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

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.


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

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. Table 2. Composition of essential oils isolated from A. urticifolia (AUF and AUL), A. millefolium (AMI), and P. gairdneri (PGI).  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.
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].

Effect of Essential Oils and Selected Component Compounds on Neutrophil Ca 2+ Influx
We evaluated the essential oils for their immunomodulatory effects on human neutrophils. In particular, the effects of the essential oils on intracellular Ca 2+ flux ([Ca 2+ ] i ) were assessed, since [Ca 2+ ] 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 [Ca 2+ ] i , with EC 50 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 [Ca 2+ ] i response to the chemoattractant f MLF with an IC 50 of 4.3 µg/mL (Figure 1), while other essential oil samples had lower inhibitory activity (Table 4).

Effect of Essential Oils and Selected Component Compounds on Neutrophil Ca 2+ Influx
We evaluated the essential oils for their immunomodulatory effects on huma trophils. In particular, the effects of the essential oils on intracellular Ca 2+ flux ([Ca 2+ ] assessed, since [Ca 2+ ]i is an important signal during neutrophil activation. Treatm neutrophils with essential oils from A. urticifolia (AUF and AUL), A. millefolium (AM P. gairdneri (PGI) activated human neutrophils, resulting in increased [Ca 2+ ]i, wit values ranging from 28.5 to 43.5 µg/mL (Table 4). Pre-incubation of neutrophils w most active of these essential oil samples (PGI) inhibited the subsequent neutrophil response to the chemoattractant fMLF with an IC50 of 4.3 µg/mL (Figure 1), while essential oil samples had lower inhibitory activity (Table 4).  The data shown are presented as the mean ± SD from one experiment that is representative of three independent experiments with similar results.
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 exclude an activity of (S)-(−)-pulegone in human neutrophils since that isomer is not commercially available.   Since dillapiole directly activated neutrophil [Ca 2+ ] i , albeit with low efficacy, it is possible that this compound could contribute to receptor desensitization and/or intracellular Ca 2+ store depletion. Indeed, pre-incubation of neutrophils with dillapiole inhibited subsequent f MLF-induced [Ca 2+ ] i , with an IC 50 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.

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 [Ca 2+ ]i is involved in neutrophil chemotaxis [9], the inhibitory effect of dillapiole on neutrophil chemotaxis is consistent with its primary effect on Ca 2+ flux.  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.
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). 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).  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 antiinflammatory activity in a carrageenan-induced rat paw edema model [49] and broad cytotoxic effects against a variety of tumor cells [50].  This is the first report on the inhibitory effects of dillapiole on hu tivation (Table 4). Dillapiole (see chemical structure in Figure 6) is found in abundance in essential oils from Piper species, Deverra triradi and in the early developmental stages of dill (Anethum graveolens L.) reported to exhibit bactericidal [45], fungicidal [46], antileishmanial tective activity [48]. Interestingly, dillapiole has also been reported to tory activity in a carrageenan-induced rat paw edema model [49] and fects against a variety of tumor cells [50]. To further characterize dillapiole, we calculated the most impor cal and ADME parameters of this compound using SwissADME [5 would be predicted to permeate the blood-brain barrier (BBB) (Table radar plot of the main characteristics, the ADME data for dillapiole p also exhibit high bioavailability (Figure 7).  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). Abbreviations: M.W., molecular weight (g/mol); MR, molar refractivity; tPSA, topological polar surface area (Å 2 ); iLogP, lipophilicity; BBB, blood-brain barrier. 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.

Materials
Dimethyl sulfoxide (DMSO), fMLF, Histopaque 1077, (−)-α-thujone, α/β-thujone, and dillapiole were purchased from Sigma-Aldrich Chemical Co.  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.

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.

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 C 8 -C 40 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.

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%.

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.

Ca 2+ Mobilization Assay
Changes in intracellular Ca 2+ concentrations ([Ca 2+ ] 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 × 10 5 cells/well). To measure the direct effects of test compound or pure essential oils on Ca 2+ flux, the compound/oil was added to the wells (final concentration of DMSO was 1%), and changes in fluorescence were monitored (λ ex = 485 nm, λ em = 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 Ca 2+ 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 f MLF. 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 f MLF 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 (EC 50 or IC 50 ) were performed by nonlinear regression analysis of the dose-response curves generated using Prism 9 (GraphPad Software, Inc., San Diego, CA, USA).

Chemotaxis Assay
Human neutrophils were resuspended in HBSS + containing 2% (v/v) heat-inactivated FBS (2 × 10 6 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 × 10 3 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 f MLF as the chemoattractant. Three lower wells were reserved for background controls (DMSO-treated cells in the upper wells and DMSO without f MLF 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% CO 2 . 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 (IC 50 ) were performed by nonlinear regression analysis of the dose-response curves generated using GraphPad Prism 9.

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 10 4 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% CO 2 . Following treatment, the substrate was added to the cells, and the samples were analyzed with a Fluoroscan Ascent FL microplate reader.

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 [Ca 2+ ] 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 [Ca 2+ ] 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.