Abstract The anti-skin inflammatory activities of rose petal extracts have been described in our previous study. Because skin inflammation is closely linked to skin aging,  our study investigated the effects of Rosa gallica petals on skin aging-related activities such as skin whitening and anti-wrinkle properties. Each sample was prepared via extraction using different ethanol ratios to evaluate optimal extraction conditions for industrial applications. Aqueous 50% (v/v) EtOH extract of R. gallica petal significantly suppressed tyrosinase activity, melanin production, and solar UV-induced matrix metalloproteinase-1, a hallmark of wrinkle formation. In addition, the aqueous 50% (v/v) EtOH extract showed the highest antioxidative effect and had the highest flavonoid contents,  consistent with the reported anti-aging effects. Overall, our findings suggest that R. gallica petals extracts exhibit anti-aging effects. Furthermore, 50% EtOH extraction, in particular, was optimal for the highest anti-aging, and antioxidative effects as well as to obtain the highest flavonoid content. 

According to relevant studies,cistanche is a common herb that is known as "the miracle herb that prolongs life". Its main component is cistanoside, which has various effects such as antioxidant, anti-inflammatory, and immune function promotion. The mechanism between cistanche and skin whitening lies in the antioxidant effect of cistanche glycosides. Melanin in human skin is produced by the oxidation of tyrosine catalyzed by tyrosinase, and the oxidation reaction requires the participation of oxygen, so the oxygen-free radicals in the body become an important factor affecting melanin production. Cistanche contains cistanoside, which is an antioxidant and can reduce the generation of free radicals in the body, thus inhibiting melanin production.

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Keywords Rosa gallica  Skin aging  Flavonoid  Antioxidative effect

Introduction

Skin plays a vital role as a protective barrier against harmful factors associated with heredity and genetics,  environmental issues, hormonal changes, and metabolic processes (Mukherjee et al., 2006). Among these, environmental factors, such as exposure to solar ultraviolet (UV) radiation, act as key mediators that contribute to premature aging (Laga and Murphy, 2009). The main symptoms of skin aging, which occurs as a result of photo-aging, included deep wrinkles, abnormal pigmentation, and elasticity (Farage et al., 2013; Wlaschek et al., 2001).

Pigmentation is a symptom of aging, which is caused by abnormal production of melanin, resulting in a variety of skin disorders including freckles, melasma, age spots,  and other hyperpigmentation syndromes (D’Mello et al., 2016; Seo et al., 2003). In the melanogenesis pathway,  tyrosinase is important as the rate-limiting enzyme that converts L-tyrosine to L-DOPA and oxidizes L-DOPA to form DOPA-quinone (Akhtar et al., 2015). Therefore,  inhibition of tyrosinase is strongly associated with melanin synthesis. Moreover, wrinkle formation, caused by the loss of collagen fibrils and elastase, is another characteristic of photoaging. Matrix metalloproteinase-1 (MMP-1), secreted by human skin fibroblasts, is mainly responsible for collagen degradation during the photo-aging process (Pandel et al., 2013). Downregulation of MMP-1 expression, which inhibits collagen degradation, may enhance wrinkle-improving functions.

For the above-stated reasons, the use of tyrosinase inhibitors or MMP inhibitors is considered a promising strategy for the alleviation of skin photo-aging. Previous studies have indicated that retinol, garlic extract (caffeine acid, and S-ally cysteine) and phytoceramide, kojic acid,  arbutin, and vitamin C may act as skin aging inhibitors (Couteau and Coiffard, 2016; Kim et al., 2013). However,  the use of these substances involves certain limitations such as various side effects including cytotoxicity, odor, and coloration (Yamakoshi et al., 2003). Therefore, current studies are focused on the development of safer, naturally derived components that confer effective skin photoprotection.

Rosa species, grown worldwide, are considered a good source of dietary supplements. Rose petal extract (RPE), which contains elements such as phenolic acid, flavonols, and anthocyanins, has been reported to exhibit many beneficial effects, such as anti-skin inflammatory activities, in addition to other biological roles (Bitis et al., 2017; Lee et al., 2018; Masek et al., 2017; Navarro-Gonzalez et al., 2015). Because these properties of RPE are known to be related to skin aging, it has been proposed as a potential candidate for skin protection. However, little is known about the effect of RPE on skin aging. Additionally, water and ethanol are considered to be the best extraction solvents because of polarity differences and safety of use (Abarca-Vargas et al., 2016). Thus, samples with different ratios of water and ethanol (0, 10, 30, 50, 70, 90, and 100% ethanol RPE) were prepared via extraction.

The purpose of this study was to investigate the effect of RPE on skin whitening and wrinkle improvement. RPE  extracts with different extraction solvent ratios (0, 10, 30, 50, 70, 90, and 100% ethanol/water solvents) were tested to determine the optimal solvent ratio for extraction.

Materials and methods

Reagent

Rosa gallica petals were imported from Turkey through GN Bio (Gyeonggi, Korea). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), Penicillin– streptomycin–neomycin, and 0.5% trypsin–EDTA were purchased from GIBCO Invitrogen (Auckland, NZ, USA). Specific antibodies against tyrosinase and b-actin were purchased from Santa Cruz Biotech (Santa Cruz, CA, USA). The primary antibody of MMP-1 was obtained from R&D systems. All other chemicals, including alpha-melanocyte stimulating hormone (a-MSH), mushroom tyrosinase, and L-DOPA (L-3,4-dihydroxyphenylalanine)  were purchased from Sigma-Aldrich Co., LLC (St. Louis, MO, USA).

Sample preparation

Rose petals were mixed with 100 mL of 0, 10, 30, 50, 70, 90, and 100% (v/v) EtOH (absolute ethanol). Soluble components were then extracted in 80°C water using a  reflux condenser. The extract was filtered through filter paper number 2 (Whatman, Maidstone, England), vacuum-concentrated, and subsequently dissolved in distilled water and freeze-dried, to be used as samples for the functional analysis test.

Cell culture

B16F10 melanoma cells were purchased from the Korean Cell Line Bank (Seoul, Korea). Human dermal fibroblast (HDF) cells were obtained from Dr. Jin Ho Chung (College of Medicine, Seoul National University, Seoul, Korea). Both cells were cultured in DMEM supplemented with 10% fetal bovine serum (v/v) and 1% (v/v) penicillin under 37  °C, and 5% CO2 conditions.

Cell viability

Cell viability was measured via MTS [3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]assay. Cells were seeded on 96-well plates and grown until confluent. Then, cells were starved with serum-free DMEM overnight and treated with the samples at indicated concentrations for 24 h. After exposure to the samples, 20 lL of MTS solution was allowed to react with the cells for 1 h. Absorbance was measured using a  microplate reader (Sunrise-Basic Tecan, Tecan Austria GmbH Gro¨dig, Austria) at 570 nm.

In vitro mushroom tyrosinase activity

The in vitro tyrosinase assay was conducted according to  methods described previously (Kim et al., 2015). In brief, 40 lL of each sample was added to the assay buffer (20 lL  of 0.1 M potassium phosphate), followed by incubation for 30 min with 20 lL of mushroom tyrosinase (0.02 mg/mL). Then, 40 lL of substrate (L-DOPA) was added to each mixture. The reaction was allowed to proceed at room  temperature for 15 min before the formation of dopachrome was analyzed by measuring absorbance at 475 nm  using a microplate reader (Infinite 2000 PRO, Tecan, Switzerland).

Evaluation of melanin production

The melanin production assay was performed according to  previously described-protocol (Friedmann and Gilchrest, 1987; Gordon et al., 1989). B16F10 cells (8 9 103 cells)  were seeded on 6-well plates with 2 mL culture media. After 24 h, samples were pretreated with the cells for 1 h,  following which a-MSH (100 nM) was exposed to the  cells. The cells were collected after 72 h, and melanin  production was measured using a microplate reader (Infinite 2000 PRO, Tecan, Switzerland) at 495 nm.

Western blot

Protein samples were obtained from cells, using 19 Cell Lysis Buffer (Cell Signaling Technology, Danvers, MA). Protein concentration was estimated using a PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific, San Jose´, CA, USA). Protein samples were loaded on to a 10% SDS– polyacrylamide gel (Bio-Rad Laboratories, Hercules, California, USA) for electrophoresis and then transferred to an Immobilon P membrane (Millipore, Billerica, MA, USA). The PVDF membrane was blocked with 5% fat-free milk  for 1 h and the membrane was treated with specific primary  antibody overnight at 4°C. Protein bands were detected  using a chemiluminescence detection kit (GE Healthcare, NJ, USA) after hybridization with an HRP-conjugated  secondary antibody (Cell Signaling).

DPPH radical scavenging assay

DPPH radical scavenging activity was measured as follows; 0.2 mL each of extract was added to 3 mL of -ethanol, to which 0.8 mL of 400 lM DPPH dissolved in  ethanol was added. This mixture was vortexed for 10 s and  maintained at room temperature for 10 min, and absorbance was measured at 517 nm (Wang et al., 1999). DPPH  radical scavenging activity was expressed as a percentage  of the absorbance of the group to which no DPPH was  added. All experiments were replicated a minimum of 3  times.

Total flavonoid content

Total flavonoid content in the extracts was measured using  the aluminum chloride method (Jia et al., 1999) which was  modificated using catechin. To 100 lL of the extract, 500 lL of distilled water and 30 lL NaNO2 were added,  following which 60 lL AlCl3 was added 6 min later. After 5 min, 200 lL of 1 M NaOH was added and the brought  up to 1 mL with distilled water. The solution was mixed  well and centrifuged at 15,928g, at 4°C, for 5 min. After  centrifugation, 200 lL of the supernatant was obtained and  absorbance was measured using a microplate reader (Infinite 2000 PRO; Tecan, Switzerland) at 510 nm.

Statistical analysis

Experiments were conducted in triplicate, and data are  expressed as the mean ± standard deviation (SD). Student’s t tests were used for single statistical comparisons. Statistical significance was set at (#) = p\ 0.05 and (##) = p\ 0.001 for comparison with the untreated control; (*) = p\ 0.05 and (**) = p \0.001 for comparison  with the a-MSH-treated group.

Results and discussion

Extraction of the petals of Rosa gallica according to different solvent ratio

Previously RPE was produced via aqueous 70% (v/v) EtOH extraction (Lee et al., 2018). However, if RPE is  intended for purposes of industrial application, extraction  conditions must be optimal. In order to investigate optimal  extraction conditions of RPE for anti-skin aging activity,  various solvents were used for RPE (Fig. 1A). Interestingly, the color of each extract varied, based on visual  observation (Fig. 1B). The color of EtOH extracts, 90% or  higher, was clear, whereas 50% (v/v) EtOH extract  appeared to be the most reddish. The result of colorimeter  revealed an a* value for redness for 50% EtOH, which  showed the highest level among the extracts (Fig. 1C).

Effect of the extracts of rose petal on skin whitening activity

Since tyrosinase is a key enzyme involved in hyperpigmentation via melanin production (Chang, 2009), we analyzed the inhibitory effect of the extracts on tyrosinase  activity in vitro. Most extracts, except 100% EtOH,  revealed dose-dependent regression on in vitro tyrosinase  activity (Fig. 2A). Furthermore, 30% and 50% EtOH  extracts showed stronger inhibitory effects than ascorbic  acid. Ascorbic acid was described as a skin whitening  agents in previous literature (Chang, 2009). To evaluate  skin whitening activity of the extracts in a cellular model,  melanin production was assessed in B16F10 melanoma  cells following treatment with each extract. A 100 nM portion of a-MSH increased melanin production and every  extract of rose petal inhibited melanin production (Fig. 2B). Furthermore, 30% and 50% EtOH extracts  showed the strongest inhibitory effet on tyrosinase activity  and melanin production in vitro, respectively. Under similar conditions, the effect of each of the extracts on  tyrosinase expression was examined. Also, a-MSH  increased tyrosinase expression and 100 lg/mL each of the  extracts attenuated tyrosinase expression (Fig. 2C). Treatment with extracts at 100 lg/mL did not cause cell cytotoxicity in B16F10 melanoma cells (Fig. 2D). Thus,  inhibition of melanin by RPE is attributed to the dual  fuction of tyrosinase activity and expression the absence of  cytotoxicity.

Effect of the RPE on solar UV (sUV)-induced MMP-1 expression in human dermal fifibroblasts

Inhibitors of MMP-1 may be regarded as anti-wrinkle  agents (Park et al., 2018; Yang et al., 2018). This is  because MMP-1 is a key enzyme, responsible for wrinkle  formation during the skin aging process (Pittayapruek  et al., 2016). To determine anti-wrinkle effects of RPE, MMP-1 expression was evaluated in human dermal  fibroblasts using Western blot analysis. MMP-1 expression  was dramatically increased by sUV irradiation (30 kJ/cm2 ),  and all extracts inhibited sUV-induced MMP-1 expression (Fig. 3A). Whereas 100% EtOH extracts exhibited mild  effect, 50% EtOH extracts displayed the strongest suppressing activity on sUV-induced MMP-1 expression. Next, we investigated MMP-1 production levels in cultured  media, because MMP-1 is a secreted protein for collagen  degradation. Similar to that shown in Fig. 3A, MMP-1 level in cultured media was suppressed by each extract (Fig. 3B). In particular, 50% EtOH extract showed the  highest suppressive activity on sUV-induced MMP-1  expression, and 100% EtOH extract exhibited a mild effect  compared to that by other extracts.

Anti-oxidative activity and total flavonoid contents  of the rose petal extracts

Antioxidants that inhibit ROS production are known to  reduce hyperpigmentation or prevent UV-induced melanin  production (Yamakoshi et al., 2003). DPPH radical scavenging activity was determined to compare the antioxidant  activity of the extracts. Interestingly, every extract showed  drastic, dose-dependent, radical scavenging activity. The 500 lg/mL samples showed anti-oxidative activity similar to vitamin C. Among the extracts, the 50% EtOH extract  showed the strongest anti-oxidative effect at the lowest  concentration (50 lg/mL) (Fig. 4A).

There are many different compounds such as terpenes,  flavonoids, and anthocyanins in rose petals (Knapp et al., 1998; Kumar et al., 2008; Oka et al., 1998; Schieber et al., 2005). These chemicals represent many biological activities (Kumar et al., 2009). In particular, flavonoids have  been described as the main group of phenolic compounds  responsible for biological properties of antioxidant and  anti-inflammatory effects (Du et al., 2016; Jung et al., 2015). Next, the total flavonoid content of each extract was  measured. Large amounts of flavonoids were present in  each extract (Fig. 4B). The results indicated that the 50% EtOH extract contained the highest flavonoid content  among the samples. This trend was similar to that observed  in the anti-oxidative assay result.

The dark red color of the 50% EtOH solvent of rose  petals is assumed to be related to anthocyanin, a flavonoid (Fig. 1). This result substantiates the findings of a previous  study (Oancea et al., 2012). According to the previous  study, anthocyanins in blueberry (Vaccinium orymbosum L.) were best-extracted in 50% EtOH solvent than in any  other solvent, including 60%, 70% and 80% EtOH (Oancea  et al., 2012). Naturally, anthocyanins occur as glycosides. Thus we surmised that the polarity of the anthocyanins may  have been accorded by the 50% EtOH solvent. The strong  anti-oxidative activity of anthocyanins is well demonstrated in various model systems (Kalt et al., 2003; RiceEvans et al., 1995). Because 50% EtOH rose petal extract  had the highest anthocyanin content, the anti-oxidative  activity of 50% EtOH extract was the strongest among the 50 lg/mL extracts.

In summary, our study demonstrated the potential of RPE as anti-skin aging ingredient. For purpose of industrial  application, optimization of extraction conditions is  essential in order to reduce production costs. The current  study investigated solvents most suitable for Rose petal  extraction, with reference to anti-skin aging activity. The  flavonoids in rose petals were extracted best by the 50% EtOH solvent. This extract showed the strongest anti-oxidative activity as well as anti-skin aging activity such as  skin whitening and wrinkle suppression activity. Clinical  studies and stability analyses may be required to test its  suitability for industrial application.

Acknowledgements This research was supported by Main Research Program (E0183112-02) of the Korea Research Food Institute (KFRI)  funded by the Ministry of Science, ICT & Future Planning and by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through High Value-added Food Technology Development Program, funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA) (118060-03-2- HD020).

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For more info: david.deng@wecistanche.com / WhatApp:86 13632399501