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Research Article
Anti-Inflammatory Activity of Spearmint (Mentha spicata) Essential Oil in Human Dermal Fibroblasts
Xuesheng Han*, Cody Beaumont and Tory L Parker*
dōTERRA International, LLC, Pleasant Grove, UT, USA

ABSTRACT
The use of Spearmint (Mentha spicata) Essential Oil (SEO) in skin care products has become popular. In this study, we evaluated the impact of a commercially available SEO on 17 protein biomarkers that perform critical functions in inflammation and tissue remodeling in a validated human dermal fibroblast system, which was designed to model the pathology of chronic inflammation. The impact of SEO on genome-wide gene expression was also evaluated. SEO showed robust antiproliferative effects in diseased human skin cells and significantly inhibited the increased production of two pro-inflammatory biomarkers Vascular Cell Adhesion Molecule-1 (VCAM-1) and interferon-inducible T-cell α chemoattractant (I-TAC). In contrast, SEO significantly increased the levels of Monocyte Chemoattractant Protein-1 (MCP-1), Epidermal Growth Factor Receptor (EGFR), Matrix Metalloproteinase-1 (MMP-1) and Tissue Inhibitor of Metalloproteinase-1 (TIMP-1). SEO significantly modulated global gene expression and altered signaling pathways, many of which are critical in the inflammatory and tissue remodeling processes. Further analysis showed that the gene expression data obtained were consistent with the observed anti-inflammatory and tissue remodeling effects of SEO. This study provided the first evidence of SEO’s anti-inflammatory activity in human dermal fibroblasts, which suggested that SEO might be a promising candidate for anti-inflammatory skincare products.
KEYWORDS
Antiproliferation; Genome-wide gene expression; Inflammation; Mentha spicata; Skin health; Spearmint essential oil

Introduction
Spearmint (Mentha spicata) Essential Oil (SEO) is rich in carvone and limonene and has a refreshing aroma. In comparison with peppermint essential oil, SEO is perceived to be a milder option for topical application in individuals with sensitive skin. This could be one of the reasons for the increased popularity of SEO in skin care, beauty and healthcare products. Studies in various models have demonstrated its therapeutic potential, which include antiproliferative, antibacteria l, antifungal, anticonvulsant and antiemetic activities [1-7]. However, studies on the effects of SEO in human skin cells are scarce. Although the anti-inflammatory properties of limonene, a major active component of SEO, have been demonstrated in both pre-clinical and clinical models; to our knowledge, no studies have been conducted to evaluate the anti-inflammatory activity of SEO in human dermal fibroblasts.

We investigated the biological activity of a commercially available SEO in a validated human dermal fibroblast cell culture line designed to model the pathology of chronic inflammation [8,9]. We analyzed the impact of SEO on 17 important protein biomarkers closely related to the inflammatory and tissue remodeling pathways. We also studied the regulatory effect of SEO on genome-wide gene expression.
Materials and Methods
All experiments were conducted in a Biologically Multiplexed Activity Profiling (Bio MAP) system HDF3CGF, a cell culture system of human dermal fibroblasts designed to model chronic inflammation and fibrosis in a robust and reproducible way. The system consists of three components: cells, stimuli to create the disease environment, and a set of biomarker (protein) readouts to examine how the treatments affect the disease environment [10]. The methodologies used in this study were essentially the same as those previously described [10-12]. The study was approved before commencement and followed the guidelines for human subject research under the regulations (45 CFR Part 46) of the US Department of health and human services.
Cell cultures
Primary human neonatal fibroblasts were prepared as previously described and plated in low-serum conditions for 24 h before stimulation with a mixture of Interleukin (IL)-1β [9], Tumor Necrosis Factor (TNF)-α, Interferon (IFN)-ϒ, 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 the assays were performed in a 96 well format [9,13].
Protein based readouts
An Enzyme Linked Immunosorbent Assay (ELISA) was used to measure the biomarker levels of cell associated and cell membrane targets. The soluble factors from the supernatants were quantified by using homogeneous time resolved fluorescence detection, bead based multiplex immunoassay, or capture ELISA. The overt adverse effects of the test agents on cell proliferation and viability (i.e., cytotoxicity) were measured by using the Sulforhodamine B (SRB) assay. For proliferation assays, the cells were cultured and then assayed after 72 h, which is optimal for the HDF3CGF system the detailed information has been described elsewhere [9]. The measurements were performed in triplicate and a glossary of the biomarkers used in this study is provided in supplementary table S1.

The quantitative biomarker data were presented as the mean log10 relative expression level (compared with the respective mean vehicle control value) ± Standard Deviation (SD) of triplicate measurements. The differences in biomarker levels between the SEO and vehicle treated cultures were tested for significance with an unpaired Student’s t-test. A p-value < 0.05, outside of the significance envelope, with an effect size of at least 10% (more than 0.05 log10 ratio units), was considered statistically significant.
RNA isolation
Total RNA was isolated from the cell lysates using the Zymo Quick-RNA MiniPrep kit (Zymo Research Corporation, Irvine, CA, USA) in accordance with the manufacturer’s instructions. The RNA concentration was measured using a NanoDrop ND-2000 (Thermo Fisher Scientific, Waltham, MA, USA) and the RNA quality was assessed by 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 an RNA integrity number score greater than 8.0.
Microarray analysis for genome-wide gene expression
A solution of 0.011% (v/v) SEO was tested for its effect on the expression of 21,224 genes in the HDF3CGF system after a 24-h treatment period. The samples for microarray analysis were processed by Asuragen, Inc. (Austin, TX, USA) in accordance with the company’s standard operating procedures. Biotin-labeled cRNA was prepared from 200 ng of total RNA with an Illumina total Prep RNA Amplification kit (Thermo Fisher Scientific) and one round of amplification. The cRNA yields were quantified via ultraviolet 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 Bead Chips (Illumina, Inc., San Diego, CA, USA). Hybridizing, washing, staining with streptavidin-conjugated cyanine-3, and scanning of the Illumina arrays were performed in accordance with the manufacturer’s instructions. Illumina bead scan software was used to produce the data files for each array and raw data were extracted using the Illumina bead studio software.

The raw data were uploaded into and analyzed for quality control metrics using the bead array package [13,14]. 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 [15,16]. The remaining probe sets comprised the data set for the rest of the analysis. Fold-change expression for each value was calculated as the log2 ratio of SEO to the vehicle control. These fold change values were uploaded into the Ingenuity Pathway Analysis (IPA, Qiagen, Redwood City, CA, USA) to generate the networks and pathway analyses.
Reagents
SEO (provided by dōTERRA International LLC, Pleasant Grove, UT, USA) was diluted in Dimethyl Sulfoxide (DMSO) to 8× the specified concentration (the final DMSO concentration in the culture media was no more than 0.1% [v/v]); 25 µL of each 8× solution was added to the cell culture to give a final volume of 200 µL DMSO (0.1%) served as the vehicle control.

The chemical composition of the oil was analyzed using Gas Chromatography-Mass Spectrometry (GC-MS). Detailed methods of GC-MS have been previously described [12]. GC-MS analysis of SEO indicated that its major chemical constitutes (i.e., >5%) are carvone (68%) and limonene (19%). The GC-MS chromatography of SEO has been included in supplementary figure 1.
Results and Discussion
Bioactivity profile of SEO in the human dermal fibroblast system HDF3CGF
We analyzed the activity of SEO in a dermal fibroblast cell system, HDF3CGF, which features the microenvironment of inflamed human skin cells with an already boosted immune response and inflammation level. Initially, four concentrations of SEO (0.011, 0.0037, 0.0012 and 0.00041%, v/v) were tested for biological activity; none of the concentrations showed clear cytotoxicity. The highest tested concentration of 0.011% was selected for further study. A key biomarker modulation activity was inferred if the biomarker values in the treated cells were significantly different (p<0.05) from those of the vehicle controls, with an effect size of at least 10% (more than 0.05 log ratio units; Figure 1).

Overall, SEO demonstrated diverse effects on multiple protein molecules, including significant antiproliferative activity in dermal fibroblasts. In addition, SEO significantly suppressed the increased production of two pro-inflammatory biomarkers, Vascular Cell Adhesion Molecule-1 (VCAM-1) and Interferon-inducible T-cell Alpha Chemoattractant (I-TAC). In contrast, SEO significantly increased the level of Monocyte Chemoattractant Protein 1 (MCP-1), which is also a pro-inflammatory chemokine. The important biomarkers related to tissue remodeling, namely Epidermal Growth Factor Receptor (EGFR), Matrix Metalloproteinase-1 (MMP-1) and Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) were also significantly increased by SEO. SEO had no significant effect on the other biomarkers tested in the experimental system.

Zhao et al., reported that SEO reduced pulmonary inflammation in rats with chronic obstructive pulmonary disease [17]. de Sousa et al., observed the spasmolytic activity of carvone and limonene, which can assist proper respiratory and cardiovascular functioning [18]. A recent human study demonstrated the beneficial effects of SEO on lung function and exercise performance [19], which may be at least partially attributed to its anti-inflammatory and spasmolytic feature. Fitsiou et al., recently showed that SEO rich in carvone (85.4%) exhibited antiproliferative activity in cancer cells [1].

Carvone and limonene were found to modulate immune response in BALB/c mice [20]. Studies on carvone derivatives also demonstrated their anti-inflammatory potential [18]. de Sousa et al., reported that orally administered hydroxydihydrocarvone exerted anti-inflammatory effects in rats and mice [21,22]. Marques et al. studied the anti-inflammatory activity of cyane-carvone, a synthetic derivative of carvone, and found that it significantly decreased IL-1β and TNF-α levels and significantly inhibited paw edema in mice [22].

Several studies of limonene showed that it demonstrated anti-inflammatory potential in a variety of disease models, possibly through the suppression of pro-inflammatory cytokines [23,24], p38 mitogen activated protein kinase, nuclear factor-κB, c-Jun N-terminal kinase , and extracellular signal-regulated kinase [25-28]. Notably, Rufino et al. observed that limonene increased the gene expression of TIMP-1 and decreased the expression of MMP-1 in a mouse model of osteoarthritis [23]. The results of the current study showed that SEO significantly increased the protein levels of TIMP-1 and MMP-1 in a human skin disease model.

Studies have also shown evidence that carvone and limonene act as transdermal permeation enhancers [29]. Limonene has been found to promote skin repair and inhibit skin tumorigenesis [30,28] .Both these properties are partly attributable to its anti-inflammatory activity. These features may further contribute to the beneficial effects of SEO on skin health.
Effects of SEO on genome-wide gene expression in the HDF3CGF system
We studied the effects of 0.011% SEO (The highest tested concentration that was not cytotoxic to the cells) on the RNA expression of 21,224 genes in the HDF3CGF system. The results show that SEO significantly modulated global gene expression; many genes were p regulated or down regulated (Table S2). Among the 200 most affected genes (log2 [expression fold change ratio relative to vehicle control] ≥ |1.5|) by SEO, the majority (148 of 200 genes) were significantly down regulated and the rest were up regulated. A cross comparison of protein and gene expression results showed that SEO significantly inhibited both the protein and gene expression of VCAM-1 and I-TAC.

Further, IPA studies indicated that the pattern of SEO bioactivity matched multiple canonical pathways listed in the literature-validated signaling pathway database (Figure 2). Many of these pathways play critical roles in the processes of inflammation, tissue remodeling, and immune modulation. The overall inhibitory effect of SEO on these genes and signaling pathways appeared consistent with its observed anti-inflammatory properties; the Supplementary Material contains more detailed information.

Collectively, these data showed that SEO, of which the main components are carvone and limonene, had significant antiproliferative and anti-inflammatory activities in human dermal fibroblasts. In addition, these findings suggested that SEO might be able to modulate immune responses during the wound healing process and promote improved wound healing. The results of the current study were consistent with those of existing studies, which indicated the therapeutic potential of SEO as an anti-inflammatory candidate for skin care products. Further research into its biological mechanism of action in specific disease models, as well as its clinical efficacy and safety, is recommended.
Conclusion
To our knowledge, this is the first study to evaluate the biological activity of SEO in pre-inflamed human dermal fibroblast cells. SEO showed significant antiproliferative activity through the inhibition of VCAM-1 and I-TAC levels and the increased expression of MCP-1, EGFR, MMP-1 and TIMP-1. Genome-wide gene expression analysis demonstrated that SEO exerted a robust and diverse impact on the expression of many genes. Many of the genes and pathways highly affected by SEO were critically involved in the inflammatory and tissue remodeling processes. These results were consistent with those of other studies that reported the anti-inflammatory and wound healing potential of SEO. Although the clinical efficacy and safety of SEO administration are still undetermined, our study indicated that SEO was a promising candidate for use in anti-inflammatory skin products.
Acknowledgment
This study was funded by dōTERRA (Pleasant Grove, UT, USA) and conducted at DiscoverX (Fremont, CA, USA). Xuesheng Han interpreted the data and drafted the manuscript. Cody Beaumont developed and conducted the GC-MS assays. Tory Parker oversaw the study design, methodology, and study agent and model selection. All authors reviewed and approved the final manuscript.
Conflicts of Interest
Xuesheng Han and Cody Beaumont and Tory Parker are employees of dōTERRA, the manufacturer of the study agent (SEO).

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Figures


Figure 1: Bioactivity profile of spearmint essential oil (SEO, 0.011% v/v) in a human dermal fibroblast system (HDF3CGF).
X-axis denotes protein-based biomarker readouts. Y-axis denotes the relative expression levels of biomarkers compared with the vehicle control values in the log form. The vehicle control values are marked in gray with a 95% confidence level.
*indicates a biomarker designated with “key activity”; the biomarker value for the treated cells was significantly different (p<0.05) from that of the vehicle control at the tested concentration, with an effect size of at least 10% (more than 0.05 log ratio units).
MCP-1 - Monocyte Chemoattractant Protein 1
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 Receptor
M-CSF - Macrophage Colony-Stimulating Factor
MMP-1 - Matrix Metalloproteinase 1
PAI-1 - Plasminogen Activator Inhibitor 1
SRB - Sulforhodamine B
TIMP - Tissue Inhibitors of Metalloproteinase



Figure 2: Top 20 canonical pathways matching the bioactivity profile of Spearmint Essential Oil (SEO, 0.011% v/v) for gene expression in the human dermal fibroblast system (HDF3CGF), produced via ingenuity pathway analysis.
The p-value is calculated using right-tailed Fisher's Exact Test. It indicates how likely the observed association between a specific pathway and the dataset would be if it resulted from random chance only. The smaller the p-value (that is, the bigger the - ln [p-value], indicated by the black bars) of a pathway, the greater the match with the bioactivity of SEO. The ratio, indicated by the gray bar, was calculated by taking the number of genes from the SEO dataset that participate in a canonical pathway and dividing it by the total number of genes in that pathway. Cdc42 - Cell division control protein 42 homolog

OX40 - Tumor necrosis factor receptor super family, member 4
RA - Rheumatoid Arthritis
IGF-1 - Insulin-Like Growth Factor 1
PPAR - Peroxisome Proliferator-Activated Receptors
VDR - Vitamin D Receptor
RXR - Retinoid X Receptor
ILK - Integrin-Linked Kinase

Citation: Han X, Beaumont C, Parker TL (2017) Anti-Inflammatory Activity of Spearmint (Mentha spicata) Essential Oil in Human Dermal Fibroblasts. J Cytol Tissue Biol 4: 015.