Antiinflammatory Activity of Cinnamon (Cinnamomum zeylanicum) Bark Essential Oil in a Human Skin Disease Model

Reagents

Cinnamon bark essential oil (dōTERRA International LLC, Pleasant Grove, UT, USA) was diluted in DMSO to 8X the specified concentrations [final DMSO concentration in culture media 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 CBEO indicated that its major chemical constitutes (i.e., >5%) were trans‐cinnamaldehyde (59%) and cinnamyl acetate (15%). The original gas chromatography–mass spectrometry chromatogram is shown in Fig. S1, and a list of all chemical constitutes found in CBEO is provided in Table S1.

Cell cultures

Primary human neonatal fibroblasts were obtained as previously described (Bergamini et al., 2012) and were plated under low serum conditions (0.125% fetal bovine serum) for 24 h. Then, the cell culture was stimulated with a mixture of interleukin‐1β, tumor necrosis factor‐α, interferon‐γ, basic fibroblast growth factor, epidermal growth factor, and platelet‐derived growth factor, for another 24 h. The cell culture and stimulation conditions for the HDF3CGF assays have been described in detail elsewhere and were performed in a 96‐well format (R Development Core Team, 2011; Bergamini et al.,2012).

Protein‐based readouts

An enzyme‐linked immunosorbent assay was used to measure the biomarker levels of cell‐associated and cell membrane targets. Soluble factors from supernatants were quantified using homogeneous time‐resolved fluorescence detection, bead‐based multiplex immunoassay, or captured enzyme‐linked immunosorbent assay. Overt adverse effects of the test agents on cell proliferation and viability (cytotoxicity) were measured using SRB assay. For proliferation assays, cells were cultured and then assayed after 72 h, which was optimized for the HDF3CGF system. Detailed information has been described elsewhere (Bergamini et al.,2012). Measurements were performed in triplicate wells, and a glossary of the biomarkers used in this study is provided in Table S2.

Quantitative biomarker data are presented as the mean log₁₀ relative expression level (compared with the respective mean vehicle control value) ± standard deviation of triplicate measurements. Differences in biomarker levels between CBEO‐treated and vehicle‐treated cultures were tested for significance with the unpaired Student's t‐test. A p‐value <0.01, outside of the significance envelope, with an effect size of at least 20% (more than 0.1 log₁₀ ratio units), was considered statistically significant.

RNA isolation

Total RNA was isolated from cell lysates using the Zymo Quick‐RNA MiniPrep kit (Zymo Research Corporation, Irvine, CA, USA), according to manufacturer's instructions. RNA concentration was determined using NanoDrop ND‐2000 (Thermo Fisher Scientific, Waltham, MA, USA). RNA quality was assessed with 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 0.0012% (v/v) concentration of CBEO was tested for its effect on expression of 21,224 genes in the HDF3CGF system after 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 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, USA). 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 software.

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). Data were normalized using quantile normalization (Bolstad et al.,2003), then re‐annotated and filtered to remove probes that were non‐specific 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. Fold‐change expression for each value was calculated as the log₂ ratio of CBEO 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 analyses.

Bioactivity profile of CBEO in pre‐inflamed human dermal fibroblasts

We analyzed CBEO's biological activity in a dermal fibroblast cell system, HDF3CGF, which mimics the disease microenvironment of inflamed human skin cells with already stimulated immune responses. Four different concentrations (0.011, 0.0037, 0.0012, and 0.00041% v/v, using DMSO as solvent) of CBEO were initially studied for their effects on cell viability. The two high concentrations were overtly cytotoxic; thus, only the 0.0012% v/v preparation, which contained the highest concentration of CBEO that was non‐cytotoxic, was used in further analyses. Key activities of biomarkers were indicated when biomarker values for test samples were significantly different (p < 0.01) from the respective values for the vehicle controls, with an effect size of at least 20% (more than 0.1 log ratio units) (Fig. 1).

Figure 1

The bioactivity profile of cinnamon bark essential oil (0.0012% v/v) in the human dermal fibroblast system HDF3CGF. The x‐axis denotes protein‐based biomarker readouts. The y‐axis denotes the relative expression levels of biomarkers when compared with the values for the vehicle control in log form. Vehicle control values are marked in gray at a 95% confidence level. * indicates a biomarker designated as having ‘key activity’, which is when a biomarker value is significantly different (p < 0.01) from the respective value for the vehicle control at a studied concentration, with an effect size of at least 20% (more than 0.1 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, the 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; TIMP, tissue inhibitor of metalloproteinase.

Cinnamon bark essential oil inhibited all the 17 biomarkers that were studied. It showed a significant anti‐proliferative activity against the dermal fibroblast cells. CBEO also significantly inhibited the production of inflammatory cytokines such as monocyte chemoattractant protein‐1, interferon gamma‐induced protein 10, interferon‐inducible T‐cell alpha chemoattractant, and monokine induced by gamma interferon (MIG). Furthermore, CBEO significantly inhibited the production of vascular cell adhesion molecule‐1 (VCAM‐1) and intercellular cell adhesion molecule‐1. The levels of several tissue remodeling molecules, including epidermal growth factor receptor (EGFR), matrix metalloproteinase 1, and plasminogen activator inhibitor‐1, were significantly decreased by CBEO. CBEO also significantly reduced the levels of macrophage colony‐stimulating factor, which is an immunomodulatory protein. The strong inhibitory effect of CBEO on the increased production of these biomarkers indicates that it may have an antiinflammatory property and, therefore, promote wound healing.

Previous studies have indicated that CBEO and its major active component cinnamaldehyde may have promising antiinflammatory properties (Tung et al.,2008; Wang et al.,2015). Chen et al. (2016) demonstrated using an animal model that trans‐cinnamaldehyde suppresses lipopolysaccharide‐induced inflammation (Chen et al.,2016). Another study has similarly reported on the antiinflammatory activity of cinnamaldehyde (Mendes et al., 2016). The observed strong antiinflammatory effect of CBEO in the human skin disease model suggests that CBEO may be used for treating inflammatory skin conditions.

In addition, the inhibitory effect of CBEO on the production of tissue remodeling biomarkers in the highly inflamed skin model suggests that CBEO and cinnamaldehyde may be beneficial for wound healing. This might be, at least, partially attributed to their antiinflammatory and antimicrobial properties (Ghosh et al.,2013; Wang et al.,2015).

Effects of CBEO on genome‐wide gene expression

To further explore the biological activities of CBEO on human skin cells, we studied the effect of the highest concentration of CBEO that was not cytotoxic to the cells (0.0012% v/v) on the RNA expression of 21,224 genes in the HDF3CGF system. The results showed a significant, strong, and diverse effect of CBEO on the regulation of the genes. Of the 200 genes that were highly regulated by CBEO [log₂ (expression fold‐change ratio relative to vehicle control) ≥ |1.5|], 148 genes were significantly downregulated, whereas 52 genes were significantly downregulated (Table S3). A cross comparison of protein and gene expression data showed that CBEO significantly inhibited the protein and gene expression levels of MIG and VCAM‐1.

The IPA showed that the bioactivity of CBEO significantly overlapped with many canonical signaling pathways from literature‐validated databases (Fig. 2). Some of these top signaling pathways (Table S4–S7), such as the hepatic fibrosis activation pathway and the antigen presentation pathway, are critically involved in inflammation and tissue remodeling. Interestingly, many pathways that play critical roles in cell cycle regulation, cancer biology, and DNA damage response were also on the list. The overall inhibitory effect of CBEO on the genes studied and the signaling pathways is not only consistent with its antiinflammatory and immune modulating potential; however, it also suggests that CBEO may play a role in modulating cell cycle regulation and cancer signaling.

Figure 2

Top 20 canonical pathways matching the bioactivity profile of cinnamon bark essential oil (0.0012% v/v) in gene expression in the HDF3CGF system, as obtained from the IPA. The p‐values were calculated using right‐tailed Fisher's exact test. Each p‐value measures how likely an observed association between a specific pathway and the dataset would be if it were only due to a random chance. The smaller the p‐value [bigger – ln (p‐value), indicated by the black bars] for a pathway is, the more significantly it matches with the bioactivity of CBEO. A ratio, indicated by the gray bar, was calculated by dividing the number of genes from the CBEO dataset that participated in a canonical pathway by the total number of genes in that pathway. NRF2, nuclear factor E2‐related factor 2; CHK, checkpoint kinase; PPAR, peroxisome proliferator‐activated receptor; Cdc 42, cell division control protein 42 homolog; BRCA1, breast cancer type 1 susceptibility protein; ERK5, extracellular signal‐regulated kinase 5.

Cinnamon essential oil has been reported to show a significant anticancer activity against head and neck squamous cell carcinoma via the suppression of EGFR tyrosine kinase (Yang et al.,2015). The EGFR tyrosine kinase signaling pathway is important for the growth, survival, proliferation, and differentiation of cells (Oda et al.,2005). Cinnamaldehyde has also been reported to be a potential anticancer drug (Hong et al.,2016), primarily due to its anti‐mutagenic, anti‐tumorigenic, anti‐proliferative, and pro‐apoptotic properties in cancer cell lines. The findings of our study are largely consistent with the anticancer properties of CBEO.

The current study has limitations. The in vitro study results cannot be directly extrapolated to more complex human skin systems. Moreover, the impact of CBEO on human global gene expression was measured over a short period. Therefore, how CBEO affects long‐term gene expression remains elusive. Nevertheless, the data from the current study provide important evidence of the biological activities of CBEO in human skin cells and suggest that CBEO is a promising antiinflammatory agent.