Chemical Composition and Immunomodulatory Activity of Essential Oils from Rhododendron albiflorum

Rhododendron (Ericaceae) extracts contain flavonoids, chromones, terpenoids, steroids, and essential oils and are used in traditional ethnobotanical medicine. However, little is known about the immunomodulatory activity of essential oils isolated from these plants. Thus, we isolated essential oils from the flowers and leaves of R. albiflorum (cascade azalea) and analyzed their chemical composition and innate immunomodulatory activity. Compositional analysis of flower (REOFl) versus leaf (REOLv) essential oils revealed significant differences. REOFl was comprised mainly of monoterpenes (92%), whereas sesquiterpenes were found in relatively low amounts. In contrast, REOLv was primarily composed of sesquiterpenes (90.9%), with a small number of monoterpenes. REOLv and its primary sesquiterpenes (viridiflorol, spathulenol, curzerene, and germacrone) induced intracellular Ca2+ mobilization in human neutrophils, C20 microglial cells, and HL60 cells transfected with N-formyl peptide receptor 1 (FPR1) or FPR2. On the other hand, pretreatment with these essential oils or component compounds inhibited agonist-induced Ca2+ mobilization and chemotaxis in human neutrophils and agonist-induced Ca2+ mobilization in microglial cells and FPR-transfected HL60 cells, indicating that the direct effect of these compounds on [Ca2+]i desensitized the cells to subsequent agonist activation. Reverse pharmacophore mapping suggested several potential kinase targets for these compounds; however, these targets were not supported by kinase binding assays. Our results provide a cellular and molecular basis to explain at least part of the beneficial immunotherapeutic properties of the R. albiflorum essential oils and suggest that essential oils from leaves of this plant may be effective in modulating some innate immune responses, possibly by inhibition of neutrophil migration.


Introduction
The genus Rhododendron belongs to the Ericaceae family of plants and includes more than 1000 identified species [1]. This genus is a source of flavonoids, tannins, essential oils, chromones, terpenoids, and steroids [2,3]. Rhododendron extracts have been reported to exhibit a diverse range of bioactivities, including antimicrobial, antioxidant, anticancer, antidiabetic, and anti-inflammatory activity [1,[4][5][6][7][8][9][10]. Additionally, extracts from various Rhododendron species have been utilized in traditional medicine for their anti-inflammatory properties. For example, R. albiflorum (cascade azalea) has been used in a poultice by the Syilx (Okanagan) and Thompson First Nations people to treat inflammatory conditions and by the Skokomish Indian Tribe as an extract for treating colds, sore throats, and cuts [11]. On the other hand, there are no publications regarding the biological activity of extracts from this plant.
Essential oils represent the volatile fraction of aromatic plants and have been actively investigated for their use in complementary or alternative medicine [12][13][14][15]. Thus, analysis of the chemical composition of essential oils from different plant species, and further evaluation of their biological properties, including immunomodulatory activity, can lead to the discovery of novel therapeutics. For example, essential oils from some Rhododendron species have been reported to be comprised of monoterpenes, sesquiterpenes, and their oxygenated derivatives (Table 1). These essential oils have also been found to be pharmacologically active [16], although little is known about their immunomodulatory activity and effects on innate immune system function. Table 1. Review of the major volatile constituents of Rhododendron essential oils.
The innate immune cells associated with most chronic neurodegenerative diseases are microglial cells [33]. These cells are resident macrophages of the central nervous system (CNS); phagocytose cellular debris; and foreign antigens, and contribute to pathological events, such as inflammation [34]. Microglial cells are capable of upregulating the synthesis and release of various inflammatory mediators [35], and excessive microglial activation can induce inflammation-mediated neuronal damage and degeneration. Nu-merous herbal compounds have been reported to suppress neurotoxicity via inhibiting microglial activation [36], and some essential oils or component compounds have been shown to have anti-inflammatory activity in microglial cells. For example, essential oils isolated from Artemisia herba-alba and Schisandra chinensis were reported to inhibit nitric oxide (NO) production induced by lipopolysaccharide (LPS) in murine BV2 microglial cells [37,38]. Similarly, linalool was reported to inhibit LPS-induced tumor necrosis factor (TNF), interleukin-1β, and NO production by BV2 cells [39]. On the other hand, the effects of R. albiflorum essential oils on microglial cells has not been evaluated.
Based on the reported anti-inflammatory properties of Rhododendron extracts, we hypothesized that some components in these extracts could have immunomodulatory activity. Additionally, the wide range of studies demonstrating immunomodulatory activity of essential oils led to the hypothesis that Rhododendron essential oils could be contributing to these therapeutic properties. Thus, we evaluate the chemical composition and immunomodulatory activity of essential oils isolated from the flowers and leaves of R. albiflorum. We show that essential oils from the leaves of R. albiflorum had a high content of sesquiterpenes, including viridiflorol, curzerene, spathulenol, bicyclogermacrene, germacrene B, and germacrone. Furthermore, we show that essential oils isolated from R. albiflorum leaves but not flowers inhibited neutrophil and microglial functional responses, including intracellular Ca 2+ mobilization and chemotaxis. Likewise, four of the major individual sesquiterpenes identified in R. albiflorum leaf essential oils also inhibited these functional responses, further defining the active components. Given the critical role of neutrophils and microglial cells in inflammation, our data support the possibility that these sesquiterpenes could be effective therapeutic compounds for the development anti-inflammatory agents.

Plant Material
R. albiflorum is found in British Columbia, Washington, Oregon, and western Montana. For these studies, we collected R. albiflorum flowers and leaves in July of 2020 during the flowering and fruiting stages on the west side of Table Mountain, Gallatin County, Montana, USA at an elevation of 2820 m above sea level. Botanical identification of the plant material was performed by botanist Robyn A. Klein from Montana State University (Bozeman, MT, USA). The samples were air-dried for 7-10 days at room temperature away from direct sunlight.

Essential Oil Isolation
Essential oils were obtained by hydrodistillation of dried plant material using a Clevenger type apparatus [31]. The yield of the essential oil was 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 (GC) 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) [40]. 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. Samples of 1 µL were injected, and the split ratio was adjusted to 40:1 to prevent overloading of the detectors. 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 the flame ionization (FI) and MS detectors, and a single injection was performed using the same column and appropriate operational conditions. The FI 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 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), as described previously [27]. Isolated neutrophils were washed and resuspended in HBSS -. Neutrophil preparations were routinely >95% pure and >98% viable. 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 ) in human neutrophils were measured with a FlexStation 3 scanning fluorometer (Molecular Devices), as previously described [27]. To assess the direct effects of test compounds or pure essential oils on Ca 2+ influx, the compounds/oils were 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+ influx, the compounds were 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%.
For analysis of Ca 2+ influx in C20 microglial cells, the cells were plated in 96-well black, clear bottom plates at 10 4 cells/well in DMEM/F12 medium containing 10% FBS. The cells were treated with 200 nM PMA for 24 h, and the medium was changed every day for 5 days. On day 5, the cells were loaded with FLIPR Calcium 5 (Molecular Devices) at a volume ratio of 1:1 for 30 min at 37 • C in the dark. The plates were then placed in a FlexStation 3 fluorometer, and basal fluorescence was measured (λ ex = 485 nm, λ em = 538 nm). Essential oils or individual compounds of interest were added manually (final concentration of DMSO was 1%), and fluorescence was monitored for 2 min to assess the direct effects of these treatments on [Ca 2+ ] i . After a 10 min incubation at 37 • C, the fluorescence baseline was recorded again, and 10 µM f MLF was added to evaluate inhibitory effects on agonist-induced [Ca 2+ ] i . Responses were normalized to the response induced by 10 µM f MLF.
For all Ca 2+ influx experiments, 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 7 (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), as described previously [27]. 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 8.

Cytotoxicity Assay
Cytotoxicity of essential oils and pure compounds was analyzed in human promyelocytic HL60 cells using a CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega), as described previously [31].

Kinase K d Determination
KINOMEscan ®® was used to determine the dissociation constant (K d ) of the indicated sesquiterpenes for selected kinases [42] (Eurofins Pharma Discovery, San Diego, CA, USA). A 12-point half-log dilution series (maximum concentration of 33 µM) was used for K d determination. Assays were performed in duplicate, and the average mean value is shown.

Molecular Modeling
PharmMapper [43] was used for identifying putative protein targets for (−)-curzerene, (+) and (−) enantiomers of spathulenol, germacrene B, germacrone, and (+)-viridiflorol. For a given small molecule, PharmMapper recognizes potential target possibilities using an "invert" pharmacophore mapping methodology. In several reference databases that 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 20 February2021) was used as a source of initial 3D structures for the investigated compounds. The structures of (−)-curzerene (CID: 12305300), (−)-spathulenol (CID: 13854255), (+)-spathulenol (CID: 92231), germacrene B (CID: 5281519), germacrone (CID: 6436348), and (+)-viridiflorol (CID: 11996452) were downloaded from PubChem in SDF format and further uploaded into PharmMapper. Up to 300 conformers of each compound were automatically generated using a corresponding option of the software. 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 selected compounds were computed using SwissADME (http://www.swissadme.ch; accessed 25 February 2021) [44]. Structures of the main sesquiterpenes found in REO Lv and used for molecular modeling are shown in Figure 1.

R PEER REVIEW
6 of 20 compound were retrieved and sorted by the normalized fit score. The physicochemical properties of selected compounds were computed using SwissADME (http://www.swissadme.ch; accessed 25 February 2021) [44]. Structures of the main sesquiterpenes found in REOLv and used for molecular modeling are shown in Figure 1.

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.

Essential Oil Composition
The yields (v/w) of essential oils obtained from R. albiflorum flowers (designated as REOFl) and leaves (designated as REOLv) were 0.4% and 0.5%, respectively. The chemical composition of these essential oils was evaluated using simultaneous GC-FID and GC-MS, and Table 2 summarizes the identified compounds, percentage composition, and relative retention indices (RRI) (compounds are listed in order of their elution). emical composition of R. albiflorum essential oils (%) isolated from flowers (REOFl) and leaves (REOLv) a .

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.

Essential Oil Composition
The yields (v/w) of essential oils obtained from R. albiflorum flowers (designated as REO Fl ) and leaves (designated as REO Lv ) were 0.4% and 0.5%, respectively. The chemical composition of these essential oils was evaluated using simultaneous GC-FID and GC-MS, and Table 2 summarizes the identified compounds, percentage composition, and relative retention indices (RRI) (compounds are listed in order of their elution). A total of 63 constituent compounds were identified in R. albiflorum essential oils. Specifically, 34 compounds were identified in REO Fl , representing~99.8% of the total essential oil composition. The main components of REO Fl were terpinolene (37.7%), limonene (14.2%), β-phellandrene (8.9%), γ-terpinene (7.1%), (Z)-β-ocimene (6.5%), p-cymene (2.8%), and curzerene (2.2%). Twenty-three other compounds were present at concentrations from 0.1% to <2.0%. In comparison, 41 compounds were identified in REO Lv , represent-ing~92.2% of the total essential oil composition. The main components of REO Lv were viridiflorol (22.0%), curzerene (17.8%), spathulenol (14.4%), bicyclogermacrene (8.9%), germacrene B (6.8%), β-elemenone (5.3%), germacrone (3.3%), γ-elemene (2.4%), and βelemene (2.2%). Fourteen other compounds were present at concentrations from 0.1% to <2%. The remaining volatile compounds identified in both essential oil samples were present in trace amounts (<0.1%). Overall, there were significant differences in essential oil composition between R. albiflorum flowers and leaves, with the major components of REO Fl being monoterpenes, such as monoterpene hydrocarbons (87%) and oxygenated monoterpenes (5.0%). In contrast, the main components of REO Lv were sesquiterpene hydrocarbons (40.9%) and oxygenated sesquiterpenes (50.0%) ( Table 3). Note that spathulenol, which we found to be present in REO Lv , was previously reported to be a major component compound in R. micranthum essential oils [21]. However, this is the first report to show that the sesquiterpenoids viridiflorol, curzerene, bicyclogermacrene, germacrene B, and germacrone are also major components of essential oils from Rhododendron spp. R. albiflorum essential oils and commercially available individual compounds were evaluated for their effects on human neutrophils and human C20 microglial cells. Specifically, we evaluated their effects on [Ca 2+ ] i , which is a key component of phagocyte activation [45,46]. We found that REO Lv treatment increased [Ca 2+ ] i , with EC 50 values of 18.6 µg/mL and 22.8 µg/mL in neutrophils and C20 microglial cells, respectively (Table 4). In addition, analysis of the major sesquiterpenes that comprised 57.5% of REO Lv (viridiflorol, spathulenol, curzerene, and germacrone) showed that these compounds also activated neutrophil Ca 2+ influx, with the most potent being viridiflorol (Table 4, Figure 2). Likewise, viridiflorol, curzerene, and germacrone also increased C20 microglial cell [Ca 2+ ] i , whereas spathulenol was inactive or had very low activity in these cells (Table 4). In any case, it is clear that viridiflorol, which is the major compound in REO Lv , is one of the principal molecules responsible for neutrophil and microglial cell activation.

Effect of R. albiflorum Essential Oils and Component Compounds on Agonist-Induced Ca 2+ Influx
Activation of Ca 2+ influx, specific receptors, or other unidentified molecular targets by agonists can result in the desensitization and subsequent downregulation of neutrophil

Effect of R. albiflorum Essential Oils and Component Compounds on Agonist-Induced Ca 2+ Influx
Activation of Ca 2+ influx, specific receptors, or other unidentified molecular targets by agonists can result in the desensitization and subsequent downregulation of neutrophil responses [29,47]. Essential oils and their components have been reported previously to modulate [Ca 2+ ] i and inhibit cell migration [29][30][31][32]. Thus, we evaluated R. albiflorum essential oils for their effects on agonist-induced neutrophil and microglial cell activation. As shown in Figure 3, REO Lv potently inhibited neutrophil Ca 2+ influx induced by the agonist f MLF, with an IC 50 of 2.7 µg/mL. Similarly, REO Lv inhibited Ca 2+ influx in f MLFactivated C20 microglial cells and FPR1-HL60 cells, as well as in WKYMVM-activated FPR2-HL60 cells ( Table 5). As expected from our results above, REO Fl had little effect on [Ca 2+ ] i in f MLF-stimulated neutrophils, even at very high REO Fl concentrations (Figure 3). Similarly, REO Fl had no effect on [Ca 2+ ] i in microglial, FPR1-HL60, and FPR2-HL60 cells (Table 5).
Molecules 2021, 26, x FOR PEER REVIEW 10 of 2 Figure 3. Effect of R. albiflorum essential oils on fMLF-induced neutrophil Ca 2+ mobilization. Human neutrophils were treated with the indicated concentrations of the REOLv, REOFl, or 1% DMSO (neg ative control) for 10 min. The cells were then activated by 5 nM fMLF, and [Ca 2+ ]i was monitored a described. The data shown are presented as the mean ± SD from one experiment that is representa tive of three independent experiments with similar results.  We next evaluated the effects of individual constituent compounds on agonist-induced Ca 2+ mobilization in human neutrophils, C20 microglial cells, and FPR-transfected HL60 cells. As shown in Table 5, the four main sesquiterpenes in REO Lv (viridiflorol, curzerene, spathulenol, and germacrone) inhibited f MLF-induced Ca 2+ influx in human neutrophils, microglial cells, and FPR1-HL60 cells and in WKYMVM-stimulated FPR2-HL60 cells, with IC 50 values in the micromolar range. As an example, the dose-dependent inhibition of f MLF-induced neutrophil Ca 2+ mobilization by viridiflorol is shown in Figure 4. These results suggest that the direct effect of these compounds on [Ca 2+ ] i (see Table 4) desensitized the cells to subsequent agonist activation. In support of this idea, we found previously that three other sesquiterpenes that are present in REO Lv (β-caryophyllene, α-humulene, and germacrene D) also inhibited agonist-induced Ca 2+ mobilization and thus desensitized human neutrophils to further agonist activation [32]. curzerene, spathulenol, and germacrone) inhibited fMLF-induced Ca 2+ influx in human neutrophils, microglial cells, and FPR1-HL60 cells and in WKYMVM-stimulated FPR2-HL60 cells, with IC50 values in the micromolar range. As an example, the dose-dependent inhibition of fMLF-induced neutrophil Ca 2+ mobilization by viridiflorol is shown in Figure  4. These results suggest that the direct effect of these compounds on [Ca 2+ ]i (see Table 4) desensitized the cells to subsequent agonist activation. In support of this idea, we found previously that three other sesquiterpenes that are present in REOLv (β-caryophyllene, αhumulene, and germacrene D) also inhibited agonist-induced Ca 2+ mobilization and thus desensitized human neutrophils to further agonist activation [32]. Evaluation of the effects of β-phellandrene, one of the principal monoterpenes in RE-OFl (8.9%), on agonist-induced neutrophil or HL60-FPR1/FPR2 Ca 2+ influx showed that it had no effect (Table 5), which is consistent with its lack of activity as a direct neutrophil agonist (see Table 4). In addition, our previous studies on a number of compounds that we determined here to comprise 77.9% of REOFl (α-pinene, camphene, β-pinene, sabinene, Evaluation of the effects of β-phellandrene, one of the principal monoterpenes in REO Fl (8.9%), on agonist-induced neutrophil or HL60-FPR1/FPR2 Ca 2+ influx showed that it had no effect (Table 5), which is consistent with its lack of activity as a direct neutrophil agonist (see Table 4). In addition, our previous studies on a number of compounds that we determined here to comprise 77.9% of REO Fl (α-pinene, camphene, β-pinene, sabinene, myrcene, α-terpinene, limonene, (E/Z)-β-ocimene, γ-terpinene, p-cymene, terpinolene, linalool, and terpinen-4-ol) showed that they had no inhibitory effect on agonist-induced neutrophil Ca 2+ mobilization [29,31,32]. Thus, these previous results together with our current analysis of β-phellandrene again serve to explain why REO Fl does not desensitize neutrophil agonist-induced activation.

Effect of R. albiflorum Essential Oils and Component Compounds on Neutrophil Chemotaxis
Various essential oils and their components have been reported previously to inhibit cell migration [29,48,49]. We found that pretreatment with REO Lv for 10 min dosedependently inhibited f MLF-induced human neutrophil chemotaxis, with an IC 50 of 3.3 µg/mL (Table 5). Likewise, the individual constituent compounds viridiflorol, curzerene, spathulenol, and germacrone also inhibited neutrophil chemotaxis, with the most potent compounds being spathulenol and germacrone ( Table 5). As an example, the dosedependent inhibition neutrophil chemotaxis by viridiflorol is shown in Figure 5. In contrast, REO Fl and the monoterpene β-phellandrene were both inactive in this assay (Table 5).

Effect of R. albiflorum Essential Oils and Component Compounds on Neutrophil Chemotaxis
Various essential oils and their components have been reported previously to inhibit cell migration [29,48,49]. We found that pretreatment with REOLv for 10 min dose-dependently inhibited fMLF-induced human neutrophil chemotaxis, with an IC50 of 3.3 μg/mL (Table 5). Likewise, the individual constituent compounds viridiflorol, curzerene, spathulenol, and germacrone also inhibited neutrophil chemotaxis, with the most potent compounds being spathulenol and germacrone ( Table 5). As an example, the dose-dependent inhibition neutrophil chemotaxis by viridiflorol is shown in Figure 5. In contrast, REOFl and the monoterpene β-phellandrene were both inactive in this assay (Table 5). To ensure that the effects of these essential oils or individual compounds on neutrophil functional activity were not influenced by possible toxicity, we evaluated cytotoxicity of REOFl and REOLv (up to 25 μg/mL) and test compounds at various concentrations (up to 25 μM) in HL60 cells during 30 min and 2 h 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 1.5 h). As shown in Figure 6, REOLv, REOFl, viridiflorol, spathulenol, curzerene, and germacrone had minimal effects on cell viability during a 30 min incuba- To ensure that the effects of these essential oils or individual compounds on neutrophil functional activity were not influenced by possible toxicity, we evaluated cytotoxicity of REO Fl and REO Lv (up to 25 µg/mL) and test compounds at various concentrations (up to 25 µM) in HL60 cells during 30 min and 2 h 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 1.5 h). As shown in Figure 6, REO Lv , REO Fl , viridiflorol, spathulenol, curzerene, and germacrone had minimal effects on cell viability during a 30 min incubation, verifying the absence of cytotoxicity during the Ca 2+ influx assay period ( Figure 6). Likewise, these samples generally had minimal cytotoxicity during the 2 h incubation, except for the highest concentrations of viridiflorol (25 µM and 2-h incubation), which exhibited a little more cytotoxicity that could have some effect on the cell migration assay.

Identification of Potential Protein Targets for Selected Sesquiterpenes
The sesquiterpene compounds identified in REO Lv have been reported to exhibit a number of biological activities. For example, viridiflorol has been shown to inhibit carrageenan-induced mouse paw edema [50]. This compound has also been shown to be a potent inhibitor of biofilm formation [51]. Curzerene has been shown to have antiproliferative effects in SPC-A1 human lung adenocarcinoma cells [52] and SKMEL-19 melanoma cells [53]. Spathulenol has been shown to inhibit formalin-induced nociceptive sensitivity and carrageenan-induced mechanical hyperalgesia in mice [54], as well as carrageenaninduced mouse paw oedema [55]. Spathulenol has also been reported to be cytotoxic for B16-F10, HepG2, K562, and HL60 cell lines (IC 50 from 18 to 52 µM after 72 h) [56]. Finally, spathulenol has been reported to exhibit spasmolytic acivity, possibly by blocking voltageoperated calcium channels [57]. Germacrone is one of the main bioactive components in the traditional Chinese medicine Rhizoma curcuma [58] and has been reported to possess anti-inflammatory, antiviral, antitumor, and immunomodulatory properties [59][60][61][62][63][64][65][66][67]. For example, germacrone has been reported to alleviate symptoms of collagen-induced arthritis by regulating the T helper type 1 and 2 (Th1/Th2) cell balance and nuclear factor κB (NF-κB) activation [68]. It has also been reported to reduce cerebral ischemia/reperfusion injury in rats via antioxidative and antiapoptotic mechanisms [69]. Finally, germacrone has been reported to inhibit Ca 2+ -activated Cl − currents and K + channel activity [70].
Despite the various biological activities reported for these compounds, little is known about their specific cellular targets. Thus, we performed reverse-pharmacophore mapping on the molecular structures of viridiflorol, curzerene, spathulenol, germacrene B, and germacrone to identify potential biological targets. Note that pharmacophore mapping of bicyclogermacrene (comprises 8.9% in REO Lv ) was previously reported [32]. 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. Specifically, we evaluated the (+)-configuration of viridiflorol [71] and the (−)-configuration of curzerene, which are the most common enantiomers found in higher plants [72]. Both the (+) and (−) enantiomers of spathulenol have been found in higher plants. For example, (+)-spathulenol was identified in essential oils from Piper species [73], Salvia hydrangea [74], Aloysia gratissima [75], Eremophila mitchellii [76], and extracts from Merremia dissecta [77]. In addition, (−)-spathulenol was found in essential oils from Elytropappus rhinocerotis [78], Annona squamosal [79], Chrysanthemum [80], Artemisia annua [81], and Cleome spinose [82]. Thus, we analyzed both enantiomers of this compound.

Identification of Potential Protein Targets for Selected Sesquiterpenes
The sesquiterpene compounds identified in REOLv have been reported to exhibit a number of biological activities. For example, viridiflorol has been shown to inhibit carrageenaninduced mouse paw edema [50]. This compound has also been shown to be a potent inhibitor of biofilm formation [51]. Curzerene has been shown to have antiproliferative effects in SPC-A1 human lung adenocarcinoma cells [52] and SKMEL-19 melanoma cells [53]. Spathulenol has been shown to inhibit formalin-induced nociceptive sensitivity and carrageenan-induced mechanical hyperalgesia in mice [54], as well as carrageenan-induced mouse paw oedema  MAPKAPK2, JNK1/3, CD11a, and PIM1 represent potential targets that could contribute to the direct inhibitory effects of REO Lv and its primary sesquiterpenes on human neutrophil function, such as chemotaxis. For example, neutrophil arrest and migration involves integrin α-L (CD11a) [83]. In neutrophils, the major substrate of MAPKAPK2 is the leukocyte specific protein 1 (LSP1), which binds to F-actin and participates directly in cell migration [84]. Imperatorin (furocoumarin) inhibits human neutrophil migration through inhibition of JNK and Ca 2+ mobilization [85]. In addition, mixed lineage kinase 3 (MLK3)-JNK signaling has been reported to play a role in the regulation of neutrophil migration [86]. Likewise, PIM kinases have been reported to promote cell migration and invasion [87].
Based on the possibility that MAPKAPK2, JNK3, and PIM1 could interfere with phagocyte migration, we evaluated the binding affinity of pure viridiflorol, curzerene, spathulenol, and germacrone toward these three kinases but did not observe any binding activity. Nevertheless, we still cannot exclude a role for integrin α-L (CD11a) as a target for viridiflorol and spathulenol in human neutrophils.
We calculated the most important physico-chemical parameters for these sesquiterpenes using SwissADME [44] (Table 7) and found that the compounds are very similar to each other in terms of many ADME properties. Nevertheless, they differed noticeably in iLogP and tPSA [88]. These descriptors are usually related to the capacity of molecules to cross cellular membranes [89]. For example, germacrene B has the highest iLogP and lowest tPSA values and was calculated to be a compound that would not permeate the blood-brain barrier (BBB). In the current studies, we evaluated effects of R. albiflorum essential oils and, more specifically, individual component compounds on innate immune cells in vitro. Since we observed potentially beneficial immunomodulatory effects and low cytotoxicity, the next step would be to evaluate the selected compounds in vivo, and these studies are being considered. Indeed, previous studies have shown that in vivo treatment with sesquiterpenes can be beneficial for various clinical problems. For example, sesquiterpenes are currently under clinical evaluation for cancer treatment (reviewed in [90,91]). Likewise, animal studies with artemisinin and its semi-synthetic sesquiterpene derivative artesunate have shown that these compounds are effective in vivo treatments using animal models of autoimmune encephalomyelitis [92] and Alzheimer's disease [93,94]. Furthermore, the sesquiterpene huperzine A is currently used clinically to improve memory and mental function in people with Alzheimer's disease or other neurodegenerative diseases [95]. Thus, the potential for clinical development of the sesquiterpenes identified here is clearly feasible.

Conclusions
Essential oils isolated from the leaves of R. albiflorum contain a high amount of sesquiterpenes (up to 91%), and these essential oils can induce human neutrophil and microglial cell Ca 2+ influx, which desensitizes these cells to subsequent agonist-induced functional responses. Moreover, the major constituents of R. albiflorum leaf essential oils (viridiflorol, curzerene, spathulenol, and germacrone) exhibited the same effects, inhibiting agonist-induced Ca 2+ mobilization and chemotaxis in human neutrophils and agonistinduced Ca 2+ mobilization in microglial cells and FPR-transfected HL60 cells. Thus, our data provide a molecular basis to explain at least part of the beneficial therapeutic effects of R. albiflorum essential oils and component compounds and suggest that inhibition of innate immune cells by component compounds of this essential oil might have anti-inflammatory effects. Future studies are now in progress to evaluate the potential of Rhododendron essential oils as therapeutic remedies for various disorders with immune and/or inflammatory mechanisms, including Alzheimer's disease, as well as to determine the molecular targets of the active compounds.