Neutrophil Immunomodulatory Activity of (−)-Borneol, a Major Component of Essential Oils Extracted from Grindelia squarrosa

Grindelia squarrosa (Pursh) Dunal is used in traditional medicine for treating various diseases; however, little is known about the immunomodulatory activity of essential oils from this plant. Thus, we isolated essential oils from the flowers (GEOFl) and leaves (GEOLv) of G. squarrosa and evaluated the chemical composition and innate immunomodulatory activity of these essential oils. Compositional analysis of these essential oils revealed that the main components were α-pinene (24.7 and 23.2% in GEOFl and GEOLv, respectively), limonene (10.0 and 14.7%), borneol (23.4 and 16.6%), p-cymen-8-ol (6.1 and 5.8%), β-pinene (4.0 and 3.8%), bornyl acetate (3.0 and 5.1%), trans-pinocarveol (4.2 and 3.7%), spathulenol (3.0 and 2.0%), myrtenol (2.5 and 1.7%), and terpinolene (1.7 and 2.0%). Enantiomer analysis showed that α-pinene, β-pinene, and borneol were present primarily as (−)-enantiomers (100% enantiomeric excess (ee) for (−)-α-pinene and (−)-borneol in both GEOFl and GEOLv; 82 and 78% ee for (−)-β-pinene in GEOFl and GEOLv), while limonene was present primarily as the (+)-enantiomer (94 and 96 ee in GEOFl and GEOLv). Grindelia essential oils activated human neutrophils, resulting in increased [Ca2+]i (EC50 = 22.3 µg/mL for GEOFl and 19.4 µg/mL for GEOLv). In addition, one of the major enantiomeric components, (−)-borneol, activated human neutrophil [Ca2+]i (EC50 = 28.7 ± 2.6), whereas (+)-borneol was inactive. Since these treatments activated neutrophils, we also evaluated if they were able to down-regulate neutrophil responses to subsequent agonist activation and found that treatment with Grindelia essential oils inhibited activation of these cells by the N-formyl peptide receptor 1 (FPR1) agonist fMLF and the FPR2 agonist WKYMVM. Likewise, (−)-borneol inhibited FPR-agonist-induced Ca2+ influx in neutrophils. Grindelia leaf and flower essential oils, as well as (−)-borneol, also inhibited fMLF-induced chemotaxis of human neutrophils (IC50 = 4.1 ± 0.8 µg/mL, 5.0 ± 1.6 µg/mL, and 5.8 ± 1.4 µM, respectively). Thus, we identified (−)-borneol as a novel modulator of human neutrophil function.


Plant Material
Plant material was collected from wild plants in August 2019 along the Madison River, approximately 10 miles east of Norris, MT, USA (45.611323 • N, 111.564253 • E). Flowers and leaves were air-dried for 7-10 days at room temperature away from direct sunlight before hydrodistillation. Botanical identification of the plant material was performed by botanist Robyn A. Klein from Montana State University, Bozeman, MT, USA.

Essential Oil Extraction
Essential oils were extracted by hydrodistillation of air-dried plant material (leaves, flowers) using a Clevenger-type apparatus, as previously described [34]. We used conditions accepted by the European Pharmacopoeia (European Directorate for the Quality of Medicines, Council of Europe, Strasbourg, France, 2014) to avoid artifacts. Yields of the essential oils 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-Flame Ionization Detector (GC-FID) and 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 [39]. 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. FID temperature was 300 • C. The 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 the FID chromatograms.

Analysis of Enantiomer Composition on Chiral Columns
Distributions of enantiomeric compounds in G. squarrosa essential oils were analyzed by using two different chiral columns, which were appropriate for each enantiomer, and an Agilent 5973 Network Mass Selective Detector on a 6890N GC system that also had an FID detector (Agilent Technologies, Santa Clara, CA, USA) (see further GC system details above). The chiral columns were Rt-βDEXse (2,3-di-O-ethyl-6-O-tert-butyl dimethylsilyl-β-cyclodextrin added into 14% cyanopropylphenyl/86% dimethyl polysiloxane, 30 m × 0.32 mm ID, 0.25 µm film thickness, USA) and Lipodex G (6-methyl-2,3pentyl-γ-cyclodextrin added into 60% polysiloxane, 25 m × 0.25 mm ID, 0.125 µm film thickness, Germany). For separation of α-pinene, the Lipodex G chiral column was used, while separation of borneol, camphor, and limonene was performed on the Rt-βDEXse column. Samples were injected (10% prepared in hexane) with a 10:1 split ratio. Injection port and detector temperatures were 250 • C. Detailed analysis parameters are provided in Supplementary Materials.

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 [40]. 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; 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 a test compound or pure essential oils on Ca 2+ influx, 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+ influx, the compound/oil was added to the wells (final concentration of DMSO was 1%) with human neutrophils. The samples were preincubated for 10 min, followed by addition of 5 nM f MLF or 5 nM WKYMVM. 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). 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 luminescence-based assay (CellTiter-Glo; Promega, Madison, WI), and luminescence measurements were converted to absolute cell numbers by comparison of the values with standard curves obtained with known numbers of neutrophils. Curve fitting (at least eight to nine points) and calculation of median effective concentration values (IC 50 ) were performed by nonlinear regression analysis of the dose-response curves generated using GraphPad Prism 9.

Cytotoxicity Assay
Cytotoxicity of essential oils and pure compounds in human neutrophils or THP-1 monocytic was analyzed with a CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega) according to the manufacturer's protocol. Briefly, human neutrophils or THP-1 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 90 min (for neutrophils) or 24 h (for THP-1 cells) 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.

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 statistically significant.
We report here the first enantioselective analysis of constituents in G. squarrosa essential oils, as all previous studies on Grindelia essential oils included identification of the essential oil constituents but not characterization of enantiomeric distribution. The enantiomeric distribution of monoterpenes in the flower and leaf essential oils of G. squarrosa was obtained using enantioselective GC analyses on chiral columns. As shown in Table 2, α-pinene, β-pinene, borneol, and camphor were present primarily or exclusively as (−)-enantiomers, while limonene was present primarily as the (+)-enantiomer. Differentiation of the enantiomeric composition of constituents is important for consideration of biological activity. Indeed, there are several examples of chiral compounds where the enantiomers had different immunomodulatory activity. For example, in vivo experiments showed that carvone enantiomers differentially modulated IgE-mediated airway inflammation in mice [52]. Likewise, the R isomer of hydroxychloroquine was found to exhibit higher antiviral activity and lower toxicity in vivo compared to the S isomer [53]. In additon, Murai et al. [54] used in silico analysis of enantioselective binding to characterize binding of immunomodulatory imide drugs to cereblon. Thus, it is clear that enantiomeric conformation of compounds has to be taken into consideration when evaluating biological effects.

Effect of the Essential Oils from G. squarrosa and (−)-Borneol on Neutrophil Ca 2+ Influx
Neutrophils are the most abundant leukocyte and are vital for innate immunity [55]. These cells are the first responders to infection and injury in various tissues, establishing the first line of defense through multiple mechanisms such as phagocytosis, cytokine secretion, and reactive oxygen species production [56,57]. 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 [29][30][31][32][33].
Since Grindelia essential oils and several component compounds 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 [60], we evaluated whether Grindelia essential oils and/or pure components could inhibit agonistinduced [Ca 2+ ] i in human neutrophils. As shown in Table 3 and Figure 2A, Grindelia essential oils inhibited [Ca 2+ ] i in f MLF-and WKYMVM-stimulated neutrophils with IC 50 values in the micromolar range. We also evaluated the effect of (−)-borneol and (+)-borneol and found that only (−)-borneol inhibited f MLF-and WKYMVM-stimulated neutrophils ( Table 3). A representative, concentration-dependent response for the inhibition of f MLF-induced neutrophil [Ca 2+ ] i by (−)-borneol is shown in Figure 2B. These results are also consistent with our data showing neutrophils were only activated by (−)-borneol (Table 3).  Since Grindelia essential oils and several component compounds stimu neutrophil [Ca 2+ ]i , and it is well recognized that agonists can down-regulat responses to subsequent treatment with heterologous or homologous agon evaluated whether Grindelia essential oils and/or pure components could inh induced [Ca 2+ ]i in human neutrophils. As shown in Table 3
To ensure that the effects of the essential oils from G. squarrosa or (−)-borneol on neutrophil functional activity were not influenced by possible toxicity, we evaluated the cytotoxicity of the essential oil samples (up to 55 µg/mL) and (−)-borneol at various concentrations (up to 50 µM) in human neutrophils. We found that the G. squarrosa essential oils were non-cytotoxic up to 55 µg/mL during a 90 min incubation period, which covers the times used to measure Ca 2+ influx (up to 30 min) and cell migration (up to 90 min) (data not shown). Likewise, (−)-borneol was non-cytotoxic in human neutrophils treated for 90 min and THP-1 monocytic cells treated for 24 h (Figure 4), confirming previous reports on the low cytotoxicity of borneol [61]. Thus, we conclude that (−)-borneol is a novel, non-cytotoxic, innate immunomodulator.  In addition to the immunomodulatory activity reported here, borneol was previously reported to exhibit a number of biological activities. For example, borneol attenuated brain neuronal and microglial inflammation in lipopolysaccharide (LPS)-induced sepsis mice with suppression of p-p65 and p38 signaling that was initially activated by LPS in the brain [62]. Borneol treatment also inhibited transient receptor potential ankyrin 1 (TRPA1), a proinflammatory and noxious pain-sensing cation channel [63], and suppressed inflammatory responses in LPS-induced acute lung injury through inhibition of the nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways [64]. Likewise, borneol-treated mice had reduced carrageenan-induced leukocyte migration to the peritoneal cavity [65], and borneol treatment suppressed proinflammatory cytokine mRNA expression in colonic inflammation [66]. Borneol attenuated asthma in mice by decreasing the CD4 + T cells' infiltration [67]. Moreover, borneol specifically induced the activation of M2 macrophages in a signal transducer and activator of transcription 3 (STAT3)-dependent manner [68].
The biological activity of (−)-borneol was also reported previously. For example, (−)borneol was recently reported to exhibit antibacterial activity [69]. (−)-Borneol was also reported to have neuroprotective effects in a middle cerebral artery occlusion (MCAO) model [70,71], as well as vasorelaxant properties, which may be attributed to Ca 2+ influx blockade through voltage-gated Ca 2+ channels, Ca 2+ mobilization from intracellular stores, and activation of K + channels [72][73][74]. Store-independent Ca 2+ channels that exist in various hematopoietic cells, including neutrophils, are pharmacologically and/or immunologically similar to voltage-gated Ca 2+ channels [75]. Thus, these storeindependent Ca 2+ channels could be potential targets of (−)-borneol in human neutrophils. Indeed, we showed previously that some essential oil compounds can modulate activity of transient receptor potential Ca 2+ channels [33,76].
We also calculated the most important physicochemical parameters for borneol using SwissADME [77]. The logP values estimated using ALOGPS 2.1 program [78] and tPSA values allowed us to predict that borneol can permeate the blood-brain barrier (BBB) ( Table 4). It should be noted that (−)-borneol itself can also increase blood-brain barrier permeability [73,79]. In addition to the immunomodulatory activity reported here, borneol was previously reported to exhibit a number of biological activities. For example, borneol attenuated brain neuronal and microglial inflammation in lipopolysaccharide (LPS)-induced sepsis mice with suppression of p-p65 and p38 signaling that was initially activated by LPS in the brain [62]. Borneol treatment also inhibited transient receptor potential ankyrin 1 (TRPA1), a proinflammatory and noxious pain-sensing cation channel [63], and suppressed inflammatory responses in LPS-induced acute lung injury through inhibition of the nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways [64]. Likewise, borneol-treated mice had reduced carrageenan-induced leukocyte migration to the peritoneal cavity [65], and borneol treatment suppressed proinflammatory cytokine mRNA expression in colonic inflammation [66]. Borneol attenuated asthma in mice by decreasing the CD4 + T cells' infiltration [67]. Moreover, borneol specifically induced the activation of M2 macrophages in a signal transducer and activator of transcription 3 (STAT3)-dependent manner [68].
The biological activity of (−)-borneol was also reported previously. For example, (−)-borneol was recently reported to exhibit antibacterial activity [69]. (−)-Borneol was also reported to have neuroprotective effects in a middle cerebral artery occlusion (MCAO) model [70,71], as well as vasorelaxant properties, which may be attributed to Ca 2+ influx blockade through voltage-gated Ca 2+ channels, Ca 2+ mobilization from intracellular stores, and activation of K + channels [72][73][74]. Store-independent Ca 2+ channels that exist in various hematopoietic cells, including neutrophils, are pharmacologically and/or immunologically similar to voltage-gated Ca 2+ channels [75]. Thus, these store-independent Ca 2+ channels could be potential targets of (−)-borneol in human neutrophils. Indeed, we showed previously that some essential oil compounds can modulate activity of transient receptor potential Ca 2+ channels [33,76].
We also calculated the most important physicochemical parameters for borneol using SwissADME [77]. The logP values estimated using ALOGPS 2.1 program [78] and tPSA values allowed us to predict that borneol can permeate the blood-brain barrier (BBB) ( Table 4). It should be noted that (−)-borneol itself can also increase blood-brain barrier permeability [73,79]. In previous research on essential oils from various plant species, we found that most essential oil compounds that inhibited f MLF-induced Ca 2+ influx were sesquiterpenes, although one was an oxygenated monoterpene (bornyl acetate) ( Table 5). In the present study, we showed that another oxygenated monoterpene, (−)-borneol, has neutrophil inhibitory activity. Grindelia essential oils also contained 2-3% spathulenol, an active, oxygenated sesquiterpene (Tables 1 and 5). Thus, this compound may also be involved in the inhibitory effect of these essential oils, although we did not evaluate this compound here.

Conclusions
We analyzed the composition of essential oils extracted from G. squarrosa leaves and flowers and report here the first determination of the enantiomeric distribution of major monoterpenes in G. squarrosa essential oils. Borneol, α-pinene, β-pinene, and camphor were primarily present as (−)-enantiomers, whereas limonene was primarily present as the (+)-enantiomer. Further analysis of the immunomodulatory activity of G. squarrosa essential oils showed that they activated human neutrophils and were able to inhibit agonist-induced neutrophil activation and chemotaxis, which might contribute to the reported anti-inflammatory activity and other pharmacological properties of extracts from this plant. The effects of essential oils from G. squarrosa might be attributable to (−)borneol, bornyl acetate, some of the minor components, or synergetic effects among these constituents. However, to verify the key targets responsible for the immunomodulatory effects of (−)-borneol, further experimental investigation is needed.