Unsaturated Fatty Acids Complex Regulates Inflammatory Cytokine Production through the Hyaluronic Acid Pathway

In this study, we aimed to develop natural and/or functional materials with antioxidant and anti-inflammatory effects. We obtained extracts from natural plants through an oil and hot-water extraction process and prepared an extract composite of an effective unsaturated fatty acid complex (EUFOC). Furthermore, the antioxidant effect of the extract complex was evaluated, and the anti-inflammatory effect was explored by assessing its inhibitory effect on nitric oxide production through its HA-promoting effect. We conducted a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay to evaluate the cell viability of the EUFOC, and the results showed that EUFOC was not cytotoxic at the test concentrations. In addition, it showed no endogenous cytotoxicity in HaCaT (human keratinocyte) cells. The EUFOC showed excellent 1,1-diphenyl-2-picrylhydrazyl- and superoxide-scavenging abilities. Moreover, it exerted an inhibitory effect on NO production at concentrations that did not inhibit cell viability. The secretion of all the cytokines was increased by lipopolysaccharide (LPS) treatment; however, this was inhibited by the EUFOC in a concentration-dependent manner. In addition, hyaluronic acid content was markedly increased by the EUFOC in a dose-dependent manner. These results suggest that the EUFOC has excellent anti-inflammatory and antioxidant properties, and hence, it can be used as a functional material in various fields.


Introduction
Different health problems have been attributed to environmental pollution and climate change [1,2]. In addition, the healthcare and anti-aging industries are growing rapidly in response to economic growth and cultural influence, and the development of new materials and additional functional exploration of existing materials are continuously increasing to meet various social demands and consumer needs [3]. Scientific evidence has increased the reliability of the pharmaceutical and healthcare industries, and the search for preventive/healthcare materials from synthetic and natural products and the application of these products have been expanding [4,5].
The interest in general healthcare is expanding to natural materials, and scientific approaches to natural materials that have been traditionally used are also increasing [6,7]. The 1,1-diphenyl-2-picrylhydrazyl (DPPH) free-radical-scavenging ability of the EUFOC was also analyzed. The cells were treated with 0.3, 0.5, and 1% EUFOC, which were not in the cytotoxic range. The results showed that the scavenging ability, which was 15-35%, increased in a concentration-dependent manner ( Figure 2).  The 1,1-diphenyl-2-picrylhydrazyl (DPPH) free-radical-scavenging ability of the EU-FOC was also analyzed. The cells were treated with 0.3, 0.5, and 1% EUFOC, which were not in the cytotoxic range. The results showed that the scavenging ability, which was 15-35%, increased in a concentration-dependent manner ( Figure 2).  Figure 3. Analysis of the superoxide (SO)-scavenging ability of EUFOC. EUFOC: effective unsaturated fatty acids complex. Control: negative control (distilled water). * p < 0.01 and ** p < 0.001 vs. control group.

Inhibition of NO Production
Oxygen is converted into a significant amount of reactive oxygen during the oxidation process in the body. It reacts with cellular components in the body, causing oxidative stress that damages cells and tissues in the body [45,46]. Continuous oxidative stress results in DNA denaturation, damages cell membranes and proteins, and induces chronic inflammatory reactions [47,48].
The inhibitory effect of the EUFOC on NO production was exhibited at concentrations that did not inhibit cell viability (Figure 4), indicating the anti-inflammatory potential of the EUFOC.

Inhibition of NO Production
Oxygen is converted into a significant amount of reactive oxygen during the oxidation process in the body. It reacts with cellular components in the body, causing oxidative stress that damages cells and tissues in the body [45,46]. Continuous oxidative stress results in DNA denaturation, damages cell membranes and proteins, and induces chronic inflammatory reactions [47,48].
The inhibitory effect of the EUFOC on NO production was exhibited at concentrations that did not inhibit cell viability (Figure 4), indicating the anti-inflammatory potential of the EUFOC.

Inhibition of NO Production
Oxygen is converted into a significant amount of reactive oxygen during the oxidation process in the body. It reacts with cellular components in the body, causing oxidative stress that damages cells and tissues in the body [45,46]. Continuous oxidative stress results in DNA denaturation, damages cell membranes and proteins, and induces chronic inflammatory reactions [47,48].
The inhibitory effect of the EUFOC on NO production was exhibited at concentrations that did not inhibit cell viability (Figure 4), indicating the anti-inflammatory potential of the EUFOC.
stress that damages cells and tissues in the body [45,46]. Continuous oxidative stress results in DNA denaturation, damages cell membranes and proteins, and induces chronic inflammatory reactions [47,48].
The inhibitory effect of the EUFOC on NO production was exhibited at concentrations that did not inhibit cell viability (Figure 4), indicating the anti-inflammatory potential of the EUFOC.

Inhibitory Effect of the EUFOC on the Production of Inflammatory Cytokines
The inflammatory response is one of the immune system responses, and cytokines, such as TNF-α and IL-1β, are produced during the initial reaction, thereby increasing the activity of immune cells in tissue wounds or infection sites. Excessive secretion of cytokines involved in inflammatory reactions can induce acute or chronic inflammatory diseases [49,50]. To determine the effect of the EUFOC on the secretion of pro-inflammatory cytokines in HaCaT cells treated with LPS, the changes in the amounts of TNF-α, IL-1β, and IL-6 were measured after treatment with different concentrations of the EUFOC. As shown in Figure 5, the levels of all the cytokines were markedly increased by LPS treatment; however, IL-6 secretion was significantly suppressed by EUFOC treatments (Figure 5c) but not TNF-α and IL-1β (Figure 5a,b). TNF-α is a major endogenous mediator in the early stage of inflammatory response, while IL-1β is a pro-inflammatory cytokine produced during the inflammatory response and a major cytokine involved in the inflammatory response [51]. Therefore, our findings indicated that the EUFOC exhibited an anti-inflammatory effect by inhibiting the late stage of inflammatory response, but not the early stage and proinflammatory response.

Inhibitory Effect of the EUFOC on the Production of Inflammatory Cytokines
The inflammatory response is one of the immune system responses, and cytokines, such as TNF-α and IL-1β, are produced during the initial reaction, thereby increasing the activity of immune cells in tissue wounds or infection sites. Excessive secretion of cytokines involved in inflammatory reactions can induce acute or chronic inflammatory diseases [49,50]. To determine the effect of the EUFOC on the secretion of pro-inflammatory cytokines in HaCaT cells treated with LPS, the changes in the amounts of TNF-α, IL-1β, and IL-6 were measured after treatment with different concentrations of the EUFOC. As shown in Figure 5, the levels of all the cytokines were markedly increased by LPS treatment; however, IL-6 secretion was significantly suppressed by EUFOC treatments ( Figure  5c) but not TNF-α and IL-1β (Figure 5a,b). TNF-α is a major endogenous mediator in the early stage of inflammatory response, while IL-1β is a pro-inflammatory cytokine produced during the inflammatory response and a major cytokine involved in the inflammatory response [51]. Therefore, our findings indicated that the EUFOC exhibited an antiinflammatory effect by inhibiting the late stage of inflammatory response, but not the early stage and pro-inflammatory response.

Inhibitory Effect of the EUFOC on COX-2 and iNOS Expression
The inflammatory response is mediated by various pathways and is closely related to cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) [15,52]. COX is an enzyme that converts arachidonic acid into prostaglandins and exists in two forms: COX-1 and COX-2. In particular, COX-2 is expressed in response to stimuli, such as cell

Inhibitory Effect of the EUFOC on COX-2 and iNOS Expression
The inflammatory response is mediated by various pathways and is closely related to cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) [15,52]. COX is an enzyme that converts arachidonic acid into prostaglandins and exists in two forms: COX-1 and COX-2. In particular, COX-2 is expressed in response to stimuli, such as cell growth factors, cytokines, tumor promoters, and reactive oxygen species, which cause inflammatory diseases [53][54][55]. iNOS generates NO to defend the body in response to external stimuli, and excessive production of NO results in inflammation, inducing tissue damage, genetic mutation, and neural damage [56].
LPS-stimulated HaCaT cells were treated with the EUFOC to observe the expression of inflammatory mediators, such as COX-2 and iNOS. As shown in Figure 6, the protein expression of COX-2 and iNOS increased when HaCaT cells were treated with LPS, and the increased protein expression tended to decrease in a concentration-dependent manner after EUFOC treatment. These findings confirmed that the EUFOC inhibited the expression of the inflammatory mediators COX-2 and iNOS in HaCaT cells, indicating its potential as an anti-inflammatory agent.
tial as an anti-inflammatory agent.
In addition to immunoglobulins, which trigger inflammatory reactions in response to external factors, macrophages are also important for immunity [56]. Inflammatory reactions are triggered by LPS, an endotoxin, and anti-inflammatory experiments are conducted at the cellular stage [57]. RAW 264.7 cells were treated with 10 ng/mL of LPS to induce an inflammatory response and increase the expression of TNF-α and iNOS, which are inflammatory mediators. Next, the cells were treated with the EUFOC to confirm the degree of cytokine inhibition [57].
As indicated in Figure 6, the cells were treated with the EUFOC (0.1, 0.3, and 0.5% (v/v), and RNA was extracted from the treated cells. The mRNA levels of COX-2 and iNOS were compared using RT-PCR ( Figure 6). COX-2 expression significantly increased in the LPS-treated group, and no suppression was observed in the EUFOC-treated groups (Figure 6a). Similarly, LPS treatment significantly increased iNOS mRNA expression. EUFOC treatment significantly reduced iNOS expression at relatively high concentrations (0.5%), but not at lower concentrations (Figure 6b). Although the EUFOC could not regulate the expression of COX-2, it exerted anti-inflammatory effects by regulating the expression of iNOS, which indicated its role in the inflammatory response.
Stimulus-induced iNOS produces a large amount of NO over a long period, and the generated NO is characterized by cytotoxicity to surrounding tissues and its ability to activate guanyl cyclase. Because NO is very small, reactive, and electrically neutral, it immediately spreads from the site of synthesis in all directions, intensifying inflammation by promoting the biosynthesis of inflammatory mediators as well as promoting inflammatory reactions, such as vascular permeability and edema [58,59].

HA Promotes Intracellular Synthesis
According to previous studies, some natural products are involved in the synthesis and maintenance of HA through various pathways [60]. HA is a glycosaminoglycan composed of D-glucuronic acid and N-acetyl-D-glucosamine, which cannot easily enter the cells due to its relatively large molecular weight [61]. In general, HA production naturally decreases with age, resulting in a decrease in water homeostasis in cells, and it may also In addition to immunoglobulins, which trigger inflammatory reactions in response to external factors, macrophages are also important for immunity [56]. Inflammatory reactions are triggered by LPS, an endotoxin, and anti-inflammatory experiments are conducted at the cellular stage [57]. RAW 264.7 cells were treated with 10 ng/mL of LPS to induce an inflammatory response and increase the expression of TNF-α and iNOS, which are inflammatory mediators. Next, the cells were treated with the EUFOC to confirm the degree of cytokine inhibition [57].
As indicated in Figure 6, the cells were treated with the EUFOC (0.1, 0.3, and 0.5% (v/v), and RNA was extracted from the treated cells. The mRNA levels of COX-2 and iNOS were compared using RT-PCR ( Figure 6). COX-2 expression significantly increased in the LPS-treated group, and no suppression was observed in the EUFOC-treated groups (Figure 6a). Similarly, LPS treatment significantly increased iNOS mRNA expression. EUFOC treatment significantly reduced iNOS expression at relatively high concentrations (0.5%), but not at lower concentrations (Figure 6b). Although the EUFOC could not regulate the expression of COX-2, it exerted anti-inflammatory effects by regulating the expression of iNOS, which indicated its role in the inflammatory response.
Stimulus-induced iNOS produces a large amount of NO over a long period, and the generated NO is characterized by cytotoxicity to surrounding tissues and its ability to activate guanyl cyclase. Because NO is very small, reactive, and electrically neutral, it immediately spreads from the site of synthesis in all directions, intensifying inflammation by promoting the biosynthesis of inflammatory mediators as well as promoting inflammatory reactions, such as vascular permeability and edema [58,59].

HA Promotes Intracellular Synthesis
According to previous studies, some natural products are involved in the synthesis and maintenance of HA through various pathways [60]. HA is a glycosaminoglycan composed of D-glucuronic acid and N-acetyl-D-glucosamine, which cannot easily enter the cells due to its relatively large molecular weight [61]. In general, HA production naturally decreases with age, resulting in a decrease in water homeostasis in cells, and it may also be reduced by external physical and chemical factors. In animal cells, different genes, such as HAS1, HAS2, and HAS3, are involved in HA synthesis, and the presence of hyaluronidase, an enzyme that decomposes HA, affects the water content of individual cells [62].
The enzymes that produce HA in animal cells are HA synthases (HAS), and HAS1, HAS2, and HAS3 have been identified to date [63,64]. HAS2 is the main gene involved in HA synthesis in animal fibroblasts and keratinocytes [65]. To confirm the effect of the EUFOC on HAS2 gene expression in keratinocytes, the cells were treated with 0.1, 0.3, or 0.5% EUFOC in 70% ethanol. After 24 h, RT-PCR was performed, and gene bands were confirmed by electrophoresis. HAS2 expression was confirmed at all the concentrations tested. In the dermal layer of animals, HA synthesis is controlled by growth factors, such as transforming growth factor β, platelet-derived growth factor BB, fibroblast growth factor, and epidermal growth factor (EGF), and it is also known to be affected by female growth factors [63,66]. EGF induces HA synthesis by activating signal transducers and activators of transcription (STAT)-3 and by inducing HAS2 gene expression [66]. Through this experiment, we confirmed that HAS2 expression in keratinocytes treated with EUFOC increased at all the test concentrations.
EUFOC treatment markedly increased the mRNA expression of HAS2 ( Figure 7a) and HAS3 (Figure 7b) in HaCaT cells in a dose-dependent manner. These results suggest that EUFOC treatment can increase HA production and may play a role in the maintenance of water molecules in the dermal layer. Therefore, we measured changes in the HA contents in HcCaT cells after treatment with the EUFOC. The EUFOC induced HA production at a level similar to that of the control group at all the concentrations, indicating that it can maintain water molecules in the dermal layer (Figure 7c). be reduced by external physical and chemical factors. In animal cells, different genes, such as HAS1, HAS2, and HAS3, are involved in HA synthesis, and the presence of hyaluronidase, an enzyme that decomposes HA, affects the water content of individual cells [62]. The enzymes that produce HA in animal cells are HA synthases (HAS), and HAS1, HAS2, and HAS3 have been identified to date [63,64]. HAS2 is the main gene involved in HA synthesis in animal fibroblasts and keratinocytes [65]. To confirm the effect of the EUFOC on HAS2 gene expression in keratinocytes, the cells were treated with 0.1, 0.3, or 0.5% EUFOC in 70% ethanol. After 24 h, RT-PCR was performed, and gene bands were confirmed by electrophoresis. HAS2 expression was confirmed at all the concentrations tested. In the dermal layer of animals, HA synthesis is controlled by growth factors, such as transforming growth factor β, platelet-derived growth factor BB, fibroblast growth factor, and epidermal growth factor (EGF), and it is also known to be affected by female growth factors [63,66]. EGF induces HA synthesis by activating signal transducers and activators of transcription (STAT)-3 and by inducing HAS2 gene expression [66]. Through this experiment, we confirmed that HAS2 expression in keratinocytes treated with EUFOC increased at all the test concentrations.
EUFOC treatment markedly increased the mRNA expression of HAS2 ( Figure 7a) and HAS3 (Figure 7b) in HaCaT cells in a dose-dependent manner. These results suggest that EUFOC treatment can increase HA production and may play a role in the maintenance of water molecules in the dermal layer. Therefore, we measured changes in the HA contents in HcCaT cells after treatment with the EUFOC. The EUFOC induced HA production at a level similar to that of the control group at all the concentrations, indicating that it can maintain water molecules in the dermal layer (Figure 7c). This study developed an EUFOC for the treatment and prevention of dermal inflammation. MTT and crystal red staining assays were conducted to evaluate the effect of the EUFOC on cell viability, and the results confirmed that no cytotoxicity occurred up to a concentration of 1.0%. In addition, cell viability was evaluated in 293T (human kidney) cells and HaCaT (human keratinocyte) cells, and cytotoxicity was not observed at 0.1, 0.3 and 0.5%. The evaluation of the inhibitory activity of the EUFOC on NO production revealed that it inhibited NO production at a concentration that did not inhibit cell viability. These results showed that LPS-induced NO production was reduced by the EUFOC, suggesting that the EUFOC is an effective anti-inflammatory agent. In order to confirm the effect of the EUFOC on the secretion of pro-inflammatory cytokines in LPS-treated HaCaT cells, the changes in the amounts of IL-1β and IL-6 induced by EUFOC treatment were measured. The LPS-induced increase in the secretion of all the cytokines was suppressed by the EUFOC in a dose-dependent manner. Moreover, the EUFOC effectively inhibited LPS-induced iNOS mRNA expression but not COX-2 expression in HaCaT cells. Although the EUFOC could not regulate the expression of COX-2, it exerted anti-inflammatory effects by regulating the expression of iNOS, suggesting its role in the inflammatory re- This study developed an EUFOC for the treatment and prevention of dermal inflammation. MTT and crystal red staining assays were conducted to evaluate the effect of the EUFOC on cell viability, and the results confirmed that no cytotoxicity occurred up to a concentration of 1.0%. In addition, cell viability was evaluated in 293T (human kidney) cells and HaCaT (human keratinocyte) cells, and cytotoxicity was not observed at 0.1, 0.3 and 0.5%. The evaluation of the inhibitory activity of the EUFOC on NO production revealed that it inhibited NO production at a concentration that did not inhibit cell viability. These results showed that LPS-induced NO production was reduced by the EUFOC, suggesting that the EUFOC is an effective anti-inflammatory agent. In order to confirm the effect of the EUFOC on the secretion of pro-inflammatory cytokines in LPS-treated HaCaT cells, the changes in the amounts of IL-1β and IL-6 induced by EUFOC treatment were measured. The LPS-induced increase in the secretion of all the cytokines was suppressed by the EU-FOC in a dose-dependent manner. Moreover, the EUFOC effectively inhibited LPS-induced iNOS mRNA expression but not COX-2 expression in HaCaT cells. Although the EUFOC could not regulate the expression of COX-2, it exerted anti-inflammatory effects by regulating the expression of iNOS, suggesting its role in the inflammatory response. In addition, the EUFOC enhanced HA production at a level similar to that of the control group at all the test concentrations, indicating its potential to effectively maintain water molecules in the dermal layers. With regard to its safety on keratinocytes, our findings showed that there was no clear cytotoxicity up to 1.0%, and its effectiveness for HA synthesis was confirmed at different concentrations. As a possible limitation, these effects were observed after a relatively long incubation time with LPS, and we should verify these functional effects in a time-dependent manner. Furthermore, these functional effects can induce molecular remodeling such as the expression of all HAS families in various time courses; however, we must investigate this hypothesis in further studies with focused materials. The EUFOC, medicinal plant oil-based combined materials, showed significant ameliorative effects on LPS-induced inflammatory damages and provided evidence of tissue regeneration via transcriptional activation of hyaluronic acid synthesis. In conclusion, these results of the present study showed that the EUFOC, a natural oil manufactured using natural plant materials, is a potential medicinal and healthcare product with excellent anti-inflammatory and antioxidant properties.

Composition of EUFOC
Essential oil components were extracted from the natural plant ingredients used in this study with an essential oil extractor (EssenLab-plus, Hanil Lab Tech, Yangju, Gyunggi-do, Republic of Korea). The essential oil obtained by mixing the essential oil extracts was named the EUFOC, and its composition is shown in Table 1. Unsaturated fatty acid content of the samples was 92.76%.

Cell Culture
All the cell lines were purchased from ATCC (Manassas, VA, USA) and cultured according to the manufacturer's instructions. The cultured cells were washed thrice with cold phosphate-buffered saline (PBS) (0.1 M, pH 7.4). The cells were completely detached from the bottom of the culture dish using Trypsin-EDTA [57]. About 10 µL of cell suspension was added to 10 mL of culture medium supplemented with 10% FBS. The mixture was mixed with the same amount of trypan blue, and then about 10 µL was taken to count the number of cells using a hemocytometer at 200× magnification [57,67]. The number of cells was calculated by repeating the experiment 10 times and then calculating the average value. Each cell was cultured under 5% CO 2 and full humidity at 37 • C. HaCaT cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin (PS, Gibco) under 5% CO 2 at 37 • C. All cells were used in 4 to 5 passages from the first stock vial and then discarded.

Analysis of DPPH-Scavenging Ability
The antioxidant activity of the EUFOC was confirmed using a commercial DPPH freeradical-scavenging activity measurement method. First, DPPH was dissolved in ethanol to prepare a 0.1 mM DPPH solution, and the EUFOC was mixed with 0.1 mM DPPH solution at a ratio of 1:1 in a 96-well plate. The mixture of the sample and the DPPH solution was incubated at 37 • C for 30 min, and then the absorbance was measured at 517 nm. The DPPH-scavenging ability was calculated using the formula below: DPPH free radical scavenging activity (%) = {1 − (A − B/C)} × 100 A: Absorbance after reacting sample with DPPH; B: Absorbance after reacting sample and ethanol; C: Absorbance after reacting ethanol and DPPH (blank).

Analysis of SO-Scavenging Ability
The SO-scavenging ability of the EUFOC was confirmed using a commercial superoxide dismutase determination kit (Sigma-Aldrich, St. Louis, MO, USA). The samples were mixed with the reaction solution in the kit, placed in a 96-well plate, and incubated at 37 • C for 20 min. After measuring the absorbance at 450 nm, the SO-scavenging ability was calculated using the following formula:
The MTT solution was added after 24 h to confirm cytotoxicity. The sample was removed after 2 h and 150 µL of DMSO was added to each well to dissolve the formed formazan, followed by the measurement of absorbance at 540 nm.
MTT was used to confirm the cytotoxicity of the EUFOC in keratinocytes. HaCaT cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA) and 1% penicillin-streptomycin (PS, Gibco, Grand Island, NY, USA) under 5% CO 2 at 37 • C. The cells were seeded in 96-well plates at a density of 2 × 10 4 /well and then stabilized for 24 h. The EUFOC, diluted in serum-free medium, was further diluted with ethanol (70%) to prepare different concentrations of the sample. After 24 h, the medium was removed from the cells and 20 µL of MTT (5 mg/mL) was added, followed by incubation in a cell incubator (37 • C, 5% CO 2 ) for 2 h. Next, MTT was removed, and 100 µL of DMSO was added. After ensuring that the crystals were dissolved in the stirrer, the absorbance was measured at 540 nm.

Identification of Inflammatory Genes Using RT-PCR
Macrophages respond to pathogens and cause an inflammatory response, producing pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β. In addition, inflammatory factors such as NO produced by iNOS and PGE2 produced by COX-2 are also secreted [68,69].

Inhibition of NO Production
NO production was measured using the Griess method. RAW 264.7 cells, pre-cultured in DMEM growth medium supplemented with 10% FBS, were seeded in 24-well tissue culture plates at a concentration of 5 × 10 4 cells/well and cultured for 1 day in a 5% CO 2 incubator. After removing the medium and starving the cells in a serum-free medium for 12 h, the EUFOC was added at the required concentration, followed by incubation for 30 min. Next, 10 ng/mL LPS (Sigma-Aldrich, St. Louis, MO, USA) was added, followed by culturing for 18 h. After incubation, the supernatant was collected and transferred to 96-well plates; Griess reagent (Sigma-Aldrich, St. Louis, MO, USA) was added, followed by incubation at room temperature for 15 min. The absorbance was measured at 540 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

ELISA Test
The cells were seeded in 6-well plates at a concentration of 2 × 10 5 /mL. After 24 h, the cells were washed twice with serum-free DMEM. The cells were re-cultured in serumfree DMEM containing 1% DMSO. After 24 h, 350 µL of medium was removed, and after another 24 h, the same amount was removed. After centrifugation at 15,000× g for 5 min, the supernatant was removed and stored at −20 • C until ELISA. An HA-ELISA kit (Echelon, Salt Lake City, UT, USA) was used to measure the HA produced by the EUFOC in keratinocytes. The 48 h treatment increased HA production in all the samples, compared with the 24 h treatment. Therefore, the EUFOC, at an appropriate concentration, can enhance HA production in keratinocytes. Further studies are necessary to explore the mechanisms of action and determine the optimal concentration of the EUFOC. All-trans retinoic acid was used as a positive control.

Quantative PCR
Keratinocytes were seeded into 6-well plates at 2 × 10 5 /well and then stabilized for 24 h. The medium was replaced with serum-free DMEM and the EUFOC was diluted with 70% ethanol. The final concentration of DMSO was adjusted to 0.1% in all the experimental groups, and 24 h after sample treatment, RNA was extracted using easy-BLUE™ Total RNA Extraction Kit (iNtRON). After measuring the RNA purity and concentration, 1 µg of RNA extracted from each 6-well plate was used to synthesize cD-NAs using a Power cDNA Synthesis Kit (iNtRON). DNA was amplified using SYBR Green Master Mix (Bio-Rad, CA, USA) in an ABI Prism 7900HT sequence-detection system (Applied Biosystems, Lennik, Belgium). Primers to HAS-2 (CTGGGACGAAGTGTGGAT-TATG and GATGAGGCTGGGTCAAGCAT) and HAS-3 (GCCCTCGGCGATTCG and TG-GATCCAGCACAGTGTCAGA) were used at 300 nM [71]. Results were expressed relative to the number of beta-actin transcripts used as an internal control.

Promotion of HA Production
We evaluated the ability of the EUFOC to promote HA production in HaCaT cells using the ELISA method. Different concentrations of the EUFOC (0.1%, 0.3%, and 0.5%) were prepared with the cell medium. HaCaT cells pre-cultured in a DMEM growth medium supplemented with 10% FBS were added to 96-well plates at a concentration of 2 × 10 4 cells/well and cultured in a 5% CO 2 incubator for 24 h. The medium was removed and the EUFOC diluted in serum-free medium was added, followed by incubation for 24 h. After incubation, the supernatant was collected and subjected to ELISA using the HA-ELISA kit (DHYAL0, R&D systems, Minneapolis, MN, USA).

Statistical Analysis
The experiment conducted in this study was repeated seven times or more in total, and the statistical significance test between the data of the experimental groups was performed using paired samples t-tests (GraphPad Prism 5). The results are expressed as mean ± standard deviation. Statistical significance is indicated as * p < 0.05, ** p value ≤ 0.01.

Data Availability Statement:
The data presented in this study are available.