1.    Introduction

Chrysothamnus viscidiflorus Nutt. (Asteraceae) is fairly widespread throughout the Great Basin of North America, ranging from British Columbia and Montana south to New Mexico, Arizona, and eastern California [1]. Shoshoni Native Americans applied a poultice of crushed stems and leaves of C. viscidiflorus to treat rheumatism; the Paiute people took an infusion of crushed leaves to treat colds [2]. There are probably three subspecies in Idaho: C. viscidiflorus subsp. lanceolatus H.M. Hall & Clem., C. viscidiflorus subsp. puberulus H.M. Hall & Clem., and C. viscidiflorus subsp. viscidiflorus (Hook.) Nutt. [3]. Chrysothamnus viscidiflorus subsp. viscidiflorus grows up to 1 m tall. The leaves are glabrous and viscid, 1 to 6 cm long and generally more than 1.5 cm wide (Fig. 1) [1,4]. The viscidiflorus subspecies ranges throughout the Great Basin (Fig. 2) [4].

Previous phytochemical investigations of C. viscidiflorus have shown the plant to be a source of flavonoids [5], labdane diterpenoids [6], benzofuranoids, chromanones [7], guaiane, germacrane, and eudesmane sesquiterpenoids, coumarins, and p-coumaric acid derivatives [8]. To our knowledge, there have been no previous reports on the essential oil composition of C. viscidiflorus. Because C. viscidiflorus was important in Native American ethnopharmacology and the essential oil had not been previously investigated, the purpose of this investigation was to obtain and analyze the essential oil of C. viscidiflorus.

2.    Materials and methods

2.1  Plant Material

Aerial parts of Chrysothamnus viscidiflorus were collected from several plants near Pine, Idaho (43°24ʹ20ʺN, 115°17ʹ33ʺW, 1426 m elevation) on June 28, 2022. The plant was identified by W.N. Setzer. Based on botanical descriptions [1,4] and comparison with herbarium samples from the New York Botanical Garden [9], the plant was identified as C. viscidiflorus subsp. viscidiflorus (Fig. 1).

 Figure 1. Chrysothamnus viscidiflorus subsp. viscidiflorus from southwestern Idaho. Photograph by K. Swor.

A voucher specimen (WNS-Cvv-5686) has been deposited in the University of Alabama in Huntsville herbarium. The fresh plant material from several plants was combined and 108.9 g was hydrodistilled using a Likens-Nickerson apparatus to give 1.221 g of a yellow essential oil.

Figure 2. Range of Chrysothamnus viscidiflorus subsp. viscidiflorus. Adapted from Anderson [4].

2.2  Gas Chromatographic Analysis

The essential oil of C. viscidiflorus subsp. viscidiflorus aerial parts was analyzed by GC-MS using a Shimadzu GCMS-QP2010 Ultra (Shimadzu Scientific Instruments, Columbia, MD, USA) operated in the electron impact (EI) mode (electron energy = 70 eV), scan range = 40–400 atomic mass units, scan rate = 3.0 scans/s, and GC-MS solution software. The GC column was a ZB-5ms fused silica capillary column (60 m length × 0.25 mm inner diameter) with a (5% phenyl)-polymethylsiloxane stationary phase and a film thickness of 0.25 μm (Phenomenex, Torrance, CA, USA). The carrier gas was helium with a column head pressure of 208.3 kPa and flow rate of 2.0 mL/min. Injector temperature was 260 °C and the ion source temperature was 260 °C. The GC oven temperature program was programmed for 50 °C initial temperature, temperature increased at a rate of 2 °C/min to 260 °C, then held at 260 °C for 5 min. A 5% w/v solution of the sample in CH₂Cl₂ was prepared and 0.1 μL was injected with a splitting mode (24.5:1). Retention index (RI) values were calculated using a homologous series of n-alkanes [10]. The essential oil components were identified by comparing their RI values and their MS fragmentation patterns with those reported in the Adams [11], FFNSC3 [12], NIST20 [13], and Satyal [14] databases.

Gas chromatography – flame ionization detection (GC-FID) was carried out using a Shimadzu GC 2010 with FID detector (Shimadzu Scientific Instruments, Columbia, MD, USA) and a ZB-5 GC column (60 m ´ 0.25 mm ´ 0.25 μm film thickness) (Phenomenex, Torrance, CA, USA), using the same operating conditions as above for GC-MS. The percent compositions were determined from raw peak areas without standardization.

The C. viscidiflorus subsp. viscidiflorus essential oil was analyzed by chiral GC-MS using a Shimadzu GCMS-QP2010S (Shimadzu Scientific Instruments, Columbia, MD, USA) instrument fitted with a Restek B-Dex 325 column (30 m ´ 0.25 mm diameter ´ 0.25 μm film thickness) (Restek Corp., Bellefonte, PA, USA). The injector and detector temperatures were 240 °C. Helium was the carrier gas with a column head pressure of 53.6 kPa and a flow rate of 1.00 mL/min. The GC oven was programmed with an initial temperature of 50 °C, held for 5 min, then increased to 100 °C at a rate of 1.0 °C/min, then increased to 220 °C at a rate of 2 °C/min. For each essential oil sample, 0.3 μL of a 5% (w/v) solution in dichloromethane was injected using a splitting mode of 24.0:1. The enantiomeric distributions were determined by comparison of retention times with authentic samples obtained from Sigma-Aldrich (Milwaukee, WI, USA). The enantiomer percentages were determined from raw peak areas.

2.3  Antimicrobial Screening

The essential oil components were screened for antibacterial activity against Staphylococcus aureus (ATCC No. 29213), Streptococcus pneumoniae (ATCC No. 49136), and Streptococcus pyogenes (ATCC No. 19615); and antifungal activity against Cryptococcus neoformans (ATCC No. 32045) using the microbroth dilution technique [15], as previously reported [16]. The individual essential oil components, (–)-β-pinene, (+)-limonene, and myrcene, were obtained from Sigma-Aldrich (St. Louis, MO) and were used as received, without additional purification. Antibacterial and antifungal positive controls were gentamicin and amphotericin B (Sigma-Aldrich, St. Louis, MO), respectively; dimethylsulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO) was the negative control.

 3.    Results and discussion

A yellow essential oil in 1.121% yield (w/w, based on fresh plant material) was obtained by hydrodistillation of the aerial parts of C. viscidiflorus. Gas chromatographic analysis (GC-MS and GC-FID) revealed a total of 66 compounds accounting for 93.6% of the total composition (Table 1). The essential oil was dominated by monoterpene hydrocarbons (82.6%), including the major components β-pinene (41.4%), limonene (18.8%), sabinene (9.1%), myrcene (4.2%), and (E)-β-ocimene (4.2%).

Very few essential oils of Chrysothamnus species have been reported. The essential oil of Chrysothamnus pulchellus Green (syn. Lorandersonia pulchella (A. Gray) Urbatsch, R.P. Robers & Neubig) from Mexico had sesquicineole (22.7%), β-phellandrene (14.9%), (Z)-β-ocimene (9–4%), β-pinene (8.8%), (E)-β-ocimene (6.4%), (E)-β-caryophyllene (3.3%), and δ-cadinene (2.8%) as major components [17]. There are three reports on the essential oil composition of Chrysothamnus nauseosus (Pall. ex Pursh) Britton (syn. Ericameria nauseosa (Pursh) G.L. Nesom & G.I. Baird) [18–20]. The major components were β-phellandrene (1.8-56.5%), β-pinene (0.3-23.3%), limonene (0.7-22.3%), (Z)-β-ocimene (0.0-29.3%), and myrcene (0.0-12.9%).

The enantiomeric distributions of chiral terpenoids in C. viscidiflorus subsp. viscidiflorus were determined by chiral GC-MS (Table 2). In the essential oil of C. viscidiflorus subsp. viscidiflorus, (+)-α-thujene (75.5%) was the dominant enantiomer, (–)-α-pinene (94.5%) predominated over (+)-α-pinene (5.5%), (+)-sabinene was the exclusive enantiomer, and (–)-β-pinene (99.8%) was the major enantiomer. Interestingly, (+)-limonene (92.3%) dominated while (–)-β-phellandrene (95.6%) was the dominant enantiomer. (–)-α-Thujone was the exclusive enantiomer observed. Both (+)-cis-sabinene hydrate (86.1%) and (+)-trans-sabinene hydrate (90.5%) were the major enantiomers, (+)-terpinen-4-ol (71.9%) was the major enantiomer while (–)-α-terpineol (87.4%) was dominant. (–)-Germacrene D was the major enantiomer. These enantiomeric distributions are very different from

Table 1. Chemical composition (percentages based on peak areas) of the essential oil from the aerial parts of Chrysothamnus viscidiflorus subsp. viscidiflorus.

those observed for Chrysothamnus nauseosus (syn Ericameria nauseosa) where (–)-α-thujene, (–)-sabinene, (–)-limonene, (–)-cis-sabinene hydrate, (–)-trans-sabinene hydrate, and (–)-terpinen-4-ol were the dominant enantiomers [20].

The major components found in C. viscidiflorus essential oil have demonstrated biological activities consistent with the traditional Native American use of the plant to treat rheumatism. β-Pinene has demonstrated both antinociceptive [21] and anti-inflammatory activities [22]. Limonene has shown both antinociceptive and anti-inflammatory activities [23, 24]. Importantly, (+)-limonene has shown antinociceptive activity [25] and anti-inflammatory activity [26,27]. Sabinene has shown anti-inflammatory effects [28,29]. Myrcene demonstrated antinociceptive activity [30, 31] as well as anti-inflammatory activity [28].

Numerous essential oil components have been identified that exhibit inhibitory activity against respiratory tract infections and pathogens [32, 33]. The commercially-available essential oil components, (–)-β-pinene, (+)-limonene, and myrcene, have been screened for antimicrobial activity against the respiratory pathogens Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, and Cryptococcus neoformans (Table 3).

Table 2. Chemical compositions (% enantiomer) of the essential oil from the aerial parts of Chrysothamnus viscidiflorus subsp. viscidiflorus.

Table 3. Antimicrobial activity (MIC, μg/mL) of Chrysothamnus viscidiflorus subsp. viscidiflorus major essential oil components^a

Both S. aureus and S. pneumoniae were particularly susceptible to the essential oil components. Furthermore, β-pinene and (+)-limonene have shown good anti-Klebsiella pneumoniae (MIC 8-64 μg/mL) [34]; β-pinene, (+)-limonene, myrcene, and sabinene have shown good activity against Mycobacterium tuberculosis (MIC 10.4, 25.0, 25.0, and 33.3 μg/mL, respectively) [35]. The antimicrobial activities of the major components of C. viscidiflorus essential oil indicate potential effectiveness against respiratory infections and may account for the Native American use of C. viscidiflorus to treat colds.

4.    Conclusions

As far as we are aware, this is the first report on the essential oil characterization of C. viscidiflorus, and adds to our understanding of the volatile phytochemistry of Chrysothamnus. The biological activities of the major components are consistent with the traditional Native American use of C. viscidiflorus to treat rheumatism and colds. Clearly, additional research is needed on other species of Chrysothamnus as well as infraspecific taxa of Chrysothamnus species in order to more fully define the volatile phytochemistry of the genus in terms of common components or marker compounds or to provide chemical profiles to differentiate species and subspecies.

Authors’ contributions

Conceptualization, W.N.S.; Methodology, P.S., N.S.D., and W.N.S.; Software, P.S.; Validation, W.N.S., Formal Analysis, P.S., N.S.D., A.P., and W.N.S.; Investigation, P.S., N.S.D., A.P., K.S., and W.N.S.; Resources, P.S. and W.N.S.; Data Curation, W.N.S.; Writing – Original Draft Preparation, W.N.S.; Writing, Review & Editing, P.S., N.S.D., A.P., K.S., and W.N.S.; Project Administration, W.N.S.

Acknowledgements

This work was carried out as part of the activities of the Aromatic Plant Research Center (APRC, https://aromaticplant.org/).

Funding

This research received no specific grant from any funding agency.

Conflicts of interest

The authors declare no conflict of interest.

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