pharmaceuticals
Article
Neutrophil Immunomodulatory Activity of Farnesene, a
Component of Artemisia dracunculus Essential Oils
Igor A. Schepetkin 1, Gulmira Özek 2 , Temel Özek 2 , Liliya N. Kirpotina 1, Andrei I. Khlebnikov 3 ,
Robyn A. Klein 4 and Mark T. Quinn 1,*
1 Department of Microbiology and Cell Biology, Montana State University, Bozeman, MT 59717, USA;
igor@montana.edu (I.A.S.); liliya@montana.edu (L.N.K.)
2 Department of Pharmacognosy, Faculty of Pharmacy, Anadolu University, Eskisehir 26470, Turkey;
gulmiraozek@gmail.com (G.Ö.); temelozek@gmail.com (T.Ö.)
3 Kizhner Research Center, Tomsk Polytechnic University, 634050 Tomsk, Russia; aikhl@chem.org.ru
4 Department of Plant Sciences and Plant Pathology, Montana State University, Bozeman, MT 59717, USA;
herbrobin@gmail.com
* Correspondence: mquinn@montana.edu; Tel.: +1-406-994-4707; Fax: +1-406-994-4303
Abstract: Despite their reported therapeutic properties, not much is known about the immunomod-
ulatory activity of essential oils present in Artemisia species. We isolated essential oils from the
flowers and leaves of five Artemisia species: A. tridentata, A. ludoviciana, A. dracunculus, A. frigida,
and A. cana. The chemical composition of the Artemisia essential oil samples had similarities
and differences as compared to those previously reported in the literature. The main compo-
nents of essential oils obtained from A. tridentata, A. ludoviciana, A. frigida, and A. cana were cam-
phor (23.0–51.3%), 1,8-cineole (5.7–30.0%), camphene (1.6–7.7%), borneol (2.3–14.6%), artemisiole
Citation: Schepetkin, I.A.; Özek, G.; (1.2–7.5%), terpinen-4-ol (2.0–6.9%), α-pinene (0.8–3.9%), and santolinatriene (0.7–3.5%). Essential oils
Özek, T.; Kirpotina, L.N.; Khlebnikov,
from A. dracunculus were enriched in methyl chavicol (38.8–42.9%), methyl eugenol (26.1–26.4%), ter-
A.I.; Klein, R.A.; Quinn, M.T.
pinolene (5.5–8.8%), (E/Z)-β-ocimene (7.3–16.0%), β-phellandrene (1.3–2.2%), p-cymen-8-ol (0.9–2.3%),
Neutrophil Immunomodulatory
Activity of Farnesene, a Component and xanthoxylin (1.2–2.2%). A comparison across species also demonstrated that some compounds
of Artemisia dracunculus Essential were present in only one Artemisia species. Although Artemisia essential oils were weak activators
Oils. Pharmaceuticals 2022, 15, 642. of human neutrophils, they were relatively more potent in inhibiting subsequent neutrophil Ca2+
https://doi.org/10.3390/ mobilization with N-formyl peptide receptor 1 (FPR1) agonist f MLF- and FPR2 agonist WKYMVM,
ph15050642 with the most potent being essential oils from A. dracunculus. Further analysis of unique compounds
found in A. dracunculus showed that farnesene, a compound with a similar hydrocarbon structure as
Academic Editors: Antonio Eduardo
2+
Miller Crotti and Eliane de lipoxin A4, inhibited Ca influx induced in human neutrophils by f MLF (IC50 = 1.2 µM), WKYMVM
Oliveira Silva (IC50 = 1.4 µM), or interleukin 8 (IC50 = 2.6 µM). Pretreatment with A. dracunculus essential oils and
farnesene also inhibited human neutrophil chemotaxis induced by f MLF, suggesting these treatments
Received: 29 April 2022 down-regulated human neutrophil responses to inflammatory chemoattractants. Thus, our studies
Accepted: 19 May 2022 have identified farnesene as a potential anti-inflammatory modulator of human neutrophils.
Published: 23 May 2022
Publisher’s Note: MDPI stays neutral Keywords: anti-inflammatory; Artemisia; calcium flux; chemotaxis; essential oils; farnesene;
with regard to jurisdictional claims in monoterpene; neutrophil
published maps and institutional affil-
iations.
1. Introduction
Copyright: © 2022 by the authors. The genus Artemisia is one of the most broadly distributed groups in the Asteraceae
Licensee MDPI, Basel, Switzerland. family [1]. Within this family, Artemisia is in the tribe Anthemideae and comprises about
This article is an open access article 530 species of plants that are found on all continents except for Antarctica. These plants
distributed under the terms and are found mainly in the Northern Hemisphere, with only 25 species in the Southern Hemi-
conditions of the Creative Commons sphere [2]. According to the latest taxonomic revisions, there are 50 Artemisia species in
Attribution (CC BY) license (https:// North America, which belong to Artemisia (Miller) Less, Absinthium (Miller) Less, Dra-
creativecommons.org/licenses/by/ cunculus Besser, and Tridentatae (Rydb.) subgenera [3]. Artemisia is most prevalent in the
4.0/). western half of the United States, with 11 species in or near the Rocky Mountain region [4].
Pharmaceuticals 2022, 15, 642. https://doi.org/10.3390/ph15050642 https://www.mdpi.com/journal/pharmaceuticals
Pharmaceuticals 2022, 15, 642 2 of 17
According to the United States Department of Agriculture, 15 Artemisia species have been
described: A. arbuscula Nutt.,A. bigelovii Gray, A. californica Less., A. cana Pursh., A. filifolia
Torr., A. frigida Willd., A. longiloba (Osterhout) Beetle, A. lugoviciana Nutt., A. nova A.Nels.,
A. pygmaea Gray, A. rigida (Nutt.) Gray, A. rothrockii Gray, A. spinescens Eaton, A. tridentata
Nutt., and A. tripartita Rydb. [5].
The importance of Artemisia species in traditional medicine is evident in a number of
ethnopharmacological reports [6–8]. Asteraceae-specific ethnobotanical reports recorded
in the Native American Ethnobotany database revealed Artemisia species as the second
most selected medicinal taxa after Achillea. A. tridentata was most commonly cited as a
pulmonary aid, whereas A. dracunculus L., A. tridentata, and A. douglasiana Besser were
cited for orthopedic uses [9]. Many Artemisia species are characterized by a strong odor
due to essential oils that are mainly concentrated in their leaves and flowers. The pharma-
cological properties of Artemisia essential oils have been reported, including antibacterial,
antiparasitic, antinociceptive, hypoglycemic, and antioxidant activities [10–17].
Essential oils from various plant species have been reported to exhibit immunomodu-
latory activity. For example, Ocimum kilimandscharicum Gürke leaf essential oils inhibited
carrageenan-induced leukocyte rolling, adhesion, and f MLF-induced leukocyte chemo-
taxis [18]. Likewise, Juniperus essential oils inhibited the production of the proinflam-
matory cytokines tumor necrosis factor (TNF), interleukin (IL)-1β, and γ-interferon by
lipopolysaccharide-stimulated leukocytes [19]. Recently, we found that essential oils from
Artemisia kotuchovii, Ferula akitschkensis, F. iliensis, Hypericum perforatum, Rhododendron albi-
florum, and Juniperus spp. can activate or inhibit human neutrophil function [20–25].
Here, we characterized the chemical composition and immunomodulatory activity
of the flower and leaf essential oils from five Artemisia species collected in Montana and
analyzed their chemical compositions and innate immunomodulatory activities. We show
that several of the Artemisia essential oils potently inhibited intracellular Ca2+ mobilization
[Ca2+]i in human neutrophils, with the most active being essential oils from A. dracun-
culus. Furthermore, we demonstrated farnesene, a unique component of A. dracunculus,
inhibited human neutrophil activation and chemotaxis and is likely one of the main active
components. Based on the critical role of neutrophils in inflammation, our data support
the possibility that farnesene could have the potential for the development of new anti-
inflammatory agents.
2. Results and Discussion
2.1. Essential Oil Composition
Essential oils were obtained from the flowers (designated by Fl) and leaves (designated
by Lv) of five Artemisia species for subsequent phytochemical and biological characteriza-
tion. Plant material was collected from wild plants in 2019 around Bozeman and Three
Forks (MT, USA) (Table 1). After botanical identification of the plant material, the flowers
and leaves were dried for 7–10 days at room temperature but protected from direct sunlight.
The yields (v/w) of Artemisia spp. essential oils ranged from 0.3 to 3.3% (Table 1). The
composition of the extracted essential oils was determined using simultaneous GC-FID
and GC/MS. Twenty-seven major compounds found in the essential oils are shown in
Table 2 and Supplementary Table S1 shows the total of all compounds identified in these
samples. The main classes of compounds in all Artemisia species were oxygenated monoter-
penes, ranging from 1.9 (ADFl) to 84.8% (ACLv), and monoterpene hydrocarbons, which
ranged from 6.5 (ACLv) to 25.8% (ADFl). Essential oils from A. tridentata, A. ludoviciana,
A. frigida, and A. cana contained high amounts of oxygenated monoterpenes (77.4–84.8%),
with 1,8-cineole and camphor being the most representative, which is consistent with
previous reports for A. ludoviciana, A. frigida, and A. cana essential oils [26]. In addition,
essential oils from the A. dracunculus were enriched in phenylpropanoids (65.3–69.0%),
including methyl chavicol (estragole) and methyl eugenol, also supporting previous reports
analyzing essential oils from aerial parts of this plant [26,27]. Note that this is the first
report comparing individual leaf and flower essential oils from these Artemisia species.
Pharmaceuticals 2022, 15, 642 3 of 17
Artemisia exhibits significant intraspecies variations in terpenes present in their essen-
tial oils [16,26,34–58]. For example, the variation in volatile components of these plants
may occur during plant maturation at different altitudes. Our studies confirmed these
findings and extended this analysis by providing the first report comparing essential oils
isolated from preparations of A. tridentata flowers and leaves prepared from the same plant
samples. Interestingly, we found that some compounds exhibited remarkable differences
in terms of their presence in different parts of the same Artemisia species. For example,
santolinatriene and artemiseole were found only in the flowers but not in the leaves of
A. ludoviciana. Likewise, santolina epoxide and the monoterpene alcohol grandisol, a
pheromone, were detected only in A. cana flowers. Similarly, trans-chrysanthenol and its
ester trans-chrysanthenyl acetate were detected only in A. frigida leaves, and fragranol was
found only in A. cana leaf essential oils and not those isolated from flowers. Therefore, it is
important to evaluate different parts of plants in terms of target compounds. A comparison
across species also demonstrated some differences in the presence of compounds, with
some compounds (e.g., (Z)-β-ocimene, methyl chavicol, (E,E)-α-farnesene, xanthoxylin),
being present in only one Artemisia species. It is also interesting that A. dracunculus essential
oils had more unique compounds that were not present in essential oils from the other
Artemisia species.
Table 1. Location and date of collection of the plant material.
Artemisia spp. Location Latitude Longitude Date of
(N) (E) Altitude (m) Plant Material Collection; Yield (%)
Specimen No.
A. ludoviciana Three Forks, MT 45.92721 111.50106 1235 leaves/flowers August 2019;
2019-IAS-A1 0.3/1.9
A. dracunculus Bozeman, MT 45.71475◦ 110.97890◦ 1646 leaves/flowers August 2019;
2019-IAS-A2 0.9/0.9
A. frigida Three Forks, MT 45.92553◦ 111.49730◦ 1240 leaves/flowers August 2019;
2019-IAS-A3 0.3/1.9
A. cana Three Forks, MT 45.92274◦ 111.49453◦ 1238 leaves/flowers August 2019;
2019-IAS-A4 1.4/1.5
A. tridentata Bozeman, MT 45.74118◦ 110.98698◦ 1415 leaves/flowers August 2019;
2019-IAS-A5 3.3/2.7
2.2. Effect of Artemisia Essential Oils and Pure Compounds on Neutrophil Ca2+ Influx
Neutrophils are the most common leukocyte in human blood and are required for
innate immune responses [59]. These cells respond rapidly to infection and injury in var-
ious tissues. They represent the first line of defense and utilize multiple mechanisms of
oxygen-dependent and oxygen-independent processes to protect against infection, includ-
ing phagocytosis, cytokine secretion, and reactive oxygen species production [60,61]. Thus,
neutrophils represent a potential target for the development of novel anti-inflammatory
therapeutics. Indeed, natural products, such as essential oils, have exhibited neutrophil
immunomodulatory activity [20–24].
Artemisia essential oils were evaluated for their immunomodulatory effects on human
neutrophils. Initially, we measured the effects of the essential oils on [Ca2+]i, which is
a key component of neutrophil activation and function [62]. We found that treatment
of neutrophils with Artemisia essential oils only modestly enhanced [Ca2+]i at relatively
high concentrations, with EC50 values ranging from in the range of 15.8 µg/mL (ALLv)
to 48.7 µg/mL (ACLv). We also evaluated some of the individual compounds present in
the various Artemisia essential oils to determine if they were responsible for neutrophil
activation, including 1,8-cineole, (+)-camphor, (−)-camphor, (±)-bornyl acetate, farnesene,
piperitone, and xanthoxylin. As shown in Table 3, 1,8-cineole, (+)-camphor, (−)-camphor,
and piperitone did not affect human neutrophil Ca2+ flux, whereas farnesene, xanthoxylin,
and (±)-bornyl acetate were weak activators, but only at relatively high concentrations.
Note that previous analysis of some of the other major compounds that we also found
here to be present in Artemisia essential oils (i.e., α-pinene, camphene, (E/Z)-β-ocimene,
Pharmaceuticals 2022, 15, 642 4 of 17
p-cymene, terpinolene, linalool, terpinen-4-ol, methyl chavicol, p-cymen-8-ol, and methyl
eugenol) showed that they had no effect on human neutrophil [Ca2+]i [24,25].
Table 2. Composition of essential oils (%) isolated from leaves and flowers of five Artemisia species.
No RRIa RRIb Compound ATLv ATFl ALLv ALFl ADLv ADFl AFLv AFFl ACLv ACFl
1 1032 1008–1039 # α-Pinene 2.1 1.8 2.5 3.9 0.1 0.1 2.2 3.3 0.8 2.0
2 1043 1011–1063 # Santolinatriene 0.7 1.7 3.5 2.2 2.4
3 1076 1043–1086 # Camphene 7.7 7.7 7.1 5.5 t t 4.0 6.0 1.6 4.1
4 1189 1179 @ Artemiseole 2.1 3.1 2.5 7.5 1.2
5 1213 1186–1231 # 1,8-Cineole 21.8 23.8 16.3 23.1 12.5 5.7 30.0 21.9
6 1218 1188–1233 # β-Phellandrene 1.3 2.2
7 1246 1211–1251 # (Z)-β-Ocimene 4.6 9.4
8 1266 1232–1267 # (E)-β-Ocimene 2.7 6.6 t
9 1280 1246–1291 # p-Cymene 1.2 1.2 0.7 0.7 0.5 0.2 3.2 2.1 0.9 0.7
10 1290 1260–1300 # Terpinolene t t 0.2 0.3 8.8 5.5 0.1 0.3 0.1 0.1
11 1329 1312 ˆ Santolina
epoxide * 3.1
12 1451 1400–1452 # β-Thujone 3.3 0.1
13 1474 1453 ** trans-
Chrysanthenol 2.0
14 1532 1481–1537 # Camphor 51.3 41.7 41.1 26.6 23.0 37.7 32.5 35.9
trans-
15 1538 1533–1590 # Chrysanthenyl 8.1
acetate
16 1553 1507–1564 # Linalool 3.8 2.5 0.1 0.2
17 1590 1549–1597 # Bornyl acetate 0.9 0.6 0.3 0.6 1.5 3.8 0.3 0.8
18 1611 1564–1630 # Terpinen-4-ol 2.7 2.9 2.4 3.8 t 3.3 6.9 2.0 2.3
19 1687 1652–1690 # Methyl chavicol 42.9 38.8
20 1719 1653–1728 # Borneol 2.4 3.3 9.6 8.1 t 6.6 14.6 2.9 2.3
21 1737 1713–1748 # (Z,E)-α-
Farnesene t
22 1748 1689–1748 # Piperitone 0.2 1.5 1.1
23 1758 # (E,E)-α-
1714–1763 Farnesene 0.2 0.5
24 1764 cis-
1751–1765 #
Chrysanthenol 0.5 0.8 1 0.5 3.9 0.5 0.2
25 1821 1807 *** Fragranol 3.0
26 1827 1827 *** Grandisol 3.9
27 1864 1813–1865 # p-Cymen-8-ol 0.2 0.1 0.1 2.3 0.9 0.4 0.3 0.1 0.1
28 2030 1961–2033 # Methyl eugenol 26.1 26.4 t 0.1
29 2637 2608 ## Xanthoxylin 2.2 1.2
Summary of the composition of Artemisia spp. essential oils
Compounds ATLv ATFl ALLv ALFl ADLv ADFl AFLv AFFl ACLv ACFl
Monoterpene Hydrocarbons 13.1 14.4 13.5 18.7 20.3 25.8 11.6 17.4 6.5 11.3
Oxygenated Monoterpenes 84.5 79.8 82.5 77.4 4.0 1.9 79.6 79.8 84.8 81.0
Sesquiterpene Hydrocarbons 2.0 0.2 0.7 1.7 2.4 0.6 0.2 0.1 0.3
Oxygenated Sesquiterpenes 0.8 1.6 1.1 1.2 1.7 1.1 2.5 0.6 0.1 0.4
Phenylpropanoids 1.3 0.2 69.0 65.3 0.1 0.1
Others t t 0.4 0.2 2.6 2.1 0.2 0.5 t 0.6
Total 98.0 97.1 98.9 97.8 98.2 97.7 94.5 98.4 91.5 93.6
Legend: The data are presented as relative % for each component identified. RRIa, relative retention index
calculated based on retention of n-alkanes. RRIb, relative retention indexes reported in the literature: # [28],
@ [26]; ˆ [29]; ** [30], *** [31,32], ## [33]. % was calculated from flame ionization detector data. Trace amounts (t)
were present at <0.1%. * Santolina epoxide was tentatively identified using Wiley and MassFinder mass spectra
libraries and published RRI. All other compounds were identified by comparison with co-injected standards.
Abbreviations for the oil samples: ATLv, A. tridentata leaves; ATFl, A. tridentata flowers; ALLv, A. ludoviciana leaves;
ALFl, A. ludoviciana flowers; ADLv, A. dracunculus leaves; ADFl, A. dracunculus flowers; AFLv, A. frigida leaves;
AFFl, A. frigida flowers; ACLv, A. cana leaves; ACFl, A. cana flowers.
Pharmaceuticals 2022, 15, 642 5 of 17
Table 3. Effect of essential oils from Artemisia spp. and pure component compounds on [Ca2+]i and
cytotoxicity in human neutrophils.
Inhibition of [Ca2+]i
Essential Oil or Activation of [Ca2+]i Cytotoxicity
Pure Compound fMLF-Induced WKYMVM-Induced
EC50 (µg/mL) IC50 (µg/mL) IC50 (µg/mL)
ATLv 23.6 ± 3.6 28.9 ± 4.1 3.4 ± 0.8 Nontoxic
ATFl 32.1 ± 4.5 19.5 ± 3.3 3.7 ± 0.1 Nontoxic
ALLv 15.8 ± 2.1 31.2 ± 3.9 8.1 ± 3.4 Nontoxic
ALFl 16.3 ± 4.2 21.4 ± 3.2 19.6 ± 6.3 Nontoxic
ADLv 25.3 ± 7.4 4.6 ± 1.3 5.3 ± 1.8 32.6 ± 4.1
ADFl 18.8 ± 3.2 2.2 ± 0.8 2.9 ± 0.8 24.7 ± 2.8
AFLv 44.7 ± 6.2 19.3 ± 4.2 32.4 ± 6.4 42.4 ± 3.2
AFFl 41.5 ± 3.5 13.0 ± 2.6 26.6 ± 6.5 31.4 ± 4.8
ACLv 48.7 ± 9.5 18.4 ± 7.6 36.5 ± 4.8 Nontoxic
ACFl 41.6 ± 11.7 22.6 ± 6.8 30.4 ± 7.1 Nontoxic
EC50 (µM) IC50 (µM) IC50 (µM)
1,8-Cineole N.A. N.A. N.A. Nontoxic
(−)-Camphor N.A. N.A. N.A. Nontoxic
(+)-Camphor N.A. N.A. N.A. Nontoxic
(±)-Bornyl acetate 50.1 ± 11.5 42.6 ± 9.7 19.1 ± 0.1 Nontoxic
Farnesene 28.5 ± 2.6 1.1 ± 0.2 1.4 ± 0.5 Nontoxic
Piperitone N.A. N.A. N.A. Nontoxic
Xanthoxylin 53.3 ± 5.0 27.2 ± 6.6 52.7 ± 11.2 Nontoxic
Legend: EC50 and IC50 values were determined by nonlinear regression analysis of the dose-response curves. For
the determination of cytotoxicity, neutrophils were incubated with indicated concentrations of the compounds for
90 min and cell viability was analyzed. N.A. indicates the samples had essentially no activity or no cytotoxicity,
respectively (EC50 or IC50 > 55 µM for pure compounds or > 55 µg/mL for the oils). Presented as the mean ± SD
of three independent experiments. Abbreviations for the oils: ATLv, A. tridentata leaves; ATFl, A. tridentata flowers;
ALLv, A. ludoviciana leaves; ALFl, A. ludoviciana flowers; ADLv, A. dracunculus leaves; ADFl, A. dracunculus flowers;
AFLv, A. frigida leaves; AFFl, A. frigida flowers; ACLv, A. cana leaves; ACFl, A. cana flowers.
Since Artemisia essential oils and individual compound components exhibited only
weak neutrophil activation, we considered whether they might have some effect on neu-
trophils activated by strong agonists, such as N-formyl peptide receptor 1 (FPR1) agonist
f MLF and FPR2 agonist WKYMVM, as ligands can downregulate or alter neutrophil re-
sponses to subsequent treatment with heterologous or homologous agonists [63]. As shown
in Table 3, Artemisia essential oils were relatively more potent inhibitors of [Ca2+]i in f MLF-
and WKYMVM-stimulated neutrophils, with some IC50 values in the low micromolar
range. Essential oils from A. dracunculus flowers (ADFl) were the most potent, with IC50
values of 2.2 and 2.9 µM for inhibition of f MLF- and WKYMVM-induced neutrophil [Ca2+]i,
respectively. Representative data showing dose-dependent inhibition of f MLF-induced
neutrophil [Ca2+]i, by ADFl are shown in Figure 1A.
We also evaluated the effect of seven compounds present in Artemisia essential oils on
f MLF- and WKYMVM-induced neutrophil [Ca2+]i and found that farnesene, xanthoxylin,
and (±)-bornyl acetate inhibited f MLF- and WKYMVM-stimulated neutrophils, while the
other compounds were inactive (Table 3). Xanthoxylin and (±)-bornyl acetate were also
quite weak inhibitors, whereas the most potent inhibitor of peptide-induced neutrophil
[Ca2+]i was farnesene (IC50 = 1.1 ± 0.2 and 1.4 ± 0.5 µM for f MLF- and WKYMVM-
activated neutrophils, respectively). Representative data showing dose-dependent inhibi-
tion of f MLF-induced neutrophil [Ca2+]i, by farnesene are shown in Figure 1B. Likewise,
farnesene also dose-dependently inhibited IL-8 induced neutrophil [Ca2+]i, with an IC50
= 2.6 ± 0.2 µM (Figure 1C). These data suggest that farnesene can modulate intracellular
signaling pathways that are common to different G protein-coupled receptors (GPCRs),
including FPR1/FPR2 and CXCR1/2 chemokine receptors resulting in down-regulation of
the response to these chemoattractants.
Pharmaceuticals 2022, 15, x FOR PEER REVIEW 6 of 17 
 
We also evaluated the effect of seven compounds present in Artemisia essential oils 
on fMLF- and WKYMVM-induced neutrophil [Ca2+]i and found that farnesene, xanthoxy-
lin, and (±)-bornyl acetate inhibited fMLF- and WKYMVM-stimulated neutrophils, while 
the other compounds were inactive (Table 3). Xanthoxylin and (±)-bornyl acetate were 
also quite weak inhibitors, whereas the most potent inhibitor of peptide-induced neutro-
phil [Ca2+]i was farnesene (IC50 = 1.1 ± 0.2 and 1.4 ± 0.5 M for fMLF- and WKYMVM-
activated neutrophils, respectively). Representative data showing dose-dependent inhibi-
tion of fMLF-induced neutrophil [Ca2+]i, by farnesene are shown in Figure 1B. Likewise, 
farnesene also dose-dependently inhibited IL-8 induced neutrophil [Ca2+]i, with an IC50 = 
2.6 ± 0.2 µM (Figure 1C). These data suggest that farnesene can modulate intracellular 
signaling pathways that are common to different G protein-coupled receptors (GPCRs), 
including FPR1/FPR2 and CXCR1/2 chemokine receptors resulting in down-regulation of 
Pharmaceuticals 2022, 15, 642 the response to these chemoattractants. 6 of 17
Figure 1. Effect of A. dracunculus flower es sential oils (ADFl) and farnesene on f MLF- and IL-8-
Figinudreu c1e. dEnffeeuctt roofp hAi.l d[Craac2u+n]c.uHluus mfloanwnere uetsrsoepnhtiialsl woielsr e(AtrDeaFtle) danfodr f1a0rnmeisnenwei tohnA fMDLF, f-a arnndes eILn-e8,-oinr-
i Fl 1%
duDceMd SnOeu(tnreogpahtiilv e[Ca2+
con]tir. oHl)u. mThaen cneellustwroeprheitlsh ewnearcet itvreaateteddw foitrh 150 nmMinf MwiLthF (AAD,BFl), ofarr2n5esneMneo, fohr u1m% an
DMSO (negative co2n+trol). The cells were then activated with 5 nM fMLF (A,B) or 25 n±M of human 
IL-8 (C), and [Ca ]i was monitored as described. The data are shown as the mean SD from one
experiment. Representative of three (for f MLF) or two (for IL-8) independent experiments with
similar results.
 
2.3. Effect of A. dracunculus Essential Oils and Pure Compounds on Neutrophil Chemotaxis
Since A. dracunculus essential oils and their unique component, farnesene, were ef-
fective inhibitors of agonist-induced neutrophil [Ca2+]i, we evaluated their effects on
neutrophil chemotaxis. Pretreatment of neutrophils with ADFl for 30 min dose-dependently
inhibited f MLF-induced human neutrophil chemotaxis toward f MLF, with an IC50 of
2.4 ± 1.1 µg/mL (Figure 2A). Likewise, pretreatment of neutrophils with farnesene also
effectively inhibited neutrophil migration toward f MLF, with an IC50 of 3.3 ± 1.4 µM. A
representative dose-dependent response for the inhibition of f MLF-induced chemotaxis
in human neutrophils by farnesene is shown in Figure 2B. Analysis of (±)-bornyl acetate
showed that this compound also inhibited f MLF-induced neutrophil chemotaxis, although
Pharmaceuticals 2022, 15, x FOR PEER REVIEW 7 of 17 
 
IL-8 (C), and [Ca2+]i was monitored as described. The data are shown as the mean ± SD from one 
experiment. Representative of three (for fMLF) or two (for IL-8) independent experiments with sim-
ilar results. 
2.3. Effect of A. dracunculus Essential Oils and Pure Compounds on Neutrophil Chemotaxis 
Since A. dracunculus essential oils and their unique component, farnesene, were ef-
fective inhibitors of agonist-induced neutrophil [Ca2+]i, we evaluated their effects on neu-
trophil chemotaxis. Pretreatment of neutrophils with ADFl for 30 min dose-dependently 
inhibited fMLF-induced human neutrophil chemotaxis toward fMLF, with an IC50 of 2.4 ± 
1.1 µg/mL (Figure 2A). Likewise, pretreatment of neutrophils with farnesene also effec-
tively inhibited neutrophil migration toward fMLF, with an IC50 of 3.3 ± 1.4 µM. A repre-
sentative dose-dependent response for the inhibition of fMLF-induced chemotaxis in hu-
Pharmaceuticals 2022, 15, 642 man neutrophils by farnesene is shown in Figure 2B. Analysis of (±)-bornyl acet7aotef  17
showed that this compound also inhibited fMLF-induced neutrophil chemotaxis, alt-
hough with much lower potency (IC50 = 11.9 ± 1.9 M). In contrast, xanthoxylin did not 
afwfeictth thmisu rcehsploownseer. potency (IC50 = 11.9 ± 1.9 µM). In contrast, xanthoxylin did not affect
this response.
 
Figure 2. Inhibition of neutrophil chemotaxis by essential oils from A. dracunculus flowers (AD )
Fi(gAu)rea n2d. Infahrinbeistieonne o(fB n).eNuteruotprhoipl hcihlecmheomtaoxtiasx biys  teoswseanrtdia1l oniMls ffrMomLF Aw. darsamcuenacsuuluresd fl.oTwheerds a(AtaDaFrl)e (A)F l
from
and farnesene (B). Neutrophil chemotaxis toward 1 nM fMLF was measured. The data are from one 
exopneeriemxepnetr itmhaetn its trheaptriessreenptraetisveen toaft itvweoo ifntdweopeinnddeepnet nedxepnetriemxpenertsim. ents.
To ensure that Artemisia essential oils or active pure compounds were not cytotoxic, we
evaTluoa etendsuthree tchyatot tAorxtiecmityisioaf ethsseeenstsieanl toiaillso oilr saacmtivpele ps u(urpe ctoom55pogu/nmdsL )waenrde tneostt ccoymtoptooxui
µ nc,d s
waet evvaarliuoautsedc otnhece cnyttroattoioxnicsit(yu poft toh5e0 eµssMen)tiinalh ouilm saamn npeleust r(oupph tiols 5d5 uµrgin/mg La)1 a.5ndh tinesctu cboamtio- n
popuenridosd ,awt vhaicrhiocuosv ceorsntcheenttrimateiosnuss e(udpto tom 5e0a sµuMre)C ian2 +huinmflaunx (nueputtroo3p0hmilsin d)uarnidngce all 1m.5i ghr ainti-on
cu(buaptitoon 1p.5erhio).d,A wshsic 2+
hohw convienrsT tahbel eti3m, eess suesnetdia tloo milseafrsoumre AC.atr iidnefnlutaxt a(,uAp. tlou d3o0v miciiann) aa,nadn d
ceAll. mcaingarahtaiodnn (oupcy ttoo 1to.5x ihc)i.t yAisn shnoeuwtnro ipnh Tilasb, lael t3h, oeussgehnetisasle onitlisa flrooimls fAro. tmridAen. dtartaac,u An.c ululudosvain- d
ciaAn.af,r aignidda Ah. acdanaa lhitatdle ncoy tcoyttooxtiocxitiycitayt itnh enehuigtrhoepsht iclos,n acletnhtoruagtiho nesss(e2n5t–i5al0 oµilMs )f,rowmh iAch. dwrae-re
cu>n1c0u-lfuosl danhdi gAh.e frritghidaan hthados ae lriettqleu icryetdottooxiincidtyu caet bthioel ohgigichaelste fcfoenctcse.nTtrhaetiopnusr e(2c5o–m50p oMun)d, s
(±)-bornyl acetate, farnesene, and xanthoxylin were nontoxic in human neutrophils during
the 90 min incubation. In addition, these compounds did not affect the viability of human
 THP-1 monocytic cells after a 24 h incubation period (Figure 3). Thus, the biological effects
of these compounds on neutrophil function are not due to compound cytotoxicity and
support the conclusion that farnesene is a novel innate immunomodulator.
2.4. Identification of Potential Protein Targets for Selected Compounds
Both farnesene and bornyl acetate inhibited neutrophil [Ca2+]i and chemotaxis, al-
though farnesene was the most effective by far. Farnesene encompasses a set of six closely
related sesquiterpenes, as the α-form can exist as four stereoisomers, and the β-isomer
exists as two stereoisomers. We identified both (Z,E)-α-farnesene and (E,E)-α-farnesene in
A. dracunculus, although (E,E)-α-farnesene was present at a higher level (Table 2). Both α-
and β- forms of farnesene have been reported previously in essential oils from Artemisia spp.
For example, (Z,E)-α-farnesene was found in A. maderaspatana L. [64] and (E,E)-α-farnesene
was present in A. ordosica Krasch. and A. dracunculus [26,65,66]. (E)-β-farnesene was re-
Pharmaceuticals 2022, 15, x FOR PEER REVIEW 8 of 17 
 
Pharmaceuticals 2022, 15, 642 which were >10-fold higher than those required to induce biological effects. The pure co8mof-17
pounds (±)-bornyl acetate, farnesene, and xanthoxylin were nontoxic in human neutro-
phils during the 90 min incubation. In addition, these compounds did not affect the via-
biplitoyr toefd htuombaenp TreHsePn-1t  imn oAn.oacnyntuica cLe.ll[s6 7a]f,teAr .am 2a4g hel liannciucabaStciho.nB piepr.i[o6d8 ](,FAig.uarbes i3n)t.h TiuhmusL, .th[6e9 ],
biAol.olgaivcaanl deuflfiefocltisa oDf Cth. e[7s0e] ,coanmdpAou. nbidesn noins  Wneiulltdr.oepshsieln ftuianlcotiiolsn [a2r6e]. nFoint adlulye, (tZo )c-βom-fapronuensedn e
cywtoatsoxfoicuitnyd ainnde ssusepnptoiarlt othiles fcroonmcluAs.iaobns itnhtahti ufamrn[2e6se],nAe .isa nan nuoav[e7l1 i]n, annadte Aim. tmouurnnoefmorotidaunalaR- e-
toric. hb. [72]. Although the α- and β-forms of farnesene are well-known pheromones [73,74],
we did not find any reports on the biological activity of these compounds in human cells.
 
Figure 3. Cytotoxicity of selected compounds. Human neutrophils (A) or human THP-1 cells (B)
Figwuerree 3in. cCuybtaotteodxwiciittyh ionfd siecaletcetdedco cmopmopuonudnsdfso.r H90ummiann( Ane)uotrro2p4hhil(sB ()A, a)n odr cheullmviaanb iTliHtyPw-1a scealnlsa l(yBz)e d.
were incubated with indicated comp±ounds for 90 min (A) or 24 h (B), and cell viability was analyzed. 
ThTeh deadtaa tparpesreesnetnedte daraer tehteh me meaena n± SDS oDf otrfiptrliipcalitcea steamsapmlepsl efrsofmro monoen eexpexepriemriemnet.n Rt.eRperepsreensetanttiavteiv oef of
twtow iondinedpeepnednednet netxepxepriemriemnetsn tws iwthit shimsiimlairla rresreuslutsl.t s.
2.4. IdenBtoifrincaytlioanc eotfa Pteothenatsiabl ePernotreeinp Toratregdetsp froerv Sioeluescltyedt Coobmepaoumndasjo r component of essential
oils isolated from Artemisia spp, including A. ludoviciana and A. frigida [17,53]. Bornyl
aceBtaottehi sfaarlnsoesaenveo laantidle bcoonrnstyilt uaecnettaptree sinenhtibinitendu mneeurotruospchoinl i[fCera2e+s]si eanntdia lchoeilms aontadxeiss,s eanltt-ial
hoouilgshf rfoarmneVsaelneeri wanaaso tfhfiec imnaolisst [e7f5fe].ctPivreev bioyu fasrs.t Fuadrineessheanvee ernecpoomrtpeadsstehsa at  bsoetr noyf lsiaxc ecltoasteelhya s
realantteid-i nsfleasqmumitaetropreynpesro, paes rtthiees αin-fdoirfmfer ecannt  eexxpiestr imase fnotualr mstoedreeolsi.soFmoreerxsa, manpdle t,hbeo rβn-yisloamceetar te
exdisotsw ansr etgwuol astteedretohiesolemveerlss.o Wf per oidineflntaimfiemda btoortyh c(Zyt,oEk)-iαn-efsarinnevsietrnoe aanndd in(Ev,Eiv)-oαa-fnadrnreesdeuncee d
in tAhe. dnruamcubnecruloufst, oatlatlhcoeullgsh,  n(Eeu,Etr)o-αp-hfailrsn, easnednem wacarso pprheasgeenst iant ma uhriginheerb rleovneclh (oTaalvbeleo l2a)r. Blaovtahg e
α-fl aunid βa-f tfeorrminstr oafn faasranlleyseinjee chtaevdel bipeoepno rleypsoarctcehda rpirdeev.iMouosrleyo ivne er,ssbeonrtniayll oaiclest fartoemin Ahribteimteidsitah e
spepx. pFroers seioxanmapndle,p (rZo,dEu)-cαti-ofanrnoefsIeLn-e1 βwaansd foTuNnFd iin hAu. mmaandeuramspbaitliacnaal Lv.e i[n64e]n adnodth (eEl,iEal)-cαe-lls
far(HneUseVnEeC ws)asa npdreRseAnWt in2 6A4.. 7ormdoascircoa pKhragscehs .[ 7a6n,d7 7A].. Bdroarcnuynlcaucluetsa [t2e6h,6a5s,6a6ls].o (Ebe)-eβn-fraerpnoersteendet o
wahsa vrepaonratelgde stoic beeff pecrtesse[7n8t ]i.n A. annua L. [67], A. magellanica Sch. Bip. [68], A. absinthium 
L. [69],T Ao .b leatvtaenrduunlidfoelrias tDanCd. t[h70e],p aronpde Art.i ebsieonfntihs eWseilcldo.m epssoeunntidasl, owiles c[2a6lc]u. lFaitneadlltyh, e(iZr)m-βo-st
farimnepsoerntea nwt pahs yfsoiuconcdh eimn iceaslsepnatriaml eotielsr sfurosimng AS.w aisbsAinDthMiuEm[ 7[92]6.]T, hAe. LaongnPuav a[l7u1e]s, easntidm aAt.e d
using ALOGPS 2.1 program [80] and tPSA values allowed us to predict that (−) bornyl
acetate, (+) bornyl acetate, (Z,E)-α-farnesene, and (E,E)-α-farnesene can all permeate the
 blood–brain barrier (BBB), according to the binary classification tree model [81] (Table 4).
Pharmaceuticals 2022, 15, 642 9 of 17
Table 4. Predicted physicochemical properties of farnesene isomers and bornyl acetate.
Property (E,E)-α-Farnesene (Z,E)-α-Farnesene Bornyl Acetate
Formula C15H24 C15H24 C12H20O2
M.W. 204.35 204.35 196.29
Heavy Atoms 15 15 14
Fraction Csp3 0.47 0.47 0.92
Rotatable Bonds 6 6 2
H-Bond Acceptors 0 0 2
H-Bond Donors 0 0 0
MR 72.32 72.32 56.33
tPSA 0.00 0.00 26.30
LogP 5.70 5.70 3.50
BBB Permeation Yes Yes Yes
Legend: M.W., molecular weight (g/mol); MR, molar refractivity; tPSA, topological polar surface area (Å2); LogP,
lipophilicity; BBB, blood–brain barrier.
Farnesene is very lipophilic (Table 4). Thus, we propose that the neutrophil signaling
inhibitory mechanisms may be based on the allosteric interaction of the farnesene chain
with the membrane portion of the receptor, and we are currently investigating this mech-
anism. Indeed, other lipophilic compounds, such as the bile acids deoxycholic acid and
chenodeoxycholic acid, have been reported to antagonize FPR1 at high concentrations
(>100 µM) [82–84]. Moreover, lipoxin A4 was also reported as an allosteric modulator
of a CB1 cannabinoid receptor and FPR2 [85,86]. To further investigate this issue, we
aligned (E,E)-α-farnesene, (Z,E)-α-farnesene, and lipoxin A4 using FieldTemplater soft-
ware (Figure 4) and found that the alignment was determined mainly by the hydrophobic
hydrocarbon skeletons of the compounds. The combined similarity measure of the super-
imposition was relatively high (S = 0.690), suggesting that the farnesene molecules could
Pharmaceuticals 2022, 15, x FOR PEER REVImEWim ic lipoxin A4 and maybe other related specialized pro-resolving mediator1s0( konf o1w7 n as
 SPMs), including resolvins, maresins, and protectins [87]. Indeed, many of these molecules
have been demonstrated to act allosterically on a number of GPCRs (reviewed in [88]).
Interestingly, we found previously that 6-methyl-3,5-heptadien-2-one (MHDO), a com-
strucptuoruanlldy sstirmuciltaurr atoll yfasrinmeisleanr eto, afalsron einsehnibei,taeldso nienuhtirboiptehdiln aecutitvroitpyh [i2l 5a]c,t iavlitthyo[u2g5h], iatlst hmoou-gh its
leculamr otalercgueltasr wtaerrgee ntsowt iedreenntiofiteidd.e ntified.
 
 
Figure 4. Chemical structure of lipoxin A and alignment of lipoxin-A (green), (E,E)-α-farnesene
Figure 4. Chemical structure of lipoxin A 4
4 and alignment of lipoxin-A4 (gre4en), (E,E)-α-farnesene 
(pink()p, ainnkd) ,(Zan,Ed)-(αZ-,fEa)r-nαe-sfaernnee (skehnaek(ik).h aki).
Not much is known about the specific cellular targets of farnesene or bornyl acetate.
NThout sm, wucehp iesr fkonromwend arebvoeurts eth-peh sapremciafcico pcehlolurelamr ataprpgientgs oonf ftahrenmesoenleec uolra brosrtrnuyclt uacreestaotfe.( Z,E)-
Thusα, -wfaer npeesrefnoerm, (eEd,E r)e-αve-frasren-pesheanrem, aacnodphthoere( −m)aapnpdin(g+ )ofno rtmhes mofobleocrunlyalr ascterutactteurteosi doef ntify
(Z,E)p-αo-tfeanrtnieaslebnioe,l o(Eg,iEca)-lαt-afragrentess.ePnhea, armndM thape p(−e)r acnodm (p+)a froerdmasd oaf tbaobransyelo afcpethaaterm toa icdoepnhtiofrye pat-
potentetiranl sbwioiltohgtihcaels etacrogmetsp.o PuhnadrsmaMndapgpeenre rcaotmedpatarergde at  idnafotarbmaaseti oonf ,pshuacrhmaascpophharomrea pcoapt-horic
terns with these compounds and generated target information, such as pharmacophoric 
characteristics and normalized fitness scores. Note, however, that PharmMapper depends 
on the availability of structures for pharmacophore mapping, and most GPCRs are not 
represented. The proper optical isomers of the compounds were submitted to the Phar-
mMapper server as mapping explicitly accounts for the three-dimensional structure of a 
molecule. The ten top-ranked potential targets found by PharmMapper are shown in Ta-
ble 5. 
Table 5. Potential protein targets for bornyl acetate and farnesene were identified by PharmMap-
per. 
Rank PDB ID Target Name Fit Score Rank PDB ID Target Name Fit Score 
(−)-Bornyl Acetate (+)-Bornyl Acetate 
1 1REU BMP2 1 1 1J96 AKR1C2 1 
2 1J96 AKR1C2 1 2 1REU BMP2 1 
3 1MX1 LCE1 0.996 3 1OKL CA2 0.9992 
4 2AO6 NR3C4 0.9925 4 2PIQ NR3C4 0.9962 
5 2PE0 PDPK1 0.9923 5 2G01 JNK1 0.9857 
6 2G01 JNK1 0.9848 6 1W8L PPIase A 0.9826 
7 1IF4 CA2 0.9815 7 2UZD Cyclin-A2 0.9821 
8 1W8L PPIase A 0.9811 8 1MX1 LCE1 0.9784 
9 1VJY TGFBR1 0.979 9 1A28 PgR 0.9771 
10 1A28 PgR 0.9636 10 2PE0 PDPK1 0.9711 
(E,E)-α-Farnesene (Z,E)-α-Farnesene 
1 1J96 AKR1C2 1 1 1PME JNK1 1 
2 3HVC p38α 1 2 3HVC p38α 1 
3 1E7A Serum albumin 1 3 3BGP Pim-1 0.9998 
4 1OJ9 MAO-B 1 4 2PG2 KIF11 0.9993 
5 1SHJ Caspase-7 1 5 1E7A Serum albumin 0.999 
 
Pharmaceuticals 2022, 15, 642 10 of 17
characteristics and normalized fitness scores. Note, however, that PharmMapper depends
on the availability of structures for pharmacophore mapping, and most GPCRs are not
represented. The proper optical isomers of the compounds were submitted to the Phar-
mMapper server as mapping explicitly accounts for the three-dimensional structure of
a molecule. The ten top-ranked potential targets found by PharmMapper are shown
in Table 5.
Table 5. Potential protein targets for bornyl acetate and farnesene were identified by PharmMapper.
Rank PDB ID Target Name Fit Score Rank PDB ID Target Name Fit Score
(−)-Bornyl Acetate (+)-Bornyl Acetate
1 1REU BMP2 1 1 1J96 AKR1C2 1
2 1J96 AKR1C2 1 2 1REU BMP2 1
3 1MX1 LCE1 0.996 3 1OKL CA2 0.9992
4 2AO6 NR3C4 0.9925 4 2PIQ NR3C4 0.9962
5 2PE0 PDPK1 0.9923 5 2G01 JNK1 0.9857
6 2G01 JNK1 0.9848 6 1W8L PPIase A 0.9826
7 1IF4 CA2 0.9815 7 2UZD Cyclin-A2 0.9821
8 1W8L PPIase A 0.9811 8 1MX1 LCE1 0.9784
9 1VJY TGFBR1 0.979 9 1A28 PgR 0.9771
10 1A28 PgR 0.9636 10 2PE0 PDPK1 0.9711
(E,E)-α-Farnesene (Z,E)-α-Farnesene
1 1J96 AKR1C2 1 1 1PME JNK1 1
2 3HVC p38α 1 2 3HVC p38α 1
3 1E7A Serum
albumin 1 3 3BGP Pim-1 0.9998
4 1OJ9 MAO-B 1 4 2PG2 KIF11 0.9993
5 1SHJ Caspase-7 1 5 1E7A Serum
albumin 0.999
6 1PME ERK2 1 6 1OJ9 MAO-B 0.9989
7 1P49 Steryl-
sulfatase 0.9989 7 1J96 AKR1C2 0.9988
8 2PIN NR1A2 0.9985 8 1L6L Apo A-II 0.9984
9 3BGP Pim-1 0.9982 9 2PIN NR1A2 0.9978
10 1L6L Apo A-II 0.9977 10 1P49 Steryl-
sulfatase 0.9975
Legend: AKR1C2, aldo-keto reductase family 1 member C2; Apo A-II, apolipoprotein A-II; BMP2, bone mor-
phogenetic protein 2; CA2, carbonic anhydrase 2; ERK2, extracellular signal-regulated kinase 2; JNK1, c-Jun
N-terminal kinase 1; KIF11, kinesin family member 11; LCE1, liver carboxylesterase 1; MAO-B, monoamine
oxidase B; NR1A2, thyroid hormone receptor β; NR3C4, androgen receptor; p38α, p38α mitogen-activated
protein kinase; PDPK1, 3-phosphoinositide-dependent protein kinase 1; PgR, progesterone receptor; Pim-1,
proto-oncogene serine/threonine-protein kinase; PPIase A, peptidyl-prolyl cis-trans isomerase A; TGFBR1, TGF-β
receptor type-1.
PharmMapper analysis indicated that kinases could be among the potential tar-
gets for bornyl acetate and farnesene. Among the top ten ranked targets for the (±)-
enantiomers of bornyl acetate were c-Jun N-terminal kinase 1 (JNK1), 3-phosphoinositide-
dependent protein kinase 1 (PDPK1), and transforming growth factor-β receptor type-1
(TGFBR1), a serine/threonine-protein kinase. Likewise, JNK1, p38α mitogen-activated
protein kinase (MAPK), extracellular signal-regulated kinase 2 (ERK2), and proto-oncogene
serine/threonine-protein kinase (Pim-1) were kinases identified among the ten top-ranked
targets for (E,E)- and (Z,E)-forms of α-farnesene (Table 5). MAPK signaling plays an
important role in neutrophil signal transduction cascades [89], and studies have shown
that members of the MAPK, JNK, and the p38 MAPK families of proteins are activated in
response to neutrophil priming/activation (reviewed in [90]). It is also clear from previous
studies that one or more of these MAPK pathways is induced by FPR agonists [91,92] and
IL-8 [93]. Thus, these compounds and especially farnesene may be general inhibitors of
Pharmaceuticals 2022, 15, 642 11 of 17
neutrophil activation through GPCRs, and further studies are in progress to evaluate this
idea and identify their specific molecular targets.
3. Materials and Methods
3.1. Materials
N-formyl-Met-Leu-Phe (f MLF), Trp-Lys-Tyr-Val-Met (WKYMVM), and farnesene (mix-
ture of isomers) were from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). (±)-Bornyl
acetate was from Cayman Chemical Company (Ann Arbor, MI, USA). Xanthoxylin was
from TargetMol (Boston, MA, USA). Piperitone (mixture of isomers), (+)-camphor, and
1,8-cineole were from TCI America (Portland, OR, USA), and (−)-camphor was from Alfa
Aesar (Ward Hill, MA, USA). Fluo-4AM was from Invitrogen (Carlsbad, CA, USA). Fetal
bovine serum was from ATCC (Manassas, VA, USA). Hanks’ balanced salt solution without
Ca2+ and Mg2+ (HBSS–) was from Life Technologies (Grand Island, NY, USA). We prepared
HBSS+ by adding 1.3 mM CaCl2 and 1.0 mM MgSO4. Human interleukin-8 (IL-8) was
purchased from Peprotech (Rocky Hill, NJ, USA).
3.2. Essential Oil Extraction
Essential oils were obtained by hydrodistillation of air-dried plant material. Hydrodis-
tillation was performed with a Clevenger-type apparatus, as previously described [25]. To
avoid artifacts, we used conditions accepted by the European Pharmacopoeia to avoid
artifacts. Essential oil yields (w/v) were calculated based on the amount of air-dried plant
material used. We prepared stock solutions of the essential oils in 10 mg/mL DMSO for
biological evaluation. For gas-chromatographic (GC) analysis, samples were prepared in
10% w/v n-hexane.
3.3. GC-Flame Ionization Detector (GC-FID) and GC-Mass Spectrometry (GC-MS) Analysis
We performed GC-MS analysis using an Agilent 5975 GC-MSD system, as previously
described [94]. An Agilent Innowax FSC column (60 m × 0.25 mm, 0.25 µm film thickness)
was used with He as the carrier gas (0.8 mL/min). The GC oven temperature was kept
at 60 ◦C for 10 min, increased to 220 ◦C at a rate of 4 ◦C/min, kept constant at 220 ◦C for
10 min, and then increased to 240 ◦C at a rate of 1 ◦C/min. The split ratio was adjusted to
40:1, and the injector temperature was 250 ◦C. MS spectra were monitored at 70 eV with
a mass range of 35 to 450 m/z. GC analysis was carried out using an Agilent 6890 N 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 flame ionization detector (FID) temperature was 300 ◦C. The
essential oil components were identified by co-injection with standards (whenever possible),
which were purchased from commercial sources or isolated from natural sources. The
identities of compounds were also using the MassFinder software 4.0 (Dr. Hochmuth
Scientific Consulting, Hamburg, Germany), Adams Library, Wiley GC/MS Library (Wiley,
Hoboken, NJ, USA), and NIST Library. Confirmation was also achieved using the in-house
“Başer Library of Essential Oil Constituents” database. The database was created using
chromatographic analysis of pure compounds run under identical conditions. Samples
were spiked with a C8–C40 n-alkane standard solution (Fluka, Buchs, Switzerland) to
determine relative retention indices (RRI). The FID chromatograms were used to calculate
the relative amounts (%) of the separated compounds.
3.4. Isolation of Human Neutrophils
We obtained human neutrophils using blood collected from healthy donors. Blood
collection was approved by the Institutional Review Board at Montana State Univer-
sity (Protocol #MQ041017). Neutrophils were isolated as described previously [95]. The
isolated neutrophils were resuspended in HBSS–. Neutrophil preparations were rou-
tinely >95% pure and >98% viable, as determined by light microscopy and trypan blue
exclusion, respectively.
Pharmaceuticals 2022, 15, 642 12 of 17
3.5. Cell Culture
Human THP-1 monocytic cells obtained from ATCC (Manassas, VA, USA) were
cultured in RPMI 1640 medium (Mediatech Inc., Herndon, VA, USA) supplemented with
10% (v/v) FBS, 100 µg/mL streptomycin, and 100 U/mL penicillin.
3.6. Ca2+ Mobilization Assay
Changes in intracellular Ca2+ concentrations ([Ca2+]i) were monitored with a FlexSta-
tion 3 (Molecular Devices, Sunnyvale, CA, USA). For these assays, neutrophils were loaded
with 1.25 µg/mL Fluo-4AM and incubated in the dark at 37 ◦C for 30 min. The cells were
then washed with HBSS-. The dye-loaded cells were resuspended in HBSS+ and pipetted
into the wells of black microtiter plates at 2 × 105 cells/well. To measure the direct effects
of samples on [Ca2+]i, the test samples were added to the wells (final concentration of
DMSO was 1%), and fluorescence was monitored (λex = 485 nm, λem = 538 nm). Changes
in fluorescence were monitored every 5 s at room temperature for 240 s. To evaluate the
inhibitory effects of the test samples, the samples were added to the wells and incubated for
10 min, with the subsequent addition of 5 nM f MLF, 5 nM WKYMVM, or 25 nM IL-8. Re-
sponses were normalized to the response induced by control 5 nM f MLF, 5 nM WKYMVM,
or 25 nM IL-8 alone without pretreatment. These responses were assigned as 100%. To
calculate median effective concentrations (EC50 or IC50), we used curve fitting (at least five
or six points) and nonlinear regression analysis of the dose–response curves. Curve fitting
was performed with Prism 9 (GraphPad Software, Inc., San Diego, CA, USA).
3.7. Chemotaxis Assay
To evaluate effects of samples on neutrophil migration, we resuspended neutrophils in
HBSS+ containing 2% (v/v) heat-inactivated FBS (2 × 106 cells/mL). We analyzed chemo-
taxis using 96-well ChemoTx chambers (Neuroprobe, Gaithersburg, MD). The neutrophils
were first preincubated with the indicated concentrations of test samples at room tem-
perature for 30 min. The pretreated cells were then pipetted into the chamber upper
wells (4 × 104 cells/well). The lower wells contained 30 µL of HBSS+ with 2% (v/v) heat-
inactivated FBS, the indicated test samples or control 1% DMSO, and 1 nM f MLF as the
chemoattractant. Three lower wells were reserved for background controls (DMSO treated
cells in the upper wells and DMSO without f MLF in the lower wells). We allowed the cells
to migrate through the polycarbonate membrane filter for 60 min at 37 ◦C/5% CO2. We
determined the number of migrated cells by measuring ATP in lysates of transmigrated
cells and comparing this to a standard curve obtained with known neutrophil numbers,
as described previously [23]. Calculation of median effective concentrations (IC50) was
performed by nonlinear regression analysis of the dose-response curves.
3.8. Cytotoxicity Assay
We analyze cytotoxicity in human neutrophils or THP-1 monocytic cells. Cytotoxi-
city was analyzed using a CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega,
Madison, WI, USA). Briefly, the cells were incubated (104 cells/well) with the indicated
concentrations of essential oil or compound for 90 min (for neutrophils) or 24 h (for THP-1
cells) at 37 ◦C/5% CO2. After incubation, we added substrate. The samples were analyzed
using a Fluoroscan Ascent FL microplate reader.
3.9. Molecular Modeling
The PharmMapper Server [96] was used to identify potential protein targets for (E,E)-
α-farnesene (CID: 5281516) and (Z,E)-α-farnesene (CID: 5362889) (CIDs are compound
identifiers in PubChem). PharmMapper recognizes potential targets based on “invert”
pharmacophore mapping. The protein biotargets are represented by sets of pharmacophore
points in reference databases incorporated in the software. We used PubChem (https:
//pubchem.ncbi.nlm.nih.gov; accessed on 20 February 2022) as a source of initial 3D
structures for our compounds. These structures were downloaded from PubChem in SDF
Pharmaceuticals 2022, 15, 642 13 of 17
format. We then uploaded the structures into PharmMapper. The system automatically
generated up to 300 conformers of each compound based on the software option. We
performed pharmacophore mapping using the “Human Protein Targets Only” database,
which contained 2241 targets. We retrieved the top 250 potential targets for each compound
evaluated. The potential targets were sorted by normalized fit score. We calculated
compound physicochemical properties using SwissADME (http://www.swissadme.ch;
accessed on 20 March 2022). Properties were calculated for the structures of (−)-bornyl
acetate (CID: 93009), (+)-bornyl acetate (CID: 6950274), (E,E)-α-farnesene (CID: 5281516),
and (Z,E)-α-farnesene (CID: 5362889). The alignment of lipoxin-A4, (E,E)-α-farnesene,
and (Z,E)-α-farnesene was made with the use of FieldTemplater software (Cresset Group,
England, UK).
3.10. Statistical Analysis
For statistical analysis, we performed a one-way analysis of variance (ANOVA), fol-
lowed by Tukey's pair-wise comparisons. We considered differences at p < 0.05 to be
statistically significant.
4. Conclusions
Our results demonstrate that: (1) Artemisia spp. essential oils contain compounds
that exhibit neutrophil immunomodulatory activity, which might contribute to the re-
ported pharmacological properties of extracts from these plants; (2) the biological ef-
fects of these compounds on neutrophil function are not due to compound cytotoxicity;
(3) essential oils from A. dracunculus flowers (ADFl) contain farnesene isomers that may
have anti-inflammatory activity due to their ability to inhibit neutrophil responses to inflam-
matory chemoattractants, establishing farnesene as a novel innate immunomodulator, and
(4) synergetic effects may be possible with other essential oil constituents, such as bornyl
acetate or xanthoxylin. Further work is in progress to define the mechanisms of farnesene
action and evaluate its therapeutic potential.
Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/ph15050642/s1, Table S1: includes all compounds found in the
essential oils from each Artemisia species.
Author Contributions: I.A.S. and M.T.Q. conceived and designed the project; I.A.S., L.N.K. and
R.A.K. collected the plant material; I.A.S., G.Ö., T.Ö. and L.N.K. performed the experiments; A.I.K.
conducted molecular modeling study; I.A.S., G.Ö., T.Ö., L.N.K. and A.I.K. analyzed and interpreted
the data; I.A.S., G.Ö. and M.T.Q. drafted and revised the manuscript. All authors have read and
agreed to the published version of the manuscript.
Funding: This research was supported in part by National Institutes of Health IDeA Program Grants
GM115371 and GM103474; USDA National Institute of Food and Agriculture Hatch project 1009546;
the Montana State University Agricultural Experiment Station; and the Tomsk Polytechnic University
Development Program (Project Priority-2030-NIP/IZ-009-0000-2022).
Institutional Review Board Statement: The study was approved by the Montana State University
Institutional Review Board (protocol 2022-168, approved 23 March 2022).
Informed Consent Statement: Informed consent was obtained from all subjects involved in
the study.
Data Availability Statement: Data are contained within the article and supplementary material.
Conflicts of Interest: The authors declare no conflict of interest.
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