Arch Dermatol Res DOI 10.1007/s00403-017-1728-1

ORIGINAL PAPER

Cosmetic applications of glucitol-core containing gallotannins from a proprietary phenolic-enriched red maple (Acer rubrum) leaves extract: inhibition of melanogenesis via down-regulation of tyrosinase and melanogenic gene expression in B16F10 melanoma cells Hang Ma1 · Jialin Xu1,2 · Nicholas A. DaSilva1 · Ling Wang3 · Zhengxi Wei1 · Liangran Guo1 · Shelby L. Johnson1 · Wei Lu1 · Jun Xu4 · Qiong Gu4 · Navindra P. Seeram1  Received: 25 September 2016 / Revised: 15 February 2017 / Accepted: 22 February 2017 © Springer-Verlag Berlin Heidelberg 2017

Abstract  The red maple (Acer rubrum) is a rich source of phenolic compounds which possess galloyl groups attached to different positions of a 1,5-anhydro-d-glucitol core. While these glucitol-core containing gallotannins (GCGs) have reported anti-oxidant and anti-glycative effects, they have not yet been evaluated for their cosmetic applications. Herein, the anti-tyrosinase and anti-melanogenic effects of a proprietary phenolic-enriched red maple leaves extract ­[Maplifa™; contains ca. 45% ginnalin A (GA) along with other GCGs] were investigated using enzyme and cellular assays. The GCGs showed anti-tyrosinase activity with ­IC50 values ranging from 101.4 to 1047.3  μM and their mechanism of tyrosinase inhibition (using GA as a Electronic supplementary material  The online version of this article (doi:10.1007/s00403-017-1728-1) contains supplementary material, which is available to authorized users. * Hang Ma [email protected] * Navindra P. Seeram [email protected] 1

Bioactive Botanical Research Laboratory, Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, University of Rhode Island, Kingston, RI 02881, USA

2

Institute of Biochemistry and Molecular Biology, College of Life and Health Sciences, Northeastern University, Shenyang 110819, China

3

Pre‑Incubator for Innovative Drugs and Medicine, School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, China

4

School of Pharmaceutical Sciences, Sun Yat-Sen University, 132 East Circle Road at University City, Guangzhou 510006, China









representative GCG) was evaluated by chelating and computational/modeling studies. GA reduced melanin content in murine melanoma B16F10 cells by 79.1 and 56.7% (at non-toxic concentrations of 25 and 50  μM, respectively), and its mechanisms of anti-melanogenic effects were evaluated by using methods including fluorescent probe (DCFDA), real-time PCR, and western blot experiments. These data indicated that GA was able to: (1) reduce the levels of reactive oxygen species, (2) down-regulate the expression of MITF, TYR, TRP-1, and TRP-2 gene levels in a timedependent manner, and (3) significantly reduce protein expression of the TRP-2 gene. Therefore, the anti-melanogenic effects of red maple GCGs warrant further investigation of this proprietary natural product extract for potential cosmetic applications. Keywords  Red maple (Acer rubrum) · Glucitol-core containing gallotannins (GCGs) · Anti-tyrosinase · Antimelanogenic · Cosmetic · Skin-whitening Abbreviations GCGs Glucitol-core containing gallotannins GA Ginnalin A GB Ginnalin B GC Ginnalin C MF Maplexin F MJ Maplexin J DCT DOPA-chrome tautomerase DOPA 3,4-dehydroxyphenylalanine DHICA 5,6-dihydroxyindol-2-carboxylic acid MITF Microphthalmia-associated transcription factor ROS Reactive oxygen species TYR Tyrosinase

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Vol.:(0123456789)



TRP-1 Tryosinase-related protein-1 TRP-2 Tryosinase-related protein-2 DCF-DA 2′,7′-Dichlorodihydrofluorescein diacetate DMEM Dulbecco’s modified Eagle medium DMSO Dimethylsulfoxide MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)2-(4-sulfophenyl)2H-tetrazolium

Introduction In mammals, skin pigments are produced in the melanosomes of melanocytes, which are situated on the basal layer between the dermis and epidermis [1]. Pigmentation plays a crucial role in protecting skin against radiation-induced damage such as exposure to ultraviolet light [2]. However, overproduction or abnormal accumulation of melanin may lead to many skin hyperpigmentation disorders including freckles, age spots, post-inflammatory hyperpigmentation, and even melanoma. Consequently, undesired excessive skin pigmentation may lead to a negative burden on the psychological well-being of patients, and thus, skin depigmentation and beauty products remain a compelling area of research for the cosmetic industry. Furthermore, the exploration of safe and effective melanogenesis inhibitors, particularly from natural sources, has attracted immense research interest [3]. The maple genus (Acer) comprises of over 120 species, most of which are found in Asia, with the remaining being native and endemic to North America. Phytochemical and biological investigations of Acer species including A. buergerianum (Chinese maple) and A. nikoense (Japanese maple) led to the discovery of several compounds with anti-melanogenic effects in B16F10 cells [4, 5]. Interestingly, A. rubrum (red maple), which is native and endemic to eastern North America, has been used traditionally as a folk medicine by the Native Americans for several ailments including skin disorders [6]. While a published study has reported on the tyrosinase inhibitory activity of a red maple bark extract, neither the bioactive compounds nor their mechanisms of inhibition have been identified [7]. Over 100 distinct genes collectively regulate the biosynthesis of melanin. In mammals, the tyrosinase (TYR), tryosinase-related protein-1 (TRP-1), and tryosinase-related protein-2 (TRP-2) enzymes are essential for overall melanin production [8]. Tyrosinase performs a pivotal role in the modulation of melanogenesis and is the rate-limiting enzyme that catalyzes the hydroxylation of l-tyrosine into 3,4-dehydroxyphenylalanine (DOPA) and consequently oxidizes DOPA into DOPA quinone. TRP-2, which serves as a DOPA-chrome tautomerase (DCT), further converts DOPA quinone into 5,6-dihydroxyindol-2-carboxylic acid

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(DHICA), whereas TRP-1 facilitates the oxidization of DHICA to form carboxylated indole-quinone [9]. Microphthalmia-associated transcription factor (MITF) is a primary transcriptional activator of the melanogenic enzymes and a principal transcription regulator that mediates the survival, proliferation, and differentiation of melanoblasts and melanocytes [10]. Given that melanin synthesis in mammals involves multiple-step reactions that are modulated by a group of enzymes and transcription factors, it is necessary to elucidate the molecular mechanisms of potential inhibitors of melanogenesis. Although many synthetic compounds, such as hydroquinone and its derivatives, have been conventionally used as inhibitors of melanin biosynthesis, their cosmetic applications have been hampered because of potential side effects including skin irritation [11]. Therefore, natural products have emerged as attractive candidates for skin-whitening cosmetic applications, since they tend to be safe and have fewer adverse effects than synthetic compounds [3, 12]. Our laboratory has conducted extensive studies on several maple species and their derived extracts, including the red maple, which has led to the identification of over 100 compounds with anti-oxidant, anti-α-glucosidase, anticancer, anti-glycation, and anti-neurodegenerative activities [13–23]. Herein, we developed and evaluated M ­ aplifa™, a proprietary standardized phenolic-enriched extract from red maple leaves, for its skin-whitening cosmetic applications.

Materials and methods Materials Leaves of red maple (Acer rubrum) were collected on the University of Rhode Island (URI) Kingston campus, (RI, USA) and identified by Mr. J. Peter Morgan. A voucher specimen (11/6/09LPMCL2) has been deposited in the URI College of Pharmacy Heber Youngken Herbarium. All solvents were purchased from Wilkem Scientific (Pawtucket, RI, USA). Mushroom tyrosinase, l-tyrosine, 4-hydroxyphenyl β-d-glucopyranoside (arbutin), and kojic acid were purchased from Sigma–Aldrich (St. Louis, MO, USA). Preparation of ­Maplifa™ Our laboratory has previously reported on the comprehensive isolation and identification of phytochemicals, primarily phenolics, from the leaves, flowers, bark, and stems of the red maple species [21–23]. Therefore, using these chemical standards, ­Maplifa™ was prepared by proprietary protocols developed in our laboratory. Briefly, the leaves of red maple were air dried and macerated in aqueous ethanol to obtain a crude extract, which was further purified on

Arch Dermatol Res

a resin column to remove chlorophyll and other plant pigments. ­Maplifa™ was obtained as an off-white free flowing powder after solvent removal in vacuo. Glucitol‑core containing gallotannins (GCG) standards All GCGs (see Fig. 1a for the chemical structures for ginnalins A–C and maplexins F and J) were generated in our laboratory as previously reported [19, 21–23]. Standardization of ­Maplifa™ by HPLC‑DAD All analyses were performed on a Hitachi Elite LaChrom system operated by the EZChrom Elite software consisting of a L2130 pump, L-2200 auto sampler, a L-2455 Diode Array Detector, and a Phenomenex Luna ­ C18 column (250 × 10 mm i.d., 5 μm). The detailed methods including solvent system for the standardization of ­Maplifa™ have previously been reported by our laboratory [22]. Tyrosinase inhibition assay The inhibitory effects of GCGs including ginnalins A–C and maplexins F and J (see Fig. 1a for structures) on mushroom tyrosinase enzyme were evaluated spectrophotometrically using l-tyrosine as a substrate according to our previously reported methods with minor modifications [24]. The tyrosinase inhibition assay was conducted in a 96-well microplate format using a SpectraMax M2 microplate reader (Molecular Devices, Sunnyvale, CA, USA). Briefly, the test samples were dissolved in 10% DMSO at

a concentration of 5.0 mg/mL and then diluted to different concentrations with phosphate buffer solution (PBS; 0.1 M, pH 6.8). Each well contained 40 μL of sample with 80 μL of PBS, 40  μL of tyrosinase (100  units/mL), and 40  μL l-tyrosine (2.5 mM). The mixture was incubated for 20 min at 37 °C, and the absorbance was measured at 490 nm. Each sample was accompanied by a blank containing all of the components except l-tyrosine. The well-known skin-whitening natural products, kojic acid, and arbutin, served as the positive controls. The results were compared with a control consisting of 10% DMSO in place of the sample. The percentage of [tyrosinase inhibition percentage was ]calculated as follows: (ΔAcontrol − ΔAsample)∕ΔAcontrol × 100% Copper ion chelating assay The copper ion chelating capacity of GA was evaluated using published UV–visible spectrometric assay with minor modifications [25]. A reaction mixture consisting of 900 µL of 0.1 M phosphate buffer solution (PBS; 0.1 M, pH 6.8), 500 µL of distilled water, 50 µL of the GA (0.05 mM) solution, and 50 µL of the mushroom tyrosinase (145 units/ mL in PBS) was incubated at room temperature for 30 min, and then, the spectra were recorded at 280–400 nm wavelength. Repeated scans were recorded after the addition of copper ion ­(CuSO4 at 50, 100, and 200 µM). Molecular docking study Kojic acid, GA, MF, and MJ were optimized using MMFF94s force field. The crystal structure of tyrosinase

Fig.  1  a Chemical structures of ginnalins A–C and maplexins F and J and b the HPLC-DAD profile of red maple leaves extract ­(Maplifa™). Compounds labelled 1–9 are ginnalin A, ginnalin B, ginnalin C, maplexin B, 4-O-galloylquinic acid, maplexin F, maplexin E, 6-O-digalloyl-2-O-galloyl1,5-anhydro-d-glucitol, and 2-O-digalloyl-6-O-galloyl1,5-anhydro-d-glucitol, respectively

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was downloaded from PDB databank (PDB ID: 2Y9X) and protonated based on Amber99 force fielded after removing water molecules. The binding site was defined as all residues within the native ligand (Tropolone). The MOE-docking module in MOE 2010.10 was used to dock the compounds against the tyrosinase structure. Other MOE-docking parameters were set to default values. During the docking study, 30 poses per compound were retained. All docked poses of kojic acid, GA, MF, and MJ were ranked based on their binding docking energies according to LondondG score. The best binding poses of each compound were selected based on the predicted affinity and interactions with the putative key residues. Cell culture Murine melanoma B16F10 cells were purchased from the American Type Culture Collection (Rock-ville, MD, USA). Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) medium that was supplemented with 10% fetal bovine serum, 1% v/v nonessential amino acids and 1% antibiotic solution at 37°C  in 5% ­ CO2. Samples were dissolved at a concentration of 10  mg/mL in dimethylsulfoxide (DMSO) to yield stock solutions, which were then diluted to the desired final concentrations with growth medium. The final DMSO concentration was <0.1%. Cell viability assay The cell viability assay was performed using the MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay as described previously with modifications [14]. Briefly, B16F10 cells (3 × 103 cells/mL) were seeded in 96-well plates and incubated for 24 h at 37 °C in a 95% air and 5% C ­ O2 atmosphere incubator. Next, the culture medium was replaced with fresh medium containing GA at various concentrations (ranging from 3 to 50 μM). At the end of 72 h of treatment with test samples, 20 μL of the MTS reagent, in combination with the electron coupling agent, phenazine methosulfate, were added to the wells and cells were further incubated at 37 °C for 3  h. Absorbance at 490  nm was monitored with a spectrophotometer (Molecular Devices, Sunnyvale, CA, USA) to obtain cell numbers relative to control populations. Inhibition of proliferation by the sample treatment cells is expressed as percentage compared to control (0.1% DMSO) cells. Data are presented as mean values ± SD and were obtained from three separate experiments.

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Cellular melanin contents The melanin content was determined using a modification of the method previously described [26]. Briefly, B16F10 cells (5 × 104 cells/well) were seeded in 24-well plates for 24 h and then the medium was changed with new DMEM medium containing the test sample at concentrations from 6 to 50  µM. After 72  h incubation, the cells were harvested through trypsinization and washed with PBS for two times. Then, the cells were lysed with 1 N NaOH containing 10% DMSO and heated at 80 °C for 1 h. After the samples were cooled to room temperature, the amount of melanin content was spectrophotometrically measured at 400 nm (Molecular Devices, Sunnyvale, CA, USA). Determination of reactive oxygen species (ROS) level in murine B16F10 melanoma cells The ROS levels in the B16F10 cells were measured using our previously published method with slight modifications [17]. Briefly, B16F10 cells were seeded into 48-well plates at a density of 10,000 cells/well in DMEM. The cells were incubated for 24 h and then treated with GA at concentrations ranging from 6 to 50  μM for 24  h. Next, fresh DMEM medium containing DCF-DA (20 µM) was added. After 20 min, the cells were washed with PBS and incubated with ­H2O2 (3.0  μg/mL) for 1  h. The fluorescence intensity was read at excitation and emission wavelengths of 485 and 525 nm, respectively, using a Spectra Max M2 spectrometer. Real‑time quantitative PCR analysis The B16F10 cells were seeded in 6-well plates at a density of 2.0 × 105  cells/well. After incubation for 24  h, cells were treated with 2 or 10 μM of GA for 48 or 72 h. Total RNA was isolated from cells using TRIzol reagent (Invitrogen; CA, USA) according to the manufacturer’s instructions. One microgram of total RNA was converted to single-stranded cDNA using oligo(dT)18 primers, and mRNA levels were quantified by quantitative real-time PCR using a Roche LightCycler detection system (Roche Applied Science, Mannheim, Germany). Samples were evaluated by using SYBR Green and compared with levels of b2m rRNA as a reference housekeeping gene. Quantitative real-time PCR conditions were optimized for each gene using appropriate forward and reverse primers. All oligonucleotides were synthesized by Invitrogen (CA, USA).

Arch Dermatol Res

Evaluation of MITF, TYR, TRP‑1, and TRP‑2 expression by western blot The expression of melanin biosynthesis-related proteins, namely, MITF, TYR, TRP-1, and TRP-2 in the B16F10 cells was measured by western blot. After incubation with GA for 72 h, proteins from B16F10 cells were resolved by SDS–PAGE and then transferred to polyvinylidene fluoride membrane. The membrane was blocked with 5% nonfat dry milk in Tris-buffered saline with Tween followed by incubation with primary antibodies overnight. Bands were visualized on X-ray film using an ECL detection kit (Amersham Biosciences, Piscataway, NJ, USA). Statistical analysis Data are presented as mean ± standard deviation of at least three separate experiments. Two-tailed unpaired student’s t test or ANOVA with Tukey post-test was used for statistical analysis of the data using the GraphPad Prism software 6.0 or Office Excel 2010 software. Significance for all tests was defined as: p ≤ 0.05 (*).

Results Development and phytochemical standardization of a phenolic‑enriched red maple leaves extract ­(Maplifa™) Using protocols and chemical standards (Fig.  1a) isolated from the red maple species as previously reported by our laboratory [21–23], a phenolic-enriched red maple leaves extract ­(Maplifa™) was developed and standardized by HPLC-DAD (Fig. 1b). ­Maplifa™ contains 40–45% ginnalin A (GA; structure shown in Fig. 1b) as well as other phenolics including ginnalin B (GB), ginnalin C (GC), maplexin B, 4-O-galloylquinic acid, maplexin F (MF), maplexin E, and 6-O-digalloyl-2-O-galloyl-1,5-anhydro-d-glucitol, 2-O-digalloyl-6-O-galloyl-1,5-anhydro-d-glucitol [22]. GCGs inhibit tyrosinase enzyme activity The inhibitory effects of five pure GCGs, including GA, GB, GC, MF, and maplexin J (MJ) (structures shown in Fig.  1a) on mushroom tyrosinase activity were evaluated ­(IC50 values shown in Table  1). The GCGs containing more than one galloyl group, namely, GA, MF, and MJ, were more active than those containing only one galloyl, namely, GB and GC. Notably, GA, which contains two galloyl groups, inhibited tyrosinase enzyme activity from 33.8 to 83.4% at concentrations ranging from 16 to 500 µM (Shown in supplementary material Fig. S1). In addition,

Table 1  Inhibitory activity ­(IC50) of five phenolics, ginnalins A–C, and maplexins F and J, on tyrosinase enzyme GCGs

# of galloyl group

IC50 (μM)a

GC GB GA MF MJ Kojic ­acidb Arbutinb

1 1 2 3 4 − −

>500 >500 101.4 ± 6.5 208.9 ± 3.1 173.8 ± 5.2 23.7 ± 0.9 61.6 ± 2.8

a

 IC50 are presented as mean ± SD from triplicate independent experiments

b

 Positive controls

GA showed the highest inhibitory activity among the GCGs with an ­IC50 value of 101.4  μM. Similarly, MF and MJ, which contain three and four galloyl groups, respectively, showed comparable I­C50 values of 208.9 and 173.8  μM. However, the GCGs with only one galloyl group, namely, GB and GC, showed weak inhibitory effects ­(IC50 = 1047.3 and 890.5  μM, respectively) on the tyrosinase enzyme. Kojic acid and arbutin which served as the positive controls had I­C50 values of 23.7 and 61.6  μM, respectively. Given that GA is the most abundant GCG in M ­ aplifa™ and it showed the highest inhibitory activity in the tyrosinase assay (Table 1), it was selected as a representative GCG, to investigate their potential mechanisms on the inhibition of tyrosinase and melanogenesis in the melanoma cells. GA chelates copper ion GA was evaluated for its copper ion chelating capacity given that its inhibitory effect on the tyrosinase enzyme may be due to its chelating ability with the copper ion at the enzyme active site. As shown in Fig. 2, GA showed a UV absorption spectrum with a characteristic peak at 276 nm, which was in agreement as previously reported [25, 27]. The UV absorption at 274 nm decreased when GA was coincubated with tyrosinase, suggesting that GA interacted with tyrosinase and altered its molecular environment. In addition, a typical bathochromic shift from 276 to 320 nm was detected in the presence of various concentrations of ­Cu2+ (ranging from 50 to 200 µM), suggesting that copper ion chelation was formed in the GA-tyrosinase complex [27]. GA‑tyrosinase binding modes analyses Next, we examined the possible binding between GA and the tyrosinase enzyme using computational modeling methods. As shown in Fig.  3a, kojic acid inserts well into the

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kojic acid, GA, MF, and MJ are −4.82, −4.79, −3.21, and −3.73 kcal/mol, respectively, which was in agreement with the results obtained from the enzyme inhibition assay and binding model analyses. Similarly, the galloyl groups of the other GCGs including MF and MJ can form hydrogen bonds with the residues of the binding site and surface of tyrosinase (Shown in supplementary material Fig. S2). GA reduces the melanin content in B16F10 melanoma cells

Fig. 2  UV–visible spectrum of GA (0.05  mM; solid line), GA (0.05 mM) with tyrosinase (145 unit; green dot), and GA (0.05 mM) with ­CuSO4 (concentrations ranging from 50 to 200  µM; blue, red, and purple dot, respectively)

binding site of tyrosinase, and can form a hydrogen bond with the side chain of Gly281 and interact with two copper ions. Compared to kojic acid, GA binds to the surface of the binding site of tyrosinase (Fig. 3b) showing lower binding affinity than that of kojic acid. The binding energies of Fig. 3  Computational docking of interactions between kojic acid or GA and tyrosinase by MOE-docking (MOE 2010.10). Ribbon model of predicted binding site. Two brown balls represent copper ions and gold represents kojic acid or GA. Red dash represents hydrogen bonds formed between kojic acid or GA and tyrosinase. a Close-up view of binding site of kojic acid and tyrosinase (left); predicted 3D kojic acid and tyrosinase (PDB: 2Y9X) complex (right). b Close-up view of binding site of GA and tyrosinase (left); predicted 3D GA and tyrosinase (PDB: 2Y9X) complex (right)

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To investigate the anti-melanogenic activities of GA, cellular-based assays were employed to measure the melanin content in murine melanoma B16F10 cells. However, to determine non-toxic test concentrations of GA, we first evaluated its effects on viability of the B16F10 cells using the MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]assay. GA was evaluated at concentrations of 6, 13, 25, and 50 μM for 72 h and cell viability was determined by comparison to untreated cells. As shown in Fig. 4, GA was non-toxic to the B16F10 cells at concentrations ranging from 6 to 13  μM (cell viabilities >90%) and slightly reduced the B16F10 cell viability to 87.0 and 89.5% at concentrations of 25 and 50  μM, respectively. Concentrations of GA tested in the current study can be considered as non-toxic in the cellular assays, since they were lower than a reported threshold of safe concentration (40 μg/mL; equivalent to 85  μM of GA) [28]. We next evaluated the inhibitory effects of GA on the biosynthesis of melanin in the B16F10 cells. As shown in Fig.  5, GA did not show

Arch Dermatol Res

GA decreases the levels of reactive oxygen species (ROS) in B16F10 melanoma cells

Fig. 4  Effects of GA on the proliferation of murine melanoma B16F10 cells. B16F10 cells (3 × 103  cells ⁄mL) were seeded in 96-well plates and incubated for 24  h at 37 °C in a 95% air and 5% ­CO2 atmosphere incubator. B16F10 cells were treated with GA (concentrations ranging from 6 to 50 μM) or without GA (as the control group). The viability of B16F10 cells was determined by the MTS assay. Each value is presented as mean ± SD from triplicate independent experiments

Fig. 5  Effects of GA on melanin biosynthesis in murine melanoma B16F10 cells. Cells were seeded at a density of 5 × 104 cells/well in a 24-well plate. After 1  day incubation, the cells were treated with GA or without GA (as control group) at concentrations ranging from 6 to 50 μM for 72 h. The melanin contents were measured at 400 nm wavelength and compared to the control group. Each value is presented as mean ± SD for triplicate independent experiments. *Statistically significant (P ≤ 0.05) difference between control and treated cells

significant inhibitory activities on melanin biosynthesis at its lower test concentrations (at 6 and 13 μM). However, at higher concentrations of 25 and 50  μM, GA significantly reduced the melanin content in B16F10 cells to 79.1 and 56.7%, respectively, compared to the control group.

The biosynthesis of melanin is promoted by oxidative stress in melanoma cells suggesting that an anti-oxidant effect could be a possible mechanism of action of GA on melanongenesis. Although our group has recently reported that GA shows potent free radical scavenging and metal chelating activities [18], its anti-oxidant effect at the cellular level has never been examined. Therefore, we evaluated the effects of GA on intracellular ROS production, induced by ­H2O2, in the B16F10 cells. As shown in Fig. 6, GA significantly decreased H ­ 2O2-induced ROS production by 23.5, 47.8, 65.2, and 55.1% at concentrations of 6, 13, 25, and 50 μM, respectively. These data indicate that GA may reduce melanin production in B16F10 cells by decreasing intracellular ROS levels. GA down‑regulates the tyrosinase‑related gene and protein expression Melanin biosynthesis involves multi-step pathways. Therefore, to explore the possible molecular mechanisms of the inhibitory effects of GCGs on melanin synthesis, the expression levels of several melanogenesis-related genes, including MITF, TYR, TRP-1, and TRP-2, in B16F10 cells were analyzed using RT-PCR. As shown in Fig.  7a–d, GA (10  µM) significantly reduced the mRNA expressions of MITF, TYR, TRP-1, and TRP-2 in B16F10 cells

Fig. 6  Effects of GA on the reduction of ­H2O2-induced intracellular ROS in B16F10 cells. The ROS production in B16F10 cells was induced by H ­ 2O2 (3.0  μg/mL) and the ROS level was indicated by the intensity of a fluorescence probe (DCF-DA) in B16F10 cells that were treated with GA (concentrations ranging from 6 to 50  μM) or without GA (control group). The fluorescence intensity was read at excitation and emission wavelengths of 485 and 525 nm, respectively. Each value is presented as mean ± SD from triplicate independent experiments. *Statistically significant (P ≤ 0.05) difference between control and treated cells

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Fig. 7  Effects of GA on the expression of melanogenesisrelated genes in murine melanoma B16F10 cells. Cells were treated with GA (10 µM) for 48 or 72 h. The expression level of genes TRP-1 (a), TYR (b), MITF (c), and TRP-2 (d) was determined by real-time PCR. B2M was used as an internal control in real-time PCR. *Statistically significant (P ≤ 0.05) difference between control and treated cells

in a time-dependent manner. After 48  h treatment, GA (10 μM) reduced the MITF, TYR, TRP-1, and TRP-2 gene expression in B16F10 cells by 83.0, 38.4, 75.2, and 53.4%, respectively. In addition, after treatment of GA for 72 h, the MITF, TYR, TRP-1, and TRP-2 gene expression in B16F10 cells was decreased by 75.2, 41.2, 54.4, and 45.3%, respectively. Next, the effects of GA on the protein expression of the melanogenesis-related enzymes in B16F10 cells were evaluated by Western blot. GA did not significantly down-regulate the protein expression of melanin biosynthesis-related enzymes at 5 and 10 μM at 48 h, nor reduce the expressions of these proteins in B16F10 cells that were co-incubated with GA for 72 h (data not shown). However, in the B16F10 cells that were treated with 10  μM of GA for 72  h, the protein expression of TRP-2 was decreased by 48.8% (see Fig. 8). Overall, these data suggest that GA reduced the biosynthesis of melanin in B16F10 cells by the down-regulation of the gene expression of MITF, TYR, TRP-1, and TRP-2 genes and that its anti-melanogenesis activity may be related to its reduction of protein levels of TRP-2.

Discussion Previous studies have shown that extracts from the plant parts of several maple (Acer) species show biological activities including anti-oxidant, anti-microbial, anti-angiogenic, and anti-melanogenic properties [29]. Maple species which are indigenous to Asia including the Chinese maple

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Fig. 8  Effects of GA on the expression of the melanogenesis-related proteins MITF and TRP-2 in murine melanoma B16F10 cells. The cells were treated with GA (10 µM) for 72 h. The expression levels of MITF and TRP-2 proteins were examined by Western blot analysis as described in the “Materials and methods” section. Equal protein loading was confirmed by β-actin expression. **Statistically significant (P ≤ 0.01) difference between control and treated cells

Arch Dermatol Res

and Japanese maple have been reported to produce several compounds with anti-melanogenic effects in B16F10 cells [4, 5]. Interestingly, a bark extract of the red maple, which is native to North America, has been reported to show tyrosinase inhibitory activity, but the bioactive compounds and their potential mechanisms of inhibition are unknown [7]. In addition, GCGs have recently been reported as regulators of skin cellular ceramide [30]. Therefore, we sought to develop a standardized phenolic-enriched extract of red maple leaves (­ Maplifa™) for potential cosmetic applications and to investigate its constituents for their potential antimelanogenic mechanism/s of action. Ginnalin A (GA), the predominant phenolic compound present in M ­ aplifa™, shows potent free radical scavenging capacity [18] and the highest inhibitory activity in the tyrosinase assay (Table  1). Therefore, GA was selected as a representative compound in Maplifa™ and evaluated in detail to provide insights into the mechanisms of anti-melanogenic activity of these compounds. Our in vitro studies showed that the gallotannins had moderate inhibitory effects against tyrosinase. In general, the inhibitory activity of the gallotannins (GA, MF, and MJ) with multiple galloyl groups (n > 2) was greater than the mono-galloyl substituted compounds (GB and GC). The inhibitory effect of GA on tyrosinase enzyme may also be possible due to its potent anti-oxidant effects including free radicals scavenging and metal (copper and ferric) chelating activities [18]. Notably, many well-known tyrosinase inhibitors are natural products which show potent anti-oxidant activity [3, 12]. In addition, data from the modeling studies suggested that the maple gallotannins may directly bind to tyrosinase and form ligandcopper ions complex. The anti-melanogenic effects of the GCGs were evaluated in B16F10 murine melanoma cells at non-toxic concentrations. At the highest test concentration of 50  μM, cell viability was >90% indicating that the compounds were, indeed, non-toxic at these concentrations. GA inhibited melanin content in B16F10 cells in a concentration-dependent manner. A decreased production of cellular ROS in B16F10 cells with the GA treatment suggested that GA served as an anti-oxidant to reduce the oxidative stress in melanocytes, which can lead to the reduction of melanin synthesis. However, it is possible that other mechanisms can also contribute to the anti-melanogenic effect of GA. For example, GA may decrease melanin production by inhibiting cellular tyrosinase activity as has been reported for other phenolic natural products [3, 12]. Next, the inhibitory effect of GA on melanin biosynthesis was further supported by examining its effect on mRNA expression of genes that regulate melanin production. The RT-PCR data demonstrated that after 48 or 72  h of treatment, GA decreased the mRNA expression of MITF, TYR, TRP-1, and TRP-2. MITF is

known for the activation of melanocyte differentiation as well as the regulation of the transcription of melanogenic enzymes including TRP-1 and TRP-2 [8]. Notably, GA decreased MITF expression more significantly compared to the other melanin synthesis genes suggesting that it down-regulated melanin biosynthesis by suppression of transcriptional activator of melanin production-related enzymes such as TRP-2. TRP-2 is a DOPA-chrome tautomerase, which is critical for the oxidation of DOPA quinone. Thus, we further examined the protein expression level of TRP-2 by Western blot analysis. A decreased protein expression level of only the TRP-2 gene suggested that GA was able to impede the transformation of DOPA quinone into 5,6-dihydroxyindol-2-carboxylic acid which could further be oxidized to form the melanin pigments. However, GA may also regulate other cell signaling pathways that are related to melanin biosynthesis. Interestingly, a structural subunit of GA, namely, gallic acid, has been reported to down-regulate melanin production associated pathways including PI3K/AKT and mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) [31]. Therefore, it is possible that GA inhibits melanin biosynthesis via multiple mechanisms but further studies would be required to confirm this. In summary, our findings suggest that GA, the major phenolic in M ­ aplifa™, could reduce the production of melanin in B16F10 cells. However, apart from GA, other GCGs, including GB and GC also reduced melanin biosynthesis (see Supplementary Material; Fig. S3). Therefore, although GA is the major constituent present in Maplifa™ largely contributing to its anti-melanogenic effects, it is possible that the other minor phenolic constituents (including GB and GC) could work in additive, synergistic and/or complementary effects towards the biological effects of the whole extract. Therefore, the current study warrants further exploration of ­Maplifa™ as a natural product derived extract for potential skin-whitening applications and/or as a therapeutic strategy for the treatment of hyperpigmentation. However, the efficacy and safety of ­Maplifa™ should be studied using in  vivo models, which will be pursued by our group in the future. Acknowledgements  HM was supported by the Omar Magnate Foundation Fellowship. The spectroscopic data were acquired from instruments located in the RI-INBRE core facility supported by Grant # P20GM103430 from the National Institute of General Medical Sciences of the National Institutes of Health. Compliance with ethical standards  Conflict of interest  HM and NPS are co-inventors on a patent application on the skin-whitening applications of maple gallotannins. The other authors declare no conflicts of interest.

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Funding  There is no funding source for this study. Ethical approval  This article does not contain any studies with human participants or animals performed by any of the authors.

References 1. Slominski A, Tobin DJ, Shibahara S, Wortsman J (2004) Melanin pigmentation in mammalian skin and its hormonal regulation. Physiol Rev 84:1155–1228 2. Costin G-E, Hearing VJ (2007) Human skin pigmentation: melanocytes modulate skin color in response to stress. FASEB J 21:976–994 3. Smit N, Vicanova J, Pavel S (2009) The hunt for natural skin whitening agents. Int J Mol Sci 10:5326–5349 4. Akazawa H, Akihisa T, Taguchi Y, Banno N, Yoneima R, Yasukawa K (2006) Melanogenesis inhibitory and free radical scavenging activities of diarylheptanoids and other phenolic compounds from the bark of Acer nikoense. Biol Pharm Bull 29:1970–1972 5. Akihisa T, Takeda A, Akazawa H, Kikuchi T, Yokokawa S, Ukiya M, Fukatsu M, Watanabe K (2012) Melanogenesisinhibitory and cytotoxic activities of diarylheptanoids from Acer nikoense bark and their derivatives. Chem Biodivers 9:1475–1489 6. Arnason T, Hebda RJ, Johns T (1981) Use of plants for food and medicine by native peoples of eastern Canada. Can J Botany 59:2189–2325 7. Royer M, Prado M, García-Pérez ME, Diouf PN, Stevanovic T (2013) Study of nutraceutical, nutricosmetics and cosmeceutical potentials of polyphenolic bark extracts from Canadian forest species. PharmaNutrition 1:158–167 8. Kwon BS (1993) Pigmentation genes: the tyrosinase gene family and the pmel 17 gene family. J Invest Dermatol 100:134S–140 9. Kobayashi T, Urabe K, Winder A, Jimenez-Cervantes C, Imokawa G, Brewington T, Solano F, Garcia-Borron J, Hearing V (1994) Tyrosinase related protein 1 (TRP1) functions as a DHICA oxidase in melanin biosynthesis. EMBO J 13:5818–5825 10. Yasumoto K-i, Yokoyama K, Shibata K, Tomita Y, Shibahara S (1994) Microphthalmia-associated transcription factor as a regulator for melanocyte-specific transcription of the human tyrosinase gene. Mol Cell Biol 14:8058–8070 11. Westerhof W, Kooyers T (2005) Hydroquinone and its analogues in dermatology—a potential health risk. J Cosmet Dermatol 4:55–59 12. Parvez S, Kang M, Chung HS, Cho C, Hong MC, Shin MK, Bae H (2006) Survey and mechanism of skin depigmenting and lightening agents. Phytother Res 20:921–934 13. González-Sarrías A, Li L, Seeram NP (2012) Effects of maple (Acer) plant part extracts on proliferation, apoptosis and cell cycle arrest of fuman tumorigenic and non-tumorigenic colon cells. Phytother Res 26:995–1002 14. González-Sarrías A, Ma H, Edmonds ME, Seeram NP (2013) Maple polyphenols, ginnalins A-C, induce S-and G2/M-cell cycle arrest in colon and breast cancer cells mediated by decreasing cyclins A and D1 levels. Food Chem 136:636–642 15. González-Sarrías A, Yuan T, Seeram NP (2012) Cytotoxicity and structure activity relationship studies of maplexins A-I, gallotannins from red maple (Acer rubrum). Food Chem Toxicol 50:1369–1376 16. Liu W, Wei Z, Ma H, Cai A, Liu Y, Sun J, DaSilva N, Johnson S, Kirschenbaum LJ, Cho B, Dain JA, Rowley DR, Shaikh ZA,

13

Arch Dermatol Res Seeram NP (2017) Anti-glycation and anti-oxidative effects of a phenolic-enriched maple syrup extract and its protective effects on normal human colon cells. Food Funct 8:757–766 17. Ma H, DaSilva NA, Liu W, Nahar PP, Wei Z, Liu Y, Pham PT, Crews R, Vattem DA, Slitt AL, Shaikh ZA, Seeram NP (2016) Effects of a standardized phenolic-enriched maple syrup extract on β-amyloid aggregation, neuroinflammation in microglial and neuronal cells, and β-amyloid induced neurotoxicity in Caenorhabditis elegans. Neurochem Res 41:2836–2847 18. Ma H, Liu W, Frost L, Kirschenbaum LJ, Dain JA, Seeram NP (2016) Glucitol-core containing gallotannins inhibit the formation of advanced glycation end-products mediated by their antioxidant potential. Food Funct 5:2213–2222 19. Ma H, Wang L, Niesen DB, Cai A, Cho BP, Tan W, Gu Q, Xu J, Seeram NP (2015) Structure activity related, mechanistic, and modeling studies of gallotannins containing a glucitol-core and α-glucosidase. RSC Adv 130:107904–107915 20. Muhsinah AB, Ma H, DaSilva NA, Tuan T, Seeram NP (2017) Bioactive glucitol-core containing gallotannins and other phytochemicals from silver maple (Acer saccharinum) leaves. Nat Prod Commun 12:83–84 21. Yuan T, Wan C, Liu K, Seeram NP (2012) New maplexins F-I and phenolic glycosides from red maple (Acer rubrum) bark. Tetrahedron 68:959–964 22. Zhang Y, Ma H, Yuan T, Seeram NP (2015) Red maple (Acer rubrum) aerial parts as a source of bioactive phenolics. Nat Prod Commun 10:1409–1412 23. Wan C, Yuan T, Li L, Kandhi V, Cech NB, Xie M, Seeram NP (2012) Maplexins, new α-glucosidase inhibitors from red maple (Acer rubrum) stems. Bioorg Med Chem Lett 22:597–600 24. Deering RW, Chen J, Sun J, Ma H, Dubert J, Barja JL, Seeram NP, Wang H, Rowley DC (2016) N-acyl dehydrotyrosines, tyrosinase inhibitors from the marine bacterium Thalassotalea sp. PP2-459. J Nat Prod 79:447–450 25. Noh J-M, Lee Y-S (2011) Inhibitory activities of hydroxyphenolic acid-amino acid conjugates on tyrosinase. Food Chem 125:953–957 26. Chou TH, Ding HY, Hung WJ, Liang CH (2010) Antioxidative characteristics and inhibition of α-melanocyte-stimulating hormone-stimulated melanogenesis of vanillin and vanillic acid from Origanum vulgare. Exp Dermatol 19:742–750 27. Kubo I, Kinst-Hori I, Chaudhuri SK, Kubo Y, Sánchez Y, Ogura T (2000) Flavonols from Heterotheca inuloides: tyrosinase inhibitory activity and structural criteria. Bioorg Med Chem 7:1749–1755 28. Curto EV, Kwong C, Hermersdörfer H, Glatt H, Santis C, Virador V, Hearing VJ, Dooley TP (1999) Inhibitors of mammalian melanocyte tyrosinase: in  vitro comparisons of alkyl esters of gentisic acid with other putative inhibitors. Biochem Pharmacol 57:663–672 29. Bi W, Gao Y, Shen J, He C, Liu H, Peng Y, Zhang C, Xiao P (2016) Traditional uses, phytochemistry, and pharmacology of the genus Acer (maple): a review. J Ethnopharmacol 189:31–60 30. Kamori A, Kato A, Miyawaki S, Koyama J, Nash RJ, Fleet GW, Miura D, Ishikawa F, Adachi I (2016) Dual action of acertannins as potential regulators of intracellular ceramide levels. Tetrahedron Asymmetr 27:1177–1185 31. Su TR, Lin JJ, Tsai CC, Huang TK, Yang ZY, Wu MO, Zheng YQ, Su CC, Wu YJ (2013) Inhibition of melanogenesis by gallic acid: possible involvement of the PI3K/Akt, MEK/ERK and Wnt/β-catenin signaling pathways in B16F10 cells. Int J Mol Sci 14:20443–20458