A New Essential Oil from the Native Andean Species Nectandra laurel Klotzsch ex Nees of Southern Ecuador: Chemical and Enantioselective Analyses

The leaves of Nectandra laurel Klotzsch ex Nees, belonging to the family, Lauraceae, were collected in the province of Loja (Ecuador), dried, and analytically steam-distilled. An unprecedented essential oil was obtained, with a 0.03% yield by weight of dry plant material. The volatile fraction was submitted to qualitative (GC-MS) and quantitative (GC-FID) chemical analysis, on two orthogonal stationary phases. Seventy-eight compounds were detected and quantified on at least one column. The essential oil was dominated by sesquiterpene hydrocarbons (53.0–53.8% on the non-polar and polar stationary phase, respectively), followed by oxygenated sesquiterpenoids (18.9–19.0%). A third group was constituted by metabolites of other origins, mainly aliphatic compounds, apparently derived from the acetate pathway (11.7–8.5%). The major components of the EO (≥3.0% with at least one column) were δ-selinene (30.5–28.8%), δ-cadinene (5.4–6.4%), epi-α-cadinol (4.9–5.2%), an undetermined compound with a molecular weight of 204 (3.4–4.2%), α-pinene (3.3–2.9%), and α-cadinol (2.9–3.0%). Finally, the essential oil was submitted to enantioselective analysis, on two β-cyclodextrin-based chiral selectors, determining the enantiomeric distribution of seven chiral terpenes. Among them, (1R,5R)-(+)-α-pinene, (1R,5R)-(+)-β-pinene, and (R)-(−)-α-phellandrene were enantiomerically pure, whereas camphene, borneol, α-copaene, and α-terpineol were present as scalemic mixtures.


Introduction
Located across the equatorial line, Ecuador is a relatively small country of the South American continent.Thanks to its orography and geographic location, it is characterized by the presence of four climatic regions: the Galapagos islands, the Pacific coast, the Andes mountains, and the Amazon Forest.Due to these very diversified climes, Ecuador possesses an extremely high biodiversity, which makes this territory a so-called "megadiverse country" [1].According to the Catalogue of Vascular Plants of Ecuador, at the date of publication, this country hosted 16,087 botanical species, of which 15,306 were natives and 4173 endemics [2].So far, from the chemical point of view, most of these native species are completely unstudied or poorly investigated, making Ecuador a potential source of new bioactive molecules and unprecedented natural products [3,4].
On these premises, our group has been investigating Ecuadorian biodiversity for more than twenty years, in search of new or rare secondary metabolites of biological interest [5][6][7].
On these premises, our group has been investigating Ecuadorian biodiversity for more than twenty years, in search of new or rare secondary metabolites of biological interest [5][6][7].During the last 6 years, the authors have been focusing on the description of new essential oils (EOs), with an emphasis in the enantiomeric composition, in the characterization of major uncommon sesquiterpenes, and in the olfactometric description of the aroma profile [8][9][10].Under these premises and with the aim of contributing to the advance in the knowledge on the phytochemistry of Ecuadorian flora, the present work perfectly fits in the described context.
The genus, Nectandra Rol.ex Rottb., belonging to the family, Lauraceae, posseses a total of 311 registered species worldwide, of which 101 are accepted [11].In Ecuador, this genus possesses 36 known species, of which 6 are endemics [2].The taxon, Nectandra laurel Klotzsch ex Nees (see Figure 1), is a tree, native to the Andean region and diffused in Venezuela, Colombia, Ecuador, Perú, and Bolivia [12].In Ecuador, this species has been described in the provinces of Azuay, Bolívar, Carchi, Chimborazo, Imbabura, Loja, Napo, and Pichincha, where it grows in the range of 1000-3500 m above sea level [2].Besides this name, this plant is also known by four others: Nectandra mollis subsp.laurel (Klotzsch ex Nees) Rohwer, Nectandra tovarensis Klotzsch & H.Karst. ex Nees, Nectandra laurel var.glabrescens Meisn., and Nectandra willdenoviana Nees [11].So far, no study has been found in the literature on the phytochemistry of this taxon, either as N. laurel or with any of its other synonyms.However, at least 10 other Nectandra spp.have been previously studied and had their EOs described.This is the case for N. amazonum, N. barbellata, N. cuspidata, N. gardneri, N. grandiflora, N. hihua, N. lanceolata, N. leucantha, N. megapotamica, and N. puberula [13].The objective of the present research is to describe the chemical and enantiomeric composition of an EO from Nectandra laurel Klotzsch ex Nees.that, to the best of the authors' knowledge, is reported here for the first time.The objective of the present research is to describe the chemical and enantiomeric composition of an EO from Nectandra laurel Klotzsch ex Nees.that, to the best of the authors' knowledge, is reported here for the first time.

Chemical Analysis of N. laurel EO
The dry leaves of N. laurel afforded an EO, with a 0.03 ± 0.002% yield by weight, analytically calculated over four repetitions.A total of seventy-eight compounds were detected in the volatile fraction and quantified on at least one column, corresponding to 94.6-91.3% of the oil mass on the non-polar and polar stationary phase, respectively.
Figure 2. GC-MS profile of N. laurel EO on a 5%-phenyl-methylpolysiloxane stationary phase.The peak numbers refer to major compounds (≥3.0% on at least one column), according to Table 1. Plants . GC-MS profile of N. laurel EO on a 5%-phenyl-methylpolysiloxane stationary phase.The peak numbers refer to major compounds (≥3.0% on at least one column), according to Table 1.
Figure 3. GC-MS profile of N. laurel EO on a polyethylene glycol stationary phase.The peak numbers refer to major compounds (≥3.0% on at least one column), according to Table 1.
Figure 3. GC-MS profile of N. laurel EO on a polyethylene glycol stationary phase.The peak numbers refer to major compounds (≥3.0% on at least one column), according to Table 1.

Discussion
As previously described, the EO from leaves of N. laurel is dominated by sesquiterpene hydrocarbons (about 50%), followed by oxygenated sesquiterpenoids (about 19%).The sesquiterpene hydrocarbon, δ-selinene, alone constitutes about 30% of the whole oil mass.According to the literature, the EOs from the genus, Nectandra, can practically be divided into five main groups: (1) EOs based on monoterpene hydrocarbons, (2) EOs based on sesquiterpene hydrocarbons, (3) EOs based on oxygenated sesquiterpenoids, (4) EOs based on both sesquiterpene hydrocarbons and oxygenated sesquiterpenoids, and (5) EOs based on phenylpropanoids and sesquiterpenes [13].The EO described in the present study clearly belongs to group (2), including species such as N. amazonum, N. cuspidata, N. hihua, some specimens of N. megapotamica, and N. leucantha.However, in all these plants, the two major components are usually (E)-β-caryophyllene and bicyclogermacrene, with other compounds such as β-selinene, α-humulene, δ-cadinene, and β-bourbonene as other important components.Apparently δ-selinene, our main constituent, is not a major compound in any other known Nectandra spp.other than N. laurel.Group (1) includes some specimens of N. megapotamica, where both pinenes are usually dominants.Group (3) contains N. grandiflora, N. lanceolata, and other specimens of N. megapotamica, where iso-bicyclogermacrenal, spathulenol, and α-bisabolol are major constituents.For what concerns group (4), N. megapotamica is once again the main representative.Finally, group ( 5) is represented by N. puberula, whose main EO component is apiole [13].Interestingly, it can be observed that the chemical composition of the EO from different specimens of N. megapotamica was so variable that the species could be located in all groups.Based on this phenomenon, it can be hypothesized that N. megapotamica EO is just the most studied among the volatile fractions of a very variable genus, and that similar results could also be obtained for N. laurel, studying the EO at different times and from different geographical regions.The same literature underlines that chemical variability is a typical feature of Lauraceae, where it is observed more because of seasonal changes than according to the vegetative stage of the plant.This fact is consistent with the need for different insect pheromones in different climes.Other Nectandra spp., reported for presenting important seasonal variations, are N. lanceolata and N. grandiflora [13].
As already mentioned, δ-selinene is absolutely the main component of this EO, reaching about 30% of the whole oil mass.Therefore, not only N. laurel EO could be considered as a source of this sesquiterpene, but its biological properties could also theoretically be predicted, at least partially, from the activities of this compound.However, no exhaustive investigations have been found in the literature about the biological activities of pure δ-selinene.Nevertheless, they can be deduced from the properties of other EOs, where this terpene predominates with an amount like the one of N. laurel.Three species were identified that produce an EO with these features: Jatropha elliptica rhizomes (Euphorbiaceae) from Brazil, Globba pendula rhizomes (Zingiberaceae) from Indochina, and Xanthium italicum flowers (Asteraceae) from Corsica [59][60][61].The amount of δ-selinene in the volatile fraction of these plants was 35.7%, 36.4%, and 22.4%, respectively; however, only for G. pendula EO were biological activities investigated.In this case, the oil showed a moderate inhibitory capacity on NO production in LPS-activated macrophages, with an IC 50 = 41.68 ± 4.51% versus 6.51 ± 0.31% of the positive control (NG-methyl-L-arginine acetate) [60].The same EO also showed a moderate in vitro cytotoxic activity against Hep3B (human hepatoma) and MCF7 (human breast carcinoma) cell lines, with an IC 50 of 35.24 ± 0.06% and 28.15 ± 1.08%, respectively versus 0.59 ± 0.19% and 6.46 ± 0.81% for the positive control (camptothecin) [60].Many different biological essays were also carried out on EOs from other Nectandra spp., such as N. amazonum (anti-leishmanial and cytotoxic), N. cuspidata (antibacterial and cytotoxic), N. gardneri (anti-leishmanial and cytotoxic), N. grandiflora (antibacterial, antifungal, and sedative in silver catfish), N. hihua (anti-leishmanial and cytotoxic), N. lanceolata (antifungal, antioxidant, anti-chemotactic, cytotoxic, and antibacterial), N. leucantha (cytotoxic), N. megapotamica (antibacterial, cytotoxic, larvicidal, anaesthetic to some fish species, antifungal, antioxidant, anti-chemotactic, and anti-leishmanial), and N. puberula (antibacterial and cytotoxic) [13].However, due to the different chemical composition of all these EOs compared to the one described here, none of these biological activities can be hypothetically extended to N. laurel.
In 2017, Oliveira et al. discovered, in N. megapotamica EO, five new oxygenated sesquiterpenoids, denominated nectandrenes.All these metabolites were characterized by the molecular formula, C 15 H 25 O, corresponding to 220 m/z [62].In Table 1, it can be observed that, among the undetermined compounds, three sesquiterpenes are isomers of nectandrenes.However, according to data reported in the literature, neither the MS spectra nor the LRIs of these molecules corresponded to any of them.
Finally, the present study was complemented with the enantiomeric composition of some chiral compounds.Ultimately, due to the difficult commercial availability of enantiomerically pure δ-selinene, no chiral information could be obtained from the enantioselective analysis on the major compound.Nevertheless, seven chiral metabolites could be analysed, determining that three of them were enantiomerically pure; one presented a very high enantiomeric excess, whereas three were determined to be scalemic mixtures.As usual, these results demonstrate the existence, in N. laurel metabolism, of different enantioselective biosynthetic pathways, devoted to the synthesis of different enantiomers for different functions.It is in fact well known that despite presenting the same physicochemical properties (except the chiroptical ones), two enantiomers can show different biological and physiological activities.It is typical among EOs that two enantiomers present different aromas or different properties as insect pheromones [63,64].In N. laurel EO, the only major constituent (≥3.0% on at least one column), whose stereochemistry could be determined, was the enantiomerically pure (1R,5R)-(+)-α-pinene.According the to literature, dextrorotatory α-pinene has been demonstrated to be an antibacterial, antimalarial, and anti-inflammatory agent.Furthermore, it was much more active as an antimycotic and anticatabolic agent compared to the laevorotatory isomer.Finally, both optical isomers are known for being active as acetylcholinesterase inhibitors [65].

Plant Material
The leaves of N. laurel were collected on 26 November 2020 (unintentional date), with the permission of the Ministry of Environment, Water, and Ecological Transition of Ecuador, with MAATE registry number MAE-DNB-CM-2016-0048. The collection site was located in the radius of 200 m from a central point, of coordinates 04 • 22 46 S and 79 • 08 46 W, at an altitude of 2350 m above sea level.The botanical identification was carried out by one of the authors (N.C.), based on collection reviews conserved at the herbarium of the Universidad Nacional de Loja (UNL), Ecuador.A botanical specimen was also deposited at the herbarium of the Universidad Técnica Particular de Loja with code 14,702.
On the day of collection, the fresh leaves were dried at 35 • C for 48 h and the dry plant material (355 g) was stored in a fresh dry place until use.

EO Distillation and Sample Preparation
The dry leaves were analytically steam-distilled in a Marcusson-type apparatus, as previously described in the literature [8].The process was repeated four times, on amounts of 80 g for 4 h.The distillation was conducted on 2 mL of cyclohexane, spiked with n-nonane as internal standard (0.71 mg/mL), producing four samples of EO in solution that could directly be injected into GC.Both cyclohexane and n-nonane were analytical purity grade and purchased from Signa-Aldrich (Saint Louis, MO, USA).During the entire investigation, the samples were always stored in the dark at −15 • C until use.

Qualitative (GC-MS) Chemical Analysis of N. laurel EO
The qualitative chemical analysis of the EO was conducted in a Trace 1310 gas chromatograph (GC), coupled with a ISQ 7000 mass spectrometer (MS) as a detector (Thermo Fisher Scientific, Walthan, MA, USA).The oven was equipped with a non-polar DB-5ms and a polar HP-INNOWax column, both 30 m long, 0.25 mm internal diameter, and 0.25 µm film thickness (Agilent Technology, Santa Clara, CA, USA).With both columns, the following thermal program was applied: 50 • C for 10 min., followed by a first gradient of 3 • C/min until 100 • C, a second gradient of 5 • C/min until 200 • C, and finally, a third gradient of 10 • C/min until 230 • C, that were maintained for 20 min.The injector and transfer line were maintained at 230 • C, with the injector operating in split mode (40:1) and the autosampler injecting 1 µL.The carrier gas was helium, flowing through the column at the constant flow of 1 mL/min, and purchased from Indura (Guayaquil, Ecuador).The MS was operated in SCAN mode, with a mass range of 40-400 m/z; the electron impact (EI) ion source was set at 70 eV and 250 • C. All the EO constituents were identified, on both columns, by comparing each mass spectrum and linear retention index (LRI) with data from the literature.The LRIs were calculated based on the retention times of a mixture of homologous n-alkanes (C 9 -C 22 from Sigma-Aldrich, Saint Louis, MO, USA), injected via the same GC method according to Van den Dool and Kratz [66].

Quantitative (GC-FID) Chemical Analysis of N. laurel EO
The quantitative chemical analysis was carried out with the same GC, columns, thermal program, and instrument configuration used for the qualitative one but with the exception of the detector, that was in this case, a FID (flame ionization detector), set to 250 • C. The EO components were quantified calculating each relative response factor (RRF) according to the corresponding combustion enthalpies [67,68].The transformed integration areas were applied to two six-point calibration curves (one for each column), obtaining two correlation coefficients greater than 0.995 [69].Isopropyl caproate was synthetized in one of the authors' laboratories (G.G.) and purified until 98.8% (GC purity).

Enantioselective GC-MS Analysis of N. laurel EO
The enantioselective analysis was performed via GC-MS, with the same instrument and MS configuration of the qualitative chemical analysis.However, in this case, the elution was conducted at the constant pressure of 70 kPa.The oven was equipped with two enantioselective columns, whose stationary phases were based on 2,3-diacetyl-6-tertbutyldimethylsilyl-β-cyclodextrin and 2,3-diethyl-6-tert-butyldimethylsilyl-β-cyclodextrin, respectively.Both columns were purchased from MEGA S.r.l., Legnano, Italy.The analyses were carried out with the following thermal program: 50 • C for 1 min, followed by a thermal gradient of 2 • C/min until 220 • C, that was maintained for 10 min.The enantiomers were identified through comparison of the MS spectra and linear retention indices with data obtained from the injection of enantiomerically pure standards.

Figure 1 .
Figure 1.Nectandra laurel Klotzsch ex Nees at the collection site; (a) fruits and leaves, (b) flowers and leaves (Photo by Nixon Cumbicus).

Figure 1 .
Figure 1.Nectandra laurel Klotzsch ex Nees at the collection site; (a) fruits and leaves, (b) flowers and leaves (Photo by Nixon Cumbicus).

a
Calculated linear retention index; b reference linear retention index; % = percent amount by weight; σ = standard deviation; MW = molecular weight.Plants 2023, 12, x FOR PEER REVIEW 5 of 11

Figure 2 .
Figure2.GC-MS profile of N. laurel EO on a 5%-phenyl-methylpolysiloxane stationary phase.The peak numbers refer to major compounds (≥3.0% on at least one column), according to Table1.

Table 1 .
Qualitative (GC-MS) and quantitative (GC-FID) analyses of N. laurel EO with two orthogonal stationary phase columns.

Table 2 .
Enantioselective analysis of N. laurel EO on two β-cyclodextrin-based chiral selectors.