.4 997 1002 1,2
10 α-Terpinene 0.6 1004 1017 1,2,3
11 o-Cymene 0.3 1013 1026 1,2
12 1,8-Cineole 31.9 1016 1031 1,2,3
13 (Z)-β-Ocimene 0.2 1028 1037 1,2
14 (E)-β-Ocimene 0.2 1038 1050 1,2
15 γ-Terpinene 1.0 1048 1059 1,2,3
16 cis-Sabinene hydrate 0.3 1057 1070 1,2
17 ρ-Mentha-3,8-diene 0.5 1077 1072 1,2
18 trans-Sabinene hydrate 10.2 1093 1098 1,2
19 Linalool 0.1 1096 1096 1,2,3
20 exo-Fenchol 0.1 1111 1121 1,2
21 allo-Ocimene 0.2 1118 1132 1,2
22 trans-Sabinol 0.2 1128 1142 1,2
23 Camphor 0.2 1133 1146 1,2,3
24 β-Pinene oxide 0.1 1147 1159 1,2
25 Isoborneol 0.5 1155 1160 1,2
26 iso-Isopulegol 0.6 1157 1159 1,2
27 neoiso-Isopulegol 2.5 1165 1171 1,2
28 α-Terpineol 3.3 1180 1188 1,2,3
29 cis-Carveol 0.2 1219 1229 1,2
30 cis-p-Mentha-1(7),8-dien-2-ol 0.1 1232 1230 1,2
31 trans-Sabinene hydrate acetate 0.7 1246 1256 1,2
32 2-(1E)-Propenyl-phenol 0.1 1265 1267 1,2
33 neo-3-Thujanol acetate 0.4 1275 1276 1,2
34 α-Terpinen-7-al 0.3 1284 1285 1,2
35 iso-Verbanol acetate 0.3 1306 1309 1,2
36 α-Terpinyl acetate 5.9 1340 1349 1,2
37 Eugenol 1.6 1347 1359 1,2,3
38 Cyclosativene 0.1 1360 1371 1,2
39 Longicyclene 0.2 1373 1374 1,2
40 β-Elemene 0.4 1381 1390 1,2
41 Methyl-eugenol 3.3 1394 1403 1,2,3
42 β-Funebrene 0.5 1408 1414 1,2
43 cis-Thujopsene 0.2 1427 1431 1,2
44 Spirolepechinene 0.1 1445 1451 1,2
45 allo-Aromadendrene 0.1 1449 1460 1,2,3
46 γ-Himachalene 0.1 1474 1482 1,2
47 a-Amorphene 0.1 1483 1484 1,2
48 δ-Amorphene 0.1 1502 1512 1,2
49 δ-Cadinene 0.2 1512 1523 1,2
50 Elemicin 0.5 1546 1557 1,2
51 Spathulenol 0.4 1563 1578 1,2,3
52 Caryophyllene oxide 0.3 1572 1583 1,2,3
Molecules 2017, 22, 930 3 of 11

53 Thujopsan-2-α-ol 0.1 1580 1587 1,2

54 Viridiflorol 0.2 1591 1592 1,2
55 Eremoligenol 0.1 1630 1631 1,2
Total 91.6
Monoterpenes hydrocarbons 34.0
Oxygenated monoterpenes 48.6
Sesquiterpene hydrocarbons 3.2
Oxygenated sesquiterpenes 0.2
Phenolic compounds 5.6
a Linear retention index on a HP-5MS column; b Linear retention index on a HP Innowax column;
c Identification method: 1 = linear retention index; 2 = identification based on the comparison of mass

spectra; 3 = Co-injection with standard compounds.

In all, 55 compounds were identified, accounting for 91.6% of the total oil. Oxygenated
monoterpenes represent 48.6% of the EO, with 1,8-cineole (31.9%), sabinene (12.2%), and linalool
(10.2%) being the main components. Other components were α-terpinyl acetate (5.9%), α-pinene
(5.8%), α-terpineol (3.3%), methyl-eugenol (3.3%), neoiso-isopulegol (2.5%), eugenol (1.6%), β-Pinene
(1.4%), and γ-terpinene (1.0%). Sesquiterpenes represent 3.4% of the oil, the hydrocarbons 3.2% (Z-
caryophyllene 0.5%, β-elemene 0.4%, spathulenol 0.4%), and the oxygenated compounds 0.2%.

2.2. Antimicrobial Activity

The antibacterial activity of the essential oil and of 1,8-cineole was tested, at different amounts,
on five bacterial strains, belonging to both Gram-positive and Gram-negative bacteria—namely
Staphylococcus aureus, Bacillus cereus (4313), and B. cereus (4384) representative of the Gram-positive;
and Escherichia coli and Pseudomonas aeruginosa characteristics Gram-negative bacteria. The results of
the activity, namely the inhibition of the diameters calculated as mm, are given in Table 2.
The essential oil showed significant antimicrobial activity against all microorganisms as early as
the lesser amount used in the test (0.4 µ L), giving inhibition zones between 6.33 and 8.66 mm. Such
results were particularly appealing, as far as we are concerned, considering the zone of inhibition
produced by tetracycline. In fact, the diameters of inhibition produced by 0.4 µ L of the essential oil
resulted in two cases almost equal (versus E. coli) or even superior (versus S. aureus) to that produced
by tetracycline. It should be also emphasized that 1 and 2 µ L of essential oil resulted in a sensitivity
by all the tester strains superior to that given by their contact with tetracycline, giving zones of
inhibition between 13.33 and 18 mm. B. cereus 4313 turned out the most sensitive organism among
those tested, and it was also more sensitive if compared the other strain of B. cereus used in the assay.
In all cases, the essential oil was more effective than 1,8-cineole, which was ineffective against E.
coli (except at 2 µ L/mL) and against P. aeruginosa and S. aureus at concentration of 0.4 µ L/mL.

Table 2. Antimicrobial activity the EO of Laurus nobilis and 1,8-cineole.

Inhibition Diameter (mm)
Bacterial
Laurus nobilis Essential Oil 1,8 Cineole Tetracycline
Strains
0.4 µL/mL 1 µL/mL 2 µL/mL 0.4 µL/mL 1 µL/mL 2 µL/mL 7 µg/mL
B.cereus
8.66 ± 1.54 b 14.66 ± 0.57 c 18 ± 0 e 5.66 ± 1.54 e 12 ± 2.64 c 14.66 ± 0.57 d 10.33 ± 0.57 a
4313
B. cereus
7.66 ± 1.54 b 12 ± 2.64 d 15.66 ± 0.57 e 5.66 ± 1.54 c 11.66 ± 1.54 c 14.66 ± 0.57 e 8.67 ± 1.67 a
4384
S. aureus 8.33 ± 0.57 c 11.66 ± 1.54 a 13.33 ± 1.54 b 0± e 7.66 ± 1.54 d 12 ± 1.54 a 11.33 ± 0.57 a
E. coli 6.33 ± 0.57 e 12 ± 0 a 16 ± 2 e 0±e 0±e 5.66 ± 1.54 e 12.70 ± 1.67 a
P. aeruginosa 8.33 ± 1.54 b 12 ± 1.73 d 15.33 ± 0.57 e 0±e 7.66 ± 1.54 c 12 ± 1.73 d 9.67 ± 0.57 a
Data are expressed in mm. Results are shown as the mean ± SD (n = 3). Means followed by different
letters in each column differ significantly to Dunnett’s multiple comparisons test, at the significance
level of p < 0.05. e: p < 0.0001; d: p < 0.001; c: p < 0.001; b: p < 0.005; a: p < 0.05 vs. tetracycline (7 µ g).

The results of halo inhibition test were confirmed by MIC test (Table 3). In fact, except on Bacillus
cereus 4313, whose growth resulted inhibited using the same volume of EO and 1,8 cineole, in all the
other cases, the essential oil was able to inhibit the growth of the microbial strains with a lower
volume than 1,8-cineole.
Molecules 2017, 22, 930 4 of 11

Table 3. Minimal inhibitory concentration (MIC, µ L) of the EO of Laurus nobilis and of 1,8-cineole.

MIC (µL/mL)
Microorganism
Laurus nobilis 1,8 Cineole
Bacillus cereus 4313 0.2 0.2
Bacillus cereus 4384 0.2 0.4
Staphylococcus aureus 0.4 1
Escherichia coli 0.8 1.5
Pseudomonas aeruginosa 0.4 1

2.3. Antifungal Activity

In Table 4 we report the widths (mm) of the inhibition halos exhibited by different volumes of
L. nobilis EO and of 1,8-cineole against different moulds (Aspergillus niger, A. versicolor, Penicillium
citrinum and P. expansum). Overall, fungal strains were sensitive both to the L. nobilis EO and 1,8-
cineole. P. expansum exhibited the most sensitivity to the action of the essential oil, just at the minimal
volume used in the test, giving halos of 8 mm. A. versicolor was more resistant only at the lower
volume used.

Table 4. Antifungal activity of L. nobilis essential oil and of 1,8-cineole.

A. niger A. versicolor P. citrinum P. expansum

Laurus nobilis EO
0.4 µ L 2±0 - 2.33 ± 0.57 8 ± 1.73
1 µL 4.33 ± 1.52 5.66 ± 1.54 4.66 ± 0.57 9.33 ± 2.08
2 µL 6±1 7.66 ± 1.54 5.66 ± 1.54 9.66 ± 0.57
1,8 cineole
2 µL - - - -
4 µL - - - -
8 µL 5.66 ± 1.54 7.66 ± 1.54 5.66 ± 1.54 9.66 ± 0.57
Data are expressed in mm. Results are shown as mean ± standard deviation (SD) of the inhibition zone (n = 3).

2.4. Cytotoxicity of 1,8 Cineole and Laurus nobilis Essential oil

The treatment of SH-SY5Y neuroblastoma cells with (1600–50 µ g/mL) of 1,8-cineole and Laurus
nobilis essential oil for 24 h resulted in a low cytotoxic activity. 1,8-Cineole and essential oil showed
an IC50 > 2000 g/mL and IC50 = 47,106 µ g/mL, respectively. However, the treatment with essential oil
resulted in a stronger cytotoxicity (IC50 < 500 µ g/mL) (Figure 1).

Figure 1. Cell viability calculated as percentage after MTT assay. Cells were treated with different
concentrations (1600–50 µ g/mL) of 1,8-cineole (A) and L. nobilis essential oil (B), for 24 h and solvent
(DMSO, 0.1%) alone. Data are the mean ± SD of three experiments ** p < 0.01, **** p < 0.0001 vs. DMSO.
Molecules 2017, 22, 930 5 of 11

2.5. Adenylate Cyclase (ADCY1): Western Blot Analysis

We investigated the effects of 1,8-cineole and L. nobilis essential oil in SH-SY5Y human
neuroblastoma cells. Representative Western blots and quantitative densitometry for adenylate
cyclase 1 (ADCY1) protein expression in SH-SY5Y following exposure to different concentrations of
1,8-cineole and the essential oil are shown in Figure 2. Treatments of SH-SY5Y neuroblastoma cells
with 1,8-cineole had no influence on ADCY1 expression (Panel A), whereas a treatment with 200 and
100 µ g/mL of L. nobilis essential oil for 24 h significantly reduced ADCY1 expression (Panel B).

Figure 2. Relative expression levels of the ADCY1 protein in SH-SY5Y cells treated with 1,8-cineole
(A) and Laurus nobilis essential oil (B). Each panel shows the densitometry of bands in the treated
groups and control. Values are the mean ± SD in each group (n = 3). * p < 0.05, compared to control
(ANOVA followed by Dunnett’s multiple comparison test).

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 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 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 [6,17–19]. 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
Molecules 2017, 22, 930 6 of 11

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 IC50 values were > 400 µ g/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 IC50 < 20 µ g/mL were considered to be cytotoxic against the treated cells
[28]. However, comparing the IC50 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 µ g/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 µ g/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.
Molecules 2017, 22, 930 7 of 11

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 N2 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 µ m 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 (C10–C35) with either those of
the literature [37–39] 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 × 108
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 µ L 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 µ g/disc; Sigma
Aldrich, Milano, Italy) was used as reference drugs. The experiments were performed in triplicate
and averaged.
Molecules 2017, 22, 930 8 of 11

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 × 105 cfu/mL. Ciprofloxacin
(3 µ M 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 106 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 µ L of culture medium and incubated
at 37 °C in humidified atmosphere of 95% O2 and 5% CO2. The day after, a 150 µ L aliquot of serial
dilutions of 1,8- cineole and the essential oil (1600–50 µ g/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 µ L 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 µ L 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].
Molecules 2017, 22, 930 9 of 11

4.11. Extraction Proteins and Western Blotting

Cells were treated with different concentrations (400–50 µ g/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.
Acknowledgments: The study was supported by The University of Salerno, FARB 2015.

Author Contributions: V.D.F. conceived and designed the experiments; L.C., L.D.M. and L.A. performed the
experiments; L.C., F.N., and L.F.S. analyzed the data; F.N. contributed to the experiments, provided reagents,
materials, and analysis tools, and helped in drafting the manuscript; F.F. and R.C. contributed to the analysis
tools; V.D.F. wrote the paper.

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

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Sample Availability: Samples of the essential oil and 1,8-cineole are available from the authors.

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