Chemical Analysis of the Essential Oil from Siparuna echinata (Kunth) A. DC. (Siparunaceae) of Ecuador and Isolation of the Rare Terpenoid Sipaucin A

The phytochemical research on the species Siparuna echinata, collected in the Province of Loja (Ecuador), led to the isolation of a rare sesquiterpenoid, called Sipaucin A. The structure was confirmed by nuclear magnetic resonance (NMR) and mass spectrometry (MS). Furthermore, the essential oil of the fruits was obtained by hydrodistillation and analyzed by gas chromatography, coupled to mass spectrometry (GC-MS) and flame ionization detector (GC-FID). Twenty-seven compounds were identified in a polydimethylsiloxane column (DB-5ms) and nineteen in a polyethylene glycol column (HP-INNOWax). Major compounds were α-pinene (24.3%, 20.3%), β-pinene (21.7%, 22.7%), β-myrcene (11.3%, 14.8%), limonene (10.0%, 11.3%), cis-ocimene (8.5%, 8.1%), and trans-ocimene (8.9%, 8.4%). In addition to the chemical analysis, the essential oil was submitted to enantioselective analysis of two major chiral monotherpenes, determining an enantiomeric excess of 100.0% for (+)-α-pinene and 6.7% for (+)-β-pinene.


Introduction
The geographic location of Ecuador, together with its geological features, makes this region one of the richest of the world in biodiversity. According to the WCMC (World Conservation Monitoring Center) of the United Nations, Ecuador is considered among the 17 megadiverse countries, counting with about 10% of the entire world plant species [1,2].
Every year, new plants are discovered and classified by botanists, while most of the known ones have never been studied so far for their metabolic composition. This fact converts Ecuador to an invaluable source of potentially new molecules of biological and pharmaceutical interest [3].
For all these reasons, the authors have been carrying on, since many years, the chemical description of the secondary metabolite profile of never studied species in Ecuador, especially in the Province of Loja. These studies are mainly focused on chemistry, with the structure elucidation of natural products, the analysis of new essential oils, and the description of biological activities [4][5][6][7].
In this work, we focused on some new terpenoids called Sipaucins, recently discovered in the species Siparuna pauciflora and described so far only once in literature [8]. Furthermore, Siparuna spp. often produce a volatile fraction that appeared to be abundant in fruits of S. echinata, justifying the study of the essential oil (EO).
The Siparunaceae, previously considered as part of the Monimiaceae, include two genera: Glossocalix and Siparuna. They have been reported for the presence of sesquiterpene volatile fractions, showing the property of inducing cell apoptosis [9][10][11].
The Siparunaceae, previously considered as part of the Monimiaceae, include two genera: Glossocalix and Siparuna. They have been reported for the presence of sesquiterpene volatile fractions, showing the property of inducing cell apoptosis [9][10][11].
The genus Siparuna includes 235 plant species distributed in tropical regions of Central and South America [12]. It is characterized by biosynthesizing interesting secondary metabolites such as sesquiterpenes [13], flavonoids [13,14], and isoquinoline alkaloids [15,16], found in plant extracts and in essential oils, with a broad spectrum of pharmacological properties, such as: antibacterial, antimalarial, and antileishmaniasis [17].

Characterization of Sipaucin A
The ethyl acetate (EtOAc) extract of S. echinata led to the isolation and characterization of Sipaucin A. After removing chlorophyll, the extract was submitted to gas chromatography-mass spectrometry (GC-MS) analysis, based on a single ion monitoring (SIM) method, that was prepared according to the ions described in literature for the electron impact MS (EIMS) spectra of Sipaucins A-C [8]. The resulting chromatogram is shown in Figure 1.   Figure 2) with 0.3 % yield (w/w referred to chlorophyll-free mixture). It was submitted to NMR spectroscopy, resulting in data identical to those from literature [8].   Figure 2) with 0.3 % yield (w/w referred to chlorophyll-free mixture). It was submitted to NMR spectroscopy, resulting in data identical to those from literature [8].
Plants 2020, 9, 187 4 of 9 Enantioselective GC Analysis The enantioselective analysis of the EO was carried out with a β-cyclodextrine-based GC column, coated with 2,3-diacethyl-6-tert-butylsilyl-β-cyclodextrin as chiral selector. The analysis was performed on two of the four major compounds (α-pinene and β-pinene), with myrcene being a non-chiral molecule and limonene enantiomers not being separable with this chiral selector. The results of the enantioselective analysis are shown in Table 2, expressed as enantiomeric distribution and enantiomeric excess (ee), and Figure 3. Table 2. Enantioselective analysis of S. echinata essential oil on diacethyl terbutylsilyl-β-cyclodextrin column.

LRIs
Enantiomers Enantioselective GC Analysis The enantioselective analysis of the EO was carried out with a β-cyclodextrine-based GC column, coated with 2,3-diacethyl-6-tert-butylsilyl-β-cyclodextrin as chiral selector. The analysis was performed on two of the four major compounds (α-pinene and β-pinene), with myrcene being a nonchiral molecule and limonene enantiomers not being separable with this chiral selector. The results of the enantioselective analysis are shown in Table 2, expressed as enantiomeric distribution and enantiomeric excess (ee), and Figure 3. Table 2. Enantioselective analysis of S. echinata essential oil on diacethyl terbutylsilyl-β-cyclodextrin column.

Discussion
Sipaucins are rare sesquiterpenoids that have been described for the first time in 2003 in S. pauciflora [8] and not yet found in other species until now. As these metabolites could be characteristic of Siparuna genus, we decided to selectively search for them in the ethyl acetate extract, performing a single ion monitoring (SIM) GC-MS analysis. In the chromatogram of Figure 1, all the peaks consistent with the selected ions are shown. In particular, peak 1, 2, and 3 represent compounds whose ions and relative abundance are, respectively, compatible with Sipaucin A, B, and C. It should be underlined that these peaks are quite evident only in SIM analysis and splitless injection, demonstrating that they actually correspond to minor constituents of the extract. This fact could justify the low yield of Sipaucin A and the non-obtention of other Sipaucins. The presence of more than three peaks, all being compatible with this family of metabolites suggests that other new related compounds are possibly produced by S. echinata, though there is no evidence of the certain presence of these molecules in the extract.

Discussion
Sipaucins are rare sesquiterpenoids that have been described for the first time in 2003 in S. pauciflora [8] and not yet found in other species until now. As these metabolites could be characteristic of Siparuna genus, we decided to selectively search for them in the ethyl acetate extract, performing a single ion monitoring (SIM) GC-MS analysis. In the chromatogram of Figure 1, all the peaks consistent with the selected ions are shown. In particular, peak 1, 2, and 3 represent compounds whose ions and relative abundance are, respectively, compatible with Sipaucin A, B, and C. It should be underlined that these peaks are quite evident only in SIM analysis and splitless injection, demonstrating that they actually correspond to minor constituents of the extract. This fact could justify the low yield of Sipaucin A and the non-obtention of other Sipaucins. The presence of more than three peaks, all being compatible with this family of metabolites suggests that other new related compounds are possibly produced by S. echinata, though there is no evidence of the certain presence of these molecules in the extract.
Nevertheless, germacrenes are constantly present, which is an important factor, as they are structurally and biogenetically related [35] to Sipaucin A.

General Information
The NMR spectra were performed in a Varian spectrometer (Walnut Creek, CA, USA, 400 MHz for 1 H and 100 MHz for 13  The enantioselective GC analysis was carried out with an enantioselective capillary column, based on 2,3-diacethyl-6-tert-butylsilyl-β-cyclodextrin as chiral selector (25 m × 0.25 mm × 0.25 µm, purchased from MEGA, Milan, Italy), installed in the same GC-MS system. Helium was used as carrier gas (Indura, Guayaquil, Ecuador).
After exposure to UV light (254 and 366 nm), the plates were treated with a mixture of sulfuric acid and vanillin as chromatic reagent for spot detection.
All organic solvents for TLC and CC were technical grade, purchased from Relubquim Cia. Ltd.a. (Quito, Ecuador), and distilled before using. All solvents for GC, alkanes for linear retention indices (LRI), and the internal standard (n-nonane) were of analytical grade (purity > 99%), from Sigma-Aldrich. The calibration standard was isopropyl caproate, obtained in the authors' laboratory by synthesis and purified to 98.8% (GC-FID purity).

Plant Material
The aerial parts of S. echinata were collected on May 2017 in the sector Guchanama, canton of Celica, province of Loja, with coordinates 4 • 05 31" S and 79 • 57 18" W, at 2220 m above sea level. The botanical sample was identified by one of the authors (N.C.) and deposited in the herbarium of the Universidad Técnica Particular de Loja, with voucher number VMZ-120. The samples were collected under governmental permission (MAE-DNB-CN-2016-0048).

Extraction and Isolation of Sipaucin A
The dried leaves of S. echinata (730 g) were extracted by Soxhlet with ethyl acetate (EtOAc) for 10 h. Soxhlet extraction was preferred to static maceration because, in a previous essay, the two methods were compared on analytical samples of plant material, producing the same chromatographic profile with higher yield in favor of Soxhlet. A yield of 27.4 % (w/w) was obtained.
An amount of 10 g of extract was eluted on C-18 reversed phase, in order to completely remove chlorophylls. The elution was performed isocratically with a mixture of methanol/water in the ratio of 85:15, producing 5 g of chlorophyll-free extract. To prove the presence of Sipaucins, 20 mg of chlorophyll-free extract was dissolved in 1 mL of dichloromethane and the solution (1 µL) was injected in splitless mode in GC-MS (DB-5ms column). The elution was conducted with a thermal gradient, according to the following program: the initial temperature was 50 • C maintained for 3 min, followed by a thermal gradient of 10 • C/min until the temperature of 250 • C, that was maintained for 17 min. The injector temperature was set at 250 • C and the carrier gas (helium) at 1 mL/min (constant flow). The SIM method is described in Table 3. The preparative purification by CC was performed from 2 g of chlorophyll-free extract. Silica gel (200 g) was packed in a glass column (50 mm × 800 mm), and the extract was eluted in fractions with varying ratios of eluent mixture hexane/EtOAc from 100% hexane until 100% EtOAc. A total of 14 fractions were obtained (JK01-JK14), according to TLC profiles.
The fraction JK11 (134 mg) was further purified by CC in isocratic conditions, eluting with a mixture of hexane/EtOAc in the ratio of 95:5. A total of five fractions were collected according to TLC and subfraction 4 (15 mg) was subjected to preparative TLC. The plate was eluted with hexane/EtOAc in the ratio of 6:4, yielding 3 mg of pure Sipaucin A.
The metabolite was characterized by NMR spectroscopy, resulting in data identical to those from literature (see Supplementary Material, Figures S1 and S2 and Table S1).

Analysis of the EO
The volatile fraction was obtained by separately hydrodistilling four amounts of 265 g of dried fruits for 5 h, using a Clevenger type glass apparatus. The essential oil was collected and dried over anhydrous sodium sulfate and stored at 4 • C until further analysis.
The analytical samples were prepared by diluting an exactly weighed amount of essential oil (corresponding to 10 µL) with 1 mL of internal standard solution, previously prepared by Plants 2020, 9, 187 7 of 9 diluting 0.7 mg of n-nonane to a total volume of 10 mL with cyclohexane. Such a preparation was repeated for each sample. These samples were directly used for the qualitative, quantitative, and enantioselective analyses.
The qualitative analysis was performed by injecting 1 µL of the EO solution (split 40:1) with the following thermal program. The initial oven temperature was 60 • C for 5 min, followed by a thermal gradient of 3 • C/min up to 180 • C and 15 • C/min up to 250 • C. The final temperature was maintained for 5 min. The injector temperature was set at 250 • C, the carrier gas (helium) was set at 1 mL/min (constant flow). Additionally, a mixture of n-alkanes (C 9 -C 25 ) was injected under the same conditions to determine the linear retention indices (LRIs).
The quantitative analysis was performed by GC-FID, applying the same method and equipment conditions described above. Quantification was performed with external calibration curve and internal standard. Two calibration curves were obtained, injecting six dilutions of isopropyl caproate (calibration standard) and n-nonane (internal standard) in both columns. The dilutions were obtained by diluting 0.8 mg, 1.8 mg, 4.1 mg, 8.3 mg, 16.9 mg, and 34.5 mg of isopropyl caproate and an exactly weighed amount of 7.6-7.8 mg of n-nonane to 10 mL with cyclohexane. All calibration curves achieved a R 2 > 0.995. The relative response factor (RRF) of each constituent of the EO versus isopropyl caproate was calculated, according to combustion enthalpy [36], prior to calibration.
The enantioselective analysis was performed by GC-MS on the same samples of the qualitative and quantitative analyses, according to the following method. The initial oven temperature was 60 • C for 2 min, followed by a thermal gradient of 2 • C/min until 220 • C, maintained for 2 min. Also, in this case, a mixture of n-alkanes (C 9 -C 25 ) was injected under the same conditions to calculate LRIs. The elution order of the enantiomeric pairs was determined according to literature data for the same chiral selector [37].

Conclusions
The rare sesquiterpenoid Sipaucin A was isolated for the second time from a natural source. The spectroscopic evidence for the presence of some other terpenes of the Sipaucin family in this species has been obtained.