Skin anti‐inflammatory activity of rose petal extract (Rosa gallica) through reduction of MAPK signaling pathway

Abstract The aim of this study was to investigate the skin anti‐inflammatory activity of rose petal extract (RPE) and the mechanisms underlying this phenomenon. Recently, flowers have been considered as dietary resources owing to their biological activities, such as inhibition of nephritis and hemorrhoids. The Rosa plant exerts various biological functions, including antioxidant and anti‐microbiological activities. Herein, we confirmed the skin anti‐inflammatory activity of RPE upon solar UV (sUV) exposure. RPE reduced sUV‐induced COX‐2 expression as well as expressions of several cytokines. Activation of MKK4‐JNK, MEK‐ERK, and MKK3‐p38 signaling pathways, which are associated with cytokine production, was also attenuated by RPE treatment. We hypothesized these RPE‐induced changes are because of its antioxidant activity, because RPE displayed drastic radical scavenging and oxygen radical absorbance capacity (ORAC). Furthermore, high anthocyanins, polyphenols, and flavonoids contents were found in RPE. Hence, these results indicated the skin anti‐inflammatory activity of RPE is because of antioxidant activity.

Here, we demonstrated the potential of rose petal extract (RPE) on sUV-induced skin inflammation via suppression of MAPK activation. Our results showed that RPE exerts strong antioxidant effects through cytokine suppression in epidermal cell lines exposed to sUV.

| Sample preparation (rose petals extract)
Rose petals (10 g) were extracted by 70% (v/v) ethanol at 70°C for 3 hr. The extracted solution was then filtered (Whatman paper).
Ethanol was subsequently evaporated, and the product was freeze-dried.

| Cell culture
The JB6 P+ cell line was kindly provided to us by Dr. Zigang Dong's laboratory in Hormel institute. Cells were cultured at 37°C and humidified atmosphere of 5% CO 2 in DMEM supplemented with 10% FBS with 0.1% penicillin/streptomycin/neomycin.

| Solar UV irradiation system
The solar UV irradiation system consists of UVA and UVB lamps.
The UVA-340 lamps were purchased from Q-Lab Corporation (Cleveland, OH); these lamps provide optimal simulated sunlight in the critical short wavelength region from 365 to 295 nm, with a peak emission of 340 nm. The ratio of UVA to UVB was measured by a UV meter at 94.5% and 5.5%, respectively.

| Cell viability
Cell viability was measured using the Cell Titer 96 Aqueous Solution (Promega). In brief, cells were culture in 96-well plates and treated with RPE for 24 hr. The 100 mg/ml of RPE stock sample was treated to the cultured media as indicated concentration (50-1,000 μg/ml).
Following the incubation period, 20 μl MTS solution was added, and cells were further incubated for 1 hr. Cell viability was measured with absorbance at 490 nm using a microplate reader (Infinite ® 2000 PRO, Tecan, Switzerland).

| Total flavonoids contents
Total flavonoid content of RPE was measured using the aluminum chloride method (Jia, Tang, & Wu, 1999) with modifications, using catechin. We used RPEs with various doses of 12.5-1,000 μg/ml.
Each sample of RPE (100 μl) was added to 500 μl DW, followed by the addition of 30 μl NaNO 2 . After 6 min, 60 μl AlCl 3 was added, and the mixture was incubated for 5 min. This was followed by the addition of 200 μl of 1 M NaOH, and the final mixture was made up to 1 ml with distilled water. The solution was mixed well again and centrifuged at 1,500 g and 4°C for 5 min. Absorbance was measured for 200 μl supernatant at 510 nm using a multiwell plate reader (Infinite ® 2000 PRO). All experiments were performed in triplicates.

| Total polyphenol content
Total polyphenol content of RPE was determined according to the Folin-Ciocalteu method using gallic acid. We used RPEs with various doses of 12.5-1,000 μg/ml. Each sample of RPE (10 μl) was mixed with 500 μl DW and 50 μl Folin-Ciocalteu's reagent. This was followed by the addition of 150 μl Na 2 CO 3 (20%, W/V), and total reaction volume was made up to 1 ml with distilled water. The solution was mixed again and incubated at room temperature for 2 hr.
Absorbance was measured at 765 nm using multi-plate readers (Infinite ® 2000 PRO). All experiments were performed in triplicates.

| Total anthocyanin content
Total anthocyanin content in RPE was measured by the pH differential method. Concentration of phenolic extracts of RPE (250, 500 μg/ ml) in 0.025 M potassium chloride buffer (pH 1.0) and 0.4 M sodium acetate buffer (pH 4.5) was determined at 510 and 700 nm, respectively, following incubation at 23°C for 15 min. The anthocyanin content was expressed in mg cyanidin-3-glucoside (CGE)/100 L RPE.

| Western blot
Following sample treatment and sUV irradiation (26 kJ/cm 2 ), proteins were collected in 1× lysis buffer (Cell signaling Biotechnology, Beverly, MA). Protein concentration was determined with a dye-binding protein assay kit (Bio-Rad Laboratories, Hercules, CA) according to manufacturer's instructions. Equal concentrations of cellular proteins were separated on polyacrylamide gels (Bio-Rad Laboratories), and were transferred to Immobilon P membranes (Millipore, Billerica, MA). Membranes were blocked with 5% fat-free milk for 1 hr, and were incubated with specific primary antibodies at 4°C overnight. Proteins were hybridized with HRP-conjugated secondary antibodies, and were visualized using the chemiluminescence detection kit (GE Healthcare, Pittsburgh, PA).

| Cytokine array
In order to confirm changes in cytokines, we used the Mouse Cytokine Array Panel A (R&D systems™). Experiments were carried out according to the manufacturer's instruction. Briefly, cells were incubated at 37°C in humidified atmosphere of 5% CO 2 ; cells were cultured in DMEM supplemented with 10% FBS and 0.1% penicillin/streptomycin/neomycin, and were treated with 400 and 800 μg/ml of RPE prior to sUV exposure. Following sUV irradiation, the media were collected, and cytokine production was determined.

| ABTS radical scavenging assay
The ABTS radical scavenging assay was performed as previously The mixture was then quickly cooled to produce the ABTS radical solution. RPE (20 μl) and vitamin C (positive control) were added to 980 μl ABTS radical solution, and the solution was allowed to react for 10 min at 37°C. Absorbance was estimated at 734 nm using multimode microplate readers (Infinite ® 2000 pro).

| ORAC assay
The ORAC (oxygen radical absorbance capacity) assay was carried out using KOMABIOTECH (Seoul, Korea) following manufacturer's instructions. First, ORAC was estimated in fluorescence units.
Experiments were performed in triplicate.

| Statistical analysis
All experiments were performed at least three times. Data are expressed as the mean ± SD. Student's t tests were used for single statistical comparisons. p < 0.05 was used to denote statistical significance.

| Rose petal extract reduces sUV-induced cytokine production through c-Jun inhibition
Chronic and excessive UV exposure causes skin deterioration; sUV over-exposed skin can result in drastic clinical changes (Lopes & Mcmahon, 2016) such as skin erythema. Sunburn (erythema) is a conspicuous result of UV exposure (Matsumura & Ananthaswamy, 2004), and is accompanied by intracellular changes such as DNA damage and cytokine production (Hruza & Pentland, 1993;Lopes & Mcmahon, 2016). Pentland et al. reported that prostaglandins (PGs) are produced from arachidonic acid in UV-irradiated human skin (Hruza & Pentland, 1993).

Therefore, we investigated the effect of RPE on sUV-induced
PGs through expression of COX-2 in the JB6 P+ cell line. RPE was obtained via the general extraction method, as shown in Figure 1a. The extract did not induce cell cytotoxicity at concentrations of ≤1,000 μg/ml in the JB6 P+ cell line (Figure 1b). Collectively, our results demonstrated that RPE inhibits sUVinduced inflammatory mediator production.

| Rose petal extract suppresses sUV-induced MAPKK-MAPK signaling pathway
Previous studies have associated the MAPK signaling pathway with sUV-related skin inflammation (Hipskind & Bilbe, 1998;Reddy & Mossman, 2002). In order to confirm whether RPE affects sUV-induced MAPK activation, we analyzed the effect of RPE on sUV-induced MAPK signaling pathway via Western blot analysis. As shown in Figure 3, sUV irradiation significantly enhanced MKK4-JNK, MEK-ERK, and MKK3-p38 signaling pathways in JB6 P+ cells. In addition, RPE pre-treatment attenuated MAPK signaling in a dose-dependent manner. Therefore, it was assumed that the inhibitory activity of RPE on inflammatory mediator production is mediated by suppression of the MAPK signaling pathway ( Figure 2).

| Rose petal extract display antioxidant activity
Oxidative stress is critical for chronic human diseases such as cancer (Valko, Rhodes, Moncol, Izakovic, & Mazur, 2006). In 2006, a study reported that treatment with the ROS scavenger N-acetyl cysteine (NAC) suppresses EGFR transactivation. Because EGFR is a transmembrane protein that stimulates several inflammatory signaling pathways F I G U R E 2 Rose petal extract (RPE) reduces sUV-induced COX-2 and cytokine production through c-Jun inhibition COX-2 expression (a) and c-Jun phosphorylation (b) was analyzed by Western blot. The band intensity was quantified and represented as the percent of sUVtreated control. (c) Effect of RPE on cytokine production in the JB6 P+ cell line was evaluated using Mouse Cytokine Array Panel A (N = 3) F I G U R E 3 Inhibitory effect of RPE on sUV-induced MAPKK and MAPK phosphorylation in the JB6 P+ cell line. Phosphorylation levels of MKK4-JNK (a), MEK-ERK (b), and MKK3-p38 (c) were estimated by Western blot analysis using specific antibodies (N = 3). RPE, rose petal extract including Ras/MAPK, we evaluated the effect of RPE on radical scavenging activity. As shown in Figures 4a and b, RPE exhibited significant ABTS and DPPH radical scavenging activity. In the case of the ABTS radical scavenging assay, nearly 80% of ABTS radicals were inhibited by 50 μg/ml RPE, which is a relative low dose (Figure 4a). To obtain more reliable antioxidant activity measurements of RPE, we tested the ORAC of RPE. As seen in Figure 4c, RPE presented dose-dependent oxygen radical capacity.

| Rose petal extract contains high amount of anthocyanins/flavonoid/polyphenols
As it is well-known that rose petals possess various polyphenols (Knapp et al., 1998;Kumar et al., 2008;Oka et al., 1998;Schieber et al., 2005), we determined the content of polyphenol, flavonoids, and anthocyanins in RPE. Polyphenol content was assayed using Folin-Ciocalteu's reagent. As shown in Figure 5a, dose-dependent increase in polyphenols was observed with increased concentration of RPE. In particular, concentration of flavonoid was high in RPE (Figure 5b).
F I G U R E 4 Antioxidant activity of RPE. ABTS (a) and DPPH (b) radical scavenging activity was measured. Vitamin C was used as the positive control. (c) Oxygen radical absorbance capacity (ORAC) assay was performed as described in Materials and methods. Data are representative of three independent experiments. The asterisks (**) denote statistical significance (p < 0.001) as compared with controls. RPE, rose petal extract  (Caliskan et al., 2017;Knapp et al., 1998;Kumar et al., 2008;Oka et al., 1998;Schieber et al., 2005). Flowers are good sources of beneficial phenolic compounds including anthocyanins (Navarro-Gonzalez et al., 2015). Anthocyanin is a naturally occurring pigment in fruits and flowers. Several previous reports have illustrated the beneficial activities of anthocyanin (Kwon et al., 2009;Matsumoto et al., 2001;Meiers et al., 2001).
We investigated the skin anti-inflammatory activity of RPE in the current study. We adopted the excessive sUV exposure-related skin inflammation model, as over-exposure of skin to UV is a physiological stimulator of skin inflammation (Lopes & Mcmahon, 2016). The JB6 P+ cell line is an optimized model for studying skin inflammation owing to its sensitivity to inflammatory enhancers such as UV, EGFR, and 12-Otetradecanoylphorbol-13-acetate (TPA) (Byum et al., 2013;Chen et al., 2001). We found that COX-2 expression induced by sUV irradiation is dose-dependently attenuated by RPE treatment. In addition, as per cytokine array results, production of several cytokines was reduced by RPE pre-treatment ( Figure 2). Furthermore, cellular signaling pathways associated with production of COX-2 and other cytokines were downregulated by RPE pre-treatment. Our results found that inhibition of MKK4-JNK, MEK-ERK, and MKK3-p38 is an underlying mechanism of RPE-mediated suppression of sUV-induced cytokine production.
The antioxidant activity of the Rosa species is well-reported in previous literatures (Kumar et al., 2009;Masek et al., 2017;Navarro-Gonzalez et al., 2015). Similar to previous studies, we found that RPE possesses strong antioxidant effects ( Figure 4). As demonstrated by the ABTS assay (Figure 4b), 100 μg/ml RPE exhibited over 90% ABTS radical scavenging activity; this was comparable to that of 10 μg/ml vitamin C (Figure 4a). As we described in the introduction section, aberrant ROS production can accelerate DNA damage and activate inflammatory signaling pathways (Chen et al., 2006;Tormos et al., 2013). Therefore, we hypothesized that suppression of MAPK signaling pathway is mediated by antioxidant activities of RPE.
While the bioactive chemical in RPE is yet to be identified, the present study suggests that 70% ethanol extract from rose petals exhibit skin anti-inflammatory and antioxidant activities via MAPK inactivation This is the first study that confirmed anti-skin inflammatory effects of RPE. Especially, we found that RPE possess anthocyanins which are well-known anti-oxidative chemicals among of polyphenols. Hence, we assumed that anthocyanins in RPE showed skin antiinflammatory activity by their anti-oxidative effect ( Figure 6).

| CON CLUS ION
We acquired ethanol extracts from the petals of R. gallica. RPE showed anti-skin inflammatory effects through suppression sUVinduced MAPK activation. Based on these findings, we have showcased the potential of RPE in novel applications.

CO N FLI C T O F I NTE R E S T
The authors declared that they have no conflict of interest.