The Leaf Essential Oil of Gynoxys buxifolia (Kunth) Cass. (Asteraceae): A Good Source of Furanoeremophilane and Bakkenolide A

The present study describes the chemical and enantiomeric composition of a new essential oil, distilled from the dry leaves of Gynoxys buxifolia (Kunth) Cass. The chemical analysis was conducted by GC-MS and GC-FID, on two orthogonal capillary columns. A total of 72 compounds were detected and quantified with at least one column, corresponding to about 85% by weight of the whole oil mass. Of the 72 components, 70 were identified by comparing the respective linear retention indices and mass spectra with data from the literature, whereas the two main constituents were identified by preparative purification and NMR experiments. The quantitative analysis was carried out calculating the relative response factor of each compound according to their combustion enthalpy. The major constituents of the EO (≥3%) were: furanoeremophilane (31.3–28.3%), bakkenolide A (17.6–16.3%), caryophyllene oxide (6.0–5.8%), and (E)-β-caryophyllene (4.4%). Additionally, the hydrolate was also analyzed with respect to the dissolved organic phase. About 40.7–43.4 mg/100 mL of organic compounds was detected in solution, of which p-vinylguaiacol was the main component (25.4–29.9 mg/100 mL). Finally, the enantioselective analysis of some chiral terpenes was carried out, with a capillary column based on β-cyclodextrin chiral stationary phase. In this analysis, (1S,5S)-(−)-α-pinene, (1S,5S)-(−)-β-pinene, (S)-(+)-α-phellandrene, (S)-(+)-β-phellandrene, and (S)-(−)-terpinen-4-ol were detected as enantiomerically pure, whereas (S)-(−)-sabinene showed an enantiomeric excess of 69.2%. The essential oil described in the present study is a good source of two uncommon volatile compounds: furanoeremophilane and bakkenolide A. The former lacks bioactivity information and deserves further investigation, whereas the latter is a promising selective anticancer product.


Introduction
Essential oils (EOs) are 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" [1]. Since these products present a wide range of applications and biological activities, together with a great commercial interest, our group is currently engaged in the study of volatile fractions from plants [2][3][4][5][6][7][8]. In particular, we are interested in EOs characterized by the presence of abundant sesquiterpene fractions, possibly dominated by new or rare sesquiterpenes. For ecological reasons, such a research line, as well phytochemistry in general, is quite promising in Ecuador. In fact, Ecuador is mentioned among the so-called "megadiverse" countries, a group of 17 countries hosting three-fourths of all higher plant species of the world, most of which remain unstudied [9][10][11]. According to some preliminary unpublished analyses, the genus Gynoxys (Asteraceae) is an excellent candidate for a systematic investigation in this sense. Though the distillation yield is usually quite low, these EOs are rich in metabolites, characterized by sesquiterpenes and, as the present study demonstrates, sometimes dominated by few uncommon major compounds. Therefore, this work is part of an unfunded project, whose aim is the description of the EOs, obtained from the species of the genus Gynoxys growing in the province of Loja (Ecuador). This systematic research began with the recent publications about the volatile fractions in the leaves of G. miniphylla and G. rugulosa and continues with the description of EOs from other species currently under investigation [12,13].
For what concerns the species G. buxifolia, only two studies have been published so far: one focusing on ecology, and the other covering the phytochemistry of the non-volatile fraction [14,15]. To the best of the authors' knowledge, no information has been reported about the EO. From the botanical point of view, Gynoxys buxifolia (Kunth) Cass. is a shrub or treelet, native to the Andean region, and growing between 3000-4000 m above sea level [16]. Furthermore, this species is known by three synonyms: Eupatorium bicolor Lam. ex DC., Gynoxys buxifolia var. brevifolia Hieron., and Senecio buxifolius Kunth [17]. With respect to the geographical distribution, G. buxifolia is almost exclusive to Ecuador, with a few exceptions in Peru and Colombia [16]. This plant, whose traditional name is Tucshi, does not possess medicinal applications; however, the leaves are used as forage for sheep and guineapigs [18].

Chemical Composition of the EO and Hydrolate
In the chemical analysis of the EO and hydrolate of G. buxifolia, a total of 72 compounds were detected and identified with at least one gas chromatographic column.
Regarding the EO, it is dominated by the sesquiterpene fraction, corresponding to 79.4-76.2% of the whole EO mass, with a non-polar and polar column. Furthermore, the monoterpene fraction accounted for 5.9-5.7% of the entire oil. Altogether, the quantified constituents corresponded to 87.9-84.3% of the whole EO. The main components in the volatile fraction of G. buxifolia (≥3.0%) were furanoeremophilane (67, 31.3-28.3%), bakkenolide A (72, 17.6-16.3%), caryophyllene oxide (61, 6.0-5.8%), and (E)-β-caryophyllene (39, 4.4%). Almost all the components of the EO were identified by mass spectrum and linear retention index (LRI), except for the rare sesquiterpenes furanoeremophilane (67) and bakkenolide A (72), whose identification was carried out through nuclear magnetic resonance (NMR) spectroscopy, with 1 H and 13 C NMR experiments. After comparing the MS and NMR spectral data with those from literature, the main constituent of the EO appeared to be identical to furanoeremophilane (67, 114.3 mg), whereas the second main constituent was identical to bakkenolide A (72, 6.6 mg) [19][20][21][22][23][24]. The original NMR and MS spectra are reported in the Supplementary Materials.
For what concerns the hydrolate, the chemical analysis was expressed as milligrams of organic compounds per 100 mL of water. The total amount of organic substances corresponded to 40.7-43.4 mg/100 mL, entirely constituted of oxygenated molecules. Though, in the EO, the non-terpene components accounted for only 2.6-2.4%, in the hydrolate they corresponded to about 75% of the organic fraction. The main constituent in the water phase was p-vinylguaiacol (29), corresponding to more than 50% of the organic fraction in solution. Of all the metabolites, (2E,4E)-heptadienal, o-tolualdehyde, linalool oxide (furanoid), p-mentha-1,5-dien-8-ol, 2-allylphenol, p-vinylguaiacol, and eremophilone were only present in the aqueous phase, whereas 1,8-cineole, linalool, terpinen-4-ol, γterpineol, caryophyllene oxide, cyclocolorenone, and bakkenolide A were detected in both phases. The results of the chemical analyses are detailed in Table 1 as well as Figures 1 and 2, whereas the major components are represented in Figure 3.

Enantioselective Analysis of the EO
The EO was submitted to enantioselective analysis, through a 2,3-diacetyl-6-tertbutyldimethylsilyl-β-cyclodextrin-based column. Seven quiral terpenes were detected, and their enantiomeric distribution analyzed by comparing the respective linear retention indices with those calculated for enantiomerically pure standards. Only sabinene appeared as an enantiomeric pair, with the levorotatory isomer showing an enantiomeric excess (e.e.) of 69%. All the other chiral metabolites were enantiomerically pure, whereas the optical isomers of limonene are inseparable with this chiral selector. The detailed results of the enantioselective analysis are reported in Table 2. Table 2. Enantiomeric separations with 2,3-diacetyl-6-tert-butyldimethylsilyl-β-cyclodextrin column.

Discussion
The chemical analyses of the EO and hydrolate were carried out through two ortogonal columns. The results are qualitatively and quantitatively comparable, with the polar column confirming most of the analytes, detected and quantified with the non-polar stationary phase. Practically, all the constituents detected as traces in the EO with the polar column were compounds close to the detection threshold on the non-polar phase. This fact resulted in a little lower total amount with the polyethylen glycol column with respect to the polysiloxane stationary phase, being 84.3% vs. 87.9%, respectively. This difference (3.6%) is spread over 72 analytes, resulting in a mean value of 0.05% for each compound. Similar considerations can be made for the hydrolate. Therefore, despite the small differences, the qualitative and quantitative analyses of both columns can be considered reciprocally consistent.
The main goal of the present study, apart from the description of an unprecedented EO and its hydrolate, is the identification of G. buxifolia volatile fraction as a good source of furanoeremophilane (67) and bakkenolide A (72). These compounds are well known, but the experience demonstrates that they are quite uncommon in the EOs, and even less so as the two main constituents (about 30% and 17% by weight, respectively, in the present EO). Furanoeremophilane (67) was discovered in 1961 by Novotny et al. in the rhizomes of Petasites officinalis and P. albus, together with some derivatives and other related sesquiterpenes [68][69][70]. Subsequently, its structure was spectroscopically confirmed through NMR experiments by other authors [19][20][21][22][23]. More recently, compound 67 and other furanoeremophilanes have been described in different botanical species, such as Lopholaena dregeana, L. platyphylla, Bedfordia salicina, Senecio inornatus, S. halimifolius, S. medley-woodii, S. inaequidens, and P. hybridus, among others [71][72][73][74]. Sometimes, nonvolatile furanoeremophilanes have been found in fixed fractions through solvent extraction. This is the case of the only existing phytochemical study about G. buxifolia, where 15 nonvolatile furanoeremophilanes were identified in G. acostae, G. buxifolia, and G. nitida [15].
Bakkenolide A (72) was discovered in 1968 in P. japonicus, a plant from the same genus in which furanoeremophilane (67) had been discovered a few years before [22,23,75]. Compound 72 was then identified as a typical metabolite of P. albus itself and, more recently, the same compound and 51 structurally related derivatives were discovered in other species of the same genus [76][77][78]. Bakkenolide A (72) has been found in some species from genera Ligularia, Homogyne, Cacalia, Cetraria, and Hertia [79][80][81][82]. It is interesting to observe that, according to the literature, furanoeremophilane (67) and bakkenolide A (72) were often found in the same plants, suggesting a common biosynthesis for these oxygenated sesquiterpenes. Though the literature usually presents only partial biosynthetic pathways for compounds 67 and 72, a quite complete scheme can be suggested, as in Figure 4. Bakkenolide A (72) was discovered in 1968 in P. japonicus, a plant from the same genus in which furanoeremophilane (67) had been discovered a few years before [22,23,75]. Compound 72 was then identified as a typical metabolite of P. albus itself and, more recently, the same compound and 51 structurally related derivatives were discovered in other species of the same genus [76][77][78]. Bakkenolide A (72) has been found in some species from genera Ligularia, Homogyne, Cacalia, Cetraria, and Hertia [79][80][81][82]. It is interesting to observe that, according to the literature, furanoeremophilane (67) and bakkenolide A (72) were often found in the same plants, suggesting a common biosynthesis for these oxygenated sesquiterpenes. Though the literature usually presents only partial biosynthetic pathways for compounds 67 and 72, a quite complete scheme can be suggested, as in Figure 4.  As usual, the allylic farnesyl cation (74) proceeds from farnesyl pyrophosphate (73), the common precursor of all sesquiterpenes. After that, the intramolecular nucleophilic addition of the terminal double bond to the allyl carbocation affords the germacryl cation (75). At this point, three subsequent carbocation transpositions produce the intermediate 76, which is converted to hydroxy eremophilane (77) by the addition of a molecule of water. Compound 77 undergoes oxidation to fukinone (78) which, through further oxidation, can be converted to the hydroxy fukinone 79 or to the fukinone epoxide 80. The intermediate 79, in a few steps, affords furanoeremophilane (67), whereas 80, after a Favorskii-like rearrangement, produces the hydroxy acid 81. Finally, compound 81, after dehydration and few more steps, produces bakkenolide A (72) [83][84][85].
For what concerns the biological activities of these major compounds, bakkenolide A (72) is clearly the most interesting. In fact, though many furanoeremophilanes have been described for possessing important biological properties, no relevant studies have been found about compound 67. On the other hand, the literature is rich in publications about bakkenolide A (72) and its biological capacities. There are two main properties of Plants 2023, 12, 1323 9 of 16 this metabolite: anticancer and antifeedant. The anticancer property was first described in 1976, when compound 72 was discovered to be cytotoxic against human and rodent cells [77]. These results were extremely promising, since bakkenolide A (72) appeared to be more toxic against human than rodent cells. Furthermore, the activity against cancerous cells was five times higher than the one against normal cells. This evidence suggested a selective cytotoxicity against human cancer, which is the main feature of an ideal anticancer drug. More recently, bakkenolide A (72) was discovered to be active against leukemia, by inhibiting the synthesis of histone deacetylase (HDAC3) [86]. This enzyme is responsible for the acetylation of proteins, and its activity is known to work improperly in human cancer. Furthermore, in leukemia cells, HDAC3 is overexpressed. For these reasons, the inhibitors of HDAC3 are considered to be potential anticancer drugs. In the cited study, bakkenolide A (72) is not a direct HDAC3 inhibitor, but it was shown to reduce the enzyme expression. The underexpression of HDAC3 passes through the inhibition of IκBα, producing the deactivation of NF-κB and, therefore, the suppression of inflammation. As a result, apoptosis is enhanced, cancer diffusion is reduced, and an indirect cytotoxic effect against healthy cells is observed [86].
Since the EO of G. buxifolia spontaneously separated from the aqueous phase, the hydrolate constitutes an interesting byproduct, suitable for investigation. The leaf hydrolate was dominated by the presence of p-vinylguaiacol (29), corresponding to about 67% by weight of the dissolved organic fraction and providing water a pleasant aroma. In 2014, p-vinylguaiacol (29) was investigated as the main degradation product of curcumin while cooking. As a result, compound 29 showed no cytotoxic properties, but it was effective as an activator of the transcription factors Nrf2 and PON1 in a dose-dependent manner. Furthermore, it produced the downregulation of interleukin-6 mRNA levels in a stain of murine macrophages. Therefore, p-vinylguaiacol (29) can be considered a non-cytotoxic, antioxidant and anti-inflammatory product [89]. On this basis, the leaf hydrolate of G. buxifolia could be potentially used as an aqueous phase in the formulation of nutraceuticals and cosmeceuticals.
Finally, the chemical description of this EO could not be considered exhaustive without the enantioselective analysis of at least some chiral components. To the best of the authors' knowledge, there are only two EOs from the genus Gynoxys whose enantioselective analyses are currently available in literature: G. miniphylla and the volatile fraction from G. rugulosa [12,13]. From the comparison between the three volatile fractions, a great difference in the enantiomeric composition emerged. In particular, we can observe that in G. miniphylla and G. rugulosa, most of the investigated chiral compounds were present as scalemic mixtures, with enantiomeric excesses that differ in between the two species. On the other hand, G. buxifolia EO was dominated by enantiomerically pure constituents. Another point was the opposite absolute configuration of the dominant enantiomers. For example, α-phellandrene and β-phellandrene were 100% levorotatory in G. miniphylla and 100% dextrorotatory in G. buxifolia. All these differences could be the result of climatic or ecological factors; however, they demonstrate the existence of different biosynthetic pathways, devoted to the biosynthesis of different enantiomers, produced by the plant for different functions [85]. In fact, despite being two enantiomers characterized by the same chemical-physical properties, they differ in a biological medium for their physiological and pharmacological activities. In the case of the EOs, whose main property is the aroma, two optical isomers of the same chiral metabolite can present different olfactory properties [90]. For this reason, enantioselective analysis is nowadays a fundamental aspect of an EO description, since different volatile fractions, characterized by similar conventional chromatographic profiles, can present a very different aroma due to their various enantiomeric compositions.

Chemicals, Materials, and Equipment
All GC analyses were carried out with a Trace 1310 gas chromatograph (Thermo Fisher Scientific, Walthan, MA, USA). For the qualitative and enantioselective analyses, the instrument was coupled to a simple quadrupole mass spectrometry detector, model ISQ 7000, also from Thermo Fisher Scientific, whereas for the quantitative analyses, a flame ionization detector (FID) was applied. 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 and transfer line were programmed at 230 • C. Two orthogonal capillary columns were used for both the qualitative and quantitative analyses: a non-polar column, based on 5%-phenylmethylpolysiloxane (DB-5ms), and a polar one (HP-INNOWax), based on polyethylene glycol. Both columns were 30 m long, with 0.25 mm internal diameter and 0.25 µm film thickness (Agilent Technology, Santa Clara, CA, USA). The enantioselective analysis was carried out through a 2,3-diacetyl-6-tert-butyldimethylsilyl-β-cyclodextrin-based capillary column (25 m × 250 µm internal diameter × 0.25 µm phase thickness), purchased from Mega, MI, Italy. The carrier gas was GC purity grade helium, purchased from Indura, Guayaquil, Ecuador. All NMR experiments were carried out with a 500 MHz Bruker spectrometer (Bruker, Billerica, MA, USA), whereas deuterated solvents were purchased from Sigma-Aldrich (St. Louis, MO, USA). The preparative chromatographic separations on column were carried out with a Reveleris ®® PREP Purification System, equipped with commercial normal phase (silica gel 60) packed columns and with both a UV-vis and a light scattering detector. The chromatograph and columns were purchased from Büchi (Büchi Labortechnik, Flawil, Switzerland). The thin-layer chromatography separations (TLC), both analytical and preparative, were conducted over silica gel 60 (0.25 mm; GF 254 , Merck) plates (from Sigma-Aldrich). After elution, the TLC plates were initially visualized under UV light (254 and 366 nm), then sprayed with a 0.5% solution of vanillin in H 2 SO 4 /ethanol 4:1, and finally heated at 200 • C. For all the GC analyses, analytical purity grade solvents were used, whereas for analytical TLC and other preparative applications, reagent grade solvents were applied. For the solid phase extraction (SPE), the cartridges were standard products, packed with 1 g of C18 reversed phase and purchased from Sigma-Aldrich. All the solvents, n-alkanes (C 9 -C 22 ) for retention indices, and internal standard (n-nonane) were purchased from Sigma-Aldrich. The calibration standard for GC-FID analyses was isopropyl caproate, obtained by synthesis in the authors' laboratory and purified to 98.8% (GC-FID).

Plant Material
The

Obtention of the EO and Hydrolate
The entire amount of dry plant (2.9 kg) was submitted to steam-distillation for 4 h, in a stainless-steel Clevenger-type apparatus, producing a dark aromatic EO that spontaneously separated from water. The yield of EO was calculated as 0.1% by weight with respect to the dry plant material. Additionally, 50 mL of hydrolate was collected for the analysis of the dissolved organic fraction. Both EO and water phase were stored at −15 • C until use.

GC Sample Preparation
Four samples were prepared by exactly weighing about 10 mg of EO and diluting them with exactly 1 mL of cyclohexane, spiked with n-nonane as an internal standard. The concentration of n-nonane was 0.7 mg/mL. For the hydrolate, four samples were prepared by passing through the reversed phase SPE cartridges, previously washed with methanol and re-conditioned with water and an exact volume of 10 mL of hydrolate. After complete removal of the water phase, the cartridge was eluted with 2 mL of acetone spiked with n-nonane as internal standard. The concentration of n-nonane in acetone was the same as in cyclohexane. Both the EO and hydrolate samples were then directly injected in GC.

Qualitative GC-MS Analyses
The qualitative analyses were conducted in GC-MS by injecting 1 µL of sample with both polar and non-polar columns (spilt ratio 40:1). In all cases, the carrier gas (He) was set at the constant flow of 1 mL/min, whereas the thermal program was as follows: 50 • C for 5 min, followed by a thermal gradient of 3 • C/min until 100 • C, then a second gradient of 5 • C/min until 180 • C, and a final ramp of 10 • C/min until 230 • C. The final temperature was maintained for 5 min. A mixture of n-alkanes, from C 9 to C 22 , was also injected under the same conditions, in order to calculate the linear retention indices (LRIs) of the sample constituents, according to van den Dool and Kratz [91]. The qualitative composition of the EO and hydrolate was determined by comparing the mass spectra and LRIs with data from literature (see Table 1).

Quantitative GC-FID Analyses
The quantitative analyses were conducted in GC-FID, with the same method, columns, and instrument configuration as GC-MS. All the analytes were quantified according to the principle that, in GC-FID, the relative response factors (RRFs) only depend on the combustion enthalpy of each compound and can be mathematically calculated [92,93]. After applying the RRFs to all the integration areas, a calibration curve was traced for each column, using isopropyl caproate as a calibration standard. The dilutions for the construction of the calibration curves were prepared as previously described in the literature, obtaining a correlation coefficient of 0.9995 [7].

Enantioselective Analysis of the EO
The enantioselective analysis was carried out in GC-MS, with the chiral-phase column described in Section 4.1 and the same instrument configuration as the qualitative analyses. The thermal program was as follows: 50 • C for 5 min, followed by a thermal gradient of 2 • C/min until 220 • C, which was maintained for 5 min. The same mixture of n-alkanes used for the qualitative analyses was also injected, and the LRIs calculated. The enantiomers of the chiral compounds, separable with this chiral selector, were identified for possessing the same mass spectrum and by comparing their LRIs with those of enantiomerically pure standards.

Purification and Identification of Furanoeremophilane and Bakkenolide A
To purify furanoeremophilane (67) and bakkenolide A (72), 1 g of pure EO was loaded onto a 220 g silica gel column and fractionated by means of the automatic chromatograph described in Section 4.1. The elution was carried out with a mixture of petroleum ether/diethyl ether, according to an increasing gradient of polarity and with a constant flow of 120 mL/min. Starting from pure petroleum ether, which was maintained for 2.5 min, the percentage of diethyl ether was increased by 2% each minute until 80% petroleum ether, which was maintained for 2 min. The diethyl ether was then raised to 25%, maintained for 5 min, followed by a new polarity gradient where the diethyl ether increased by 10% every 2 min. The gradient ended when the composition of the mobile phase reached 80% diethyl ether. This process produced 130 fractions of different volumes, which were submitted to TLC analysis (petroleum ether/diethyl ether 80:20). The tubes of similar composition were reunited, and the solvent evaporated at reduced pressure, finally obtaining 12 fractions (A1-A12 in order of increasing polarity). To identify the fractions containing the two major components, the 12 samples were injected in GC-MS with the non-polar column, under the same conditions as the qualitative analysis. On the one hand, the fraction A1 contained the first major compound, corresponding to peak 67, in an almost pure form, and it was directly submitted to 1 H and 13 C NMR. On the other hand, the second major component, corresponding to peak 72, was detected in fractions A8-A10, as a mixture with other compounds. Therefore, the three fractions were reunited, and 20 mg was fractionated on normal phase preparative TLC, eluting with a mixture of petroleum ether/dichloromethane/methanol in the ratio 50:45.5:0.5. The purified metabolite was submitted to 1 H and 13 C NMR spectroscopy.

Conclusions
The leaves of Gynoxys buxifolia (Kunth) Cass. produce an EO and an hydrolate, which are described in this study for the first time. Two biosynthetically related uncommon oxygenated sesquiterpenes are the main constituents of the EO: furanoeremophilane and bakkenolide A. Thanks to the good yield, the high amount of the two components, and the easiness of their purification, this EO is a good source of both metabolites for further bioactivity research. Furthermore, the enantioselective analysis supported the existence of different biosynthetic pathways, to produce different enantiomers in this and other EOs. Finally, the presence of p-vinylguaiacol as the major organic component of the hydrolate has been described, together with a possible application.