Arborvitae (Thuja plicata) essential oil signiﬁcantly inhibited critical
inﬂammation- and tissue remodeling-related proteins and genes in human
Xuesheng Han*, Tory L. Parker
oTERRA International, LLC, 389 S. 1300 W., Pleasant Grove, UT 84062, USA
Received 21 December 2016; revised 6 February 2017
Available online 20 February 2017
Arborvitae (Thuja plicata) essential oil (AEO) is becoming increasingly popular in skincare, although its biological activity in human skin
cells has not been investigated. Therefore, we sought to study AEO's effect on 17 important protein biomarkers that are closely related to
inﬂammation and tissue remodeling by using a pre-inﬂamed human dermal ﬁbroblast culture model. AEO signiﬁcantly inhibited the expression
of vascular cell adhesion molecule 1 (VCAM-1), intracellular cell adhesion molecule 1 (ICAM-1), interferon gamma-induced protein 10 (IP-10),
interferon-inducible T-cell chemoattractant (I-TAC), monokine induced by interferon gamma (MIG), and macrophage colony-stimulating factor
(M-CSF). It also showed signiﬁcant antiproliferative activity and robustly inhibited collagen-I, collagen-III, plasminogen activator inhibitor-1
(PAI-1), and tissue inhibitor of metalloproteinase 1 and 2 (TIMP-1 and TIMP-2). The inhibitory effect of AEO on increased production of
these protein biomarkers suggests it has anti-inﬂammatory property. We then studied the effect of AEO on the genome-wide expression of
21,224 genes in the same cell culture. AEO signiﬁcantly and diversely modulated global gene expression. Ingenuity pathway analysis (IPA)
showed that AEO robustly affected numerous critical genes and signaling pathways closely involved in inﬂammatory and tissue remodeling
processes. The ﬁndings of this study provide the ﬁrst evidence of the biological activity and beneﬁcial action of AEO in human skin cells.
©2017 The Authors. Published by Elsevier B.V. on behalf of Socie
´Franc¸aise de Biochimie et Biologie Mole
´culaire (SFBBM). This is an open
access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Keywords: Arborvitae essential oil; Vascular cell adhesion molecule 1; Intracellular cell adhesion molecule 1; Interferon gamma-induced protein 10; Interferon-
inducible T-cell chemoattractant; Collagen-III
Arborvitae (Thuja plicata), also known as western red
cedar, and its essential oils have been traditionally used as a
natural insect repellent and wood preservative, primarily
because of its insecticidal and antimicrobial property [1e3].
Recently, the topical use of Arborvitae essential oil (AEO) for
skincare has gained popularity. However, a literature search
revealed no existing studies of the biological activities of AEO
in human cells. Therefore, we evaluated the biological activ-
ities of a commercially available AEO in a pre-inﬂamed
human dermal ﬁbroblast culture model, which was designed
to model the disease biology of chronic skin inﬂammation.
First, we analyzed the effect of AEO on 17 important protein
biomarkers that are closely related to inﬂammation and tissue
remodeling. Then, we studied its effect on genome-wide gene
expression in the same cell culture.
2. Materials and methods
All experiments were conducted using a BioMAP system
HDF3CGF, which was designed to model the pathology of
chronic inﬂammation in a robust and reproducible manner.
Nonstandard abbreviations: AEO, Arborvitae (Thuja plicata) essential oil.
E-mail address: firstname.lastname@example.org (X. Han).
Available online at www.sciencedirect.com
Biochimie Open 4 (2017) 56e60
2214-0085/©2017 The Authors. Published by Elsevier B.V. on behalf of Socie
´Franc¸aise de Biochimie et Biologie Mole
´culaire (SFBBM). This is an open access
article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
The system comprises three components: a cell type, stimuli to
create the disease environment, and a set of biomarker (pro-
tein) readouts to examine how the treatments affected the
disease environment .
2.1. Cell culture
Primary human neonatal ﬁbroblasts (HDFs) were prepared
as previously described  and were plated under low serum
conditions for 24 h before stimulation with a mixture of
interleukin (IL)-1b, tumor necrosis factor (TNF)-a, interferon
(IFN)-Y, basic ﬁbroblast growth factor (bFGF), epidermal
growth factor (EGF), and platelet-derived growth factor
(PDGF). The cell culture and stimulation conditions for the
HDF3CGF assays have been described in detail elsewhere and
were performed in a 96-well plate [5,6].
2.2. Protein-based readouts
Direct enzyme-linked immunosorbent assay (ELISA) was
used to measure the biomarker levels of cell-associated and
cell membrane targets. Soluble factors in the supernatants
were quantiﬁed using either homogeneous time-resolved
ﬂuorescence (HTRF) detection, bead-based multiplex immu-
noassay, 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 pro-
liferation assays, the cells were cultured and measured after
72 h, which is optimal for the HDF3CGF system, and the
detailed procedure has been described in a previous study .
Measurements were performed in triplicate wells, and a
glossary of the biomarkers used in this study is provided in
Supplementary Table S1.
2.3. RNA isolation
Total RNA was isolated from cell lysates using the Zymo
Quick-RNA MiniPrep kit (Zymo Research Corp., Irvine, CA,
USA) according to the manufacturer's instructions. RNA con-
centration was determined using a NanoDrop ND-2000 system
(Thermo Fisher Scientiﬁc). RNA quality was assessed using a
Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA,
USA) and an Agilent RNA 6000 Nano kit. All samples had an
A260/A280 ratio between 1.9 and 2.1 and a RIN score >8.0.
2.4. Microarray analysis of genome-wide gene
The effect of 0.011% AEO on the expression of 21,224
genes was evaluated in the HDF3CGF system after a 24-h
treatment. Samples for microarray analysis were processed by
Asuragen, Inc. (Austin, TX, USA) according to the company's
standard operating procedures. Biotin-labeled cRNA was
prepared from 200 ng of total RNA using an Illumina Total-
Prep RNA Ampliﬁcation kit (Thermo Fisher Scientiﬁc) and
one round of ampliﬁcation. The cRNA yields were quantiﬁed
using ultraviolet (UV) spectrophotometry, and the distribution
of the transcript sizes was assessed using the Agilent Bio-
analyzer 2100. Labeled cRNA (750 ng) was used to probe
Illumina human HT-12 v4 expression bead chips (Illumina,
Inc., San Diego, CA, USA). Hybridization, washing, staining
with streptavidin-conjugated cyanine-3, and scanning of the
Illumina arrays were carried out according to the manufac-
turer'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  and analyzed for
quality-control metrics using the beadarray package . The
data were normalized using quantile normalization , and
then re-annotated and ﬁltered to remove probes that were non-
speciﬁc or mapped to intronic or intragenic regions . 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
ratio of AEO to the vehicle control.
These fold-change values were uploaded onto Ingenuity
Pathway Analysis (IPA, QIAGEN, Redwood City, CA, USA,
www.qiagen.com/ingenuity) to generate the networks and
oTERRA Intl., UT, USA) was diluted in dimethyl
sulfoxide (DMSO) to 8the speciﬁed concentrations (ﬁnal
DMSO concentration in culture media was no more than 0.1%
[v/v]). Then, 25 mL of each 8solution was added to the cell
culture to obtain a ﬁnal volume of 200 mL, and DMSO (0.1%)
served as the vehicle control. The gas chromatography-mass
spectrometry (GCeMS) analysis of AEO indicated that it
mainly contained methyl thujate (53%) and smaller amounts
of numerous other aromatic molecules.
3. Results and discussion
3.1. Bioactivity proﬁle of AEO in pre-inﬂamed HDFs
We analyzed the biological activity of AEO by using an
HDF3CGF cell system, which simulated the microenvironment
of inﬂamed human skin cells with already boosted immune
responses and inﬂammatory levels. None of the four studied
concentrations (0.011, 0.0037, 0.0012, and 0.00041%, v/v) was
overtly cytotoxic, and therefore, the activity of 0.011% con-
centration was included for analysis. Key activities of bio-
markers were designated if biomarker values were signiﬁcantly
different (p <0.05) from those of vehicle controls, outside of
the signiﬁcance envelope, with an effect size of at least 10%
(>0.05 log ratio units, Fig. 1) and are discussed below.
The expressions of several inﬂammatory biomarkers, such
as vascular cell adhesion molecule 1 (VCAM-1), intracellular
cell adhesion molecule 1 (ICAM-1), interferon gamma-
induced protein 10 (IP-10), interferon-inducible T-cell che-
moattractant (I-TAC), and monokine induced by interferon
gamma (MIG), signiﬁcantly decreased in response to AEO
(Fig. 1). Speciﬁcally, the levels of these protein biomarkers
were already highly elevated in the pre-stimulated inﬂamed
57X. Han, T.L. Parker / Biochimie Open 4 (2017) 56e60
dermal ﬁbroblasts. The inhibitory effects of AEO on the
increased production of proinﬂammatory biomarkers suggest
that it might possess anti-inﬂammatory properties.
AEO also showed signiﬁcant antiproliferative activity in
dermal ﬁbroblasts, as measured using the SRB proliferation assay
72 h after treatment. The levels of ﬁve tissue remodeling mole-
culesdcollagen-I, collagen-III, plasminogen activator inhibitor-1
(PAI-1), and tissue inhibitor of metalloproteinase 1 and 2 (TIMP-
1 and TIMP-2)dsigniﬁcantly decreased in response to AEO
treatment. AEO also signiﬁcantly inhibited the level of macro-
phage colony-stimulating factor (M-CSF), a cytokine that me-
diates macrophage differentiation and thus, immunomodulation.
It is noteworthy that the inhibitory effects of AEO on the
increased production of these protein biomarkers were
concentration-dependent. AEO inhibited all these factors, which
suggests that it might play important roles in tissue remodeling
and immunomodulation, and thus, the wound healing processes.
These effects of AEO are presumably mediated by slowing down
the tissue repair process, which reduces the chance of scar for-
mation or improper chronic wound healing [10,11].
Recent studies on the essential oils of T. plicata-related
species, their major active components, or both have shown
preliminary evidence of their therapeutic efﬁcacy and safety in
disease models [12e14]. We conducted a literature search and
found that no study has been conducted on the effects of AEO
or its major component methyl thujate in human cells or
similar models. Therefore, to the best of our knowledge, the
current study is the ﬁrst evidence of the biological activities of
AEO in a human skin disease model, which suggests their
anti-inﬂammatory, immunomodulatory, and tissue-remodeling
properties in the human skin.
3.2. Effects of AEO on genome-wide gene expression
We then analyzed the effect of 0.011% AEO (the highest
studied non-cytotoxic concentration in these cells) on the RNA
expression of 21,224 genes in the same cells. The results
showed the signiﬁcantly diverse regulatory effect of AEO on
human genes, with numerous genes being either upregulated
or downregulated. Among the 200 most-regulated genes (with
a fold-change ratio of expression over the vehicle control of
j1.5j) by AEO, the majority (121 out of 200 genes) were
signiﬁcantly downregulated (Table S2). A cross-comparison of
the protein and gene expression data revealed that AEO
signiﬁcantly inhibited both the protein and gene expression
levels of VCAM-1,IP-10, and I-TAC. This suggests that AEO
might play a profound role in regulating these three important
IPA showed that the bioactivity of AEO signiﬁcantly over-
lapped with numerous canonical pathways from the literature-
validated database analysis (Fig. 2). Many of these signaling
pathways are closely related to inﬂammation, immunomodu-
lation, and tissue remodeling. Overall, AEO appeared to inhibit
these signaling pathways in the highly inﬂamed human skin
cells, suggesting it has potential anti-inﬂammatory and
immunomodulatory effects (see Supplementary Materials for
To the best of our knowledge, this study provides the ﬁrst
evidence of the biological activities of AEO in highly
Fig. 1. The bioactivity proﬁle of Arborvitae (Thuja plicata) essential oil (AEO, 0.011%, v/v in dimethyl sulfoxide, DMSO) in human dermal ﬁbroblast
culture (HDF3CGF). X-axis denotes protein-based biomarker readouts. Y-axis denotes the relative expression levels of biomarkers compared with those of
vehicle controls, in log form. Vehicle control values are shaded gray, with 95% signiﬁcance envelope. * indicates a biomarker designated with “key activity”:
biomarker value was signiﬁcantly different (p <0.05) from that of vehicle controls at the studied concentration, outside of the signiﬁcance envelope, with an effect
size of at least 10% (>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 gamma-induced protein 10; I-TAC, interferon-inducible T-cell alpha chemoattractant; IL-8, interleukin-8; MIG, monokine
induced by gamma interferon; EGFR, epidermal growth factor; M-CSF, macrophage colony-stimulating factor; MMP-1, matrix metalloproteinase 1; PAI-1,
plasminogen activator inhibitor 1; TIMP, tissue inhibitor of metalloproteinase.
58 X. Han, T.L. Parker / Biochimie Open 4 (2017) 56e60
inﬂamed human skin cells. The ﬁndings show that AEO
signiﬁcantly inhibited numerous protein and genes involved
in inﬂammation, immune responses, and tissue remodeling.
In addition, AEO diversely and signiﬁcantly modulated
global gene expression. Furthermore, AEO robustly affected
various important signaling pathways in human cells. These
ﬁndings provide the ﬁrst evidence for the therapeutic po-
tential of AEO in human skin cell inﬂammation. Further
studies on the mechanism of action and clinical efﬁcacy of
AEO are required before drawing deﬁnite conclusions about
its therapeutic properties.
Conﬂict of interest
X.H. and T.P. are employees of d
oTERRA, where the study
agent AEO was manufactured.
This study was funded by d
oTERRA (Pleasant Grove,
UT, USA), and conducted at DiscoverX (Freemont, CA,
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