Essential Oil Composition and Enantioselective Profile of Agastache urticifolia (Lamiaceae) and Monardella odoratissima (Lamiaceae) from Utah

Two species within the Lamiaceae (mint) family, Agastache urticifolia and Monardella odoratissima, are aromatic plants that are native to the Intermountain Region (USA). Essential oil produced through steam distillation was examined to establish the essential oil yield and both the achiral and chiral aromatic profiles of both plant species. The resulting essential oils were analyzed by GC/MS, GC/FID, and MRR (molecular rotational resonance). For A. urticifolia and M. odoratissima, achiral essential oil profiles were largely composed of limonene (71.0%, 27.7%), trans-β-ocimene (3.6%, 6.9%), and pulegone (15.9%, 4.3%), respectively. Between the two species, eight chiral pairs were analyzed and, interestingly, the dominant enantiomer (calculated as ee%) of limonene and pulegone switched between the two species. Where enantiopure standards were not commercially available, MRR was used as a reliable analytical technique for chiral analysis. This study verifies the achiral profile for A. urticifolia and, for the first time to the authors’ knowledge, establishes the achiral profile for M. odoratissima and chiral profile for both species. Additionally, this study confirms the utility and practicality of using MRR for determining chiral profiles in essential oils.


Introduction
The Lamiaceae (mint) family is comprised of over seven thousand species and within the mentheae tribe are two small genera, Agastache and Monardella [1]. Both genera contain plant species that are native to the state of Utah, Agastache urticifolia and Monardella odoratissima [2][3][4][5].
Agastache urticifolia (Benth.) Kuntze is a perennial flowering mint that is strongly aromatic [5,6]. Two varieties of the species exist, var. urticifolia and var. glaucifolia, however, only native populations of the former exist in Utah mountain ranges [6]. Though native to the Intermountain West (USA), this species has been cultivated throughout the world [7][8][9] and even used for ethnobotanical practices in Bangladesh [10]. Scanning electron microscopy has shown that adaxial portions of the leaf contain both simple non-glandular and glandular (capitate and subsessile) trichomes from which essential oil can be extracted [11]. Previously analyzed A. urticifolia essential oil samples from cultivated (Russia) and native (Oregon, USA) populations have been shown to be primarily composed of the volatile compounds limonene, menthone, and pulegone [8,12], all of which are found as enantiomers in nature. To the authors' knowledge, samples from Utah have not been previously analyzed.
Monardella species are endemic to western North America [13]. Monardella odoratissima Benth. is a small, fragrant perennial flowering mint [5,6] found in California, Colorado, Idaho, Montana, Nevada, Oregon, Utah, and Washington state [14][15][16][17]. The morphological variation of this species from distinct locations has led some researchers to classify these variations as ecotypes while others have classified them as either subspecies or varieties, so additional botanical clarification appears necessary [6,14,15,18]. To the authors' knowledge, the essential oil composition of this species has not been previously established.
Plants in the mint family are the topic of frequent research and are of economic importance [19]. The essential oil extracted from aromatic plants in the mint family are used for flavor, fragrance, medicines, as well as other industries [20]. Due to the abundant variety of species within the mint family and the variety of volatile compounds they produce, they are widely distributed and cultivated throughout the world [20,21]. Research on the essential oil of novel plants in the mint family, as well as the development of analytical techniques to test those essential oils, is of both academic and economic interest.
The present study establishes and compares the essential oil composition, enantiomeric profile of prominent chiral compounds, and essential oil yield of A. urticifolia and M. odoratissima from Utah mountains. These data are determined utilizing conventional analytical techniques of GC/MS and GC/FID, and the novel application of chiral tagging molecular rotational resonance (MRR). MRR characterizes volatile compounds in the gas phase through their characteristic pure rotational momentum transitions [22]. As MRR is an extremely high-resolution spectroscopic technique and is highly sensitive to small changes in a molecule's three-dimensional mass distribution, distinct compounds, including isomers, can be resolved in a mixture without chromatographic separation or the need for enantiopure reference standards [23,24]. To the authors' knowledge, this is the first time A. urticifolia essential oil of Utahn origin has been researched as well as the first time the composition of M. odoratissima essential oil and enantiomeric profile of either species from any location have been researched. Results provide a foundation for future pharmacological research and taxonomy-chemotaxonomic clarification.

KI
Between the two species, eight chiral pairs were analyzed: α-pinene, sabinene, βpinene, limonene, menthone, pulegone, α-terpineol, and citronellol (Table 2). Enantiopure reference standards were commercially available for all prominent (defined as averages ≥ 1%) chiral monoterpenoids found in these essential oils, except for sabinene. Chiral tagging molecular rotational resonance (MRR) was used to determine ee% of sabinene in M. odoratissima essential oil. To illustrate the similarity between values from conventional techniques (GC/MS and GC/FID) and the novel application of chiral tagging MRR for determining chiral profiles, ee% was determined for limonene in both essential oils using both techniques (Table 3). Values (ee%) compared between each technique agree within 4% or less. Table 2. Enantiomeric excess of chiral monoterpenoids that had an average area % (achiral profile) ≥ 1% for Agastache urticifolia and Monardella odoratissima. Chiral ratios (calculated as ee%) determined by GC/FID for all compounds except for sabinene, which was determined by molecular rotational resonance (MRR) (average values, n = 3 per species). Each essential oil sample was analyzed in triplicate to ensure repeatability (standard deviation for analysis by GC/FID < 0.5 for all compounds; standard deviation for analysis by MRR 3.6 for sabinene).

Enantiomer
Agastache urticifolia ee% Monardella odoratissima ee% Table 3. Enantiomeric excess (ee%) of limonene in essential oils of Agastache urticifolia and Monardella odoratissima determined by GC/FID and molecular rotational resonance (MRR) (average values, n = 3 per species). Each essential oil sample was analyzed in triplicate to ensure repeatability (standard deviation for analysis by GC/FID < 0.5 for repeat injections; standard deviation for analysis by MRR 2.8 for repeat injections). Values (ee%) between techniques agree within 4% or less.

Method Enantiomer
Agastache urticifolia ee%  Table 4. The average essential oil yield for A. urticifolia and M. odoratissima are 0.14% and 0.16%, respectively. Table 4. Yield data, including weight of plant material distilled (g), essential oil yield (g), and calculated essential oil yield (%). Average calculated yields for Agastache urticifolia and Monardella odoratissima are 0.14% and 0.16%, respectively. The relative standard deviation (RSD) is provided for essential oil yield for both species.
Enantiopure reference standards were commercially available for all prominent chiral monoterpenoids found in these essential oils, except for sabinene. Given the distinct capabilities of molecular rotational resonance (MRR) to resolve enantiomers through chiral tagging and quantify specific compounds, including isomers, without chromatographic separation or the need for enantiopure reference standards, this technique was used to determine ee% of sabinene in M. odoratissima essential oil. To illustrate the similarity of values between the conventional techniques of GC/MS and GC/FID [28][29][30] and the novel application of MRR for determining chiral profiles, ee% was determined for limonene in both essential oils using both techniques. Values (ee%) compared between each technique agree within 4% or less. The standard deviation calculated on repeat injections of the MRR measurements was higher than that of the GC measurements ( Table 2, Table 3). In MRR, the precision of the measurement is determined by the integration time of the method. The methods in this study were approximately 18 min in length (including time for cleaning between samples). A longer method could be developed in the future to improve the measurement precision as needed.
The essential oils of A. urticifolia and M. odoratissima display characteristics common to the more well-known essential oils of peppermint and pennyroyal mint, in terms of both the achiral and chiral aromatic profiles. The detailed profiles provide a foundation for future pharmacological research and taxonomy-chemotaxonomic clarification; however, additional research is needed to investigate both fields. of the method. The methods in this study were approximately 18 min in length (including time for cleaning between samples). A longer method could be developed in the future to improve the measurement precision as needed.

Plant
The essential oils of A. urticifolia and M. odoratissima display characteristics common to the more well-known essential oils of peppermint and pennyroyal mint, in terms of both the achiral and chiral aromatic profiles. The detailed profiles provide a foundation for future pharmacological research and taxonomy-chemotaxonomic clarification; however, additional research is needed to investigate both fields. Plant material was prepared for laboratory-scale distillation as follows: plant material from each species was collected, bagged, and stored at -20 ± 2 °C until steam distilled. Steam distillation was performed in triplicate, resulting in three distillations per plant species and six distillations over the course of this research project. Plant material was prepared for laboratory-scale distillation as follows: plant material from each species was collected, bagged, and stored at -20 ± 2 • C until steam distilled. Steam distillation was performed in triplicate, resulting in three distillations per plant species and six distillations over the course of this research project.

Plant
Laboratory-scale distillation was as follows (using a custom distillation unit): 1.5 L of water added to 2 L steam generator that fed to a 2 L distillation chamber, plant material accurately weighed and added to the distillation chamber, distillation for 1.5 h from passover by indirect steam, essential oil separated by a cooled condenser and Florentine flask. Each essential oil sample was filtered and stored at room temperature in a sealed amber glass bottle until analysis.
The percent yield was calculated as the ratio of the mass of processed plant material immediately before distillation to the mass of essential oil produced, multiplied by 100.
Essential oils were analyzed, and volatile compounds identified, by GC/MS using an Agilent 7890B GC/5977B MSD (Agilent Technologies, Santa Clara, CA, USA) and Agilent J&W DB-5, 60 m × 0.25 mm, 0.25 µm film thickness, fused silica capillary column. Operating conditions: 0.1 µL of sample (20% soln. for essential oils in ethanol), 150:1 split ratio, initial oven temperature of 40 • C with an initial hold time of 5 min, oven ramp rate of 4.5 • C per minute to 310 • C with a hold time of 5 min, helium carrier gas. The electron ionization energy was 70 eV, scan range 35-650 amu, scan rate 2.4 scans per second, source temperature 230 • C, and quadrupole temperature 150 • C. Volatile compounds were identified using the Adams volatile oil library [25] using Chemstation library search in conjunction with retention indices. Note that limonene/1,8-cineole elute as unresolved peaks. Their ratios were determined by the ratio of masses 41, 68, 93 (limonene), 43, 71, 81 (1,8-cineole). Volatile compounds were quantified and are reported as a relative area percent by GC/FID using an Agilent 7890B and Agilent J&W DB-5, 60 m × 0.25 mm, 0.25 µm film thickness, fused silica capillary column. Operating conditions: 0.1 µL of sample (20% soln. for essential oils in ethanol, 1% for reference compounds in ethanol, 0.1% soln. for C7-C30 alkanes in hexane), 25:1 split injection, initial oven temperature at 40 • C with an initial hold time of 2 min, oven ramp rate of 3.0 • C per minute to 250 • C with a hold time of 3 min, helium carrier gas. Essential oil samples were analyzed in triplicate by GC/FID to ensure repeatability (standard deviation < 1 for all compounds). Compounds were assigned using retention indices coupled with the retention time data of reference compounds (MilliporeSigma, Sigma-Aldrich, St. Louis, MO, USA).
Enantioselective analysis was performed on chiral monoterpenoids that had an average area % (achiral profile) ≥ 1 for each species. Essential oils were analyzed, and chiral pairs identified, by GC/MS using an Agilent 7890B GC/5977B MSD (Agilent Technologies, Santa Clara, CA, USA) and Restek Rt-β, 30 m × 0.32 mm, 0.25 µm film thickness, fused silica capillary column. Operating conditions: 0.2 µL of sample (0.5% soln. for essential oils in ethanol), 25:1 split ratio, initial oven temperature of 40 • C with an initial hold time of 20 min, oven ramp rate of 2.0 • C per minute to 140 • C with a hold time of 35 min, second oven ramp rate of 30.0 • C per minute to 230 • C with a hold time of 2 min, helium carrier gas. The electron ionization energy was 70 eV, scan range 35-650 amu, scan rate 2.4 scans per second, source temperature 230 • C, and quadrupole temperature 150 • C. Volatile compounds were identified using the Adams volatile oil library [25] using Chemstation library search. Chiral pairs were quantified and are reported as enantiomeric excess (ee %) (Equation (1)) by GC/FID using an Agilent 7890B and Restek Rt-β, 30 m × 0.32 mm, 0.25 µm film thickness, fused silica capillary column. Operating conditions: 0.2 µL of sample (0.5% or 2% soln. for essential oils in ethanol, 0.1% for enantiopure reference compounds in ethanol), 10:1 split injection, initial oven temperature at 40 • C with an initial hold time of 20 min, oven ramp rate of 2.0 • C per minute to 140 • C with a hold time of 35 min, second oven ramp rate of 30.0 • C per minute to 230 • C with a hold time of 2 min, helium carrier gas. Essential oil samples were analyzed in triplicate by GC/FID to ensure repeatability (standard deviation < 0.5 when calculating ee % for each chiral pair). Enantiopure reference standards were used for α-pinene, β-pinene, limonene, pulegone, α-terpineol Assignment of (R)-(+)-sabinene and (S)-(-)-sabinene was determined by the isoMRR spectrometer (BrightSpec, Inc., Charlottesville, VA, USA). Duplicate determination of ee% was calculated for limonene to demonstrate technique reliability. For molecular rotational resonance (MRR), high-purity neon carrier gas, seeded with the volatile compound(s) under study, was injected into a high vacuum chamber through a pulsed supersonic expansion nozzle to create a rotationally cold sample for analysis. For resolution of enantiomers, the chiral tagging technique was used, where a small chiral molecule of known enantiomeric purity was added into the carrier gas stream. In the pulsed expansion, noncovalent diastereomeric complexes were formed between the analyte and tag, which were used to distinguish the enantiomers of the analyte by MRR [31,32]. The structures of the non-covalent complexes formed in the experiment are determined by comparison of the experimentally determined rotational constants for each of the diastereomeric complexes to those calculated from quantum chemical calculations using dispersion-corrected density functional theory. After normalization of the instrument response to the two complexes using a racemic chiral tag sample, both the absolute configuration (AC) and ee% of a volatile, chiral compound can be determined by measuring the relative signals of the two complexes. In this study, the targeted isoMRR spectrometer [24,31] was used to perform the chiral tagging measurements, with a measurement cycle time of approximately 18 min for each essential oil sample. Analytes were characterized in a procedure very similar to in the previous studies [31,32]. For each measurement, 5 µL of undiluted oil was injected, and heated to 30 • C to generate vapor of the intended analytes. The tag used was (R)-propylene oxide (PO) for the limonene ee% determination, and (R)-1,1,1-trifluoropropan-2-ol (TFIP) for the sabinene ee% determination.

Conclusions
To the authors' knowledge, this is the first time Agastache urticifolia essential oil of Utahn origin has been researched as well as the first time the composition of Monardella odoratissima essential oil and enantiomeric profiles of either species from any location have been researched.
The essential oils of A. urticifolia and M. odoratissima share similar compounds in their achiral composition, with both essential oils being largely composed of limonene, trans-β-ocimene, and pulegone. Pulegone, which is a prominent compound in both plant species, is not a common component of many essential oils. However, it is the prominent compound of Mentha pulegium (pennyroyal mint), another plant species in the Lamiaceae (mint) family.
Between the two species, eight chiral pairs were analyzed. These data were determined utilizing conventional analytical techniques of gas chromatography (GC), and, where commercial enantiopure reference standards were not available, the novel application of chiral tagging molecular rotational resonance (MRR). Upon investigating analytical technique repeatability, the standard deviation calculated on repeat injections of the MRR measurements was higher than that of the GC measurements. In MRR, the precision of the measurement is determined by the integration time of the method. The methods in this study, including time for cleaning between samples, were approximately 18 min in length. This is in stark contrast to the chiral GC method, which run time is 110 min, not including the rinse/clean between sample injections. A longer MRR method could be developed in the future to improve the measurement precision as needed. Notwithstanding, this study confirms the utility and practicality of using MRR for determining chiral profiles in essential oils. In future studies on these two mint species, additional chiral pairs (monoterpenoids < 1%; sesquiterpenoids) could also be researched to determine a complete enantiomeric profile.
The essential oils of A. urticifolia and M. odoratissima display characteristics common to two well-known essential oils in the mint family, peppermint and pennyroyal mint, in terms of both the achiral and chiral aromatic profiles. Additional research could lead to the commercial cultivation and application of A. urticifolia and M. odoratissima essential oils. Research on the essential oils of these novel plants in the mint family, as well as the development of analytical techniques to test the essential oils, is of both academic and economic interest. The essential oil extracted from these aromatic plants could potentially be used for flavor, fragrance, medicines, as well as other industries. The detailed profiles established herein provide a foundation for future use and applications of these plants, as well as taxonomy-chemotaxonomic clarification. However, additional research is needed to investigate these fields.  Informed Consent Statement: Not applicable for studies not involving humans.

Data Availability Statement:
The data presented in this study are available upon request from the corresponding author.