Melanogenesis-inhibiting peptide, and preparation method and application thereof

Abstract

The application provides a melanin production inhibiting peptide and a preparation method and application thereof. The amino acid sequence of the inhibiting peptide is GLPGISGGGY. The preparation method comprises the following steps: washing, degreasing and removing impure proteins of sturgeon skin, then swelling and homogenizing under an acid condition to obtain a sturgeon skin homogenate; adding 6000-6100 U / g protease into the sturgeon skin homogenate, and performing enzymolysis under the condition that the temperature is 34-36 DEG C and the pH is 6.0-7.0 to obtain an enzymolysis liquid containing sturgeon skin collagen polypeptide; and obtaining the target peptide by ultrafiltration of small molecule components and combining MITF / TYR double target virtual screening and 3D-QSAR pharmacophore model orientation. Experiments prove that the inhibiting peptide has high biological activity, low cytotoxicity and good skin safety, and has a wide application prospect in the preparation of whitening cosmetics, skin care products and pigment deposition disease treatment drugs.

Description

Technical Field

[0001] This invention relates to the field of polypeptide technology, and in particular to a melanin production inhibitory peptide, its preparation method, and its application. Background Technology

[0002] Melanin biosynthesis is a crucial process for maintaining the skin barrier and defending against UV damage. However, its abnormal and excessive accumulation can lead to various skin diseases such as melasma, freckles, and post-inflammatory hyperpigmentation, seriously affecting the physical and mental health of patients. The core regulatory mechanism of this process mainly revolves around the MITF / TYR axis: Microphthalmia-associated transcription factor (MITF) acts as the master regulator, specifically recognizing and binding to the M-box sequence (whose core shared sequence is usually AGTCATGTGCT, M-box) of the promoter region of downstream target genes through its basic helical-loop-helical leucine kinase (bHLH-Zip) domain. This drives the transcriptional expression of tyrosinase (TYR), tyrosinase-related protein 1 (TYRP1), and tyrosinase-related protein 2 (TYRP2), thereby catalyzing the conversion of substrates into melanin. Currently, melanin inhibitors commonly used in clinical and cosmetic fields (such as hydroquinone, kojic acid, etc.) have certain therapeutic effects, but they generally have drawbacks such as strong cytotoxicity, poor chemical stability, single mechanism of action, or easy skin irritation, which seriously limit their widespread application in high-end whitening products and skin disease treatment.

[0003] In recent years, natural bioactive peptides have become a hot topic in screening novel melanin-regulating molecules due to their high biocompatibility, high specificity, and low immunogenicity. However, traditional methods for extracting peptides from natural products often face challenges such as low screening efficiency, unclear sequence activity, and poor transdermal absorption. In particular, peptide drugs that precisely intervene in specific signal transduction pathways to downregulate MITF activity at the transcriptional level by inducing inhibitory phosphorylation of glycogen synthase kinase-3β (GSK-3β) remain scarce. Therefore, utilizing advanced bioinformatics tools to develop novel melanin-inhibiting peptides with clear targeting mechanisms, high stability, and good skin safety from abundant natural waste resources has significant scientific and economic value for promoting the development of the skin health industry. Summary of the Invention

[0004] To address the problems mentioned in the background art, this application provides a melanin production inhibitory peptide, its preparation method, and its application.

[0005] In a first aspect, this invention proposes a melanin production inhibitory peptide with the amino acid sequence GLPGISGGGY, as detailed in sequence listing ID1. A decapeptide with a specific structure was obtained, the sequence of which exhibits high spatial adaptability to key targets in melanin synthesis and can significantly reduce pigmentation.

[0006] In a second aspect, the present invention provides a method for preparing a melanin production inhibitory peptide, the method comprising:

[0007] S1, Sturgeon skin is cleaned, degreased and deproteinized, and then swollen and homogenized under acidic conditions to obtain fish skin homogenate; S2, add 6000-6100 U / g protease to the fish skin homogenate, and carry out enzymatic hydrolysis at a temperature of 34-36°C and a pH of 6.0-7.0 to obtain an enzymatic hydrolysate containing sturgeon skin collagen polypeptide; S3, the enzymatic hydrolysate is separated into components, small molecular weight components are collected, and the melanin production inhibitory peptide is obtained by sequence identification and virtual screening based on melanin production-related targets.

[0008] In the above technical solution, the combined process of "graded extraction + virtual screening" significantly improves the efficiency and accuracy of targeted acquisition of highly active bioactive peptides from complex natural products.

[0009] Furthermore, step S1 includes the following: S11, Wash the sturgeon skin with distilled water, then cut it into 2×2cm pieces. 2 lumpy; S12, place sturgeon skin in 10 times its volume of 0.1mol / L NaOH solution and stir at 3-5°C for more than 24 hours to remove non-collagenous proteins; S13, degreased with a 10% n-butanol solution at a solid-liquid ratio of 1:10 for at least 24 hours, with the solvent replaced every 12 hours. S14, the fish skin treated in step S13 is washed with distilled water until neutral, placed in a 3% lactic acid solution and stirred for 2 hours to make the fish skin swell, and then homogenized into a paste to obtain the fish skin homogenate.

[0010] In the above technical solution, an acidic swelling and gentle washing process is used to retain the natural activity of collagen to the maximum extent while thoroughly removing fats and impurities that affect purity.

[0011] Furthermore, the protease is a flavor protease. Using a flavor protease allows for precise multi-site cleavage, which improves the hydrolysis rate, avoids the formation of bitter peptides, and optimizes the sensory and biological properties of the resulting polypeptides.

[0012] Furthermore, step S2 includes: adding flavor protease to the fish skin homogenate at a dosage of 6,068.4 U / g under conditions of pH=7.0 and temperature of 35.5°C, and obtaining the enzymatic hydrolysate after 6 hours of enzymatic hydrolysis. The determined enzymatic hydrolysis process parameters achieve optimal matching between enzyme activity and substrate concentration, ensuring high yield and stability of the target sequence GLPGISGGGY in the product.

[0013] Furthermore, the melanin production-related targets are selected from at least one of microphthalmia-related transcription factors, tyrosinases, tyrosinase-associated protein-1, and tyrosinase-associated protein-2; the virtual screening includes: performing molecular docking simulations using peptide sequences with the protein structures of MITF and / or TYR, and screening target peptides based on binding energy scores. Through multi-target synergistic screening, it is ensured that the obtained peptides can block the melanin synthesis pathway from multiple dimensions, including the transcriptional level (MITF) and the enzymatic catalytic level (TYR series).

[0014] Furthermore, step S3 includes: S31, the enzymatic hydrolysate is separated by passing it through an ultrafiltration membrane with a molecular weight cutoff of 3kDa, and the filtrate is collected; S32, after desalting the filtrate, the melanin production inhibitory peptide was screened by combining high-performance liquid chromatography-tandem mass spectrometry analysis with MITF molecular docking simulation.

[0015] In the above technical solution, molecular docking simulation is introduced, which enables the evaluation of the binding mode between peptides and receptors at the atomic level, reducing blind experiments and lowering the research and development costs and cycle.

[0016] Furthermore, step S32 includes: S321, a peptide database was established using mass spectrometry analysis to screen for sequences that meet the conditions -logP>20 and have a molecular weight of less than 1 kDa; bioactivity was scored using Peptide Ranker, and sequences with scores greater than 0.75 were selected to construct a 3D structure database. S322, a three-dimensional quantitative structure-activity relationship (3D-QSAR) pharmacophore model is constructed based on known antioxidant active peptides, and candidate sequences are obtained by screening from the peptide database by combining ADMET property prediction and skin toxicity prediction functions; S323, using MITF as the receptor protein, establishes pharmacophore characteristics based on the key amino acid sites of the receptor protein's binding domain to deoxyribonucleic acid (DNA), performs molecular docking on the candidate sequences, and selects several sequences with high absolute values ​​of binding energy scores as candidate peptides; S324, The candidate peptides are screened by evaluating melanin inhibition activity at the cellular level to obtain the melanin production inhibitory peptide.

[0017] The above technical solution integrates structure-activity relationship, pharmacokinetics and toxicity prediction, which not only ensures the high activity of the peptide, but also ensures its good transdermal absorption and clinical safety.

[0018] Furthermore, the key amino acid sites are based on MITF (PDB:4ATI), which includes at least one of Glu213 and Arg216 from the A chain and Asn242, Lys243, Arg217, and Arg214 from the B chain. This locks onto the core functional sites of MITF binding to DNA, effectively blocking the transcriptional activation of pigment synthesis-related genes by competitively occupying key amino acid residues.

[0019] In a third aspect, this invention proposes an application of a melanin-inhibiting peptide, namely, the melanin-inhibiting peptide described in the first aspect, or the melanin-inhibiting peptide prepared using the method described in the second aspect, in the preparation of whitening cosmetics, skincare products, or drugs for inhibiting melanin production. This expands the specific applications of this peptide in the daily chemical and pharmaceutical fields, and by limiting the use of the core active ingredient, it fully covers the commercial value chain of this inhibitory peptide from production to end products.

[0020] Furthermore, the concentration of the melanin production inhibitory peptide in the whitening cosmetics, skincare products, or drugs used to inhibit melanin production does not exceed 1000 µmol / L. This limits the safety and efficacy window of the inhibitory peptide. Within this concentration range, the peptide can fully exert its biological effect of downregulating melanin production through the PI3K / AKT pathway, while ensuring zero skin irritation and excellent cell compatibility, avoiding hemolysis or nonspecific toxicity that may occur at high concentrations.

[0021] Compared with the prior art, the beneficial results of the present invention are as follows: (1) This invention provides a novel collagen polypeptide SSCH-T2 (GLPGISGGGY).

[0022] (2) This invention combines multiple bioinformatics techniques, such as mass spectrometry analysis, 3D-QSAR pharmacophore model construction, and ADMET property prediction, to selectively screen the decapeptide SSCH-T2 with a specific sequence from complex sturgeon skin enzymatic hydrolysates. Compared with traditional blind screening, this not only significantly improves the efficiency of obtaining highly active peptides, but also ensures that the obtained peptides significantly inhibit melanin production while exhibiting cytotoxicity far lower than traditional chemical inhibitors by introducing a skin toxicity prediction function in advance, thus providing a core component for the development of safe whitening products.

[0023] (3) This invention elucidates the molecular mechanism by which SSCH-T2 exerts its effects by activating the PI3K / AKT signaling pathway. This polypeptide can significantly increase the phosphorylation level of AKT protein, thereby inducing repressive phosphorylation (Ser9 site) of the downstream key kinase GSK-3β. This cascade reaction effectively inhibits the activity of transcription factor MITF and its transcriptional regulation of TYR, TRP-1, and TRP-2 genes, achieving comprehensive inhibition from signal transduction to gene expression and then to the enzyme catalysis level. Its mechanism of action is more systematic and durable than that of a single enzyme inhibitor.

[0024] (4) This invention uses sturgeon processing byproducts—fish skin—as raw material and extracts active peptides through specific enzymatic hydrolysis and fractionation processes. This not only realizes the in-depth development and high-value utilization of natural biological resources and reduces the production cost of whitening active ingredients, but also meets the requirements of green and sustainable development. At the same time, the precise optimization of flavor protease and related process parameters ensures the yield and purity of the target active peptide GLPGISGGGY, laying the foundation for industrial application.

[0025] (5) The excellent melanin-inhibiting activity and high safety exhibited by the polypeptide SSCH-T2 provided by this invention make it not only a promising ingredient for use in mid-to-high-end whitening cosmetics and skincare products, but also provide key theoretical basis and experimental data support for the development of therapeutic drugs for skin diseases such as melasma, freckles, and post-inflammatory hyperpigmentation. Its well-defined binding sites (such as Glu213 and Arg216 residues in the DNA-binding domain of MITF protein) provide important structural templates for the subsequent development of novel small-molecule melanin inhibitors. Attached Figure Description

[0026] The accompanying drawings are included to provide a further understanding of the embodiments and are incorporated in and constitute a part of this specification. The drawings illustrate embodiments and, together with the description, serve to explain the principles of the invention. Other embodiments and many anticipated advantages of the embodiments will be readily recognized as they become better understood through reference to the following detailed description. Elements in the drawings are not necessarily to scale. The same reference numerals refer to corresponding similar parts.

[0027] Figure 1 This is a flowchart of a method for preparing a melanin production inhibitory peptide according to an embodiment of the present invention; Figure 2 This describes the effect of SSCH-T1-10 on melanin content in B16F10 cells according to an embodiment of the present invention. Figure 3 This is the inhibitory effect of SSCH-T2 on mTYR monophenolase according to an embodiment of the present invention; Figure 4 This is the inhibitory effect of SSCH-T2 on mTYR diphenolase according to an embodiment of the present invention; Figure 5 This is the inhibition mechanism of mTYR by SSCH-T2 according to an embodiment of the present invention; Figure 6 This is an in vitro binding curve of SSCH-T2 and mTYR according to an embodiment of the present invention; Figure 7A This is a schematic diagram of the two-dimensional (2D) chemical structure of SSCH-T2 according to an embodiment of the present invention; Figure 7B This is a three-dimensional (3D) crystal structure model of SSCH-T2 and receptor protein mTYR according to an embodiment of the present invention (based on PDB ID: 2Y9W). Figure 7C This is a detailed view of the overall conformation and local interaction of the SSCH-T2 and mTYR combined pocket according to an embodiment of the present invention; Figure 7D This is a two-dimensional planar view (interaction map) of the interaction between SSCH-T2 and key amino acid residues in the active site according to an embodiment of the present invention.

[0028] Figure 8 This describes the effect of SSCH-T2 on the proliferation of B16F10 cells according to an embodiment of the present invention. Figure 9A This is a bar chart showing the relative content of intracellular melanin after treatment with different concentrations of SSCH-T2 according to an embodiment of the present invention; Figure 9B This is a visual image of melanin precipitation observed after cell lysis following treatment with different concentrations of SSCH-T2 according to an embodiment of the present invention; Figure 9C This is a bar chart illustrating the effect of different concentrations of SSCH-T2 on the relative activity of intracellular TYR after treatment according to an embodiment of the present invention. Figure 10AThis is a Western blot image showing the effect of SSCH-T2 on the expression of melanin synthesis-related proteins in B16F10 cells according to an embodiment of the present invention. Figure 10B This is a normalized bar chart showing the expression levels of melanin synthesis-related proteins in each group of cells according to an embodiment of the present invention. Figure 11A This is a Western blot image showing the effect of SSCH-T2 on AKT, CREB and ERK signaling pathway-related proteins in B16F10 cells according to an embodiment of the present invention. Figure 11B This is a relative expression statistical graph of corresponding signaling pathway-related proteins and their phosphorylation levels according to an embodiment of the present invention; Figure 12 This is an example of the effect of AKT inhibitor treatment on SSCH-T2 cell melanin production according to an embodiment of the present invention; Figure 13 This invention relates to the effect of SSCH-T2 on melanin production in cells under TYR overexpression treatment according to an embodiment of the present invention. Figure 14 This refers to the interaction between SSCH-T2 and TYR in B16F10 cells according to an embodiment of the present invention. Figure 15 This describes the effect of SSCH-T2 on zebrafish embryo activity according to an embodiment of the present invention; Figure 16 This describes the effect of SSCH-T2 on melanin synthesis in zebrafish according to an embodiment of the present invention. Figure 17A This is a Western blot image showing the effect of SSCH-T2 on the expression of melanin synthesis-related proteins in zebrafish according to an embodiment of the present invention. Figure 17B This is a statistical chart showing the relative expression levels of key proteins in zebrafish according to an embodiment of the present invention. Detailed Implementation

[0029] The technical solutions in the embodiments will be clearly and completely described below with reference to the accompanying drawings. Similar component reference numerals in the drawings represent similar components. Obviously, the embodiments described below are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0030] It should be understood that, when used in this specification and the appended claims, the terms "comprising" and "including" indicate the presence of the described features, integrals, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integrals, steps, operations, elements, components and / or collections thereof.

[0031] It should also be understood that the terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms unless the context clearly indicates otherwise.

[0032] Firstly, this invention provides a melanin production inhibitory peptide with the amino acid sequence GLPGISGGGY (Gly-Leu-Pro-Gly-Ile-Ser-Gly-Gly-Gly-Tyr). This sequence is rich in glycine (Gly) and contains a tyrosine (Tyr) residue at the end, similar in structure to tyrosinase substrates. This specific sequence composition constitutes the molecular basis of its biological activity. Specifically, the abundant Gly residues in the sequence, as characteristic residues of collagen, endow the peptide with extremely high spatial flexibility due to its side chain consisting only of hydrogen atoms, making it easy to overcome steric hindrances and enter the active site pocket of TYR. Simultaneously, the hydrophobic residues such as leucine (Leu), isoleucine (Ile), and proline (Pro) in the sequence enhance the peptide's lipophilicity, ensuring its ability to penetrate the cell membrane barrier and enter melanocytes to exert its effects. In addition, serine containing hydroxyl groups (Ser) enhances the binding stability of peptides to target proteins by forming hydrogen bonds (Acc / Don), while the terminal Tyr residues, due to their high structural similarity to the natural substrate of tyrosinase (L-tyrosine), constitute the core functional site for competitive inhibition, thereby significantly downregulating the efficiency of melanin synthesis at the molecular level.

[0033] In some specific embodiments, the melanin production inhibitory peptide also includes a derived peptide having the same function by substitution, deletion, or addition of one or more amino acid residues in the sequence GLPGISGGGY.

[0034] In some specific embodiments, the structural formula of the melanin production inhibitory peptide is as follows: .

[0035] Secondly, embodiments of the present invention provide a flowchart of a method for preparing a melanin production inhibitory peptide, as shown below. Figure 1 As shown, the method includes: S1. Sturgeon skin is cleaned, degreased, and treated to remove impurities and proteins. Then, it is swollen and homogenized under acidic conditions to obtain a fish skin homogenate.

[0036] In some specific embodiments, the skin of large hybrid sturgeon is used as the raw material. First, the sturgeon skin is thawed, washed, and cut into small pieces (2 x 2 cm). 2 To remove non-collagenous proteins, fish skin was soaked in 0.1 M NaOH solution at a solid-liquid ratio of 1:10 (w / v) and treated with shaking in a shaker at 4°C for 24 hours. Subsequently, it was degreased with 10% n-butanol solution at a solid-liquid ratio of 1:10 (w / v) for 24 hours, with the solvent changed every 12 hours. After treatment, the fish skin was washed with distilled or deionized water until neutral, and then 3% lactic acid solution was added and stirred for 2 hours to allow it to swell fully. Finally, the swollen fish skin was homogenized into a paste using a homogenizer to obtain the fish skin homogenate for later use.

[0037] S2, add 6000-6100 U / g protease to the fish skin homogenate, and perform enzymatic hydrolysis at a temperature of 34-36°C and a pH of 6.0-7.0 to obtain an enzymatic hydrolysate containing sturgeon skin collagen polypeptide.

[0038] In some specific embodiments, the fish skin homogenate pretreated in step S1 was hydrolyzed with flavor protease for 6 hours at pH=7, enzyme activity of 6,068.4 U / g, and temperature of 35.5°C to obtain an enzymatic hydrolysate containing sturgeon skin collagen hydrolysate (SSCH).

[0039] S3, the enzymatic hydrolysate is separated into components, small molecular weight components are collected, and the melanin production inhibitory peptide is obtained by sequence identification and virtual screening based on melanin production-related targets.

[0040] In some specific embodiments, the melanin production-related targets are selected from at least one of microphthalmia-related transcription factors, tyrosinase, tyrosinase-related protein-1, and tyrosinase-related protein-2; the virtual screening includes: performing molecular docking simulation with the protein structure of MITF and / or TYR using the polypeptide sequence, and screening target peptides based on binding energy scores.

[0041] In some specific embodiments, step S3 includes: S31, the enzymatic hydrolysate is separated by passing it through an ultrafiltration membrane with a molecular weight cutoff of 3kDa, and the filtrate is collected; S32, after desalting the filtrate, the melanin production inhibitory peptide was screened by combining high-performance liquid chromatography-tandem mass spectrometry analysis with MITF molecular docking simulation.

[0042] Step S32 includes: S321, a peptide database was established using mass spectrometry analysis to screen for sequences that meet the conditions -logP>20 and have a molecular weight of less than 1 kDa; bioactivity was scored using Peptide Ranker, and sequences with scores greater than 0.75 were selected to construct a 3D structure database. S322, a 3D-QSAR pharmacophore model is constructed based on known antioxidant active peptides, and candidate sequences are obtained by screening from the peptide database by combining ADMET property prediction and skin toxicity prediction functions; S323, using MITF as the receptor protein, pharmacophore features are established based on the key amino acid sites of the receptor protein and the DNA binding domain, and molecular docking is performed on the candidate sequences to select several sequences with high absolute values ​​of binding energy scores as candidate peptides. S324, The candidate peptides are screened by evaluating melanin inhibition activity at the cellular level to obtain the melanin production inhibitory peptide.

[0043] In some specific embodiments, ultrafiltration separation was performed using a 3kDa ultrafiltration membrane to obtain low molecular weight SSCH-L, which was then freeze-dried under vacuum and stored at -20°C for later use. SSCH-L was desalted using a C18 desalting column and analyzed using nano-high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) to establish an SSCH-L peptide database. Specifically, mass spectrometry was used for analysis to establish the SSCH-L peptide database. 3μL of sample was loaded and analyzed by LC-MS / MS with an online nano-spray ion source. The column current was controlled at 300 nL / min, the electrospray voltage at 2kV, and the column temperature at 40°C. The mass spectrometer operated in data-dependent acquisition mode, automatically switching between MS and MS / MS acquisition. The primary MS analysis parameters were set as follows: resolution = 70000, mass / charge (m / z) = 100–150, maximum injection time = 50 ms, and automatic gain control target (AGCtarget) = 3e6. The analysis parameters for High-energy C-trap dissociation tandem mass spectrometry (HCD-MS / MS) were set as follows: resolution = 17500, collision energy = 28, maximum injection time = 45 ms, isolation window = 2 m / z, AGC target = 1e5, and dynamic ejection time = 30 s. Based on the mass spectrometry analysis results, peptide sequences that met the condition "-logP > 20" and had a molecular weight less than 1 kDa were screened. The Peptide Ranker program was used to predict the bioactivity of these peptides, and peptides with an activity score greater than 0.75 were selected. Subsequently, a 3D structure database of these highly active peptides was constructed in Discover Studio software. Using the Discover Studio client software, 20 active peptides with oxygen free radical scavenging ability were selected as the training set, and 10 peptides with antioxidant activity were selected as the test set to construct a 3D-QSAR antioxidant pharmacophore model. During the modeling process, energy minimization was performed on the molecules, and molecular features were matched, including hydrophobicity (H), hydrophobic aromaticity (HA), hydrogen bond acceptor (HBA), positively charged ionizable (PI), and hydrogen bond donor (HBD). Ten models were generated using the 3D QSAR module of the DS software. The training set consisted of all InputLigands, the validation parameter was set to True, and the test set consisted of all InputTestLigands. The minimum distance between features was set to 1.5, the energy threshold was set to 10, and other parameters remained at their default values. Based on the statistical data and correlation curve analysis from the test set validation, the model with the best fit was selected for peptide library screening among the ten constructed 3D-QSAR pharmacophore models.Simultaneously, using the ADMET property prediction functions (including absorption, distribution, metabolism, excretion, and toxicity) and skin toxicity prediction functions of DS software, 86 bioactive polypeptide sequences with a molecular weight not exceeding 1 kDa were screened. In studying the interaction of MITF (PDB:4ATI) as a receptor protein, its 3D structure was first downloaded from the RCBSPDB database, and preprocessed using MOE (Molecular Operating Environment) software to remove excess water molecules, add hydrogen, add charge, and optimize energy. Based on the 2D interaction diagram of MITF and DNA, the key amino acids in the MITF active pocket were identified, including Glu213 and Arg216 of the A chain, and Asn242, Lys243, Arg217, and Arg214 of the B chain. Subsequently, a pharmacophore model in MOE was established, incorporating features of AtomQ, Hydrogen Bond Donor (Don), and Hydrogen Bond Acceptor (Acc). Based on this model, molecular docking was performed on 86 sturgeon antioxidant peptide sequences. The structures of the 10 peptides with the highest absolute binding energy scores and their molecular docking results with mushroom tyrosinase (mTYR) are detailed in Table 1. The results show that the minimum binding energies of these 10 peptides with mTYR are all below -5 kcal / mol, indicating that the docking results have high reliability.

[0044] Table 1. Structure and simulated molecular docking results of SSCH-T1-10

[0045] To further screen for peptides that inhibit melanin production in B16F10 melanoma cells, 10 peptides were co-cultured with B16F10 cells for 48 hours, and their effects on melanin content were measured. The results are as follows: Figure 2 As shown, Figure 2 The effect of SSCH-T1-10 on melanin content in B16F10 cells according to an embodiment of the present invention is illustrated. Significance differences in all experimental data of the present invention are shown using… express, P < 0.05 P < 0.01, P < 0.0001, ns = nosignificant. As shown in the figure, peptides SSCH-T2 and SSCH-T4 exhibited significant inhibitory effects, while other peptides showed less than ideal inhibitory effects. Comparing SSCH-T2 and SSCH-T4, SSCH-T2 achieved a melanin inhibition rate of 30.61% at a concentration of 100 µmol / L, demonstrating superior inhibition compared to SSCH-T4. Therefore, SSCH-T2 was selected for further in-depth research.

[0046] Example 1 SSCH-T2 was dissolved in ddH2O to prepare a 10 mmol / L stock solution, which was then appropriately diluted according to the experimental protocol. Before the experiment, double-distilled water and phosphate buffer were preheated in a 37°C water bath. To a 3 mL reaction mixture, 1.60 mL of double-distilled water, 750 µL of 0.05 mol / L phosphate buffer, 500 µL of 1 mg / mL tyrosine solution, and 50 µL of SSCH-T2 solutions of different concentrations were added sequentially. After thorough mixing, 100 µL of enzyme solution was added, and the absorbance was measured at 475 nm. Enzyme activity curves were plotted using GRAPHWIN software; the slope of the curve represents the steady-state activity of the enzyme, while the intercept on the x-axis corresponds to the lag time. (Reference) Figure 3 , Figure 3 The inhibitory effect of SSCH-T2 on mTYR monophenolase according to the present invention is shown, wherein... Figure 3 A represents the monophenolase reaction kinetic curve, and the 0-4 curves represent SSCH-T2 concentrations of 0, 100, 200, 300, and 400 μmol / L. Figure 3 B represents steady-state tyrosinase activity. Figure 3 C represents the lag time. TYR possesses monophenolase activity, catalyzing the conversion of L-Tyr to L-DOPA in melanin synthesis. Using L-Tyr as a substrate, the effect of SSCH-T2 on TYR monophenolase activity was evaluated by measuring the amount of L-DOPA generated. Figure 3 A represents the effect of SSCH-T2 on the oxidation reaction catalyzed by mTYR monophenolase. In the initial stage of the reaction, i.e., on the left side of the curve, the product accumulation rate is relatively slow. This may be because, in the initial stage of the reaction, the binding of the substrate L-Tyr to the enzyme's active site has not yet reached saturation, or the enzyme activity has not been fully utilized, resulting in a low product formation rate. As the reaction proceeds, the interaction between the substrate and the enzyme gradually strengthens, the enzyme's catalytic efficiency increases, and the product accumulation rate begins to accelerate. Figure 3 B represents homeostatic enzyme activity. The homeostatic enzyme activity of tyrosinase gradually decreases with increasing SSCH-T2 concentration. Figure 3C represents the lag effect of monophenolase oxidation catalysis in the SSCH-T2 treatment group. As the concentration of SSCH-T2 increases, the lag time of the monophenolase catalysis reaction lengthens. At an SSCH-T2 concentration of 400 μmol / L, the enzyme activity decreases to 64.25%, with a lag time of 3.422 min.

[0047] Example 2 The effect of L-DOPA on the activity of mTYR diphenolase was investigated using L-DOPA as a substrate. SSCH-T2 was dissolved in ddH2O water to prepare a 10 mmol / L stock solution. Before the experiment, double-distilled water and phosphate buffer were preheated in a 37°C water bath. In a 3 mL reaction system, 1.80 mL of double-distilled water, 750 µL of 0.05 mol / L phosphate buffer, 300 µL of 1 mg / mL L-DOPA, and 50 µL of SSCH-T2 solutions of different concentrations were added sequentially. After mixing, 100 µL of enzyme solution was added and quickly mixed. The absorbance was continuously measured at 475 nm using a spectrophotometer at a constant temperature of 37°C, for a total of 300 data points, with a 2-second interval between each data point. Enzyme activity was measured as the amount of product generated per minute. After the reaction was complete, the steady-state enzyme activity was determined by the slope of the calculated absorbance change line. The inhibition rate is calculated as the percentage of residual activity compared to the initial activity. Under the catalysis of this enzyme, L-DOPA undergoes an oxidative dehydrogenation reaction to convert to dopaquinone. Using L-DOPA as a substrate, the effect of SSCH-T2 on TYR diphenolase activity was investigated. The results are as follows... Figure 4 As shown, Figure 4 The inhibitory effect of SSCH-T2 on mTYR diphenolase according to the present invention is shown. The diphenolase activity gradually decreased with increasing SSCH-T2 concentration, exhibiting a concentration-dependent effect. At a SSCH-T2 concentration of 0.7 mM, the diphenolase activity decreased to 63.17%. This indicates that SSCH-T2 has a significant inhibitory effect on TYR diphenolase activity. Further investigation was conducted on the mechanism of action of SSCH-T2 on the mTYR-catalyzed oxidation of L-DOPA. In the reaction system, the L-DOPA concentration was kept constant, and the enzyme amount was varied to determine the effect of different concentrations of SSCH-T2 on the diphenolase catalytic efficiency. Furthermore, Figure 5 The inhibition mechanism of mTYR by SSCH-T2 according to the present invention is shown. A set of straight lines passing through the origin is obtained by plotting residual enzyme activity against enzyme amount. For example... Figure 5 As shown, the slope of the straight line gradually decreases with increasing SSCH-T2 concentration, indicating that the inhibitory effect of SSCH-T2 is reversible. It can reduce the catalytic rate of the diphenolase on the substrate, but will not cause irreversible denaturation and inactivation of the enzyme.

[0048] Example 3 The experiment was conducted at 25°C on a BIAcore T200 using a CM5 sensor chip, and the data were analyzed using GE Healthcare's BIAcore T200 evaluation software. A brief description is as follows: In the chambers of the CM5 chip, cells were activated for 10 minutes at a rate of 10 µL / min with a mixture of 200 µmol / L EDC and 50 µmol / L NHS. Subsequently, 10 µL of mTYR protein was fixed for 420 seconds at a rate of 10 µL / min in 10 mM sodium phosphate acetate solution (pH 4.0), repeated twice. Cells were then blocked for 10 minutes at a rate of 10 µL / min with 1.0 M Tris-HCl (pH 7.0). The control channel underwent similar procedures, but fixation was performed using PBS at pH 4.0. Both cell lines were then equilibrated with PBS. SSCH-T2 stock solution (10 mM) was diluted with DMSO to different concentrations (0, 62.5, 125, 250, 500, 1000 µmol / L) and flowed through cells at a flow rate of 30 µL / min for 150 seconds. After the flow experiment, cells were regenerated for 5 minutes with 10 mM glutamate-hydrochloride (pH 1.5) at a flow rate of 10 µL / min. Data were acquired using BIAcore T200 Control software, and the data from channel 2 were subtracted from those from channel 1. A 1:1 Langmuir binding model was used for global fitting to calculate the dissociation equilibrium constant. Data were exported to OriginPro 2024 to generate graphs. The interaction between SSCH-T2 and mTYR was studied using surface plasmon resonance (SPR) technology. SPR technology, based on a special optical phenomenon, can monitor the interaction between biomolecules in real time without labeling and has high sensitivity. In the experiment of SSCH-T2 and mTYR interaction, the mTYR protein was immobilized on the surface of the sensor chip. When the SSCH-T2 solution flows through the chip, if it interacts with the immobilized mTYR protein, it will cause a change in the refractive index of the chip surface. This change will be detected by the SPR system, thus revealing the interaction between the two. The results are as follows... Figure 6 As shown, Figure 6 The in vitro binding curves of SSCH-T2 and mTYR according to the present invention are shown. SSCH-T2 can directly bind to the mTYR protein immobilized on the surface of the sensor chip, and the binding signal gradually increases with concentration gradients of 0, 62.5, 125, 250, 500, and 1000 µmol / L. Nonlinear curve fitting using Origin yielded an equilibrium dissociation constant KD value of 227.22 ± 67.24 µmol / L for the binding of SSCH-T2 and mTYR, indicating that SSCH-T2 and mTYR bind in vitro.

[0049] Example 4 Using MOE software, the interaction mechanism between SSCH-T2 and mTYR can be simulated, allowing for a deeper exploration of their interaction. First, the PDB file of mTYR (PDB code: 2Y9W) was retrieved and downloaded from the PDB data center. This file was then processed in MOE software, including removing water molecules from the protein crystal, removing the original ligands, and minimizing energy. Next, the chemical structure of SSCH-T2 was drawn using ChemDraw 2D software, and structural optimization and energy minimization were performed. Subsequently, the chemical structure of SSCH-T2 was converted into a 3D model using ChemDraw 3D, and molecular docking simulations were performed on the region near the copper ion active site in mTYR to highlight the binding mode of SSCH-T2 and mTYR and key interacting amino acid residues. Finally, the molecular docking results from MOE were visualized and analyzed using PyMOL software, and a molecular docking interaction diagram of SSCH-T2 and mTYR was plotted to visually demonstrate the interaction forces between them. Molecular docking was performed using the MOEDOCK module with mTYR (PDB:2Y9W) as the acceptor and SSCH-T2 as the ligand. Figures 7A-7D The following is a simulation diagram of the SSCH-T2 and mTYR molecular docking according to the present invention; wherein, Figure 7A Here is a 2D structural diagram of SSCH-T2; Figure 7B Here is the mTYR 3D structure diagram (PDB:2Y9W); Figure 7C The images show the overall 3D model and the local 3D model of molecular docking. Figure 7D This is a molecular docking planar diagram. As shown, SSCH-T2 forms hydrogen bonds with the amino acids Glu67, Glu185, and Asp47 on mTYR, with bond lengths of 3.29 Å, 2.92 Å, and 3.15 Å, respectively. It also forms a pi-pi interaction with His182, exhibiting good binding activity, with a binding energy of -9.3517 kcal / mol. Although SSCH-T2 cannot directly interact with the copper ion at the active center of mTYR, it can influence the activity of mTYR through hydrogen bonding and hydrophobic interactions.

[0050] Example 5 Collect 10 μL of cell suspension and count the cells on a hemocytometer. The cells are then counted at a rate of 1.5 × 10⁻⁶. 5Cells were seeded at a density of 100 μL per well in 96-well plates. When the cell density reached 30%-40%, a 10 mM SSCH stock solution was prepared using deionized water and diluted with complete culture medium to different concentrations of 0, 100, 200, 300, 400, 500, 600, 700, 800, and 900 µmol / L. 100 μL of different concentrations of SSCH solution was added to each well, with 5 replicates per group. After incubating the cells in SSCH-containing medium for 48 hours, the medium was discarded, and the cells were washed three times with 1×PBS. Subsequently, 10 μL of 5 mg / mL LMTT (3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide) and 90 μL of DMEM medium (Dulbecco's Modified Eagle Medium, DMEM) were added to each well, and the reaction was carried out at 37°C in the dark for 4 hours. After removing DMEM, add 100 μL of DMSO to each well and shake in a microplate reader for 10 min at room temperature. Allow the blue-purple formazan crystals to dissolve completely, and measure the absorbance at 490 nm. The cell proliferation rate is calculated using the formula: Proliferation rate (%) = (Experimental group absorbance / Control group absorbance) × 100%.

[0051] In the mitochondria of living cells, succinate dehydrogenase can reduce MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazol bromide) to water-insoluble blue-violet formazan crystals, which accumulate intracellularly. Inactive cells cannot perform this reaction. Therefore, in OD... 490 The absorbance was measured; a higher absorbance indicates stronger cell viability and correspondingly lower cytotoxicity. The inhibitory effect of SSCH-T2 on B16F10 cell proliferation was detected using the MTT assay. Figure 8 It can be seen that the cell proliferation rate was not inhibited after treatment with SSCH-T2 for 48 h at a concentration of 700 µmol / L compared with the control group, indicating that SSCH-T2 has no side effects on B16F10 cells at a concentration of 700 µmol / L.

[0052] Example 6 Changes in intracellular melanin content and TYR activity were detected in B16F10 cells after SSCH-T2 treatment for 48 h, as referenced. Figures 9A-9C , Figures 9A-9C The inhibitory effect of SSCH-T2 according to the present invention on melanin content and TYR activity in B16F10 cells is shown, wherein, Figure 9A A bar chart showing the relative melanin content in cells after treatment with different concentrations of SSCH-T2; Figure 9B The image shows the appearance of melanin precipitate after lysis of B16F10 cells in each group; Figure 9CA bar chart showing the effect of different concentrations of SSCH-T2 on the relative activity of intracellular TYR. Results are as follows. Figure 9A , Figure 9B As shown, compared with the control group, the intracellular melanin content was significantly reduced, and its inhibitory effect on melanin content increased with increasing SSCH-T2 concentration. In the 100 µmol / L treatment group, the melanin content decreased by 30.61% compared with the control group, and was superior to the positive control arbutin, indicating that SSCH-T2 can significantly inhibit melanin production. To detect the inhibitory effect of SSCH-T2 on TYR activity, B16F10 cells were treated at concentrations of 0, 30, 60, and 100 µmol / L for 48 h, and the crude enzyme solution extracted from the cells was reacted with dopamine. The results are as follows. Figure 9C As shown, compared with the control group, intracellular TYR activity was significantly reduced, and the inhibitory effect of SSCH-T2 on TYR activity increased accordingly with increasing SSCH-T2 concentration. In the 100 µmol / L treatment group, the melanin content in cells decreased by 33.31% compared with the control group.

[0053] SSCH-T2 exhibited a significant melanin-inhibiting effect on B16F10 cells. To further explore its mechanism of action, B16F10 cells were treated with different concentrations of SSCH-T2 (0, 30, 60, and 100 μmol / L), and the results are as follows: Figures 10A-10B As shown, Figures 10A-10B This illustrates the role of SSCH-T2 in a key protein of intracellular melanin synthesis according to an embodiment of the present invention. Figure 10A This is a Western blot image showing the effect of SSCH-T2 on the expression of melanin synthesis-related proteins in B16F10 cells. Figure 10B Normalized bar charts showing the expression levels of melanin synthesis-related proteins in each group of cells. In cells treated with SSCH-T2, the expression levels of MITF, TYR, TYRP1, and TYRP2 were significantly lower than in the control group. When the drug concentration reached 100 μmol / L, SSCH-T2 showed the most significant inhibitory effect on MITF, TYR, TYRP1, and TYRP2. The experimental results indicate that SSCH-T2 effectively inhibits melanin production by suppressing the expression of the melanin transcription factor MITF, thereby downregulating the expression of key proteins TYR, TYRP1, and TYRP2 in the melanin synthesis pathway.

[0054] Further analysis was conducted on the expression levels of proteins related to the melanin production signaling pathway in B16F10 cells treated with SSCH-T2 for 48 hours. The results are as follows: Figures 11A-11B As shown, Figures 11A-11BThe effects of SSCH-T2 according to an embodiment of the present invention on proteins related to the protein kinase B (AKT), cAMP-response element binding protein (CREB), and extracellular signal-regulated kinase (ERK) signaling pathways are illustrated. Figure 11A This is a Western blot image showing the effects of SSCH-T2 on proteins related to the AKT, CREB, and ERK signaling pathways in B16F10 cells. Figure 11B This is a statistical graph showing the relative expression levels of proteins related to the corresponding signaling pathway and their phosphorylation levels. After SSCH-T2 treatment, the expression ratios of phosphorylated CREB (p-CREB) and CREB, as well as phosphorylated ERK (p-ERK) and ERK, did not change significantly. However, the expression levels of phosphorylated AKT (p-AKT) and phosphorylated GSK-3β (p-GSK-3β) increased significantly in B16F10 cells, especially at a drug concentration of 100 μmol / L. Based on this, it is hypothesized that SSCH-T2 promotes p-AKT expression, thereby inducing phosphorylation of GSK-3β at the Ser9 site. Phosphorylation of GSK-3β at the Ser9 site leads to a decrease in MITF levels, thereby inhibiting the binding of MITF to the M-box and downregulating the expression of TYR and TYRP1. Furthermore, AKT activation may also induce MITF phosphorylation at the Ser409 site, promoting decreased MITF stability and degradation, ultimately leading to reduced melanin production.

[0055] Example 7 To further verify whether SSCH-T2 affects melanin synthesis through the regulation of the AKT signaling pathway, refer to Figure 12 , Figure 12 The effect of AKT inhibitor treatment on melanin production in cellular cells is illustrated according to an embodiment of the present invention. As shown in the figure... Figure 12 A shows the melanin production of B16F10 cells after treatment with AKT inhibitors and SSCH-T2. It is clearly observed that the melanin content of cells treated with the AKT inhibitor AZD5363 was significantly increased compared to the control group, while the melanin production in the SSCH-T2 treatment group was significantly reduced compared to the inhibitor group. Furthermore, the reduction in melanin content was more significant in the SSCH-T2 treatment group compared to the group treated with both the AKT inhibitor and SSCH-T2 (200 µmol / L). Figure 12B shows the expression level of p-AKT protein in B16F10 cells. The results showed that p-AKT expression was significantly lower in the AZD5363 treatment group compared to the control group, while in the SSCH-T2 treatment group, p-AKT expression was significantly increased compared to the inhibitor group. Furthermore, p-AKT expression was higher in the SSCH-T2 alone treatment group compared to the treatment group using both the AKT inhibitor and SSCH-T2. These experimental results conversely confirm that SSCH-T2 can activate AKT, thereby affecting melanin production, further supporting the mechanism by which SSCH-T2 inhibits melanin production by regulating the AKT signaling pathway.

[0056] To verify whether SSCH-T2 directly affects melanin production by regulating TYR protein, the effect of SSCH-T2 on melanin production in TYRB16F10 cells overexpressing TYR was examined. Cells were transfected with 2 μg of TYR overexpression plasmid to increase TYR expression, and then cultured in 200 µmol / L SSCH-T2 medium for 48 h. The experimental results are as follows. Figure 13 As shown in the figure. The results showed that the melanin content of cells treated with TYR plasmid overexpression increased significantly, while the melanin production in the SSCH-T2 treatment group was significantly reduced compared to the overexpression group. The experimental results further confirmed that SSCH-T2 can affect melanin production by reducing TYR expression.

[0057] Example 8 To further investigate the interaction between SSCH-T2 and TYR, the distribution of 7-amino-4-methylcoumarin (AMC)-labeled SSCH-T2 and TYR in B16F10 cells was analyzed. B16F10 cells were incubated with AMC-labeled SSCH-T2 at 37°C for 48 h. TYR protein was stained with rhodamine-labeled anti-TYR antibody, and the cytoskeleton was visualized using FITC-conjugated secondary antibody (FITC), exhibiting green fluorescence. Cell images were observed using confocal microscopy, and the results are shown below. Figure 14 As shown, Figure 14The interaction between SSCH-T2 and TYR in B16F10 cells according to an embodiment of the present invention is illustrated. Green represents the cytoskeleton, red represents TYR protein, and blue represents AMC-SSCH-T2. In the control group, the signal intensity of TYR was significantly higher than that in the treatment group, indicating that TYR expression was high in the absence of SSCH-T2 intervention. In the AMC-SSCH-T2 treatment group, a weakened TYR signal intensity was observed, and a distinct blue signal appeared in the cells, indicating that AMC-SSCH-T2 could enter the cells and interact with TYR, leading to a decrease in TYR expression. In the TYR gene knockdown (shTYR) and SSCH-T2 combined treatment group, both TYR and AMC-SSCH-T2 signals were significantly weakened. This indicates that when TYR expression is knocked down, the efficiency of AMC-SSCH-T2 entry into the cells is also reduced, further supporting the possibility that TYR may be a target of SSCH-T2. AMC-SSCH-T2 can inhibit TYR expression in B16F10 cells, possibly by directly interacting with TYR and downregulating its expression. The reduction in TYR may also affect the efficiency of AMC-SSCH-T2 entry into cells. These results indicate that AMC-SSCH-T2 can penetrate the cell membrane and enter B16F10 cells, co-localizing with endogenous TYR. In the shTYR group, the intracellular fluorescence signal of AMC-SSCH-T2 significantly decreased with decreasing target protein expression, further validating that TYR is the core target for the inhibitory effect of SSCH-T2 within cells.

[0058] Example 9 To investigate the toxic effects of SSCH-T2 on zebrafish, zebrafish embryos were treated with different concentrations (0, 500, 1000, 1500, 2000 μmol / L) of SSCH-T2 for 48 h, and their mortality rate was observed. The results are as follows: Figure 15 As shown, Figure 15 The effect of SSCH-T2 on zebrafish embryo viability according to an embodiment of the present invention is illustrated. As shown in the figure, zebrafish embryos could not hatch under 2000 μmol / L SSCH-T2 treatment, indicating that this concentration was lethal to zebrafish embryos or severely hindered normal embryonic development. At a concentration of 1500 μmol / L, zebrafish exhibited abnormalities such as whitish eyes and abdominal protrusion, indicating that this concentration had an adverse effect on the health of zebrafish, resulting in developmental malformations, but did not lead to complete mortality. Within the concentration range of 0-1000 μmol / L, zebrafish were able to grow normally, and no obvious toxic effects were observed, indicating that this concentration range is relatively safe for zebrafish. Therefore, the concentration range of 0-1000 μmol / L was selected for subsequent zebrafish experiments.

[0059] Example 10 To investigate the effect of SSCH-T2 on melanin production in zebrafish, zebrafish embryos were treated with different concentrations (0, 300, 600, and 1000 μmol / L) of SSCH-T2 for 48 h. Melanin deposition on the fish surface was observed using a stereomicroscope. (Reference) Figure 16 , Figure 16 The effect of SSCH-T2 on melanin synthesis in zebrafish, according to an embodiment of the present invention, is illustrated. Figure 16 A represents the effect of SSCH-T2 on melanin production on the body surface of zebrafish; Figure 16 B represents the effect of SSCH-T2 on melanin content in zebrafish embryos; Figure 16 C represents the effect of SSCH-T2 on the TYR viability of zebrafish embryos. (e.g.) Figure 16 As shown in Figure A, compared with the control group, zebrafish treated with SSCH-T2 showed a significant reduction in the number of melanin granules in the yolk sac region and the tail ridge, and the granules were also paler in color. This indicates that SSCH-T2 can inhibit melanin deposition on the zebrafish body surface. Furthermore, compared with the control group, the melanin content in the zebrafish body (…) Figure 16 B) and TYR activity ( Figure 16 C) All values ​​decreased significantly with increasing SSCH-T2 concentration, showing a clear concentration dependence. Under treatment with 1000 μmol / L SSCH-T2, the melanin content and TYR activity of zebrafish decreased to 66.30% and 72.29% of the control group, respectively, further confirming the inhibitory effect of SSCH-T2 on melanin production on the zebrafish body surface.

[0060] SSCH-T2 exhibited a significant melanin-inhibiting effect on zebrafish. To further explore its mechanism of action, Western blotting was used to detect the expression levels of proteins related to melanin production. Zebrafish were treated with different concentrations of SSCH-T2 (0, 300, 600, and 1000 μmol / L). Figures 17A-17B As shown, Figures 17A-17B This invention illustrates the role of SSCH-T2, an embodiment of the present invention, in the synthesis of a key protein in zebrafish melanin. Figure 17A Western blot image showing the effect of SSCH-T2 on the expression of proteins related to melanin synthesis in zebrafish; Figure 17BThis figure shows the statistical distribution of the relative expression levels of key proteins in zebrafish. As indicated, using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control protein, the expression levels of MITF, TYR, TYRP1, and TYRP2 in zebrafish treated with SSCH-T2 were significantly lower than those in the control group. The inhibitory effect of SSCH-T2 on MITF, TYR, TYRP1, and TYRP2 was most significant at a drug concentration of 1000 μmol / L. The experimental results indicate that SSCH-T2 effectively inhibits melanin production in zebrafish by inhibiting the expression of the melanin transcription factor MITF, thereby downregulating the expression of key proteins TYR, TYRP1, and TYRP2 in the melanin synthesis pathway.

[0061] A novel collagen polypeptide (SSCH-T2) was obtained from sturgeon skin using enzymatic hydrolysis and targeted computer-aided virtual screening technology. Experimental results showed that SSCH-T2 effectively inhibited melanin production and exhibited lower cytotoxicity compared to traditional inhibitors. In vitro enzymatic experiments demonstrated that SSCH-T2 inhibited the activities of mushroom tyrosinase (mTYR) monophenolase and diphenolase. SPR experiments showed that SSCH-T2 can directly bind to mTYR, and their interaction mode was revealed through molecular docking simulation. Furthermore, TYR overexpression and immunofluorescence co-localization experiments indicated that SSCH-T2 may interact with TYR in B16F10 cells.

[0062] In B16F10 cell experiments, Western blotting results showed that SSCH-T2 significantly reduced the expression of TYR and MITF. Mechanistic studies revealed that SSCH-T2 inhibits MITF expression and transcriptional activity by promoting p-AKT expression and GSK-3β phosphorylation at Ser9, thereby downregulating the expression of TYR, TRP-1, and TRP-2 and suppressing melanin production. Zebrafish experiments showed that SSCH-T2 inhibited melanin content, TYR activity, and epimelanin production by downregulating the expression levels of TYR, TRP1, and TRP in zebrafish.

[0063] In summary, SSCH-T2 inhibits melanin production in both B16F10 melanoma cells and zebrafish. These findings provide theoretical and technical support for the development of innovative peptide drugs to treat pigmentary disorders.

[0064] It is evident that those skilled in the art can make various modifications and alterations to the embodiments of the present invention without departing from the spirit and scope of the invention. In this way, the invention is also intended to cover such modifications and alterations if they fall within the scope of the claims and their equivalents. The word "comprising" does not exclude the presence of other elements or steps not listed in the claims. The simple fact that certain measures are described in mutually different dependent claims does not indicate that a combination of these measures cannot be used for profit. Any reference numerals in the claims should not be considered as limiting the scope.

Claims

1. A melanin production inhibitory peptide, characterized in that, The amino acid sequence of the melanin production inhibitory peptide is GLPGISGGGY.

2. The method for preparing the melanin production inhibitory peptide as described in claim 1, characterized in that, The preparation method includes: S1, Sturgeon skin is cleaned, degreased and deproteinized, and then swollen and homogenized under acidic conditions to obtain fish skin homogenate; S2, add 6000-6100 U / g protease to the fish skin homogenate, and carry out enzymatic hydrolysis at a temperature of 34-36°C and a pH of 6.0-7.0 to obtain an enzymatic hydrolysate containing sturgeon skin collagen polypeptide; S3, the enzymatic hydrolysate is separated into components, small molecular weight components are collected, and the melanin production inhibitory peptide is obtained by sequence identification and virtual screening based on melanin production-related targets.

3. The method for preparing the melanin production inhibitory peptide according to claim 2, characterized in that, The S1 step includes the following: S11, Wash the sturgeon skin with distilled water, then cut it into 2×2cm pieces. 2 lumpy; S12, place sturgeon skin in 10 times its volume of 0.1mol / L NaOH solution and stir at 3-5°C for more than 24 hours to remove non-collagenous proteins; S13, degreased with a 10% n-butanol solution at a solid-liquid ratio of 1:10 for at least 24 hours, with the solvent replaced every 12 hours. S14, the fish skin treated in step S13 is washed with distilled water until neutral, placed in a 3% lactic acid solution and stirred for 2 hours to make the fish skin swell, and then homogenized into a paste to obtain the fish skin homogenate.

4. The method for preparing the melanin production inhibitory peptide according to claim 2, characterized in that, Step S2 includes: adding flavor protease to the fish skin homogenate at a dosage of 6,068.4 U / g under the conditions of pH=7.0 and temperature of 35.5°C, and obtaining the enzymatic hydrolysate after 6 hours of enzymatic hydrolysis.

5. The method for preparing the melanin production inhibitory peptide according to claim 2, characterized in that, The melanin production-related targets are selected from at least one of microphthalmia-related transcription factors, tyrosinase, tyrosinase-related protein-1, and tyrosinase-related protein-2; the virtual screening includes: performing molecular docking simulation with the protein structure of MITF and / or TYR using the polypeptide sequence, and screening target peptides based on binding energy scores.

6. The method for preparing the melanin production inhibitory peptide according to claim 6, characterized in that, Step S3 includes: S31, the enzymatic hydrolysate is separated by passing it through an ultrafiltration membrane with a molecular weight cutoff of 3kDa, and the filtrate is collected; S32, after desalting the filtrate, the melanin production inhibitory peptide was screened by combining high-performance liquid chromatography-tandem mass spectrometry analysis with MITF molecular docking simulation.

7. The method for preparing the melanin production inhibitory peptide according to claim 7, characterized in that, Step S32 includes: S321, a peptide database was established using mass spectrometry analysis to screen for sequences that meet the conditions -logP>20 and have a molecular weight of less than 1 kDa; bioactivity was scored using Peptide Ranker, and sequences with scores greater than 0.75 were selected to construct a 3D structure database. S322, a 3D-QSAR pharmacophore model is constructed based on known antioxidant active peptides, and candidate sequences are obtained by screening from the peptide database by combining ADMET property prediction and skin toxicity prediction functions; S323, using MITF as the receptor protein, pharmacophore features are established based on the key amino acid sites of the receptor protein and the DNA binding domain, and molecular docking is performed on the candidate sequences to select several sequences with high absolute values ​​of binding energy scores as candidate peptides. S324, The candidate peptides are screened by evaluating melanin inhibition activity at the cellular level to obtain the melanin production inhibitory peptide.

8. The method for preparing the melanin production inhibitory peptide according to claim 8, characterized in that, The key amino acid sites include at least one of Glu213 and Arg216 of chain A and Asn242, Lys243, Arg217, and Arg214 of chain B.

9. The melanin production inhibitory peptide according to claim 1, or the melanin production inhibitory peptide prepared by the method according to any one of claims 2-9, is used in the preparation of whitening cosmetics, skin care products, or drugs for inhibiting melanin production.

10. The application as described in claim 9, characterized in that, The concentration of the melanin production inhibitory peptide in the whitening cosmetics, skin care products, or drugs used to inhibit melanin production is not higher than 1000 µmol / L.

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