Essential Oil Compositions of Pinus Species (P. contorta Subsp. contorta, P. ponderosa var. ponderosa, and P. flexilis); Enantiomeric Distribution of Terpenoids in Pinus Species

Pinus species are important in traditional medicine throughout their ranges, and pine essential oils are of interest in aromatherapy and as topical treatments. In this work, the leaf (needle) essential oils of Pinus ponderosa var. ponderosa and Pinus contorta subsp. contorta from Oregon and Pinus flexilis growing in Idaho, have been obtained by hydrodistillation and analyzed by gas chromatographic techniques. The leaf essential oil of P. ponderosa was dominated by β-pinene (21.5–55.3%), methyl chavicol (8.5–41.5%), α-pinene (3.6–9.6%), δ-3-carene (3.6–6.2%), and α-terpineol (1.4–5.3%). The major components of P. contorta essential oil were β-phellandrene (23.8%), terpinen-4-ol (11.0%). The essential oil of P. flexilis was dominated by α-pinene (37.1%), β-pinene (21.9%), bornyl acetate (12.8%), and camphene (8.5%). Chiral gas chromatography revealed the enantiomeric ratios of α-pinene and limonene to be variable, but (−)-β-pinene predominated in Pinus essential oils.


Introduction
Numerous members of the genus Pinus (Pinaceae) are used in traditional medicine in their native ranges [1] and several essential oils derived from the genus are commercially important for use in aromatherapy and topical therapy applications, such as Scots pine (Pinus sylvestris L.), black pine (Pinus nigra J.F. Arnold), jack pine (Pinus bansksiana Lamb.), and white pine (Pinus strobus L.) [2]. In this work, the leaf essential oils of Pinus ponderosa Douglas ex C. Lawson var. ponderosa, Pinus contorta Douglas ex Loudon subsp. contorta, and Pinus flexilis E. James have been investigated for their chemical compositions and terpenoid enantiomeric distributions. In the case where essential oils are used therapeutically (e.g., aromatherapy) the different compositions and enantiomers may have very different biological activities. For commercial essential oils, the chemical compositions and enantiomeric distribution can be valuable for assessing the quality and consistency of the essential oil as well as a potential screen for adulteration or contamination.
Pinus ponderosa, the ponderosa pine (Figure 1), is the most widespread species of pine in western North America and ranges from British Columbia, south through the Cascade Range, the Sierra Nevada range of California, the Rocky Mountains and into the southwestern mountains of Utah, Arizona, and New Mexico. World Flora Online currently lists 11 subtaxa for the species [3], but the taxonomy is not resolved [4]. However, two varieties of the species are generally recognized: Pinus ponderosa var. ponderosa, the Pacific ponderosa pine, which ranges from southern British Columbia, south through the mountains of Washington, Oregon, and California, and Pinus ponderosa var. scopulorum Engelm., the Rocky Mountain ponderosa pine, found in eastern Montana, western North and South Dakota and Nebraska, Wyoming, Nebraska, northern and central Colorado and Utah [5]. Flathead Native Americans used the boughs of P. ponderosa in sweat lodges to treat muscular pains, while the Navajo people took a decoction of the needles for coughs and fever [6].
Molecules 2022, 27, x FOR PEER REVIEW 2 of 17 Dakota and Nebraska, Wyoming, Nebraska, northern and central Colorado and Utah [5]. Flathead Native Americans used the boughs of P. ponderosa in sweat lodges to treat muscular pains, while the Navajo people took a decoction of the needles for coughs and fever [6]. The native range of P. contorta is western North America, where there are three recognized subspecies: P. contorta subsp. latifolia (Engelm.) Critchf., the Rocky Mountain lodgepole pine, is found in the Rocky Mountains from the Yukon, south through Colorado; P. contorta subsp. murrayana (Balf.) Engelm., the Sierra lodgepole pine, found along the Cascade Range from Washington, through Oregon, and into northern California, and the Sierra Nevada Range in California; and P. contorta subsp. contorta, the shore pine ( Figure  2), which ranges along the Pacific coast from southern Alaska, south to northwestern California [7,8]. The Haisla and Hanaksiala Native Americans used smoldering twigs of P. contorta subsp. contorta to alleviate pain and swelling of arthritic or injured joints [6]. The native range of P. contorta is western North America, where there are three recognized subspecies: P. contorta subsp. latifolia (Engelm.) Critchf., the Rocky Mountain lodgepole pine, is found in the Rocky Mountains from the Yukon, south through Colorado; P. contorta subsp. murrayana (Balf.) Engelm., the Sierra lodgepole pine, found along the Cascade Range from Washington, through Oregon, and into northern California, and the Sierra Nevada Range in California; and P. contorta subsp. contorta, the shore pine ( Figure 2), which ranges along the Pacific coast from southern Alaska, south to northwestern California [7,8]. The Haisla and Hanaksiala Native Americans used smoldering twigs of P. contorta subsp. contorta to alleviate pain and swelling of arthritic or injured joints [6].
Pinus flexilis (Figure 3) naturally ranges in the Rocky Mountains of western North America, from southwest Alberta and southeast British Columbia, south through Colorado and New Mexico. It is also found in the mountains of Utah, Idaho, Nevada, and California [9]. The Navajo people used P. flexilis as cough medicine and to reduce fever [6]. As part of our investigation into the essential oil compositions of Pinus species [10,11], we have examined the compositions of the leaf essential oils of P. ponderosa var. ponderosa from La Pine, Oregon, P. contorta subsp. contorta from Ona Beach, Oregon, and Pinus flexilis from Boise, Idaho. As far as we are aware, this is the first report on the leaf oil composition of P. flexilis and the first report on the enantiomeric distributions of terpenoids in these Pinus species. Pinus flexilis (Figure 3) naturally ranges in the Rocky Mountains of western North America, from southwest Alberta and southeast British Columbia, south through Colorado and New Mexico. It is also found in the mountains of Utah, Idaho, Nevada, and California [9]. The Navajo people used P. flexilis as cough medicine and to reduce fever [6]. As part of our investigation into the essential oil compositions of Pinus species [10,11], we have examined the compositions of the leaf essential oils of P. ponderosa var. ponderosa from La Pine, Oregon, P. contorta subsp. contorta from Ona Beach, Oregon, and Pinus flexilis from Boise, Idaho. As far as we are aware, this is the first report on the leaf oil composition of P. flexilis and the first report on the enantiomeric distributions of terpenoids in these Pinus species.

Chemical Composition of Pinus flexilis
Hydrodistillation of the fresh leaves (needles) of P. flexilis gave a colorless essential oil in 0.273% (w/w) yield. There have been no previous reports on P. flexilis essential oil yields. However, essential oils from Pinus species have been obtained in yields ranging from 0.08% (P. rigida) to 2.33% (P. pumila) [14]. The essential oil composition is presented in Table 3. A total of 102 compounds were identified in the leaf essential oil of P. flexilis, accounting for 99.7% of the composition. The major components in the essential oil were α-pinene (37.1%), β-pinene (21.9%), bornyl acetate (12.8%), and camphene (8.5%).   RI calc = Retention index calculated with respect to a homologous series of n-alkanes on a ZB-5ms column. RI db = Reference retention index obtained from the databases [15][16][17][18]. tr = trace (<0.05%). a Identification tentative; the MS is a good match (93% similarity match), but there is no reference RI available.

Enantiomeric Distribution of Terpenoids
The enantiomeric distributions of several terpenoid essential oil components have been determined by chiral gas chromatography-mass spectrometry. The enantiomeric distributions of terpenoid components of P. ponderosa var. ponderosa, P. contorta subsp. contorta, and P. flexilis essential oils are summarized in Table 4.

Gas Chromatography-Mass Spectrometry
Gas chromatographic-mass spectral (GC-MS) analysis of the Pinus essential oils was carried as previously described [31]: Shimadzu GCMS-QP2010 Ultra, ZB-5ms fused silica capillary column (60 m length, 0.25 mm diameter, 0.25 µm film thickness), He carrier gas, 2.0 mL/min flow rate, injection and ion source temperatures 260 • C; GC oven program 50 • C to 260 • C at 2.0 • C/min; 0.1 µL of a 5% (w/v) sample of essential oil in CH 2 Cl 2 injected, split mode, 24.5:1 split ratio. Retention index (RI) values were calculated using a linear equation by Van den Dool and Kratz [32]. Identification of the essential oil components was carried out by comparison of MS fragmentation and comparison of retention indices (RI) with those available in the databases [15][16][17][18]. Representative gas chromatograms of the Pinus species are shown in supplementary Figure S1.

Chiral Gas Chromatography-Mass Spectrometry
Chiral GC-MS of the leaf essential oils was carried out, as reported previously [34]: Shimadzu GCMS-QP2010S, electron impact (EI) mode, electron energy = 70 eV; scan range = 40-400 amu, scan rate = 3.0 scans/s; Restek B-Dex 325 chiral capillary GC column (30 m length × 0.25 mm inside diameter × 0.25 µm film thickness). Oven temperature program: starting temperature = 50 • C, temperature increased 1.5 • C/min to 120 • C, then 2 • C/min to 200 • C, and kept at 200 • C for an additional 5 min; carrier gas was helium, flow rate = 1.8 mL/min. For each essential oil sample, a 3% w/v solution in CH 2 Cl 2 was prepared, and 0.1 µL was injected using a split ratio of 1:45. The enantiomers of the monoterpenoids were identified by comparison of retention times with authentic samples obtained from Sigma-Aldrich (Milwaukee, WI, USA). The enantiomer percentages were determined from peak areas.

Conclusions
The leaf essential oil compositions of P. ponderosa var. ponderosa and P. contorta subsp. contorta from Oregon, USA, have been determined. The enantiomeric distributions of these two Pinus species are reported for the first time. The chemical composition as well as the enantiomeric distribution for P. flexilis from Idaho, USA, are reported for the first time. Both α-pinene and limonene show considerable variation in enantiomeric distribution between and within Pinus species, but (−)-β-pinene is consistently the more dominant enantiomer. This work adds to our knowledge of the essential oil compositions of the genus Pinus. Additional studies on chemical compositions as well as enantiomeric distributions of members of the Pinaceae are underway in our laboratories.