Abstract
The essential oils from the leaves and twigs of Juniperus horizontalis and Juniperus scopulorum, growing in southwestern Idaho, were obtained by hydrodistillation and analyzed by gas chromatography–mass spectrometry (GC-MS) and gas chromatography–flame ionization detection (GC(FID)). The major components in the essential oil of J horizontalis were α-pinene, 16.9%, predominantly ( + )-α-pinene; sabinene, 37.1%, exclusively ( + )-sabinene; myrcene, 5.0%; and terpinene-4-ol, predominantly ( + )-terpinen-4-ol. The essential oil of J scopulorum was rich in sabinene, 29.8%, exclusively ( + )-sabinene; terpinene-4-ol, 13.2%, nearly racemic; α-elemol, 9.7%; and 8α-acetoxyelemol, 6.4%. In addition, 12 commercial Juniperus communis fruit essential oils were analyzed by chiral GC-MS. α-Pinene showed considerable variation in enantiomeric distribution, depending on geographical location. The dominant enantiomer of sabinene in J communis essential oils was ( + )-sabinene.
Introduction
Juniperus L. is the largest genus in terms of number of species in the Cupressaceae. World Flora Online currently lists 75 species of Juniperus. 1 Eighteen species of Juniperus are known to occur in North America. 2 Several species of Juniperus have commercial importance, including Juniperus communis L. (common juniper), used to flavor gin, Juniperus oxycedrus L. (cade juniper), the source of antiparasitic oil of Cade, and Juniperus virginiana L. (eastern red cedar), used as an insect repellent. 3 Many Juniperus species are used in local traditional medicine and several are important sources of essential oils. 4
Juniperus horizontalis Moench (creeping juniper) ranges naturally across Canada and south into northern United States (eg Montana, Wyoming, North and South Dakota, Minnesota, Wisconsin, Michigan, New York Vermont, and Maine). 2 In Cheyenne traditional medicine, the plant was used as a remedy for colds, coughs, and fevers. 5 The leaf essential oil of J horizontalis, cultivated in Tehran, Iran, and rich in limonene and sabinene, showed good antibacterial activity against Staphylococcus aureus, Staphylococcus epidermidis, Bacillus anthracis, Bacillus subtilis, Listeria monocytogenes, and Escherichia coli with minimum inhibitory concentration (MIC) values ≤ 64 μg/mL. 6 Likewise, a p-cymene and linalool-rich J horizontalis leaf essential oil from Van, Turkey, showed good antibacterial activity against Enterococcus faecalis and Salmonella typhimurium. 7
Juniperus scopulorum Sarg. (Rocky Mountain juniper) is found in western North America, including British Columbia, Washington, Oregon, Nevada, through Idaho, Montana, Wyoming, Utah Colorado, Arizona, and New Mexico, and east to North and South Dakota and western Nebraska. 2 Several Native North American tribes used J scopulorum in their traditional medicine to treat coughs, colds, and fevers (Cheyenne, Flathead, Kutenai, Navajo, Nez Perce, Sioux, Thompson), arthritis and rheumatism (Blackfoot, Montana, Navajo, Okanagan, Swinomish, Thompson). 5 The essential oil of J scopulorum, cultivated in Hoyo, Argentina, was dominated by sabinene (86.5%) and demonstrated antifungal effectiveness against Fusarium verticillioides, Aspergillus flavus, Aspergillus parasiticus, and Rhodotorula sp. 8
The purpose of this work was to examine the essential oil compositions of J horizontalis and J scopulorum growing in southwestern Idaho, with particular emphasis on the enantiomeric distribution of monoterpenoid components. A survey of enantiomeric monoterpenoid distributions in Juniperus is also presented for comparison.
Results and Discussion
J horizontalis and J scopulorum Essential Oil Compositions
The essential oils from the leaves and twigs of J horizontalis and J scopulorum were obtained in 0.318% and 0.543% yield, respectively, as colorless essential oils. The chemical compositions of the essential oils are compiled in Table 1. The compositions are qualitatively similar to those previously reported for J horizontalis and J scopulorum from Wyoming. 9 Key differences in the compositions of J horizontalis are the large concentration of α-pinene in this work (16.9%) compared to those found in the Wyoming collection (2.4%-6.6%) and the lower concentration of sabinene (37.1%) found in the Idaho sample compared to those found in the Wyoming collection (56.6%-61.0%). Pregeijerene B was found in 2.3% to 4.4% in the Wyoming samples, but was not detected in the Idaho sample.
Chemical Compositions of the Essential Oils From the Leaves and Twigs of Juniperus horizontalis and Juniperus scopulorum Growing in Southwestern Idaho.
The Idaho J scopulorum essential oil had higher concentrations of terpinen-4-ol (13.2%), α-elemol (9.7%), and 8α-acetoxyelemol (6.4%) compared to the essential oils from Wyoming (3.3%-5.9%, 0.0%-5.6%, and 0.0%-4.3%, respectively). The limonene concentration was lower in the Idaho sample (2.0%) than the samples from Wyoming (2.5%-22.0%).
Monoterpenoid Enantiomeric Distributions
A chiral gas chromatography–mass spectrometry (GC-MS) analysis was carried out on J horizontalis and J scopulorum essential oils to assess the enantiomeric distributions of monoterpenoids (Table 2). Additionally, 2 commercial J communis fruit essential oils from Nepal and 10 samples from Albania were analyzed for comparison. Enantiomeric distributions of monoterpenoids in J communis, J oxycedrus, and Juniperus phoenicea reported in the literature14-16 are also included in Table 2 for comparison.
Enantiomeric Distribution [%( + ):%(–)] of Monoterpenoids in Juniperus Essential Oils.
Note: aJuniperus essential oil from Idaho.
Commercial Juniperus communis fruit essential oils from the Aromatic Plant Research Center (APRC) collection.
The reported percentages to not add up to 100%.
The monoterpene hydrocarbons α-thujene, sabinene, β-pinene, and δ-3-carene were found to occur in both J horizontalis and J scopulorum as their pure ( + ) enantiomers. ( + )-α-Thujene dominated most of the J communis fruit essential oils examined with the exception of 2 samples from Albania (Table 2, samples #2 and #3). Pure ( + )-sabinene and ( + )-δ-3-carene were found in J communis leaf essential oils from Poland. 17 ( + )-Sabinene dominated the fruit essential oils of commercial J communis (Table 2), but was the minor enantiomer in J oxycedrus and J phoenicea leaf essential oils. 16 ( + )-δ-3-Carene also dominates the essential oils of J oxycedrus, J phoenicea, and J communis fruit essential oils. In contrast to J horizontalis and J scopulorum essential oils, pure (–)-β-pinene was found in J communis leaf oil, 17 which is consistent with J communis fruit essential oils (Table 2).
The major α-pinene enantiomer in J horizontalis and J scopulorum essential oils was ( + )-α-pinene, consistent with that observed in J communis leaf essential oil15,17,18 J oxycedrus, 14 and J phoenicea 16 leaf essential oils. Interestingly, commercial J communis fruit essential oils from Nepal showed ( + )-α-pinene predominating, but those from Albania had (–)-α-pinene as the major enantiomer.
( + )-Limonene generally seems to predominate in Juniperus essential oils, but there are some exceptions in J communis leaf 17 and fruit (Albania samples #2 and #3) essential oils. ( + )-Linalool predominated in the leaf essential oils of J horizontalis, J scopulorum, and J phoenicea, whereas (–)-linalool predominated in J oxycedrus leaf oil and the fruit essential oils of J communis. There was a slight excess of ( + )-terpinen-4-ol in J horizontalis and J scopulorum essential oils, whereas a slight excess of (–)-terpinen-4-ol was observed in J communis, J oxycedrus, and J phoenicea leaf essential oils. Commercial J communis fruit essential oils also showed a slight excess of ( + )-terpinen-4-ol with the exception of two samples from Albania (#2 and #3).
Conclusions
The essential oil compositions of J horizontalis and J scopulorum growing in Idaho showed qualitative similarities to those Juniperus essential oils growing in Wyoming, but with some quantitative differences. The enantiomeric distributions of the major components, α-pinene, sabinene, and terpinene-4-ol, were comparable to the enantiomeric distributions observed in commercial J communis fruit essential oils.
Materials and Methods
Plant Material
Samples of J horizontalis and J scopulorum were obtained from the Idaho Botanical Garden (43°36′04″N, 116°09′35″W, 862 m elevation) on July 29, 2021. The plants were identified by Daniel Murphy, Collections Curator, Idaho Botanical Garden. Voucher specimens (WNS22192758 and WNS22114504) have been deposited in the herbarium of the University of Alabama in Huntsville (HALA). For J horizontalis, fresh leaves and twigs (167.80 g) were hydrodistilled using a Likens-Nickerson apparatus with continuous extraction with dichloromethane for 3 h to give a colorless essential oil (533.8 mg). Fresh leaves and twigs of J scopulorum (93.90 g) were hydrodistilled as above to give a colorless essential oil (510.1 mg).
Commercial Essential Oils from APRC
Commercial samples of J communis fruit essential oils from Nepal and from Albania were obtained from the Aromatic Plant Research Center (APRC) collection and analyzed as received.
GC-MS
The essential oils of J horizontalis and J scopulorum were analyzed by GC-MS as reported previously 19 : Shimadzu GCMS-QP2010 Ultra with a ZB-5 ms GC column (60 m length, 0.25 mm diameter, 0.25 μm film thickness), injector and detector temperatures 260 °C, He carrier gas with column head pressure of 208 kPa and flow rate of 2.00 mL/min, GC oven temperature program 50 °C to 260 °C (2 °C/min), and 1.0 μL injection of 5% solution of each essential oil in dichloromethane (split mode, 24:1). The retention indices (RIs) were determined using a series of reference n-alkanes. The compounds listed in Table 1 were identified by comparing the mass spectrum fragmentation data and calculated RIs with those in the databases.10-13
GC(FID)
The essential oils of J horizontalis and J scopulorum were analyzed by gas chromatography–flame ionization detection (GC(FID)) as reported previously 20 : Shimadzu GC 2010 with FID detector, ZB-5 GC column, same operating conditions as above for GC-MS. The percent compositions were determined from raw peak areas without standardization.
Chiral GC-MS
The J horizontalis, J scopulorum, and J communis essential oils were analyzed by chiral GC-MS as previously reported 20 : Shimadzu GCMS-QP2010S instrument, Restek B-Dex 325 column (30 m length, 0.25 mm diameter, 0.25 μm film thickness), injector and detector temperatures 240 °C, He carrier gas with column head pressure of 53.8 kPa and flow rate of 1.00 mL/min, GC oven temperature program 50 °C, held for 5 min, increased 1.0 °C/min to 100 °C, then increased 2.0 °C/min to 220 °C; and 0.3 μL injection of 5% solution of essential oil samples in dichloromethane (split mode, 24:1) was injected. The enantiomeric distributions were determined by comparison of retention times with authentic samples obtained from Sigma-Aldrich (Milwaukee, WI, USA). The relative enantiomer percentages were calculated from peak areas.
Footnotes
Acknowledgments
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