A new leaf essential oil from the Andean species Gynoxys szyszylowiczii Hieron. of southern Ecuador: chemical and enantioselective analyses

The essential oil obtained from the dry leaves of Gynoxys szyszylowiczii Hieron. was described in this study for the first time. The chemical analysis, conducted on two stationary phases of different polarity, permitted to identify sixty-four compounds, that were quantified with at least one column. The main components, on a non-polar and polar stationary phase respectively, were germacrene D (21.6–19.2%), α-pinene (4.4–4.9%), n-tricosane (4.3% on both columns), (E)-β-caryophyllene (3.3–4.3%), 1-docosene (3.2–2.8%), α-cadinol (2.8–3.1%), and cis-β-guaiene (2.6–3.0%). This investigation was complemented by the enantioselective analysis of some major chiral compounds, carried out on two chiral selectors based on β-cyclodextrines. As a result, (S)-( +)-α-phellandrene, (S)-( +)-β-phellandrene, and (1S,2R,6R,7R,8R)-( +)-α-copaene appeared enantiomerically pure, whereas α-pinene, β-pinene, terpinen-4-ol, and germacrene D were detected as scalemic mixtures. Finally, linalool was practically racemic. The distillation yield, analytically calculated by weight of dry plant material, was 0.03%.

During the last decades, the increasing and almost exhaustive information about phytochemistry in the boreal hemisphere, shifted the interest of natural product chemists from temperate countries to tropical regions, where most of the botanical species are often still unstudied.In this regard, Ecuador is an ideal place to investigate secondary metabolites since it has been mentioned by the United Nations among the seventeen "megadiverse" countries 1 .So far, mainly for historical reasons, Ecuador's outstanding biodiversity has been very poorly investigated, and most of its native and endemic plants still lack a systematic chemical description 2,3 .For this reason, and with the aim of contributing to the chemotaxonomic description and the biological enhancement of the Ecuadorian biodiversity, our group has been searching for new natural products in this region during the last twenty years 4,5 .More recently, our interest focused on the description of new essential oils (EOs), with emphasis on their chemical compositions, enantioselective analyses, and gas chromatography-olfactometry (GC-O) profiles [6][7][8][9] .In particular, the present research is a part of an unfunded project, dealing on the systematic description of volatile fractions from the main species of genus Gynoxys Cass.(Asteraceae) in southern Ecuador.In this respect, based on their accessibility on the territory, twelve unprecedented species were selected for their EOs to be described: Gynoxys miniphylla Cuatrec., Gynoxys rugulosa Muschl., Gynoxys buxifolia (Kunth) Cass., Gynoxys laurifolia (Kunth) Cass., Gynoxys szyszylowiczii Hieron., Gynoxys sancti-antonii Cuatrec., Gynoxys calyculisolvens Hieron., Gynoxys pulchella (Kunth) Cass., Gynoxys cuicochensis Cuatrec., Gynoxys reinaldii Cuatrec., Gynoxys hallii Hieron., and Gynoxys azuayensis Cuatrec.All these species grow wild in the highlands of the province of Loja, and they are native or endemic of this region.So far, the chemical and enantioselective analyses of the EOs from G. miniphylla, G. rugulosa, G. buxifolia, and G. laurifolia have been published, G. szyszylowiczii is the object of the present study, whereas all the other species are currently under investigation [10][11][12][13] .The species Gynoxys szyszylowiczii Hieron. is a quite uncommon taxon, with no botanical synonyms and it is absent in the Catalogue of Vascular Plants of Ecuador [14][15][16] .However, despite the diffusion area of this species corresponds to Peru, the only available reference specimen was collected in Ecuador in 1998, in the same collection place of the samples www.nature.com/scientificreports/ that were used in this research (see plant material, Section "Plant material") 15 .Botanically, G. szyszylowiczii is a tree, 5 m tall, with hexagonal branches when young.The plant is densely reddish-tomentose, with opposite petiolate leaves.Blades are elliptical-oblong or ovate-oblong, with rounded or slightly cordate base, obtuse and pointed apex, denticulate margin, subcoriaceous, sparsely tomentose above when young.The inflorescences are corymbose and branched 17 .
No traditional use has been reported for this species in Ecuador or elsewhere.To the best of the authors' knowledge, this report represents the first description of an EO obtained from G. szyszylowiczii.The study has been completed with the enantioselective analysis of some major chiral terpenes and terpenoids.

Chemical analysis
The dry leaves of G. szyszylowiczii analytically produced an EO with a 0.03 ± 0.004% yield (w/w), as a mean value over four distillations.The volatile fraction, despite in cyclohexane solution, was characterized by a mild sweet, hay like odour, very similar to the ones of most of the Gynoxys EOs so far studied.The qualitative analysis permitted to identify and quantify sixty-four components with at least one of the two columns used.The quantification corresponded to 86.1-84.1% of the whole oil mass, on the non-polar and polar stationary phases respectively.The EO was dominated by the sesquiterpene fraction, with sesquiterpenes and oxygenated sesquiterpenoids accounting for 37.5-35.6%and 10.8-10.0% of the total mass respectively.Secondly, as usual in Gynoxys EOs, a heavy aliphatic fraction is present, corresponding to 27.5-28.5% of the entire oil composition.Finally, a monoterpene fraction was identified, with monoterpenes and oxygenated monoterpenoids corresponding together to 10.1-9.8% of the whole volatile fraction.Major constituents of G. szyszylowiczii EO (≥ 3.0% on at least one column) were germacrene D (21.6-19.2%,peak 29), α-pinene (4.4-4.9%,peak 1), n-tricosane (4.3% on both columns, peak 57), (E)-β-caryophyllene (3.3-4.3%, peak 24), 1-docosene (3.2-2.8%,peak 54), α-cadinol (2.8-3.1%,peak 43), and cis-β-guaiene (2.6-3.0%,peak 30).The complete chemical analysis is reported in Table 1, whereas the gas chromatographic profiles with both stationary phases are reported in Figs. 1 and 2.

Enantioselective analysis
The enantioselective analysis permitted to determine the distribution and enantiomeric excess (ee) of height chiral terpenes.Among them, (S)-( +)-α-phellandrene, (S)-( +)-β-phellandrene, and (1S,2R,6R,7R,8R)-( +)-α-copaene were enantiomerically pure, linalool was practically racemic, whereas α-pinene, terpinen-4-ol, and germacrene D were present as scalemic mixtures.The analysis was based on two enantioselective columns to ensure the enantiomer separation of all the detected chiral compounds.As usual, the enantioselective analysis is limited to the chiral components for which enantiomerically pure standards were available.The results of the enantioselective analysis are detailed in Table 2.

Discussion
The EO from the leaves of G. szyszylowiczii presented a GC pattern similar to the one of other already known Gynoxys EOs [10][11][12][13] .In fact, we can observe in Figs. 1 and 2, that three main fractions were evident: a minoritarian monoterpene fraction, a majoritarian sesquiterpene fraction, and an important heavy aliphatic fraction.The compared chemical compositions of major components in G. buxifolia, G. laurifolia, G. rugulosa, G. miniphylla, and G. szyszylowiczii EOs are plot in Fig. 3 where compounds, whose abundance is ≥ 3.0% in at least one EO, are represented.
According to Fig. 3, it can be observed that G. buxifolia EO was completely different from the others, from both the qualitative and quantitative points of view.On the other hand, G. laurifolia, G. rugulosa, G. miniphylla, and G. szyszylowiczii produced more similar volatile fractions.In fact, in all the species other than G. buxifolia, germacrene D, α-pinene, (E)-β-caryophyllene, β-pinene, α-humulene, δ-cadinene, and α-cadinol were main components, common to all the EOs in a quite similar amount.For what concerns the heavy aliphatic fraction, G. szyszylowiczii was similar to G. rugulosa, despite the relative abundances differed.Only in G. miniphylla EO, δ-3-carene and trans-myrtanol acetate were among the main compounds.The potential anti-inflammatory and cholinergic activities of these EOs, among many others based on their chemical analyses and according to literature, were discussed in our previous studies.These properties could speculatively be extended also to G. szyszylowiczii, due to their similar compositions 11,13 .Finally, the enantioselective analysis of and G. szyszylowiczii EO can also be compared with those conducted on G. buxifolia, G. laurifolia, G. rugulosa, and G. miniphylla, as indicated in Table 3.
As we can observe, the EOs did not apparently shared a common distribution pattern.In fact, in different volatile fractions, different enantiomers of the same compound could be detected in an enantiomerically pure form.In other cases, where scalemic mixtures were observed, the dominant enantiomer was not always the same.On the other hand, β-phellandrene always appeared as a pure enantiomer, whereas β-pinene, sabinene, α-phellandrene, linalool, terpinen-4-ol, and α-terpineol were sometimes observed as almost racemic.As usual, the presence of different enantiomers in a natural source can be justified by the different biological properties that they exert in living organisms, due to the chiral character of enzymes and receptors 49,50 .About G. szyszylowiczii EO, we could observe the presence of α-copaene as an abundant compound (about 1%).This sesquiterpene is not common in this botanical genus and has been observed so far only in G. rugulosa, where it is present in his almost enantiomerically pure dextrorotatory form.This enantiomer has been described for being a chemical rendezvous cue for the Mediterranean fruit fly Ceratitis capitata 51 .Furthermore, according to literature, (1S, 2R, 6R, 7R, 8R)-( +)-α-copaene affected C. capitata virgin females, inducing "pseudomale" courtship behaviour in a short-range bioassay.Another issue is the different enantiomeric excess of α-pinene and β-pinene within the same species (see Table 3).In fact, both α-pinene and β-pinene derive from the same pinyl cation, by effect of the same enzyme pinene synthase 52 .Since the final stereochemistry of pinenes depends on the configuration of the common carbocation, the same enantiomeric excess is expected to be observed, for the same enantiomer, in both pinenes.However, a different experimental evidence is reported in the present study.This phenomenon could theoretically be explained, for example through the enantiospecific transformation of only one enantiomer into another unidentified compound.

Plant material
The leaves of G. szyszylowiczii were collected on 9 March 2020 in the province of Loja (Ecuador), from different trees and treelets located within the distance of 200 m from a central point, whose coordinates were 03° 40′ 52'' S

Distillation and sample preparation
Four amounts of dry leaves (80.7 g, 81.5 g, 80.7 g, and 80.8 g respectively) were analytically steam distilled for four hours each, as previously described in literature, in a modified Dean-Stark apparatus 11 .The volatile fraction, after condensation, was extracted in situ through 2 mL of cyclohexane, previously spiked with n-nonane Figure 2. GC-MS profile of G. szyszylowiczii EO on a polyethylene glycol stationary phase.The peak numbers refer to Table 1.Black numbers: major terpenes and terpenoids (≥ 3.0%); green numbers: heavy n-alkanes; red numbers: heavy 1-alkenes.
Table 2. Enantioselective analysis of some chiral terpenes from G. szyszylowiczii EO.LRI = calculated linear retention index; ee = enantiomeric excess; § partially resolved peaks; * extracting ion 111 m/z. as internal standard (0.7 mg/mL).An internal standard was necessary because the EO was obtained as a solvent solution of unknown concentration.The four repetitions, obtained in this way, were directly injectable into GC and they were stored at -15 °C until use.Both cyclohexane and n-nonane were analytical grade and purchased from Sigma-Aldrich (St. Louis, MO, USA).Both qualitative and quantitative analyses were conducted on two different columns (30 m long, 0.25 mm internal diameter and 0.25 μm film thickness), purchased from Agilent Technology (Santa Clara, CA, USA).One column was based on 5%-phenyl-methylpolysiloxane (HP-5) as a non-polar stationary phase and the other one was based on polyethylene glycol (HP-INNOWax) as a polar stationary phase.For the qualitative analysis, the columns were installed in a gas chromatograph (GC) model Trace 1310, coupled to a simple quadrupole detector (MS) model ISQ 7000 (Thermo Fisher Scientific, Walthan, MA, USA).The ion source was an electron ionization (EI) device, with 70 eV ionization energy.The MS was operated in SCAN mode, with the mass analyser set at the mass range of 40-400 m/z.Both ion source and transfer line were heated at 230 °C.For the quantitative analysis, the same GC as the qualitative one was used.However, a flame ionization detector (FID), set at 230 °C, was employed instead of MS.In both qualitative and quantitative analyses, the injection volume (1 μL), injector conditions, carrier gas, and thermal program were the same.The injector was operated in split mode (40:1 for GC-MS and 10:1 for GC-FID) and it was heated at 230 °C, whereas the carrier gas was helium (purchased from Indura, Guayaquil, Ecuador), that was set at the constant flow of 1 mL/min.In the qualitative analysis, the EO components were identified by comparison of each MS spectrum and linear retention index (LRI) with data from literature (see Table 1).The LRIs were calculated according to Van den Dool and Kratz, based on a mixture of homologues n-alkanes in the range C 9 -C 27 (Sigma-Aldrich, St. Louis, MO, USA) 53 .In the quantitative analysis, each constituent was quantified through a six-point calibration curve, using n-nonane (Sigma-Aldrich, St. Louis, MO, USA) as internal standard and isopropyl caproate as calibration standard, as previously described in literature 54 .Isopropyl caproate was synthetised in one of the authors' laboratories (G.G.) and purified until 98.8% (GC-FID purity).Both calibration curves afforded a correlation coefficient of 0.999.The EO components were quantified calculating each relative response factor versus isopropyl caproate, based on its combustion enthalpy, according to literature 55,56 .

Enantioselective analysis
The enantioselective analysis was conducted on two different GC columns, whose stationary phases were based on β-cyclodextrins (2,3-diacetyl-6-tert-butyldimethylsilyl-β-cyclodextrin and 2,3-diethyl-6-tert-butyldimethylsilylβ-cyclodextrin), purchased from MEGA S.r.l., Legnano (MI), Italy.The columns were 25 m long, 0.25 mm internal diameter and 0.25 μm phase thickness, installed in the same GC-MS instrument described for the qualitative analysis.The analytical method was based on the following thermal program: 50 °C for 1 min, a thermal gradient of 2 °C/min until 220 °C, that were maintained for 10 min.The carrier gas (He) was set at the constant pressure of 70 kPa.Sample volumes, injector temperature, transfer line temperature, and MS parameters were the same as the qualitative analyses, whereas split ratio was 20:1 in order to significantly increase sensitivity.The enantiomers of the main chiral components were identified by mass spectrum and injection of enantiomerically pure standards (Sigma-Aldrich, St. Louis, MO, USA).

Policy statement about plant investigation
The authors declare that all experimental research and field studies on plants (either cultivated or wild), including the collection of plant material, were carried out in accordance with relevant institutional, national, and international guidelines and legislation.Furthermore, this study was conducted under permission of the Ministry of Environment, Water and Ecological Transition of Ecuador, with MAATE registry number MAE-DNB-CM-2016-0048. Finally, the authors declare that a minimal quantity of plant material was collected to carry on the present investigation, avoiding any injury to the shrubs at the collection site.

Conclusions
The dry leaves of Gynoxys szyszylowiczii Hieron.produce an EO, with a yield of 0.03% by weight.On the one hand, the chemical profile is consistent with the ones previously described for G. laurifolia, G. rugulosa, and G. miniphylla, but very different from G. buxifolia.The chemical profile showed a volatile fraction dominated by sesquiterpenes and heavy aliphatic compounds (hydrocarbons and alkenes).On the other hand, the enantiomeric composition of the Gynoxys spp.studied so far did not apparently present a common pattern.For a more objective interpretation of Gynoxys metabolism, a statistical comparison of all the EOs will be the object of a future study (Supplementary Information file 1).

Figure 3 .Table 3 .
Figure 3. Percent abundance of major components in G. buxifolia, G. laurifolia, G. rugulosa, G. miniphylla, and G. szyszylowiczii EOs.The values correspond to constituents whose abundance is ≥ 3.0% in at least one plant EO, as a mean value between the two GC columns.