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Ishophloroglucin A Isolated From Ishige Okamurae Suppresses Melanogenesis Induced By α-MSH: In Vitro And In Vivo Part 2

Apr 03, 2023

3. Discussion 

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The compound DPHC isolated from IOE already had reported tyrosinase inhibitory activity and protective effect against UV-B radiation-induced cell damage in vitro [8]; however, the anti-melanogenesis effect of the components derived from IOE in silico by interaction with tyrosinase, in vivo in phenotype studies in animal models, and their underlying molecular mechanisms, have not yet been examined. In the present study, we determined the anti-melanogenesis and tyrosinase inhibitory activities of IPA, a phlorotannin isolated from IO and IOE, in a vertebrate model of zebrafish in vivo and in B16F10 melanoma cells in vitro, after induction with α-MSH.


Treatment with α-MSH is known to induce melanin synthesis and tyrosinase activity [20]. In addition, tyrosinase has been demonstrated to be essential for melanogenesis [29]. Previous studies have shown that molecular docking can be used to evaluate tyrosinase inhibitory activity [30,31]. Thus, molecular docking calculations were performed to understand the binding model of IPA and DPHC, a known polyphenol isolated from IO, which has revealed higher binding energy in anti-melanogenesis activity than arbutin, as a positive control. According to the results (Figure 1), IPA revealed the lowest docking scores, which indicated that the interaction of IPA with the target protein tyrosinase was stronger than the other two compounds, DPHC, and arbutin. However, there is a limitation in correlating the inhibition of mushroom tyrosinase activity with that of cellular tyrosinase or melanin production in cultured melanocytes [32]. Thus, the inhibitory effects of IPA on tyrosinase activity and melanogenesis were examined in a zebrafish in vivo model and murine B16F10 melanoma cells. 


Melanin pigments accumulate on the surface of zebrafish, allowing microscopic observation of the pigmentation process without complicated experimental procedures, making them a suitable model for screening melanogenesis inhibitors [33,34]. We evaluated the melanin inhibitory effects of IPA and IOE in a zebrafish larva model stimulated by α-MSH through melanin content determination. All the tested samples exerted profound inhibitory effects on zebrafish pigmentation with no signifificant toxicity (Figure S2). The inhibitory effects of pigmentation were observed via morphological analysis of zebrafish larvae related to the different treatments (Figure 2). In addition, the use of early-stage larvae rather than the adult stage provides another advantage in testing the percutaneous effects of medicinal or cosmetic compounds [33,35]. In this case, we chose α-MSH as an inducer both in zebrafish in vivo and in B16F10 melanoma cells in vitro. According to both results in zebrafish embryos and B16F10 melanoma cells, the melanin content increased by α-MSH stimulation.


Melanin content correlates directly with the activity and protein levels of tyrosinase [36]. Therefore, we determined the inhibitory effects of IPA and IOE on the tyrosinase activity induced by α-MSH on B16F10 cells. We found that the IPA-treated group had a decreased tyrosinase activity and melanin content stimulated by α-MSH, with a reduction of approximately 35% tyrosinase activity and 40% melanin content (Figure 4A, C). IOE inhibited the activity of tyrosinase in a dose-dependent manner and significantly decreased melanogenesis in B16F10 cells (Figure 4B, D). Compared with arbutin, IPA and IOE have signifificant inhibitory effects on melanin production and tyrosinase activity, which was to the results of previous molecular docking studies. 


To investigate the mechanism of the inhibitory effects on α-MSH-induced melanin synthesis in cells, Western blotting was performed. The expression levels of melanin-related proteins, including ERK, JNK, and p38, following treatment with IPA or IOE, were evaluated. Activated phosphorylation of ERK can promote the degradation of MITF via the ubiquitin–proteasome-dependent pathway [28]. This suggests that potential melanogenesis inhibitors may suppress melanin synthesis by promoting the proteasomal degradation of MITF, which was related to the activation of the ERK signaling pathways [37]. The ERK, JNK, and p38 MAPKs belong to the MAPK family [38–40]. In addition, activation of the p38 MAPK pathway induced MITF expression [41]. In this study, Western blot analysis revealed that IPA promotes p-JNK and p-p38 (Figure 5B, C). In contrast, the levels of p-ERK did not change with the treatment of IPA and IOE (Figure 5A). These results imply that the inhibitory effects of IPA and IOE on tyrosinase activity and melanogenesis may be related to the JNK and p38 signaling pathways. 

4. Materials and Methods 

4.1. Chemicals and Reagents 

Dimethylsulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), L-DOPA, alpha-melanocyte stimulating hormone (α-MSH) and phosphate-buffered saline (PBS) were purchased from Sigma–Aldrich Chemical Co. (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from Invitrogen–Gibco (Grand Island, NY, USA). The extracellular signal-regulated kinase (ERK1/2), phosphorylated ERK1/2 (p-ERK1/2), c-Jun N-terminal kinase (JNK), phosphorylated JNK (p-JNK), p38, phosphorylated p38 (p-p38), cAMP response element-binding protein (CREB), phosphorylated CREB (p-CREB), microphthalmia-associated transcription factor (MITF), tyrosinase-related protein-1 (Trp-2), tyrosinase-related protein-1 (Trp-1), tyrosinase (TYR), anti-mouse and anti-rabbit IgG antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). All other reagents, including α-MSH, were purchased from Sigma–Aldrich Chemical Co. 

4.2. Molecular Docking of Tyrosinase 

For the docking study, the crystal structure of tyrosinase (PDB: 3NM8) was obtained from the Protein Data Bank. The docking studies were performed using CDOCKER in Accelrys Discovery Studio 3.0 (Accelrys, Inc., San Diego, CA, USA). When a whole nucleotide sequence is covered by the receptor grid, ligands are presumed to select the best docking position [42]. The docking procedure was mentioned in the previous study. Briefly, three steps were followed: (1) conversion of the 2D structure into a 3D structure; (2) calculation of charges; and (3) addition of hydrogen atoms using the flexible docking program [31,43]. 

4.3. Preparation of IOE and Isolation of IPA 

IO was harvested in June 2018 along the east coast of Jeju Island, Korea. The alga was washed twice with tap water to remove the salt, epiphytes, and sand attached to the surface. Next, it was carefully rinsed with fresh water and was maintained in a medical refrigerator at −20 ◦C. Thereafter, the frozen alga was lyophilized and homogenized with a grinder before extraction. The IOE was extracted in 50% ethanol (v/v, in water) under stirring for 24 h at room temperature, then it was filtered. The filtrate extract was concentrated under decompression and freeze-dried to powder (IOE). A 50% ethanol extract of IO was conducted by Shinwoo Co. Ltd. (Lot No. SW9E29SA, Gyeonggi-do, Korea). IPA was isolated from IOE as previously described [9]. Briefly, the IOE was fractionated using centrifugal partition chromatography. All fractions were collected and the IPA was eventually purified by a semi-preparative HPLC column (YMC-Pack ODS-A, 10 mm, 250 mm, 5 m). IPA was determined as a polyphenol and its chemical structure (Figure S1, Supplementary Materials) was identified via LC/MS analysis with a mass m/z of 992.1315, thus indicating a molecular formula of C96H66O48 (1986.26 of calculated molecular weight, ∆0.6, [M − 2H] 2−). 

4.4. Origin and Maintenance of Parental Zebrafish

Adult zebrafish were obtained from a commercial dealer (Seoul Aquarium, Seoul, Korea) and 10 fish were preserved in a 3-L acrylic tank at 28.5 ◦C, with a 14:10 h light: dark cycle. Zebrafish were fed twice a day, for 6 days⁄week, with supplementary Tetramin flake food (SEWHAPET Food Co., Seoul, Korea). Embryos were collected within 30 min by natural spawning and induced in the morning by turning on the light. The zebrafish experiment received approval from the Animal Care and Use Committee of Jeju National University (Approval No. 2017-0001). 

4.5. Measurement of Melanin Content in Zebrafish Larvae

IPA and IOE and stimulator α-MSH concentrations were used to examine the effects of concentration on embryo development. Fifteen zebrafish embryos (3–4 hpf) were seeded in each well, comprising 1.9 mL embryo medium, in a 12-well seeding plate. Test samples were dissolved in 1% DMSO with 1× PBS and mixed well. Every morning for the first 5 pdf, viable embryos were enumerated to obtain a survival measure. To determine the melanin content, embryos at 7–9 hpf were seeded into a 6-well plate with 30 embryos in each well into a 2.7-mL embryo medium. After 3 days, the larvae were rinsed twice with 1× PBS to remove any residual reagents or particles, and similar amounts of larvae were placed into the e-tubes. Before measuring the melanin content, several larvae of each group were captured with a microscope, and the remaining were centrifuged.


After centrifugation, the pellet was dissolved in 1 mL of 1 N NaOH at 90 ◦C for 60 min. The mixture was then vigorously vortexed to solubilize the melanin pigment. The Absorbance of the supernatant was measured at 490 nm. The result was compared with the control which was considered to represent one hundred. The melanin content was calibrated by protein amount, and the observations were repeated in triplicate. 

4.6. Cytoxicity of IPA and IOE in B16F10 Cells

B16F10 mouse melanoma cells were obtained from ATCC (American Type Culture Collection, Manassas, VA, USA). B16F10 cells were cultured in DMEM supplemented with 100 U/mL of penicillin, 100 µg/mL of streptomycin, and 10% FBS. The cells were then incubated in an atmosphere of 5% CO2 at 37 ◦C and were then subcultured every 3–5 days. The cytotoxicity of IPA and IOE against B16F10 cells was investigated via colorimetric MTT assay. Briefly, the cells were seeded in 24-well plates at a

concentration of 2 × 104cells/mL. About 16 h after seeding, the cells were incubated with IPA and IOE at different concentrations for 72 h, and their viability was determined. 

4.7. Determination of Cellular Melanin Content

Cellular melanin content was measured using a previously described method [33]. The cells (2 × 104 cells/mL) were incubated with various concentrations of IPA and IOE for 72 h; therefore, they were washed in ice-cold PBS. Briefly, the cells were incubated at 80 ◦C for 1 h in 1 mL of 1 N NaOH/10% DMSO and were then vortexed to solubilize the melanin: the absorbance was measured at 450 nm. The optical density of the inhibition in the control was considered to be 100%. The data are presented in the form of mean percentages and the results were repeated in triplicate.

















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