The Flower Essential Oil of Dalea mutisii Kunth (Fabaceae) from Ecuador: Chemical, Enantioselective, and Olfactometric Analyses

An essential oil was distilled with 0.25% yield from fresh flowers of Dalea mutisii Kunth, a native species mainly growing in the Andean region of Ecuador. A total of 50 compounds were identified, and most of them were quantified. The chemical composition was characterized by the prevalence of monoterpene hydrocarbons (>90%). Major components were α-pinene (42.9%), β-pinene (15.1%), β-phellandrene (12.6%), myrcene (6.7%), and (Z)-β-ocimene (5.4%). The essential oil was then submitted to enantioselective analysis, with a 2,3-diethyl-6-tert-butyldimethylsilyl-β-cyclodextrin-based capillary column. An enantiomeric excess was measured for (1R,5R)-(+)-α-pinene (91.6%), (1R,5R)-(+)-β-pinene (15.2%), (R)-(−)-α-phellandrene (4.8%), and (R)-(−)-β-phellandrene (88.8%), whereas (R)-(+)-limonene was enantiomerically pure. A gas chromatography–olfactometry (GC–O) analysis was additionally carried out on this pleasantly fragrant essential oil, following an aroma extract dilution analysis (AEDA) approach. Main odorants were α-pinene, β-pinene, α-phellandrene, and (Z)-β-ocimene, with dilution factors (FD) of 8, 4, 2, and 2, respectively.


Introduction
The study of natural products has been one of the leading ways of finding new molecules of biological interest since the beginning of 19th century. Currently, most European and many North American botanical species have been phytochemically investigated, resulting in a quite wide knowledge of their secondary metabolite composition and biological activity. As a consequence, despite these studies not yet being exhaustive, the discovery of new molecules within the European and North American flora is becoming more and more difficult. This is the reason why, during the next 30 years, the interest in looking for new metabolites shifted from temperate to tropical countries, where an impressive biodiversity, together with a more recent scientific history, makes phytochemical studies very important. Belonging to a group of 17 megadiverse countries [1], Ecuador is one of the most promising places in the world for carrying out these studies. For this reason, the authors have been investigating the endemic flora of Ecuador for many years, in order to contribute to the phytochemical knowledge of the Ecuadorian biodiversity [2].

Chemical Analysis and Physical Properties
The distillation of the fresh flowers of D. mutisii afforded an EO, with a mean distillation yield of 0.25% (w/w). The mean relative density of the EO was 0.891 g/cm 3 , whereas the mean refraction index was 1.4773. In the chemical analysis, 50 compounds were identified and 35 were quantified, corresponding to 98.3% of the whole sample mass. The EO mainly constituted monoterpene hydrocarbons, α-pinene (42.9%), β-pinene (15.1%), β-phellandrene (12.6%), myrcene (6.7%), and (Z)-β-ocimene (5.4%). The monoterpene fraction contributed to 91.8% of the whole EO. Only traces of one oxygenated monoterpene (cryptone) were detected. On the other hand, many sesquiterpenes and sesquiterpenoids were identified, being 4.8% and 1.6% of the respective sesquiterpene hydrocarbon and oxygenated fractions. Small amounts of phenylpropanoids and aliphatic esters were also observed, with (E)-iso-amyl cinnamate as the only quantifiable one (0.1%). The complete chemical analysis is shown in Table 1.

Enantioselective Analysis
The enantioselective analysis [28,29] was performed with a capillary column, using 2,3-diethyl-6-tert-butyldimethylsilyl-β-cyclodextrin as a chiral selector. A total of four monoterpene enantiomeric pairs were identified, calculating the respective enantiomeric distribution and enantiomeric excess (ee). Only (R)-(+)-limonene was identified as an enantiomerically pure component. The complete enantioselective analysis is presented in Table 2 and Figure 1, where the baseline separation of all the identified enantiomers can be observed. Table 2. Enantioselective analysis of some chiral constituents of D. mutisii EO with 2,3-diethyl-6-tert-butyldimethylsilyl-β-cyclodextrin column.

Olfactometric Analysis
The olfactometric analysis was carried out through gas chromatography-olfactometry (GC-O) [30], following an aroma extract dilution analysis (AEDA) protocol [31][32][33]. Four olfactory relevant compounds were detected, with α-pinene being the principal sensory component of the EO, with a dilution factor (FD) of 8. The results of the GC-O analysis are represented in Table 3 and Figure 2.
With regard to the sensory properties of D. mutisii EO, a strong and pleasant woody-resinous odor motivated us to study the aromatic profile using gas chromatography-olfactometry. An AEDA evaluation of the mixture was performed, which afforded four components as mainly responsible for the olfactory properties. According to this analysis, α-pinene, β-pinene, α-phellandrene, and (Z)-β-ocimene were the aroma-determining components, with a dilution factor (FD) of 8, 3, 2, and 2, respectively. In order to obtain a more comprehensive information on the chemical composition in general and the aromatic profile in particular, the study was complemented with the enantioselective analysis of the monoterpene fraction. In fact, it is well known that the enantiomeric composition of a chiral mixture influences its biological properties, including sensory perception [34]. In D. mutisii EO, α-pinene, β-pinene, α-phellandrene, and β-phellandrene were present as enantiomeric mixtures, whilst (R)-(+)-limonene was enantiomerically pure. The enantiomeric excess of β-pinene and α-phellandrene was close to the racemic mixture, whilst, for (1R,5R)-(+)-α-pinene and (R)-(−)-β-phellandrene, it was 91.6% and 88.8%, respectively. All these results were consistent with the perceived aroma of the whole EO.
Despite the different focus of the present study, some consideration could be afforded to the chemical composition and the biological properties of this EO. In order to formulate some consistent hypotheses, we should compare our volatile fraction with that from D. strobilacea [23], whose chemical composition is the most similar among the EOs described in literature for this genus. D. strobilacea is an aromatic plant, growing wild in the highlands from Peru to Chile. It is used in traditional medicine as "hierba de chil", in the form of a decoction, for treating gastrointestinal disorders. No application has been described that correlates the traditional use of this plant with that of D. mutisii; however, the EO from D. strobilacea was submitted to some interesting antibacterial essays. In particular, for its minimum inhibitory concentration (MIC), it resulted very active against Enterococcus faecalis (MIC = 7.81 µg/mL vs. >125 µg/mL for vancomycin) and not very active against Klebsiella pneumoniae (MIC = 59.5 µg/mL vs. 15.4 µg/mL for vancomycin) [23]. These results are consistent with the traditional use of D. strobilacea but do not support the main traditional use of D. mutisii against pneumonia. Four hypotheses can be formulated in this case: (1) the EOs are similar in composition but not identical, which may explain the difference; (2) the enantiomeric composition of the active components could be different; (3) the anti-pneumonia activity of D. mutisii could be due to its nonvolatile fraction; (4) the traditional use of D. mutisii to treat pneumonia could be scientifically unsupported. Nevertheless, all these considerations are inconclusive in the face of a lack of direct bioactivity-based evidence, merely constituting some working hypotheses.

Distillation of the EO and Sample Preparation
A total of three samples of pure EO were obtained by preparative steam distillation of each fresh sample of plant material. Each distillation was carried out for 3 h, inside a stainless-steel Clevenger-type apparatus. After recovery of the organic layer, which spontaneously separated from water, the EO was dried over anhydrous sodium sulfate and immediately stored in amber vials at −15 • C. For all GC injections, about 10 mg of EO were exactly weighted and diluted with 1 mL of cyclohexane, containing n-nonane as internal standard (0.7 mg/mL).

Qualitative Chemical Analysis
The qualitative analysis was run with a gas chromatography-mass spectrometry (GC-MS) system, consisting of an Agilent Technologies gas chromatograph 6890N, coupled with a quadrupole Mass Spectrometry Detector (MSD) 5973 (Santa Clara, CA, USA). The MSD was operated in SCAN mode and electronic ionization (70 eV), set at a mass range detection of 35-350 m/z. The MS transfer line temperature was set at 280 • C, while the ion source temperature was set at 200 • C. The gas chromatograph was equipped with a nonpolar stationary phase capillary column DB-5ms (5% phenyl-methylpolysiloxane, 30 m length, 0.25 mm internal diameter, and 0.25 µm film thickness; J & W Scientific, Folsom, CA, USA). The GC-MS analyses were performed as follows: the carrier gas was helium, set at a constant flow rate of 1 mL/min. The injection volume was 1 µL, with the injector operated in split mode (split ratio of 40:1) at the temperature of 250 • C. The elution was conducted from 50 • C (1 min) to 250 • C (10 min) at a gradient rate of 3 • C/min.
The EO components were identified by comparing both their linear retention indices (LRIs), calculated according to van den Dool and Kratz [26], and their mass spectra to those reported in literature (see Table 1). The linear retention indices were calculated using the homologous series of linear alkanes from n-nonane to n-pentacosane (C 9 purity 99% from BDH, Dubai, UAE and C 10 -C 25 purity 99% from Sigma-Aldrich, St. Louis, MO, USA). The identification of major components (>5%) was confirmed by injection of pure reference standard samples. All solvents and standards used in this study (analytical grade, purity > 99%) were purchased from Sigma-Aldrich.

Quantitative Chemical Analysis
The quantitative analysis was carried out with the same GC system as the qualitative one, coupled with a flame ionization detector (FID), and equipped with an Agilent Technologies 7683 series autoinjector (Little Falls, DE, USA).
The instrumental conditions were the same as the qualitative analyses but with a different thermal program: 50 • C for 1 min, a first thermal gradient to 180 • C at a rate of 3 • C/min, and then a second gradient to 250 • C at a rate of 15 • C/min. At the end, the oven temperature was kept at 250 • C for 15 min. The FID was alimented as follows: hydrogen flow 30 mL/min, air flow 300 mL/min. The temperature of the detector was set at 250 • C. The quantitative composition was obtained by using relative response factors (RRFs), calculated on the basis of the combustion enthalpy [35,36]. The RRFs were directly applied to the internal standard peak, which served for both normalization and quantification. The original method was modified, since n-nonane instead of methyl octanoate was used as internal standard. In order to carry out the quantitative analysis, the preparation described in Section 4.2 was applied twice to each pure EO, affording a total of six analytical samples. The quantitative results (Table 1) were obtained as mean values and standard deviations.
The following thermal program was used: 50 • C, held for 5 min, rising to 220 • C at a rate of 2 • C/min, and kept at this temperature for 5 min. The elution order was established according to literature, where enantiomerically pure standards were injected in the same column and conditions [3,10].

GC-O Analysis
The GC-O analyses were carried out with the same GC-FID system described for the quantitative ones, coupled to a sniffing port device model ODP 3, from Gerstel GmbH & Co.KG., Mülheim an der Ruhr, Germany. The GC-O system was configured with a 50% split ratio between sniffing port and detector. The olfactometric evaluations were performed by a panel of four trained people, presenting no anosmia for common monoterpenes and following an AEDA approach [31][32][33]. The samples were prepared as solutions of the EO in cyclohexane, at the concentration of 200 µL/mL, corresponding to a dilution factor (FD) of 1. The injection volume was 1 µL. The AEDA method was applied by acting on the split ratio, according to the following sequence: splitless, 1:1, 2:1, 3:1, 4:1, 5:1, and 6:1. After a preliminary qualitative screening at the highest concentration, the sniffing procedure of each panelist was carried out until 16 min. The acceptance criteria for the detected odors and FD values was that each perception had to be confirmed by at least three panelists in at least two following dilutions or, alternatively, once by all the panelists at the same dilution. During the analysis, the panelists were asked to give a descriptor for each perceived odor, utilizing the adjectives commonly used for terpenes and terpenoids.

Conclusions
The flowers of D. mutisii Kunth produce an EO, characterized by a pleasant woody-resinous odor and a quite good yield of 0.25% (w/w). The chemical composition is dominated by monoterpenes, which contribute to more than 90% by weight. The main chiral components of the EO are present as enantiomeric pairs, except for the case of the enantiomerically pure (R)-(+)-limonene. With regard to the olfactory profile, four monoterpene hydrocarbons appear to be determinant. In fact, α-pinene, β-pinene, α-phellandrene, and (Z)-β-ocimene are the main odorous constituents, in order of decreasing importance. The descriptors assigned to these compounds during the GC-O analysis are consistent with the perceived fragrance of the whole EO.