Chemical Composition and In Vitro Antioxidant Activity of Salvia aratocensis (Lamiaceae) Essential Oils and Extracts

Salvia aratocensis (Lamiaceae) is an endemic shrub from the Chicamocha River Canyon in Santander (Colombia). Its essential oil (EO) was distilled from the aerial parts of the plant via steam distillation and microwave-assisted hydrodistillation and analyzed using GC/MS and GC/FID. Hydroethanolic extracts were isolated from dry plants before distillation and from the residual plant material after distillation. The extracts were characterized via UHPLC-ESI(+/−)-Orbitrap-HRMS. The S. aratocensis essential oil was rich in oxygenated sesquiterpenes (60–69%) and presented τ-cadinol (44–48%) and 1,10-di-epi-cubenol (21–24%) as its major components. The in vitro antioxidant activity of the EOs, measured via an ABTS+• assay, was 32–49 μmol Trolox® g−1 and that measured using the ORAC assay was 1520–1610 μmol Trolox® g−1. Ursolic acid (28.9–39.8 mg g−1) and luteolin-7-O-glucuronide (1.16–25.3 mg g−1) were the major S. aratocensis extract constituents. The antioxidant activity of the S. aratocensis extract, obtained from undistilled plant material, was higher (82 ± 4 μmol Trolox® g−1, ABTS+•; 1300 ± 14 μmol Trolox® g−1, ORAC) than that of the extracts obtained from the residual plant material (51–73 μmol Trolox® g−1, ABTS+•; 752–1205 μmol Trolox® g−1, ORAC). S. aratocensis EO and extract had higher ORAC antioxidant capacity than the reference substances butyl hydroxy toluene (98 μmol Trolox® g−1) and α-tocopherol (450 μmol Trolox® g−1). S. aratocensis EOs and extracts have the potential to be used as natural antioxidants for cosmetics and pharmaceutical products.


Introduction
The genus Salvia L. belongs to the Lamiaceae family and includes approximately 1000 species distributed throughout the world [1]. Salvia plants, in the form of infusions and decoctions, have been traditionally used to treat colds, pain, infections, bronchitis, and insomnia [2]. In the Vademecum Colombiano de planta medicinales, only two Salvia species have been documented, i.e., S. paliifolia Kunth, which is a native plant, and S. officinalis L., which was introduced [3]. In Colombia, 20 sections and more than 75 taxa of Salvia have been described, which makes the genus Salvia the most diverse of the Lamiaceae family in Colombia [4,5]. S. aratocensis (J.R.I. Wood & Harely) Fern. Alonso, a species assigned to the Section Angulatae Epling (Figure 1), is a bushy and resistant aromatic plant that grows up to two meters in height and is found mainly between 900 and 2500 MAMSL on the southern slope of the Chicamocha River Canyon in Boyacá and Santander (Colombia) [4,5]. S. aratocensis has an essential oil (EO) rich in the oxygenated sesquiterpene τ-cadinol, which possesses antibacterial activity against Mycobacterium tuberculosis (MIC = 125 µg mL −1 ) [6].
During the distillation of aromatic plants, two byproducts are generated, i.e., hydrolate and residual biomass; the latter can represent more than 98% of the weight of all the plant material used. The residual biomass contains compounds of biological interest such as flavonoids [7], which are plant secondary metabolites classified as phenolic compounds.
During the distillation of aromatic plants, two byproducts are generated, i.e., hydrolate and residual biomass; the latter can represent more than 98% of the weight of all the plant material used. The residual biomass contains compounds of biological interest such as flavonoids [7], which are plant secondary metabolites classified as phenolic compounds. Flavonoids participate in the processes of pigmentation, immunization, protection against UV radiation, and nitrogen fixation, among others [8].
The flavones luteolin, cirsimaritin, and eupatilin have been found in extracts isolated from Salvia spp. [9][10][11]. The biological activities of flavones are as follows: neuroprotective, anti-inflammatory, antiulcer, antimicrobial, antimalarial, antidiabetic, anticancer, and antioxidant activity, among others [12]. Antioxidants such as flavones are used to prevent cancer, cardiovascular disease, and neurodegenerative disorders because they contribute to the balance between antioxidant defense and the generation of radicals through genetic and environmental means [12]. In a previous study, the antioxidant activity of S. aratocensis extracts, measured using the DPPH • assay, was ten times lower than that of vitamin E [13]; however, to date, no reports on the substances present in the extracts have been published.
The main objective of this research was to determine the effect of the distillation processes of S. aratocensis via steam distillation (SD) and microwave-assisted hydrodistillation (MWHD) on the EO and extract chemical compositions and on the antioxidant activity of the extracts obtained from plant material before and after distillation.

Essential Oil and Extract Yields
The yields of essential oils distilled from S. aratocensis via SD and MWHD and those of the hydroalcoholic extracts obtained from dry plant material before and after distillation are shown in Table 1. The flavones luteolin, cirsimaritin, and eupatilin have been found in extracts isolated from Salvia spp. [9][10][11]. The biological activities of flavones are as follows: neuroprotective, anti-inflammatory, antiulcer, antimicrobial, antimalarial, antidiabetic, anticancer, and antioxidant activity, among others [12]. Antioxidants such as flavones are used to prevent cancer, cardiovascular disease, and neurodegenerative disorders because they contribute to the balance between antioxidant defense and the generation of radicals through genetic and environmental means [12]. In a previous study, the antioxidant activity of S. aratocensis extracts, measured using the DPPH • assay, was ten times lower than that of vitamin E [13]; however, to date, no reports on the substances present in the extracts have been published.
The main objective of this research was to determine the effect of the distillation processes of S. aratocensis via steam distillation (SD) and microwave-assisted hydrodistillation (MWHD) on the EO and extract chemical compositions and on the antioxidant activity of the extracts obtained from plant material before and after distillation.

Essential Oil and Extract Yields
The yields of essential oils distilled from S. aratocensis via SD and MWHD and those of the hydroalcoholic extracts obtained from dry plant material before and after distillation are shown in Table 1. The EO yields from S. aratocensis distilled via SD (0.07%) and MWHD (0.08%) were lower than those reported by Bueno et al. [6] (0.5%). The yields of the S. aratocensis extracts obtained from the dry plants before distillation and those from the dry plant material after SD or MWHD were 19%, 4.6%, and 4.5%, respectively. No previous reports were found on the S. aratocensis extraction yields either from dry plants before distillation or from residual biomass after distillation.

Essential Oil Chemical Characterization
S. aratocensis EO analysis identified 28 compounds (Table 2), including 14 sesquiterpene hydrocarbons, 10 oxygenated sesquiterpenes, 2 benzoic acid derivatives, 1 oxygenated monoterpene, and 1 diterpene. Oxygenated sesquiterpenes (60-69%) were the most abundant compounds, followed by sesquiterpene hydrocarbons (24-31%). Figure 2 shows the GC/FID chromatographic profiles of the S. aratocensis EOs obtained via SD and MWHD.   Figure S1 (Supplementary Materials). e Mass spectrum is shown in Figure S2   -0.189 ± 0.005 a Tentative identification based on retention times (tR) and linear retention indices measured using DB-5 (nonpolar) and DB-WAX (polar) columns [6,[14][15][16]. b Tentative identification based on mass spectra (MS; electron ionization, 70 eV, >95% coincidence), study of fragmentation patterns, and comparison with MS spectra from NIST (2017) [15], Adams (2007) [16], and Wiley (2008) [17] spectral databases. c Confirmatory identification based on standard substances, namely, (E)-β-caryophyllene  Figure S1 (Supplementary Materials). e Mass spectrum is shown in Figure S2 Table 2. Figure 3 shows the relative amount variation as a function of the employed distillation technique of the eight major S. aratocensis EO components measured as the GC/FID area relative to that of the internal standard (n-tetradecane). The relative amount of τcadinol found in the S. aratocensis EO under study (45-49%) was two time higher than that  Table 2. Figure 3 shows the relative amount variation as a function of the employed distillation technique of the eight major S. aratocensis EO components measured as the GC/FID area relative to that of the internal standard (n-tetradecane). The relative amount of τ-cadinol found in the S. aratocensis EO under study (45-49%) was two time higher than that reported by Bueno et al. [6] (20%). The A i /A ISTD ratio ( Figure 3) showed that there was no significant difference in the τ-cadinol amount in the EOs obtained via SD or MWHD. The A i /A ISTD ratios of (E)-β-caryophyllene, germacrene D, and benzyl benzoate were higher in the SD-EO than in the MWHD-EO. However, 1,10-di epi-cubenol was higher in the MWHD-EO ( Figure 3). reported by Bueno et al. [6] (20%). The Ai/AISTD ratio ( Figure 3) showed that there was no significant difference in the τ-cadinol amount in the EOs obtained via SD or MWHD. The Ai/AISTD ratios of (E)-β-caryophyllene, germacrene D, and benzyl benzoate were higher in the SD-EO than in the MWHD-EO. However, 1,10-di epi-cubenol was higher in the MWHD-EO ( Figure 3).   Table 3. The extracted ionic currents (EICs) of the protonated molecules [M+H] + of the substances in the extracts obtained from the plant material before and after distillation are shown in Figure 4.
A total of 21 compounds were identified in the S. aratocensis extract from the dry plant material before distillation. Standard substances allowed for the confirmatory identification of 14 compounds. A comparison of exact masses, the fragmentation patterns' isotopic ratios of characteristic ions, and retention times (elution order) with those reported in the scientific literature led to presumptive identifications [9,10,[18][19][20][21][22][23][24][25][26].
The presumptive LC/MS identification was conducted in two stages. First, the exact masses detected in full-scan mode and their corresponding elemental formulas were used to perform a search in databases such as PUBCHEM [23], FOODB [24], and Phenol-Explorer [27] to obtain a list of possible flavonoid-type molecules. The [M+H] + and [M−H] − ions were fragmented in the higher-energy dissociation chamber (HCD) to obtain their mass spectra at 10, 20,30, or 40 eV.
In the second stage, selected ion monitoring (SIM) was performed on those protonated or deprotonated molecules detected in the full scan; these ions were filtered by the quadrupole and stored in the C-trap, from whence they were sent to the HCD. The use of the quadrupole filter to isolate the ions of interest allowed for "cleaner" mass spectra and the execution of the quantification in a more exact and reproducible manner because, depending on the acquisition mode, several substances may eventually coelute and generate ions from different protonated or deprotonated molecules.   Table 3. The extracted ionic currents (EICs) of the protonated molecules [M+H] + of the substances in the extracts obtained from the plant material before and after distillation are shown in Figure 4.
A total of 21 compounds were identified in the S. aratocensis extract from the dry plant material before distillation. Standard substances allowed for the confirmatory identification of 14 compounds. A comparison of exact masses, the fragmentation patterns' isotopic ratios of characteristic ions, and retention times (elution order) with those reported in the scientific literature led to presumptive identifications [9,10,[18][19][20][21][22][23][24][25][26].
The presumptive LC/MS identification was conducted in two stages. First, the exact masses detected in full-scan mode and their corresponding elemental formulas were used to perform a search in databases such as PUBCHEM [23], FOODB [24], and Phenol-Explorer [27] to obtain a list of possible flavonoid-type molecules. The [M+H] + and [M−H] − ions were fragmented in the higher-energy dissociation chamber (HCD) to obtain their mass spectra at 10, 20,30, or 40 eV.
In the second stage, selected ion monitoring (SIM) was performed on those protonated or deprotonated molecules detected in the full scan; these ions were filtered by the quadrupole and stored in the C-trap, from whence they were sent to the HCD. The use of the quadrupole filter to isolate the ions of interest allowed for "cleaner" mass spectra and the execution of the quantification in a more exact and reproducible manner because, depending on the acquisition mode, several substances may eventually coelute and generate ions from different protonated or deprotonated molecules.   Table 3.
The mass spectrum of the S. aratocensis extract obtained from plant material before distillation contained a signal at m/z 449.10780 occurring at tR = 4.82 min. This ion was filtered in SIM mode, and mass spectra were obtained in HCD at 10, 20, 30, and 40 eV. The   Table 3.     The loss of water from protonated flavonoid glycoside molecules may indicate the existence of a C-glycoside [28]. Considering the exact masses of the protonated [M+H] + molecules and those of the product ions, the compound was initially identified as luteolin-C-hexoside. If the sugar was attached at the C-8 carbon, the molecule would be orientin, but if it was at the C-6 position, it would be iso-orientin.
Li et al. [18] showed that during the fragmentation of iso-orientin via the accelerated atom bombardment (FAB) method, the ion [(M+H)-3H 2 O] + was detected with an abundance above 1%, while for orientin, the abundance of the same ion was below 1%. In the present work, the product ion [(M+H)-3H 2 O] + was observed with a relatively high abundance (25%), which supports the identification of the compound as iso-orientin. Three fragments observed in the iso-orientin mass spectrum were products of sugar cleavages, as shown in Figure 5. The loss of water from protonated flavonoid glycoside molecules may indicate t existence of a C-glycoside [28]. Considering the exact masses of the protonated [M+H molecules and those of the product ions, the compound was initially identified as luteoli C-hexoside. If the sugar was attached at the C-8 carbon, the molecule would be orient but if it was at the C-6 position, it would be iso-orientin.
Li et al. [18] showed that during the fragmentation of iso-orientin via the accelerat atom bombardment (FAB) method, the ion [(M+H)-3H2O] + was detected with an abu dance above 1%, while for orientin, the abundance of the same ion was below 1%. In t present work, the product ion [(M+H)-3H2O] + was observed with a relatively high abu dance (25%), which supports the identification of the compound as iso-orientin. Thr fragments observed in the iso-orientin mass spectrum were products of sugar cleavag as shown in Figure 5.
In the iso-orientin mass spectrum obtained using ESI (  The parameters of the calibration curves used for the quantification of the S. aratoce sis extract components are presented in Table 4. The substance amounts (expressed as m of substance g −1 extract) in the S. aratocensis extracts obtained from plant material befo or after distillation are shown in Table 5. The parameters of the calibration curves used for the quantification of the S. aratocensis extract components are presented in Table 4. The substance amounts (expressed as mg of substance g −1 extract) in the S. aratocensis extracts obtained from plant material before or after distillation are shown in Table 5.

Antioxidant Activity
The antioxidant activities of the EOs and extracts of S. aratocensis measured using the ABTS +• and ORAC methods are shown in Table 6. Table 6. Antioxidant activity (measured using the ABTS +• and ORAC methods) of the essential oils distilled from S. aratocensis via steam distillation (EO-SD) and via hydrodistillation assisted by microwave radiation (EO-MWHD) and of the S. aratocensis extracts isolated from dry plants and after their distillation via SD or MWHD.
The following biological activities have been reported for τ-cadinol: spasmolytic activity, according to a test incorporating guinea pig ileum [43]; muscle relaxant via calcium antagonist, according to investigations employing rat aorta [44]; bactericidal, as determined in tests with Staphylococcus aureus; and fungicidal activity, as determined in trials with Trichophyton mentagrophytes Robin [45]. τ-Cadinol regulates dendritic cells' differentiation from human monocytes, which may have interesting applications for cancer treatment [46]. Due to its τ-cadinol content, S. aratocensis EO has great potential for use as a natural ingredient of phytopharmaceutical products. Additional experiments are necessary to increase EO distillation yields and τ-cadinol content.
Ursolic acid, a pentacyclic triterpene, was the most abundant compound identified in the extract from dry plant material before distillation (37 ± 3 mg g −1 ). Its concentrations in the extracts from dry residual biomass after SD or MWHD were 28.9 ± 0.6 mg g −1 and 39.8 ± 0.6 mg g −1 . Thus, SD resulted in a 22% decrease in ursolic acid content in the extract, while hydrodistillation had no significant effect.
Salvigenin, a compound that is more polar than ursolic acid, had larger content variations. The extract from dry plant material before distillation had a salvigenin content of 0.8 ± 0.1 mg g −1 , while the post-distillation extracts had decreases of 67% (SD) and 46% (MWHD). The extract composition analysis showed that the post-distillation changes of low-polarity compounds were smaller than those of phenolic compounds. The phenolic compound losses may be a combination of thermal decomposition and their dissolution into the hydrosol, which is typically discarded. The substance amount decreases were greater in the residual biomass after SD than after MWHD.
The hydroalcoholic extraction yield from dry S. aratocensis plants before distillation was 19%. This means that approximately 703 mg of ursolic acid can be obtained from 100 g of this plant material. The ursolic acid amount would be reduced to 133 or 167 mg if plant residue after SD or MWHD was employed. It is possible to obtain four S. aratocensis harvests per year and 280 ± 38 g (aerial parts) of each harvested plant, offering a 64% moisture content. This translates into the production of approximately 2.8 g of ursolic acid per plant every year.
Ursolic acid is present in a large number of Lamiaceae species [47]. It has important biological properties, including cytotoxic activity against HL-60, BGC, BEL-7402, and HeLa cancer lines [48]. It has anti-inflammatory activity, according to studies concerning the enzymes involved in the inflammatory cascade [49]. Additionally, it is an apoptosis inducer, according to results obtained with A431 squamous cell carcinoma model cell lines and those derived from HaCat keratinocytes [50]. It is also an active compound against Mycobacterium tuberculosis [51]. It inhibits cholesterol synthesis, according to in vivo studies with mice [52]. The DPPH • assay revealed its antiradical activity [53]. A review described other biological activities of ursolic acid, extraction methods, and a collection of patents on its uses in cosmetics (49) and pharmaceuticals (97) [47].
The flavones identified in the S. aratocensis extracts, i.e., apigenin, cirsimaritin, jaceosidin, eupatilin, luteolin, and their glycosylated derivatives, have been found in other species of the same genus, e.g., S. officinalis [9], S. plebeia R. Br. [10], and S. nemorosa L. [11]. Luteolin-7-O-glucuronide was the most abundant flavone in the S. aratocensis extract from pre-distillation plant material (25.3 ± 0.3 mg g −1 extract). Dapkevicius et al. [54] studied the antiradical activity of a Thymus vulgaris L. extract using a DPPH online HPLC method and found that luteolin-7-O-glucuronide was active. In vitro assays employing human lymphocytes that were conducted by Orhan et al. [55] showed that luteolin-7-O-glucuronide at 40 µM was not toxic. These authors found that it has great potential as an antigenotoxic agent against aflatoxin B1. Based on in vitro cellular assays, Cho et al. [56] demonstrated that luteolin-7-O-glucuronide exhibits anti-inflammatory and antioxidant properties.

Antioxidant Activity of Essential Oil and Extracts
The extract from dry plant material before distillation showed the highest antioxidant activity among the samples examined with the ABTS +• assay. S. aratocensis EOs, and the extract from dry plant material before distillation, had about twice the ORAC antioxidant activity of the extract from the post-SD residual biomass. The distillation technique had a small effect on the Eos' antioxidant activity (1520 ± 9 µmol Trolox ® g −1 for SD; 1610 ± 67 µmol Trolox ® g −1 for MWHD). The lower number of phenolic compounds in the SD residual biomass was consistent with the reduced antioxidant activity of its extract. The measured antioxidant activities of S. aratocencis EOs and extracts were higher than those of commercial antioxidants such as BHT (98 ± 5 µmol Trolox ® g −1 ) and α-tocopherol (450 ± 50 µmol Trolox ® g −1 ).

Plant Material
S. aratocensis was cultivated-from cuttings collected in the field-at the CENIVAM Research Center located on the central campus of Universidad Industrial de Santander, Bucaramanga, Santander, Colombia (07 • 08.422 N 073 • 06.960 W). Botanical identification was carried out in the National Herbarium of the Institute of Natural Sciences of the National University of Colombia, Bogotá, Colombia (voucher number COL517740). The S. aratocensis plants collected for distillation and extraction were in a flowering stage and only their aerial undamaged parts were used. The collection permit for gathering S. aratocensis in Chicamocha Canyon (control sheets for herbarium and cuttings for cultivation) was obtained through the contract for access to genetic resources and derived products at N • 270 signed between Universidad Industrial de Santander and the Ministry of Environment and Sustainable Development.

Essential Oil Distillation
EOs were distilled from fresh S. aratocensis plants via two methods: (1) hydrodistillation assisted by microwave radiation (MWHD) in a modified household microwave oven (Model MS32J5133AG, Samsung, Negerin Sembilan, Malaysia) according to the method reproted by Stashenko et al. [57] and (2) steam distillation (SD) using a 0.1 m 3 stainless-steel distiller. Briefly, for MWHD distillation, freshly cut S. aratocensis aerial parts (350 g) were suspended in water (500 mL) in a 2 L flask attached to a Clevenger-type apparatus with a Dean-Stark distillation reservoir and spiral and spherical condensers. The aqueous mixture was subjected to microwave radiation for 1.5 h (15 min × 6). Regarding SD, the freshly cut plant material (19 kg) was compacted to a charge density of 316.3 kg m −3 in a 0.1 m 3 still. Steam was generated in a TECNIK N-553 6 BHP boiler (Tecnik ® Ltd., Bogotá, Colombia), which was operated at 5 × 10 5 Pa with a condensate flow of 180 mL min −1 . Distillation was carried out for 3 h. The EOs were dried with anhydrous sodium sulfate and stored in a refrigerator at 4 • C until analysis and use.

Extraction
S. aratocensis extracts were obtained from dry plant material before and after distillation according to the method reported by Durling et al. [58] with some modifications. Briefly, the plant material (100 g) was deposited in a 4 L glass container, to which an aqueous solution of ethanol in water (2 L, 70% v v −1 ) was added. The extractions were carried out in an Elmasonic S15H ultrasonic bath (Elmasonic, Signen, Germany) (37 kHz) for 1 h at 50 • C. The extracts were filtered (Whatman N • 1 paper), concentrated in a Heidolph ® Basis Hel-Vap HL rotary evaporator (Schwabach, Germany), dried in a VirTis ® AdVantage Plus lyophilizer (New York, USA), and stored in amber glass containers under a nitrogen atmosphere until analysis.

GC/FID and GC/MS Essential Oil Analysis
The EOs of S. aratocensis were analyzed via GC/FID/MS in a gas chromatograph (GC 6890 System Plus, Agilent Technologies, AT, Palo Alto, CA, USA) with a mass-selective detector (AT, MSD 5973 Network) and a flame ionization detection system (FID 250 • C) using electron ionization (EI, 70 eV). Sample introduction (EO in CH 2 Cl 2 at 1.2%) was performed using an automatic injector, which was operated in split mode (ratio 1:30), and the injection port temperature was 250 • C. Capillary DB-5 and DB-WAX (J & W Scientific, Folsom, CA, USA) columns with the same dimensions, namely, 60 m × 0.25 mm, I.D. × 0.25 µm, d f ., were used. The initial pressure at the head of the column was 113 × 10 3 Pa, and a constant flow (1.0 mL min −1 ) of the helium carrier gas (99.995%, Messer, Bucaramanga, Colombia) was maintained. The temperature of the chromatographic oven was programmed to increase from 50 • C (5 min) to 150 • C (2 min) at 5 • C/min and then to 230 • C (10 min) at 5 • C/min. When the DB-5 column was used, ramping with additional heating was employed (up to 275 • C (15 min) at 10 • C/min). The transfer line temperature in the GC/MS system was 285 • C. The mass range used for the acquisition in full-scan mode was m/z 40-450 u, with an acquisition speed of 3.58 scan s −1 , which was set using the MSD ChemStation Ver. G1701DA AT software. Compound identification was based on the linear retention indices (LRIs) and by comparison of the experimental mass spectra with those reported in the Adams 2004 [16], NIST 2017 [15] and Wiley 2008 [17] databases. Standard substances were used, i.e., (E)-β-caryophyllene, α-humulene, (2E, 6Z)-farnesol, benzyl benzoate, and benzyl salicylate, which were analyzed under the same chromatographic conditions as the EOs.

UHPL-ESI (+/−) -Orbitrap-HRMS Analysis of the Extracts
The S. aratocensis hydroethanolic extracts were analyzed in a Vanquish TM ultrahighperformance liquid chromatograph (UHPLC, Thermo Scientific, Waltham, MA, USA), equipped with a degassing unit, a gradient binary pump, and an autosampler, kept at 10 • C. A Zorbax Eclipse XDB C 18 column (Sigma Aldrich, St. Louis, MO, USA) of 50 mm L × 2.1 mm I.D. and a 1.8 µm particle size was used. The column compartment was kept at 40 • C. The flow rate of the mobile phase containing Type I water (A) and MeOH (B), both incorporating formic acid (0.1%) and ammonium formate (5 mM), was 300 µL/min. The initial gradient condition was 100% A, which was changed linearly to 100% B after 8 min, held for 4 min, returned to 100% A after 1 min, and then held for 3 min. The injection volume was 2 µL. The UHPLC was connected to a Q-Exactive Plus Orbitrap mass spectrometer (Thermo Scientific, Bremen, Germany) with a heated electrospray ionization source (HESI-II) and polarity exchangers for periods <500 ms with fragmentation at the HCD. The capillary voltage was 3.5 kV. The nebulizer and capillary temperatures were 350 • C and 320 • C, respectively. Nitrogen (>99% purity) was obtained from a nitrogen generator (Peak Scientific, Scotland, UK). The sheath gas and auxiliary gas (N 2 ) were set at 40 and 10 arbitrary units, respectively. During the full scan of the MS, the mass resolution was set at 70,000 (full width at half maximum (at m/z 200)-FWHM) with an automatic gain control target of 3 × 10 6 , a maximum injection time of the C-trap of 200 ms, and a mass range of m/z 80-1000. Ions injected into the higher-energy dissociation chamber (HCD) via the C-trap were fragmented with normalized collision energies through steps from 10 to 70 eV. Mass spectra were recorded in the all-ion fragmentation mode for each collision energy, employing a mass resolution of 35,000. Full instrument calibration was performed every fortnight using Pierce LTQ Velos ESI positive ion calibration solution and a Pierce ESI negative ion calibration solution (Thermo Scientific, Rockford, IL, USA). Data were analyzed using Thermo Xcalibur 3.1 software (Thermo Scientific, San José, CA, USA).

ABTS +• Assay
The in vitro antioxidant activity of the S. aratocensis EOs and extracts was evaluated using the ABTS +• assay, which was performed according to the methodology described by Re et al. [59] with some modifications. In brief, in an aqueous sodium acetate solution (50 mL, 20 mM, and pH = 4.5), ABTS (7 mM) and potassium persulfate (PDS) (2.45 mM) were reacted for 24 h in the absence of light to produce the radical cation ABTS +• . Absorbance readings were taken at a wavelength corresponding to λ = 750 nm and at a temperature of 25 • C. Antioxidant activity was expressed as µmol Trolox ® . All measurements were performed in triplicate, and the results were expressed as the mean ± standard deviation.

ORAC Assay
The in vitro antioxidant activity of the S. aratocensis EOs and extracts was measured using the ORAC assay, which was performed according to the procedure described by Huang et al. [60] with some modifications. A Varioskan LUX VL0000D0 spectrophotometer (Thermo Scientific, Singapore), equipped with 200 µL 96-well poly(styrene) black microplates, was used under the fluorescence module. Diluted samples of the EOs and extracts (25 µL) were added to each well, and a fluorescein solution (150 µL and 8.16 × 10 −5 mM in phosphate buffer) was added. The mixture was incubated for 18 min at 37 • C and was completed with an AAPH solution (25 µL, 153 mM, in phosphate buffer). Fluorescence was measured for 80 min with excitation wavelengths of λ = 490 nm and emission wavelengths of λ = 520 nm. The antioxidant capacity was determined according to the difference between the area under the curve of the sample and the blank of the phosphate buffer reaction. All measurements were performed in triplicate, and the results were expressed as the mean ± standard deviation.