The Essential Oil of Salvia rosmarinus Spenn. from Italy as a Source of Health-Promoting Compounds: Chemical Profile and Antioxidant and Cholinesterase Inhibitory Activity

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), 2,2′-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.


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][6][7].
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].

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 (IC 50 ) values and selectivity index (SI) are reported in Table 2. R2 sample exhibited a good inhibitory activity against AChE (IC 50 of 41.86 µg/mL), 2.1-times higher in comparison to R1 (IC 50 of 85.96 µg/mL). 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 the best results were observed for samples collected in Matmata and Dj. Khamess with IC 50 value of 64.7 against AChE.
A promising AChE inhibitory activity was observed also with Spanish S. rosmarinus essential oil with IC 50 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 IC 50 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.
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.

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), 2,2 -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 IC 50 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 (IC 50 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. Data are expressed as means ± S.D (n = 3). a At concentration of 1000 µg/mL. 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 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 IC 50 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 IC 50 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 IC 50 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 IC 50 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 (SC 50 ) 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 IC 50 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 IC 50 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 (IC 50 of 27.28 µg/mL). The half maximal effective concentration (EC 50 ) (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 (IC 50 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 > 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 for ABTS radical cation scavenging. Thus, the antioxidant activity was correlated to essential oil composition and, consequently, to oxygenated monoterpenes and mixture of mono-and sesquiterpene hydrocarbons [33]. In addition, according to Bajalan et al. [49], the antioxidant activity was positively correlated with the most abundant compounds, such as β-pinene or 1,8-cineole. In a previous study, 1,8-cineole and α-pinene demonstrated to prevent lipid peroxidation [56]. Comparing both compounds, α-pinene showed the major capacity to inhibit lipid peroxidation. The higher lipophilic character of this monoterpene drives strong interactions with cell membranes. Interestingly, α-pinene and 1,8-cineole increased the activity and protein expression of the main antioxidant enzymes including catalase, glutathione peroxidase, glutathione reductase, and superoxide dismutase. However, it is important to know that minor compounds may act in synergism with the most abundant compounds. 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 > βpinene > 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 > 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 for ABTS radical cation scavenging. Thus, the antioxidant activity was correlated to essential oil composition and, consequently, to oxygenated monoterpenes and mixture of mono-and sesquiterpene hydrocarbons [33]. In addition, according to Bajalan et al. [49], the antioxidant activity was positively correlated with the most abundant compounds, such as β-pinene or 1,8-cineole. In a previous study, 1,8-cineole and α-pinene demonstrated to prevent lipid peroxidation [56]. Comparing both compounds, α-pinene showed the major capacity to inhibit lipid peroxidation. The higher lipophilic character of this monoterpene drives strong interactions with cell membranes. Interestingly, α-pinene and 1,8-cineole increased the activity and protein expression of the main antioxidant enzymes including catalase, glutathione peroxidase, glutathione reductase, and superoxide dismutase. However, it is important to know that minor compounds may act in synergism with the most abundant compounds.

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.

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 (C 8 -C 24 ) 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.

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. Physostigmine was used as positive control. Results are calculated as IC 50 values (µg/mL).

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

DPPH and ABTS Tests
The radical scavenging potential was examined using two different spectrophotometric methods, such as DPPH and ABTS tests.
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 (%) = [(A 0 − A)/A 0 ] × 100, where A 0 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 IC 50 values (µg/mL).

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 FeCl 30 (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.

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 = [(A 0 − A t ) /(A 0 * − A t *)] × 100, where A 0 and A 0 * are the absorbance values obtained at the time 0 for samples and control, respectively, while A t and A t * are the absorbance values obtained after 30 and 60 min of incubation for samples and control, respectively.

Statistical Analysis
All experiments were performed in triplicate (n = 3). Data are expressed as means ± standard deviation (SD). The concentration giving 50% inhibition (IC 50 ) 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 deviation. Moreover, for each sample, the average of T-scores was used to calculate global antioxidant score (GAS) value [60].

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.