Biological Activity of an Essential Oil Blend in Human Dermal Fibroblasts
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Citation: Biological Activity of an Essential Oil Blend in Human Dermal Fibroblasts. American Research Journal of Dermatology. 2017; 1(1): 1-22.
Copyright This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Research on thebiological activities of essential oilsin human skin cells is limited. This studyfirst analyzedthe effect of an essential oil blend (EOB) on 17 important protein biomarkers in cytokine-stimulated human dermal fibroblasts. The EOB was composed of essential oils of frankincense resin, sweet orange peel, litsea fruit, thyme plant, clove bud, summer savory plant, and niaouli leaf. The results showed that EOBhad excellentantiproliferativeactivity. It significantlyincreased vascular cell adhesion molecule 1 levels and slightly increased themonocyte chemoattractant protein 1 and epidermal growth factor receptor production. We then studied theeffect of the EOB on the expression levels of 21,224 genes in the same cells. We found that the EOBmarkedly affected genome-wide gene expression. Further analysis revealed that the EOBpleiotropically regulated multiple signaling pathways in human cells including hepatic fibrosis activation, antigen presentation, mitotic roles of the polo-like kinase, and cyclin and cell cycle regulation. Many pathways significantly affected by the EOB are closely related toinflammatory and immune responses. The results suggest thatthe EOBmayaffectbiological processes and global gene expression in human skin cells. Further research into the underlying mechanism of action of the EOB is needed.
Keywords: Essential oil; frankincense; sweet orange;litsea; thyme;inflammation; immune response; signaling pathway; tumor; genome-wide gene expression
Essential oils are complex mixtures of aromatic compounds naturally produced in plants. Theyhave been used historically as well as currently for treating a variety of diseases and maintaining health in humans(Lv et al., 2013; Perry & Perry, 2006). Recent pre-clinical and clinical studies have provided evidence supporting the benefits of essential oils to human health (Kozioł et al., 2014; Navarra et al., 2015), resulting in a wider acceptance and use of essential oils in the US and worldwide. Despite this trend, very few studies have elucidated the mechanisms of action of essential oils inhuman cells.
Thousands of distinct terpene compounds have been identified in essential oils, many of which are known for possessing diverse biological activities. Because every essential oil is primarily composed of a unique mixture of just a few of these compounds, it is hypothesized that each oilhas its own unique array of biological activities. For example, Oregano essential oil is known to have powerful anti-fungal and anti-microbial effects due to its high phenylpropene content, while Lavender’s main constituents, linalool and linalyl acetate, are known to calm the CNS by activating GABAA receptors.
The tendency for essential oil compounds to exhibit synergy and antagonism is another phenomenon that is receiving growing attention. A recent study on membrane dynamics suggested that the ratios of constituents might affect an oil’s activity just as much as the identity of the constituents (Hac-Wydro et al., 2017). The possibility of synergy and antagonism has sparked an interest in blending, or creating mixtures of essential oils, to achieve an oil combination with novel effects. Therefore, we studiedthe biological effect of an industrial essential oil blend (EOB)on a human skin disease model, the HDF3CGF pre-inflamed dermal fibroblast system, which we have used previously to study the effects of individual essential oils. The current findings will allow comparison of this blend’s activitywith that of the individual essential oils and possibly other blends, aiding in future research on synergy, antagonism, and additive effects of essential oil blends.
The EOBcontains a mixture of essential oils from frankincense (Boswellia carterii, Boswellia frereana, and Boswelliasacra) resin, sweet orange (Citrus sinensis) peel, listea (Litsea cubeba) fruit, thyme (Thymus vulgaris) plant, clove (Eugenia caryophyllata) bud, the summer savory (Satureja hortensis) plant, and niaouli (Melaleuca quinquinervia) leaf.Although many of these individual essential oils and their active constituents are known to have various therapeutic benefits, this was the first study to examine the effect of a commercial blend of these oilson human genome-wide gene expression in the HDF3CGF model system. We also studiedthe EOB’s effect on biomarkers related to inflammation, immune responses, and tissue remodeling.
MATERIALS AND METHODS
All experiments were conducted using a biologically multiplexed activity profiling (Bio MAP) system HDF3CGF designed to model the pathology of chronic inflammation robustly and reproducibly. The system comprised three components: a cell type, stimuli to create the disease environment, and a set of biomarker (protein) readouts to examine the treatment effects on the disease environment (Berg et al., 2010). The methodologies used in this study were essentially the same as those previously described (Han & Parker, 2017a, 2017b; Kunkel et al., 2004)”container-title”:”Cogent Medicine”,”page”:”1306200”,”volume”:”4”,”issue”:”1”,”source”:”Taylor and Francis+NEJM”,”abstract”:”Although juniper (Juniperus communis.
The EOB (dōTERRA, Pleasant Grove, UT, USA) was diluted in dimethyl sulfoxide (DMSO) to 8× the specified concentrations (final DMSO concentration was no more than 0.1%). Then, 25 μL of each 8× solution was added to the cell culture to a final volume of 200 μL whileDMSO (0.1%) served as the vehicle control.
The composition of the EOBwas as follows: frankincense (a mixture of B. carterii, B. frereana, and B. sacra) resin oil, sweet orange (C. sinensis) peel oil, lemongrass (Cymbopogon flexuosus) leaf oil, thyme (T. vulgaris) plant oil, clove (E. caryophyllata) bud oil, summer savory (S. hortensis) plant oil, and niaouli (M. quinquinervia) leaf oil. The exact percentage composition is proprietary to the supplying company. Aromatic compounds distilled from the plant material comprised 100% of the EOB. Each essential oil originated from a country where the plant is grown. The essential oils were shipped to the US, where they were blended into the EOB. Gas chromatography-mass spectrometry analysis of the EOB showed that it contained 23–27% limonene, 11–14% alpha-pinene, 6–8% eugenol, 6–8% thymol, 5–7% carvacrol, 5–7% eucalyptol, 4–6% gamma-terpinene, and smaller amounts of other aromatic compounds.
Primary human neonatal fibroblasts were prepared 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 (IL)-1β, tumor necrosis factor (TNF)-α, interferon (IFN)-ϒ, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and platelet-derived growth factor (PDGF) for another 24 h. The cell culture for the HDF3CGF assays was performed in a 96-well plate,and the stimulation conditions were described in detail elsewhere (Bergamini et al., 2012; R Development Core Team, 2011).
An 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 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 measured after 72 h, which is optimal for the HDF3CGF system, and the detailed procedure was described in a previous study (Bergamini et al., 2012). The measurements were performed in triplicate wells, and a glossary of the biomarkers used in this study is provided in Supplementary Table S1.
This difference could be attributed to one of the other oils in the blend or perhaps the unique combination of oils. Finally, the blend had virtually no effect on collagen levels, which were significantly downregulated by Frankincense, Clove, and Lemongrass. These observations support the hypothesis that the EOB has unique biological activity that may perhaps be more than a simple sum of effects from the individual essential oils included in the blend. One obvious limitation to comparing the blend with these oils, however, is our lack of biomarker data on Niaouli, Summer Savory, Thyme, and Orange. Future research will make it possible to conduct a more comprehensive comparison of the effects of the EOB compared to its individualcomponent oils.