plants

Article
The Essential Oil of Salvia rosmarinus Spenn. from
Italy as a Source of Health-Promoting Compounds:
Chemical Profile and Antioxidant and Cholinesterase
Inhibitory Activity
Mariarosaria Leporini 1 , Marco Bonesi 1 , Monica Rosa Loizzo 1 ,
Nicodemo Giuseppe Passalacqua 2 and Rosa Tundis 1, *
1 Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036 Rende (CS), Italy;
[email protected] (M.L.); [email protected] (M.B.); [email protected] (M.R.L.)
2 Museum of Natural History of Calabria and Botanic Garden, University of Calabria, 87036 Rende (CS), Italy;
[email protected]
* Correspondence: [email protected]; Tel.: +39-0984-493246




Received: 10 June 2020; Accepted: 24 June 2020; Published: 26 June 2020


Abstract: The chemical composition of the essential oil from Salvia rosmarinus Spenn. collected
in Calabrian Ionian (R1) and Tyrrhenian (R2) coast (Southern Italy) was examined by gas
chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). Essential oils are
mainly characterized by monoterpene hydrocarbons (39.32–40.70%) and oxygenated monoterpenes
(36.08-39.47%). The 1,8-cineole, α-pinene, camphor, and trans-caryophyllene are the most
representative compounds. S. rosmarinus essential oils were investigated for their antioxidant activity
by using 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,20 -azino-bis (3-ethylbenzothiazoline-6-sulfonic
acid) (ABTS), ferric reducing ability power (FRAP), and β-carotene bleaching tests. Additionally,
acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activity assays were used to
screen the neuroprotective effects of S. rosmarinus. R2 showed the highest antioxidant potential as
confirmed by relative antioxidant capacity index (RACI) and exhibited a selective activity against
AChE (half maximal inhibitory concentration, IC50 , value of 41.86 µg/mL). These results suggest
S. rosmarinus essential oil as a potential source of bioactive compounds.

Keywords: Salvia rosmarinus; essential oil; GC; GC-MS; antioxidant potential; cholinesterase
inhibitory activity

1. Introduction
Salvia rosmarinus Spenn., also known as rosemary, is a plant that belongs to the Lamiaceae family [1].
Rosemary is an evergreen, generally erect, rounded shrub with aromatic, needle-like, grey-green leaves
and tiny, two-lipped, pale blue to white flowers. S. rosmarinus is native to dry scrub and rocky places in
the Mediterranean areas of southern Europe to western Asia. Rosemary grows on loam soil with good
drainage in an open, sunny position. It grows best in neutral to alkaline conditions with average fertility.
S. rosmarinus is a well-known aromatic plant, used for thousands of years for ornamental, culinary,
medicinal, and ritual proposes [2]. The most used name, Rosmarinus officinalis L., has to be considered
a synonym of the actual name, Salvia rosmarinus, because molecular investigations evidenced as
Rosmarinus L. is nested in Salvia L. [3]. The distribution area is manly in the western-central part of the
Mediterranean basin, although in the eastern part it is considered introduced in several places [4]. S.
rosmarinus has a remarkable morphological variability which led botanists to the taxonomic recognition
of several specific and infraspecific taxa that are considered in the variability range of the species [5–7].

Plants 2020, 9, 798; doi:10.3390/plants9060798 www.mdpi.com/journal/plants

Plants 2020, 9, 798 2 of 13

The analysis of the genetic variation pattern in the Mediterranean area [3] highlighted the presence
of a basal haplotype from which four branches derived, consisting of a total of nine haplotypes clustered
in two co-ancestry groups of populations. This work included one Calabrian population, which was
found with the two widely shared basal haplotypes. Nonetheless, plants outside the core distribution
of the species were proven to show a different chemical profile and biological activity [8]. Calabrian
populations are in the eastern side of the species’ distribution range and differ from plants commonly
cultivated by the prostrate habitus of individuals.
Phytochemical studies on this Salvia species have reported the presence of different classes of
bioactive compounds mainly including polyphenols, phenol diterpenes, and triterpenes. Rosmarinic
acid, carnosic acid, carnosol, caffeic acid, betulinic acid, and ursolic acid are the dominant constituents.
S. rosmarinus is also a rich source of essential oil [9]. Taking into account that numerous aspects, such
as area of collection, time of harvest, and environmental and agronomic factors, influence the chemical
composition of rosemary essential oil, several studies analysed the chemical profile of rosemary
essential oil in different stages of ontogenesis in Greece [10] and Turkey [11], under the influence of
different climatic factors in Tunisia [12–15] and Turkey [16], at different altitudes in Spain [17], and at
different latitudes and longitudes in Sardinia (Italy) [18], as well as in dependence of pedological
characteristics in France [19].
The essential oil of S. rosmarinus was demonstrated to possess antibacterial, antioxidant, antifungal,
and anti-inflammatory properties [20]. Additionally, it is traditionally recognized to alleviate
muscle pain and to improve cognitive diseases including Alzheimer disease (AD) [21]. AD is a
neurodegenerative disease associated with loss of cholinergic neurons in parts of the brain and
deposition of β-amyloid in the form of neurofibrillary tangles and amyloid plaques [22,23]. At present,
there is no a therapeutic approach that can delay the AD progression, but available treatment has
provided symptomatic benefits. Nowadays, two main classes of drugs are available for AD patients:
Cholinesterase inhibitors, such as donepezil, galantamine, and rivastigmine, and the glutamate
antagonist memantine [24]. Two cholinesterases, acetylcholinesterase (AChE) and butyrylcholinesterase
(BChE), participate in cholinergic neurotransmission by hydrolysing acetylcholine (Ach) in the central
and peripheral nervous system. AChE is responsible for the degradation of Ach in the synaptic cleft
of cholinergic synapses and neuromuscular junctions into choline and acetate. It has a crucial role
in regulating many functions such as learning, memory, cerebral blood flow control, and cortical
organization of movement. On the other hand, BChE has a higher activity in liver, heart, intestine,
kidney, and lung. The enzymes share 65% amino acids’ sequence homology and show similar molecular
forms and active sites. Oxidative stress has been identified as a contributing factor in the progression
of neurodegenerative diseases. The abnormal cellular metabolism caused by the excessive production
of reactive oxygen species (ROS) could affect both production and accumulation of β-amyloid that
could exacerbate cellular dysfunction and ROS production, thus contributing to a vicious cycle [25].
Thus, restoring the level of acetylcholine and the use of natural antioxidants may be proposed in the
treatment and management of AD [26,27].
Following our previous studies [28–30], the present work aimed to analyse the chemical
composition, the antioxidant effects, and the anti-cholinesterase activities of Italian S. rosmarinus
essential oils. The chemical profile, the antioxidant properties, and acetylcholinesterase (AChE) and
butyrylcholinesterase (BChE) inhibitory activity have been investigated.

2. Results and Discussion

2.1. Chemical Profile

The essential oils obtained by hydrodistillation of S. rosmarinus aerial parts collected in two areas of
Calabria (Southern Italy) were herein investigated. The main constituents are listed in Table 1. Thirty-one
compounds were identified, accounting for 97.48 and 98.78% of the total composition of essential oil for R1
Plants 2020, 9, 798 3 of 13

(essential oil of S. rosmarinus from Ionian coast) and R2 (essential oil of S. rosmarinus from Tyrrhenian coast),
respectively. Chromatograms are reported in Supplementary Materials (Figures S1 and S2).

Table 1. The main constituents (%) of Salvia rosmarinus essential oils.

Nr. Compound Class RI a R1 R2 I.M b Sign

1 Thujene MH 926 2.34 ± 0.03 a 0.88 ± 0.05 b 1,2 **
2 α-Pinene MH 938 10.37 ± 0.01 a 10.96 ± 1.33 b 1,2,3 **
3 Camphene MH 953 6.30 ± 0.24 a 6.87 ± 0.21 b 1,2,3 **
6 Sabinene MH 973 2.82 ± 0.11 a 1.01 ± 0.06 b 1,2,3 **
4 β-Pinene MH 980 7.89 ± 1.03 b 8.23 ± 0.62 a 1,2,3 **
5 Myrcene MH 993 1.32 ± 0.06 b 2.73 ± 0.12 a 1,2,3 **
7 α-Phellandrene MH 1005 0.70 ± 0.02 a 0.39 ± 0.04 b 1,2 **
8 δ-3-Carene MH 1009 nd 0.34 ± 0.02 a 1,2 **
9 α-Terpinene MH 1012 1.19 ± 0.08 b 1.35 ± 0.03 a 1,2,3 **
10 o-Cymene MH 1020 0.25 ± 0.2 a tr 1,2 **
11 p-Cymene MH 1025 1.80 ± 0.18 a 0.67 ± 0.07 b 1,2 **
12 Limonene MH 1030 1.78 ± 0.06 b 2.30 ± 0.09 a 1,2,3 **
13 1,8-Cineole OM 1034 16.98 ± 2.11 b 21.89 ± 2.32 a 1,2,3 **
14 γ-Terpinene MH 1057 2.76 ± 0.25 a 2.42 ± 0.06 b 1,2,3 **
15 Terpinolene MH 1086 1.18 ± 0.05 a 1.17 ± 0.01 a 1,2,3 ns
16 Linalool OM 1098 0.35 ± 0.06 a nd 1,2,3 **
17 α-Thujone OM 1106 0.10 ± 0.01 a nd 1,2 **
18 Camphor OM 1145 7.27 ± 0.23 b 11.08 ± 0.76 a 1,2 **
19 Borneol OM 1167 5.30 ± 0.26 a 3.31 ± 0.08 b 1,2 **
20 Terpinen-4-ol OM 1176 2.03 ± 0.09 a nd 1,2 **
21 α-Terpineol OM 1189 4.05 ± 0.26 a 3.19 ± 0.10 b 1,2,3 **
22 (-)-Bornyl acetate SH 1286 2.40 ± 0.11 b 4.26 ± 0.12 a 1,2 **
23 α-Cubebene SH 1352 nd 0.43 ± 0.03 a 1,2 **
24 α-Copaene SH 1377 0.24 ± 0.03 a nd 1,2 **
25 trans-Caryophyllene SH 1415 10.58 ± 1.98 a 8.62 ± 0.17 b 1,2,3 **
26 Aromadendrene SH 1437 0.30 ± 0.03 a 0.25 ± 0.04 a 1,2 ns
27 α-Humulene SH 1455 1.95 ± 0.05 a 1.49 ± 0.10 b 1,2 **
28 γ-Muurolene SH 1478 0.32 ± 0.05 a 0.32 ± 0.01 a 1,2 ns
29 α-Amorphene SH 1487 0.25 ± 0.02 a tr 1,2 **
30 δ-Selinene SH 1493 tr 0.58 ± 0.02 a 1,2 **
31 β-Bisabolene SH 1508 tr 0.41 ± 0.01 a 1,2 **
32 γ-Cadinene SH 1515 tr 0.44 ± 0.05 a 1,2 **
33 δ-Cadinene SH 1526 0.46 ± 0.03 b 1.41 ± 0.01 a 1,2 **
Caryophyllene
34 OS 1580 0.65 ± 0.04 a 0.54 ± 0.02 b 1,2 **
oxide
35 Viridiflorol OS 1591 1.65 ± 0.07 a tr 1,2 **
36 Manool OS 2055 1.90 ± 0.08 a 1.24 ± 0.06 b 1,2 **
MH 40.70 39.32
OM 36.08 39.47
SH 16.50 18.21
OS 4.20 1.78
Total identified 97.48 98.78
R1: S. rosmarinus from Ionian coast; R2: S. rosmarinus from Tyrrhenian coast. Monoterpene hydrocarbons: MH;
oxygenated monoterpenes: OM; sesquiterpene hydrocarbons: SH; oxygenated sesquiterpenes: OS. Data are reported
as the mean ± standard deviation (n = 3). a RI: Retention indices on the HP 5MS (5%-phenyl)-methylpolysiloxane
nonpolar column. b IM, identification method: 1. Comparison of retention times, 2. Comparison of mass spectra
with MS libraries, 3. Comparison with authentic compounds; tr: Trace (<0.1%); nd: Not detected. Differences were
evaluated by one-way analysis of variance (ANOVA) completed with a multicomparison Tukey’s test; ** p < 0.05.
Means in the same row with different small letters differ significantly (p < 0.05). Sign: Significant; ns: Not significant.

The main volatiles were monoterpene hydrocarbons (40.7 and 39.32%, respectively per R1
and R2), followed by oxygenated monoterpenes (36.08 and 39.47%, respectively per R1 and R2)
and sesquiterpene hydrocarbons (16.5 and 18.21%, respectively per R1 and R2). The 1,8-cineole,
α-pinene, trans-caryophyllene, and β-pinene are the most abundant compounds of both rosemary
essential oils (Figure S3). The 1,8-cineole content was highest in R2 (21.89%) compared to R1 (16.98%).
Trans-caryophyllene content was greater in R1 (10.58) than R2 (8.62%). Conversely, a similar value was
observed for α-pinene (10.96 and 10.37%, for R2 and R1, respectively). Significant amounts of camphor
Plants 2020, 9, 798 4 of 13

(11.08 and 7.27%, respectively for R2 and R1) and camphene (6.87% and 6.30%, respectively for R2
and R1) were found. The δ-3-sarene, α-cubebene, δ-selinene, β-bisabolene, and γ-cadinene were not
detected in R1 sample. On the other hand, linalool, α-thujone, terpinen-4-ol, and α-copaene were not
identified or were absent in sample R2.
The relative quantities, presence, and/or absence of rosemary essential oils’ constituents are
strongly affected by environmental conditions and agronomic management practices, as shown in
literature data. According to Pintore et al. [31], Angioni et al. [10] demonstrated that Sardinian rosemary
essential oil is an α-pinene/borneol/bornyl acetate/verbenone chemotype. Compared with our essential
oils, Sardinian samples presented more content in α-pinene (~23%), borneol (~16%), bornyl-acetate
(~10.4%), and camphene (~7.6%) but lower camphor content in camphor (~4.5%). Verbenone compound
was not detected in our essential oils. In Corsican rosemary essential oil, the same trend and similar
values were observed: α-pinene > borneol > bornyl acetate > verbenone.
Forty-one samples of S. rosmarinus collected in different locations of Sicily were analysed by
Napoli et al. [32]. Monoterpenes, both hydrocarbons (range of 21–68%) and oxygenated (range of
29–79%), were the most highly represented components. Three essential oils obtained from aerial parts
of S. rosmarinus growing in Tunisia were analysed with detection of 38 components. Among them,
1,8-cineole and camphor were found in more content (33.08–37.75% and 13.55–18.13%, respectively)
than our essential oils, but similar values for α-pinene (8.58–9.32%), α-terpineol (6.79–8.17%), camphene
(5.07–5.58%), and borneol (4.08–5.48%) were observed [33].
The different chemical composition of the essential oils is subject to change under the influence of
various factors including climatic conditions, environmental factors, and time of collection [34].

2.2. Cholinesterase Activity

In order to examine the in vitro neuroprotective effects of rosemary essential oils, the inhibitory
activity of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes was assessed.
Indeed, in spite of the multi-factorial nature of AD, the available therapeutic approach currently
applied is based on the cholinergic hypothesis, which postulates that at least some of the cognitive decline
experienced by patients affected by AD results from a deficiency in neurotransmitter acetylcholine
and, thus, in a reduced cholinergic neurotransmission in brain cortical or hippocampal regions.
Therefore, restoring the levels of acetylcholine through the inhibition of both cholinesterases is a
useful therapeutic approach to treat AD. Both rosemary essential oils inhibited AChE and BChE in
a concentration-dependent manner. The half maximal inhibitory concentration (IC50 ) values and
selectivity index (SI) are reported in Table 2. R2 sample exhibited a good inhibitory activity against
AChE (IC50 of 41.86 µg/mL), 2.1-times higher in comparison to R1 (IC50 of 85.96 µg/mL).

Table 2. Cholinesterases’ inhibitory activity of rosemary essential oils.

AChE BChE
Sample SI (BChE/AChE)
IC50 (µg/mL) IC50 (µg/mL)
R1 85.96 ± 3.12 **** 46.71 ± 1.85 **** 0.54
R2 41.86 ± 1.63 **** 48.29 ± 1.90 **** 1.15
Positive control
Physostigmine 0.12 ± 0.01 0.21 ± 0.03 2.0
Data are expressed as means ± S.D (n = 3). SI: selective index. Differences within and between groups were
evaluated by one-way ANOVA followed by a multicomparison Dunnett’s test α = 0.05): **** p < 0.0001 compared
with the positive control.

Interestingly, R2 Italian essential oil showed stronger activity against AChE enzyme compared to
other rosemary essential oils previously investigated. Ben Jemia et al. [20] confirmed the neuroprotective
activity of S. rosmarinus essential oil from Tunisia. In this study, eight essential oils were screened and
Plants 2020, 9, 798 5 of 13

the best results were observed for samples collected in Matmata and Dj. Khamess with IC50 value of
64.7 against AChE.
A promising AChE inhibitory activity was observed also with Spanish S. rosmarinus essential
oil with IC50 values of 68.4 µg/mL [35]. The results are in accord with Mata et al. [36] who assessed
the AChE inhibitory activity of rosemary essential oil from Portugal, finding an IC50 of 69.8 µg/mL.
Less activity was reported for Tunisian S. rosmarinus essential oil with inhibition percentage of 36.2%
at concentration of 50 mg/mL against AChE [37]. Turkish S. rosmarinus essential oil was previously
investigated by Orhan et al. [38]. An inhibitions’ percentage of 63.7 and 74% at concentration of 1
mg/mL, respectively for AChE and BChE enzymes, was observed.
Among identified compounds, α-pinene, 1,8-cineole, and camphor exhibited IC50 values of 0.63,
0.67 mM, and >10 mM and showed to be competitively reversible inhibitors of AChE [39]. More
recently, 1,8-cineole and camphor were investigated against AChE. IC50 values of 2.27 and 21.43 µM,
respectively, were found [40]. A percentage of AChE inhibition of 48.5% at 1 mM was found for
β-pinene [41]. In another work, trans-caryophyllene revealed an AChE inhibition of 32% at the final
concentration of 0.06 mM with a more selective activity against BChE (IC50 value of 78.6 µM) [21].
Some studies have analysed structure–activity relationships for monoterpenes and AChE inhibitory
activity [42]. One of these works proposed that the interactions of the hydrocarbon skeletal of terpenes
are with the AChE hydrophobic active center. Since the hydrophobicity degree of the active site on the
AChE is described to be different among the globular forms of AChE, this may further explain the
variation in inhibition seen between studies. In conclusion, the inhibitory activity of the essential oil
likely results from a complex interaction of its chemical components, ultimately producing synergistic
or antagonistic inhibitory responses [20,43]. Indeed, essential oils are mixtures of components that
exhibit generally higher activities than their pure constituents. Their final activities are due to the
combined effects of components.

2.3. Antioxidant Activity

Literature data evidenced a correlation between oxidative stress and degenerative diseases.
Indeed, the oxidative stress is a key hallmark of generation and accumulation of ROS that promote
neuronal cell death [17]. Additionally, an elevated level of lipid peroxidation marker in the brain of
AD patients, especially in the region of the temporal lobe, was found [44]. Iron-induced oxidative
stress, as evidenced by iron accumulation in the brain of AD, is responsible for neurodegeneration in
patients diagnosed with AD. This iron accumulation in the brain of AD patients leads to the formation
of hydroxyl radicals through the Fenton reaction [44]. The search of new cholinesterases’ inhibitors
capable of exerting antioxidant effects is, therefore, desirable.
Herein, in order to estimate the antioxidant potential effects, S. rosmarinus essential oils
were investigated by using different in vitro assays, namely 2,2-diphenyl-1-picrylhydrazyl (DPPH),
2,20 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), ferric reducing ability power (FRAP),
and β-carotene bleaching test (Table 3). An approach based on the use of different tests is required
because antioxidants can exert their capacity through different mechanisms of radicals’ inactivation [45].
This scientific evidence may justify the diversity of results in relation to the applied test.
In DPPH test, R1 sample exhibited a greater radicals’ scavenging activity with a percentage of
33.21%. Conversely, the ABTS+ radical cation was more sensible to R2 oil that showed an IC50 value of
22.61 µg/mL. This evidence can be justified because ABTS+ and DPPH radicals possessed a different
stereochemistry and a different training mechanism and, after reaction with essential oils, these two
radicals can produce different responses.
In the β-carotene bleaching test after 30 min of incubation, R2 demonstrated the highest capacity
to inhibit lipid peroxidation (IC50 of 33.18 µg/mL). The ability to induce the reduction of iron ion,
measured with FRAP test, was greater for R2 with value of 5.59 µM Fe (II)/g.
Plants 2020, 9, 798 6 of 13

Table 3. In Vitro antioxidant activity of rosemary essential oils.

DPPH Test a ABTS Test β-carotene Bleaching Test FRAP Test

Sample
IC50 (µg/mL) IC50 (µg/mL) IC50 (µg/mL) µM Fe (II)/g
R1 33.21% 35.43 ± 2.83 **** 45.21 ± 2.76 **** 49.48 ± 3.08 **** 2.95 ± 1.46 ****
R2 29.84% 22.61 ± 1.91 **** 33.18 ± 2.11 **** 44.81 ± 2.75 **** 5.59 ± 1.95 ****
Positive control
Ascorbic acid 5.02 ± 0.80 1.70 ± 0.06
Propyl gallate 0.09 ± 0.004 0.09 ± 0.004
BHT 63.22 ± 4.3
Data are expressed as means ± S.D (n = 3). At concentration of 1000 µg/mL. Differences within and between
a

groups were evaluated by one-way ANOVA followed by a multicomparison Dunnett’s test α = 0.05): **** p < 0.0001
compared with the positive controls.

Our results differed from those recently reported by Chraibi et al. [46] who analysed the radical
scavenging power against DPPH of S. rosmarinus essential oil from Morocco and found a better activity
with IC50 value of 2.77 mg/mL. El Kamli et al. [47] reported the potential of Moroccan rosemary
essential oils to inhibit lipid peroxidation. However, all samples demonstrated less antioxidant
capacity compared to our samples, showing IC50 values in the range of 18.12–24.23 mg/mL. Previously,
Bouyahya et al. [48] assessed the antioxidant activity of Moroccan S. rosmarinus essential oils but, in
comparison with our results, this essential oil showed major activity with IC50 value of 85.74 and
523.41 µg/mL, respectively, for FRAP and DPPH test. The antioxidant ability of essential oils obtained
from seven Iranian populations of S. rosmarinus was analysed, finding lower inhibition percentages
ranging from 28.73–73.69%, tested at concentration of 3.6 mg/mL in DPPH test [49].
Greater antioxidant activity was previously reported for rosemary, investigated at three different
stages of development, collected near Lake Garda (Italy) [50]. The authors compared the essential oils’
radical scavenging capacity at flowering, post-flowering, and vegetative stage. The best activity was
observed at flowering stage with IC50 values of 36.78 µL, followed by post-flowering, and vegetative
state (79.69 and 111.94 µg/mL, respectively).
Fifteen essential oils from the aerial parts of Algerian S. rosmarinus were investigated by Hendel
et al. [51]. All the samples showed moderate antioxidant activity, being the half-maximal scavenging
concentration (SC50 ) values in the range of 120.4–326.1 µL/mL. The antioxidant activity of S. rosmarinus
essential oil from France was confirmed by Miladi et al. [52] who found a greater activity with IC50
value of 189 µg/mL in DPPH test.
Previously, Kadri et al. [53] screened the S. rosmarinus essential oil from Tunisia for its in vitro
antioxidant activities using DPPH, β-carotene bleaching test, and FRAP assay. The results of the DPPH
assay showed an IC50 value of 110.20 µg/mL, better than our results. In the β-carotene bleaching
test, Tunisian essential oil showed a higher inhibition of lipid peroxidation (IC50 of 27.28 µg/mL).
The half maximal effective concentration (EC50 ) (concentration at which the absorbance is 0.5) value of
S. rosmarinus essential oil was 38.68 µg/mL. Interestingly, Hussain et al. [54] reported that essential oil
of S. rosmarinus, cultivated in Pakistan, showed strong antioxidant activity (IC50 of 20.9 µg/mL).
Relative antioxidant capacity index (RACI) and global antioxidant score (GAS) analyses were
applied to evaluate the antioxidant potential of essential oils (Figure 1). Based on RACI (36.06 and
−35.11 for R1 and R2, respectively) and GAS data (−17.14 and −13.04 for R1 and R2, respectively),
S. rosmarinus essential oil from the Tyrrhenian coast was more active than that from the Ionian coast.
Nie et al. [55] evaluated the radical scavenging activity of active compounds of rosemary essential
oil in DPPH and ABTS test. In the first test, the active order was camphor > bornyl acetate > p-cymene
> 3-carene > o-cymene > α-pinene > terpinen-4-ol > camphene > linalool oxide acetate > β-pinene >
α-bisabolene. The authors suggested that the carbonyl group is important to free radical scavenging
activity and the presence of a double bond conjugated to a carbonyl group will further enhance the
antioxidant activity. In ABTS test, the active order was o-cymene > camphene > α-pinene > camphor
 Plants 2020, 9, 798 7 of 13

> bornyl acetate > α-bisabolene. It was observed that camphor and bornyl acetate showed strong
activity in scavenging DPPH radical but less activity against ABTS radical cation due to different
kinds of free radicals and the way antioxidants interact with free radicals. Indeed, both affected
the scavenging effect of antioxidants. In this case, the cyclic ether group was particularly important
Plants
for 2020,
ABTS 9, xradical
FOR PEER REVIEW
cation scavenging. Thus, the antioxidant activity was correlated to essential 7 of 13
oil
composition and, consequently, to oxygenated monoterpenes and mixture of mono- and sesquiterpene
test, Tunisian essential
hydrocarbons [33]. In oil showedaccording
addition, a higher inhibition
to Bajalan of
et lipid peroxidation
al. [49], (IC50 activity
the antioxidant μg/mL).
of 27.28 was The
positively
half maximalwith
correlated effective concentration
the most (EC50) (concentration
abundant compounds, at which or
such as β-pinene the1,8-cineole.
absorbanceInisa0.5) value of
previous S.
study,
rosmarinus essential oil was 38.68 μg/mL. Interestingly, Hussain et al. [54] reported
1,8-cineole and α-pinene demonstrated to prevent lipid peroxidation [56]. Comparing both compounds, that essential oil
ofα-pinene
S. rosmarinus,
showedcultivated
the majorin Pakistan,
capacity showed
to inhibitstrong
lipid antioxidant
peroxidation. activity 20.9 μg/mL).
(IC50 oflipophilic
The higher character
Relative antioxidant capacity index (RACI) and global antioxidant score
of this monoterpene drives strong interactions with cell membranes. Interestingly, α-pinene (GAS) analyses wereand
applied to evaluate the antioxidant potential of essential oils (Figure 1). Based on
1,8-cineole increased the activity and protein expression of the main antioxidant enzymes including RACI (36.06 and
−35.11 for R1
catalase, and R2, respectively)
glutathione and GAS data
peroxidase, glutathione (−17.14 and
reductase, and−13.04 for R1dismutase.
superoxide and R2, respectively),
However, itS.is
rosmarinus
importantessential
to knowoil from
that minorthecompounds
Tyrrhenian may
coastact
was
in more activewith
synergism thanthe
that from
most the Ionian
abundant coast.
compounds.

a b

Figure 1. Global antioxidant score (GAS) (a) and Relative antioxidant capacity index (RACI); (b) values
of rosemary
Figure essential
1. Global oils. score (GAS) (a) and Relative antioxidant capacity index (RACI); (b) values
antioxidant
of rosemary essential oils.
3. Materials and Methods

3.1.Nie et al. and

Chemicals [55]Reagents
evaluated the radical scavenging activity of active compounds of rosemary
essential oil in DPPH and ABTS test. In the first test, the active order was camphor > bornyl acetate >
p-cymeneSolvents of analytical
> 3-carene grade> were
> o-cymene α-pineneobtained from VWR >International
> terpinen-4-ol camphene >s.r.l. (Milan,
linalool oxideItaly). Ascorbic
acetate > β-
acid, propyl gallate, butylated hydroxytoluene (BHT), 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic)
pinene > α-bisabolene. The authors suggested that the carbonyl group is important to free radical
acid (ABTS)
scavenging solution,
activity and2,2-diphenyl-1-picrylhydrazyl
the presence of a double bond(DPPH), conjugatedtripyridyltriazine
to a carbonyl group(TPTZ), β-carotene,
will further
Tween 20, linoleic acid, acetylcholinesterase (AChE) from Electrophorus electricus
enhance the antioxidant activity. In ABTS test, the active order was o-cymene > camphene > α-pinene (EC 3.1.1.7, Type VI-S)
> and butyrylcholinesterase
camphor > bornyl acetate >(BChE) from equine
α-bisabolene. It was serum
observed (ECthat
3.1.1.8),
camphoracetylthiocholine
and bornyl acetateiodide (ATCI),
showed
butyrylthiocholine iodide (BTCI), 5,5 0 -dithio-bis(2-nitrobenzoic acid) (DTNB), and physostigmine were
strong activity in scavenging DPPH radical but less activity against ABTS radical cation due to
purchased
different from
kinds ofSigma-Aldrich
free radicals andS.p.a.
the(Milan, Italy).
way antioxidants interact with free radicals. Indeed, both
affected the scavenging effect of antioxidants. In this case, the cyclic ether group was particularly
3.2. Plant Materials
important for ABTS radical cation scavenging. Thus, the antioxidant activity was correlated to
S. rosmarinus
essential oil compositionwere collected in Calabriato
and, consequently, (Southern
oxygenated Italy) in Cirò Marina,
monoterpenes andIonian coast
mixture of [R1
mono-(Crotone,
and
latitude: 39 22◦ 0 43” N, longitude: ◦ 0
17addition,
01 52” E,according
360 m above the sea),
sesquiterpene hydrocarbons [33]. In to Bajalan etvoucher specimen
al. [49], the n. CLU
antioxidant 23969],
activity
andpositively
in Praia acorrelated
Mare, Tyrrhenian ◦ 520 14” N, longitude: 15◦ 470 26” E, 30
was with thecoast
most[R2 (Cosenza,
abundant latitude: 39such
compounds, as β-pinene or 1,8-cineole. In a
previous study, 1,8-cineole and α-pinene demonstrated to prevent lipid peroxidation
m above the sea), voucher specimen n. CLU 23974]. Both sites are characterized by a Mediterranean [56].
climate. However,
Comparing the R1 siteα-pinene
both compounds, results with a dry-thermo-Mediterranean
showed the major capacity to inhibit bioclimate, whereas in the
lipid peroxidation. TheR2
site the
higher bioclimate
lipophilic is sub humid-meso-Mediterranean.
character of this monoterpene drivesPlants strongofinteractions
rosemary grow withincell
a shrubby habitat
membranes.
with an openα-pinene
Interestingly, canopy structure and some
and 1,8-cineole bare ground
increased (garrigue)
the activity and onprotein
a rockyexpression
limestone soil.
of the main
Aerial enzymes
antioxidant parts of a including
single plantcatalase,
per eachglutathione
locality wereperoxidase,
harvested inglutathione
order to obtain an adequate
reductase, and
quantity for the analysis. Plant materials were examined for integrity
superoxide dismutase. However, it is important to know that minor compounds may act in and absence of dust and insect
synergism with the most abundant compounds.

3. Materials and Methods

Plants 2020, 9, 798 8 of 13

contamination. The authentication was carried out by Dr. Nicodemo G. Passalacqua at the Natural
History Museum of Calabria and the Botanic Garden, University of Calabria (Italy).

3.3. Extraction Procedure

Fresh S. rosmarinus areal parts (0.74 and 1.20 kg for R1 and R2, respectively) were subject to
hydrodistillation for 3 h using a Clevenger-type apparatus [57]. The white-yellow, obtained essential
oils (3.21 and 7.01 mL) were dried over anhydrous sodium sulphate, stored in hermetically sealed
brown glass bottles, and kept at 4 ◦ C before analysis. Yields (w/w) of 0.58 and 0.41% for R1 and R2,
respectively, were obtained.

3.4. Gas Chromatography (GC) and Gas Chromatography-Mass Spectrometry (GC-MS) Analyses
The chemical composition of S. rosmarinus essential oils was screened by gas chromatography
associated with mass spectrometry, using a Hewlett-Packard gas chromatograph (Agilent, Milan,
Italy) equipped with a non-polar HP-5 capillary column (30 m × 0.25 mm, 0.25 µm), associated with a
Hewlett-Packard mass spectrometer (Agilent, Milan, Italy). The ionization of the sample constituents
was performed in electronic impact (EI, 70 electronvolt). The analyses were carried out with the
following temperature schedule: Isotherm at 50 ◦ C for 5 min, temperature increase from 50 to 250 ◦ C of
5 ◦ C/min, and finally isotherm at 250 ◦ C for 10 min. Helium was used as carrier gas (1.0 mL/min). One
µL of diluted essential oil (1/10 v/v, in n-hexane) was injected (split ratio 1:20). Essential oils were also
analyzed using a Shimadzu GC17A gas chromatograph, equipped with an ionization flame detector
(FID) and an HP-5 capillary column (30 m × 0.25 mm, 0.25 µm) (Shimadzu, Milan, Italy). Nitrogen was
used as transport gas. The conditions used were the same as those described for the GC-MS analyses.
Retention index were determined in relation to a homologous series of n-alkanes (C8 -C24 ) under the
same operating conditions.
The identification of compounds was based on the comparison of their RI, either with those
in literature or with those of available authentic standards (Sigma-Aldrich, Milan, Italy), on the
comparison of the mass spectral data with the Wiley 138 library, and referring to the spectral data of
pure compounds.

3.5. In Vitro Anti-Cholinesterase Activity

The inhibition of AChE and BChE enzymes was measured by using a modified colorimetric
Ellman’s method [20] based on the reaction of released thiocholine to give a colored product with a
chromogenic reagent such as AChE from E. electricus (EC 3.1.1.7, Type VI-S) and BChE equine serum
(EC 3.1.1.8). Acetylthiocholine (ATCI) iodide and butyrylthiocholine iodide (BTCI) were employed as
the substrates of the reaction. In brief, enzyme, essential oils, and phosphate buffer were mixed in
microplates and incubated in an ice bath at 4 ◦ C. After 30 min, physostigmine was added. The reaction
started by adding 5,5’-dithiobis(2-nitrobenzoic-acid) (DTNB) solution and substrate. The microplate
was placed in a thermostatic water bath (Branson model 3800-CPXH, Milan, Italy) for 20 min at 37
◦ C. The reaction was stopped by placing the microplate in an ice bath and adding physostigmine.

The absorbance was measured at 405 nm. The percentage inhibition was calculated by the equation:

Inhibition (%) = [(Blank − Blank positive control) − (Experiment −

Experiment control)]/[Blank − Blank positive control] × 100.
Physostigmine was used as positive control. Results are calculated as IC50 values (µg/mL).

3.6. In Vitro Antioxidant Activity

Four in vitro assays, namely DPPH, ABTS, FRAP, and β-carotene bleaching tests, were herein
applied to investigate the antioxidant effects of R1 and R2 essential oils. Often natural antioxidants may
Plants 2020, 9, 798 9 of 13

be multifunctional and may act in vivo through different mechanisms. Therefore, no single technique
can completely evaluate the antioxidant potential of a plant extract.

3.6.1. DPPH and ABTS Tests

The radical scavenging potential was examined using two different spectrophotometric methods,
such as DPPH and ABTS tests.
DPPH assay was performed according to the procedure previously reported [58]. The DPPH
solution (1.0 × 10−4 M) and rosemary essential oils at different concentrations (62.5–1000 µg/mL) were
mixed. After 30 min, the absorbance was measured at 517 nm.
The DPPH radicals’ scavenging activity was calculated by using the following equation: DPPH
radicals’ scavenging activity (%) = [1 − (sample absorbance with DPPH − sample absorbance without
DPPH)] × 100. ABTS test was employed following the procedure previously used by [59]. ABTS
solution (7 mM) and potassium persulphate (2.45 mM) were mixed in order to obtain ABTS radical
cation solution (ABTS+ ). After 12 h, ABTS+ was diluted with ethanol to final absorbance of 0.70 at 734
nm. Successively, 2 mL of diluted ABTS+ solution was added to extracts (25 µL) at concentrations
(1–400 µg/mL). After 6 min the absorbance was read at 734 nm. The ABTS scavenging capacity was
calculated following the equation: ABTS radicals’ scavenging activity (%) = [(A0 − A)/A0 ] × 100, where
A0 is the absorbance of the control reaction and A is the absorbance in the presence of extract.
In both tests, ascorbic acid was used as positive control. Results are calculated as IC50 values (µg/mL).

3.6.2. FRAP Assay

The ability to reduce iron ions was assessed using FRAP test [58]. A solution of tripyridyltriazine
(TPTZ, 10 mM), 2.5 mL of FeCl30 (20 mM), HCl (40 mM), and 25 mL of acetate buffer (0.3 M) at pH 3.6
was prepared in order to obtain FRAP reagent. The latter (2.0 mL), water (900 µL), and essential oil
(100 µL) at a concentration of 2.5 mg/mL were mixed. After 30 min of incubation, the absorbance was
measured at 595 nm. Butylated hydroxytoluene (BHT) was used as a positive control. The FRAP value
was expressed as µM Fe (II)/g.

3.6.3. β-Carotene Bleaching Test

The ability of rosemary essential oils to protect lipid peroxidation was assessed by using the
β-carotene bleaching test [59]. Propyl gallate was the positive control. In brief, a mixture of β-carotene,
linoleic acid, and 100% Tween 20 was prepared. The obtained emulsion was added to a 96-well
microplate containing sample at concentrations ranging from 100 to 2.5 µg/mL. The absorbance was
measured at 470 nm against a blank at t = 0 and after 30 and 60 min of incubation. The antioxidant
activity was calculated as follows: AA = [(A0 − At ) /(A0 * − At *)] × 100, where A0 and A0 * are the
absorbance values obtained at the time 0 for samples and control, respectively, while At and At * are
the absorbance values obtained after 30 and 60 min of incubation for samples and control, respectively.

3.7. Statistical Analysis

All experiments were performed in triplicate (n = 3). Data are expressed as means ± standard
deviation (SD). The concentration giving 50% inhibition (IC50 ) was calculated by nonlinear regression
with the use of Prism GraphPad Prism version 4.0 for Windows (GraphPad Software, San Diego, CA,
USA). The concentration-response curve was obtained by plotting the percentage inhibition versus
concentration. Differences within and between groups were evaluated by one-way analysis of variance
test (ANOVA) followed by a multi-comparison Dunnett’s test that was used to compare each group
with the positive control in biological assays. Differences were considered to be significant at p < 0.05
in the biological test. Relative antioxidant capacity index (RACI) is an integrated statistical application
to analyse the antioxidant capacity values generated by different in vitro methods [60].
Data obtained from antioxidant assays were employed to calculate RACI value by using the
following equation: RACI = (x − µ)/σ, where x is the raw data, µ is the mean, and σ is the standard
Plants 2020, 9, 798 10 of 13

deviation. Moreover, for each sample, the average of T-scores was used to calculate global antioxidant
score (GAS) value [60].

4. Conclusions
In this work, two Calabrian rosemary populations (collected on the Ionian and Tyrrhenian coasts)
were studied. Chemical analyses revealed 1,8-cineole, α-pinene, camphor, and trans-caryophyllene as
the most representative compounds. However, differences occurred between samples, suggesting that
a different chemical composition of the essential oils is subject to change under the influence of various
factors including climatic conditions and environmental factors. Our results confirmed S. rosmarinus
essential oils, in particular, the Tyrrhenian sample, as a promising source of natural cholinesterase
inhibitors useful in the management and treatment of neurodegenerative diseases.
Obtained data provide the basis for further in vivo studies in order to establish synergistic and
antagonistic effects, and the pharmacokinetic parameters, such as dose and administration route, that
could corroborate these first results on the potential health benefits of S. rosmarinus essential oils and
their pure constituents.

Supplementary Materials: The following are available online at http://www.mdpi.com/2223-7747/9/6/798/s1,

Figure S1: Chromatogram of S. rosmarinus essential oil from Ionian coast (sample R1), Figure S2: Chromatogram
of S. rosmarinus essential oil from Tyrrhenian coast (sample R2), Figure S3: Mass spectra of the most representative
constituents of rosemary essential oil.
Author Contributions: Conceptualization, R.T. and N.G.P.; chemical analysis, M.B. and R.T.; biological
investigation, M.L. and M.R.L.; writing-original draft preparation, M.L. and M.B.; writing-review and editing, R.T.,
M.R.L., and N.G.P.; supervision, R.T. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Acknowledgments: Authors wish to thank Maria Rosaria Rose for her technical support.
Conflicts of Interest: The authors declare not conflicts of interest.

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