Innate Immunomodulatory Activity of Cedrol, a Component of Essential Oils Isolated from Juniperus Species

Little is known about the immunomodulatory activity of essential oils isolated from Juniperus species. Thus, we isolated essential oils from the cones and leaves of eight juniper species found in Montana and in Kazakhstan, including J. horizontalis, J. scopolorum, J. communis, J. seravschanica, J. sabina, J. pseudosabina, J. pseudosabina subsp. turkestanica, and J. sibirica. We report here the chemical composition and innate immunomodulatory activity of these essential oils. Compositional analysis of the 16 samples of Juniper essential oils revealed similarities and differences between our analyses and those previously reported for essential oils from this species. Our studies represent the first analysis of essential oils isolated from the cones of four of these Juniper species. Several essential oil samples contained high levels of cedrol, which was fairly unique to three Juniper species from Kazakhstan. We found that these essential oils and pure (+)-cedrol induced intracellular Ca2+ mobilization in human neutrophils. Furthermore, pretreatment of human neutrophils and N-formyl peptide receptor 1 and 2 (FPR1 and FPR2) transfected HL60 cells with these essential oils or (+)-cedrol inhibited agonist-induced Ca2+ mobilization, suggesting these responses were desensitized by this pretreatment. In support of this conclusion, pretreatment with essential oils from J. seravschanica cones (containing 16.8% cedrol) or pure (+)-cedrol inhibited human neutrophil chemotaxis to N-formyl peptide. Finally, reverse pharmacophore mapping predicted several potential kinase targets for cedrol. Thus, our studies have identified cedrol as a novel neutrophil agonist that can desensitize cells to subsequent stimulation by N-formyl peptide.


Introduction
The genus Juniperus L. consists of 75 species, which grow mainly in the northern hemisphere [1]. Juniperus is one of the main genera of the Cupressaceae family [2], and according to the generally accepted classification, the genus is divided into three sections distributed throughout the world [1,3,4]. These evergreen coniferous trees or shrubs thrive under a variety of environmental conditions. Some juniper species grow in the Arctic zone and the alpine belt, while others are found in subtropical and tropical zones [1,[5][6][7][8].
Among the least studied Juniperus are those growing in Kazakhstan. In Kazakhstan, the local names for Juniperus species are "arsha" (in Kazakh) and "mojjevelnik" (in Russian) [9,10]. Kazakhstan's latitude ranges from 40 • 7 N to 54 • 9 N, and therefore, is geographically similar to the northern states of the USA, including Montana (44 • 2 N to found in various regions of the world is necessary to expand our understanding of the regional differences in these species.
In the present studies, we compared the chemical composition and immunomodulatory activity of essential oils obtained from the cones and leaves of seven juniper species collected in Kazakhstan and Montana, including J. horizontalis, J. scopolorum, J. communis, J. seravschanica, J. sabina, J. pseudosabina, J. pseudosabina subsp. turkestanica, and J. sibirica and analyzed their chemical composition and innate immunomodulatory activity. We show that the Juniper essential oils inhibited intracellular Ca 2+ mobilization in neutrophils. Furthermore, we demonstrated that cedrol, which was present at high levels in some Juniper species from Kazakhstan, also inhibited human neutrophil functional responses and is likely one of the active components in these essential oils. Given the critical role of neutrophils in inflammation, our data support the possibility that cedrol could be effective therapeutic compound for the development anti-inflammatory agents.

Essential Oil Composition
Leaves and cones were collected from mature Juniperus shrubs and trees (Table 1) and extracted, as described under Materials and Methods. The extraction yields (v/w) of essential oils obtained from Juniperus spp. ranged from 0.4 to 2.0% ( Table 1). The chemical composition of these essential oils was evaluated using simultaneous GC-FID and GC/MS. Major compounds (>2%) of the essential oils are shown in Table 2, and Supplementary Table  S1 summarizes all identified compounds. The main class of compounds in all samples was monoterpenes, which ranged from 52.5 to 98.3% of the essential oil composition isolated from various Juniper species (Table 3). In addition, essential oils from the cones of J. sibirica (JSI Con ) were enriched in sesquiterpenes (46.4%). The leaf oils of J. horizontalis from Turkey, Iran, USA, Egypt, and Canada (Saskatchewan) have been previously reported to contain linalool, limonene, sabinene, and bornyl acetate as their major volatile constituents [44,[62][63][64]79]. In the present study, we found that JH Lv also had a high content of sabinyl acetate (>16%), which was not found in previous reports ( Table 2). The cone oil of J. horizontalis from Iran was reported to contain myrcene (22.6%), limonene (27.8%), and sabinene (38.0%) [79]. In our investigation, JH Con had predominantly sabinene (>77.0%) ( Table 2), which may reflect the differences in ecology where these plants were collected.

Effect of the Juniperus Essential Oils and Cedrol on Neutrophil [Ca 2+ ] i
The innate immune system is essential for host defense and provides immediate defense against infection. Neutrophils are the most abundant leukocytes in the blood and constitute key components of the innate immunity that perform an important effector and regulatory functions in the development of human inflammatory response [84,85]. Thus, neutrophils represent an ideal pharmacological target for therapeutic development, and numerous natural products, including essential oils, have been shown to exhibit neutrophil immunomodulatory activity [86][87][88].
Juniperus essential oils were evaluated for their immunomodulatory effects on human neutrophils. Specifically, we evaluated their effects on [Ca 2+ ] i , which is a key component of phagocyte activation [45,46]. We found that treatment of neutrophils with Juniperus essential oils increased [Ca 2+ ] i , with EC 50 values ranging from 13.9 µg/mL (JT Con ) to 49.8 µg/mL (JH Con ). In addition, analysis of (+) cedrol, a sesquiterpene present at levels of 13.1% (JSE Lv ) to 16.8% (JSE Con ) showed that this compound also activated neutrophil [Ca 2+ ] i ( Figure 1 and Table 4). Thus, cedrol represents a novel essential oil component with innate immunomodulatory activity. Note that we evaluated (+)-cedrol (Figure 2), because this isoform is present in Juniperus essential oils [89].   Since Juniperus essential oils and cedrol stimulated human neutrophil [Ca 2+ ] i , and it is well recognized that agonists can down regulate neutrophil responses to subsequent treatment with heterologous or homologous agonists [90], we evaluated whether Juniperus essential oils and/or cedrol could inhibit agonist-induced Ca 2+ mobilization in human neutrophils and FPR-transfected HL60 cells. As shown in Table 4, Juniperus essential oils inhibited [Ca 2+ ] i in f MLF-stimulated FPR1-HL60 cells and in WKYMVM-stimulated FPR2-HL60 cells with IC 50 values in the micromolar range. Most of the essential oils also inhibited f MLF-induced Ca 2+ flux in human neutrophils. However, essential oils from J. horizontalis cones (JH Con ) were inactive. Note that JH Con has a minimal content of sesqueterpenes (Tables 2 and 3), which seem to contribute to the observed biological activities of the other Juniperus essential oils. Analysis of cedrol, the unique sesquiterpene in essential oils from J. seravschanica, J. sabina, and J. pseudosabina (Table 2) showed that it also inhibited f MLF-induced neutrophil [Ca 2+ ] i (Table 4), and this effect was dose-dependent ( Figure 3), indicating that cedrol desensitized the neutrophils and FPR-transfected HL60 cells to subsequent agonist activation. Previously, we analyzed the effects of a number of essential oil compounds on human neutrophil Ca 2+ flux and found that many of the same compounds that are present in Juniperus essential oils, including α-pinene, β-pinene, sabinene, myrcene, α-terpinene, limonene, γ-terpinene, p-cymene, linalol, and terpinene-4-ol, had no effect on human neutrophils [86,87]. The only exception was germacrene D, which is present in JC Con , JC Lv , and JSI Con and was shown previously to active human neutrophil [Ca 2+ ] i [91]. On the other hand, these are the first studies to evaluate the effects of cedrol on neutrophil function.

Effect of Cedrol on Neutrophil Chemotaxis
Various essential oils and their components have been reported previously to inhibit neutrophil migration [86,87,91]. We found that pretreatment with JSE Con for 10 min dose-dependently inhibited f MLF-induced human neutrophil chemotaxis, with an IC 50 of 3.1 ± 1.1 µg/mL ( Figure 4A). Likewise, cedrol also inhibited f MLF-induced neutrophil chemotaxis, with an IC 50 of 10.6 ± 3.4 µM ( Figure 4B). These results are consistent with the ability of these treatments to inhibit agonist-induced [Ca 2+ ] i in human neutrophils and again support the conclusion that cedrol is a novel innate immunomodulator. To ensure that our analyses of neutrophil functional activity were not due to toxic effects, we evaluated cytotoxicity of the essential oils at a fairly high concentration (25 µg/mL) and cedrol (25 and 50 µM) in HL60 cells during 30 min and 90 min incubation periods. These incubation periods are comparable to the times used to measure Ca 2+ mobilization (up to 30 min) and cell migration (up to 90 min). As shown in Figure 5, some of the leaf essential oils had fairly high cytotoxic effects, especially during longer incubation times (e.g., JH Lv , JS Lv , and JSE Lv ). However, cedrol and JSE Con , the essential oil containing the highest concentration of cedrol, had little cytotoxicity after 30 or 90 min, verifying the absence of their cytotoxicity during the Ca 2+ flux and chemotaxis assays.

Identification of Potential Protein Targets for Cedrol
Cedrol has been reported to exhibit a number of biological activities, including anticancer [92][93][94] and anti-inflammatory. For example, cedrol was shown to have analgesic and anti-inflammatory effects in complete Freund's adjuvant (CFA)-induced arthritis in rats [95] and in mice with collagen-induced arthritis (CIA) [96]. This sesquiterpene also attenuated neuropathic pain in chronic constriction injury of rats by inhibiting the inflammatory response [97]. Despite the various biological activities reported for cedrol, little is known about its specific cellular targets. Thus, we performed reverse-pharmacophore mapping on the molecular structures of (+)-cedrol to identify potential biological targets. PharmMapper was used to compare a large database of pharmacophore patterns with these compounds and generate target information, including normalized fitness scores and pharmacophoric characteristics. It is important to submit a compound to the PharmMapper server in the form of the proper optical isomer, as this methodology explicitly accounts for 3D structure of a molecule. Thus, we evaluated the (+)-configuration of cedrol, which is the most common enantiomer found in Juniper plants [89]. The results of PharmMapper analysis indicated that three kinases could be potential targets for cedrol: proviral integration Moloney virus kinase (PIM1), vascular endothelial growth factor receptor 2 (VEGFR2), and c-Jun N-terminal kinase 1 (JNK1) ( Table 5). They are present among the 20 top-ranked targets found by PharmMapper. We also calculated the most important physico-chemical parameters for cedrol using SwissADME [98] (Table 6 and Figure 6) and found that cedrol would be predicted to permeate the blood-brain barrier (BBB). According to the radar plot, the ADME characteristics of (+)-cedrol predict that is would exhibit high bioavailability.

BBB permeation Yes
Abbreviations: M.W., molecular weight (g/mol); MR, molar refractivity; tPSA, topological polar surface area (Å 2 ); iLogP, lipophilicity; BBB, blood-brain barrier.   Table 1). The collected samples were air-dried for 7-10 days at room temperature away from direct sunlight before hydrodistillation. Botanical identification of the plant material collected in Kazakhstan was performed by botanist Anna A. Ivashchenko from Ile-Alatau National Park, Almaty, Kazakhstan, who has an extensive experience in the taxonomical identification of Cupressaceae species. Botanical identification of the plant material collected in Montana was performed by botanist Robyn A. Klein from Montana State University, Bozeman, MT, USA.

Essential Oil Extraction
Essential oils were obtained by hydrodistillation of dried plant material using a Clevenger-type apparatus, as previously described [86]. We used conditions accepted by the European Pharmacopoeia (European Directorate for the Quality of Medicines, Council of Europe, Strasbourg, France, 2014) to avoid artifacts. The essential oils collected in the graduated tube of the Clevenger-type apparatus were stored in amber vials and used for subsequent analyses. Yields were calculated based on the amount of air-dried plant material used. Stock solutions of the essential oils were prepared in DMSO (10 mg/mL) for biological evaluation and in n-hexane (10% w/v) for gas-chromatographic analysis.

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis
GC-MS analysis was performed with an Agilent 5975 GC-MSD system (Agilent Technologies, Santa Clara, CA, USA), as reported previously [99]. 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 carried out 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 appropriate operational conditions. 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.

Isolation of Human Neutrophils
For isolation of human neutrophils, blood was collected from healthy donors in accordance with a protocol approved by the Institutional Review Board at Montana State University (Protocol #MQ041017). 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 [100]. Isolated neutrophils were washed twice and resuspended in HBSS -. Neutrophil preparations were routinely >95% pure, as determined by light microscopy, and >98% viable, as determined by trypan blue exclusion. Neutrophils were obtained from multiple different donors (n = 8); however, the cells from different donors were never pooled during experiments.

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). 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 assess 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. To evaluate inhibitory effects of the compounds on FPR1/FPR2-dependent Ca 2+ flux, the compound/oil was added to the wells (final concentration of DMSO was 1%) with cells (human neutrophils or FPR1/FPR2 HL60 cells). The samples were preincubated for 10 min, followed by addition of 5 nM f MLF (for human neutrophils or FPR1-HL60 cells) or 5 nM WKYMVM (for FPR2-HL60 cells). 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 or 5 nM WKYMVM, which 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 fetal bovine serum (2 × 10 6 cells/mL), and chemotaxis was analyzed in 96-well ChemoTx 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. The lower wells were loaded with 30 µL of HBSS + containing 2% (v/v) fetal bovine serum and the indicated concentrations of test sample, DMSO (negative control), or 1 nM f MLF as a positive control. 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 luminescencebased 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 HL60 monocytic cells was analyzed with a CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega), according to the manufacturer's protocol. Briefly, HL60 cells were cultured at a density of 10 4 cells/well with different concentrations of essential oil or compound (final concentration of DMSO was 1%) for 30 min or 2 h at 37 • C and 5% CO 2 . Following treatment, substrate was added to the cells, and the samples were analyzed with a Fluoroscan Ascent FL microplate reader.

Molecular Modeling
The PharmMapper Server [101] was used for identifying the protein targets for (+)-cedrol. For a given small molecule, PharmMapper recognizes potential target possibilities using an "invert" pharmacophore mapping methodology. In several reference databases which are incorporated in the software, the protein biotargets are represented by sets of pharmacophore points that provide faster mapping. The PubChem database (https://pubchem.ncbi.nlm.nih.gov; accessed on 20 September 2021) was used as a source of initial 3D structures of the investigated compounds. The structure of (+)-cedrol (CID: 65575) was downloaded from PubChem in SDF format and further uploaded into the PharmMapper web server. Up to 300 conformers of each compound were automatically generated using a corresponding option of the software. The pharmacophore mapping was performed with the "Human Protein Targets Only" database containing 2241 targets. The top 250 potential targets per compound were retrieved and sorted by the normalized fit score. The physicochemical properties of (+)-cedrol were computed using SwissADMEe (http://www.swissadme.ch; accessed on 20 September 2021).

Statistical Analysis
One-way analysis of variance (ANOVA) was performed on the data sets, followed by Tukey's pair-wise comparisons. Pair-wise comparisons with differences at p < 0.05 were considered to be statistically significant.

Conclusions
Compositional analysis of 16 essential oils isolated from Juniper plants revealed similarities and differences between our analyses and those previous reported for essential oils from this species. Notably, our studies represent the first time that essential oils isolated from the cones of many of these Juniper species have been analyzed, including cones from J. sabina (JSA Con ), J. pseudosabina (JPS Con ), and J. pseudosabina subsp. turkestanica (JT Con ). Interestingly, several of the essential oil samples from Juniper collected in Kazakhstan but not in Montana contained high levels of cedrol, which was fairly unique among the samples and was enriched in JSE, JSA, and JPS. Evaluation of the biological activities of the parent essential oils or pure cedrol itself showed that they induced intracellular Ca 2+ mobilization in human neutrophils, which is a key component of neutrophil activation.
Indeed, pretreatment of cells with these essential oils or cedrol inhibited subsequent heterologous agonist-induced Ca 2+ mobilization, which is a common feature of many neutrophil agonists. Likewise, pretreatment with JSE Con (16.8% cedrol content) or pure cedrol inhibited human neutrophil chemotaxis toward N-formyl peptide. Thus, these data suggest that cedrol is a neutrophil agonist that can desensitize cells to subsequent stimulation by N-formyl peptide. However, further studies are necessary to define the precise mechanisms involved in the anti-inflammatory properties of cedrol.  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.