Laurus nobilis, Salvia sclarea and Salvia officinalis Essential Oils and Hydrolates: Evaluation of Liquid and Vapor Phase Chemical Composition and Biological Activities

Laurus nobilis, Salvia officinalis and Salvia sclarea essential oils (EOs) and hydrolates (HYs) were investigated to define their chemical compositions and biological properties. Gas-chromatography/Mass-spectrometry (GC/MS) and Headspace-GC/MS (HS-GC/MS) techniques were used to characterize the liquid and vapor phase chemical composition of EOs and HYs. 1,8-Cineole (42.2%, 33.5%) and α-pinene (16.7%, 39.0%) were the main compounds of L. nobilis EO; 1,8-cineole (30.3%, 48.4%) and camphor (17.1%, 8.7%) were for S. officinalis EO; linalyl acetate (62.6%, 30.1%) and linalool (11.1%, 28.9%) were for S. sclarea EO for the liquid and vapor phase, respectively. Chemical profile of HYs was characterized by 1,8-cineole (65.1%, 61.4%) as a main constituent of L. nobilis and S. officinalis HYs, while linalool (89.5%) was the main constituent of S. sclarea HY. The antioxidant activity of EOs and HYs was carried out by DPPH and ABTS assays and antimicrobial properties were also investigated by microdilution and the disc diffusion method for liquid and vapor phase against five different bacterial strains such as Escherichia coli ATCC 25922, Pseudomonas fluorescens ATCC 13525 and Acinetobacter bohemicus DSM 102855 among Gram-negative and Bacillus cereus ATCC 10876 and Kocuria marina DSM 16420 among Gram-positive. L. nobilis and S. officinalis EOs demonstrated considerable antibacterial activity, while S. sclarea EO proved to be less effective. Agar diffusion method and vapor phase test showed the EOs activity with the biggest halo inhibition diameters against A. bohemicus and B. cereus. A remarkably high antioxidant activity was determined for L. nobilis showing low EC50 values and also for S. sclarea; good EO results were obtained in both of the used assays. S. officinalis EC50 values were slightly higher to which corresponds to a lower antioxidant activity. Concerning the HYs, the EC50 values for L. nobilis, S. officinalis and S. sclarea were remarkably high corresponding to an extremely low antioxidant activity, as also obtained by expressing the values in Trolox equivalent antioxidant capacity (TEAC).


Introduction
Medicinal and aromatic plants, characterized by a particular aroma and flavor, are among the first remedies that humans used to treat diseases and are a source of biologically active molecules [1][2][3]. Essential oils (EOs) are produced by the plant secondary metabolism and mainly obtained by the steam distillation process [4]. In the EOs isolation process, Hydrolates (HYs) are also obtained as products of the steam distillation, and a small amount of EO constituents dissolve in HYs. Precious oxygenated compounds, which provide specific organoleptic properties and flavor, as well as biological activity, make them useful for food and cosmetic industries [5,6].
Laurus nobilis, known as laurel, is a perennial shrub which is widespread in temperate and warm regions, especially in the Mediterranean area where is cultivated as an ornamental plant reaching considerable size [7]; it grows spontaneously in the environment and is a diagnostic taxon of many habitats such as Lauro nobilis-Sambucion nigrae, Lauro nobilis-Tilion platyphylli, and Lauro nobilis-Ulmion minoris [8,9]. Laurel belongs to the Lauraceae family, which comprises approximately 2500 species, and was well-known in antiquity for being a symbol of peace and victory in Greece and ancient Rome, both in the military and in sport competitions, with its intertwined branches to create crowns. The use of laurel in foods is nowadays diffused and its particularly odorous leaves and berries are added to various dish preparations. In previous studies, the chemical composition of liquid phase L. nobilis EO was investigated and 1,8-cineole, α-terpinyl acetate, α-pinene and α-terpineol resulted among the main components [10][11][12]. During time laurel gained a considerable importance in traditional medicine [13] and its leaf extracts or EO were used as gastroprotective and antidiarrheal [14,15], analgesic and anti-inflammatory [16], as antidiabetic and to decrease the risk factors of cardiovascular diseases [17,18]. The cytotoxic, antibacterial and antioxidant activities were also investigated, and different authors reported anticancer, antioxidant, insecticidal and molluscidal, antidiabetic, antimicrobial and antifungal activities by L. nobilis [19].
Among the Lamiaceae family, Salvia genus, whose name comes from the Latin "salvare" which means "to heal, to cure," has always been considered to possess numerous medicinal properties in traditional medicine of Asia and the Middle East. About 1000 species belong to this genus and they are herbaceous, suffruticosus or shrubby perennial plants displaying a remarkable diversity in growth forms, in floral morphology and pollination biology, and in secondary metabolites production [20]. The chemical composition of Salvia species is particularly rich in sesquiterpenoids, diterpenoids, triterpenoids, steroids, polyphenols and others [21,22], and bioactive molecules were reported to exert proapototic and anticancer activities, to act in prevention and treatment of cardiovascular diseases, to be therapeutic in liver diseases, to be active in the central nervous system as a potential cognitive protective, and to prevent or disaggregate beta amyloid and fibrils as inhibitors of acetylcholinesterase [23][24][25][26][27][28][29][30][31].
To our knowledge this is the first study on the liquid and vapor phase chemical characterization of L. nobilis, S. officinalis and S. sclarea EOs and their HYs carried out by the headspace-gas-chromatography/mass-spectrometry (HS-GC/MS) technique. The antibacterial activity of EOs and HYs was also investigated by microdilution and disc diffusion methods; furthermore, DPPH and ABTS assays were performed to evaluate their antioxidant activities.

Vapor Phase HYs Chemical Composition
By HS-GC/MS analysis, the composition of the vapor phase of all HYs was described. Seven compounds were identified in L. nobilis and in S. officinalis and only two in S. sclarea, and their percentage values are reported in Table 4. 1,8-Cineole (65.1%, 61.4%) was the main compound in both L. nobilis and S. officinalis HYs, followed by α-thujone (11.1%) and camphor (22.5%) in the first and second HY, respectively. On the other hand, linalool (89.5%) and α-terpineol (10.5%) were the only two compounds detected in S. sclarea HY. Sesquiterpenes compounds were missing in all the HYs (Figure 1). The chromatograms of HYs analyses were also reported (Figures 2-4).

Antibacterial Activity of L. nobilis, S. officinalis and S. sclarea EOs and HYs
To define the bactericidal and inhibitory activities of the investigated aromatic plants, microdilution assay, expressed as MIC (Minimum Inhibitory Concentration) and MBC (Minimal Bactericidal Concentration) and agar diffusion method and vapor phase test expressed as mm of inhibition diameter of the halo for the liquid (IZ) and vapor phase (VIZ), respectively, were carried out. Furthermore MBC/MIC ratios, which consider an agent as bacteriostatic when the MBC/MIC ratio > 4 and as bactericidal when the MBC/MIC ratio ≤ 4 [32], were reported. In Table 5  In the vapor phase test, S. sclarea EO did not inhibit the bacterial growth for all the tested strains, and both the liquid and the vapor phase HY were not active.

Discussion
GC/MS and HS-GC/MS techniques were carried out to describe the chemical composition of EOs (liquid and vapor phase) and HYs. In particular, using the headspace autosampler, as carried out in this case, no solvent extraction process is necessary for the analysis of HYs. The chemical characterization of L. nobilis EO showed a high eucalyptol content for both the liquid and vapor phase (30.4% and 48.4%) as well as in the HY (65.1%), and these data agreed with the reported values [33][34][35].
Variations in the chemical composition of EOs depend on many factors such as the quality of the plant material, the part of the plant used for extraction, the characteristics of the climate and soil, the harvest time, as well as methods and times extraction used for the production and analysis [39,[43][44][45][46][47]. Depending on the distillation time and method, the chemical profile of HYs can also vary [48].
Our biological assays, the microwell dilution method, agar diffusion method and vapor phase test, showed antibacterial activity of the investigated aromatic plants against the tested bacterial strains. L. nobilis EO showed a high antibacterial activity with MIC values ranging from 0.78% to 3.13% and MBC values from 1.56% to 3.13%. For all bacterial strains the IZ halos were detected (from 37.67 ± 2.08 mm to 7.33 ± 0.58 mm) and the vapor phase proved to be even more active against A. bohemicus (VIZ 45.67 ± 4.04 mm), K. marina (VIZ 26.67 ± 2.52 mm) and B. cereus (VIZ 47.33 ± 2.52 mm). No VIZs were detected against the E. coli and P. fluorescens.
Concerning S. officinalis EO, the obtained results confirmed the antibacterial properties of the EO, showing MIC values from 0.39% to 6.25%, MBC values from 0.78% to 6.25% and IZ values from 38.33 ± mm to 8.00 ± mm. As for L. nobilis, a high antibacterial activity for the EO vapor phase was recorded against A. bohemicus, K. marina and B. cereus with the inhibition halos of 21.67 ± 1.53 mm, 26.67 ± 1.15 mm and 23.00 ± 3.61 mm; E. coli and P. fluorescens were not affected by the treatment.
Interestingly, the larger inhibition halos were obtained in L. nobilis EO against A. bohemicus and B. cereus in vapor phase tests and in S. officinalis EO against K. marina, both in the liquid and vapor phase. The agar diffusion test showed activities for L. nobilis EO against E. coli and P. fluorescens with a smaller diameter.
These findings highlighted that L. nobilis and S. officinalis EOs were active against E. coli and P. fluorescens growth; Gram-negative bacteria, usually more resistant than Grampositive for the presence of a complex outer membrane rich in lipopolysaccharide, does not allow the diffusion of hydrophobic molecules present in the essential oils [49]; however, some exceptions were reported [50,51].
S. sclarea EO was less effective, showing MIC and MBC values from 12.5% and 1.56%, and no activity was reported against P. fluorescens. The agar diffusion test revealed that the EO was active against A. bohemicus, K. marina and B. cereus with smaller IZ halos. On the contrary, the vapor phase test for S. sclarea revealed no activity for all bacterial strains.
On the other hand, the investigated aromatic plant HYs were not active against the bacterial strains at tested concentrations. However, it is known that HYs have a lower biological activity than the corresponding essential oils [52] and their potentially antimicrobial activity is also significantly influenced by the distillation method used [33].
As reviewed by Alejo-Armijo et al. [19], L. nobilis possess numerous biological properties and the antimicrobial activities were demonstrated against a broad spectrum of human pathogenic microorganisms, pathogens and spoilage bacteria associated with food [37,53,54]. Its antimicrobial activity is probably related to the high percentage of 1,8cineole (42.2%) and α-pinene (39.0%) detected in the liquid and vapor phase. 1,8-Cineole was found to be active against the development of biofilms formed by the methicillinresistant strains [55], and its antibacterial activity is enhanced when combined with carvacrol [56]. α-Pinene also demonstrated antibacterial activity [57].
In the present investigation, S. officinalis EO was also characterized by 1,8 cineole (30.4% and 48.4%) as the most abundant compound both in the liquid and in vapor phase, while S. sclarea EO, which was less active among the aromatic plants investigated, was rich in linalyl acetate (62.6%) and linalool (28.9%) in the liquid and vapor phase, respectively.
The chemical composition of different aromatic and medicinal herb EOs and their antimicrobial activities against human pathogenic bacteria were studied and a scale of antibacterial activities of their main compounds was defined; the linalyl acetate and linalool were active to a minor extent [58,59]. The liquid and the vapor phase antibacterial activity reported in this study for L. nobilis, S. officinalis and S. sclarea EOs was probably due to the synergistic action of their constituent compounds. In this regard, we assumed that the observed antibacterial action may be explained by the synergistic effect of the different components and/or by the presence of other active molecules, even in small amounts.
The development of antibiotic resistant strains caused the failure of antibiotic therapies with serious consequences on animal and human health. The use of EOs as antibacterial agents opens the prospects for new molecules and new strategies to overcome the infectious; similarly, in crop production the use of such compounds could be an alternative to chemicals against various bacteria and fungi [60,61]. In this view, deeper considerations should be given regarding the EOs mechanism of action based on long-term exposure to pathogens and the synergistic effects for the use of EOs-based antibacterial complexes [62].
In regards to the antioxidant activity, L. nobilis EO was characterized by low EC 50 values, 0.18 ± 0.04 µg/mL and 2.58 ± 0.08 µg/mL (DPPH and ABTS assays, respectively), which revealed a very high antioxidant power. S. sclarea EO gave good results (7.79 ± 1.06 µg/mL and 2.26 ± 0.05 µg/mL) in both assays, respectively. Regarding S. officinalis, IC 50 values were slightly higher (7.79 ± 1.06 µg/mL and 2.26 ± 0.05 µg/mL for DPPH and ABTS, respectively) which correspond to lower antioxidant activity. Concerning the HYs, the obtained EC 50 values were remarkably high in the DPPH and ABTS assays which correspond to an extremely low antioxidant activity, as also obtained by expressing the values in TEAC.
Different papers reported the antioxidant activities of aromatic plant EOs due to the richness of their chemical composition [2,63,64]. L. nobilis EOs from different regions possess antioxidant properties [65][66][67], as well as Salvia species EOs [68].
Particularly, in our investigation oxygenated monoterpenes were the most abundant in L. nobilis (liquid phase) and S. sclarea (liquid and vapor phase) EOs, known for their high antioxidant activity [69][70][71].

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis
To describe the chemical composition of the EOs, a gas chromatograph with a flame ionization detector (FID) directly coupled to a mass spectrometer (MS) Perkin Elmer Clarus 500 model (Waltham, MA, USA) was used. The GC was equipped with a Varian Factor Four VF-1 capillary column. Helium was used as carrier gas at a flow rate of 1 mL/min. The injector was set to a 280 • C and the oven temperature program was as follows: from 60 • C ramped up to 220 • C at a rate of 6 • C min −1 , and finally isothermal at 220 • C for 20 min. For liquid injections, 1 µL of each EO was diluted in 1 mL of methanol and 1 µL of the solution was injected. The Electron Impact-Mass Spectrometer (EI-MS), mass spectra were recorded at 70 eV (EI) and were scanned in the range 40-500 m/z. Ion source and the connection parts temperature was 220 • C. The GC-TIC mass spectra were obtained by the TurboMass data analysis software (Perkin Elmer). The identification of components was performed by matching their mass spectra with those stored in the Wiley and NIST 02 mass spectra libraries database. Furthermore, the linear retention indices (LRIs), (relative to C8-C30 aliphatic hydrocarbons, injected in the column at the same operating conditions described above) were calculated and compared with available retention data present in the literature. Relative percentages of all identified components were obtained by peak area normalization from GC-FID chromatograms without the use of an internal standard or correction factors and were expressed in percentages. All analyses were performed three times.

Head Space GC-MS Analysis
To describe the volatile fraction of the EOs and HYs vapor phase, a Perkin-Elmer Headspace Turbomatrix 40 (Waltham, MA, USA) autosampler connected to GC-MS was used for the headspace analysis. The operative conditions were performed as previously described [72][73][74]. About 1 mL of EO and 2 mL of HY were placed separately in 20 mL vials sealed with headspace PTFE-coated silicone rubber septa and caps. Quantification of identified compounds was performed by GC-FID in the same conditions described in Section 4.2.

Antibacterial Activities
To

Microwell Dilution Method
To screen antimicrobial activities of EOs and their corresponding HYs, Minimum Inhibitory Concentration (MIC) was carried out according to the microwell dilution method in triplicate. All the matrices were diluted in LB broth (from 6.25% to 0.003%). DMSO controls, growth controls without treatments, positive controls with gentamicin (100 µg/mL to 0.049 µg/mL) and sterility controls without bacteria were added to 96 microwell plates. Bacteria at 10 6 CFU/mL were added to each well, except to the sterility control, and were two-fold diluted. The microplates were incubated for 24 h at the corresponding growth temperature. The visualization of the bacterial growth was observed by adding 20 µL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (200 µg/mL, MTT) to each well [75,76].
Minimum Bactericidal Concentration (MBC) was carried out. Plated 10 µL of the last four dilutions from the microwell dilution method in which no bacteria growth was observed on LB agar following 24 h of incubation. The MBC value was determined by an antibacterial agent concentration for which no growth on agar was observed. The assay was carried out in triplicate. To determine the activity of the EOs and the corresponding HYs, the MBC/MIC ratio was reported. The ratio MBC/MIC > 4 reveled bacteriostatic activity, while the ratio MBC/MIC ≤ 4 revealed bactericidal activity for the tested antimicrobial agent [32].

Agar Diffusion Method and Vapor Phase Test (VPT)
The antibacterial activities of L. nobilis, S. officinalis and S. sclarea EOs and HYs liquid and vapor phase were investigated, determining the diameter of the inhibition halo of the bacteria growth on agar petri dishes. Concerning the agar diffusion method, sterile disks (6 mm diameter, Oxoid), impregnated with 10 µL of samples, were placed on LB broth agar surface where the bacterial strains (approximately 10 8 Colony Forming Unit/mL-CFU/mL) had been sown. As a positive control, 2 µL of gentamicin (10 mg/mL) was used.
The petri plates were incubated for 24 h at the corresponding growth temperature, and the diameter of the inhibition halos was recorded by a vernier caliper rule [75].
The antibacterial activity of the vapor phases was evaluated by the disk volatization method. LB agar was poured into Petri plates where each bacterial suspension (10 8 CFU/mL) was plated once it had solidified. A lower amount of agar was poured into Petri plate cover where 6 mm sterile disks had been placed and soaked with 10 µL of the EOs and the corresponding HYs. To seal the Petri plates in order to prevent any vapor leakage, liquid agar was added in the space between the cover and the base. The Petri plates were incubated for 24 h in the upside-down position at the bacterial growth temperature. The inhibition halos were measured by a vernier caliper rule. Negative controls were carried out without the essential. Agar diffusion method and VPTs were carried out in triplicate for each bacterial strain; the means and the respective standard deviations (SD) of the measured halo were reported [72,77].

DPPH Radical Scavenging Activity Assay
To examine the radical scavenging capacity of the L. nobilis, S. sclarea, S. officinalis EOs and their HYs, a DPPH (2,2-diphenyl-1-picrylhydrazyl radical) assay was used [78]. The working solution was made by mixing 3.9 mg of DPPH reagent with 50 mL of methanol. The assay was carried out in 96-well plates in which wells with 100 µL of 12 geometric dilutions in methanol of each sample were added to 100 µL of DPPH solution. As controls, geometric dilutions of the samples without DPPH, Trolox geometric dilutions and blank DPPH were also measured. The plates were incubated for 30 min in dark conditions. The absorbance decreases were measured at 517 nm using a Tecan Sunrise™ UV-vis spectrophotometer. DPPH radical scavenging capacity was considered by the following relationship: where % A.A-percentage of antioxidant activity, A D -absorbance of blank DPPH control, A S -absorbance of the sample. The IC 50 values of the samples (mg/L) and Trolox (µM) were calculated using calibration curves by plotting the % A.A against sample concentrations. The IC 50 parameter of the samples were reported as the required concentration to scavenge 50% of DPPH radical [79]. The activity assay was repeated three times.
TEAC value, which correlates to the concentration of Trolox with the dry weigh of the tested sample, was calculated from the Trolox IC 50 (µM) and the sample IC 50 (mg/L):

ABTS Radical Scavenging Activity Assay
The radical activities of the L. nobilis, S. sclarea and S. officinalis EOs and their HYs were also calculated using the pre-formed radical monocation of 2,2 -azinobis-(3ethylbenzothiazoline-6-sulfonic acid) (ABTS+•), as reported by Pellegrini et al. [80], with some variations. The working aqueous solution was made by mixing potassium persulfate (140 mM) with ABTS reagent (7 mM). The solution was kept for 16 h at room temperature in the dark. Before use, the ABTS+• solution was diluted in ethanol 100% to give an absorbance of 0.70 ± 0.02 at 734 nm. In total, 20 µL of each sample was added to 1980 µL of working solution; as a control, 20 µL of ethanol were used. Therefore, the obtained solutions were incubated for 5 min at room temperature and the absorbances were measured at 734 nm by a Jasco V-630 UV-Visible spectrophotometer and operating Spectra Manager™ software. The final values expressed as a percentage of inhibition against samples concentrations were likened against a standard curve of Trolox [81]. The concentrations used for Trolox range from 0.15625 µM to 2.5 µM.

Statistical Analysis
The results were expressed as means ± standard deviation (SD) and the ANOVA test (one-way analysis of variance test) using GraphPad Prism software (GraphPad Prism 5.0, GraphPad Software Inc., San Diego, CA, USA) was used to evaluate statistical discrepancies between the groups (p values < 0.05).

Conclusions
To the best of our knowledge, this is the first work reporting the liquid and vapor phase chemical composition of L. nobilis, S. sclarea and S. officinalis EOs from the Tuscany region (Italy) investigated by GC-MS and HS-GC/MS techniques to describe the volatile profile of the respective HYs by HS-GC/MS. The antibacterial activity was also evaluated by microdilution and the disc diffusion method, as well as the antioxidant activity by DPPH and ABTS assays. The tested EOs exhibited antibacterial and antioxidant activities, particularly L. nobilis vapor phase, which determined the major inhibition halo. HYs showed to not be antibacterial at the tested concentrations, while the antioxidant properties were extremely low compared to that of the EOs. The obtained results show the potential use of EOs as possible natural antibacterial substances and sources of bioactive molecules. To assess HY biological properties further, tests will be run on other environmental bacterial strains. From a general point of view, our findings confirm the importance of bioactive plant molecules, and as a consequence it will be important to maintain productivity and stability of cultivated plants. In this view, in situ and ex situ conservation of the progenitors of domesticated plants, crop-wild relatives, become necessary, and the gene bank to maintain biodiversity should be improved [82][83][84].