Rose Petal Extract (Rosa Gallica) Exerts Skin Whitening And Anti-Skin Wrinkle Effects
Mar 26, 2022
Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com
Young-Ran Song,1,* Won-Chul Lim,1,* Ahram Han,1 Myung-hee Lee,1 Eun Ju Shin,1
Kwang-Min Lee,1,2 Tae-Gyu Nam,1 and Tae-Gyu Lim1,3
ABSTRACT
We sought to investigate the effect of extracts from Rosa gallica petals (RPE) on skin whitening and anti-wrinkle activity. Tyrosinase activity was attenuated by RPE treatment, concomitant with the reduction of melanin accumulation in human B16F10 melanoma. Treatment of the facial skin of volunteers in a clinical trial with an RPE-containing formulation enhanced skin brightness (L* value) significantly. The underlying mechanism responsible was determined to be associated with mitogen-activated protein kinase (MAPK) activation. In addition, RPE exhibited anti-wrinkle formation activity of human dermal fibroblasts by suppressing matrix metalloproteinase (MMP)-1 level. In vivo study, RPE also inhibited solar ultraviolet-stimulated MMP-1 level by c-Jun regulation. Overall, our findings indicate that RPE evokes skin whitening and anti-wrinkle formation activity by regulating intracellular signaling, supporting its utility as an ingredient for skin whitening and anti-wrinkle cosmetic products.
KEYWORDS: melanin, MMP-1, Rosa gallica, skin whitening, wrinkle formation
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INTRODUCTION
Although skin aging has a genetically determined baseline rate, it is a complex and multifactorial process that can be accelerated by various environmental factors.1 Normal skin aging is a naturally occurring chronological process over time that is exacerbated by exogenous aging, triggered by external stresses owing to sunlight, atmospheric pollutants, and extreme temperatures.2 Of these, solar ultraviolet (SUV) exposure (in particular, ultraviolet B [UVB]) is a primary cause of photoaging of the skin, marked by hyperpigmentation (melanin overproduction) and wrinkle formation.3 SUV radiation stimulates the generation of reactive oxygen species (ROS)/reactive nitrogen species and DNA damage, leading to various aging-related processes in skin cells such as inflammation, remodeling of the extracellular matrix (ECM), cell death, and hyperpigmentation.4 Melanin (black or brown pigmentation) protects the skin from UV-induced damage.5 However, dysregulated or excessive melanin production in the skin can result in hyperpigmentation disorders including chloasma, freckles, and senile lentigines.6 Arbutin and kojic acid, which have been developed to treat these diseases, are known to pose a risk for intestinal cancer or liver cancer, respectively.7 For this reason, there has been interest in the utilization of natural materials that are excellent in inhibiting melanin production without serious side effects. Melanogenesis is stimulated by tyrosinase, which plays a crucial role in the initial rate-determining step of the melanin biosynthetic pathway, and this enzyme is stabilized and activated by TRP-1 and TRP2.8 These melanogenic proteins transcription is modulated by microphthalmia-associated transcription factor (MITF). UV exposure activates the MITF promoter and induces tyrosinase gene expression, thereby resulting in the accumulation of melanin in melanocytes. In addition, mitogen-activated protein kinases (MAPKs), consisting of the extracellular signal-regulated kinase (ERK), c-Jun-N-terminal kinase ( JNK), and p38, leads to transcriptional control of melanogenesis.8 In particular, ERK and JNK are known to negatively regulate MITF expression,9 whereas p38 MAPK induces the proteasomal decomposition of melanogenic enzymes. Phosphorylated p38 MAPK, in turn, activates the expression of MITF and melanogenic enzymes.10
ECM proteins such as collagen, elastin, and proteoglycans are present in the dermal matrix, which gives strength and resiliency to the skin.11 During the skin aging process, dermal matrix alterations including changes to collagen proportions occur, ultimately leading to wrinkling formation. Furthermore, UV irradiation also triggers collagenase decomposition, during which the oxidative stress generated facilitates proteolytic matrix metalloproteinase (MMPs) expression that reduces collagen synthesis and accelerates collagen degradation.2 Oxidative stress stimulates various photoaging-related biological pathways, as well as cytoplasmic kinases and MAPKs.4 MAPKs simulate activator protein 1 (AP-1; c-Fos/c-Jun), a transcriptional factor that further upregulates the level of MMPs.12 These proteins are primarily responsible for collagen degradation.7 Of the MMPs, MMP-1 is known as an important enzyme involved in the breakdown of the ECM by decomposing type I collagen, a key component in the dermis.3 Thus, MMP-1 inhibition has been considered a viable strategy for anti-skin aging therapy.
The Rosa (R.) species is included in the family Rosaceae and the genus Rosa is one of the most widely distributed plants. These are important commercial crops used for gardening, decorations, and perfumes. As edible flowers, petals are also commonly used as ingredients in functional foods because of their color, wealthy flavor, and elevated nutritional value.13 Meanwhile, roses have been utilized as herbal remedies for treating dysmenorrhea, diarrhea, and nephritis, improving blood circulation, managing pain, and hemostasis.13,14 The physiological properties observed may be attributable to their abundance of phytochemicals including phenolic compounds, flavonoids, and anthocyanins.15 Indeed, some studies have demonstrated biological functionalities containing strong anti-oxidation, anti-microbiology, anti-inflammation, and anti-allergy.15–17 Recently, we reported that rose petal ethanol extract elicits skin anti-inflammatory potential.18 In addition, optimal extraction conditions of rose petals having an anti-aging effect on the skin have been investigated for industrial applications.19 However, despite their wide availability, the potential anti-aging properties of rose petal extracts (RPEs) for skincare remain poorly understood. In this study, we assessed the potential applications of RPE in skincare and their molecular mechanisms responsible.
MATERIALS AND METHODS
Reagents
Rose petals were purchased from Turkey through GN Bio (Gyeonggi-do, Korea). MTS (CellTiter 96 Aqueous One Solution Cell Proliferation Assay) solution was purchased from Promega (Madison, WI, USA). Polyclonal anti-tyrosinase, anti-b-actin, anti-ERK, anti-MKK4, and anti-JNK were purchased from Santa Cruz Biotechnology (CA, USA). Monoclonal anti-phospho-MEK (Ser217/221), anti-MEK, anti-phospho-MKK4, anti-phospho-MKK3/6, anti-phosphoERK, anti-phospho-JNK, anti-MKK3, anti-phospho-p38, anti-p38, and anti-phospho-c-Jun were purchased from Cell Signaling Technology (Danvers, MA, USA). Monoclonal anti-MMP-1 and anti-type I collagen were obtained from R&D Systems (Minneapolis, MN, USA) and Ab cam (Cambridge, United Kingdom), respectively. Alpha melanocyte-stimulating hormone (a-MSH), mushroom tyrosinase, and l-3,4-dihydroxyphenylalanine (l-DOPA) were obtained from Sigma-Aldrich (St. Louis, MO, USA).
Preparation of RPE
Rose petals (10 g) were air-dried, ground, and mixed with 1000 mL of 70% (v/v) ethanol and extracted at 70C for 3 h using a soxhlet extractor. The product was then filtered through No. 2 filter paper (Whatman, Maidstone, United Kingdom). The solvent was continuously vacuum concentrated, and the residue was lyophilized using a freeze dryer.
In vitro mushroom tyrosinase activity assay
The enzymatic assay was carried out by measuring DOPA chrome formation as described previously.20 In brief, 40 lL of RPE sample (12.5, 25, 50, and 100 LG/mL) was diluted to 20 lL of PBS buffer. After adding 20 lL of mushroom tyrosinase (0.02 mg/mL), incubation was conducted for 30 min. Substrate l-DOPA was added to the mixture containing enzyme and then incubated for 15 min. The dopachrome product was then measured at 475 nm. Arbutin and vitamin C were used as the positive control.
Cistanche extract reduces tyrosinase's activity
Cell culture
Murine melanoma B16F10 cells and primary human dermal fibroblasts (HDFs) were obtained from the Korean Cell Line Bank (Seoul, Korea) and ScienCell Research Laboratories (Carlsbad, CA, USA), respectively. Both cells were maintained in Dulbecco’s modified Eagle medium (DMEM) (GIBCO Invitrogen, Auckland, New Zealand) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% streptomycin–penicillin (GIBCO Invitrogen) at 37C and 5% CO2 incubator.
Cell viability assay
The MTS assay was used to measure the RPE-induced proliferation of human melanoma B16F10 cells. In brief, the cells were seeded and cultured with DMEM containing 10% FBS and 1% antibiotics in 96-well plates. The cells were then serum-starved for 12 h and added with RPE samples (50–1000 LG/mL) for 24 h. Subsequently, 10% MTS solution including phenazine methosulfate was treated to the cells for 1 h. The degree of viable cells was measured at 490 nm.
Melanin production assay
Melanin formation in human melanoma B16F10 cells was assessed as reported previously.20 In brief, the cells (8 · 103 cells/well) were cultured in six-well plates in 2 mL DMEM. The next day, the cells were pretreated with RPE (100 and 200 lg/mL) for 1 h before a-MSH 100 nM was added and then further incubated for 72 h. Extracellular melanin was investigated at 495 nm.
Western blot analysis
Protein samples from B16F10 cells, HDFs, and human skin tissue were lysed with RIPA buffer [20 mM Tris-HCl (pH 7.5) 150 mM NaCl, 1 mM Na2EDTA 1 mM EGTA 1% NP-40 1% sodium deoxycholate 2.5 mM sodium pyrophosphate 1 mM bglycerophosphate 1 mM Na3VO4 1 lg/ml leupeptin] and quantified by Pierce BCA Protein Assay Kit. The proteins were separated by 10% sodium dodecyl sulfate acrylamide gels based on molecular weight and then transferred to nitrocellulose membranes. The membranes were blocked with 1 · bovine serum albumin solution for 1 h and incubated with the indicated primary antibody at 4C for 12 h. After reacting with horseradish peroxidase-conjugated secondary antibody for 1 h, the intensity of protein expression was visualized using a chemiluminescence reader (UVITEC Cambridge).
Subjects and treatments
The ability of RPE to improve facial skin whitening was assessed in a double-blind clinical trial in 10 women (>20 years old). The basic formulation was prepared using purified water, polyacrylic acid, 1,2-hexanediol, and sodium hydroxide. RPE was added at a concentration of 0.05% (v/v). Over a 6-week supplementation period, the subjects applied the RPE formula to one side of their face, and the other side of the face was treated once daily with only the base formulation as a control. During the trial period, the volunteers were banned from using other cosmetics.
Evaluation of skin condition
Skin condition was evaluated according to the standard operating protocol of the Korea Institute of Dermatological Sciences (Seoul, Korea) and was performed at 22C – 2C and 50% – 5% humidity. After washing the face for 30 min, the skin state of each subject was investigated using SkinColorCatch (Delfin Technologies, Kuopio, Finland) and comments were collected. The scores were determined by L* value (brightness) and measured immediately before the test and at 3 and 6 weeks later.
SUV irradiation
The SUV irradiation was conducted by combining ultraviolet A (UVA) and UVB lamps (Q-Lab Corporation, Cleveland, OH, USA); their lights emit at 340 nm, which mimics optimal sunlight emitted between 365 and 295 nm. The ratio of UVA to UVB produced by this lamp was 94.5% and 5.5%, respectively. HDFs and human skin-equivalent tissue in serum-free media were stimulated by SUV irradiation at 23 and 46 kJ/m2 using a UVB lamp, respectively.
Human skin sample preparation
Abdominal skin sections were obtained from a female Caucasian patient (Biopredic International, Saint Gregoire, France). In brief, the tissue samples were collected from the abdomen after plastic surgery in accordance with French Law L.1245-2 CSP. This study followed all principles set out in the Declaration of Helsinki. The skin tissue was incubated in 10% FBS proper medium provided from Biopredic International at 37C under 5% CO2 for 10 days in which the media was exchanged every 2 days. During the running period, the tissue was also exposed with an RPEsample 1 h before SUV irradiation of 46 kJ/m2 twice a day. At the end of the treatment, the tissue was used for immunohistochemical (IHC) analysis or immunoblot analysis.
IHC observation
IHC analysis was conducted by contract research agency ABION (Seoul, Korea). Skin tissue was soaked in 10% formaldehyde, dehydrated through a gradient of ethanol concentration, and embedded in paraffin wax. The endogenous peroxidase of the section was inactivated with 0.3% H2O2 for 15 min and then incubated with an anti-MMP-1 antibody (R&D Systems, Inc., USA, Canada) for an hour. After washing five times, the sections were incubated with secondary antibodies for 20 min. The sections were stained with DAB (3,30 -diaminobenzidine) detection reagent (K3468; DAKO, Glostrup, Denmark) and observed and photographed using a light microscope (BX40; Olympus, Tokyo, Japan).
Statistical analysis
All experiments were performed at least three times and data were expressed as mean – standard deviation. Statistical significance was evaluated using Student’s t-test in the SPSS program (Chicago, IL, USA) and significant differences were expressed as P < .05.
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RESULTS
RPE suppresses melanin production through tyrosinase inhibition
Tyrosinase is the only rate-limiting enzyme for melanogenesis present in human skin cells.8 In melanocytes, DOPA is produced from tyrosine by tyrosinase, which is further oxidized into dopaquinone in the melanin synthesis pathway.7 In this study, a mushroom tyrosinase assay was used to determine the anti-melanogenic activity of RPE. As given in Figure 1A, RPE elicited a significant dose-dependent inhibitory effect on tyrosinase activity and the inhibitory effect at 50–100 lM of RPE was higher than that of arbutin and vitamin C. These results were further confirmed by evaluating the effect of RPE on melanin secretion and tyrosinase expression in a-MSH-treated B16F10 melanoma cells. Keratinocytes secrete a-MSH to protect the skin against excessive UV exposure, which induces the activation of melanocytes to produce melanin.5 It was confirmed that a-MSH treatment-induced tyrosinase expression and melanin production in melanocytes (Fig. 1B, C). However, the tyrosinase expression in a-MSH-induced B16H10 cells was suppressed dose-dependently by RPE treatment (Fig. 1B), consistent with the results described previously. Moreover, RPE (100 and 200 LG/mL) significantly reduced melanin content that was increased in a-MSH-stimulated melanocytes (Fig. 1C). In addition, we confirmed that the extract did not induce cell cytotoxicity at concentrations of £1000 LG/mL in B16F10 cells (Fig. 1D).
RPE downregulates MITF expression by inducing MAPK activation in B16F10 melanoma cells
Various signaling pathways can mediate melanin pigment formation. Melanogenesis is controlled by MAPKs that are categorized into three subtypes consisting of ERK, JNK, and p38, and the upstream activators MEK, MKK4, or MKK3/6, by transcriptionally regulating the expression of MITF and tyrosinase.21 The phosphorylation of p38 MAPK regulates melanogenesis by stimulating the proteasomal degradation of melanogenic enzymes.6 ERK is known to downregulate MITF expression and melanogenesis through the phosphorylation of more than 160 proteins.10 In addition, JNK is involved in the proliferation and apoptosis of cancer cells and also controls melanogenesis by downregulating MITF expression.
To clarify the mechanism responsible for the biological activity of RPE, we investigated the effect of RPE on the MAPK signaling pathway through western blot analysis. As given in Figure 2, RPE treatment was also shown to phosphorylate the MEK-ERK, MKK4/7-JNK, and MKK3/ 6-p38 MAPK pathways in B16F10 cells. In addition, the phosphorylation of MAPKKs-MAPKs was significantly enhanced with increasing RPE concentrations. Furthermore, we observed that RPE reduced the expression of MITF. These results suggest that RPE inhibits MITF and tyrosinase expression by activating the MAPK signaling pathways.
RPE elicits a skin whitening effect on human skin
In this study, the effect of RPE on human skin brightness was further confirmed in a double-blind clinical trial. Each subject received the application of two different formulations, one with 0.05% (v/v) RPE and one without RPE on each half of their faces once daily for 6 weeks. Skin brightness as assessed by the L* value was evaluated using SkinColorCatch (Delfin Technologies). As given in Figure 3, the skin brightness in the presence of treatment with the 0.05% (v/v) RPE-containing formulation was significantly increased compared with the controls. RPE treatment for 3 weeks resulted in a significant increase in L* values, and the effect further increased as treatment time continued.
RPE prevents collagen degradation through MAPK-MMP-1 inhibition in HDFs
UV irradiation generates oxidative stress and thus triggers collagen decomposition by inducing the activation of MMPs.2 MMP-1 is an important factor in the photoaging process because it specifically cleaves type I collagen.3 The expression of MMP-1 is mediated by MAPKs such as ERK, p38 MAPK, and JNK, subsequently increasing the activation of the AP-1 transcription factor.22 As a subunit of AP-1, c-Jun is also induced in response to MAPKs activation stimulated by UV radiation.23 We sought to investigate whether the wrinkle-reducing effect of RPE was determined by the degree of suppression of MMP-1 and MMP-1-mediated upstream signaling transduction in SUV-irradiated HDFs. The expression level of type I collagen that reflects the quantity of collagen synthesized in the cells was also evaluated. As given in Figure 4A, B, SUV irradiation resulted in reduced type I collagen expression and increased MMP-1 expression in HDFs. However, the expression of type I collagen levels reduced by SUV irradiation was recovered dramatically by RPE treatment, which also significantly attenuated the increased levels of MMP-1 expression induced by SUV irradiation. In addition, the effect of RPE on SUV-induced phosphorylation of AP-1 and MAPKs was examined to elucidate the signaling pathways involved in the SUV-mediated expression of MMP-1. The results showed that the increased phosphorylation of c-Jun in SUV-induced HDFs was markedly suppressed by RPE in a dose-dependent manner (Fig. 4C). Furthermore, RPE inhibited the SUV-induced phosphorylation of MEKERK, MKK4/7-JNK, and MKK3/6-p38 (Fig. 4D–F). These results suggest that RPE alleviates SUV-induced collagen degradation by suppressing MMP-1 expression through downregulation of the upstream AP-1 and MAPKs signaling pathways in HDFs.
RPE reduces SUV-induced MMP-1 expression by downregulating AP-1 and MAPKK-MAPK signaling in human skin tissue
To further confirm the anti-photoaging properties of RPE in vivo, we pretreated human skin tissue samples for 1 h, followed by 46 kJ/m2 of SUV irradiation. As given in Figure 5A, MMP-1 expression was markedly increased by SUV exposure, whereas RPE treatment suppressed SUV-induced MMP-1 expression levels. In addition, IHC staining analysis revealed the protective effect of RPE on epidermal thickness by SUV exposure by indicating the level of MMP-1 expression in the skin tissue. MMP-1 expression in the SUV-treated human skin model was noticeably increased, whereas RPE treatment reduced SUV-induced MMP-1 expression (Fig. 5B). AP-1 stimulates the expression of MMP-1,24 and MAPKs are major mediators of UV-induced MMP-1 expression by regulation of AP-1 transactivation.25 As given in Figure 5C, RPE inhibited SUV-induced phosphorylation of c-Jun in human skin tissue, indicating downregulation of AP-1 transcriptional activation by RPE. Moreover, RPE suppressed SUV-induced phosphorylation of MEK-ERK, MKK4/7-JNK, and MKK3/6-p38 (Fig. 6). Collectively, these results suggest that RPE suppresses SUV-induced collagen production in dermal cells and skin tissue.
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DISCUSSION
Many of the ornamental rose cultivars that exist today have long been used for food and medicinal purposes. Indeed, various bioactive compounds of interest are present in the rose plant, including phenolic compounds.26 Previously, we reported on the anti-skin inflammatory activity of RPE using mouse epidermal cells, in which RPE reduced SUV-induced COX-2 expression by inhibiting the MAPK signaling pathway.
The close relationship between inflammation and the aging process is well established,28 and we sought to investigate this relationship in the context of skin whitening and anti-wrinkle formation activities. As given in Figure 1, RPE dose-dependently suppressed in vitro tyrosinase activity and its expression in B16F10 cells. Furthermore, the final outcome of skin pigmentation, melanin production, was inhibited by RPE treatment in B16F10 cells. RPE also induced the phosphorylation of MAPKs and its upstream activator MAPKKs, followed by a decrease in MITF expression (Fig. 2). MAPKs are known to be key signaling factors that regulate melanogenesis in melanocytes.21 The ERK and JNK pathways are involved in numerous signal transduction steps, and also participate in MITF regulation, which sequentially leads to tyrosinase and melanogenesis inhibition.9 Meanwhile, the activation of p38 MAPK leads to inhibition of melanin synthesis by inducing degradation of the tyrosinase protein.6 Taken together, these results demonstrate that RPE inhibits melanogenesis in B16F10 melanoma cells through inhibition of MITF and tyrosinase transcription, and the effect of RPE occurs in association with activation of the MAPKK-MAPK signaling pathways.
To confirm whether the skin whitening and anti-wrinkle formation activities of RPE are valid for human skin, we treated volunteers with RPE for 6 weeks to evaluate skin whitening activity. We also evaluated ex vivo human skin tissue for 10 days to assess the anti-wrinkle formation effects. Treatment with the RPE-containing formulation significantly enhanced the L* values, a unit of measure for skin whiteness (Fig. 3). Furthermore, the majority of volunteers answered that their skin condition, including whiteness, was improved by the formulation containing RPE, based on a questionnaire survey (data not shown).
SUV radiation is considered to be a primary factor in skin aging-related processes.4 In this study, RPE not only restored procollagen synthesis but also suppressed SUV-induced MMP-1 expression in HDFs (Fig. 4). Furthermore, IHC staining analysis showed that RPE could attenuate SUV-induced MMP1 expression in skin tissue (Fig. 5B). In addition, we investigated whether transcription factors such as MAPK and AP-1 are influenced by RPE. We found that protein levels were elevated in SUV-irradiated HDF and skin tissue, whereas RPE significantly inhibited the MAPKK-MAPK pathway and AP-1 activation. After SUV exposure, the resultant ROS can act as activators for several molecular pathways, including the MAPK pathway that activates AP-1 and nuclear factor-kappa B transcription.29 These pathways contribute to the expression of numerous genes including MMPs, leading to collagen deficiency and wrinkling.12 In a previous study, we also demonstrated that RPE contains high concentrations of antioxidants such as anthocyanins, polyphenols, and flavonoids, thereby providing some level of anti-inflammatory capacity against SUV exposure.27 Therefore, the protective effect of RPE on skin damage by SUV radiation shown in this study may be attributable to its antioxidant capacity.
In conclusion, we observed that RPE exhibits skinwhitening and anti-wrinkle effects while inhibiting tyrosinase activity and melanin formation in melanocytes. The anti-melanogenic properties of RPE may be elicited through the activation of the MAPKK-MAPK signaling pathway. In addition, RPE not only promotes procollagen synthesis but also inhibits MMP expression that is induced by SUV exposure in skin fibroblasts and tissue, by suppressing the MAPKK-MAPK pathway and AP-1 activation. Moreover, no negative effects associated with formulation stability were detected in the volunteers. Our findings suggest that RPE may be an effective skin-protecting biomaterial, with applications in the clinical and/or cosmetic treatment of skin pigmentation and wrinkles.
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