Inonotus Obliquus extracts as an inhibitor of α-MSH-induced melanogenesis in B 16 F 10 mouse melanoma cells

Melanogenesis is a biosynthetic pathway for producing of the pigment melanin in human skin. Tyrosinase, a key enzyme, catalyzing is the first step in melanogenesis and the downregulation of the tyrosinase enzyme activity is the most reported method for anti-melanogenesis. According to the hyperpigmentation as an important issue in cosmetic industry, there is a big demand for melanogenesis inhibitors. In the present study, we identified the anti-melanogenic effect of Inonotus Obliquus in α-MSH-induced B16F10 mouse melanoma cells as a new inhibitor. Comparing with control and Inonotus Obliquus extracts treated B16F10, we identified melanin contents, tyrosinase activity, tyrosinase mRNA and protein expression, MITF activity using a constructed plasmid. As shown in these results, we demonstrated that Inonotus Obliquus extracts down-regulated melanin synthesis using down-regulating activity and expression of tyrosinase which is key enzyme to produce melanin. In addition, we revealed expression of tyrosinase is regulated by MITF through repressing MITF transcriptional activity. Inonotus Obliquus extracts has potential to repress melanogenesis and decreased of hyperpigmentation and to use as cosmetic ingredient.


Introduction
The skin has epidermal units that are composed of keratinocytes and melanocytes.The melanocytes are responsible for melanin production and distribution which process called melanogenesis [15].These units are composed of a melanocytes surrounded by keratinocytes and regulated by a closed paracrine system [18].Melanin is the primary determinant of skin, hair and eye color [16].Melanogenesis is a complex process with different stages [14].When the melanin is abnormal deposited, it may cause different types of pigmentation disorders, which are classified as hypo or hyperpigmentation [17].Melanocytes produce melanin via melanogenesis and controlled by tyrosinase [20].The reason that tyrosinase activity is important on skin-pigmentation.Since this enzyme has an important impact on skin color or pigmentation defects treatment, cosmetic and pharmaceutical industries have been continuously seeking new candidates of ingredient regulating tyrosinase activity [5,6].New tyrosinase inhibitors are continually required to deal with melanogenesis caused problems effectively with a few side effects.Therefore, the effects of various natural source extracts on human skin whitening and anti-melanogenesis have drawn significant attention, and numerous studies have been conducted to verify their benefits in cosmetic formulations [8,9].
In this study, we found that the Inonotus Obliquus extracts as a potent melanogenesis inhibitors by decreasing the activity of tyrosinase, a key enzyme in the synthetic pathway of melanin.Inonotus Obliquus are commonly known as Chaga mushroom, which used to natural medicine in many other countries such as Japan, China, Russia and Baltics [1].Inonotus Obliquus extracts have been evaluated as a traditional and natural source of bioactive compounds over many centuries and have recently been used for potential components in the cosmetic and pharmaceutical industries.Inonotus Obliquus extracts have been reported to have numerous physiological functions such effects on anticancer, homeostasis, anti-virus and antioxidant actions in in vitro and in vivo models [2,3,7,10,11].We paid attention to melanogenesis as one of the new physiological functions of Inonotus Obliquus extracts.
In the present study, Inonotus Obliquus extracts evaluated its anti-melanogenesis effects by monitoring the melanin contents, intracellular tyrosinase activity and MITF expression in α-MSHinduced melanogenesis in B16F10 mouse melanoma cells.

Measurement of melanin content
B16F10 cells were harvested 48 h after treatment, washed with PBS and lysed in 1 N sodium hydroxide (NaOH; Sigma-Aldrich) at 95 ℃ for 15 min.Melanin content was determined by absorbance at 450 nm using a Microplate Reader (SpectraMax ® i3x; Molecular Deviced, USA).The amount of melanin was calculated using a melanin standard curve.The protein concentration of each lysate was determined by Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions.The cellular melanin content was normalized to total protein concentration.The B16F10 cells were washed with PBS and lysed with lysis buffer containing 1% Triton X-100 (Biopure), 150 mM NaCl (Biopure), 50 mM HEPES (pH 7.5, Biopure) and 5 mM EDTA (Biopure).The supernatant was separated from the cell lysate by centrifugation and mixed with 2 mM L-DOPA(Sigma-Aldrich) in 0.1 M sodium phosphate buffer (pH 7.4) solution.After 30 min incubation at 37℃, the absorbance at 450 nm was read using a Microplate Reader (SpectraMax ® i3x; Molecular Deviced, USA).The protein concentration of each lysate was determined by Bradford protein assay (Thermo Fisher Scientific, USA) according to the manufacturer's instructions.The activity of intracellular tyrosinase was normalized to total protein concentration.

Expression of tyrosinase mRNA
Total RNA was isolated using TRIzol TM (Invitrogen, Thermo Fisher Scientific) according to the manufacturer's protocol.The purity and concentration of the RNA was evaluated based on using MaestroNano®, a micro volume spectrophotometer (Maestrogen Inc., USA).The cDNAs were synthesized from total RNAs by using the miScript Reverse Transcription kit (Qiagen) according to the manufacturer's protocol.Quantitative real-time PCR was performed using StepOnePlus TM Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific).Tyrosinase mRNA expression was performed using the following Tyrosinase specific primers: Tyrosinase forward 5'-CAAGTACAGGGATCGGCCAAC-3'; Tyrosinase reverse 5'-GGTGCATTGGCTTCTGGGTAA-3'.PCR was performed using the HOT FIREPol EvaGreen® qPCR Mix Plus (ROX).The expression of mRNA was analyzed by 2 -ΔΔCT calculate method and nomalized with β-actin.

MITF transcription activity
To determined transcription activity of MITF when B16F10 cells were treated with Inonotus Obliquus extracts.B16F10 melanoma cells were transfected with pGL3 (Invitrogen) and pGL3-MITF plasmids using Lipofectamine 2000 (Invitrogen) and co-transfected with the pCMV-β-galactosidase plasmid to normalize the transfection efficiency.After incubation for 24h, the transfected cells were treated with Inonotus Obliquus extracts.After treatment, the cells were lysed at Passive Lysis Buffer (Promega Corp., USA).The luciferase activity was determined by Veritas TM Microplate Luminometer (Veritas, USA).The results are the averages of three independent experiments.

Statistical analysis.
All data were presented as means ± standard deviation of different measurements obtained by three independent experiments at least.Significance of the data was estimated by student's t-test.p value < 0.05 was considered statistically significant.(p values: * < 0.05).We first examined whether Inonotus Obliquus is cytotoxic to B16F10 melanoma cells using MTT assay.B16F10 cells were treated with Inonotus Obliquus extracts at indicated concentration respectively and measured 48 h later.Treatment with Inonotus Obliquus extracts caused no significant change in cell viability at final concentrations of 100 μg/mL, but markedly reduced it at 200 μg/mL as revealed by short-term (48 h) cell viability assay (Figure 1).Therefore, the following experiments about Inonotus Obliquus extracts were performed within the concentration that have no cytotoxicity.

Inonotus Obliquus extracts decreased melanin contents in the B16F10 mouse melanoma cells
To measure of melanin contents, after the treatment with the α-MSH (200 ng/ml) and Inonotus Obliquus extracts simultaneously, and thereafter compared to α-MSH as a negative control (Figure 2).The Inonotus Obliquus extracts dose-dependently reduced the melanin contents of the B16F10 cells at the concentrations that did not influence the cell viability.The Inonotus Obliquus extracts (100 μg/mL) was found to have a greater inhibitory effect on melanin contents in the cells.

Inonotus Obliquus extracts directly inhibit the tyrosinase activity
Tyrosinase is a rate-limiting enzyme in melanin synthesis.Many melanin synthesis inhibitors reduce melanogenesis by directly inhibiting the tyrosinase activity.We examined the direct effect of Inonotus Obliquus extracts on tyrosinase activity by using mushroom tyrosinase, because the inhibitory effect of the Inonotus Obliquus extracts on melanin synthesis was remarkable.Figure 3A shows the change in cellular tyrosinase activity when the Obliquus extracts treated in the range of 0-100 μg/mL on the α-MSH treated B16F10 mouse melanoma cells.The tyrosinase activity was dose-dependently decreased when adding the Inonotus Obliquus extracts on the B16F10 mouse melanoma cells compared with the control.The mushroom tyrosinase also decreased at concentration of 100 μg/mL Inonotus Obliquus extracts.These results indicate that the inhibitory effect of the Inonotus Obliquus extracts on melanin synthesis was related to the direct inhibitory effect on tyrosinase activity.

Inonotus Obliquus extracts downregulate α-MSH-induced tyrosinase expression
To evaluate the effect of Inonotus Obliquus extracts on tyrosinase expression, B16F10 cells were pre-treated with Inonotus Obliquus extracts before stimulation with α-MSH and tyrosinase protein levels were examined using western blot analysis.Pre-treatment with Inonotus Obliquus extracts at the 100 and 200 μg/mL concentrations dose-dependently inhibited the α-MSH-induced accumulation of tyrosinase proteins (Figure 4A).RT-PCR analysis shows similar results in 50 and 100 μg/mL concentrations (Figure 4B).These data demonstrated that Inonotus Obliquus extracts significantly decreased α-MSH-induced tyrosinase expression at the mRNA level without eliciting cytotoxic effect in B16F10 melanoma cells.

Inonotus Obliquus extracts
To determine whether Inonotus Obliquus extracts affect the MITF promoter activity and if so, which regulatory region is required for Inonotus Obliquus extracts mediated suppression of the MITF promoter expression, we constructed a series of reporter plasmid containing a MITF gene promoter.Luciferase reporter activities of the MITF region constructs were increased by α-MSH stimulation, which was respectably reduced by treated to Inonotus Obliquus extracts (Figure 5).These data suggest that Inonotus Obliquus extracts downregulate MITF expression at the promoter level and that the Inonotus Obliquus extracts response element responsible for the suppression of the MITF transcriptional level.

Discussion
Melanogenesis is a biosynthetic pathway for producing the pigment melanin in for the most of colors human has such as eyes, hair, skin [15].Melanogenesis is a complex process with different steps.A key enzyme, tyrosinase, catalyzes the first step of melanogenesis and the down-regulation of the enzyme activity is the most investigated method for the inhibition of melanogenesis [16].According to the hyperpigmentation as an important issue in cosmetic industry, there is a big demand for melanogenesis inhibitors.[14].
There are many requirements for cosmetics products, but the most important that they should be use safety with no side effects and have positive effects on the skin.Recently, natural products have attracted extensive attention in cosmetic industry.There are numerous products from natural source such as foods, fruits, plants that are being exploited in pharmaceutical or cosmetics, and many potential products are yet to be used [8][9][12][13].Inonotus Obliquus is one of the medicinal mushrooms and also use in traditional oriental therapy and several nutrient functional foods [1,13].Inonotus Obliquus extracts have been reported to have physiological functions such effects on anticancer, homeostasis, anti-virus and antioxidant actions [2][3]7,[10][11].In this study, we paid attention to antimelanogenesis as one of the new physiological functions of Inonotus Obliquus extracts.Over the last years, understanding of melanocyte biology and the melanin synthesis processes have made remarkable progress as increased interest in whitening and hyperpigmentation, opening paths in the discovery of new melanogenesis inhibitors [5].Many other approaches to inhibition of melanogenesis include accelerating the tyrosinase degradation and inhibiting tyrosinase mRNA transcription through a reduction of MITF activity in addition to the direct inhibition of tyrosinase activity.
We investigated the effects of Inonotus Obliquus extracts on melanogenesis by using mushroom tyrosinase in a cell-free system and cellular tyrosinase in cultured B16F10 melanoma cells with α-MSH as a negative control.In advance of following experiments, we measured melanin contents.The melanin contents in the Inonotus Obliquus extracts treated cells was significantly reduced, and then we examined the effect of the Inonotus Obliquus extracts on the cellular tyrosinase level.B16F10 cells treated with the Inonotus Obliquus extracts showed the cellular tyrosinase activity to be significantly decreased as well.When using mushroom tyrosinase, the inhibition effect of the Inonotus Obliquus extracts was also considerable.These results indicate that the Inonotus Obliquus extracts significantly inhibited not only melanin production but also intracellular tyrosinase activity in α-MSH-induced B16F10 mouse melanoma cells.The results of the western blot and the real-time RT-PCR analysis indicated that the tyrosinase protein and mRNA levels decreased by the Inonotus Obliquus extracts.We suggest that the Inonotus Obliquus extracts inhibited melanogenesis at the transcriptional level and also directly inhibited tyrosinase activity.Tyrosinase gene expression is regulated by the MITF [20].MITF (microphthalmia-associated transcription factor) is a melanocyte-specific transcription factor and can regulate biological process in melanoma and melanocytes cells including pigmentation, proliferation and survival [21].MITF binds and activated melanogenic gene, tyrosinase, promoters, thereby increasing their expression, which results in increased melanin synthesis.To determine whether Inonotus Obliquus effects on MITF activity, we constructed plasmid that has MITF promoter region and performed Luciferase assay using the plasmid.In this experiment, Inonotus Obliquus extracts also decreased MITF expression in a concentration-dependent manner.Thus, we expected that anti-melanogenesis effect of Inonotus Obliquus extracts was contributed by a mechanism related to MITF expression.
In conclusion, it is suggested that the Inonotus Obliquus extracts was a melanogenesis inhibitor in B16F10 mouse melanoma cells which stimulated α-MSH and that the mechanism might include decreasing of intracellular tyrosinase at the mRNA level by inhibiting MITF transcriptional activity.Taken together, Inonotus Obliquus extracts may be useful in anti-melanogenic ingredient for therapeutic treatment of hyperpigmentation and whitening cosmetic products as a natural source.

Figure 1 .
Figure 1.Cytotoxicity of extracts of Inonotus Obliquus in B16F10 mouse melanoma cells.Effects of Inonotus Obliquus extracts on the cell viability.There is no significant difference between control and Inonotus Obliquus extracts treated cells.Cytotoxicity was exhibited as a percentage of control at the indicated concentrations.Values are M±S.D. from triplicate experiments.M±S.D., mean±standard deviation.

Figure 2 .
Figure 2. Effects of Inonotus Obliquus extracts on melanin contents in B16F10 mouse melanoma cells.Inonotus Obliquus extracts decreased melanin contents in B16F10 mouse melanoma cells.Melanin contents were exhibited as a percentage of control at the indicated concentrations.Values are M±S.D. from triplicate experiments.#p <0.05 compared with non-treated cells; *p <0.05 compared with α-MSH-treated cells; IO

Figure 4 .
Figure 4. Effects of Inonotus Obliquus extracts on tyrosinase expression.Inonotus Obliquus extracts decreased tyrosinase expression in B16F10 mouse melanoma cells.(A) Protein level of tyrosinase, (B) mRNA level of tyrosinase.mRNA level of tyrosinase were exhibited as a percentage of control at the indicated concentrations.Values are M±S.D. from triplicate experiments.#p<0.05 compared with non-treated cells; *p<0.05 compared with α-MSH-treated cells; IO ext., Inonotus Obliquus extract extracts; α-MSH, melanocyte-stimulating hormone; M±S.D., mean±standard deviation.