Pharmaceutical use of a cyanidin-3-O-glucoside-4-vinylphenol-mannoprotein complex

Abstract

This invention discloses cyanidin-3- O The use of a complex (C3GVP–MP) consisting of glucoside-4-vinylphenol (C3GVP) and mannose protein (MP) in the preparation of a medicament for treating and / or preventing melanin synthesis-related diseases. This invention provides a medicament for treating and / or preventing melanin synthesis-related diseases, the medicament containing cyanidin-3- O γ-Glucoside-4-vinylphenol-mannose protein complex. Experiments of this invention show that cyanidin-3- O The 4-glucosinolate-4-vinylphenol-mannose protein complex (C3GVP-MP) can provide nutritional intervention for melanin synthesis and is therefore used to prepare drugs for the treatment and / or prevention of melanin synthesis-related diseases.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the use of cyanidin-3-O-glucoside-4-vinylphenol-mannose protein complex in the preparation of medicines, particularly in medicines for treating and / or preventing melanin synthesis. Background Technology

[0002] Melanin is a pigment produced by melanocytes in the basal layer of the epidermis. Under normal physiological conditions, it protects the skin from damage caused by solar radiation. However, excessive production, secretion, and accumulation of melanin can darken the skin and even lead to skin diseases such as freckles, melasma, and skin cancer. This not only negatively impacts appearance but can also cause emotional and psychological distress, resulting in a decline in quality of life. Considering the adverse reactions such as dermatitis and irritation caused by many traditional medications, the search for harmless natural compounds that can inhibit melanin production has become a highly anticipated research direction.

[0003] Cyanidin-3-O-glucoside-4-vinylphenol (C3GVP) is a novel polyphenol obtained by reacting cyanidin-3-O-glucoside with p-coumaric acid. The pyran ring formed between the C4 and C5 positions endows this substance with good structural stability and biological activity. However, C3GVP is prone to self-polymerization in salt ion solutions, forming precipitates, and its normal color may be affected by non-covalent interactions after prolonged storage at room temperature. These characteristics not only limit its functional activity research but also hinder its practical application in production and processing. Mannoprotein (MP), a soluble protein derived from the yeast cell wall, can bind to various polyphenols, improving the stability and solubility of target substances.

[0004] Current research has not reported any inhibitory effects of C3GVP on melanin synthesis, and its protective mechanism remains unclear. Summary of the Invention

[0005] The primary objective of this invention is to provide the use of cyanidin-3-O-glucoside-4-vinylphenol-mannose protein complex (C3GVP-MP) in the preparation of medicaments for the treatment and / or prevention of melanin synthesis-related diseases.

[0006] A further feature of the application according to the present invention is that the drug is a drug that interferes with melanin synthesis.

[0007] Optionally, the drug that intervenes in melanin synthesis is a drug that intervenes in key factors of melanin synthesis.

[0008] The key factors for melanin synthesis are preferably tyrosinase, tyrosine-associated protein 1, and microphthalmia-associated transcription factor.

[0009] Optionally, the drug that intervenes in melanin synthesis is a drug that intervenes in melanin synthesis-related pathways.

[0010] The preferred melanin synthesis pathway is the Wnt / β-catenin signaling pathway.

[0011] Another object of the present invention is to provide a medicament for treating and / or preventing diseases related to melanin synthesis.

[0012] The therapeutic and / or preventive medicines of the present invention contain cyanidin-3-O-glucoside-4-vinylphenol-mannose protein complex.

[0013] This invention improves the stability and solubility of C3GVP by preparing a C3GVP-MP complex from cyanidin-3-O-glucoside-4-vinylphenol (C3GVP) and mannoprotein (MP).

[0014] Experiments in this invention demonstrate that the C3GVP-MP complex can reduce tyrosinase activity and inhibit melanin production in zebrafish, with the complex showing a stronger effect than the C3GVP monomer. C3GVP-MP intervenes in melanin synthesis by downregulating the mRNA expression of some factors in the Wnt / β-catenin signaling pathway, thereby downregulating the gene expression of key melanin synthesis factors such as tyrosinase, tyrosine-associated protein 1, and microphthalmopathy-associated transcription factor, thus inhibiting melanin synthesis. Therefore, the cyanidin-3-O-glucoside-4-vinylphenol-mannose protein complex (C3GVP-MP) can provide nutritional intervention for melanin synthesis and can thus be used to prepare drugs for the treatment and / or prevention of melanin synthesis-related diseases. Attached Figure Description

[0015] Figure 1 This is the molecular structure diagram of C3GVP.

[0016] Figure 2 This image shows the melanin synthesis in zebrafish. A represents photographs of different groups of zebrafish, and B represents the statistical analysis of melanin area in the heads of different groups of zebrafish.

[0017] Figure 3 This refers to the tyrosinase activity in zebrafish.

[0018] Figure 4 The results show the KEGG enrichment of differentially expressed genes.

[0019] Figures 5A to 5C The results show the GO enrichment of differentially expressed genes.

[0020] Figure 6 The values ​​represent the mRNA expression levels of key factors in melanin synthesis. Specifically, A represents the mRNA expression level of Tyr, B represents the mRNA expression level of Tyrp1a, C represents the mRNA expression level of Mitfb, and D represents the mRNA expression level of Dct.

[0021] Figure 7 The mRNA expression levels of genes in the Wnt / β-catenin signaling pathway are represented. Specifically, A represents the mRNA expression level of Gnao1a, B represents the mRNA expression level of β-catenin, C represents the mRNA expression level of Tcf712, and D represents the mRNA expression level of Lef1. Detailed Implementation

[0022] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0023] Experimental materials:

[0024] 1. Experimental reagents

[0025] Black soybean husk extract (Bozhou, Anhui); p-coumaric acid (Shanghai Yuanye Biotechnology Co., Ltd.); citric acid, sodium citrate (Shanghai Maclean Biochemical Technology Co., Ltd.); sodium benzoate, trifluoroacetic acid (Shanghai Aladdin Biochemical Technology Co., Ltd.); mannoprotein (Laflude, France); methanol (Tianjin Damao Chemical Reagent Factory); chromatographic grade formic acid (Tianjin Kemeio Chemical Reagent Co., Ltd.); chromatographic grade acetonitrile (Merck Group GmbH, Germany).

[0026] 2. Laboratory animals

[0027] Zebrafish, wild-type AB, reaching sexual maturity (3-4 months), were purchased from the National Zebrafish Resource Center. The experimental animals were housed in a zebrafish recirculating aquaculture system, with the temperature controlled at 27-28.5℃, pH maintained at 7-8, and a photocycle of 14 hours of light and 10 hours of darkness. They were fed brine shrimp twice daily. Osmolarity and pH levels were measured periodically.

[0028] 3. Animal grouping

[0029] Under a stereomicroscope, embryos with normal development 24 hours after fertilization were selected using a plastic dropper and randomly assigned to 96-well plates. The basic animal culture medium was embryo culture medium (7.0 g NaCl, 0.4 g NaHCO3, 0.1 g KCl, and 0.235 g CaCl2 dissolved in 2 L of ultrapure water, then vacuum filtered and sterilized). C3GVP-MP or C3GVP groups were supplemented with C3GVP-MP or C3GVP to a concentration of 80 μM (based on C3GVP). MP groups were supplemented with MP solution of the same concentration as the C3GVP-MP groups. The plates were incubated in a biochemical incubator (28 ± 0.5 °C) for 24 h.

[0030] 4. Primer usage information

[0031] The primer sequences involved in the examples are shown in Table 1.

[0032] Table 1: PCR Primer Information

[0033]

[0034]

[0035] All primers were synthesized by Shanghai Sangon Biotech Co., Ltd.

[0036] Experimental methods

[0037] I. Preparation and Identification of C3GVP and C3GVP-MP Complexes

[0038] Black soybean husk extract was dissolved in a 0.1M citrate-sodium citrate buffer solution at pH 3.2 to achieve a final concentration of 1.0 mM for cyanidin-3-O-glucoside. Coumaric acid was added to achieve a molar ratio of 10:1 to cyanidin-3-O-glucoside, and 200 mg / mL sodium benzoate was added and mixed thoroughly. The reaction system was placed in a constant temperature shaker at 30℃ and 100 rpm in the dark for 30 days. After the reaction, the solution was centrifuged at 10,000 rpm for 5 min to remove the precipitate. The supernatant was purified by medium-pressure liquid chromatography. A medium-pressure liquid chromatography column (H100 cm × φ6 cm) was packed using a wet packing method, with approximately 1.2 kg of C18 packing material with a particle size of 22 μm. The column was equilibrated with methanol before use. The chromatographic settings were as follows: mobile phase A was 0.5% trifluoroacetic acid aqueous solution, mobile phase B was 100% methanol, the sample loading volume was 60 mL, and the flow rate was 30 mL / min. The detection wavelength was 504 nm, the monitoring wavelength was 520 nm, and the collection threshold was the peak fragments with detection wavelengths greater than the monitoring wavelength. The specific gradient program is as follows: 0-5 min, 6% B; 5-10 min, 6%-15% B; 10-18 min, 15%-32% B; 18-22 min, 32%-33% B; 22-32 min, 33%-34% B; 32-47 min, 34%-35% B; 47-72 min, 35%-44% B; 72-107 min, 44%-45% B; 107-127 min, 45%-65% B; 127-197 min, 65%-65% B; 197-207 min, 65%-100% B; 207-237 min, 100% B; 237-239 min, 100%-50% B; 239-259 min, 50%-6% B; 259-264 min, 6% B. After removing methanol from the collected fraction by vacuum concentration, C3GVP powder was obtained by freeze drying. 1.0 mg / mL of MP was added to a 50 μM C3GVP solution, and the mixture was stirred thoroughly and freeze-dried for 48 h to obtain a powder containing C3GVP–MP.

[0039] Anthocyanin samples were dissolved in ultrapure water and filtered through a 0.22 μm filter membrane. Qualitative analysis of anthocyanins was performed using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS). A C18 reversed-phase column (2.1 × 100 mm, 1.8 μm) was used. The mobile phase consisted of 2% formic acid in water (phase A) and 100% acetonitrile (phase B). The injection volume was 2.0 μL, the flow rate was 0.10 mL / min, the column temperature was 30 °C, and the detection wavelengths were 280 nm and 499 nm. The gradient program was as follows: 0–3 min, 6% B; 3–15 min, 6%–28% B; 15–20 min, 28%–90% B; 20–22 min, 90% B; 22–23 min, 90%–6% B; 23–28 min, 6% B. The mass spectrometry conditions were set as follows: ESI-MS analysis was performed in positive and negative ion scanning modes, with an interface voltage of 4.0 kV, an interface temperature of 300 °C, a desolvation tube temperature of 250 °C, and a heating block temperature of 400 °C. Nitrogen was used as the nebulizer, dryer, and heater, with a flow rate of 3.0 L / min for the nebulizer, 10.0 L / min for the dryer, and 10.0 L / min for the heater. Argon was used as the collision gas, and the collision chamber pressure was 230 kPa. Precursor ion scanning was performed in the range of 100–1000 m / z. After selecting the target ion, a secondary mass spectrometry product ion scan was performed to obtain the molecular structure of C3GVP (see [link to relevant documentation]). Figure 1 ).

[0040] II. Quantitative Analysis of Melanin on Zebrafish Body Surface

[0041] Under a stereomicroscope, 24 hpf normally developing embryos were selected using a plastic dropper and randomly assigned to 96-well plates, 5 embryos / well. After the original culture water was aspirated with a pipette, 200 μL of C3GVP-MP solution, or C3GVP solution (80 μM based on C3GVP), or MP solution with the same MP concentration as the C3GVP-MP group, was added to each well. The embryo culture water served as a control group. The plates were incubated in a biochemical incubator (28±0.5℃) for 24 h and photographed under a stereomicroscope. Zebrafish were fixed with 3% methylcellulose, head to the left, abdomen down, and photographed with fixed shooting parameters, focusing on the head of the embryo from a top-down position. The melanin area of ​​the zebrafish head was analyzed using ImageJ image analysis software.

[0042] III. Tyrosinase Activity Detection

[0043] 1. Tyrosinase activity assay

[0044] (1) Collect 45 zebrafish juveniles, wash them twice with embryo culture water and PBS, and then transfer them to 1.5mL EP tubes. Absorb the liquid, add 300μL PBS to each tube, and grind them thoroughly on ice with a hand grinder to extract the enzyme solution.

[0045] (2) Centrifuge at 12000 rpm for 10 min at 4℃ and take the supernatant.

[0046] (3) The protein concentration of the supernatant was determined by the BCA method. The determination method is shown in 2.

[0047] (4) Add 100 μL of enzyme solution and 100 μL of dopa solution to a 96-well plate, and take 100 μL of PBS and 100 μL of dopa solution as controls. Incubate at 37°C for 1 h.

[0048] (5) Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance at 475 nm.

[0049] 2. Protein concentration quantification

[0050] (1) After the protein standard is restored to room temperature and thawed, the 5 mg / mL protein standard is diluted with PBS to a final concentration of 0.5 mg / mL.

[0051] (2) Prepare BCA working solution according to the sample quantity with A:B = 50:1 and mix thoroughly.

[0052] (3) Dilute the 0.5 mg / mL protein standard with PBS to obtain protein concentration gradients of 0, 0.025, 0.05, 0.1, 0.2, 0.3 and 0.4 mg / mL.

[0053] (4) Dilute the lysed supernatant 10 times and add 20 μL to a 96-well plate, with 3 replicates for each sample.

[0054] (5) Add 200 μL of BCA working solution to each well, shake, and incubate at 37°C for 20 min.

[0055] (6) Measure the absorbance at 562 nm using an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the protein concentration based on the standard curve and dilution factor.

[0056] IV. Transcriptomics Analysis

[0057] 1. Sample grouping and processing

[0058] Under a stereomicroscope, normally developing embryos at 24 hpf were selected using a plastic dropper and randomly assigned to 6-well plates, 45 embryos per well. After removing the original culture water with a pipette, 3 mL of C3GVP-MP solution, or C3GVP solution (based on a C3GVP concentration of 80 μM), or MP solution with the same MP concentration as the C3GVP-MP group, was added to each well. The embryo culture water was used as a control group. The plates were incubated in a biochemical incubator (28 ± 0.5℃) for 24 h. After washing with PBS, the embryos were transferred to 1.5 mL EP tubes, flash-frozen in liquid nitrogen, and stored at -80℃. Samples were delivered on dry ice. Transcriptome sequencing was performed by Wuhan Metawell Biotechnology Co., Ltd.

[0059] 2. Transcriptome sequencing

[0060] Sample testing: Agarose gel electrophoresis was used to analyze RNA integrity and the presence of DNA contamination; a NanoPhotometer spectrophotometer was used to detect RNA purity (OD260 / 280 and OD260 / 230); a Qubit 4.0 fluorometer / MD microplate reader was used to measure RNA concentration with high precision; and a Qsep400 bioanalyzer was used to accurately detect RNA integrity.

[0061] Library construction: mRNA with polyA tails was enriched using Oligo(dT) magnetic beads. Fragmentation buffer was then added to break the RNA into short fragments. Using these short fragments as templates, first-stranded cDNA was synthesized using six-base random primers. Second-stranded cDNA was synthesized using buffer, dNTPs (dTTP, dATP, dGTP, and dCTP), and DNA polymerase I. The double-stranded cDNA was then purified using DNA purification magnetic beads. The purified double-stranded cDNA underwent end repair, A-tailing, and ligation with sequencing adapters. Fragment size selection was then performed using DNA purification magnetic beads, and finally, PCR enrichment was performed to obtain the final cDNA library.

[0062] Library quality control: (1) Use the Qubit dye method for preliminary quantification and use a fragment analyzer to detect the insert size of the library. If the insert size meets the expectations, proceed to the next step of the experiment; (2) Use the Q-PCR method to accurately quantify the effective concentration of the library (effective concentration of the library > 2nM) and complete the library inspection.

[0063] Sequencing: After the library passes the library inspection, different libraries are pooled according to the target amount of data to be sequenced, and then sequenced using the Illumina platform.

[0064] 3. Bioinformatics Analysis

[0065] FPKM (Fragments Per Kilobase of transcript per Million fragmentsmapped) was used as an indicator to measure gene expression levels. After standardization, p-values ​​and FC values ​​were calculated. The differential gene screening criteria were p < 0.05 and |Log2FC| ≥ 0.5. KEGG enrichment analysis and GO enrichment analysis were performed on the genes, and corresponding images were plotted.

[0066] V. RNA Extraction and qRT-PCR

[0067] 1. Zebrafish RNA extraction

[0068] (1) Collect 45 zebrafish juveniles, remove the original liquid and add 500 μL of lysis buffer. Use a hand grinder to grind them at low temperature on ice until there are no visible solids.

[0069] (2) Add 200 μL of enzyme-free water, then invert the container to mix well and let stand for 5 minutes.

[0070] (3) Centrifuge at room temperature and 12000g for 15 min, and take 500 μL of the upper aqueous phase and place it in a new EP tube.

[0071] (4) Add the same volume (500 μL) of isopropanol, then invert the container to mix well and let stand for 10 min.

[0072] (5) Centrifuge at 12000g for 10 min at room temperature, carefully discard the supernatant, and obtain a white precipitate.

[0073] (6) Add 500 μL of freshly prepared 75% ethanol with DEPC water to the precipitate and gently shake to wash the precipitate.

[0074] (7) Centrifuge at room temperature and 8000g for 5 min.

[0075] (8) Repeat (6) and (7).

[0076] (9) Invert the EP tube onto the filter paper and let it air dry for about 15 minutes.

[0077] (10) Add 20 μL of DEPC water, and after the precipitate dissolves, take 2.0 μL of RNA stock solution and determine the RNA concentration and A260 / 280 purity using Nanodrop.

[0078] 2. RNA reverse transcription

[0079] The reagents used for RNA reverse transcription were all from the Novizan reverse transcription kit.

[0080] (1) DNA removal reaction

[0081] Add an appropriate amount of template RNA to a 200 μL centrifuge tube, add 4 μL of 4×gDNAwiper Mix, and add an appropriate amount of RNase-free ddH2O to bring the reaction volume to 16 μL. Mix well and incubate at 42℃ for 2 min in a PCR instrument.

[0082] (2) Reverse transcription reaction

[0083] Add 4 μL of 5×HiScript III qRT SuperMix to the above system and mix thoroughly, bringing the final reaction volume to 20 μL. Perform reverse transcription in a PCR instrument at 37℃ for 15 min; 85℃ for 5 s; and 4℃ for 5 min. After the reaction, aliquot the apparatus and store at -80℃ for later use.

[0084] (3) Real-time quantitative PCR

[0085] Prepare the reaction system according to Table 4-4 and perform amplification under the following conditions: Pre-denaturation (repeat: 1 time): 95℃, 30s; PCR reaction (repeat: 40 times): 95℃, 5s; 60℃, 30s; melting.

[0086] Table 2: Real-time PCR reaction system

[0087] reagents Usage SYBR GreenI 5.0μL PCR forward primer (10 μM) 0.4μL PCR reverse primers (10 μM) 0.4μL cDNA reaction solution 1.0μL <![CDATA[DEPC H2O]]> 3.2μL total 10μL

[0088] (4) Processing of qPCR results

[0089] Based on the Ct value obtained from the reaction, Ct is calculated. (目的) -Ct (内参) =ΔCt, and then obtain ΔCt and 2 through inter-group calculations. -ΔΔCt .

[0090] IV. Particle size analysis of C3GVP-MP complex in embryo culture medium

[0091] Using zebrafish embryo culture medium as the dispersion medium, the final concentration of C3GVP was set to 80 μM. The particle size distribution of C3GVP and C3GVP-MP was determined under two conditions. The measurements were performed at room temperature, and the refractive index of the sample particles was set to 1.4.

[0092] Results and Analysis

[0093] 1. Effects of C3GVP-MP on melanin production in zebrafish body surface

[0094] Microscopic observation of juvenile zebrafish revealed a decrease in melanin production on their body surface after treatment with different drugs. Quantitative analysis of the melanin signal intensity in the zebrafish head was performed; the analysis areas and results are as follows. Figure 2As shown, MP treatment did not significantly affect melanin production on the zebrafish body surface, while both the C3GVP-MP complex and C3GVP monomers significantly reduced melanin production, with the complex showing a significantly stronger effect than the monomer. Two hypotheses exist for this phenomenon: first, the introduction of MP allows more C3GVP to remain stably in the culture medium rather than polymerizing and forming precipitates, thus enabling more C3GVP to interfere with melanin production; second, the formation of the complex may also have created a combined effect of inhibiting melanin production between C3GVP and MP.

[0095] 2. Effects of C3GVP-MP on tyrosinase activity in zebrafish

[0096] like Figure 2 As shown, the results of administering MP alone to zebrafish showed an opposite trend to the increase in melanin area, significantly increasing tyrosinase activity. However, for C3GVP-MP and C3GVP, the results for both groups were largely consistent with the trend of melanin area on the zebrafish body surface, both significantly reducing tyrosinase activity, with C3GVP-MP showing a significantly stronger inhibitory effect than C3GVP. These results indicate that C3GVP itself can inhibit tyrosinase activity to reduce melanin production, but its unstable nature limits this activity. MP stabilizes C3GVP in the embryo culture medium, thus fully demonstrating C3GVP's ability to inhibit tyrosinase activity.

[0097] 3. Enrichment analysis of C3GVP-MP on RNA expression levels in zebrafish

[0098] KEGG enrichment analysis was performed on the upregulated and downregulated genes in the three differentially expressed groups: C3GVP-MP vs control, C3GVP vs control, and MP vs control. The results are as follows: Figure 3 As shown. Focusing on the pathways enriched by the downregulated genes, for C3GVP-MP vs control, KEGG enriched four pathways, including oxidative phosphorylation and melanogenesis. Oxidative phosphorylation was the most enriched pathway, and melanogenesis is directly related to melanin synthesis in zebrafish. For C3GVP vs control, KEGG only enriched one pathway, oxidative phosphorylation. For MP vs control, KEGG enriched three pathways, with oxidative phosphorylation also being the most enriched.

[0099] GO enrichment analysis was performed on the upregulated and downregulated genes in the three differentially expressed groups: C3GVP-MP vs control, C3GVP vs control, and MP vs control. The results are as follows: Figure 4As shown in the figure, focusing on the pathways enriched by the downregulated genes revealed that the C3GVP-MP vs control group had the most enriched pathways, including the canonical Wnt signaling pathway. For both the C3GVP vs control and MP vs control groups, fewer GO pathways were enriched, and no melanin synthesis-related pathways were found in either group.

[0100] The intervention of C3GVP-MP downregulates the classical Wnt signaling pathway in zebrafish, which is noteworthy. The Wnt / β-catenin signaling pathway is an important pathway in melanin production. On the one hand, Wnt can participate in the proliferation of melanocyte stem cells. On the other hand, Wnt can inhibit the complex composed of adenomatous polyposis coli protein (APC), cytoskeleton axon protein (Axin), and glycogen synthase kinase-3β (GSK3β) by activating receptor protein (Frizzled protein). The reduction of this complex will lead to a decrease in the phosphorylation ratio of β-catenin. Unphosphorylated β-catenin is translocated to the nucleus and directly binds to DNA-associated T cell factor / lymhid enhancing factor (Tcf / Lef) and regulates the Mitf gene, thereby activating Mitf target genes including Tyr, Tyrp1, and Dct

[146] , promoting melanin production. The enrichment of this pathway in the differentially expressed genes of C3GVP-MP vs control but not in the other two groups corresponds to the enrichment of KEGG in the melanin synthesis pathway. Based on this, it is speculated that the complex has a stronger inhibitory effect on melanin production than the monomer.

[0101] 4. Effects of C3GVP-MP on RNA expression levels in zebrafish

[0102] (1) Effects of C3GVP-MP on genes of key factors in melanin synthesis

[0103] Based on the Melanogenesis pathway observed in the KEGG enrichment analysis of differentially downregulated genes between C3GVP-MP and the control group, a subset of key melanin synthesis genes with significant differential expression in transcriptomic analysis were selected for qPCR validation. These genes were Tyr, Dct, Tyrp1a, and Mitfb. The results are as follows: Figures 5A to 5CAs shown in the diagram, in the early stages of zebrafish development, neural crest cells transform into melanocyte precursor cells. The key transcription factor Mitf is activated, further activating the differentiation of other melanocytes and the expression of related proteins such as Tyr, Dct, and Tyrp1, ultimately leading to the migration and differentiation of melanocyte precursor cells into melanocytes rich in melanin. Compared to the control group, the C3GVP group showed significantly decreased expression levels of Mitfb and Dct genes, and slightly decreased expression levels of Tyr and Tyrp1a genes. The MP group showed significantly decreased Mitfb gene expression, slightly decreased Tyr and Tyrp1a gene expression, and increased Dct gene expression. The C3GVP–MP group showed significantly decreased expression levels of Tyr, Tyrp1a, and Mitfb genes, while significantly increased Dct gene expression. The above results indicate that C3GVP-MP, C3GVP, and MP all have a certain inhibitory effect on some key genes in zebrafish melanin synthesis. Among them, the C3GVP-MP complex combines the effects of C3GVP and MP, downregulates the most key factors, and has the strongest melanin inhibition ability. Therefore, it was observed in the KEGG enrichment analysis. This result is also consistent with the statistics of melanin area on the zebrafish body surface.

[0104] (2) Effects of C3GVP-MP on genes in the Wnt / β-catenin signaling pathway

[0105] Studies have shown that the Wnt / β-catenin signaling pathway plays an important role in melanin production. Based on the classic Wnt pathway observed in GO enrichment analysis of differentially downregulated genes in C3GVP-MP vs control groups, several differentially expressed Wnt / β-catenin signaling pathway genes in transcriptomic analysis were screened and validated by qPCR. These genes were Gnao1a, β-catenin, Tcf712, and Lef1. The results are as follows: Figure 6As shown, C3GVP-MP significantly reduced the expression of three genes: Gnao1a, β-catinin, and Tcf712. This suggests that C3GVP-MP may promote β-catenin phosphorylation by reducing the inhibition of APC, Axin, and GSK3β complex formation, thereby decreasing the proportion of unphosphorylated β-catenin and preventing it from binding to Tcf to alter DNA conformation. This, in turn, reduces Mitf gene expression and activation of Tyr and Tyrp1. Therefore, C3GVP-MP can downregulate melanin production by participating in the Wnt / β-catenin signaling pathway, which is consistent with the transcriptome results. For C3GVP, only β-catinin mRNA expression was significantly downregulated; Gnao1a and Tcf712 expression did not show significant differences. This suggests that the tendency of C3GVP to polymerize and precipitate in complex solution environments limits its regulation of melanin-related genes. After MP intervention, the expression of three genes, Gnao1a, β-catinin and Tcf712, decreased slightly, but no significant difference was observed.

[0106] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. Cyanide-3- O The use of γ-glucosinolate-4-vinylphenol-mannose protein complex in the preparation of medicaments for the treatment and / or prevention of melanin synthesis-related diseases, said complex being cyanidin-3- O It is prepared by γ-glucoside-4-vinylphenol (C3GVP) and mannoprotein (MP), and the melanin synthesis-related diseases are selected from: melanin-induced freckles, melasma, and skin cancer.

2. The application according to claim 1, characterized in that: The drug in question is one that interferes with melanin synthesis.

3. The application according to claim 2, characterized in that: The drug that intervenes in melanin synthesis is a drug that intervenes in key factors of melanin synthesis.

4. The application according to claim 3, characterized in that, The key factors in melanin synthesis are tyrosinase, tyrosine-associated protein 1, and microphthalmia-associated transcription factor.

5. The application according to claim 2, characterized in that: The drugs that interfere with melanin synthesis are those that interfere with pathways related to melanin synthesis.

6. The application according to claim 5, characterized in that: The melanin synthesis pathway is the Wnt / β-catenin signaling pathway.

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