Biological Activities of Frankincense Essential Oil in Human Dermal Fibroblasts
Xuesheng Han, Damian Rodriguez, Tory L. Parker
Reference: BIOPEN 34
To appear in: Biochimie Open
Received Date: 12 December 2016
Revised Date: 25 January 2017
Accepted Date: 27 January 2017
Please cite this article as: X. Han, D. Rodriguez, T.L Parker, Biological Activities of Frankincense
Essential Oil in Human Dermal Fibroblasts, Biochimie Open (2017), doi: 10.1016/j.biopen.2017.01.003.
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Title: Biological Activities of Frankincense Essential Oil in Human Dermal Fibroblasts
Authors and Affiliations:
, Damian Rodriguez, and Tory L Parker
dōTERRA International, LLC, 389 S. 1300 W. Pleasant Grove, UT 84062, USA
Corresponding author: Xuesheng Han, dōTERRA International, LLC, 389 S. 1300 W. Pleasant
Grove, UT 84062, USA. Email: firstname.lastname@example.org.
Running title: Effect of Frankincense essential oil in human skin cells
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Although frankincense essential oil (FREO
) has become increasingly popular in skin care,
research on its biological activities in human skin cells is scarce, if not completely absent. In the
current study, we explored the biological activities of FREO in pre-inflamed human dermal
fibroblasts by analyzing the levels of 17 important protein biomarkers pertinent to
inflammation and tissue remodeling. FREO exhibited robust anti-proliferative activity in these
skin cells. It also significantly inhibited collagen III, interferon gamma-induced protein 10, and
intracellular cell adhesion molecule 1. We also studied its effect in regulating genome-wide
gene expression. FREO robustly modulated global gene expression. Furthermore, Ingenuity®
Pathway Analysis showed that FREO affected many important signaling pathways that are
closely related to inflammation, immune response, and tissue remodeling. This study provides
the first evidence of the biological activities of FREO in human dermal fibroblasts. Consistent
with existing studies in other models, the current study suggests that FREO possesses promising
potential to modulate the biological processes of inflammation and tissue remodeling in human
Keywords: inflammation; immune response; tissue remodeling; alpha-pinene; anti-
proliferation; skin health
FREO, frankincense essential oil
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Frankincense is a resin obtained from trees of the genus Boswellia. Historically, frankincense
whole resin, its extract, and essential oil have been extensively used for a number of health
purposes in Chinese and Ayurvedic medicine. FREO has been traditionally used for its anti-
inflammatory property. Recently, FREO has become increasingly popular for promoting skin
health. However, a literature search showed no published study of the biological activities of
FREO in human skin cells.
In this study, we explored the biological activities of a commercially available FREO in
human dermal fibroblasts in vitro. We first studied the effect of FREO on the levels of 17
important biomarkers related to inflammation, immune response, and tissue remodeling in the
skin cells. Then, we studied the effect of FREO on the expression levels of 21,224 genes, using
genome-wide analysis of the same cells. The results showed that FREO was biologically active
and significantly affected expression of these biomarkers and genes.
2. Materials and Methods
All experiments were conducted in a BioMAP HDF3CGF system, a cell culture of human
dermal fibroblasts that is designed to model chronic inflammation and fibrosis in a robust and
reproducible way. The system consists of three components: a cell type, stimuli to create the
disease environment, and set of biomarker (protein) readouts to examine how treatments
affect that disease environment .
2.1. Cell cultures
Primary human neonatal foreskin fibroblasts (HDFn) were obtained as previously described
. HDFn were plated in low serum conditions, 24-h before stimulation with cytokines. Cell
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culture and stimulation conditions for HDF3CGF assays have been described in detail
elsewhere, and were performed in a 96-well format [2,3].
2.2. Protein-based readouts
Direct ELISA was used to measure the biomarker levels of cell-associated and cell
membrane targets. Soluble factors from supernatants were quantified using HTRF® detection,
bead-based multiplex immunoassay, or capture ELISA. Overt adverse effects of the test agents
on cell proliferation and viability (i.e., cytotoxicity) were measured using SRB
proliferation assays, cells were cultured and then assayed after 72 h, which was optimized for
the HDF3CGF system. Detailed information has been described elsewhere . Measurements
were performed in triplicate wells. See Table S1 in Supplementary Materials for a glossary of
the biomarkers used in this study.
2.3. RNA isolation
Total RNA was isolated from cell lysates using the Zymo Quick-RNA™ MiniPrep kit (Zymo
Research Corporation, Irvine, CA), according to manufacturer’s instructions. RNA concentration
was determined using NanoDrop ND-2000 (Thermo Fisher Scientific). RNA quality was assessed
with 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 Number
score greater than 8.0.
2.4. Microarray analysis for genome-wide gene expression
A 0.003% (v/v) concentration of FREO was tested for its effect on expression of 21,224
genes in the HDF3CGF system after 24-h treatment. Samples for microarray analysis were
SRB, sulforhodamine B
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processed by Asuragen, Inc. (Austin, TX), according to the company’s standard operating
procedures. Biotin-labeled cRNA was prepared from 200 ng of total RNA with an Illumina®
TotalPrep™ RNA Amplification kit (Thermo Fisher Scientific) and one round of amplification. The
cRNA yields were quantified via UV spectroscopy, and the distribution of transcript sizes was
assessed using the Agilent Bioanalyzer 2100. Labeled cRNA (750 ng) was used to probe Illumina
Human HT-12 v4 Expression BeadChips (Illumina, Inc., San Diego, CA). Hybridizing, washing,
staining with streptavidin-conjugated Cyanine-3, and scanning of the Illumina arrays was
performed according to the manufacturer’s instructions. Illumina BeadScan software was used
to produce the data files for each array; raw data were extracted using Illumina BeadStudio
Raw data were uploaded into R  and analyzed for quality-control metrics using the
beadarray package . Data were normalized using quantile normalization , 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. Fold-change expression for each value was calculated as the log
ratio of FREO to
vehicle control. These fold-change values were uploaded to Ingenuity® Pathway Analysis (IPA®
QIAGEN, Redwood City, CA, www.qiagen.com/ingenuity) to generate the network and pathway
FREO (lot number 143504A, dōTERRA International LLC, Pleasant Grove, UT, USA) was
diluted in DMSO to 8X the specified concentrations (final DMSO concentration in culture media
IPA, Ingenuity® Pathway Analysis
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was no more than 0.1% (v/v)); 25 µL of each 8X solution was added to the cell culture to a final
volume of 200 µL. DMSO (0.1% (v/v)) served as the vehicle control. Gas chromatography–mass
spectrometry analysis of FREO indicated that its major chemical constitutes (i.e., >5%) were
alpha-pinene (57%), limonene (8%), and caprylyl acetate (7%).
3.1. Bioactivity profile of FREO in pre-inflamed human dermal fibroblasts
Four different concentrations (0.003, 0.001, 0.00033, and 0.00011% (v/v)) of FREO were
initially tested for biological activity in the dermal fibroblasts. None of the four concentrations
was overtly cytotoxic, and, therefore, the highest concentration (i.e., 0.003%) was analyzed
further. FREO showed significant anti-proliferative activity in dermal fibroblasts. Biomarkers
were designated if FREO values were significantly different (p < 0.05) from vehicle controls,
with an effect size of at least 10% (more than 0.05 log ratio units) (Figure 1). The level of a
tissue remodeling biomarker, collagen III, decreased in response to FREO. FREO significantly
reduced levels of
interferon gamma-induced protein 10 (
intracellular cell adhesion
molecule 1 (
), both important inflammatory biomarkers. FREO also slightly lowered the
levels of PAI-I, serine proteinase inhibitor and inhibitor of tissue plasminogen activator (tPA)
and urokinase (uPA), which is involved in tissue remodeling.
IP-10, interferon gamma-induced protein 10
ICAM-1, intracellular cell adhesion molecule 1
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Figure 1. The bioactivity profile of FREO (0.003% (v/v) in DMSO) in BioMAP System HDF3CGF. X-
axis denotes protein-based biomarker readouts. Y-axis denotes the relative expression levels of
biomarkers compared to vehicle control values, in log form. Vehicle control values are shaded
in gray, denoting the 95% confidence level. A * indicates a biomarker designated with “key
activity,” i.e., biomarker values were significantly different (p < 0.05) from vehicle controls, with
an effect size of at least 10% (more than 0.05 log ratio units).
3.2. Effects of FREO on gene expression: a genome-wide total RNA expression assay
To further explore the effect of 0.003% (v/v) FREO on human skin cells, we analyzed its
effect on the RNA expression of 21,224 genes. The results show a robust effect of FREO on
regulating human genes, with many genes being upregulated and many others being
downregulated. Among the top 83 regulated genes (absolute value of the fold-change ratio of
gene expression over vehicle control ≥ 1.5) by FREO, 42 were upregulated, and 41 were
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downregulated (Table S2). IPA analysis showed that the bioactivity of FREO significantly
overlapped with many canonical pathways (Figure 2) from the literature-validated database
(IPA®, QIAGEN, Redwood City, CA, www.qiagen.com/ingenuity). Many of these signaling
pathways are closely related to the biological processes of inflammation, immune response,
and tissue remodeling in human cells. See Supplementary Materials for more details.
Figure 2. Top 20 canonical pathways matching FREO’s bioactivity profile of gene expression in
the HDF3CGF system, produced via Ingenuity® Pathway Analysis (IPA®, QIAGEN, Redwood City,
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CA, www.qiagen.com/ingenuity). Each p-value is calculated with the 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 - log (p-
value), indicated by the black bars) the pathway has, the more significantly it matches with the
bioactivity of FREO. A ratio, indicated by each gray bar, is calculated by taking the number of
genes from the FREO dataset that participate in a canonical pathway, and dividing it by the total
number of genes in that pathway.
4.1. The anti-inflammatory and immune-modulating properties of FREO
Inflammation is a protective response that involves immune cells, blood vessels, and
molecular mediators. The purpose of inflammation is to eliminate the initial cause of cell injury,
remove necrotic cells and tissues damaged from the injury and inflammatory process, and
initiate tissue repair. Chronic inflammation may lead to a variety of diseases, such as hay fever,
periodontitis, atherosclerosis, rheumatoid arthritis, and cancer [7,8].
FREO significantly reduced the levels of IP-10 and ICAM-1, important pro-inflammatory
biomarkers, suggesting its anti-inflammatory potential. Alpha-pinene, the top constituent of
FREO, is widely recognized as the major anti-inflammatory component of FREO. Gayathri et al.
showed that alpha-pinene showed anti-inflammatory properties in human peripheral blood
mononuclear cells and mouse macrophages through inhibition of tumor necrosis factor-α,
interleukin-1β, nitric oxide, and mitogen activated protein kinases . An in vitro study showed
that isolated alpha-pinene had the ability to reduce the expression of pro-inflammatory
cytokines . Another study found that alpha-pinene inhibited the nuclear translocation of
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NF-kB induced by
in THP-1 cells, explaining its benefits in the treatment of
upper and lower airway diseases .
Recent research has also provided evidence that alpha-pinene has some immune-enhancing
properties, particularly regarding enhanced T-cell activity. In two related studies, the effects on
human immune function of essential oils from trees were investigated [12,13]. In both studies,
it was found that exposure to alpha-pinene increased T-cell activity and decreased stress
hormone levels. Consistent with these studies, microarray results of current study showed that
FREO affected some important inflammation- and immune-related signaling pathways. Gene
expression of many cytokines and other important players in inflammation and immune
responses was significantly inhibited in pre-stimulated, inflamed skin cells, indicating that FREO
has potential immune modulating properties.
4.2. Potential roles of FREO in the wound healing process
Wound healing is an intricate process, where the skin or other body tissue repairs itself
after injury. This process is composed of several phases: blood clotting (hemostasis),
inflammation, growth of new tissue (proliferation), and remodeling of tissue (maturation) .
The wound healing process is not only complex, but also fragile, and it is susceptible to
disruption, leading to the formation of non-healing, chronic wounds . Collagen III is
secreted by fibroblasts during the wound remodeling or repairing process, prior to the
deposition of collagen I. FREO dramatically lowered the level of collagen III, and therefore, it
would likely improve healing by reducing the chance of scar formation or wound persistence.
Additionally, the robust, anti-proliferative activity of FREO in skin cells could also contribute to
better wound healing.
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The significant, anti-proliferative activity of FREO observed in this study may have important
implications in skin and other cells. An in vitro study showed that FREO induced cell death in J82
bladder cancer cells via NRF-2-mediated oxidative stress . FREO and/or its top constituent,
alpha-pinene, has also been shown to be anti-proliferative and pro-apoptotic toward several
other types of cancer cells [17–21]. Further research into FREO’s activities in cancer cells will
better our understanding of its biological mechanism of action.
To our knowledge, this was the first study of the biological activities of FREO in human
dermal fibroblasts. FREO was significantly anti-proliferative to these cells. FREO significantly
inhibited the production of collagen III, IP-10, and ICAM-1. Genome-wide gene expression
analysis showed that FREO modulated global gene expression. It also robustly affected signaling
pathways which are relevant to inflammation and tissue remodeling.
This study was funded by dōTERRA Intl (UT, USA). We would like to thank Editage (www.editage
.com) for English language editing.
Conflicts of Interest
X.H., D.R., and T.P. are employees of dōTERRA.
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Please consider as potential referees:
Zhenhua Liu, Ph.D.
Dr. Liu is an expert in the research of health benefits of natural products, and their impact on
molecular pathways such as Wnt-pathway.
Sreejayan Nair, Ph.D.
Dr. Nair is an expert in studying the health benefits of natural compounds, including essential
Runzhi Lai, Ph.D.
Dr. Lai is an expert in molecular biology and biochemistry.
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Frankincense essential oil
(FREO) was anti-proliferative to human dermal fibroblasts.
• FREO significantly inhibited collagen III, interferon gamma-induced protein 10, and
intracellular cell adhesion molecule 1.
• FREO robustly modulated global gene expression.
• FREO affected many important signaling pathways that are closely related to
inflammation, immune response, and tissue remodeling.