Chemical Composition and Immunomodulatory Activity of Hypericum perforatum Essential Oils

Hypericum L. (Hypericaceae) extracts have been used for their therapeutic effects; however, not much is known about the immunomodulatory activity of essential oils extracted from this plant. We isolated essential oils from the flowers and leaves of H. perforatum and analyzed their chemical composition and innate immunomodulatory activity. Analysis of flower (HEOFl) versus leaf (HEOLv) essential oils using gas chromatography–mass spectrometry revealed that HEOFl was comprised mainly of monoterpenes (52.8%), with an abundance of oxygenated monoterpenes, including cis-p-menth-3-en-1,2-diol (9.1%), α-terpineol (6.1%), terpinen-4-ol (7.4%), and limonen-4-ol (3.2%), whereas the sesquiterpenes were found in trace amounts. In contrast, HEOLv was primarily composed of sesquiterpenes (63.2%), including germacrene D (25.7%) and β-caryophyllene (9.5%). HEOLv also contained oxygenated monoterpenes, including terpinen-4-ol (2.6%), while monoterpene hydrocarbons were found in trace amounts. Both HEOFl and HEOLv inhibited neutrophil Ca2+ mobilization, chemotaxis, and reactive oxygen species (ROS) production, with HEOLv being much more active than HEOFl. Furthermore, the pure sesquiterpenes germacrene D, β-caryophyllene, and α-humulene also inhibited these neutrophil responses, suggesting that these compounds represented the active components of HEOLv. Although reverse pharmacophore mapping suggested that potential protein targets of germacrene D, β-caryophyllene, bicyclogermacrene, and α-humulene could be PIM1 and mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MAPKAK2), a kinase binding affinity assay did not support this finding, implying that other biological targets are involved. Our results provide a cellular and molecular basis to explain at least part of the beneficial immunotherapeutic properties of the H. perforatum essential oils.


Introduction
Hypericum L. (Hypericaceae) is comprised of approximately 500 species that can be found around the world. Various products from Hypericum species have been used as antidepressant, sedative, diuretic, antiphlogistic, analgesic, astringent, and antipyretic remedies in Europe, America,

Essential Oil Extraction
Essential oils were obtained by hydrodistillation of dried plant material using a Clevenger apparatus, as previously described [26]. 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 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 analysis.

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis
GC-MS analysis was performed using an Agilent 5975 GC-MSD system (Agilent Technologies, Santa Clara, CA, USA), as reported previously [27]. 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 on 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. The ionization detector (FID) temperature was 300 • C. Essential oil components were identified by co-injection with standards (whenever possible), which were purchased commercially 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
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 #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 [22]. 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 neutrophil intracellular Ca 2+ concentrations ([Ca 2+ ] i ) were measured using a FlexStation 3 scanning fluorometer (Molecular Devices, Sunnyvale, CA, USA). Briefly, human neutrophils suspended in HBSS − were loaded with Fluo-4AM at a final concentration of 1.25 µg/mL and incubated for 30 min in the dark at 37 • C. The cells were then washed with HBSS − , resuspended in HBSS + , and aliquoted into the wells of flat-bottom, half-area 96-well black microtiter plates (2 × 10 5 cells/well). Essential oils or pure compounds diluted in DMSO were added to the wells (final concentration of DMSO was 1%). The samples were preincubated for 10 min, followed by addition of 5 nM f MLF. 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/oil. The maximum change in fluorescence, expressed in arbitrary units over baseline, was used to determine the response. Responses were normalized to the response induced by 5 nM f MLF, which was assigned a value of 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 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). After preincubation with the indicated concentrations of the test sample (essential oil or pure compound) or DMSO (1% final concentration) for 30 min at room temperature, the cells were 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 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 8.

ROS Production Assay
ROS production was determined by monitoring L-012-enhanced chemiluminescence, which is a reliable method for detecting superoxide anion (O 2 − ) production [22]. Human neutrophils were resuspended at 2 × 10 5 cells/mL in HBSS + supplemented with 40 µM L-012. Cells (100 µL) were aliquoted into wells of 96-well flat-bottomed microtiter plates containing essential oil or compounds at different concentrations (final DMSO concentration of 1%). Cells were preincubated for 10 min, and 200 nM PMA was added to each well to stimulate ROS production. Luminescence was monitored for 120 min (2-min intervals) at 37 • C using a Fluoroskan Ascent FL microtiter plate reader (Thermo Electron, Waltham, MA, USA). The curve of light intensity (in relative luminescence units) was plotted against time, and the area under the curve was calculated as total luminescence. Compound concentrations that inhibited ROS production by 50% of the PMA-induced response (positive control) were determined by graphing the percentage inhibition of ROS production versus the logarithm of concentration of test sample (IC 50 ). Each curve was determined using five to seven concentrations.

Kinase K d Determination
Selected sesquiterpenes were submitted for dissociation constant (K d ) determination toward PIM1 and MAPKAPK2 using KINOMEscan [28] (Eurofins Pharma Discovery, San Diego, CA, USA).
For dissociation constant K d determination, a 12-point half-log dilution series (a maximum concentration of 33 µM) was used. Assays were performed in duplicate, and their average mean value is displayed.

Human Neutrophil Elastase (HNE) Inhibition Assay
Essential oils and individual compounds were dissolved in 100% DMSO at 5 mM stock concentrations. The final concentration of DMSO in the reactions was 1%, and this level of DMSO had no effect on enzyme activity. Sivelestat, a known HNE inhibitor, was used as a positive control. The inhibition assay was performed, as described previously [29]. Briefly, a solution containing 200 mM Tris-HCl (pH 7.5), 0.01% bovine serum albumin, 0.05% Tween-20, and 20 mU/mL of human neutrophil elastase was added to black, flat-bottom 96-well microtiter plates containing different concentrations of test compounds. Reactions were initiated by addition of 25 µM elastase substrate N-methylsuccinyl-Ala-Ala-Pro-Val-7-amino-4-methylcoumarin in a final reaction volume of 100 µL/well. Kinetic measurements were obtained every 30 s for 10 min at 25 • C using a Fluoroskan Ascent FL fluorescence microplate reader (Thermo Electron, MA, USA) with excitation and emission wavelengths at 355 and 460 nm, respectively. The concentration of compound that caused 50% inhibition of the enzymatic reaction (IC 50 ) was calculated by plotting % inhibition versus logarithm of inhibitor concentration.

Cytotoxicity Assay
Human promyelocytic leukemia HL-60 cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated FBS, 10 mM HEPES, 100 µg/mL streptomycin, and 100 U/mL penicillin. Cytotoxicity was analyzed with a CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega, Madison, WI, USA), according to the manufacturer's protocol. Briefly, HL-60 cells were cultured at a density of 1 × 10 5 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 Fluoroskan Ascent FL microplate reader.

Molecular Modeling
Structures of the main sesquiterpenes found in HEO Lv and used for molecular modeling are shown in Figure 1. The protein targets for β-caryophyllene, (−)-germacrene D, (+)-bicyclogermacrene, and α-humulene were analyzed using the PharmMapper Server [30]. This online tool is intended to recognize potential target possibilities for a given small molecule through an "invert" pharmacophore mapping approach. The software uses several built-in reference databases of protein drug targets encoded by sets of pharmacophore points for faster mapping. Initial 3D structures of the investigated compounds were downloaded from the PubChem database (https://pubchem.ncbi.nlm.nih.gov) and saved in Tripos MOL2 format. The MOL2 files of (−)-β-caryophyllene, (−)-germacrene D, (+)-bicyclogermacrene, and α-humulene (PubChem compound CIDs: 5281515, 5317570, 5315347, and 5281520, respectively; see Figure 1) were uploaded into the PharmMapper web server. Automatic generation of up to 300 conformers for each compound was switched on. The "Human Protein Targets Only" database containing 2241 targets was selected for pharmacophore mapping. The top 250 potential targets were retrieved and sorted by normalized fit score value. The physicochemical properties of selected compounds were computed using SwissADME (http://www.swissadme.ch) [31].

Human Neutrophil Elastase (HNE) Inhibition Assay
Essential oils and individual compounds were dissolved in 100% DMSO at 5 mM stock concentrations. The final concentration of DMSO in the reactions was 1%, and this level of DMSO had no effect on enzyme activity. Sivelestat, a known HNE inhibitor, was used as a positive control. The inhibition assay was performed, as described previously [29]. Briefly, a solution containing 200 mM Tris-HCl (pH 7.5), 0.01% bovine serum albumin, 0.05% Tween-20, and 20 mU/mL of human neutrophil elastase was added to black, flat-bottom 96-well microtiter plates containing different concentrations of test compounds. Reactions were initiated by addition of 25 µ M elastase substrate N-methylsuccinyl-Ala-Ala-Pro-Val-7-amino-4-methylcoumarin in a final reaction volume of 100 µ L/well. Kinetic measurements were obtained every 30 s for 10 min at 25 °C using a Fluoroskan Ascent FL fluorescence microplate reader (Thermo Electron, MA, USA) with excitation and emission wavelengths at 355 and 460 nm, respectively. The concentration of compound that caused 50% inhibition of the enzymatic reaction (IC50) was calculated by plotting % inhibition versus logarithm of inhibitor concentration.

Cytotoxicity Assay
Human promyelocytic leukemia HL-60 cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated FBS, 10 mM HEPES, 100 µ g/mL streptomycin, and 100 U/mL penicillin. Cytotoxicity was analyzed with a CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega, Madison, WI, USA), according to the manufacturer's protocol. Briefly, HL-60 cells were cultured at a density of 1 × 10 5 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% CO2. Following treatment, substrate was added to the cells, and the samples were analyzed with a Fluoroskan Ascent FL microplate reader.

Molecular Modeling
Structures of the main sesquiterpenes found in HEOLv and used for molecular modeling are shown in Figure 1. The protein targets for β-caryophyllene, (−)-germacrene D, (+)-bicyclogermacrene, and α-humulene were analyzed using the PharmMapper Server [30]. This online tool is intended to recognize potential target possibilities for a given small molecule through an "invert" pharmacophore mapping approach. The software uses several built-in reference databases of protein drug targets encoded by sets of pharmacophore points for faster mapping. Initial 3D structures of the investigated compounds were downloaded from the PubChem database (https://pubchem.ncbi.nlm.nih.gov) and saved in Tripos MOL2 format. The MOL2 files of (−)-βcaryophyllene, (−)-germacrene D, (+)-bicyclogermacrene, and α-humulene (PubChem compound CIDs: 5281515, 5317570, 5315347, and 5281520, respectively; see Figure 1) were uploaded into the PharmMapper web server. Automatic generation of up to 300 conformers for each compound was switched on. The "Human Protein Targets Only" database containing 2241 targets was selected for pharmacophore mapping. The top 250 potential targets were retrieved and sorted by normalized fit score value. The physicochemical properties of selected compounds were computed using SwissADME (http://www.swissadme.ch) [31].

Essential Oil Composition
Although the chemical composition of H. perforatum essential oils has been reported previously in several publications [9,11,[32][33][34][35][36][37][38][39], there is a wide variation in the reported levels of secondary metabolites from different H. perforatum plant samples (see Table 1 for a summary of results from recent studies since 2010). This variability can impact the specific pharmacological activity of essential oils/extracts [40,41]. In addition, few studies have investigated flower and leaf essential oils separately, and there are no publications on the chemical composition of essential oils from H. perforatum collected in the Rocky Mountain region of the United States. Thus, we analyzed the essential oil composition of flowers and leaves from H. perforatum samples collected in this region. Table 1. Review of the major volatile constituents of H. perforatum essential oils (2010-2020).

Effect of the Essential Oils and Their Components on Neutrophil Functional Responses
Essential oils and their components have been reported previously to modulate intracellular Ca 2+ flux and inhibit cell migration [24][25][26]. We screened Hypericum essential oils for neutrophil immunomodulatory activity and evaluated the effects of HEO Fl and HEO Lv and selected compounds on neutrophil activation.
As shown in Table 4, both HEO Fl and HEO Lv inhibited intracellular Ca 2+ flux in f MLF activated neutrophils, although HEO Lv was~5-fold more potent than HEO Fl . A representative time course for the inhibition of f MLF-stimulated Ca 2+ flux by HEO Fl and HEO Lv (25 µg/mL each) is shown in Figure 2. We next considered the effects of individual constituent compounds on neutrophil Ca 2+ mobilization in an effort to identify the active component(s). Previously, we analyzed the effect of a number of these same compounds on neutrophil Ca 2+ flux, including 16 compounds that we found here to comprise 24.0% of HEO Fl and 29.6% of HEO Lv [24,26]. These data are included in Table 4 for reference. As shown in Table 4, β-pinene, sabinene, and γ-terpinene, which represent 11.3% of HEO Lv , were found previously to have no effect on neutrophil Ca 2+ mobilization and thus are likely not involved in the inhibitory effects of Hypericum essential oils. In contrast, we found previously that 6-methyl-3,5-heptadien-2-one (MHDO) inhibited neutrophil Ca 2+ flux, although it is present in only trace amounts in HEO Fl (<1.0%). Thus, it is possible that MHDO contributes to the inhibition observed with HEO Fl treatment, but it is more likely that there are other inhibitory compounds in HEO Fl . Unfortunately, pure samples of the main compounds in HEO Fl , such as 3-methoxy-2,3-dimethylcyclobutene (MDCB, 9.8%), cis-p-menth-3-en-1,2-diol (9.1%), and 4-hydroxy-4-methyl-cyclohex-2-enone (HMCH, 3.4%), are not commercially available for testing. In HEO Fl , most of the unidentified compounds are oxygenated constituents, which we could not identify by MS data alone, and their relative amounts were <0.5% except for a few at~1.0%. Since we identified 71.3% of the HEO Fl components, the unidentified active components may also be present in the remaining 28.7% of unknown compounds, and further investigation will be needed to identify these components.  Human neutrophils were pretreated for 10 min with 25 µ g/mL of the indicated essential oil or 1% DMSO (negative control), followed by stimulation with 5 nM fMLF (indicated by arrow), and Ca 2+ flux was monitored for the indicated times. The data are from one experiment that is representative of three independent experiments.
We also evaluated the effect of the sesquiterpenes germacrene D, α-humulene (also known as αcaryophyllene), and β-caryophyllene, which are principal components of HEOLv, and the monoterpenoid geraniol, a minor component of both HEOFl and HEOLv, on neutrophil Ca 2+ Figure 2. Effect of HEO Lv and HEO Fl on f MLF-induced Ca 2+ mobilization in human neutrophils. Human neutrophils were pretreated for 10 min with 25 µg/mL of the indicated essential oil or 1% DMSO (negative control), followed by stimulation with 5 nM f MLF (indicated by arrow), and Ca 2+ flux was monitored for the indicated times. The data are from one experiment that is representative of three independent experiments.
We also evaluated the effect of the sesquiterpenes germacrene D, α-humulene (also known as α-caryophyllene), and β-caryophyllene, which are principal components of HEO Lv , and the monoterpenoid geraniol, a minor component of both HEO Fl and HEO Lv , on neutrophil Ca 2+ mobilization induced by f MLF. As shown in Table 4, geraniol had no effect on f MLF-stimulated Ca 2+ flux in human neutrophils. In contrast, all three sesquiterpenes potently inhibited f MLF-stimulated Ca 2+ mobilization, with IC 50 values in the sub-micromolar range. A representative concentration-dependent response for the inhibition of f MLF-induced neutrophil Ca 2+ mobilization by germacrene D is shown in Figure 3.
Human neutrophils were pretreated for 10 min with 25 µ g/mL of the indicated essential oil or 1% DMSO (negative control), followed by stimulation with 5 nM fMLF (indicated by arrow), and Ca 2+ flux was monitored for the indicated times. The data are from one experiment that is representative of three independent experiments.
We also evaluated the effect of the sesquiterpenes germacrene D, α-humulene (also known as αcaryophyllene), and β-caryophyllene, which are principal components of HEOLv, and the monoterpenoid geraniol, a minor component of both HEOFl and HEOLv, on neutrophil Ca 2+ mobilization induced by fMLF. As shown in Table 4, geraniol had no effect on fMLF-stimulated Ca 2+ flux in human neutrophils. In contrast, all three sesquiterpenes potently inhibited fMLF-stimulated Ca 2+ mobilization, with IC50 values in the sub-micromolar range. A representative concentrationdependent response for the inhibition of fMLF-induced neutrophil Ca 2+ mobilization by germacrene D is shown in Figure 3.   Various essential oils and their components have been reported previously to inhibit cell migration [24,50,51]. We found that pretreatment with HEO Fl or HEO Lv for 30 min concentration-dependently attenuated f MLF-induced neutrophil chemotaxis with IC 50 values of 5.7 and 5.2 µg/mL, respectively (Table 4). In this case, the inhibitory effect of HEO Lv was approximately the same as that of HEO Fl . A representative concentration-dependent response for the inhibition of neutrophil chemotaxis by HEO Lv is shown in Figure 4. In our previous studies [24,26], we found that pretreatment with β-pinene, sabinene, and γ-terpinene inhibited neutrophil migration with IC 50 values of 23.9, 39.1, and 32.5 µM, respectively. These active compounds compose 8.3% and 11.3% of HEO Fl and HEO Lv , respectively. We also tested other commercially available components of the essential oils, including geraniol; myrtenol; terpinen-4-ol; α-terpineol; and the sesquiterpenes germacrene D, α-humulene, and β-caryophyllene, and found that only these three sesquiterpenes inhibited neutrophil migration, whereas the other compounds tested were inactive (Table 4). A representative concentration-dependent response for the inhibition of neutrophil chemotaxis by germacrene D is shown in Figure 5.
Several essential oils have been reported to modulate ROS production by neutrophils [26,52,53]. Thus, we evaluated the effect of HEO Fl and HEO Lv on PMA-induced ROS production by human neutrophils and found that, similar to their effects on Ca 2+ mobilization and chemotaxis, Hypericum essential oils inhibited ROS production, with HEO Lv being~8-fold more potent than HEO Fl . We also evaluated geraniol; myrtenol; terpinen-4-ol; α-terpineol; γ-terpinene; and the sesquiterpenes germacrene D, α-humulene, and β-caryophyllene in the same test-system and found that only the three sesquiterpenes inhibited ROS production in neutrophils, with IC 50 values in the micromolar range ( Table 3). As examples, representative concentration-dependent responses for inhibition of PMA-induced ROS production in human neutrophils treated by germacrene D are shown in Figure 6. Note also that none of the essential oils, monoterpenes, or sesquiterpenes that we evaluated directly activated ROS production by human neutrophils. Although various essential oils have been reported to modulate ROS production and Ca 2+ mobilization in neutrophils [24,26,52,53], this is the first study to report the neutrophil immunomodulatory effects of essential oils isolated from Hypericum species, as well as the selected sesquiterpenes found in HEO Lv .
Various essential oils and their components have been reported previously to inhibit cell migration [24,50,51]. We found that pretreatment with HEOFl or HEOLv for 30 min concentrationdependently attenuated fMLF-induced neutrophil chemotaxis with IC50 values of 5.7 and 5.2 µ g/mL, respectively (Table 4). In this case, the inhibitory effect of HEOLv was approximately the same as that of HEOFl. A representative concentration-dependent response for the inhibition of neutrophil chemotaxis by HEOLv is shown in Figure 4. In our previous studies [24,26], we found that pretreatment with β-pinene, sabinene, and γ-terpinene inhibited neutrophil migration with IC50 values of 23.9, 39.1, and 32.5 µ M, respectively. These active compounds compose 8.3% and 11.3% of HEOFl and HEOLv, respectively. We also tested other commercially available components of the essential oils, including geraniol; myrtenol; terpinen-4-ol; α-terpineol; and the sesquiterpenes germacrene D, α-humulene, and β-caryophyllene, and found that only these three sesquiterpenes inhibited neutrophil migration, whereas the other compounds tested were inactive (Table 4). A representative concentration-dependent response for the inhibition of neutrophil chemotaxis by germacrene D is shown in Figure 5.   Several essential oils have been reported to modulate ROS production by neutrophils [26,52,53]. Thus, we evaluated the effect of HEOFl and HEOLv on PMA-induced ROS production by human neutrophils and found that, similar to their effects on Ca 2+ mobilization and chemotaxis, Hypericum essential oils inhibited ROS production, with HEOLv being ~8-fold more potent than HEOFl. We also PMA-induced ROS production in human neutrophils treated by germacrene D are shown in Figure  6. Note also that none of the essential oils, monoterpenes, or sesquiterpenes that we evaluated directly activated ROS production by human neutrophils. Although various essential oils have been reported to modulate ROS production and Ca 2+ mobilization in neutrophils [24,26,52,53], this is the first study to report the neutrophil immunomodulatory effects of essential oils isolated from Hypericum species, as well as the selected sesquiterpenes found in HEOLv.  To ensure that the results regarding inhibition of neutrophil functional activity (Ca 2+ flux, cell migration, and ROS production) were not significantly influenced by potential toxicity, we evaluated cytotoxicity of HEO Fl , HEO Lv , germacrene D, α-humulene, and β-caryophyllene at various concentrations in HL-60 cells. As shown in Figure 7A, HEO Fl and HEO Lv had minimal cytotoxicity during a 30-min incubation. After 2 h, HEO Fl and HEO Lv exhibited some cytotoxic effects at 12.5 µg/mL. On the other hand, HEO Lv demonstrated inhibitory activity in all three cell-based assays, and HEO Fl inhibited chemotaxis assay at much lower concentrations (1-5 µg/mL, see Table 4). In addition, the inhibitory effects of HEO Fl and HEO Lv on neutrophil ROS production were observed during the first 30 min after PMA activation. Thus, it is unlikely that the inhibition of neutrophil responses by Hypericum essential oils was due to cytotoxicity at the concentrations and time periods tested. Furthermore, analysis of the pure sesquiterpenes showed that they had little or no cytotoxicity at all concentrations when tested over a 2 h incubation time ( Figure 7B), again indicating that the inhibition of neutrophil responses by germacrene D, α-humulene, and β-caryophyllene was not due to cytotoxicity.
Although some essential oils and their components were previously identified as HNE inhibitors [48,49], evaluation of HEO Fl ; HEO Lv ; and the sesquiterpenes germacrene D, α-humulene, and β-caryophyllene showed that they did not inhibit HNE, even at high tested concentrations (up to 50 µg/mL for the essential oils and 50 µM for the pure sesquiterpenes, data not shown).
Clearly, HEO Lv was most potent essential oil in our biological screening. Thus, we focused our biological evaluation on the major compounds in HEO Lv that had not been investigated previously (i.e., the sesquiterpene compounds). Germacrene D, β-caryophyllene, α-humulene, and bicyclogermacrene are the main sesquiterpenes, representing 38.9% of HEO Lv . Although the germacrene D receptor was identified in neuronal cells of insects [54], biological activity of this compound in mammalian cells is completely unknown. In contrast, β-caryophyllene and α-humulene have been investigated in terms of their biological activity. For example, β-caryophyllene is a type 2 cannabinoid (CB2) receptor agonist and has been reported to inhibit α-glucosidase [55,56]. It also has anti-inflammatory activity in vitro and in vivo [57][58][59]. The reported immunomodulatory effects of β-caryophyllene include inhibition of microglial cells, CD4 + and CD8 + T lymphocytes, and expression of proinflammatory cytokines [59]. In addition, β-caryophyllene was reported to exert neuroprotective effects by modulating the expression of inflammatory mediators [60,61]. Furthermore, this sesquiterpene was reported to induce tumor cell apoptosis [62], prevent attachment of monocytic THP-1 cells to endothelial cells in vitro [63], and impair Mycobacterium bovis (BCG)-induced neutrophil accumulation in mouse pleurisy [64]. Although there are no direct data regarding inhibitory effects of this compound on ROS production, the caryophyllene-related sesquiterpenoids rumphellols A and B were reported to inhibit ROS production in human neutrophils [65]. Although some essential oils and their components were previously identified as HNE inhibitors [48,49], evaluation of HEOFl; HEOLv; and the sesquiterpenes germacrene D, α-humulene, and βcaryophyllene showed that they did not inhibit HNE, even at high tested concentrations (up to 50 μg/mL for the essential oils and 50 μM for the pure sesquiterpenes, data not shown).
Clearly, HEOLv was most potent essential oil in our biological screening. Thus, we focused our biological evaluation on the major compounds in HEOLv that had not been investigated previously (i.e., the sesquiterpene compounds). Germacrene D, β-caryophyllene, α-humulene, and bicyclogermacrene are the main sesquiterpenes, representing 38.9% of HEOLv. Although the α-Humulene is an isomer of β-caryophyllene, and these two compounds are often present as a mixture in some plants [66,67]. α-Humulene has been reported to inhibit nuclear factor (NF)-κB and activating protein (AP)-1 activity, the expression of P-selectin, and the increased mucus secretion in the lung in experimental airway allergic inflammation [68]. Both α-humulene and β-caryophyllene have been reported to inhibit lipopolysaccharide-induced NF-κB activation and neutrophil migration, although only α-humulene had the ability to prevent the production of the proinflammatory cytokines tumor necrosis factor (TNF) and interleukin (IL)-1β in a model of acute inflammation in rat paw [69]. α-Humulene and β-caryophyllene have also been reported to have acaricidal activities against Dermatophagoides farinae and D. pteronyssinus [70]. Finally, these sesquiterpenes inhibited cytochrome P4503A activity in rats and in human hepatic microsomes in vitro [71]. Thus, it is clear that the sesquiterpene compounds found in H. perforatum essential oils have a number of biological activities, including the neutrophil immunomodulatory activity reported here.

Identification of Potential Protein Targets for Selected Sesquiterpenes
Although we have not performed enantiomeric investigation of the main active sesquiterpenes of H. perforatum oils, (−)-β-caryophyllene was reported to be the most commonly found form of β-caryophyllene in many essential oils [72]. This enantiomeric form was also reported in essential oils of Hypericum species, including H. perforatum [73]. In nature, germacrene D occurs in two enantiomer forms, although the (−)-enantiomer is the most prevalent one found in higher plants [54,74]. Likewise, the (+)-configuration of bicyclogermacrene is the most common enantiomer in higher plants [75,76]. Thus, we performed reverse pharmacophore mapping on α-humulene and the enantiomeric structures of (−)-β-caryophyllene, (−)-germacrene D, and (+)-bicyclogermacrene to identify their potential biological targets. PharmMapper compared a large database of pharmacophore patterns with our test compounds and generated target information, including normalized fitness scores and pharmacophoric characteristics.
Among these potential protein targets, only CD11a, MAPKAPK2, and PIM1 could explain the direct inhibitory effect of HEO Lv and its primary sesquiterpenes on human neutrophil functional activities, including inhibition of ROS production and chemotaxis. Indeed, neutrophil arrest and migration involves integrin α-L (CD11a) [77]. Upon activation of p38 MAPK, MAPKAPK2 binds to p38 MAPK, leading to phosphorylation of Hsp27, Akt, and Cdc25, which are involved in regulation of various essential cellular functions [78]. In support of this idea, MAPKAPK2 −/− neutrophils generated less O 2 − , and both NADPH-oxidase activation and p47 phox phosphorylation were decreased [79].
PIM kinases have been reported to promote cell migration and invasion [80], and participation of PIM1 in p22 phox -dependent signaling also was reported [81]. Since MAPKAPK2 and PIM1 could interfere with NADPH-oxidase activation and suppress phagocyte migration and ROS production, we evaluated the binding affinity of pure β-caryophyllene, α-humulene, and germacrene D toward these two kinases using KINOMEscan but did not find any binding activity.
It should be noted that β-caryophyllene oxide was completely inactive in human neutrophils. We also conducted PharmMapper analysis for this compound and found that CD11a (fit score = 0.998) was its best ranked potential protein target. Other potential protein targets for β-caryophyllene oxide are KIF11 (fit score = 0.980), AKR1C2 (0.979), BMP-2 (0.966), MAPKAPK2 (0.965), steroid sulfatase (0.936), caspase-7 (0.930), and PIM1 (0.923). Thus, because CD11a is a potential target for both β-caryophyllene oxide and β-caryophyllene, this protein is unlikely to be a relevant target for β-caryophyllene in human neutrophils. Table 5. Identification of potential protein targets of (−)-β-caryophyllene, (−)-germacrene D, α-humulene, and bicyclogermacrene. Using the SwissADME online tool [31], we calculated the most important physicochemical parameters for the sesquiterpenes, including β-caryophyllene oxide (Table 6), and found that the compounds are very similar to each other in terms of many ADME properties. Nevertheless, they differed noticeably in topological polar surface area (tPSA) and Log P. These descriptors are usually related to the capacity of molecules to cross cellular membranes [82]. For example, it was reported earlier that compounds with LogP > 4 and TPSA < 40 Å 2 had optimal antimycobacterial activity [83]. Thus, it is possible that the inactivity of β-caryophyllene oxide in human neutrophils could be explained by low cell membrane permeability, as this compound is less lipophilic and more polar than the investigated sesquiterpenes of purely hydrocarbon nature. Table 6. Physicochemical properties of (−)-germacrene D, bicyclogermacrene, α-humulene, (−)-β-caryophyllene, and β-caryophyllene oxide.

Conclusions
We report here that essential oils isolated from leaves of H. perforatum contain a high amount of sesquiterpenes and that these essential oils are potent inhibitors of human neutrophil functional responses. Moreover, the essential oil constituents germacrene D, β-caryophyllene, and α-humulene were also potent inhibitors of f MLF-induced Ca 2+ mobilization, chemotaxis, and ROS production by human neutrophils. Thus, our data provide a molecular basis to explain at least part of the beneficial therapeutic effects of essential oils from H. perforatum and suggest that suppression of neutrophils by the essential oil components of this plant might have anti-inflammatory effects. Future studies are now in progress to evaluate the potential of Hypericum essential oils as therapeutic remedies for various disorders with immune and/or inflammatory mechanisms, as well as to determine molecular targets of their active components.

Conflicts of Interest:
The authors declare no competing financial interest.