Anti-inflammatory activity of clove (Eugenia caryophyllata) essential oil in human dermal fibroblasts

Abstract

Context: Clove (Eugenia caryophyllata Thunb. [Myrtaceae]) essential oil (CEO) has been shown to possess antimicrobial, antifungal, antiviral, antioxidant, anti-inflammatory and anticancer properties. However, few studies have focused on its topical use.
Objective: We investigated the biological activity of a commercially available CEO in a human skin disease model.
Materials and methods: We evaluated the effect of CEO on 17 protein biomarkers that play critical roles in inflammation and tissue remodelling in a validated human dermal fibroblast system, which was designed to model chronic inflammation and fibrosis. Four concentrations of CEO (0.011, 0.0037, 0.0012, and 0.00041%, v/v) were studied. The effect of 0.011% CEO on genome-wide gene expression was also evaluated.
Results and discussion: CEO at a concentration of 0.011% showed robust antiproliferative effects on human dermal fibroblasts. It significantly inhibited the increased production of several proinflammatory biomarkers such as vascular cell adhesion molecule-1 (VCAM-1), interferon γ-induced protein 10 (IP-10), interferon-inducible T-cell α chemoattractant (I-TAC), and monokine induced by γ interferon (MIG). CEO also significantly inhibited tissue remodelling protein molecules, namely, collagen-I, collagen-III, macrophage colony-stimulating factor (M-CSF), and tissue inhibitor of metalloproteinase 2 (TIMP-2). Furthermore, it significantly modulated global gene expression and altered signalling pathways critical for inflammation, tissue remodelling, and cancer signalling processes. CEO significantly inhibited VCAM-1 and collagen III at both protein and gene expression levels.
Conclusions: This study provides important evidence of CEO-induced anti-inflammatory and tissue remodelling activity in human dermal fibroblasts. This study also supports the anticancer properties of CEO and its major active component eugenol.

Full text

SHORT COMMUNICATION
Anti-inflammatory activity of clove (Eugenia caryophyllata) essential oil in human
dermal fibroblasts
Xuesheng Han and Tory L. Parker
d
oTERRA International, LLC, Pleasant Grove, UT, USA
ABSTRACT
Context: Clove (Eugenia caryophyllata Thunb. [Myrtaceae]) essential oil (CEO) has been shown to possess
antimicrobial, antifungal, antiviral, antioxidant, anti-inflammatory and anticancer properties. However, few
studies have focused on its topical use.
Objective: We investigated the biological activity of a commercially available CEO in a human skin disease
model.
Materials and methods: We evaluated the effect of CEO on 17 protein biomarkers that play critical roles
in inflammation and tissue remodelling in a validated human dermal fibroblast system, which was
designed to model chronic inflammation and fibrosis. Four concentrations of CEO (0.011, 0.0037, 0.0012,
and 0.00041%, v/v) were studied. The effect of 0.011% CEO on genome-wide gene expression was also
evaluated.
Results and discussion: CEO at a concentration of 0.011% showed robust antiproliferative effects on
human dermal fibroblasts. It significantly inhibited the increased production of several proinflammatory
biomarkers such as vascular cell adhesion molecule-1 (VCAM-1), interferon c-induced protein 10 (IP-10),
interferon-inducible T-cell achemoattractant (I-TAC), and monokine induced by cinterferon (MIG). CEO
also significantly inhibited tissue remodelling protein molecules, namely, collagen-I, collagen-III, macro-
phage colony-stimulating factor (M-CSF), and tissue inhibitor of metalloproteinase 2 (TIMP-2). Furthermore,
it significantly modulated global gene expression and altered signalling pathways critical for inflammation,
tissue remodelling, and cancer signalling processes. CEO significantly inhibited VCAM-1 and collagen III at
both protein and gene expression levels.
Conclusions: This study provides important evidence of CEO-induced anti-inflammatory and tissue remod-
elling activity in human dermal fibroblasts. This study also supports the anticancer properties of CEO and
its major active component eugenol.
ARTICLE HISTORY
Received 25 January 2017
Revised 29 March 2017
Accepted 29 March 2017
KEYWORDS
Anti-inflammation; skin
health; vascular cell
adhesion molecule-1;
collagen III; cancer
signalling; immune
response; eugenol;
interferon c-induced
protein 10; interferon-
inducible T-cell a
chemoattractant; monokine
induced by cinterferon
Introduction
Clove (Eugenia caryophyllata Thunb. [Myrtaceae]) essential oil
(CEO) is topically used for a variety of health purposes. Scientific
studies have evaluated its antimicrobial, antifungal, antiviral, anti-
oxidant, anti-inflammatory and anticancer properties in a variety
of models. However, research regarding its biological activity in
human skin cells is scarce. Prashar et al. (2006) reported that
CEO and its major active component eugenol displayed cytotox-
icity against human fibroblasts and endothelial cells, at concen-
trations as low as 0.03%(v/v). Koh et al. (2013) showed the anti-
inflammatory activity of eugenol in human gingival fibroblast
and pulp cells.
In this study, we investigated the biological activity of a
commercially available CEO in a well-validated human skin
disease model. We studied the effect of CEO on 17 protein
biomarkers that are closely related to inflammation, immune
response, and tissue remodelling processes. We also analyzed
the effect of CEO on genome-wide gene expression. The data
provide important evidence of the biological activity of CEO
in human dermal fibroblasts. The study supports the anti-
inflammatory and anticancer properties of CEO, and will likely
facilitate the future study of its mechanisms of action, clinical
efficacy, and safety.
Materials and methods
All the experiments were conducted using a Biologically
Multiplexed Activity Profiling (BioMAP) human dermal fibro-
blast system HDF3CGF (Kunkel et al. 2004a,2004b; Berg et al.
2010), which was designed to model the pathology of chronic
inflammation in a robust and reproducible manner. The system
comprises three components: a cell type, stimuli to create the dis-
ease environment, and a set of biomarker (protein) readouts to
examine how the treatments affected the disease environment
(Berg et al. 2010). The methodologies used in this study were
essentially the same as those previously described (Kunkel et al.
2004a,2004b; Han & Parker 2017a,2017b).
Reagents
CEO (d
oTERRA Intl., Pleasant Grove, UT) was diluted in
dimethyl sulfoxide (DMSO) to 8 the specified concentrations
CONTACT Xuesheng Han lhan@doterra.com d
oTERRA International, LLC, 389 S. 1300 W. Pleasant Grove, UT 84062, USA
Supplemental data for this article can be accessed here.
ß2017 doterra international.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/Licenses/by/4.0/), which permits unrestricted use, distri-
bution, and reproduction in any medium, provided the original work is properly cited.
PHARMACEUTICAL BIOLOGY, 2017
VOL. 55, NO. 1, 1619–1622
http://dx.doi.org/10.1080/13880209.2017.1314513

(final DMSO concentration in culture media was no more than
0.1%[v/v]). Then, 25 lL of each 8 solution was added to the
cell culture to obtain a final volume of 200 lL; DMSO (0.1%)
served as the vehicle control. Chemical analysis of CEO by gas
chromatography-mass spectrometry indicated that its major
chemical constitutes (i.e., >5%) are eugenol (81%), eugenol acet-
ate (10%), and trans-caryophyllene (7%).
Cell cultures
Primary human neonatal fibroblasts were prepared as previously
described (Bergamini et al. 2012) and were plated under low
serum conditions for 24 h before stimulation with a mixture of
interleukin (IL)-1b, tumour necrosis factor (TNF)-a, interferon
(IFN)-c, basic fibroblast growth factor (bFGF), epidermal growth
factor (EGF), and platelet-derived growth factor (PDGF). The
HDF3CGF assays were performed in a 96-well plate and cell cul-
ture and stimulation conditions have been described in detail
elsewhere (Bergamini et al. 2012).
Protein-based readouts
An enzyme-linked immunosorbent assay (ELISA) was used to
measure the biomarker levels of cell-associated and cell mem-
brane targets. Soluble factors in the supernatants were quantified
using either homogeneous time-resolved fluorescence detection,
bead-based multiplex immunoassay, or capture ELISA. The
adverse effects of the test agents on cell proliferation and viability
(cytotoxicity) were measured using the sulforhodamine B (SRB)
assay. For proliferation assays, the cells were cultured and meas-
ured after 72 h, which is optimal for the HDF3CGF system; the
detailed procedure was described by Bergamini et al. (2012).
Measurements were performed in triplicate wells, and a glossary
of the biomarkers used in this study is provided in
Supplementary Table S1.
Quantitative biomarker data are presented as the mean log
10
relative expression level (compared to the respective mean vehicle
control value) ± standard deviation (SD) of triplicate measure-
ments. Differences in biomarker levels between CEO- and
vehicle-treated cultures were tested for significance with the
unpaired Student’st-test. A p-value <0.05, outside of the signifi-
cance envelope, with an effect size of at least 10%(more than
0.05 log
10
ratio units), was regarded as statistically significant.
RNA isolation
Total RNA was isolated from cell lysates using the Zymo Quick-
RNA MiniPrep kit (Zymo Research Corp., Irvine, CA) according
to the manufacturer’s instructions. RNA concentration was deter-
mined using a NanoDrop ND-2000 system (Thermo Fisher
Scientific, Waltham, MA). The RNA quality was assessed using a
Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA) and an
Agilent RNA 6000 Nano kit. All samples had an A260/A280 ratio
between 1.9 and 2.1 and an RNA integrity score >8.0.
Microarray analysis of genome-wide gene expression
The effect of 0.011%CEO on the expression of 21,224 genes was
evaluated in the HDF3CGF system after treatment for 24 h.
Samples for microarray analysis were processed by Asuragen,
Inc. (Austin, TX) according to the company’s standard operating
procedures. Biotin-labelled cRNA was prepared from 200 ng of
total RNA using an Illumina TotalPrep RNA Amplification kit
(Thermo Fisher Scientific, Waltham, MA) and one round of
amplification. The cRNA yields were quantified using ultraviolet
spectrophotometry, and the distribution of the transcript sizes
was assessed using the Agilent Bioanalyzer 2100. Labelled cRNA
(750 ng) was used to probe Illumina human HT-12 v4 expression
bead chips (Illumina, Inc., San Diego, CA). Hybridization, wash-
ing, staining with streptavidin-conjugated cyanine-3, and scan-
ning of the Illumina arrays were carried out according to the
manufacturer’s instructions. The Illumina BeadScan software was
used to produce the data files for each array; the raw data were
extracted using Illumina BeadStudio software.
The raw data were uploaded into R (R Development Core
Team 2011) and analyzed for quality-control metrics using the
beadarray package (Dunning et al. 2007). The data were normal-
ized using quantile normalization (Bolstad et al. 2003), and then
re-annotated and filtered to remove probes that were nonspecific
or mapped to intronic or intragenic regions (Barbosa-Morais
et al. 2010). The remaining probe sets comprised the data set
for the remainder of the analysis. The fold-change expression
for each set was calculated as the log
2
ratio of CEO to the
vehicle control. These fold-change values were uploaded onto
Ingenuity Pathway Analysis (IPA, Qiagen, Redwood City, CA,
www.qiagen.com/ingenuity) to generate the networks and path-
way analyses.
Results and discussion
Bioactivity profile of CEO in pre-inflamed human dermal
fibroblasts
We studied the activity of CEO in a dermal fibroblast system,
HDF3CGF, which simulates the disease microenvironment of
inflamed human skin cells. None of the four investigated CEO
concentrations (0.011, 0.0037, 0.0012, and 0.00041%, v/v) was
overly cytotoxic; hence, all were used for further analysis. Key
biomarker activities were designated if biomarker values were
significantly different (p<0.05) from vehicle controls at the
0.011%concentration with an effect size of at least 10%(more
than 0.05 log ratio units, Figure 1).
Overall, CEO inhibited many of these 17 important bio-
markers. CEO showed significant antiproliferative activity in
human dermal fibroblasts. CEO also significantly decreased the
levels of inflammatory biomarkers such as vascular cell adhesion
molecule-1 (VCAM-1), interferon gamma-induced protein 10
(IP-10), interferon-inducible T-cell achemoattractant (I-TAC),
and monokine induced by cinterferon (MIG). Furthermore, it
significantly inhibited tissue remodelling protein molecules,
namely, collagen I, collagen III, macrophage colony-stimulating
factor (M-CSF), and tissue inhibitor of metalloproteinase 2
(TIMP-2). The effect of CEO on these biomarkers appeared to be
concentration-dependent. The significant inhibitory effects
exerted by CEO on these biomarkers indicate that CEO may pos-
sess anti-inflammatory and pro-wound-healing properties.
A number of studies have reported the anti-inflammatory
properties of CEO and its major active component eugenol. Both
CEO and eugenol exhibited anti-inflammatory activities in mur-
ine macrophages, inhibiting the production of pro-inflammatory
cytokines (Rodrigues et al. 2009; Bachiega et al. 2012). The cur-
rent study provides further support for the anti-inflammatory
properties of CEO and eugenol.
Interestingly, Koh et al. (2013) found that eugenol inhibited
increased IL-8 production in human gingival fibroblasts (HGF),
but not in periodontal ligament fibroblasts (HPLF) or skin
1620 X. HAN AND T. L. PARKER

keratinocytes (HaCat) (Koh et al. 2013). Our study demonstrated
that CEO significantly inhibited many pro-inflammatory cyto-
kines in pre-inflamed human dermal fibroblast cells, but did not
significantly affect IL-8 levels. Further research is needed to
evaluate the biological mechanism underlying the effect of CEO
in different human cells.
Effects of CEO on genome-wide gene expression
To further explore the biological activities of CEO in human skin
cells, we studied the effect of 0.011%CEO (the highest tested
non-cytotoxic concentration) on the RNA expression of 21,224
genes in the HDF3CGF system. The results showed that CEO
Figure 1. Bioactivity profile of clove essential oil (CEO, 0.011% v/v) in human dermal fibroblast culture HDF3CGF. X-axis denotes protein-based biomarker readouts.
Y-axis denotes the relative expression levels of biomarkers compared to vehicle control values, in log
10
form. Vehicle control values are shaded in grey, denoting the
95% significance envelope. Error bars represent the standard deviations from triplicate measurements. A indicates a biomarker designated with ‘key activity,’i.e., bio-
marker values were significantly different (p<0.05) from vehicle controls, outside of the significance envelope, with an effect size of at least 10% (more than 0.05 log
ratio units). MCP-1, monocyte chemoattractant protein; VCAM-1, vascular cell adhesion molecule 1; ICAM-1, intracellular cell adhesion molecule 1; IP-10, interferon
c-induced protein 10; I-TAC, interferon-inducible T-cell achemoattractant; IL-8, interleukin-8; MIG, monokine induced by cinterferon; EGFR, epidermal growth factor
receptor; M-CSF, macrophage colony-stimulating factor; MMP-1, matrix metalloproteinase 1; PAI-1, plasminogen activator inhibitor 1; TIMP, tissue inhibitor of
metalloproteinase.
Figure 2. Top 20 canonical pathways matching the bioactivity profile of clove essential oil (CEO, 0.011% v/v) in gene expression in the HDF3CGF system produced
using Ingenuity Pathway Analysis (IPA, Qiagen, www.qiagen.com/ingenuity). Each p-value was calculated using right-tailed Fisher's exact test. The p-value measures the
likelihood that the observed association between a specific pathway and the dataset is due to random chance. The smaller p-value (the bigger - ln [p-value], indicated
by the black bars) the pathway has, the more significantly it matches the bioactivity of CEO. A ratio, indicated by each grey bar, was calculated by taking the number
of genes from the CEO dataset that participate in a canonical pathway, and dividing it by the total number of genes in that pathway. GADD45: growth arrest and
DNA-damage-inducible protein 45.
PHARMACEUTICAL BIOLOGY 1621

exerted robust and diverse effects on gene regulation, with many
genes being downregulated and others being upregulated. Of the
200 most affected genes (log
2
[expression fold-change ratio rela-
tive to vehicle control] j1.5j), the majority (142 out of 200
genes) were significantly downregulated, and the rest were upre-
gulated (Table S2). A cross comparison of protein and gene
expression data revealed that CEO significantly inhibited collagen
III and VCAM-1 at both protein and gene expression levels.
IPA studies showed that CEO bioactivity significantly matched
that of many canonical signalling pathways from the literature-
validated database (Figure 2). Many of these pathways are
involved in inflammation, tissue remodelling, stress response, cell
cycle regulation, cancer signalling, or cellular metabolism. This
indicates that CEO may play an important role in a variety of bio-
logical and physiological processes. For example, the top matched
pathway was hepatic fibrosis activation, and this was inhibited by
CEO, further supporting its anti-inflammatory properties.
In addition to their anti-inflammatory properties, CEO and
eugenol have been shown to possess anticancer properties against
breast, colorectal, lung, and leukaemia cancer cells (Yoo et al.
2005; Kouidhi et al. 2010; Kumar et al. 2014). These previous
findings are largely consistent with the findings of the current
study, which indicate that CEO affected cell cycle control and
cancer biology.
Conclusions
Taken together, the results of this study demonstrated the anti-
inflammatory, immune-modulating, and tissue remodelling activ-
ities of CEO in a human skin disease model. The microarray study
also showed evidence suggesting the role of CEO in modulating
important signalling pathways related to immune function, cell
cycle control, cellular stress responses, and even cancer biology.
These data largely support the anti-inflammatory and anticancer
properties of CEO and its major active component eugenol.
Disclosure statement
X.H. and T.P. are employees of d
oTERRA, where the study agent
CEO was manufactured.
Funding
The study was funded by d
oTERRA (Pleasant Grove, UT) and con-
ducted at DiscoverX (Fremont, CA).
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