Pseudoalteromone A, a Ubiquinone Derivative from Marine Pseudoalteromonas spp., Suppresses Melanogenesis

An ubiquinone derivative, pseudoalteromone A (1), has been isolated from two marine-derived Pseudoalteromonas spp., APmarine002 and ROA-050, and its anti-melanogenesis activity was investigated. The anti-melanogenic capacity of pseudoalteromone A was demonstrated by assessing the intracellular and extracellular melanin content and cellular tyrosinase activity in the B16 cell line, Melan-a mouse melanocyte cell line, and MNT-1 human malignant melanoma cell line. Treatment with pseudoalteromone A (40 μg/mL) for 72 h reduced α-melanocyte-stimulating hormone (α-MSH)-induced intracellular melanin production by up to 44.68% in B16 cells and 38.24% in MNT-1 cells. Notably, pseudoalteromone A induced a concentration-dependent reduction in cellular tyrosinase activity in B16 cell, and Western blot analyses showed that this inhibitory activity was associated with a significant decrease in protein levels of tyrosinase and tyrosinase-related protein 1 (Tyrp-1), suggesting that pseudoalteromone A exerts its anti-melanogenesis activity through effects on melanogenic genes. We further evaluated the skin-whitening effect of pseudoalteromone A in the three-dimensional (3D) pigmented-epidermis model, MelanoDerm, and visualized the 3D distribution of melanin by two-photon excited fluorescence imaging in this human skin equivalent. Collectively, our findings suggest that pseudoalteromone A inhibits tyrosinase activity and expression and that this accounts for its anti-melanogenic effects in melanocytes.


Introduction
There is little doubt that UV-radiation affects all people in their daily life. Harmful effects of solar radiation cause skin damage and skin aging, but the body counters these harmful influences through the production of the pigment, melanin [1,2]. Despite the considerable virtue of melanin, irregular production of this pigment underlies several skin diseases and conditions, including melasma, leukoplakia, lentigo, albinism, moles, and freckles [3]. To address the consequences of abnormal melanogenesis processes, numerous researchers have investigated melanogenesis mechanisms. The shared mechanism of melanin production is initiated as an oxidative progress in which tyrosine is converted first to 1-3,4-dihydroxyphenylalanine (DOPA), and then to dopaquinone, dopachrome and finally the pigment melanin, primarily by tyrosinase and related enzyme systems [4]. Given that catalysis is central to the oxidative action of tyrosinase, the capacity to suppress tyrosinase activity and expression is important for anti-melanogenesis activity. A number of medications that possess this capacity, such as kojic acid, 4-n-butylresorcinol, hydroquinone and tretinoin, are currently available [5]. Although these synthetic agents are very effective, they are accompanied by undesirable side-effects. For example, hydroquinone causes blistering, skin cracking, and dryness in individuals with sensitive skin. Therefore, recent research on anti-melanogenesis in the cosmetic industry has focused on the development of novel, natural sources-derived agents that are free from adverse effects.
Marine microorganisms are known to produce many bioactive molecules, with additional secondary metabolites exhibiting significant activities in various molecular and biochemical pathways [6]. In our continuing research effort to discover anti-melanogenic agents from marine microorganisms for use as constituents of cosmetic products, we have focused on screening crude extracts from libraries of cultures produced by dozens of marine microorganisms. In the process, we discovered that crude extracts of Pseudoalteromonas spp., APma-rine002 and ROA-050, exhibit moderate inhibitory activity on melanogenesis in α-melanocytestimulating hormone (α-MSH) stimulated B16 melanoma cells. Using assay-guided purification of crude extracts of APmarine002 and ROA-050 based on anti-melanogenic activity, we identified the pseudoalteromone A (1) and p-coumaric acid as such anti-melanogenic agents ( Figure 1). Pseudoalteromone A, first isolated from Pseudoalteromonas sp. CGH2XX and subsequently isolated from two other Pseudoalteromonas spp., Pseudoalteromonas rubra QD1-2 and Pseudoalteromonas sp. P1-9 [7,8], has been reported to exhibit cytotoxicity against MOLT-4 human acute lymphoblastic leukemia cells and inhibit the release of elastase by human neutrophils [9]. However, anti-melanogenic effects of pseudoalteromone A have not been previously reported. Here, we describe the inhibitory effects of pseudoalteromone A on melanogenesis both in vitro and in a 3D pigmented-epidermis model (MelanoDerm).
Marine microorganisms are known to produce many tional secondary metabolites exhibiting significant activi ochemical pathways [6]. In our continuing research effo agents from marine microorganisms for use as constituen focused on screening crude extracts from libraries of cult rine microorganisms. In the process, we discovered th omonas spp., APmarine002 and ROA-050, exhibit modera genesis in α-melanocyte-stimulating hormone (α-MSH) Using assay-guided purification of crude extracts of APm anti-melanogenic activity, we identified the pseudoaltero as such anti-melanogenic agents ( Figure 1). Pseudoaltero doalteromonas sp. CGH2XX and subsequently isolated fr spp., Pseudoalteromonas rubra QD1-2 and Pseudoalteromo ported to exhibit cytotoxicity against MOLT-4 human ac and inhibit the release of elastase by human neutrophils effects of pseudoalteromone A have not been previously inhibitory effects of pseudoalteromone A on melanogene mented-epidermis model (MelanoDerm).

Identification of pseudoalteromone A
A liquid chromatography-mass spectrometry (LC-M culture broths of APmarine002 and ROA-050 revealed th ric acid, which has been reported to exhibit anti-melano tion of tyrosinase activity [10]. Interestingly, we found t rate from the polar fraction containing p-coumaric acidalso exerted potent inhibitory activity on melanogenesis. tigated the chemical components of the non-polar fractio ural products with the observed anti-melanogenic efficac of crude extracts of culture broths of APmarine002 and strains exhibited a peak with a retention time of 11.65 mi

Identification of Pseudoalteromone A
A liquid chromatography-mass spectrometry (LC-MS) analysis of crude extracts of culture broths of APmarine002 and ROA-050 revealed that both strains produce p-coumaric acid, which has been reported to exhibit anti-melanogenic effects through the inhibition of tyrosinase activity [10]. Interestingly, we found that the non-polar fraction, separate from the polar fraction containing p-coumaric acid-based anti-melanogenic activity, also exerted potent inhibitory activity on melanogenesis. Therefore, we intensively investigated the chemical components of the non-polar fraction to identify the distinctive natural products with the observed anti-melanogenic efficacy. The expanded LC-MS analysis of crude extracts of culture broths of APmarine002 and ROA-050 also revealed that both strains exhibited a peak with a retention time of 11.65 min and each peak showed the m/z 343 [M + Na] + in the MS spectra (Figures 2 and 3).
Assay-guided isolation of the extract from large-scale cultures of ROA-050 yielded compound 1. The 1 H nuclear magnetic resonance (NMR) spectrum (CD 3    A comparison of 1 H, 13 C NMR and MS spectra with previously reported data [9] identified compound 1 as pseudoalteromone A (Figures S1 & S2).

Anti-Melanogenic Efficacy of pseudoalteromone A in B16, Melan-a and MNT-1 Cells
Before assessing the anti-melanogenic efficacy of pseudoalteromone A, we measured its cytotoxicity towards B16 cells using CCK-8 assays. B16 cells were cultured for 24 h and then treated with pseudoalteromone A at concentrations of 5-40 μg/mL for 72 h. As shown in Figure 4A, pseudoalteromone A was not cytotoxic at concentrations of 5-10 μg/mL. Accordingly, we used 2.5-10 μg/mL pseudoalteromone A in the subsequent experiments.
The maximum cytotoxicity-free concentration of pseudoalteromone A in B16 cells was 10 μg/mL. Thus, to assess the effect of pseudoalteromone A on the melanogenesis in B16 cells, we co-treated α-MSH-stimulated cells with pseudoalteromone A at concentrations of 2.5-10 μg/mL. After 72 h, we confirmed the increased production of α-MSH-    A comparison of 1 H, 13 C NMR and MS spectra with previously reported data [9] identified compound 1 as pseudoalteromone A (Figures S1 & S2).

Anti-Melanogenic Efficacy of pseudoalteromone A in B16, Melan-a and MNT-1 Cells
Before assessing the anti-melanogenic efficacy of pseudoalteromone A, we measured its cytotoxicity towards B16 cells using CCK-8 assays. B16 cells were cultured for 24 h and then treated with pseudoalteromone A at concentrations of 5-40 μg/mL for 72 h. As shown in Figure 4A, pseudoalteromone A was not cytotoxic at concentrations of 5-10 μg/mL. Accordingly, we used 2.5-10 μg/mL pseudoalteromone A in the subsequent experiments.
The maximum cytotoxicity-free concentration of pseudoalteromone A in B16 cells was 10 μg/mL. Thus, to assess the effect of pseudoalteromone A on the melanogenesis in B16 cells, we co-treated α-MSH-stimulated cells with pseudoalteromone A at concentrations of 2.5-10 μg/mL. After 72 h, we confirmed the increased production of α-MSHstimulated cells compared with controls that were not treated with α-MSH and showed A comparison of 1 H, 13 C NMR and MS spectra with previously reported data [9] identified compound 1 as pseudoalteromone A (Figures S1 and S2).

Anti-Melanogenic Efficacy of Pseudoalteromone A in B16, Melan-a and MNT-1 Cells
Before assessing the anti-melanogenic efficacy of pseudoalteromone A, we measured its cytotoxicity towards B16 cells using CCK-8 assays. B16 cells were cultured for 24 h and then treated with pseudoalteromone A at concentrations of 5-40 µg/mL for 72 h. As shown in Figure 4A, pseudoalteromone A was not cytotoxic at concentrations of 5-10 µg/mL. Accordingly, we used 2.5-10 µg/mL pseudoalteromone A in the subsequent experiments. pendent manner without causing cytotoxicity in either cell line. Specifically, treatment of Melan-a cells with pseudoalteromone A at a concentration of 5 μg/mL for 72 h reduced intracellular melanin content by 44.68 % ( Figure 4E), and at 40 μg/mL reduced melanin content in MNT-1 cells by 38.24 % after 72 h ( Figure 4G). Treatment with the positive control kojic acid at 25-100 μg/mL also decreased intracellular melanin content. However, these anti-melanogenic effects were lower than those of pseudoalteromone A under the same concentration conditions, indicating that pseudoalteromone A exerts a greater inhibitory effect than kojic acid in the present study. Results are presented as means ± SD, expressed as a percentage relative to the control group (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. control group). "-"means non-treated or without any treatment and "+" means treated with the indicated compound (α-MSH (0.1 µM)). ### p < 0.001.
The maximum cytotoxicity-free concentration of pseudoalteromone A in B16 cells was 10 µg/mL. Thus, to assess the effect of pseudoalteromone A on the melanogenesis in B16 cells, we co-treated α-MSH-stimulated cells with pseudoalteromone A at concentrations of Mar. Drugs 2021, 19, 612 5 of 12 2.5-10 µg/mL. After 72 h, we confirmed the increased production of α-MSH-stimulated cells compared with controls that were not treated with α-MSH and showed that both pseudoalteromone A and kojic acid, an anti-melanogenesis agent included as a positive control, inhibited, not only α-MSH-induced intracellular melanin synthesis ( Figure 4B), but also extracellular melanin levels ( Figure 4C) in B16 cells, effects that were concentration dependent. We further assessed the effects of pseudoalteromone A on the viability of a Melan-a mouse melanocyte cell and a MNT-1 human malignant melanoma cell as described for B16 cells, and found that pseudoalteromone A showed cytotoxicity toward Melan-a cells ( Figure 4D) beginning at a concentration of 10 µg/mL and against MNT-1 at 50 µg/mL ( Figure 4F). Therefore, for subsequent experiments, we selected 5 µg/mL and 40 µg/mL as sub-toxic concentrations of pseudoalteromone A for use in Melan-a cells and MNT-1 cells, respectively. Treatment of α-MSH-stimulated Melan-a cells and MNT-1 cells with pseudoalteromone A reduced intracellular melanin content in a concentrationdependent manner without causing cytotoxicity in either cell line. Specifically, treatment of Melan-a cells with pseudoalteromone A at a concentration of 5 µg/mL for 72 h reduced intracellular melanin content by 44.68% ( Figure 4E), and at 40 µg/mL reduced melanin content in MNT-1 cells by 38.24% after 72 h ( Figure 4G). Treatment with the positive control kojic acid at 25-100 µg/mL also decreased intracellular melanin content. However, these anti-melanogenic effects were lower than those of pseudoalteromone A under the same concentration conditions, indicating that pseudoalteromone A exerts a greater inhibitory effect than kojic acid in the present study.
The enzymatic activity tyrosinase is essential in the regulation of melanogenesis. Thus, inhibitory effect on this activity of the enzyme could explain the anti-melanogenic effect of pseudoalteromone A. To directly estimate the inhibitory effect of pseudoalteromone A on tyrosinase activity, we tested it against two different sources of tyrosinase: mushroom tyrosinase and cellular tyrosinase extracted from B16 cells. As shown in Figure 5A, pseudoalteromone A inhibited mushroom tyrosinase activity in a concentration-dependent manner. In this assay, we used L-tyrosine and L-3,4-dihydroxyphenylalanine (L-DOPA) as substrates to check both the tyrosine hydroxylase activity and DOPA oxidase activity. To more precisely examine the tyrosinase-inhibitory efficacy of pseudoalteromone A, we determined intracellular tyrosinase activity by measuring the oxidation rate of L-3,4dihydroxyphenylalanine (L-DOPA) to dopachrome in B16 cells incubated with or without pseudoalteromone A. This analysis showed that pseudoalteromone A inhibited cellular tyrosinase activity in a concentration-dependent manner ( Figure 5B). Results are presented as means ± SD, expressed as a percentage relative to the control group (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. control group). "-"means non-treated or without any treatment and "+" means treated with the indicated compound (α-MSH (0.1μM)). ### p < 0.001.
The enzymatic activity tyrosinase is essential in the regulation of melanogenesis. Thus, inhibitory effect on this activity of the enzyme could explain the anti-melanogenic effect of pseudoalteromone A. To directly estimate the inhibitory effect of pseudoalteromone A on tyrosinase activity, we tested it against two different sources of tyrosinase: mushroom tyrosinase and cellular tyrosinase extracted from B16 cells. As shown in Fig.  5A, pseudoalteromone A inhibited mushroom tyrosinase activity in a concentration-dependent manner. In this assay, we used L-tyrosine and L-3,4-dihydroxyphenylalanine (L-DOPA) as substrates to check both the tyrosine hydroxylase activity and DOPA oxidase activity. To more precisely examine the tyrosinase-inhibitory efficacy of pseudoalteromone A, we determined intracellular tyrosinase activity by measuring the oxidation rate of L-3,4-dihydroxyphenylalanine (L-DOPA) to dopachrome in B16 cells incubated with or without pseudoalteromone A. This analysis showed that pseudoalteromone A inhibited cellular tyrosinase activity in a concentration-dependent manner. ( Figure 5B) To investigate the effects of pseudoalteromone A on expression levels of melanogenesis-related protein, tyrosinase, Tyrp-1 and Tyrp-2, we performed Western blotting. This analysis showed that pseudoalteromone A decreased protein levels of tyrosinase and Tyrp-1 in B16 cells ( Figure 6A,B); it also dramatically inhibited expression of tyrosinase in Melan-a cells (Figure 6C,D) and tyrosinase, Tyrp-1 and Tyrp-2 in MNT-1 cells ( Figure  6E,F). Collectively, these results suggest that the anti-melanogenic effects of pseudoalter- showed that pseudoalteromone A decreased protein levels of tyrosinase and Tyrp-1 in B16 cells ( Figure 6A,B); it also dramatically inhibited expression of tyrosinase in Melan-a cells ( Figure 6C,D) and tyrosinase, Tyrp-1 and Tyrp-2 in MNT-1 cells (Figure 6E,F). Collectively, these results suggest that the anti-melanogenic effects of pseudoalteromone A are mediated by downregulation of protein levels of melanogenic genes.

Whitening Effect of pseudoalteromone A on a 3D Human Skin Equivalent
To further investigate the anti-melanogenic efficacy of pseudoalteromone A, we evaluated its skin whitening effect on MelanoDerm (MEL-300-B, MatTek Corp., Ashland, MA, USA), a 3D pigmented epidermis model. MelanoDerm was cultured in the presence or absence of pseudoalteromone A for 14 days, and the skin-whitening effect was evaluated through optical and histological examinations. The activity of melanocytes causes Mela-noDerm to spontaneously darken according to the duration of the incubation period. Notably, pseudoalteromone A-treated cultures were brighter than non-treated controls, a finding confirmed by the demonstration of a significant lightening effect, measured by image analysis as a change in skin lightness (ΔL) ( Figure 7A). We were also able to confirm the whitening effect of pseudoalteromone A through histological examination ( Figure  7B,C). Fontana Masson's (F-M) staining revealed that pseudoalteromone A treatment significantly reduced epidermal melanin, and hematoxylin and eosin (H&E) staining showed no significant epidermal damage. These results demonstrate the potential skin whitening effect of pseudoalteromone A through inhibition of epidermal pigmentation. Furthermore, visualization of the 3D distribution of melanin in the basal layer of MelanoDerm by TPEF microscopy ( Figure 8A) confirmed that the extent of melanin decrease depended on the pseudoalteromone A concentration ( Figure 8B,C).

Figure 6. Inhibitory effect of pseudoalteromone A on the expression of melanogenic enzymes. (A,C,E) Protein levels of melanogenic enzymes were determined by Western blot analysis in B16 cells (1), Melan-a cells (C) and MNT-1 (E). Cells were treated with pseudoalteromone A for 72 h.
Equal amounts of proteins from each cell type were resolved by SDS-PAGE on 10% gels and detected using specific antibodies; β-actin was detected as a control for equal loading. (B,D,F) Protein bands were quantified densitometrically and analysed using ImageJ software. Results for B16 cells (B), Melan-a cells (D) and MNT-1 cells (F) are presented as means ± SD, expressed as a percentage relative to controls (** p < 0.01, *** p < 0.001 vs. control group).; ### p < 0.001 vs. the α-MSH-untreated control group).

Whitening Effect of Pseudoalteromone A on a 3D Human Skin Equivalent
To further investigate the anti-melanogenic efficacy of pseudoalteromone A, we evaluated its skin whitening effect on MelanoDerm (MEL-300-B, MatTek Corp., Ashland, MA, USA), a 3D pigmented epidermis model. MelanoDerm was cultured in the presence or absence of pseudoalteromone A for 14 days, and the skin-whitening effect was evaluated through optical and histological examinations. The activity of melanocytes causes Melano-Derm to spontaneously darken according to the duration of the incubation period. Notably, pseudoalteromone A-treated cultures were brighter than non-treated controls, a finding confirmed by the demonstration of a significant lightening effect, measured by image analysis as a change in skin lightness (∆L) ( Figure 7A). We were also able to confirm the whitening effect of pseudoalteromone A through histological examination ( Figure 7B,C). Fontana Masson's (F-M) staining revealed that pseudoalteromone A treatment significantly reduced epidermal melanin, and hematoxylin and eosin (H&E) staining showed no significant epidermal damage. These results demonstrate the potential skin whitening effect of pseudoalteromone A through inhibition of epidermal pigmentation. Furthermore, visualization of the 3D distribution of melanin in the basal layer of MelanoDerm by TPEF microscopy ( Figure 8A) confirmed that the extent of melanin decrease depended on the pseudoalteromone A concentration ( Figure 8B,C).

Discussion
We showed that two different marine-derived Pseudoalteromonas strains, APmarine002 (isolated from soft coral in Jeju island) and ROA-050 (isolated from marine sediments of Pohang, South Korea), produced the same anti-melanogenic agents, pseudoalteromone A (1) and p-coumaric acid. We also demonstrated that pseudoalteromone A clearly inhibits melanin synthesis in different cell lines (B16, Melan-a and MNT-1 cells) and exerts a whitening effect in a human skin equivalent.
To begin defining the action mechanism of pseudoalteromone A, we tested the inhibitory effect of pseudoalteromone A on tyrosinase activity. First, we determined whether pseudoalteromone A directly inhibits tyrosinase activity. Simultaneously, we performed the total cellular tyrosinase activity after pseudoalteromone A treatment in α-MSH-treated B16 cells. In Figure 5A, we found that the de-pigmentary effect of pseudoalteromone A on melanocytes mainly depends on reduced tyrosinase activity, as supported by the significant (albeit weak) direct inhibition of mushroom tyrosinase activity and robust inhibition of cellular tyrosinase activity by pseudoalteromone A (Figure 5). Based on above data, we supposed that the decreased total cellular tyrosinase activity by pseudoalteromone A treatment may be mainly caused by the decreased level of tyrosinase expression rather than the direct inhibition of tyrosinase activity. In addition, further support is provided by the observation that pseudoalteromone A reduced expression of tyrosinase as well as that of the tyrosine-related enzymes, Tyrp-1 and Tyrp-2 ( Figure 6). Collectively, these findings suggest that pseudoalteromone A inhibits melanogenesis by suppressing both tyrosinase activity and expression.
Despite finding that pseudoalteromone A suppressed tyrosinase, Tyrp-1 and Tyrp-2 expression, our evidence for an underlying molecular action mechanism of pseudoalteromone A is incomplete. To establish a detailed molecular mechanism, we plan future studies to determine whether pseudoalteromone A suppresses expression of melanocyte-inducing transcription factor (MiTF) through inhibition of mitogen-activated protein kinase (MAPK) or cAMP-depend on protein kinase (PKA) signaling pathways in melanocytes, reflecting the fact that MAPK and PKA signaling are intimately linked with MiTF-induced tyrosinase expression during melanogenesis [11][12][13][14][15][16][17]. Collectively, the data from this study provide preliminary evidence supporting the possibility of pseudoalteromone A as a potent and effective whitening agent that could be used in cosmetics and medicinal formulations.

General Experimental Procedures
UV spectra were recorded in methanol (MeOH) on a Cinco UVS-2100 spectrophotometer. 1D and 2D NMR spectra were recorded on a Varian Unity-Enova at 400 MHz using signals of the residual solvent as internal references (p h. 3.31 ppm and a ct 49.00 ppm for methanol-d 4 (CD 3 OD-d 4 ). Low-resolution LC/MS measurement was performed at the National Research Facilities and Equipment Center (Nano Bioenergy Materials Center) at Elwha Woman's University using an Agilent Technologies 1260 quadrupole and Waters micromassZQ LC/MS system with a reversed-phase column (Phenomenex Luna C18 (2) 100 Å, 50 mm × 4.6 mm, 5 µm) at a flow rate 1.0 mL/min. Column chromatography separation was performed using a C18 column eluting with a gradient of MeOH and H 2 O. Fractions were purified using a WATERS 1525 binary HPLC pump with a reversed-phase C18 column (Phenomenex Luna C18 (2), 250 mm × 10 mm, 5 µm) eluting with 48% CH 3 CN in H 2 O at flow rate of 2.0 mL/min.

Strain Isolation
Pseudoalteromonas strain APmarine002 was isolated from coral collected at Jeju island. Pseudoalteromonas strain ROA-050 was isolated from the marine sediments of Pohang, South Korea. An NCBI blast analysis of 16S rRNA gene sequences identified Strain APmarine002 as Pseudoalteromonas rubra QD1-2 and strain ROA-050 as Pseudoalteromonas piscicida, with 100.0% identity in both cases.

Fermentation, Extraction and Purification
Strains APmarine002 and ROA-050 were independently cultured in 1 × 2.5 L Ultra Yield Flasks, each containing 1 L of seawater-based medium (10 g starch, 2 g yeast extract, 4 g peptone, 34.75 g artificial sea salt dissolved in distilled H 2 O), and at 27 • C with shaking (150 rpm). After 7 days of cultivation, each broth was extracted using ethyl acetate (EtOAc; 1 L overall), and the solvent was removed in vacuo to yield 23.1 mg and 84.2 mg of EtOAc crude extract, respectively. Both extracts were used to perform the LC-MS analysis. For the large cultivation to isolate compound 1, strain ROA-050 was cultured in 40 × 2.5 L Ultra Yield Flasks, each containing 1 L of seawater-based medium (10 g starch, 2 g yeast extract, 4 g peptone, 34.75 g artificial sea salt dissolved in distilled H 2 O), and at 27 • C with shaking (150 rpm). After 7 days of cultivation, the broth was extracted using ethyl acetate (EtOAc; 40 L overall), and the solvent was removed in vacuo to yield 3.

Cell Viability
B16 cells, Melan-a cells and MNT-1 cells were seeded on 96-well plates (1 × 10 4 cells/well) and cultured for 24 h, after which the medium was replaced with the medium containing pseudoalteromone A, diluted to the indicated concentrations, and plates were incubated for 72 h. The medium was then removed and the fresh culture medium containing 10% CCK-8 solution (DOJINDO, Tokyo, Japan) was added. After incubating 1 h, supernatants of each cell were transferred to 96-well plates, and the absorbance was measured at 450 nm using a Synergy™ HTX Multi-Mode Microplate Reader (Bioteck, Winooski, VT, USA).

Measurement of Melanin Content
B16 cells were seeded overnight in 48-well plates (2 × 10 4 cells/well) and then treated for 72 h with increasing concentrations of pseudoalteromone A. The extracellular melanin content in the phenol red-free cell culture medium was measured at 405 nm with Synergy™ HTX Multi-Mode Microplate Reader (Bioteck, Winooski, VT, USA). After removing the medium, 200 µL of 1 N NaOH was added, and plates were heated to 60 • C for 30 min to solubilize intracellular melanin. Next, 100 µL aliquots of lysate were transferred to a 96-well plate and absorbance was measured at 405 nm. Melan-a cells and MNT-1 cells were seeded in 24-well plates (10 × 10 4 cells/well) for 24 h, and treated with pseudoalteromone A. Then, the intracellular melanin contents were measured. Results were normalized to total protein content, determined using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Intracellular melanin levels are presented relative to those in the control group, expressed as a percentage.

Cell-Free Enzymatic Assay for Tyrosinase Activity
The inhibitory effects of pseudoalteromone A on tyrosinase activity, tested using a mushroom tyrosinase enzyme, was assessed as a function of DOPA oxidase activity and t and tyrosine hydroxylase activity. For these experiments, a 5 µL sample was added to a 96-well plate to which was added 50 µL of 0.1 M sodium phosphate buffer (pH 6.8), 40 µL distilled water, and 5 µL of enzyme solution (2000-unit mushroom tyrosinase). After mixing, 50 µL of 20 mM L-DOPA or 0.03% L-tyrosine (tyrosinase substrate) was added to each well and the amount of products formed in the reaction mixture was determined by kinetically measuring absorbance values at 475 nm at 10 min intervals for at least 1 h at 37 • C using a spectrophotometer.

Assay of Cellular Tyrosinase Activity
The activity of tyrosinase enzyme was evaluated by measuring the oxidase rate of L-DOPA to dopachrome, an intermediate in melanin biosynthesis. B16 melanoma cells were plated in 6-well plates (20 × 10 4 cells/well) and incubated overnight, then treated with increasing concentrations of pseudoalteromone A for 3 days in a humidified incubator. Cells were then washed twice in ice-cold phosphate-buffered saline (PBS) and lysed in 1 mL of 0.1 M sodium phosphate buffer (pH 6.8) containing 1% (w/v) Triton X-100. The resulting whole-cell lysates were microcentrifuged at 13,000 rpm for 20 min at 4 • C, and the resulting supernatants were placed in a 96-well plate for reactions with L-DOPA (2 mg/10 mL). Tyrosinase activity was analyzed spectrophotometrically by kinetically measuring absorbance values at 475 nm at 10 min intervals for at least 1 h at 37 • C using a spectrophotometer.

Western Blotting
B16, Melan-A and MNT-1 cells were seeded onto 6-well plates (20 × 10 4 cells/well) and incubated for 24 h. The medium was then removed and fresh culture medium containing different concentrations of pseudoalteromone A was added, after which plates were incubated for the appropriate time. After washing twice with ice-cold PBS, intracellular proteins were extracted from each cell type by lysing cells in 1 × RIPA buffer (Cell Signaling Technology, Danvers, MA, USA), diluted with distilled water containing protease inhibitor cocktail III and phosphatase inhibitor Cocktail set III (Merck, Darmstadt, Germany) at a ratio of 200:1. Cell lysates were centrifuged at 13,000 rpm at 4 • C, and protein concentration was measured using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Appropriate amounts of protein from each cell type were resolved by SDS-PAGE on 10% gels and then transferred onto a nitrocellulose membrane (Bio-Rd, Hercules, CA, USA). Membranes were blocked by incubating in 1 × Tris-buffered saline (TBS) containing 5% skim milk for at least 1 h, then incubated at 4 • C overnight with primary antibodies, diluted as recommended by the manufacturer. Membranes are washed three times in TBS containing Tween 20 and incubated with species-appropriate secondary antibodies at recommended dilutions for 1 h at room temperature. Immuno-reactive proteins were visualized using Clarity™ Western ECL substrate (Bio-Rad, Hercules, CA, USA). The densities of target protein bands were measured using iBright™ CL750 Imaging System (Invitrogen, Carlsbad, CA, USA).

Skin-Whitening Assay Using a Pigmented 3D Skin Model
MelanoDerm (MEL-300-B, MatTek Corp., Ashland, MA, USA), used for the skinlightening study, was cultured in EPI-100-NMM-113-PRF medium (MatTek Corp., Ashland, MA, USA) at 37 • C in a humidified 5% CO 2 incubator. Different concentrations of pseudoalteromone A were added to the culture medium every other day for 14 days. Thereafter, epidermal pigmentation was examined by performing optical and histological analyses. The epidermal pigmentation level was calculated by comparing variations in L* values (a lightness/darkness index) on days 1 and 14 and estimating the difference between them (∆L). For the histological examination, tissues were fixed in 10% neutral buffered formalin (BBC Biochemical, Mount Vernon, WA, USA) and embedded in paraffin. Paraffin-embedded samples were then sliced into 5 µm sections, stained with hematoxylin and eosin (H and E) and Fontana Masson's (F-M) staining (for melanin), and then examined histologically.
4.11. Two-Photon Excitation Fluorescence (TPEF) Imaging TPEF imaging was used to visualize the three-dimensional distribution of melanin in the human skin equivalent, as described in our previous studies [18,19]. Intracellular melanin (green in Figure 8) in the melanocyte-rich layer was measured by imaging the basement membrane side of MelanoDerm samples in a total volume of 400 (x) × 400 (y) × 40 (z) µm 3 . Relative TPEF signal intensities and melanin volume were quantified using Image-Pro Premier 3D software (Media Cybernetics, Inc., Bethesda, MD, USA).

Conclusions
In this paper, we identified a new anti-melanogenic agent, pseudoalteromone A, from cultures of the marine microorganism, Pseudoalteromonas spp. APmarine002 and ROA-050. We showed that pseudoalteromone A suppresses melanin synthesis in vitro, reducing tyrosinase activity and expression, and confirmed its skin-whitening effect in MelanoDerm, a 3D pigmented epidermis model. On the basis of these results, we conclude that marine microorganisms are a good source of biologically active compounds and further suggest that pseudoalteromone A is a promising anti-melanogenic agent in the cosmetic field.