A New Essential Oil from the Leaves of Gynoxys rugulosa Muschl. (Asteraceae) Growing in Southern Ecuador: Chemical and Enantioselective Analyses

An essential oil, distilled from the leaves of the Andean species Gynoxys rugulosa Muschl., is described in the present study for the first time. The chemical composition was qualitatively and quantitatively determined by GC–MS and GC–FID, respectively. On the one hand, the qualitative composition was obtained by comparing the mass spectrum and the linear retention index of each component with data from literature. On the other hand, the quantitative composition was determined by calculating the relative response factor of each constituent, according to its combustion enthalpy. Both analyses were carried out with two orthogonal columns of nonpolar and polar stationary phases. A total of 112 compounds were detected and quantified with at least one column, corresponding to 87.3–93.0% of the whole oil mass. Among the 112 detected components, 103 were identified. The main constituents were α-pinene (5.3–6.0%), (E)-β-caryophyllene (2.4–2.8%), α-humulene (3.0–3.2%), germacrene D (4.9–6.5%), δ-cadinene (2.2–2.3%), caryophyllene oxide (1.6–2.2%), α-cadinol (3.8–4.4%), 1-nonadecanol (1.7–1.9%), 1-eicosanol (0.9–1.2%), n-tricosane (3.3–3.4%), 1-heneicosanol (4.5–5.8%), n-pentacosane (5.8–7.1%), 1-tricosanol (4.0–4.5%), and n-heptacosane (3.0–3.5%). Furthermore, an enantioselective analysis was carried out on the essential oil, by means of two cyclodextrin-based capillary columns. The enantiomers of α-pinene, β-pinene, sabinene, α-phellandrene, β-phellandrene, linalool, α-copaene, terpinen-4-ol, α-terpineol, and germacrene D were detected, and the respective enantiomeric excess was calculated.


Introduction
During the last 40 years, the phytochemical investigation has shifted from temperate climates to tropical countries, where most of the botanical species are still unstudied [1,2]. In this sense, a great importance is given to the so-called "megadiverse" countries, a group of 17 countries, including Ecuador, characterized by possessing three-fourths of all higher plant species of the world [3]. For this reason, our group has been investigating the phytochemistry of the Ecuadorian flora for more than 20 years, by describing the major metabolites of unprecedented botanical species [4][5][6][7][8][9]. Together with nonvolatile compounds, we are very interested in essential oils (EOs), defined by the European Pharmacopoeia as "odorous products, usually of complex composition, obtained from a botanically defined plant raw material by steam distillation, dry distillation, or a suitable mechanical process without heating" [10][11][12][13][14][15][16][17]. Our interest in the EOs derives from the commercial importance of these mixtures and, overall, from the fact that they can be sources of new or rare sesquiterpenoids, often biologically active, together with enantiomeric compounds. As discussed in a previous paper, we selected the poorly studied genus Gynoxys as a Since this plant is little known and quite rare, no traditional use exists to the best of the authors' knowledge. From the legal point of view, probably due to the lack of botanical information, G. rugulosa is not a protected species, and it does not even appear in the reference publication for threatened taxa (The Red Book of the Endemic Plants in Ecuador). Therefore, the present study presents the first description of an EO distilled from Gynoxys rugulosa Muschl., together with the enantiomeric composition of some chiral terpenes.

Chemical Analysis of the EO
The detailed amount of each component and fraction is represented in Table 1. Overall, with respect to the polar and nonpolar column, the monoterpene fraction ranged between 12.9% and 10.3% of the whole EO respectively, the sesquiterpene fraction ranged between 39.1% and 43.3%, and the other non-terpene compounds ranged between 35.3% and 39.4%. A total of 87.3-93.0% of the oil mass was quantified. The distillation yield of this EO, analytically calculated over four repetitions, was 0.02% ± 0.004% by weight of dry plant material. Since this plant is little known and quite rare, no traditional use exists to the best of the authors' knowledge. From the legal point of view, probably due to the lack of botanical information, G. rugulosa is not a protected species, and it does not even appear in the reference publication for threatened taxa (The Red Book of the Endemic Plants in Ecuador). Therefore, the present study presents the first description of an EO distilled from Gynoxys rugulosa Muschl., together with the enantiomeric composition of some chiral terpenes.

Chemical Analysis of the EO
The detailed amount of each component and fraction is represented in Table 1. Overall, with respect to the polar and nonpolar column, the monoterpene fraction ranged between 12.9% and 10.3% of the whole EO respectively, the sesquiterpene fraction ranged between 39.1% and 43.3%, and the other non-terpene compounds ranged between 35.3% and 39.4%. A total of 87.3-93.0% of the oil mass was quantified. The distillation yield of this EO, analytically calculated over four repetitions, was 0.02% ± 0.004% by weight of dry plant material. According to its chromatographic profiles (Figures 2 and 3), the EO from leaves of G. rugulosa was composed of three main groups of components: a poor monoterpene fraction, an important sesquiterpene fraction, and an abundant heavy fraction, characterized by long-chained alcohols and alkanes.      Table 1.

Enantioselective Analysis of the EO
For almost all the identified enantiomers, the enantioselective analysis was carried out through a 2,3-diacetyl-6-tert-butyldimethylsilyl-β-cyclodextrin capillary column, with the exception of α-copaene and germacrene D. For these compounds, a 2,3-diethyl-6-tertbutyldimethylsilyl-β-cyclodextrin column was used since their enantiomers are inseparable with the other chiral selector. As a result, eight enantiomeric pairs and two enantiomerically pure terpenes were detected. On the one hand, most of the chiral metabolites were present as scalemic mixtures, whereas α-terpineol was practically a racemate. On the other hand, (1R,5R)-(+)-β-pinene and (S)-(+)-β-phellandrene were enantiomerically pure. All the enantiomers were identified through the MS spectrum and by comparing their linear retention indices (LRI) with those of a mixture of enantiomerically pure standards. The enantiomeric distribution and the enantiomeric excess (e.e.) of the detected enantiomers are reported in Table 2.

Chemical Composition and Main Components
The chemical analyses were carried out through two orthogonal columns, affording reciprocally consistent results. Most of the components identified through the nonpolar column were confirmed on the polar one, with few exceptions for some minority compounds. According to our experience, this is not an unusual phenomenon, due to the higher baseline that is sometimes observed with polyethylene glycol stationary phases. As a result, the total quantitative analysis on the nonpolar column resulted a little higher than the one with the polar stationary phase (93.0% vs. 87.3%). This discrepancy is actually acceptable if we consider that it was a 6% difference, spread over 112 compounds. On the other hand, the polar column permitted to separate some constituents that were physically inseparable with the nonpolar phase. Among them, a major compound, corresponding to peak 99, was included.

Chemical Composition and Main Components
As previously mentioned in Section 2.1, the EO distilled from the leaves of G. rugulosa can be described as composed of three main fractions: a monoterpene fraction, a sesquiterpene fraction, and a heavy fraction, the latter constituting long-chained alcohols and alkanes. This last fraction, despite being very abundant, is not common in most EOs, and its constituents are not known for presenting interesting biological activities or constituting important toxicological issues. For these reasons, the discussion of the present volatile fraction focuses on its terpene components. With this respect, the chemical composition of this EO is coherent with the one discussed, in a previous paper, for the entire genus Gynoxys and especially for the species G. miniphylla [18]. In fact, we can find many common major components, which can be better visualized normalizing each amount to the only terpene fraction, in order to neglect the contribution of the heavy components. The results of this approach are shown in Table 3. It can be observed that these two EOs share, with the same order of magnitude, α-pinene, (E)-β-caryophyllene, α-humulene, germacrene D, δ-cadinene, and α-cadinol, whereas α-phellandrene and β-phellandrene are only typical of G. miniphylla. Furthermore, on the one hand, trans-myrtanol acetate is only present in G. miniphylla, whereas, on the other hand, caryophyllene oxide was only detected in G. rugulosa.

Biological Activities of Major Components
According to the chemical composition, we could hypothetically expect for G. rugulosa EO some of the properties expectable for the volatile fraction of G. miniphylla. For example, due to the high amount of α-pinene, the anti-inflammatory, bronchodilator, antibacterial, antifungal, and antileishmanial activities must be considered [92][93][94][95][96][97]. Likewise, a potential cholinergic capacity could be expected [98,99].
For what concerns germacrene D, to the best of the authors' knowledge, no important biological activities have been described in the literature. This sesquiterpene is mainly known for its ecological role as an attractive for the moths of genus Heliothis and Helicoverpa [100][101][102].
Another important component is (E)-β-caryophyllene, which is probably the most common sesquiterpene hydrocarbon in EOs. This metabolite is known to possess a very wide range of biological activities, such as neuroprotective, anti-inflammatory, sedative, anxiolytic, antidepressant, anticonvulsant, and antitumor. Despite the most important activity probably being the anti-inflammatory one, exerted by (E)-β-caryophyllene via countless different mechanisms, this metabolite became quite known for being a nonpsychogenic selective agonist of type 2 cannabinoid receptors (CB2-R) [103].
Another major component is α-humulene, relatively more abundant than (E)-βcaryophyllene in this EO. This metabolite is biogenetically related to (E)-β-caryophyllene, and that is the reason why we often found both sesquiterpenes together in many EOs. Like (E)-β-caryophyllene, the very common α-humulene has also been the object of pharmacological studies [104]. The main biological activity reported for α-humulene is its anticancer property, which it shares with its isomer (E)-β-caryophyllene. Furthermore, α-humulene also synergically enhances the antitumor activity of typical cytotoxic drugs (e.g., paclitaxel), by increasing their bioavailability. Anti-inflammatory, antimicrobial, antileishmanial, antiparasitic, cicatrizing, and gastroprotective activities, among others, have also been demonstrated. Of all these latter activities, the anti-inflammatory one is probably the most promising [104].
Another very common but quite less studied sesquiterpene is δ-cadinene. This metabolite is very abundant in some EOs, such as the one obtained from Kadsura longipedunculata (21.8%) and Cedar atlantica (36.3%) [105,106]. According to the literature, both EOs presented a strong antioxidant and antibacterial activity against Gram-positive bacteria. In addition, on the one hand, the EO from K. longipedunculata demonstrated a potential in vitro anti-inflammatory activity, a pro-apoptosis capacity, and a poor cytotoxic activity [105]. On the other hand, the EO from C. atlantica was mainly interesting for its anti-insect and antibiofilm activities [106].
Lastly, an interesting biological property must be mentioned for α-cadinol. In 2007, Wen et al. investigated the antiviral activity of more than 200 natural products against the severe acute respiratory syndrome coronavirus (SARS-CoV). Of all the assayed products, only 22 showed a strong activity; α-cadinol was among them [107].

Significance of the Enantiomeric Composition
The description of the enantiomeric profile for a new EO is currently a key aspect of its chemical analysis. The importance of the enantioselective analysis is evident if we consider that two enantiomers, chemically indistinguishable in a nonchiral medium, usually show dramatically different in vivo biological properties. In particular, the optical isomers can present different olfactory properties. For this reason, two EOs, showing a very similar chemical composition, can be characterized by two completely different aromas [108]. This phenomenon cannot be explained by a classical chemical analysis but can be understand comparing the enantioselective profiles.
Comparing the EO from G. rugulosa with the volatile fraction of G. miniphylla, the two enantiomeric profiles appear dramatically different [18]. This variability, which can also depend on ecological and climatic factors, attests to the existence in plants of different biosynthetic pathways, where diverse enzymes catalyze the synthesis of different enantiomers for possibly different functions.

Plant Material
The leaves of G. rugulosa were collected on 29 July 2020, from many shrubs in the

EO Distillation and Sample Preparation
The dry, whole leaves were analytically steam-distilled in a glass Marcusson-type apparatus, where the plant material was placed in a separated reservoir, installed between the water heater and the condenser. The bottom of the collection tube was connected to the vapor conduct, such that the aqueous phase was recycled during the process (see Figure 4). Moreover, the collection tube was refrigerated, to avoid overheating of the EO. A volume of 2 mL of cyclohexane, containing n-nonane as an internal standard (0.70 mg/mL), was placed over the aqueous phase in the collection tube. With this configuration, the condensed vapors passed through the cyclohexane layer before collection, and the EO was concentrated in the organic phase. The distillation was repeated four times, for 4 h each, obtaining four samples of EO in cyclohexane, which were directly injected into GC (injection volume: Plants 2023, 12, x FOR PEER REVIEW 10 of 15

Qualitative (GC-MS) and Quantitative (GC-FID) Chemical Analyses
The qualitative analysis of G. rugulosa EO was carried out with gas chromatographymass spectrometry (GC-MS) equipment, consisting of a Trace 1310 gas chromatograph, coupled to a simple quadrupole mass spectrometry detector, model ISQ 7000 (Thermo Fisher Scientific, Walthan, MA, USA). The mass spectrometer was operated in SCAN mode (scan range 40-400 m/z), with the electron ionization (EI) source set at 70 eV, the ion source at 230 °C, and the transfer line at 200 °C. A nonpolar column, based on 5% phenylmethylpolysiloxane, and a polar one, based on a polyethylene glycol stationary phase, were applied to both the qualitative and the quantitative analyses. The nonpolar column was DB-5ms (30 m long, 0.25 mm internal diameter, and 0.25 μm film thickness), whereas the polar one was HP-INNOWax (30 m × 0.25 mm × 0.25 μm), both purchased from Agilent Technology (Santa Clara, CA, USA). For the nonpolar column, the GC oven was operated according to the following program: 50 °C for 10 min., followed by a first thermal gradient of 2 °C/min until 170 °C, and then a second gradient of 10 °C/min until 250 °C, which was maintained for 20 min (total time 98 min). With the polar column, the same thermal program was applied, except that the final temperature was set at 230 °C, due to the lower stability of the polyethylene glycol stationary phase. The injector was operated in split mode (40:1), and its temperature was set at 230 °C. The carrier gas (GC grade helium, from Indura, Guayaquil, Ecuador) was maintained at a constant flow of 1 mL/min. The components of the EO were identified by calculating the linear retention indices (LRIs) according to Van den Dool and Kratz, and by comparing these values and the respective mass spectra with data from literature (see Table 1) [109].
The quantitative analysis was conducted with the same instrument, equipped with a flame ionization detector (FID), and the same two columns used for the qualitative one. The injector parameters, carrier gas flow, and thermal programs were the same as the GC-MS analyses, except for the final temperature time, which was set at 30 min. The constituents of the EO were quantified by external calibration, using iso-propyl caproate as the calibration standard and n-nonane as the internal standard. A six-point calibration curve was traced for each column, as previously described in the literature, with a correlation coefficient of 0.998 [16]. The use of iso-propyl caproate as a quantification standard is based on the principle that, with FID detection, the relative response factors (RRFs) of different analytes versus a unique standard only depend on the combustion enthalpy and,

Qualitative (GC-MS) and Quantitative (GC-FID) Chemical Analyses
The qualitative analysis of G. rugulosa EO was carried out with gas chromatographymass spectrometry (GC-MS) equipment, consisting of a Trace 1310 gas chromatograph, coupled to a simple quadrupole mass spectrometry detector, model ISQ 7000 (Thermo Fisher Scientific, Walthan, MA, USA). The mass spectrometer was operated in SCAN mode (scan range 40-400 m/z), with the electron ionization (EI) source set at 70 eV, the ion source at 230 • C, and the transfer line at 200 • C. A nonpolar column, based on 5% phenyl-methylpolysiloxane, and a polar one, based on a polyethylene glycol stationary phase, were applied to both the qualitative and the quantitative analyses. The nonpolar column was DB-5ms (30 m long, 0.25 mm internal diameter, and 0.25 µm film thickness), whereas the polar one was HP-INNOWax (30 m × 0.25 mm × 0.25 µm), both purchased from Agilent Technology (Santa Clara, CA, USA). For the nonpolar column, the GC oven was operated according to the following program: 50 • C for 10 min., followed by a first thermal gradient of 2 • C/min until 170 • C, and then a second gradient of 10 • C/min until 250 • C, which was maintained for 20 min (total time 98 min). With the polar column, the same thermal program was applied, except that the final temperature was set at 230 • C, due to the lower stability of the polyethylene glycol stationary phase. The injector was operated in split mode (40:1), and its temperature was set at 230 • C. The carrier gas (GC grade helium, from Indura, Guayaquil, Ecuador) was maintained at a constant flow of 1 mL/min. The components of the EO were identified by calculating the linear retention indices (LRIs) according to Van den Dool and Kratz, and by comparing these values and the respective mass spectra with data from literature (see Table 1) [109].
The quantitative analysis was conducted with the same instrument, equipped with a flame ionization detector (FID), and the same two columns used for the qualitative one. The injector parameters, carrier gas flow, and thermal programs were the same as the GC-MS analyses, except for the final temperature time, which was set at 30 min. The constituents of the EO were quantified by external calibration, using iso-propyl caproate as the calibration standard and n-nonane as the internal standard. A six-point calibration curve was traced for each column, as previously described in the literature, with a correlation coefficient of 0.998 [16]. The use of iso-propyl caproate as a quantification standard is based on the principle that, with FID detection, the relative response factors (RRFs) of different analytes versus a unique standard only depend on the combustion enthalpy and, consequently, on the molecular formula of each compound. Therefore, the RRF of each EO component was calculated as described in the literature [110,111]. The total amount of EO, against which the percentage of each component was calculated, was analytically determined through the total area of the chromatogram, to which a mean RRF value was applied. All the analytical-grade solvents, the n-alkanes (C 9 -C 30 ) for retention indices, and the internal standard (n-nonane) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The calibration standard was isopropyl caproate, obtained via synthesis in the authors' laboratory and purified to 98.8% (GC-FID).

Enantioselective Analyses
The enantioselective analyses were carried out by GC-MS, through two enantioselective capillary columns. They were based on 2,3-diethyl-6-tert-butyldimethylsilyl-βcyclodextrin and 2,3-diacetyl-6-tert-butyldimethylsilyl-β-cyclodextrin as chiral selectors (25 m × 250 µm internal diameter × 0.25 µm phase thickness, from Mega, MI, Italy). The GC-MS was operated with the same injector and MS parameters of the qualitative ones. With both enantioselective columns, the following thermal program was applied: 50 • C for 1 min, followed by a thermal gradient of 2 • C/min until 220 • C, which was maintained for 10 min (total time 96 min). Unlike the qualitative and quantitative analyses, a carrier gas constant pressure of 70 kPa was used instead of the constant flow of 1 mL/min. The enantiomers present in the EO, which were separable on the chiral selectors, were identified through the injection of enantiomerically pure standards (1 mg/mL, split 40:1, 1 µL injected). In this case, a mixture of n-alkanes (C 9 -C 21 ) was also injected to calculate the retention indices.

Conclusions
The leaves of the Andean species Gynoxys rugulosa Muschl. produce an essential oil, whose chemical and enantiomeric composition was described in the present study for the first time. Despite the low distillation yield, this volatile fraction could possess some interesting biological properties, due to its chemical composition. In fact, thanks to the presence of (E)-β-caryophyllene, α-humulene, and δ-cadinene, the EO of G. rugulosa could be promising as an antibacterial agent against Gram-positive bacteria and as an anti-inflammatory product. Furthermore, the presence of different biosynthetic pathways, selective for the biosynthesis of specific enantiomers, was proposed. The biological activities, suggested in the present work, should be experimentally verified in future.