Gymnosperms of Idaho: Chemical Compositions and Enantiomeric Distributions of Essential Oils of Abies lasiocarpa, Picea engelmannii, Pinus contorta, Pseudotsuga menziesii, and Thuja plicata

Conifers are of great economic value in terms of lumber production, important for construction and other uses such as pulp and paper. They are also important sources of essential oils. Conifer species have been vital to the ethnobotany and traditional herbal medicine of many different Native American groups. The objective of this work was to obtain and analyze the essential oils of several conifer species (Abies lasiocarpa, Picea engelmannii, Pinus contorta, Pseudotsuga menziesii, and Thuja plicata) growing in Idaho. The foliar essential oils were obtained by hydrodistillation and then analyzed by gas chromatographic methods, including GC-MS, GC-FID, and chiral GC-MS. The essential oils were obtained in varying yields from 0.66% up to 4.70%. The essential oil compositions were largely dominated by monoterpene hydrocarbons and oxygenated monoterpenoids. The chiral monoterpenoids were generally rich in the (−)-enantiomers for members of the Pinaceae, but the (+)-enantiomers predominated in the Cupressaceae. The essential oil compositions obtained in this work are qualitatively similar, but quantitatively different, to previously reported compositions and confirm and complement the previous reports. However, this is the first comprehensive analysis of the chiral terpenoid components in these conifer species. Additional research on essential oils of the Pinaceae and Cupressaceae is needed to describe the chemical profiles, chemical compositions, and enantiomeric distributions more reliably in the various species and infraspecific taxa of these two families.


Introduction
Idaho, and western North America in general, is home to great habitat diversity, including mountains, canyons, and Great Basin deserts, and is also home to a large number of conifer species. Many of these trees are important sources of timber and other forest products; they have been important in Native American cultures in traditional medicine, and in addition to wood and wood products, are sources of essential oils. As part of our ongoing investigation into the essential oils of Idaho, we have collected samples of Rocky Mountain subalpine fir (Abies lasiocarpa var. lasiocarpa) (Pinaceae), Engelmann spruce (Picea engelmannii subsp. engelmannii) (Pinaceae), Rocky Mountain lodgepole pine (Pinus contorta subsp. latifolia) (Pinaceae), Rocky Mountain Douglas fir (Pseudotsuga menziesii var. glauca) (Pinaceae), and western red cedar (Thuja plicata) (Cupressaceae) growing in Idaho. The foliar essential oils have been obtained by hydrodistillation and the essential oils analyzed by gas chromatographic (GC-MS and GC-FID) methods. The enantiomeric distributions of monoterpenoid components have also been examined using chiral GC-MS. analyzed by gas chromatographic (GC-MS and GC-FID) methods. The enantiomeric distributions of monoterpenoid components have also been examined using chiral GC-MS.
Abies lasiocarpa (Hook.) Nutt. (subalpine fir, Pinaceae) is native to the mountains of western North America ( Figure 1) [1]. On young trees, the bark is smooth and gray with resin blisters, but appears rough and fissured on older trees. The leaves are flat needles, 1.5-3 cm long ( Figure 2). The infraspecific taxonomy of A. lasiocarpa has been debated and three varieties have been suggested: Abies lasiocarpa (Hook.) Nutt. var. lasiocarpa (coastal subalpine fir, ranging from British Columbia south through the Cascade Mountains of Washington and Oregon); Abies lasiocarpa var. bifolia (A. Murray bis) Eckenw. (Rocky Mountain subalpine fir, ranging from British Columbia south through the Rocky Mountains of Idaho, Montana and Colorado); and Abies lasiocarpa var. arizonica (Merriam) Lemmon (corkbark fir, found in high mountains of Arizona and New Mexico) based on morphological and monoterpenoid profiles [2,3]. However, based on DNA data, there is little support for the recognition of A. l. var. bifolia as a distinct variety, but rather a chemotype of A. l. var. lasiocarpa due to geographical selection differences [2]. The foliar essential oil compositions of the three varieties have been investigated previously by Hunt and von Rudloff [4] and by Adams and co-authors [2]. The essential oil of coastal subalpine fir has been characterized by relatively high concentrations of β-phellandrene (36.8-58.8%), while Rocky Mountain subalpine fir essential oil is rich in camphene (7.3-16.2%) and bornyl acetate (13.0-31.6%) [4]. Corkbark fir also has high concentrations of camphene (15.2%) and bornyl acetate (34.4%) [2]. The Shoshoni people took an infusion of the needles of A. lasiocarpa to treat colds [5].  [6]. Figure 1. Natural range of Abies lasiocarpa [6].  [7]. Two subspecies of P. engelmannii have been recognized [8], P. engelmannii subsp. engelmanii and Picea engelmannii subsp. mexicana (Martínex) P.A. Schmidt, which is found on the high mountains of northern Mexico [9]. The bark of P. engelmannii is thin and flaky; the needles are 15-30 mm long ( Figure 4).   [7]. Two subspecies of P. engelmannii have been recognized [8], P. engelmannii subsp. engelmanii and Picea engelmannii subsp. mexicana (Martínex) P.A. Schmidt, which is found on the high mountains of northern Mexico [9]. The bark of P. engelmannii is thin and flaky; the needles are 15-30 mm long ( Figure 4).  Pinus contorta Douglas ex Loudon subsp. latifolia (Engelm. ex S. Watson) Critchf. (Rocky Mountain lodgepole pine, Pinaceae) is found in the Rocky Mountains of western North America, from the Yukon, south through Colorado ( Figure 5). There are two other subspecies of P. contorta, P. contorta subsp. contorta Douglas ex Loudon (the shore pine), which ranges along the Pacific coast from southern Alaska, south to northwestern California, and P. contorta subsp. murrayana (Balf.) Engelm. (the Sierra lodgepole pine), which ranges in the Cascade Range in Washington and Oregon, south into northern California and the Sierra Nevada Range ( Figure 5) [10,11]. The gray-brown bark of P. contorta subsp. latifolia is thin and scaly, while the needles are 4-8 cm long and in pairs ( Figure 6). P. contorta subsp. contorta. P. contorta subsp. murrayana. P. contorta subsp. latifolia [6].  Pinus contorta Douglas ex Loudon subsp. latifolia (Engelm. ex S. Watson) Critchf. (Rocky Mountain lodgepole pine, Pinaceae) is found in the Rocky Mountains of western North America, from the Yukon, south through Colorado ( Figure 5). There are two other subspecies of P. contorta, P. contorta subsp. contorta Douglas ex Loudon (the shore pine), which ranges along the Pacific coast from southern Alaska, south to northwestern California, and P. contorta subsp. murrayana (Balf.) Engelm. (the Sierra lodgepole pine), which ranges in the Cascade Range in Washington and Oregon, south into northern California and the Sierra Nevada Range ( Figure 5) [10,11]. The gray-brown bark of P. contorta subsp. latifolia is thin and scaly, while the needles are 4-8 cm long and in pairs ( Figure 6).     Pseudotsuga menziesii (Mirb.) Franco (syn. Abies menziesii Mirb.) (Rocky Mountain Douglas fir, Pinaceae) is an important timber tree native to western North America [12]. The tree has been introduced to many temperate regions throughout the world. There are two varieties of Douglas fir, P. menziesii var. menziesii (coastal Douglas fir), which ranges from coastal British Columbia south through the Cascades into the Coastal and Sierra Nevada mountains of northern California, and P. menziesii var. glauca (Mayr) Franco (Rocky Mountain Douglas fir), which ranges from central British Columbia south into Arizona and New Mexico (Figure 7) [13]. There are populations of P. menziesii in Mexico that are morphologically similar to P. menziesii var. glauca that have been referred to as Pseudotsuga menziesii var. oaxacana Debreczy & I. Rácz [14], but there is little support for this particular taxon [15]. The bark on young trees is thin, smooth, gray, and covered with resin blisters. On mature trees, it is thicker (3-6 cm) and furrowed. The leaves are needles (2-3 cm long) spirally arranged around the branch (Figure 8). P. menziesii var. menziesii. P. menziesii var. glauca [6]. Pseudotsuga menziesii (Mirb.) Franco (syn. Abies menziesii Mirb.) (Rocky Mountain Douglas fir, Pinaceae) is an important timber tree native to western North America [12]. The tree has been introduced to many temperate regions throughout the world. There are two varieties of Douglas fir, P. menziesii var. menziesii (coastal Douglas fir), which ranges from coastal British Columbia south through the Cascades into the Coastal and Sierra Nevada mountains of northern California, and P. menziesii var. glauca (Mayr) Franco (Rocky Mountain Douglas fir), which ranges from central British Columbia south into Arizona and New Mexico (Figure 7) [13]. There are populations of P. menziesii in Mexico that are morphologically similar to P. menziesii var. glauca that have been referred to as Pseudotsuga menziesii var. oaxacana Debreczy & I. Rácz [14], but there is little support for this particular taxon [15]. The bark on young trees is thin, smooth, gray, and covered with resin blisters. On mature trees, it is thicker (3-6 cm) and furrowed. The leaves are needles (2-3 cm long) spirally arranged around the branch (Figure 8). Pseudotsuga menziesii (Mirb.) Franco (syn. Abies menziesii Mirb.) (Rocky Mountain Douglas fir, Pinaceae) is an important timber tree native to western North America [12]. The tree has been introduced to many temperate regions throughout the world. There are two varieties of Douglas fir, P. menziesii var. menziesii (coastal Douglas fir), which ranges from coastal British Columbia south through the Cascades into the Coastal and Sierra Nevada mountains of northern California, and P. menziesii var. glauca (Mayr) Franco (Rocky Mountain Douglas fir), which ranges from central British Columbia south into Arizona and New Mexico (Figure 7) [13]. There are populations of P. menziesii in Mexico that are morphologically similar to P. menziesii var. glauca that have been referred to as Pseudotsuga menziesii var. oaxacana Debreczy & I. Rácz [14], but there is little support for this particular taxon [15]. The bark on young trees is thin, smooth, gray, and covered with resin blisters. On mature trees, it is thicker (3-6 cm) and furrowed. The leaves are needles (2-3 cm long) spirally arranged around the branch ( Figure 8).   Pseudotsuga menziesii (Mirb.) Franco (syn. Abies menziesii Mirb.) (Rocky Mountain Douglas fir, Pinaceae) is an important timber tree native to western North America [12]. The tree has been introduced to many temperate regions throughout the world. There are two varieties of Douglas fir, P. menziesii var. menziesii (coastal Douglas fir), which ranges from coastal British Columbia south through the Cascades into the Coastal and Sierra Nevada mountains of northern California, and P. menziesii var. glauca (Mayr) Franco (Rocky Mountain Douglas fir), which ranges from central British Columbia south into Arizona and New Mexico (Figure 7) [13]. There are populations of P. menziesii in Mexico that are morphologically similar to P. menziesii var. glauca that have been referred to as Pseudotsuga menziesii var. oaxacana Debreczy & I. Rácz [14], but there is little support for this particular taxon [15]. The bark on young trees is thin, smooth, gray, and covered with resin blisters. On mature trees, it is thicker (3-6 cm) and furrowed. The leaves are needles (2-3 cm long) spirally arranged around the branch ( Figure 8). Pseudotsuga menziesii (Mirb.) Franco (syn. Abies menziesii Mirb.) (Rocky Mountain Douglas fir, Pinaceae) is an important timber tree native to western North America [12]. The tree has been introduced to many temperate regions throughout the world. There are two varieties of Douglas fir, P. menziesii var. menziesii (coastal Douglas fir), which ranges from coastal British Columbia south through the Cascades into the Coastal and Sierra Nevada mountains of northern California, and P. menziesii var. glauca (Mayr) Franco (Rocky Mountain Douglas fir), which ranges from central British Columbia south into Arizona and New Mexico (Figure 7) [13]. There are populations of P. menziesii in Mexico that are morphologically similar to P. menziesii var. glauca that have been referred to as Pseudotsuga menziesii var. oaxacana Debreczy & I. Rácz [14], but there is little support for this particular taxon [15]. The bark on young trees is thin, smooth, gray, and covered with resin blisters. On mature trees, it is thicker (3-6 cm) and furrowed. The leaves are needles (2-3 cm long) spirally arranged around the branch ( Figure 8).  The essential oils of both varieties (menziesii and glauca) have been extensively investigated by von Rudloff [16] and by Adams and co-workers [15]. The coastal Douglas fir has been characterized by relatively high concentrations of β-pinene (20-35%), terpinolene (5-20%), and terpinen-4-ol (5-15%), while the Rocky Mountain Douglas fir has shown large concentrations of camphene (20-30%), bornyl acetate (20-30%), and α-pinene (15-20%) [16]. In this work, the leaf essential oils from three individuals collected in southern Idaho have been obtained and the essential oil compositions determined using gas chromatographic methods. A comparison with Douglas fir essential oils from coastal, Rocky Mountain, and samples cultivated outside North America has also been carried out.
Thuja plicata Donn ex D. Don (western red cedar, Cupressaceae) is a large to very large evergreen tree native to western North America, ranging along the Cascade-Coastal Mountain Ranges from southeastern Alaska to northern California, and inland in the Rocky Mountains from British Columbia to the panhandle of northern Idaho ( Figure 9) [17]. Western red cedar is an important timber-producing tree and has been introduced to other temperate zone locations, including Europe, Great Britain, Australia, and New Zealand [18][19][20][21][22][23][24][25]. The thin, gray-brown bark forms vertical bands of fissures; the branch termini form flat boughs with scale-like leaves; the cones are 10-18 mm long and 4-5 mm wide with overlapping scales ( Figure 10). The Nez Perce people used an infusion of the foliage to treat colds and coughs [5].  The essential oils of both varieties (menziesii and glauca) have been extensively investigated by von Rudloff [16] and by Adams and co-workers [15]. The coastal Douglas fir has been characterized by relatively high concentrations of β-pinene (20-35%), terpinolene (5-20%), and terpinen-4-ol (5-15%), while the Rocky Mountain Douglas fir has shown large concentrations of camphene (20-30%), bornyl acetate (20-30%), and α-pinene (15-20%) [16]. In this work, the leaf essential oils from three individuals collected in southern Idaho have been obtained and the essential oil compositions determined using gas chromatographic methods. A comparison with Douglas fir essential oils from coastal, Rocky Mountain, and samples cultivated outside North America has also been carried out.
Thuja plicata Donn ex D. Don (western red cedar, Cupressaceae) is a large to very large evergreen tree native to western North America, ranging along the Cascade-Coastal Mountain Ranges from southeastern Alaska to northern California, and inland in the Rocky Mountains from British Columbia to the panhandle of northern Idaho ( Figure 9) [17]. Western red cedar is an important timber-producing tree and has been introduced to other temperate zone locations, including Europe, Great Britain, Australia, and New Zealand [18][19][20][21][22][23][24][25]. The thin, gray-brown bark forms vertical bands of fissures; the branch termini form flat boughs with scale-like leaves; the cones are 10-18 mm long and 4-5 mm wide with overlapping scales ( Figure 10). The Nez Perce people used an infusion of the foliage to treat colds and coughs [5]. The essential oils of both varieties (menziesii and glauca) have been extensively investigated by von Rudloff [16] and by Adams and co-workers [15]. The coastal Douglas fir has been characterized by relatively high concentrations of β-pinene (20-35%), terpinolene (5-20%), and terpinen-4-ol (5-15%), while the Rocky Mountain Douglas fir has shown large concentrations of camphene (20-30%), bornyl acetate (20-30%), and α-pinene (15-20%) [16]. In this work, the leaf essential oils from three individuals collected in southern Idaho have been obtained and the essential oil compositions determined using gas chromatographic methods. A comparison with Douglas fir essential oils from coastal, Rocky Mountain, and samples cultivated outside North America has also been carried out.
Thuja plicata Donn ex D. Don (western red cedar, Cupressaceae) is a large to very large evergreen tree native to western North America, ranging along the Cascade-Coastal Mountain Ranges from southeastern Alaska to northern California, and inland in the Rocky Mountains from British Columbia to the panhandle of northern Idaho ( Figure 9) [17]. Western red cedar is an important timber-producing tree and has been introduced to other temperate zone locations, including Europe, Great Britain, Australia, and New Zealand [18][19][20][21][22][23][24][25]. The thin, gray-brown bark forms vertical bands of fissures; the branch termini form flat boughs with scale-like leaves; the cones are 10-18 mm long and 4-5 mm wide with overlapping scales ( Figure 10). The Nez Perce people used an infusion of the foliage to treat colds and coughs [5].

Essential Oil Composition
Essential oils of the conifer species were obtained by hydrodistillation and the essential oil compositions determined using gas chromatography (GC-MS and GC-FID).

Abies lasiocarpa var. lasiocarpa
The foliage (branch tips and leaves, no cones) from two individual mature A. lasiocarpa var. lasiocarpa trees (A.l.l. #1 and A.l.l. #2) from southern Idaho were hydrodistilled to give colorless essential oils in 1.611% and 1.857% yield based on masses of fresh/frozen plant material). Gas chromatographic analysis of the essential oils was carried out to assess the chemical compositions (Table 1).

Essential Oil Composition
Essential oils of the conifer species were obtained by hydrodistillation and the essential oil compositions determined using gas chromatography (GC-MS and GC-FID).

Abies lasiocarpa var. lasiocarpa
The foliage (branch tips and leaves, no cones) from two individual mature A. lasiocarpa var. lasiocarpa trees (A.l.l. #1 and A.l.l. #2) from southern Idaho were hydrodistilled to give colorless essential oils in 1.611% and 1.857% yield based on masses of fresh/frozen plant material). Gas chromatographic analysis of the essential oils was carried out to assess the chemical compositions (Table 1). The major components in A. lasiocarpa essential oils were limonene (20.3% and 34.6%), bornyl acetate (24.7% and 18.5%), β-pinene (13.6% and 9.3%), camphene (10.9% and 7.4%), and α-pinene (5.0% and 4.5%). The compositions are consistent with those reported by Adams and co-authors for Rocky Mountain subalpine fir from Montana and Utah [2].

Picea engelmannii subsp. engelmannii
Hydrodistillation of the branch tips and leaves of P. engelmannii subsp. engelmannii (P.e.e.) gave a yellow essential oil in 0.912% yield based on mass of fresh/frozen plant material. The essential oil composition is listed in Table 2. The essential oil was rich in oxygenated monoterpenoids (50.2%), including camphor (22.8%), borneol (8.3%), and camphene hydrate (6.0%), as well as monoterpene hydrocarbons, (38.2%) myrcene (11.7%) and camphene (6.0%). There have been previous examinations of P. engelmannii from Arizona [31] and from Poland [32]. An agglomerative hierarchical cluster (AHC) analysis was carried out to reveal the similarities between these essential oil samples (Figure 11). The samples from Arizona (sampled on 6 June, 20 June, and 25 July of 1984) showed wide variation in essential oil composition (<70% similarity) for the three dates. Mardarowicz and co-workers sampled a mature tree and saplings of cultivated trees in Poland [32]. The juvenile and mature foliar essential oils were very different in composition, but the composition of the mature foliar essential oil is similar (>80% similarity) to the P. engelmannii essential oil from Idaho. Thus, for example, the major components in the mature foliar essential oil from Poland were camphor (14.9%), borneol (5.2%), camphene hydrate (5.0%), myrcene (12.2%), and camphene (3.5%). Interestingly, the Poland sample had 5.6% benzaldehyde, which was not observed in the Idaho sample. foliar essential oils were very different in composition, but the composition of the m foliar essential oil is similar (>80% similarity) to the P. engelmannii essential oil from I Thus, for example, the major components in the mature foliar essential oil from Po were camphor (14.9%), borneol (5.2%), camphene hydrate (5.0%), myrcene (12.2%) camphene (3.5%). Interestingly, the Poland sample had 5.6% benzaldehyde, which not observed in the Idaho sample.

Pinus contorta subsp. latifolia
Leaves (needles) of P. contorta subsp. latifolia from two mature trees (P.c.l. #1 and #2) were hydrodistilled to give colorless essential oils in 3.105% and 1.702% yield b on masses of fresh/frozen plant material. The gas chromatographic results are sum rized in Table 3. The major components in the essential oils were β-pinene (27.0%
Molecules 2023, 28, x FOR PEER REVIEW 1 subsp. murrayana from Oregon [10], and P. contorta subsp. contorta from Oregon [1 AHC analysis was carried out (Figure 12). The three P. contorta subsp. latifolia sa show > 90% similarity, while P. contorta subsp. murrayana shows 87% similarity to th folia subspecies. The least similar in essential oil composition is P. contorta subsp. co with only 45% similarity. Although β-phellandrene was the major component in all P. contorta essential oils, β-pinene was only a minor component (0.5%) in P. contorta s contorta, but terpinen-4-ol was a major component (11.0%) in P. contorta subsp. co which account for the lack of similarity of this essential oil.

Pseudotsuga menziesii var. glauca
Hydrodistillation of the leaves (needles) of P. menziesii from three individua (P.m.g. #1, P.m.g. #2, and P.m.g. #3) from southern Idaho gave pale-yellow essential 0.658-1.462% yield based on masses of fresh/frozen plant material. The chemical co sitions of the three P. menziesii samples are compiled in Table 4.
The major components in the essential oils were bornyl acetate (38.7-41.1%), camphene (15.0-19.5%), α-pinene (6.3-11.2%), and limonene (3.9-5.4%), confirming the identification of these samples as Rocky Mountain Douglas fir (P. menziesii var. glauca) [15,16]. In order to complement the volatile phytochemical differences between P. menziesii var. menziesii [15,34] and P. menziesii var. glauca as well as place samples from outside North America [31,[35][36][37][38][39] into chemical context, both agglomerative hierarchical cluster (AHC) analysis ( Figure 13) and principal component analysis (PCA, Figure 14) were carried out using the percent compositions of the major components (Supplementary Table S1).   There are two well-defined clusters based on the AHC. Cluster 1 is a cluster made up of samples from Idaho (this work), Yellowstone, Arizona, and New Mexico; dominated by bornyl acetate and camphene; and is clearly P. menziesii var. glauca based on the volatile phytochemicals and the geographical locations. Cluster 2 is made up of samples from Washington state (P. menziesii var. menziesii) as well as cultivated samples from Serbia, Romania, Austria, Bulgaria, Argentina, and New Zealand, and is defined by large concentrations of β-pinene, terpinolene, and sabinene. The chemical compositions of the non-North American cultivated samples are consistent with the menziesii variety and are likely derived from P. menziesii var. menziesii parents. There is one sample from Arizona [31] that does not fit into either the glauca or the menziesii varieties, and likely represents an "Interior Intermediate" chemotype [16].
The PCA verifies the AHC with the P. menziesii var. glauca group positively correlating with bornyl acetate and camphene. The P. menziesii var. menziesii group, on the other hand, positively correlates with β-pinene, terpinolene, and sabinene. The "Interior Intermediate" sample from Arizona correlates most strongly with camphene, α-pinene, β-pinene, and limonene.

Thuja plicata
Hydrodistillation of T. plicata foliage from five different trees (T.p. #1-T.p. #5) growing near Coeur d'Alene, Idaho, gave pale-yellow essential oils in yields ranging from 0.99% to 4.70% based on masses of fresh/frozen plant material. The essential oils were analyzed by gas chromatographic methods (GC-MS and GC-FID, Table 5).  The essential oils were dominated by α-thujone (72.5-77.8%) and β-thujone  [24]. That is, the foliar essential oils of T. plicata, regardless of geographical location, have been dominated by α-thujone, with lesser amounts of β-thujone, sabinene, and terpinen-4-ol [40]. Samples from Poland, however, showed relatively high concentrations of fenchone (7.1-11.3%), which were not reported in the samples from Serbia or from Idaho. Thuja plicata has shown low genetic diversity [41], which is consistent with the low variation in essential oil composition.
The foliar essential oil of T. plicata has shown insecticidal [42], insect antifeedant [25], as well as antibacterial and antifungal [25,43] activities. The biological activities of T. plicata essential oil can be attributed to the major component, α-thujone. The toxicity of α-thujone has been determined to be due to modulation of the γ-aminobutyric acid (GABA) type A receptor [44]. α-Thujone, and to a lesser extent, β-thujone have shown antinociceptive activities in a rodent model [45]. In addition, thujone has shown anti-inflammatory activity due to inhibition of induced interleukin (IL-6 and IL-8) release [46]. Thus, the biological properties of α-thujone are consistent with the Native American herbal medicinal uses of the plant.
A comparison of essential oil compositions between the five species of conifers in this study (see Supplementary Table S2) shows that A. lasiocarpa var. lasiocarpa and P. menziesii var. glauca have similar compositions, both species are rich in bornyl acetate, camphene, and limonene. On the other hand, P. engelmanii var. engelmanii (dominated by camphor and myrcene), Pinus contorta subsp. latifolia (rich in β-pinene and β-phellandrene), and Thuja plicata (dominated by thujones), are completely dissimilar in composition with all of the other species.

Gas Chromatographic Analyses
Gas chromatography-mass spectrometry (GC-MS) was carried out using the instrumentation and conditions previously reported [51]: Shimadzu GC-MS-QP2010 Ultra (Shimadzu Scientific Instruments, Columbia, MD, USA), ZB-5ms GC column (5% phenyl polydimethylsiloxane, 60 m × 0.25 mm × 0.25 µm film thickness) (Phenomenex, Torrance, CA, USA), injector and detector temperatures = 260 • C, helium carrier gas (column head pressure = 208.5 kPa, flow rate = 2.00 mL/min), GC oven temperature program = 50 • C start, ramp to 260 • C at 2 • C/min. For each essential oil sample, 1.0 µL of a 5% (w/v) solution in CH 2 Cl 2 was injected (splitting mode of 24.5:1). Retention index (RI) values were determined using a homologous series of n-alkanes [26]. The essential oil compositions were ascertained by comparison of their RI values and MS fragmentation patterns with those reported in the databases [27][28][29][30] using the LabSolutions GCMS solution software version 4.45 (Shimadzu Scientific Instruments, Columbia, MD, USA). ferent geographical locations) to describe the chemical profiles, chemical compositions and enantiomeric distributions more reliably in the various species and infraspecific taxa of these two families.