Next Article in Journal
The Comparative Root System Architecture of Declining and Non-Declining Trees in Two Apple Orchards in New York
Next Article in Special Issue
Eucalyptus camaldulensis Dehnh Leaf Essential Oil from Palestine Exhibits Antimicrobial and Antioxidant Activity but No Effect on Porcine Pancreatic Lipase and α-Amylase
Previous Article in Journal
Plant-CNN-ViT: Plant Classification with Ensemble of Convolutional Neural Networks and Vision Transformer
Previous Article in Special Issue
Variation in the Chemical Composition of Endemic Specimens of Hedychium coronarium J. Koenig from the Amazon and In Silico Investigation of the ADME/Tox Properties of the Major Compounds
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Plants
Volume 12
Issue 14
10.3390/plants12142643
plants-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Composition and Biological Activity of the Essential Oils from Wild Horsemint, Yarrow, and Yampah from Subalpine Meadows in Southwestern Montana: Immunomodulatory Activity of Dillapiole

by
Igor A. Schepetkin
1,
Gulmira Özek
2,
Temel Özek
2,
Liliya N. Kirpotina
1,
Robyn A. Klein
3,
Andrei I. Khlebnikov
4 and
Mark T. Quinn
1,*
1
Department of Microbiology and Cell Biology, Montana State University, Bozeman, MT 59717, USA
2
Department of Pharmacognosy, Faculty of Pharmacy, Anadolu University, Eskisehir 26470, Turkey
3
Department of Plant Sciences and Plant Pathology, Montana State University, Bozeman, MT 59717, USA
4
Kizhner Research Center, Tomsk Polytechnic University, 634050 Tomsk, Russia
*
Author to whom correspondence should be addressed.
Plants 2023, 12(14), 2643; https://doi.org/10.3390/plants12142643
Submission received: 23 June 2023 / Revised: 8 July 2023 / Accepted: 12 July 2023 / Published: 14 July 2023
(This article belongs to the Special Issue Advances in Essential Oils from Medicinal Plants)
Browse Figures
Versions Notes

Abstract

:
Agastache urticifolia (Benth.) Kuntze (horsemint), Achillea millefolium L. (yarrow), and Perideridia gairdneri (Hook. & Arn.) Mathias (yampah) are native, culturally important plants that grow in the subalpine meadows of Montana. Analysis of the composition of essential oils extracted from these plants showed that the main components of essential oils obtained from flowers and leaves of A. urticifolia (designated as AUF/AUL) were menthone (2.7/25.7%), isomenthone (2.6/29.1%), pulegone (78.9/28.8%), and limonene (4.2/6.2%), whereas essential oils obtained from the inflorescence of A. millefolium (designated as AMI) were high in α-thujone (17.1%) and β-thujone (14.9%), 1,8-cineole (17.0%), camphor (13.0%), sabinene (7.0%), guaia-3,9-dien-11-ol (3.2%), and terpinen-4-ol (2.5%). Essential oils obtained from the inflorescence of P. gairdneri (designated as PGI) contained high amounts of dillapiole (30.3%), p-cymen-8-ol (14.1%), terpinolene (12.0%), 4-hydroxy-4-methyl-cyclohex-2-enone (6.2%), and γ-terpinene (2.4%). Evaluation of their immunomodulatory activity demonstrated that essential oils extracted from all of these plants could activate human neutrophils with varying efficacy. Analysis of individual components showed that dillapiole activated human neutrophil intracellular Ca2+ flux ([Ca2+]i) (EC50 = 19.3 ± 1.4 μM), while α-thujone, β-thujone, menthone, isomenthone, and pulegone were inactive. Since dillapiole activated neutrophils, we also evaluated if it was able to down-regulate neutrophil responses to subsequent agonist activation and found that pretreatment with dillapiole inhibited neutrophil activation by the chemoattractant fMLF (IC50 = 34.3 ± 2.1 μM). Pretreatment with P. gairdneri essential oil or dillapiole also inhibited neutrophil chemotaxis induced by fMLF, suggesting these treatments could down-regulate human neutrophil responses to inflammatory chemoattractants. Thus, dillapiole may be a novel modulator of human neutrophil function.

1. Introduction

Agastache urticifolia (Benth.) Kuntze (horsemint), Achillea millefolium L. (yarrow), and Perideridia gairdneri (Hook. & Arn.) Mathias (yampah) are native, culturally important plants that can be found in the subalpine meadows of Montana. The leaves of all three species are strongly aromatic, especially when crushed. Analysis of ethnobotanical reports recorded in the Native American Ethnobotany database indicated that a decoction of A. urticifolia leaves was used for rheumatism and rhinitis [1]. Likewise, the Cheyenne people have used roots, stems, and leaves of P. gairdneri for different medicinal purposes [1], Blackfoot people have used roots of P. gairdneri to draw inflammation from swellings and as a nostril wash for rhinitis [1], and the Cherokee people have used A. millefolium for treating bloody hemorrhoids, bloody urine, and bowel complaints [1,2]. Achillea millefolium L. has also been widely used as a wound-healing agent and to treat gastrointestinal complaints [2,3], and infusions of the herb have been used as a treatment for fever [1,2]. Lastly, yarrow extract has been reported to exhibit spasmogenic effects in murine and human gastric antrum [4].
Essential oils are one of the bioactive components present in medicinal plant extracts and are currently recognized for their medicinal properties. For example, essential oils have been reported to exhibit immunomodulatory and anti-inflammatory effects [5,6,7]. Some of the earliest innate immune cell types that respond to the presence of pathogenic organisms are neutrophils [8]. Neutrophils are recruited to inflammatory sites of injury or infection by a variety of factors, including N-formyl-Met-Leu-Phe (fMLF), a bacterial or mitochondria-derived peptide, and chemokines [9]. Chemokines activate neutrophil G-protein coupled receptors (GPCRs) and stimulate chemotaxis, as well as the production of inflammatory mediators, including reactive oxygen species, cytokines, and proteases [9]. We recently found that essential oils from Populus balsamifera, Grindelia squarrosa, Rhododendron albiflorum, Juniperus, and Artemisia spp. can modulate human neutrophil functions [10,11,12,13,14]. Likewise, essential oils of A. millefolium have been clinically recognized as a treatment for wounds and other skin-inflammatory conditions [15]. In contrast, not much is known about the therapeutic properties of the essential oils from the other two plant species studied in this research, A. urticifolia, and P. gairdneri [16,17,18].
Based on the reported therapeutic effects of extracts of horsemint, yarrow, and yampah, this work aimed to evaluate the composition and innate immunomodulatory activity of essential oils from these plants. Essential oils were isolated from these plants and analyzed for their chemical compositions and innate immunomodulatory activities. Interestingly, these essential oils exhibited immunomodulatory activity and inhibited intracellular Ca2+ mobilization ([Ca2+]i) in activated human neutrophils. Furthermore, dillapiole, which was present at high levels in essential oils of P. gairdneri also inhibited human neutrophil functional responses. Thus, dillapiole is likely one of the main active components in these essential oils. Since neutrophils play an important role in inflammation, these data suggest that dillapiole could be considered in the development of new anti-inflammatory agents.

2. Results and Discussion

2.1. Plant Material

Plant material was collected in July 2021 near Bozeman, MT, USA (Table 1). The plant material was air-dried at room temperature for 7–10 days in the dark before hydrodistillation. Botanical identification of the plant material was performed at Montana State University, Bozeman, MT, USA.

2.2. Essential Oil Composition

The distillation yields (v/w) of essential oils obtained from the three plant species were 0.2 to 1.9% (Table 1). Simultaneous GC-FID and GC/MS were used to evaluate the chemical composition of these essential oils (Table 2), and a summary of their chemical composition is shown in Table 3. A total of 55/44, 65, and 43 compounds, accounting for 97.4%/98.5%, 98.7%, and 80.0% of the essential oils from flowers and leaves of A. urticifolia (designated as AUF/AUL), inflorescences of A. millefolium (designated as AMI), and inflorescences of P. gairdneri (designated as PGI) respectively, were identified and quantified.
Major compounds of AUF were pulegone (78.9%), limonene (4.2%), menthone (2.7%), isomenthone (2.6%), piperitenone (1.6%), and trans-p-mentha-8-methylthio-3-one (1.1%). Similarly, the major compounds of AUL were isomenthone (29.1%), pulegone (28.8%), menthone (25.7%), and limonene (6.2%). Thus, these results are consistent with previous findings suggesting that A. urticifolia essential oils are primarily composed of limonene, menthone, and pulegone, although isomenthone was found as a minor compound in native Oregon and Utah, USA plant populations [17]. Biological characteristics and dynamics of essential oil content of A. urticifolia in Moldova have also been reported [16]. In the essential oil of A. urticifolia from Moldova, 17 compounds were identified, with the basic ones being phenylpropanoids, estragole (41.1%) and methyl eugenol (5.1%), as well as monoterpenes, pulegone (20.4%), limonene (15.3%), isomenthone (12.0%), and menthone (1.7%) [19]. The essential oils of A. urticifolia cultivated in the Middle Ural (Russia) contained a high abundance of monoterpenes, including menthone (23.0%), isomenthone (9.9%), and pulegone (5.6%). Sesquiterpenes were also present, including spathulenol (5.4%), α-cadinol (1.8%), and caryophyllene-4(12)8(13)-diene-5α-ol (1.5%) [20]. In general, a literature survey and comparative evaluation of Agastache profiles revealed that the composition of essential oils is relatively variable, but with phenylpropanoids and oxygenated monoterpenes predominating. Namely, estragole (syn. methylchavicol), methyleugenol, and (E)-anethole are usually the most abundant constituents. Other chemotypes of Agastache are rich in menthone, isomenthone, pulegone, and limonene [18].
We also found sulfur-containing monoterpenes [trans-p-mentha-8-methyl-thio-3-one (1.1% and 0.8%) and cis-p-mentha-8-methyl-thio-3-one (0.5% and 0.4%)], in flower and leaf essential oils of A. urticifolia. The thio-compounds are perhaps responsible for the characteristic scent of these oils. Notably, this is the first report of thio-monoterpenes in Agastache essential oils. Previously, different representatives of the Lamiaceae family, e.g., Agathosma and Calamintha species, have been reported to contain sulfur-monoterpenes [21,22]. Likewise, a sulfur derivative of pulegone was reported to be a major constituent of buchu (Agathosma betulina) essential oils, as well as methylthio- and acetylthio-derivatives of pulegone and other p-menthane constituents [23].
Essential oils from inflorescences of A. millefolium (AMI) contained high amounts of α-thujone (17.1%), β-thujone (14.9%), 1,8-cineole (17.0), camphor (13.0%), sabinene (7.0%), guaia-3,9-dien-11-ol (3.2%), and terpinen-4-ol (2.5%). High amounts of α-thujone and β-thujone were previously reported in essential oils extracted from A. millefolium collected in Europe and Chile [24,25,26]. High levels of oxygenated monoterpenes (53.9–76.1%), mainly α- and β-thujone (up to 26.8%), camphor (up to 24.5%), 1,8-cineole (up to 20.3%) and artemisia ketone (up to 10.1%), were identified in essential oils of A. millefolium from France, Belgium, Spain, Italy, Russia, and Armenia. The content of chamazulene in these samples was only 0–0.8%. In the literature, relatively high amounts of the above-mentioned terpenes were reported to be typical for hexaploid yarrow plants. Additionally, 1,8-cineole and camphor were primary components of A. millefolium essential oils from Serbia, France, and Eastern Turkey [27,28,29]. According to “Millefolii Herba” from the European Pharmacopoeia, the content of proazulenes expressed as chamazulene should be a minimum of 0.02% (dried drug) in yarrow [30]. However, proazulenes were not detected in our samples. A literature survey revealed that yarrow essential oils from Chile were rich in β-thujone (96.2%), while other compounds identified were α-thujone, 1,8-cineole, p-cymene, and 4-terpineol (all < 1.0%) [26]. Significant variations in essential oil content and composition in commercial samples of yarrow were reported by Raal et al. [31], with the most important components of yarrow essential oils being chamazulene (0.8–44.3%), β-pinene (tr—23.3%), sabinene (0–16.5%), bornyl acetate (tr—15.8%), (E)-β-caryophyllene (2.5–14.3%), (E)-nerolidol (tr—9.6%), 1,8-cineole (trace—9.6%), and germacrene D (0.2–7.8%). Chemotypes containing chamazulene, chamazulene + bornyl acetate, chamazulene + β-pinene + (E)-β-caryophyllene, sabinene + 1,8-cineole, and β-pinene + α-terpinyl acetate have also been reported [31]. Such variation in the composition of yarrow essential oils may be due to various factors related to chemotype, ecotype, phenophases, altitude, and variations in environmental conditions, such as temperature, photoperiod, relative humidity, and irradiance. Moreover, genetic background may be the factor responsible for affecting the chemistry of secondary metabolites of these plants. The chemical composition also varies strongly due to different ploidy (di-, tetra-, hexa-, octoploid), and frequent hybridization within this group but also with different Achillea species. For example, the major constituents of tetraploid A. millefolium plants include chamazulene, β-pinene, and caryophyllene, while octoploid plants contain ~80% oxygen-containing monoterpenes, with linalool as the major constituent [32].
Essential oils from inflorescences of P. gairdneri (PGI) contained high amounts of dillapiole (30.3%), p-cymen-8-ol (14.1%), terpinolene (12.0%), 4-hydroxy-4-methyl-cyclohex-2-enone (6.2%), and γ-terpinene (2.4%). This is the first report on the composition of essential oils extracted from P. gairdneri. The high content of 4-hydroxy-4-methyl-cyclohex-2-enone is interesting since this compound was reported in flower essential oils from Hypericum perforatum [33] and Ledum palustre var. nipponicum [34] and can be metabolized to pulegone [35].

2.3. Effect of Essential Oils and Selected Component Compounds on Neutrophil Ca2+ Influx

We evaluated the essential oils for their immunomodulatory effects on human neutrophils. In particular, the effects of the essential oils on intracellular Ca2+ flux ([Ca2+]i) were assessed, since [Ca2+]i is an important signal during neutrophil activation. Treatment of neutrophils with essential oils from A. urticifolia (AUF and AUL), A. millefolium (AMI), and P. gairdneri (PGI) activated human neutrophils, resulting in increased [Ca2+]i, with EC50 values ranging from 28.5 to 43.5 µg/mL (Table 4). Pre-incubation of neutrophils with the most active of these essential oil samples (PGI) inhibited the subsequent neutrophil [Ca2+]i response to the chemoattractant fMLF with an IC50 of 4.3 µg/mL (Figure 1), while other essential oil samples had lower inhibitory activity (Table 4).
Previously, several of the compounds that are present in the essential oils evaluated here were shown to have no activation and (or) inhibitory effects on human neutrophil [Ca2+]i, including camphor, 1,8-cineole, p-cymene, p-cymen-8-ol, elemicine, hexanal, limonene, linalool, myrcene, (E/Z)-β-ocimene, β-phellandrene, α-pinene, β-pinene, piperitenone, sabinene, spathulenol, α-terpinene, terpinen-4-ol, and terpinolene [12,13,36,37]. In contrast, (±)-bornyl acetate, (−)-borneol, germacrene D, and nerolidol were found previously to inhibit agonist-induced activation of human neutrophils [10,11,12,13,33]. Thus, the inhibitory effects of AMI essential oils on human neutrophil Ca2+ flux are likely due to the presence of bornyl acetate, (−)-borneol, germacrene D, and nerolidol, whereas germacrene D and some other minor components could be responsible for the biological activity of AUF/AUL.
We evaluated the activity of additional constituent compounds from our essential oil samples that have not been evaluated previously in human neutrophils, including α-thujene, α/β-thujone, menthone, isomenthone, pulegone, and dillapiole. The results showed that only dillapiole, a major component of PGI, was active (Table 4, Figure 2). Indeed, dillapiole effectively activated human neutrophil [Ca2+]i, with an EC50 of 19.3 μM. Note that the addition of control fMLF caused a rapid increase in [Ca2+]i that peaked by 1 min and gradually declined to basal values, reflecting the rapid clearance of Ca2+ from the cytosol. The time course of [Ca2+]i induced by dillapiole is different from that observed in fMLF-stimulated cells and likely reflects activation of a different pathway in neutrophils. Further studies will be important to define the specific receptor or target of dillapiole.
Since dillapiole directly activated neutrophil [Ca2+]i, albeit with low efficacy, it is possible that this compound could contribute to receptor desensitization and/or intracellular Ca2+ store depletion. Indeed, pre-incubation of neutrophils with dillapiole inhibited subsequent fMLF-induced [Ca2+]i, with an IC50 of 13.9 μM (Figure 3). Note that essential oils from A. urticifolia contained predominantly the (S)-(−) enantiomer of pulegone [17]. Here, we evaluated the activity of commercially available (R)-(+)-pulegone. Thus, we cannot exclude an activity of (S)-(−)-pulegone in human neutrophils since that isomer is not commercially available.

2.4. Effect of PGI Essential Oil and Dillapiole on Neutrophil Chemotaxis

Various essential oils and their components have been reported to inhibit neutrophil chemotaxis [38,39,40]. In the present study, the effects of PGI and its major component compound dillapiole (30.3%) on human neutrophil chemotaxis were evaluated. Pretreatment with PGI dose-dependently inhibited fMLF-induced neutrophil chemotaxis (IC50 = 10.5 ± 3.3 μg/mL) (Figure 4A). Likewise, pretreatment with dillapiole also inhibited fMLF-induced human neutrophil chemotaxis (IC50 = 91.3 ± 22.2 µM) (Figure 4B). Because [Ca2+]i is involved in neutrophil chemotaxis [9], the inhibitory effect of dillapiole on neutrophil chemotaxis is consistent with its primary effect on Ca2+ flux.
To evaluate the toxicity of essential oils from P. gairdneri and dillapiole, we incubated neutrophils with PGI (up to 100 µg/mL) and pure dillapiole at various concentrations (up to 100 µM) and evaluated cell viability. As shown in Figure 5, PGI had little to no cytotoxicity during a 30-min incubation period but was cytotoxic during a 90-min incubation period at concentrations of 50 and 100 µg/mL. Note that the inhibitory effects of PGI on neutrophil functional activity were found at much lower concentrations (<10 µg/mL). Dillapiole had no neutrophil cytotoxicity at all concentrations and times tested (Figure 5).
This is the first report on the inhibitory effects of dillapiole on human neutrophil activation (Table 4). Dillapiole (see chemical structure in Figure 6) is a phenylpropanoid found in abundance in essential oils from Piper species, Deverra triradiata Hochst. ex Boiss, and in the early developmental stages of dill (Anethum graveolens L.) [41,42,43,44]. It has been reported to exhibit bactericidal [45], fungicidal [46], antileishmanial [47], and gastroprotective activity [48]. Interestingly, dillapiole has also been reported to have anti-inflammatory activity in a carrageenan-induced rat paw edema model [49] and broad cytotoxic effects against a variety of tumor cells [50].
To further characterize dillapiole, we calculated the most important physico-chemical and ADME parameters of this compound using SwissADME [51] and found that it would be predicted to permeate the blood–brain barrier (BBB) (Table 5). According to the radar plot of the main characteristics, the ADME data for dillapiole predict that it would also exhibit high bioavailability (Figure 7).
One of the issues noted for this research is that DMSO was required for solubilizing our samples, which may be problematic in the development of new therapeutics. However, recent studies by Carneiro et al. [52] indicate that nanoemulsions and nanostructured lipid carriers could be used for the delivery of essential oils and dillapiole. Thus, nanocarriers loaded with dillapiole could potentially represent an interesting strategy for developing this compound for the treatment of inflammation.

3. Materials and Methods

3.1. Materials

Dimethyl sulfoxide (DMSO), fMLF, Histopaque 1077, (−)-α-thujone, α/β-thujone, and dillapiole were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Menthone, isomenthone, and pulegone were from Toronto Research Chemicals (North York, ON, Canada). n-Hexane was purchased from Merck (Darmstadt, Germany). Fluo-4AM was purchased from Invitrogen (Carlsbad, CA, USA). Hanks’ balanced salt solution (HBSS; 0.137 M NaCl, 5.4 mM KCl, 0.25 mM Na2HPO4, 0.44 mM KH2PO4, 4.2 mM NaHCO3, 5.56 mM glucose, and 10 mM HEPES, pH 7.4) was purchased from Life Technologies (Grand Island, NY, USA). HBSS without Ca2+ and Mg2+ is designated as HBSS–; HBSS containing 1.3 mM CaCl2 and 1.0 mM MgSO4 is designated as HBSS+.

3.2. Essential Oil Extraction

Essential oils were obtained from the air-dried plant material by hydrodistillation using a Clevenger-type apparatus, as previously described [37]. We used conditions accepted by the European Pharmacopoeia (European Directorate for the Quality of Medicines, Council of Europe, Strasbourg, France, 2014) to avoid artifacts. Yields of essential oils were calculated based on the amount of air-dried plant material used.

3.3. Gas Chromatography (GC-FID) and Gas Chromatography-Mass Spectrometry (GC-MS) Analysis

Stock solutions of the essential oils were prepared in n-hexane (10% w/v), and GC-MS analysis was performed with an Agilent 5975 GC-MSD system (Agilent Technologies, Santa Clara, CA, USA), as reported previously [53]. An Agilent Innowax FSC column (60 m × 0.25 mm, 0.25 μm film thickness) was used with He as the carrier gas (0.8 mL/min). The GC oven temperature was kept at 60 °C for 10 min, increased to 220 °C at a rate of 4 °C/min, kept constant at 220 °C for 10 min, and then increased to 240 °C at a rate of 1 °C/min. The split ratio was adjusted to 40:1, and the injector temperature was 250 °C. MS spectra were monitored at 70 eV with a mass range of 35 to 450 m/z. GC analysis was performed using an Agilent 6890N GC system. To obtain the same elution order as with GC-MS, the line was split for FID and MS detectors, and a single injection was performed using the same column and operational conditions. The flame ionization detector (FID) temperature was 300 °C. The essential oil components were identified by co-injection with standards (whenever possible), which were purchased from commercial sources or isolated from natural sources. In addition, compound identities were confirmed by comparison of their mass spectra with those in the Wiley GC/MS Library (Wiley, NY, USA), MassFinder software 4.0 (Dr. Hochmuth Scientific Consulting, Hamburg, Germany), Adams Library, and NIST Library. Confirmation was also achieved using the in-house “Başer Library of Essential Oil Constituents” database, obtained from chromatographic runs of pure compounds performed with the same equipment and conditions. A C8–C40 n-alkane standard solution (Fluka, Buchs, Switzerland) was used to spike the samples for the determination of relative retention indices (RRI). Relative percentage amounts of the separated compounds were calculated from the FID chromatograms.

3.4. Sample Preparation for Biological Studies

Stock solutions of the essential oils and pure compounds were prepared in DMSO (10 mg/mL and 10 mM, respectively) for biological evaluation and stored at −20 °C. For dose-response analysis, all dilutions of the essential oils and pure compounds were in DMSO. The final concentration of DMSO in cell media was 1%.

3.5. Human Neutrophil Isolation

Human neutrophils were isolated from blood that was collected from healthy donors in accordance with a protocol approved by the Institutional Review Board at Montana State University (Protocol #2022-168). Neutrophils were purified from the blood using dextran sedimentation, followed by Histopaque 1077 gradient separation and hypotonic lysis of red blood cells, as described previously [54]. Neutrophil preparations were routinely >95% pure, as determined by light microscopy, and >98% viable, as determined by trypan blue exclusion.

3.6. Ca2+ Mobilization Assay

Changes in intracellular Ca2+ concentrations ([Ca2+]i) were measured with a FlexStation 3 scanning fluorometer (Molecular Devices, Sunnyvale, CA, USA), as described previously [53]. Briefly, human neutrophils were suspended in HBSS-, loaded with Fluo-4AM at a final concentration of 1.25 μg/mL, and incubated for 30 min in the dark at 37 °C. After dye loading, the cells were washed with HBSS-, resuspended in HBSS+, separated into aliquots, and loaded into the wells of flat-bottom, half-area well black microtiter plates (2 × 105 cells/well). To measure the direct effects of test compound or pure essential oils on Ca2+ flux, the compound/oil was added to the wells (final concentration of DMSO was 1%), and changes in fluorescence were monitored (λex = 485 nm, λem = 538 nm) every 5 s for 240 s at room temperature after addition of the test compound or control agonist for comparison. To evaluate the inhibitory effects of the compounds on Ca2+ flux, the compound/oil was added to the wells (the final concentration of DMSO was 1%). The samples were preincubated for 10 min, followed by the addition of 5 nM fMLF. The maximum change in fluorescence, expressed in arbitrary units over baseline, was used to determine the agonist response. Responses were normalized to the response induced by 5 nM fMLF alone without pretreatment, and these responses were assigned as 100%. Curve fitting (at least five or six points) and calculation of median effective concentration values (EC50 or IC50) were performed by nonlinear regression analysis of the dose–response curves generated using Prism 9 (GraphPad Software, Inc., San Diego, CA, USA).

3.7. Chemotaxis Assay

Human neutrophils were resuspended in HBSS+ containing 2% (v/v) heat-inactivated FBS (2 × 106 cells/mL), and chemotaxis was analyzed in 96-well ChemoTx#105-5 chemotaxis chambers (Neuroprobe, Gaithersburg, MD, USA). In brief, neutrophils were preincubated with the indicated concentrations of the test sample (essential oil or pure compound) or DMSO (1% final concentration) for 30 min at room temperature and added to the upper wells of the ChemoTx chemotaxis chambers (40 × 103 cells/well). The lower wells were loaded with 30 µL of HBSS+ containing 2% (v/v) heat-inactivated FBS, the indicated concentrations of the test sample or control DMSO, and 1 nM fMLF as the chemoattractant. Three lower wells were reserved for background controls (DMSO-treated cells in the upper wells and DMSO without fMLF in the lower wells). Neutrophils were added to the upper wells and allowed to migrate through the 5.0-µm pore polycarbonate membrane filter for 60 min at 37 °C and 5% CO2. The number of migrated cells was determined by measuring ATP in lysates of transmigrated cells using a luminescence-based assay (CellTiter-Glo; Promega, Madison, WI, USA), and luminescence measurements were converted to absolute cell numbers by comparison of the values with standard curves obtained with known numbers of neutrophils. Curve fitting (at least eight to nine points) and calculation of median effective concentration values (IC50) were performed by nonlinear regression analysis of the dose–response curves generated using GraphPad Prism 9.

3.8. Cytotoxicity Assay

Cytotoxicity of essential oils and pure compounds in human neutrophils was analyzed using a CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega), according to the manufacturer’s protocol. Briefly, human neutrophils were incubated at a density of 104 cells/well with different concentrations of essential oils or compounds (the final concentration of DMSO was 1%) for 90 min at 37 °C and 5% CO2. Following treatment, the substrate was added to the cells, and the samples were analyzed with a Fluoroscan Ascent FL microplate reader.

4. Conclusions

Analysis of the composition of essential oils extracted from A. urticifolia, A. millefolium, and P. gairdneri collected in Montana subalpine meadows showed that the main components of essential oils obtained from A. urticifolia were menthone, isomenthone, pulegone, and limonene; whereas essential oils obtained from A. millefolium were high in α-thujone and β-thujone, 1,8-cineole, camphor, sabinene, guaia-3,9-dien-11-ol, and terpinen-4-ol; and essential oils obtained from P. gairdneri contained high amounts of dillapiole, p-cymen-8-ol, terpinolene, 4-hydroxy-4-methyl-cyclohex-2-enone, and γ-terpinene. Essential oils from these plants inhibited [Ca2+]i in human neutrophils, with varying potency. The biological effects of A. urticifolia and A. millefolium essential oils might be attributable primarily to previously reported active constituents, including bornyl acetate, borneol, germacrene D, and nerolidol. Dillapiole, which was present at high levels in essential oils of P. gairdneri, inhibited [Ca2+]i in neutrophils and chemotaxis. Thus, dillapiole is likely one of the main active components in these essential oils. Given the critical role of neutrophils in inflammation, these data support the possibility that dillapiole or its structural analogs could be considered in the development of new anti-inflammatory agents. To verify the key targets responsible for the immunomodulatory effects of dillapiole, further experimental investigation is needed.

Author Contributions

I.A.S. and M.T.Q. conceived and designed the project. I.A.S., L.N.K. and R.A.K. collected and identified the plant material. I.A.S., G.Ö., T.Ö. and L.N.K. performed the experiments. A.I.K. conducted the molecular modeling study. I.A.S., G.Ö., T.Ö., R.A.K., L.N.K. and A.I.K. analyzed and interpreted the data. I.A.S., G.Ö. and M.T.Q. drafted and revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by National Institutes of Health IDeA Program Grants GM115371 and GM103474, USDA National Institute of Food and Agriculture Hatch project 1009546, the Montana State University Agricultural Experiment Station, and the Tomsk Polytechnic University Development Program (project Priority-2030-NIP/IZ-009-375-2023).

Institutional Review Board Statement

The study was approved by the Montana State University Institutional Review Board (protocol 2022-168, approved 23 March 2022).

Informed Consent Statement

Informed consent was obtained from all subjects involved in the study.

Data Availability Statement

The data that support the findings of this study are available from the authors upon reasonable request.

Conflicts of Interest

The authors declare no competing financial interest.

References

  1. Moerman, D.E. Native American Medicinal Plants: An Ethnobotanical Dictionary; Timber Press: Portland, OR, USA, 2009; p. 799. [Google Scholar]
  2. Chandler, R.F.; Hooper, S.N.; Harvey, M.J. Ethnobotany and phytochemistry of yarrow, Achillea millefolium, Compositae. Econ. Bot. 1982, 36, 203–223. [Google Scholar] [CrossRef]
  3. Ali, S.I.; Gopalakrishnan, B.; Venkatesalu, V. Pharmacognosy, phytochemistry and pharmacological properties of Achillea millefolium L.: A review. Phytother. Res. 2017, 31, 1140–1161. [Google Scholar] [CrossRef]
  4. Borrelli, F.; Romano, B.; Fasolino, I.; Tagliatatela-Scafati, O.; Aprea, G.; Capasso, R.; Capasso, F.; Bottazzi, E.C.; Izzo, A.A. Prokinetic effect of a standardized yarrow (Achillea millefolium) extract and its constituent choline: Studies in the mouse and human stomach. Neurogastroenterol. Motil. 2012, 24, E164–E190. [Google Scholar] [CrossRef] [PubMed]
  5. Sandner, G.; Heckmann, M.; Weghuber, J. Immunomodulatory activities of selected essential oils. Biomolecules 2020, 10, 1139. [Google Scholar] [CrossRef] [PubMed]
  6. Gandhi, G.R.; Vasconcelos, A.B.S.; Haran, G.H.; Calisto, V.; Jothi, G.; Quintans, J.S.S.; Cuevas, L.E.; Narain, N.; Junior, L.J.Q.; Cipolotti, R.; et al. Essential oils and its bioactive compounds modulating cytokines: A systematic review on anti-asthmatic and immunomodulatory properties. Phytomedicine 2020, 73, 152854. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  7. Valdivieso-Ugarte, M.; Gomez-Llorente, C.; Plaza-Diaz, J.; Gil, A. Antimicrobial, antioxidant, and immunomodulatory properties of essential oils: A systematic review. Nutrients 2019, 11, 2786. [Google Scholar] [CrossRef] [Green Version]
  8. Beutler, B. Innate immunity: An overview. Mol. Immunol. 2004, 40, 845–859. [Google Scholar] [CrossRef]
  9. Bokoch, G.M. Chemoattractant signaling and leukocyte activation. Blood 1995, 86, 1649–1660. [Google Scholar] [CrossRef] [Green Version]
  10. Schepetkin, I.A.; Ozek, G.; Ozek, T.; Kirpotina, L.N.; Kokorina, P.I.; Khlebnikov, A.I.; Quinn, M.T. Neutrophil Immunomodulatory activity of nerolidol, a major component of essential oils from Populus balsamifera buds and propolis. Plants 2022, 11, 3399. [Google Scholar] [CrossRef]
  11. Schepetkin, I.A.; Ozek, G.; Ozek, T.; Kirpotina, L.N.; Khlebnikov, A.I.; Quinn, M.T. Neutrophil immunomodulatory activity of (−)-borneol, a major component of essential oils extracted from Grindelia squarrosa. Molecules 2022, 27, 4897. [Google Scholar] [CrossRef]
  12. Schepetkin, I.A.; Ozek, G.; Ozek, T.; Kirpotina, L.N.; Khlebnikov, A.I.; Quinn, M.T. Chemical composition and immunomodulatory activity of essential oils from Rhododendron albiflorum. Molecules 2021, 26, 3652. [Google Scholar] [CrossRef]
  13. Schepetkin, I.A.; Ozek, G.; Ozek, T.; Kirpotina, L.N.; Khlebnikov, A.I.; Klein, R.A.; Quinn, M.T. Neutrophil immunomodulatory activity of farnesene, a component of Artemisia dracunculus essential oils. Pharmaceuticals 2022, 15, 642. [Google Scholar] [CrossRef] [PubMed]
  14. Ozek, G.; Schepetkin, I.A.; Yermagambetova, M.; Ozek, T.; Kirpotina, L.N.; Almerekova, S.S.; Abugalieva, S.I.; Khlebnikov, A.I.; Quinn, M.T. Innate immunomodulatory activity of cedrol, a component of essential oils isolated from Juniperus species. Molecules 2021, 26, 7644. [Google Scholar] [CrossRef]
  15. Tadic, V.; Arsic, I.; Zvezdanovic, J.; Zugic, A.; Cvetkovic, D.; Pavkov, S. The estimation of the traditionally used yarrow (Achillea millefolium L. Asteraceae) oil extracts with anti-inflamatory potential in topical application. J. Ethnopharmacol. 2017, 199, 138–148. [Google Scholar] [CrossRef]
  16. Bogdan, A.; Colţun, M. Agastache urticifolia (Benth.) Kuntze–aromatic plant introduced and researched in the Botanical Garden. Rev. Bot. 2020, 21, 158–160. [Google Scholar] [CrossRef]
  17. Wilson, T.M.; Davis, A.; Sonstrom, R.E.; Neill, J.L.; Ziebarth, E.A.; Poulson, A.; Carlson, R.E. Essential oil composition and enantioselective profile of Agastache urticifolia (Lamiaceae) and Monardella odoratissima (Lamiaceae) from Utah. Molecules 2023, 28, 2249. [Google Scholar] [CrossRef]
  18. Zielinska, S.; Matkowski, A. Phytochemistry and bioactivity of aromatic and medicinal plants from the genus Agastache (Lamiaceae). Phytochem. Rev. 2014, 13, 391–416. [Google Scholar] [CrossRef] [Green Version]
  19. Bogdan, A.; Colţun, M.; Gille, E.; Necula, R.D.; Grigoraş, V. The biology and the chemical composition of the essential oil of the specie Agastache urticifolia (Benth). Kuntze. In Proceedings of the V International Scientific and Practical Conference within the Framework of the VI Scientific Forum “Science Week in Kruty”, Kruty, Ukraine, 11 March 2021; pp. 38–44. [Google Scholar]
  20. Myadelets, M.A.; Vorobyeva, T.A.; Domrachev, D.V. Composition of the essential oils of some species belonging to genus Agastache clayton ex Gronov (Lamiaceae) cultivated under the conditions of the Middle Ural. Chem. Sustain. Dev. 2013, 21, 397–401. [Google Scholar]
  21. Viljoen, A.M.; Moolla, A.; van Vuuren, S.F.; van Zyl, R.L.; Baser, K.H.C.; Demirci, B.; Ozek, T.; Trinder-Smith, T.H. The biological activity and essential oil composition of 17 Agathosma (Rutaceae) species. J. Essent. Oil Res. 2006, 18, 2–16. [Google Scholar] [CrossRef]
  22. Alan, S.; Kurkcuoglu, M.; Baser, K.H.C. Composition of essential oils of Calamintha nepeta (L.) Savi Subsp nepeta and Calamintha nepeta (L.) Savi Subsp glandulosa (Req.) PW Ball. Asian J. Chem. 2011, 23, 2357–2360. [Google Scholar]
  23. Kaiser, R.; Lamparsky, D.; Schudel, P. Analysis of Buchu leaf oil. J. Agr. Food Chem. 1975, 23, 943–950. [Google Scholar] [CrossRef]
  24. Orav, A.; Arak, E.; Raal, A. Phytochemical analysis of the essential oil of Achillea millefolium L. from various European countries. Nat. Prod. Res. 2006, 20, 1082–1088. [Google Scholar] [CrossRef]
  25. Falconieri, D.; Piras, A.; Porcedda, S.; Marongiu, B.; Goncalves, M.J.; Cabral, C.; Cavaleiro, C.; Salgueiro, L. Chemical composition and biological activity of the volatile extracts of Achillea millefolium. Nat Prod Commun 2011, 6, 1527–1530. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  26. Tampe, J.; Parra, L.; Huaiquil, K.; Mutis, A.; Quiroz, A. Repellent effect and metabolite volatile profile of the essential oil of Achillea millefolium against Aegorhinus nodipennis (Hope) (Coleoptera: Curculionidae). Neotrop. Entomol. 2015, 44, 279–285. [Google Scholar] [CrossRef]
  27. Toplan, G.G.; Taskin, T.; Iscan, G.; Goger, F.; Kurkcuoglu, M.; Civas, A.; Ecevit-Genc, G.; Mat, A.; Baser, K.H.C. Comparative studies on essential oil and phenolic content with in vitro antioxidant, anticholinesterase, antimicrobial activities of Achillea biebersteinii Afan. and A. millefolium subsp. millefolium Afan. L. growing in Eastern Turkey. Molecules 2022, 27, 1956. [Google Scholar] [CrossRef]
  28. Acimovic, M.; Zoric, M.; Zheljazkov, V.D.; Pezo, L.; Cabarkapa, I.; Jeremic, J.S.; Cvetkovic, M. Chemical characterization and antibacterial activity of essential oil of medicinal plants from Eastern Serbia. Molecules 2020, 25, 5482. [Google Scholar] [CrossRef]
  29. El-Kalamouni, C.; Venskutonis, P.R.; Zebib, B.; Merah, O.; Raynaud, C.; Talou, T. Antioxidant and antimicrobial activities of the essential oil of Achillea millefolium L. grown in France. Medicines 2017, 4, 30. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  30. Millefolii Herba, Y. European Pharmacopoeia. European Directorate for the Quality of Medicines & HealthCare of the Council of Europe (EDQM), 9th ed.; Council of Europe: Strasbourg, France, 2016; Volume 1, pp. 1564–1565. [Google Scholar]
  31. Raal, A.; Orav, A.; Arak, E. Essential oil content and composition in commercial Achillea millefolium L. herbs from different countries. J. Essent. Oil Bear. Plants 2012, 15, 22–31. [Google Scholar] [CrossRef]
  32. Rauchensteiner, F.; Nejati, S.; Saukel, J. The Achillea millefolium group (Asteraceae) in Middle Europe and the Balkans: A diverse source for the crude drug Herba Millefolii. J. Trad. Medi. 2004, 21, 113–119. [Google Scholar]
  33. Schepetkin, I.A.; Ozek, G.; Ozek, T.; Kirpotina, L.N.; Khlebnikov, A.I.; Quinn, M.T. Chemical composition and immunomodulatory activity of Hypericum perforatum essential oils. Biomolecules 2020, 10, 916. [Google Scholar] [CrossRef] [PubMed]
  34. Naya, Y.; Nagahama, Y.; Kotake, M. Volatile components of Ledum palustre Var Nipponicum Et Yesoense. Heterocycles 1978, 10, 29–36. [Google Scholar] [CrossRef]
  35. Madyastha, K.M.; Raj, C.P. Stereoselective hydroxylation of 4-methyl-2-cyclohexenone in rats: Its relevance to R-(+)-pulegone-mediated hepatotoxicity. Biochem. Biophys. Res. Comm. 2002, 297, 202–205. [Google Scholar] [CrossRef] [PubMed]
  36. Schepetkin, I.A.; Kushnarenko, S.V.; Ozek, G.; Kirpotina, L.N.; Sinharoy, P.; Utegenova, G.A.; Abidkulova, K.T.; Ozek, T.; Baser, K.H.; Kovrizhina, A.R.; et al. Modulation of Human neutrophil responses by the essential oils from Ferula akitschkensis and their constituents. J. Agric. Food Chem. 2016, 64, 7156–7170. [Google Scholar] [CrossRef] [Green Version]
  37. Schepetkin, I.A.; Kushnarenko, S.V.; Ozek, G.; Kirpotina, L.N.; Utegenova, G.A.; Kotukhov, Y.A.; Danilova, A.N.; Ozek, T.; Baser, K.H.; Quinn, M.T. Inhibition of human neutrophil responses by the essential oil of Artemisia kotuchovii and its constituents. J. Agric. Food Chem. 2015, 63, 4999–5007. [Google Scholar] [CrossRef] [Green Version]
  38. Kreutz, T.; Carneiro, S.B.; Soares, K.D.; Limberger, R.P.; Apel, M.A.; Veiga, V.F.; Koester, L.S. Aniba canelilla (Kunth) Mez essential oil-loaded nanoemulsion: Improved stability of the main constituents and in vitro antichemotactic activity. Ind. Crops Prod. 2021, 171, 113949. [Google Scholar] [CrossRef]
  39. Maciel, A.J.; Lacerda, C.P.; Danielli, L.J.; Bordignon, S.A.L.; Fuentefria, A.M.; Apel, M.A. Antichemotactic and antifungal action of the essential oils from Cryptocarya aschersoniana, Schinus terebinthifolia, and Cinnamomum amoenum. Chem. Biodiver. 2019, 16, 204. [Google Scholar] [CrossRef] [PubMed]
  40. Soares, K.D.; Bordignon, S.A.L.; Apel, M.A. Chemical composition and anti-inflammatory activity of the essential oils of Piper gaudichaudianum and Piper mikanianum. J. Ethnopharmacol. 2022, 297, 115533. [Google Scholar] [CrossRef] [PubMed]
  41. Santana, A.I.; Vila, R.; Canigueral, S.; Gupta, M.P. Chemical composition and biological activity of essential oils from different species of Piper from Panama. Planta Med. 2016, 82, 986–991. [Google Scholar] [CrossRef]
  42. Ferreira, O.O.; Cruz, J.N.; de Moraes, A.A.B.; Franco, C.D.P.; Lima, R.R.; Dos Anjos, T.O.; Siqueira, G.M.; do Nascimento, L.D.; Cascaes, M.M.; de Oliveira, M.S.; et al. Essential oil of the plants growing in the Brazilian Amazon: Chemical composition, antioxidants, and biological applications. Molecules 2022, 27, 4373. [Google Scholar] [CrossRef]
  43. Vila, R.; Tomi, M.; Mundina, M.; Santana, A.I.; Solis, P.N.; Arce, J.B.L.; Iclina, J.L.B.; Iglesias, J.; Gupta, M.P.; Casanova, J.; et al. Unusual composition of the essential oils from the leaves of Piper aduncum. Flavour Fragr. J. 2005, 20, 67–69. [Google Scholar] [CrossRef]
  44. Guetat, A.; Abdelwahab, A.T.; Yahia, Y.; Rhimi, W.; Alzahrani, A.K.; Boulila, A.; Cafarchia, C.; Boussaid, M. Deverra triradiata Hochst. ex Boiss. from the Northern Region of Saudi Arabia: Essential oil profiling, plant extracts and biological activities. Plants 2022, 11, 1543. [Google Scholar] [CrossRef] [PubMed]
  45. Brazao, M.A.B.; Brazao, F.V.; Maia, J.G.S.; Monteiro, M.C. Antibacterial activity of the Piper aduncum oil and dillapiole, its main constituent, against multidrug-resistant strains. Bol. Latinoam. Caribe Plantas 2014, 13, 517–526. [Google Scholar]
  46. de Almeida, R.R.P.; Souto, R.N.P.; Bastos, C.N.; da Silva, M.H.L.; Maia, J.G.S. Chemical variation in Piper aduncum and biological properties of its dillapiole-rich essential oil. Chem. Biodiver. 2009, 6, 1427–1434. [Google Scholar] [CrossRef] [PubMed]
  47. Parise, R.; Pasqualoto, K.F.M.; Magri, F.M.M.; Ferreira, A.K.; da Silva, B.A.V.G.; Damiao, M.C.F.C.B.; Tavares, M.T.; Azevedo, R.A.; Auada, A.V.V.; Polli, M.C.; et al. Dillapiole as antileishmanial agent: Discovery, cytotoxic activity and preliminary SAR studies of dillapiole analogues. Arch. Pharm. 2012, 345, 934–944. [Google Scholar] [CrossRef]
  48. Rojas-Martinez, R.; Arrieta, J.; Cruz-Antonio, L.; Arrieta-Baez, D.; Velazquez-Mendez, A.M.; Sanchez-Mendoza, M.E. Dillapiole, isolated from Peperomia pellucida, shows gastroprotector activity against ethanol-induced gastric lesions in Wistar rats. Molecules 2013, 18, 11327–11337. [Google Scholar] [CrossRef] [Green Version]
  49. Parise, R.; Pastrello, M.; Camerlingo, C.E.P.; Silva, G.J.; Agostinho, L.A.; de Souza, T.; Magri, F.M.M.; Ribeiro, R.R.; Brandt, C.A.; Polli, M.C. The anti-inflammatory activity of dillapiole and some semisynthetic analogues. Pharmaceut. Biol. 2011, 49, 1173–1179. [Google Scholar] [CrossRef]
  50. Ferreira, A.K.; de-Sa, P.L.; Pasqualoto, K.F.M.; de Azevedo, R.A.; Camara, D.A.D.; Costa, A.S.; Figueiredo, C.R.; Matsuo, A.L.; Massaoka, M.H.; Auada, A.V.V.; et al. Cytotoxic effects of dillapiole on MDA-MB-231 cells involve the induction of apoptosis through the mitochondrial pathway by inducing an oxidative stress while altering the cytoskeleton network. Biochimie 2014, 99, 195–207. [Google Scholar] [CrossRef]
  51. Daina, A.; Michielin, O.; Zoete, V. SwissADME: A free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules. Sci. Rep. 2017, 7, 42717. [Google Scholar] [CrossRef] [Green Version]
  52. Carneiro, S.B.; Kreutz, T.; Limberger, R.P.; Teixeira, H.F.; da Veiga Júnior, V.F.; Koester, L.S. Piper aduncum essential oil rich in dillapiole: Development of hydrogel-thickened nanoemulsion and nanostructured lipid carrier intended for skin delivery. Pharmaceutics 2022, 14, 2525. [Google Scholar] [CrossRef]
  53. Ozek, G.; Ishmuratova, M.; Tabanca, N.; Radwan, M.M.; Goger, F.; Ozek, T.; Wedge, D.E.; Becnel, J.J.; Cutler, S.J.; Can Baser, K.H. One-step multiple component isolation from the oil of Crinitaria tatarica (Less.) Sojak by preparative capillary gas chromatography with characterization by spectroscopic and spectrometric techniques and evaluation of biological activity. J. Sep. Sci. 2012, 35, 650–660. [Google Scholar] [CrossRef]
  54. Schepetkin, I.A.; Kirpotina, L.N.; Khlebnikov, A.I.; Quinn, M.T. High-throughput screening for small-molecule activators of neutrophils: Identification of novel N-formyl peptide receptor agonists. Mol. Pharmacol. 2007, 71, 1061–1074. [Google Scholar] [CrossRef] [PubMed] [Green Version]
Plants 12 02643 g001 550
Figure 1. Effect of the PGI essential oil on fMLF-induced neutrophil [Ca2+]i. Human neutrophils were incubated with the indicated concentrations of the essential oil or 1% DMSO (negative control) for 10 min. The cells were then activated with 5 nM fMLF, and [Ca2+]i was monitored as described. The data shown are presented as the mean ± SD from one experiment that is representative of three independent experiments with similar results.
Figure 1. Effect of the PGI essential oil on fMLF-induced neutrophil [Ca2+]i. Human neutrophils were incubated with the indicated concentrations of the essential oil or 1% DMSO (negative control) for 10 min. The cells were then activated with 5 nM fMLF, and [Ca2+]i was monitored as described. The data shown are presented as the mean ± SD from one experiment that is representative of three independent experiments with similar results.
Plants 12 02643 g001
Plants 12 02643 g002 550
Figure 2. Effect of dillapiole on neutrophil [Ca2+]i. Human neutrophils were treated with dillapiole (50 and 100 µM), 5 nM fMLF (positive control), or 1% DMSO (negative control), and [Ca2+]i was monitored for the indicated times (arrow indicates when treatment was added). Data are from one experiment that is representative of three independent experiments.
Figure 2. Effect of dillapiole on neutrophil [Ca2+]i. Human neutrophils were treated with dillapiole (50 and 100 µM), 5 nM fMLF (positive control), or 1% DMSO (negative control), and [Ca2+]i was monitored for the indicated times (arrow indicates when treatment was added). Data are from one experiment that is representative of three independent experiments.
Plants 12 02643 g002
Plants 12 02643 g003 550
Figure 3. Effect of dillapiole on fMLF-induced neutrophil [Ca2+]i. Human neutrophils were treated with the indicated concentrations of the dillapiole or 1% DMSO (negative control) for 10 min. The cells were then activated with 5 nM fMLF, and [Ca2+]i was monitored as described. The data shown are presented as the mean ± SD from one experiment that is representative of three independent experiments with similar results.
Figure 3. Effect of dillapiole on fMLF-induced neutrophil [Ca2+]i. Human neutrophils were treated with the indicated concentrations of the dillapiole or 1% DMSO (negative control) for 10 min. The cells were then activated with 5 nM fMLF, and [Ca2+]i was monitored as described. The data shown are presented as the mean ± SD from one experiment that is representative of three independent experiments with similar results.
Plants 12 02643 g003
Plants 12 02643 g004 550
Figure 4. Effect of the PGI essential oil and dillapiole on human neutrophil chemotaxis. Neutrophils were pretreated with the indicated concentrations of the essential oil (A) or dillapiole (B), and neutrophil migration toward 1 nM fMLF was measured, as described. The data are from one experiment that is representative of two independent experiments.
Figure 4. Effect of the PGI essential oil and dillapiole on human neutrophil chemotaxis. Neutrophils were pretreated with the indicated concentrations of the essential oil (A) or dillapiole (B), and neutrophil migration toward 1 nM fMLF was measured, as described. The data are from one experiment that is representative of two independent experiments.
Plants 12 02643 g004
Plants 12 02643 g005 550
Figure 5. Cytotoxicity of the essential oils from P. gairdneri (PGI) and dillapiole. Human neutrophils were preincubated with the indicated concentrations of the essential oils or pure dillapiole for 30 min or 90 min, and cell viability was analyzed, as described. Values are the mean ± SD of triplicate samples from one experiment that is representative of three independent experiments with similar results.
Figure 5. Cytotoxicity of the essential oils from P. gairdneri (PGI) and dillapiole. Human neutrophils were preincubated with the indicated concentrations of the essential oils or pure dillapiole for 30 min or 90 min, and cell viability was analyzed, as described. Values are the mean ± SD of triplicate samples from one experiment that is representative of three independent experiments with similar results.
Plants 12 02643 g005
Plants 12 02643 g006 550
Figure 6. Chemical structure of dillapiole.
Figure 6. Chemical structure of dillapiole.
Plants 12 02643 g006
Plants 12 02643 g007 550
Figure 7. Bioavailability radar plot of dillapiole. The plot depicts the LIPO (lipophilicity), SIZE (molecular weight), POLAR (polarity), INSOLU (insolubility), INSATU (unsaturation), and FLEX (rotatable bond flexibility) parameters.
Figure 7. Bioavailability radar plot of dillapiole. The plot depicts the LIPO (lipophilicity), SIZE (molecular weight), POLAR (polarity), INSOLU (insolubility), INSATU (unsaturation), and FLEX (rotatable bond flexibility) parameters.
Plants 12 02643 g007
Table
Table 1. Location, date of collection of the plant material, and distillation yields of essential oils.
Table 1. Location, date of collection of the plant material, and distillation yields of essential oils.
PlantLocationLatitude
(N)		Longitude
(E)		Altitude (m)Plant MaterialDate of
Collection		Yield (%)
A. urticifoliaHyalite Canyon, Bozeman, MT, USA45.48990°111.00091°2272leaves/flowers07/20210.2/0.5
A. millefoliumHyalite Canyon, Bozeman, MT, USA45.48346°110.97882°2042inflorescence07/20210.2
P. gairdneriHyalite Canyon, Bozeman, MT, USA45.49671°110.98859°1978inflorescence07/20211.9
Table
Table 2. Composition of essential oils isolated from A. urticifolia (AUF and AUL), A. millefolium (AMI), and P. gairdneri (PGI).
Table 2. Composition of essential oils isolated from A. urticifolia (AUF and AUL), A. millefolium (AMI), and P. gairdneri (PGI).
RRICompoundAUFAULAMIPGIRRICompoundAUFAULAMIPGI
1032α-Pinenett1.20.21704γ-Muurolene t
1035α-Thujenetttt1706α-Terpineol t1.91.1
10482-Methyl-3-buten-2-oltt 1715γ-Terpineol t
1076Camphene 2.3t1719Borneol 1.2
1093Hexanalt t 1720trans-Sabinol 0.2
1118β-Pinenett1.9t1726Germacrene D0.1 0.1
1132Sabinene0.10.17.00.11748Piperitone0.10.7
1136Isoamyl acetatet0.4 1751Carvone0.10.2 t
1174Myrcene0.40.4t0.21773δ-Cadinene 0.2
1176α-Phellandrene t0.11797p-Methyl acetophenonet 0.6
1185Isobutyl 2-methyl butyrate tt 1802Cumin aldehyde 0.2
1188α-Terpinene 0.2t1811p-Mentha-1,3-dien-7-al 0.4
1195Dehydro-1,8-cineolet 0.2 1814p-Mentha-1,5-dien-7-ol 0.7
12032-Methyl butyl isobutyrate t 18382-Phenylethyl acetate0.10.2
1203Limonene4.26.20.21.31845trans-Carveolt
12131,8-Cineolett17.0 1849Pulegone epoxide 0.4
1218β-Phellandrene0.10.1 0.51864p-Cymen-8-olt 14.1
1225(Z)-3-Hexenalt0.2 1865Isopiperitenone0.50.1
1244Amyl furan t 1877TMMT1.10.8
1246(Z)-β-Ocimene0.10.1t0.41894CMMT0.50.4
1255γ-Terpinenet 0.42.418981,11-Oxidocalamenene 0.3
1266(E)-β-Ocimene0.50.3 0.31949Piperitenone1.60.8
12663-Octanonet 1969cis-Jasmone 0.2
1280p-Cymenet 0.71.119988,9-dehydrothymol0.1t
1285Isoamyl isovaleratet t 2008Caryophyllene oxide0.30.40.5
1286MBMB 0.1 2016Isoamyl phenyl acetate 0.1
1290Terpinolenet 0.112.02045Carotol 0.2
1384α-Pinene oxide0.1 2050(E)-Nerolidol 0.2
1386Octenyl acetatet 2068Hexahydro-farnesyl acetone0.1
1400Nonanal t 2074Caryophylla-2(12),6(13)-dien-5-one 0.3
14081,3,8-p-Menthatriene 0.12094p-Cresol 0.2
1413Rose furant 2096Elemol 1.7
1437α-Thujone 17.10.32096(E)-Methyl cinnamate 0.3
14432,5- Dimethylstyrenet 2100Heneicosane 0.3
1451β-Thujone0.1 14.9 2103Guaiol 0.1
1452α,p-Dimethylstyrenet 0.62113Cumin alcohol 0.3
14521-Octen-3-olt 21154-Hydroxy-4-methyl-cyclohex-2-enone 6.2
1458cis-1,2-Limonene epoxidet 2144Spathulenol0.10.2
1474trans-Sabinene hydrate 0.3 2181Isothymol0.3
1475Menthone2.725.7 2183γ-Decalactone 0.6
1497α-Copaene 0.2 2184cis-p-Menth-3-en-1,2-diol 0.4
1497Menthofuran0.1 2185γ-Eudesmol 0.9
1503Isomenthone2.629.1 2192Nonanoic acid 0.1
1532Camphor 13.0 2195Fokienol 0.1
1541Benzaldehyde0.20.4 2209T-Muurolol t
1553Linalool 0.3 2221Isocarvacrol 0.3
1556cis-Sabinene hydrate 0.2 2228Eremoligenol 0.1
1571trans-p-Menth-2-en-1-ol 0.1 2245Elemicine 1.2
1583cis-Isopulegone0.50.2 2250α-Eudesmol 0.5
1590Bornyl acetate 1.7 2250Fukinanolide 0.6
1598trans-Isopulegone0.5 2255α-Cadinol t
1611Terpinen-4-ol 2.50.42257β-Eudesmol 1.1
1612β-Caryophyllene0.50.30.30.12272Copaborneol t
1618Camphene hydrate 0.2 2290Guaia-3,9-dien-11-ol 3.2
1626MMO t 2296Myristicine 0.1
1638cis-p-Menth-2-en-1-ol 0.1 2303Menthofurolactone0.2t
1639trans-p-Mentha-2,8-dien-1-ol0.50.6 2316Caryophylladienol I 0.6
1642Thuj-3-en10-al 0.2 2324Caryophylladienol II 0.2
1648Myrtenal 0.6 2368Eudesma-4(15),7-diene-1-β-ol t
1651Sabinaketone 0.1 2384Dillapiole 30.3
1658Sabinyl acetate 0.10.2 24202-Methyl isoborneol * 1.1
1662Pulegone78.928.8 0.12622Phytol t
1678cis-p-Mentha-2,8-dien-1-ol 0.5 2655Benzyl benzoate 1.9
1682δ-Terpineol 0.6 2758Artedouglasia oxide B 0.6
1690Cryptone0.10.3 0.2
The data are presented as relative % calculated from flame ionization detector data for each component identified. RRI, relative retention index calculated based on retention of n-alkanes. Trace amounts (t) were present at <0.1%. * Identified tentatively using the Wiley and MassFinder mass spectra libraries and published RRI. All other compounds were identified by comparison with co-injected standards. Abbreviations: AUF, essential oil from flowers of A. urticifolia; AUL, essential oil from leaves of A. urticifolia; AMI, essential oil from inflorescences of A. millefolium; PGI, essential oil from inflorescences of P. gairdneri. MMO, 2-methyl-6-methylene-3,7-octadien-2-ol; MBMB, 2-methyl butyl 2-methyl butyrate; TMMT, trans-p-mentha-8-methylthio-3-one; CMMT, cis-p-mentha-8-methylthio-3-one.
Table
Table 3. Summary of the chemical composition (%) of essential oils from A. urticifolia, A. millefolium, and P. gairdneri.
Table 3. Summary of the chemical composition (%) of essential oils from A. urticifolia, A. millefolium, and P. gairdneri.
CompoundsAUFAULAMIPGI
Monoterpene hydrocarbons5.47.214.019.3
Oxygenated monoterpenes90.688.773.618.4
Sesquiterpene hydrocarbons0.60.30.80.1
Oxygenated sesquiterpenes0.40.69.81.4
Oxygenated diterpenes t
Phenylpropanoids 31.6
Substituted cyclohexanones 6.2
Others0.41.70.53.6
Total97.498.598.280.6
Abbreviations: AUF, essential oil from flowers of A. urticifolia; AUL, essential oil from leaves of A. urticifolia; AMI, essential oil from inflorescences of A. millefolium; PGI, essential oil from inflorescences of P. gairdneri.
Table
Table 4. Effect of essential oils and their selected component compounds on Ca2+ flux in human neutrophils.
Table 4. Effect of essential oils and their selected component compounds on Ca2+ flux in human neutrophils.
Essential Oil or Pure CompoundDirect ActivationInhibition of fMLF-Induced Response a
EC50 (µg/mL); (Efficacy, %)IC50 (µg/mL)
AUF 28.5 ± 2.1 (130)43.0 ± 2.8
AUL 43.5 ± 6.2 (150)25.0 ± 4.2
AMI41.5 ± 0.7 (140)24.5 ± 2.1
PGI30.6 ± 4.2 (70)4.3 ± 2.2
EC50 (µM); (Efficacy, %)IC50 (µM)
α-ThujeneN.A.N.A.
α/β-ThujoneN.A.N.A.
MenthoneN.A.N.A.
IsomenthoneN.A.N.A.
PulegoneN.A.N.A.
Dillapiole19.3 ± 1.4 (65)13.9 ± 4.2
a Inhibition of neutrophil Ca2+ flux induced by 5 nM fMLF. N.A.: no activity was observed, even at the highest concentration tested (50 µM). EC50 and IC50 values are presented as the mean ± S.D. of three independent experiments. Efficacy is the maximum response to an essential oil (or compound) compared to that induced by control 5 nM fMLF (100%). AUF, essential oil from flowers of A. urticifolia; AUL, essential oil from leaves of A. urticifolia; AMI, essential oil from inflorescences of A. millefolium; PGI, essential oil from inflorescences of P. gairdneri.
Table
Table 5. Predicted physicochemical properties of dillapiole according to SwissADME results.
Table 5. Predicted physicochemical properties of dillapiole according to SwissADME results.
Molecular DescriptorProperty
FormulaC12H14O4
M.W.222.24
Heavy atoms16
Fraction Csp30.33
Rotatable bonds4
H-bond acceptors4
H-bond donors0
MR59.59
tPSA36.92
iLogP2.82
BBB permeationYes
Abbreviations: M.W., molecular weight (g/mol); MR, molar refractivity; tPSA, topological polar surface area (Å2); iLogP, lipophilicity; BBB, blood–brain barrier.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Schepetkin, I.A.; Özek, G.; Özek, T.; Kirpotina, L.N.; Klein, R.A.; Khlebnikov, A.I.; Quinn, M.T. Composition and Biological Activity of the Essential Oils from Wild Horsemint, Yarrow, and Yampah from Subalpine Meadows in Southwestern Montana: Immunomodulatory Activity of Dillapiole. Plants 2023, 12, 2643. https://doi.org/10.3390/plants12142643

AMA Style

Schepetkin IA, Özek G, Özek T, Kirpotina LN, Klein RA, Khlebnikov AI, Quinn MT. Composition and Biological Activity of the Essential Oils from Wild Horsemint, Yarrow, and Yampah from Subalpine Meadows in Southwestern Montana: Immunomodulatory Activity of Dillapiole. Plants. 2023; 12(14):2643. https://doi.org/10.3390/plants12142643

Chicago/Turabian Style

Schepetkin, Igor A., Gulmira Özek, Temel Özek, Liliya N. Kirpotina, Robyn A. Klein, Andrei I. Khlebnikov, and Mark T. Quinn. 2023. "Composition and Biological Activity of the Essential Oils from Wild Horsemint, Yarrow, and Yampah from Subalpine Meadows in Southwestern Montana: Immunomodulatory Activity of Dillapiole" Plants 12, no. 14: 2643. https://doi.org/10.3390/plants12142643

APA Style

Schepetkin, I. A., Özek, G., Özek, T., Kirpotina, L. N., Klein, R. A., Khlebnikov, A. I., & Quinn, M. T. (2023). Composition and Biological Activity of the Essential Oils from Wild Horsemint, Yarrow, and Yampah from Subalpine Meadows in Southwestern Montana: Immunomodulatory Activity of Dillapiole. Plants, 12(14), 2643. https://doi.org/10.3390/plants12142643

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop