A new essential oil from the native Ecuadorian species Steiractinia sodiroi (Hieron.) S.F. Blake (Asteraceae): chemical and enantioselective analyses

In the present study, the essential oil from dry leaves of Steiractinia sodiroi (Hieron.) S.F. Blake is described for the first time. The plant material, collected in the Province of Loja (Ecuador), was analytically steam-distilled in a Marcusson-type apparatus, affording an essential oil with a 0.2 ± 0.12% yield. The volatile fraction was submitted to GC–MS and GC–FID analyses, on two stationary phases of different polarity. A total of sixty-seven compounds, corresponding to 95.6–91.2% by weight of the whole oil mass, on the two columns respectively, were detected and quantified with at least one column. The quantification was carried out calculating the relative response factors of each constituent according to their combustion enthalpy. The major components were limonene (25.6–24.9%), sabinene (11.7–12.4%), germacrene D (7.7–7.0%), α-pinene (7.8–6.9%), δ-cadinene (7.3–7.0%), (E)-β-caryophyllene (4.8–4.5%), and bicyclogermacrene (3.6–3.0%). The chemical composition was complemented with the enantioselective analysis of some major chiral compounds, conducted by means of two β-cyclodextrin-based capillary columns. Three constituents, (S)-(+)-α-phellandrene, (R)-(−)-1-octen-3-ol, and (S)-(−)-limonene were enantiomerically pure, whereas (1R,5R)-(+)-β-pinene, (1S,5S)-(−)-sabinene, (R)-(−)-terpinen-4-ol, (R)-(+)-α-terpineol, and (R)-(+)-germacrene D presented an enantiomeric excess. Finally, α-pinene was present as a racemic mixture.

stereactinolides; whereas a more recent article was published by Gamboa-Carvajal et al. about the biological activity of hydro-ethanolic extracts from S. aspera [11][12][13] .On the other hand, only one article has recently been published about a volatile fraction from the genus Steiractinia, citing the chemical composition and biological activities of S. aspera EO 14 .Therefore, the present study constitutes the first investigation about the chemical and enantiomeric composition of an EO from S. sodiroi.

Chemical analysis
The dry leaves of S. sodiroi afforded, after steam-distillation, an EO with an estimated yield of 0.2 ± 0.12% as an analytical mean value over four repetitions.The EO, analysed on two stationary phases of different polarity, permitted to detect sixty-seven compounds, that were quantified on at least one column.The volatile fraction was dominated by monoterpene hydrocarbons, corresponding to 95.6-91.2%by weight of the whole oil mass, on a non-polar and polar column respectively.After that, the sesquiterpene hydrocarbons represented the second main fraction, with a content of 26.9-24.0%on the two columns.The major components of the EO (≥ 3.0% with at least one column) were limonene (25.6-24.9%),sabinene (11.7-12.4%),germacrene D (7.7-7.0%),α-pinene (7.8-6.9%),δ-cadinene (7.3-7.0%),(E)-β-caryophyllene (4.8-4.5%), and bicyclogermacrene (3.6-3.0%).The gas chromatographic profile with both stationary phases is represented in Figs. 1 and 2, whereas the complete chemical analysis is reported in Table 1.The mass spectra of the undetermined compounds are represented in Fig. 3.

Discussion
The chemical analysis was carried out on two columns with stationary phases of different polarity, that afforded reciprocally consistent results from both the qualitative and quantitative point of view.The main components are some common mono and sesquiterpenes, with limonene as the dominant constituent accounting for about one quarter of the whole oil.According to the chemical composition, some biological activities could be expected.
In particular, due to the very high abundance of limonene (more than one quarter of the total composition), the anti-inflammatory, antioxidant, anticancer, antinociceptive, and antidiabetic activities should be considered as suitable to be investigated in further studies 65 .About the chemical composition, the only EO described in literature from the genus Steiractinia is the one obtained from S. aspera 14 .The comparison between the major component abundance of the two volatile fractions is represented in Fig. 4. As it can be observed, both EOs are dominated by some common monoterpenes and many of them are shared by both volatile fractions.However, despite the similar qualitative composition, the relative abundances are quite different.In fact, in S. aspera EO, the very major component is α-pinene, whereas S. sodiroi EO is dominated by limonene (both about 25%).Furthermore, of the other S. aspera important components, β-phellandrene and α-copaene are absent in S. sodiroi whereas, on the other hand, bicyclogermacrene, δ-cadinene, and spathulenol are absent or minority compounds in S. aspera EO.
The enantioselective analyses showed that some chiral components were enantiomerically pure, others were present as scalemic mixtures, whereas α-pinene was detected as a racemic mixture.These results demonstrated that, as usual, different enantiomers are produced by S. sodiroi metabolism, with the aim of carrying on different biological functions.The enantiomeric excess of a chiral compound in nature can be explained with the enantiospecific kinetic resolution of a racemic mixture or because of enzymatic enantioselective reactions on a non-chiral precursor.The latter case is represented in Fig. 5, where each enantiomer of limonene can be obtained from a specific conformer of the non-chiral precursor geranyl pyrophosphate, enzymatically stabilized respect to the other one.It is well known that the enantiomers of the same metabolite are often characterized by enantioselective biological properties such as, for example, different aromas 66 .These differences, mainly due to the chiral character of receptors and enzymes, often determine enantioselective biological activities.In the present EO, the dominant component is present in an enantiomerically pure form, being (S)-(−)-limonene the only detected enantiomer.According to literature, the laevorotatory form of limonene is sometimes more active than the dextrorotatory one as an antibacterial agent, for instance against Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Moraxella catarrhalis, and Cryptococcus neoformans 67 .

Distillation and sample preparation
Four amounts (50 g, 35 g, 35 g, and 35 g respectively) of dry leaves were separately steam-distilled for four hours, in the Marcusson-type apparatus previously described in literature 68 .The distillation was conducted analytically, over 2 mL of cyclohexane spiked with n-nonane as internal standard (0.69 mg/mL).After distillation, the EO solutions were recovered, stored in the dark at − 14 °C and directly injected into GC for the analyses.The solvent and internal standard were purchased by Sigma-Aldrich (St. Louis, MO, USA).

Qualitative and quantitative chemical analyses
The qualitative chemical analyses were conducted through gas chromatography-mass spectrometry (GC-MS), in a Trace 1310 gas chromatograph, coupled to a simple quadrupole detector model ISQ 7000 (Thermo Fisher Scientific, Walthan, MA, USA).The MS was operated in SCAN mode, with a mass range of 40-400 m/z.The electron ionization (EI) ion source was set at 70 eV, whereas both ion source and transfer line were programmed at the temperature of 230 °C.The injections were repeated in two columns with stationary phases of different polarity, a DB-5 ms and a HP-INNOWax, both purchased from Agilent Technology (Santa Clara, CA, USA).The stationary phases were based on 5%-phenyl-methylpolysiloxane and polyethylene glycol respectively, whereas the columns were 30 m long and characterized by 0.25 mm internal diameter and 0.25 μm film thickness.The oven was programmed according to the following thermal gradient, that was applied to both columns: 50 °C for 5 min, followed by a ramp of 3 °C/min until 100 °C, then a second ramp of 5 °C/min until 180 °C, and finally 10 °C/ min until 230 °C.The final temperature was maintained for 10 min.The injector was operated in split mode, set at 230 °C, and injecting 1 μL of EO solution with a split ratio of 40:1.The carrier gas was GC grade helium, set at the constant flow of 1 mL/min, and purchased from Indura, Guayaquil, Ecuador.All the EO components were identified comparing the respective linear retention index and mass spectra with data from literature (see Table 1).The linear retention indices (LRIs) were calculated according to Van den Dool and Kratz, with respect to a series of homologous n-alkanes in the range C 9 -C 22 69 .All the alkanes were purchased from Sigma-Aldrich.The quantitative analyses were carried out with the same GC, columns, configuration, and thermal program of the qualitative ones, except for the use of a flame ionization detector (FID) instead of MS and the value of split ratio (10:1).All the detected compounds were quantified calculating each relative response factor (RRF) versus isopropyl caproate, according to their combustion enthalpy 70,71 .A six-point calibration curve was built for each column, using isopropyl caproate as calibration standard and n-nonane as internal standard, according to what was previously described in literature 72 .Both curves produced a correlation coefficient of 0.998.The internal standard was purchased from Sigma-Aldrich, whereas isopropyl caproate was synthetised in the authors' laboratory and purified until 98.8% (GC-FID purity).

Enantioselective analysis
The S. sodiroi EO was also submitted to enantioselective analysis, through two enantioselective capillary columns, based on 2,3-diacetyl-6-tert-butyldimethylsilyl-β-cyclodextrin and 2,3-diethyl-6-tert-butyldimethylsilylβ-cyclodextrin respectively.Both columns were 25 m long, 250 μm internal diameter and 0.25 μm phase thickness, purchased from Mega, MI, Italy.The analysis was conducted in the same GC-MS instrument used for the qualitative ones, with 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 (total time 96 min).The carrier gas (He) was set at the constant pressure of 70 kPa.Sample volume, split ratio, injector temperature, transfer line temperature, and MS parameters were the same as the qualitative analyses.The enantiomers were identified by means of the respective MS spectra and linear retention indices, and through the injection of enantiomerically pure standards, that were purchased from Sigma-Aldrich.

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.The species Steiractinia sodiroi (Hieron.)S.F.Blake appears neither in the IUCN Red List of Threatened Plants nor in the Red Book of the Endemic Plants of Ecuador.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
) and quantitative (GC-FID) chemical composition of S. sodiroi EO on 5%-phenyl-methylpolysiloxane and polyethylene glycol stationary phases.a Calculated linear retention index.b Reference linear retention index, trace = < 0.1% % = percent by weight, σ = standard deviation, MW = molecular weight, § = identified only by mass spectrum in the polar column.

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S)-(−)-limonene 1049** 100.0 100.0 (R)-(+)-germacrene D 1502** 98.2 96.3 (S)-(−)-germacrene D 1509** 1.8 Ministry of Environment, Water and Ecological Transition of Ecuador, with MAATE registry number MAE-DNB-CM-2016-0048. The identification of the botanical species was carried out by one of the authors (N.C.), according to botanical specimens with code 3319939 conserved at the National Museum of Natural History, Smithsonian Institution, Washington, DC, USA.A voucher was deposited at the herbarium of the Universidad Técnica Particular de Loja with code 14666.The plant material was dried the same day of collection, at 35 °C for 48 h, and stored in a fresh, dark place until use.

Figure 4 .
Figure 4. Compared abundance of major EO components of S. aspera and S. sodiroi.

Figure 5 .
Figure 5. Possible mechanism for the enantioselective biosynthesis of limonene.