**1. Introduction**

Melanin is a macromolecule biological pigment, mainly composed of eumelanin and pheomelanin. Eumelanin is brown-black, and pheomelanin is red-brown. The different proportions of the two pigments cause the different colors of hair and skin [1]. Melanin can protect the skin from the harmful effects of environmental pollution factors, such as ultraviolet radiation and oxidative stress. However, excessive accumulation of melanin can lead to the appearance of many skin diseases, including melasma, freckles, age spots, and other pigmentation syndromes. The process of melanin synthesis is extremely complicated, mainly involving three enzymes in the tyrosinase gene family, namely tyrosinase (Tyrosinase, TYR), tyrosinase-related protein-1 (Tyrosinase-related protein-1, TRP-1), and tyrosinase-related protein-2 (Tyrosinase-related protein-2, TRP-2), in which tyrosinase is the key enzyme in this reaction [2].

It Is well known that melanocortin 1 receptor (MC1R)/α-melanocyte, stimulating hormone (α-MSH), phosphatidylinositol 3-kinase (PI3K)/activation of phosphorylation of protein kinase B (Akt), mitogen-activated protein kinase (MAPK), Wingless/Integrated (Wnt)/β-catenin, nitric oxide (NO), and other signaling pathways are involved in melanin production of melanocytes [3,4]. Microphthalmia-associated transcription factor (MITF) are the most important molecular targets in these pathways, and the change of MITF expression

**Citation:** Hu, Z.; Sha, X.; Zhang, L.; Huang, S.; Tu, Z. Effect of Grass Carp Scale Collagen Peptide FTGML on cAMP-PI3K/Akt and MAPK Signaling Pathways in B16F10 Melanoma Cells and Correlation between Anti-Melanin and Antioxidant Properties. *Foods* **2022**, *11*, 391. https://doi.org/10.3390/ foods11030391

Academic Editors: Rotimi Aluko, Jianhua Xie, Yanjun Zhang and Hansong Yu

Received: 8 December 2021 Accepted: 27 January 2022 Published: 29 January 2022

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is directly related to abnormal skin and hair pigments [5]. MITF is a basic helix-loop-helixleucine zipper transcription factor, which can share a highly conserved sequence with the m-box motif of the promoter region (TYR, TRP-1, TRP-2 in the promoter region; that is, 5- -AGTCATGTGCT-3- ), and regulates the expression of TYR, TRP-1, and TRP-2; thus, regulating the production of melanin [6]. Previous studies have found that some peptides inhibit melanin synthesis mainly through ERK in the mitogen-activated protein kinase (MAKP) signaling pathway [7,8]. In the previous experiments of this study, we found a peptide Phe-Thr-Gly-Met-Leu (FTGML) could promote cell apoptosis, which was related to the other two pathways of MAKP (i.e., p38 and JNK) [9], but there was no relevant report that the peptide could inhibit melanin synthesis through these two pathways.

In recent years, people have tried to develop new natural-derived whitening cosmetics or skin bleaching agents, as natural products have the advantages of being less toxic and having fewer side effects compared to chemicals. Examples include tea [10], mung bean seed [2], etc. Fucoidan derived from hizikia fusiforme can significantly inhibit the expression pathway of tyrosinase and tyrosinase-related proteins in B16F10 cells by regulating the extracellular signal-regulated kinase mitogen-activated protein kinase (ERK-MAPK), and downregulating the MITF [11]. Tea catechins downregulate MITF expression by inhibiting cyclic adenosine monophosphate (cAMP), leading to subsequent phosphorylation of CREB, and a decrease in the levels of tyrosinase, TRP-1, and TRP-2, thereby reducing melanin synthesis [12]. However, at present, most research on natural products that inhibit melanin mostly focus on plant sources. Natural products from animal sources, especially fish, have less research in this area, and most of them focus on basic index evaluation. The previous research of our group found that grass carp fish scale gelatin hydrolysate has a good whitening effect, and a new peptide consisting of five amino acids (phenylalaninethreonine-glycine-methionine-leucine, FTGML) was obtained, with good in vitro tyrosinase inhibitory activity (IC50 value was 1.89 mM). However, the melanin inhibitory mechanism is still unclear.

In addition, there are reports that hydrogen peroxide (H2O2) and other ROS and RS are produced during melanogenesis, leading to advanced oxidative stress in melanocytes. Therefore, the use of free radical scavengers in this process can play a role in feedback regulation. Studies have shown that ROS scavengers and ROS generation inhibitors may inhibit ultraviolet-induced melanogenesis [13]. Antioxidants, such as ascorbic acid derivatives and reduced glutathione (GSH), have also been used as inhibitors of melanin production [14,15]. Therefore, it is necessary to pay attention to the antioxidant effect and melanin inhibitory effect of compounds or natural products at the same time, and explore the relationship between the two effects, but there are few studies on the relevant mechanism.

In terms of cell structure and melanin synthesis, the mouse melanoma cell line B16F10 is highly consistent with human melanoma cell. Furthermore, it is difficult to culture human melanoma cells when evaluating the efficacy of active whitening, due to its stricter requirements for cell culture and handling condition. The mouse melanoma cell line B16F10 has the advantages of multiple passages, rapid development, relatively simple culture conditions, high malignancy, and good tumorigenicity. As such, B16F10 cells are widely used as the effective cells for cell evaluation of whitening activity substances [16–18]. In this study, we aim to further analyze the anti-melanin effect and potential mechanism of FTGML based on the previous stage in the mouse melanoma cell line B16F10. It is also assumed that FTGML functionally causes cAMP-PI3K/Akt- and MAPK-mediated downregulation of MITF, which is the main cascade of melanin production, leading to the decrease of tyrosinase in melanocytes. In addition, we also studied the correlation between anti-melanin and anti-oxidation in melanocytes, and provided the possibility of predicting its anti-melanin potential through antioxidant activity in the future. Our research contributes to the development of new cosmetic ingredients, food supplements, and functional foods containing collagen peptides.

#### **2. Materials and Methods**

#### *2.1. Experimental Materials*

FTGML was obtained from the grass carp fish scale gelatin hydrolysate. In brief, grass carp fish scale gelatin was hydrolyzed by alcalase and gastrointestinal simulated digestion to obtain a mixture of peptides. The peptides with tyrosinase inhibitory activity were screened and identified by bioaffinity ultrafiltration-mass spectrometry, and peptide FT-GML showed the best tyrosinase inhibitory activity. FTGML was synthesized in Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China). The purity of all synthetic peptides was above 95%. Mushroom tyrosinase (EC1.14.18.1, with specific activity 6680 U/mg) and L-DOPA were produced by Sigma-Aldrich (St. Louis, MO, USA). The murine melanoma B16F10 cells (CL0039) were acquired from Fenghui Biological Technology Co., Ltd. (Changsha, China). All other reagents were analytical grade.

#### *2.2. Cell Culture*

B16F10 cells was cultured by the medium of RPMI-1640, with 10% fetal bovine serum (FBS), and 1% penicillin/streptomycin at 37 ◦C in 5% CO2.

#### *2.3. Cell Viability Assay*

Cell viability was assessed by Cell Counting Kit-8 (CCK-8) assay. The cells in the logarithmic growth phase were digested by trypsin, and counted under the microscope to make a cell suspension of 1~5 × <sup>10</sup><sup>4</sup> cells/mL. A 100 <sup>μ</sup>L cell suspension was taken to a 96-well culture plate with 1~5 × <sup>10</sup><sup>3</sup> cells/well. Wells added with 100 <sup>μ</sup>L medium were used as blank control, and wells added with different concentrations (0, 0.1, 0.2, 0.4, 0.8, 1.6 mg/mL) of FTGML solution and 0.75 mg/mL of kojic acid were used as the experimental groups and positive control groups, respectively. After 0 h, 24 h, and 48 h of incubation at 37 ◦C, the 1:10 volume ratio mixed CCK-8 and serum-free essential basic medium was added to the test well at 100 μL per well, and incubated for 1 h at 37 ◦C in a 5% CO2 incubator. The absorbance at 450 nm was measured with a microplate reader.

#### *2.4. Determination of Apoptosis Rate of B16F10 Cells*

Cells in 2.3 were collected and performed according to the Annexin V-FITC kit (Thermo Fisher Scientific, Carlsbad, CA, USA). The apoptosis of B16F10 cells was analyzed by flow cytometry (Ex = 488 nm, FL1 Em = 525 ± 20 nm, FL2 Em = 585 ± 21 nm) and FlowJo software.
