Melanogenesis inhibitory activity of Korean Undaria pinnatifida in mouse B16 melanoma cells

A number of seaweed species are used as traditional foods and medicine in different parts of the world, including Asian countries. However, very few data on the anti-melanogenic effect of seaweed have been published. Undaria pinnatifida (Dolmiyeok), a brown alga, is a traditional food in Jeju Island, the southern regions of the Korea peninsula. In this study, ethylacetate extracts of U. pinnatifida (UPE) were examined for their anti-melanogenic potentials. Our results supports the finding that UPE down-regulated melanin content in a dose-dependent pattern. To clarify the target of UPE action in melanogenesis, we performed Western blotting for tyrosinase and microphthalmia-associated transcription factor (MITF), which are key melanogenic enzymes. UPE inhibited tyrosinase and MITF expressions in a dose-dependent manner. These results indicate that treatment with UPE significantly inhibits the melanogenesis in B16 cells, and may be effective in the whitening agent for the skin.


Introduction
There are many traditional foodstuffs in Korea. Undaria pinnatifida, called "Dolmiyeok or Miyeok" is one of them and is considered to be a healthy foodstuff in Korea. It is a very large brown alga, golden-brown in colour, related to the Laminaria species and other kelps. Adult specimens can grow to an overall length of between 1.5 and 3 m in less than a year -a rate of growth of up to one centimetre per day. Undaria pinnatifida is widely distributed along the coasts of Korea, Japan, China and Russia. Considering the traditional concept, several studies have focused on the beneficial effects of U. pinnatifida on antiinflammatory (Khan et al., 2009;Yoo et al., 2012), antioxidant (Han et al., 2004;Hu et al., 2010;Moreira et al., 2010), antihypertensive (Suetsuna et al., 2004), antiviral (Thompson & Dragar, 2004;Hayashi et al., 2013), anticoagulating , and antiobesity (Okada et al., A variety of seaweeds provide an interesting, largely unexplored source for the development of potential new drugs and skin-care cosmetics (Yang et al., 2010a;b;d;Yoon et al., 2010a;d). They have existed from antiquity to the present and have played significant roles in skin health and drug discovery, especially anti-inflammatory agents against skin diseases (Moon et al., 2011, Yang et al., 2010cYoon et al., 2010b). The seaweed extracts are also known to be inhibitors of melanin production, sometimes more potent than the classical inhibitors hydroquinone/ arbutin or kojic acid, and not associated with side effects. Therefore, the present study focused on whether the ethyl acetate fraction from U. pinnatifida (UPE) inhibited melanin production and melanogenic protein expression in mouse B16 melanoma cells.

Materials and solvent extraction
U. pinnatifida specimens were collected in April 2010 from Gapa Island, Korea. The specimen voucher (no. CSC-201) is deposited with Cosmetic Science Center, Department of Chemistry, Jeju National University, frozen and stored at -20 °C until use. For extraction, the material was first ground into a fine powder and freezedried using a vacuum freeze-dryer. The dried powder (90 g) was extracted with 80% ethanol (EtOH; 2 L) at room temperature for 24 h and then evaporated under vacuum. The evaporated EtOH extract (5 g) was suspended in water (1 L) and fractionated with ethyl acetate (EtOAc; 500 mL). The yield and recovery of EtOAc fractions were 0.6535 g and 13.1%, respectively.

Cell viability assay
Cell viability assay was measured as described previously, with slight modification (Yoon et al., 2010c). Cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay. Cells were seeded on 96-well plates, and drug treatment began 18 h after seeding, B16F10 murine melanoma cells were incubated with various concentrations of UPE for 72 h at 37 °C in a humidified 95% air and 5% CO 2 atmosphere. MTT (1 mg/mL in phosphate-buffer saline, PBS) was added to each well in a 1/10 volume of medium. Cells were incubated at 37 °C for 4 h. Finally, the supernatant was removed and the formazan crystals were dissolved in DMSO. Absorbance was measured at 570 nm. Percent of cells showing cytotoxicity was determined relative to the control group.

Measurement of melanin contents
Extracellular melanin release was measured as described previously, with slight modification (Yoon et al., 2010c). Cells of the murine melanoma cell line, B16F10, plated at 1.0 × 10 5 cells/mL, were stimulated with α-MSH (50 nM) and then incubated with aliquots of UPE (3.125, 6.25 and 12.5 μg/mL) at 37 °C for 72 h; the cells were then washed in ice-cold phosphate-buffered saline. Briefly, the samples were incubated at 80 °C for 1 h in 1 mL of 1 N NaOH/10% DMSO and then vortexed to solubilize the melanin; the absorbance at 405 nm was then measured. Further, the melanin content was determined based on the absorbance/μg of protein in the extract from each cell. The protein concentration of the cells was determined by using a protein assay kit (Pierce, Rockford, IL, USA).

Determination of cellular tyrosinase activity
Cellular tyrosinase activity was measured by the method of Yen et al. with some modification (Yen et al., 2012). Briefly, the culture method for determining cellular tyrosinase assay was similar to that for determining melanin content. After treatment with different concentrations of UPE for 72 h, the cells were collected after treatment with trypsin-EDTA and centrifuged at 15,000 rpm for 15 min to obtain cell pellets. The pellet solutions were frozen and thawed twice and then centrifuged at 15,000 rpm for 15 min. We added 80 μL of the supernatant in a 96-well plate and mixed it with 20 μL of 0.2% L-DOPA. After incubation for 1 h, the optical densities were measured at 475 nm using a microplate spectrophotometer. The inhibitory activity of the UPE treated cells was presented as percentage against that of the untreated cells.

Measurement of tyrosinase and MITF in melanoma B16/F10 cells by Western blot
To determine the amount of tyrosinase and MITF protein, Western blotting analysis was performed. B16 melanoma cells that had been stimulated by α-MSH (50 nM) were treated with UPE (3.125, 6.25 and 12.5 μg/mL) for 3 days. After treatment, the cells were collected and lysed with cell lysis buffer [50 mM Tris-HCl (pH 6.8), 2% SDS, 6% mercaptoethanol, 1% glycerol]. Whole cell lysates (5 × 10 4 cells equivalents per lane) were separated by 7.5% SDSpolyacrylamide gel electrophoresis as described previously and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% skimmed milk in phosphate-buffered saline containing 0.05% Tween 20. Tyrosinase and MITF bands were detected with rabbit polyclonal anti-tyrosinase antibody (dilution 1:1000) and rabbit polyclonal anti-MITF antibody (dilution 1:500), respectively, purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and then further incubated with horseradish peroxidase-conjugated antirabbit IgG antibody at a 1:5000 dilution. Bound antibodies were detected using an enhanced chemiluminescence kit (Amersham Biosciences, Buckinghamshire, UK), following the manufacturer's instructions. Loading control was assessed using anti-β-actin antibody. Positive bands were analysed using a gel image analysis instrument.

Results and discussion
Melanin plays a crucial role in protecting the skin against harmful ultraviolet light, but overproduction and accumulation of melanin could create serious skin problems such as freckles, age pigment, and melasma. Thus, the inhibition of melanogenesis has been the focus on medicinal and cosmetic treatments for skin depigmenting and lightening. Therefore, this study focused on whether the ethyl acetate fraction from UPE inhibited melanin production and melanogenic protein expression in mouse B16 melanoma cells. In the present study, the changes in the melanin contents in the cells treated with UPE were evaluated for anti-melanogenesis activity. The melanin contents of cells were significantly attenuated by UPE in a dose-dependent manner ( Figure 1A). In spite of a number of studies reporting on anti-melanogenic agents, such as hydroquinone, kojic acid, and arbutin, sometimes side effects such as irritation of the skin and exhibition of cell toxicity were observed. Therefore it is necessary to find potent natural products that act as antimelanogenic agents without side effects. To investigate the cytotoxicity of UPE on cell proliferation, B16 murine melanoma cells were treated with various concentrations (3.125-25 μg/mL) of UPE for 72 h. As shown in Figure 1B, there was no significant difference in cell proliferation between control and UPE-treated cells until 12.5 μg/mL, suggesting that the inhibitory effects of UPE on melanin biosynthesis were not attributable to its cytotoxicity. Since cellular tyrosinase activity is also the major factor that stimulates melanin synthesis and ultimately induces melanogenesis, we determined to assess cellular tyrosinase activity for investigating the antimelanogenesis activity of UPE on B16 murine melanoma cells. B16 murine melanoma cells were pretreated with UPE at doses of 3.125-12.5 μg/mL. UPE treatment significantly reduced the cellular tyrosinase activity in a dose-dependent manner compared to the control (Figure 2).
Human melanocytes are known to express tyrosinase and MITF is a factor that effectively transactivates tyrosinase. The expression of these proteins was evaluated using /mL) were preincubated for 18 h and the melanin content was assayed after incubation of the B16F10 cells treated with α-MSH (100 nM), melasolv (40 μM), and UPE (3.125, 6.25 and 12.5 μg/mL) for 72 h at 37 °C in a 5% CO 2 atmosphere. The absorbance was measured at 405 nm by ELISA. MTT assay was performed after incubation of the B16F10 cells treated with varying concentrations of UPE (3.125, 6.25 and 12.5 μg/mL) for 24 h at 37 °C in a 5% CO 2 atmosphere. The absorbance was measured at 570 nm with a spectrophotometer (Power Wave; Bio-tek, Winooski, VT). Values are the mean ± SEM of triplicate experiments. *p<0.05; **p<0.01.
Western blot analysis. As compared with the untreated control values, UPE-treated cells showed dose-dependent decreases in tyrosinase and MITF expression (Figure 3).
In summary, we investigated the anti-melanogenic effects of UPE and related melanogenic activity. The present results suggest that MITF protein levels are reduced by UPE. The hypopigmentation effect of UPE may be the result of down-regulation of MITF gene expression, which would then repress the protein and gene expressions of tyrosinase. Therefore we suggest that UPE can be a useful inhibitor of melanogenesis and has beneficial effects in the treatment of hyperpigmentation disorders such as ephelis and oedema. However, the inhibitory mechanism of melanin production in B16 murine melanoma cells by UPE remains unclear. The investigation of the exact mechanisms and further in vivo experiments are needed to evaluate the possible use of UPE as a natural skinwhitening agent.