A whitening polypeptide compound and a preparation method and application thereof

Abstract

The present application relates to the technical field of polypeptide compounds, and particularly relates to a whitening polypeptide compound, a preparation method and application thereof, wherein the whitening polypeptide compound is obtained by free arrangement and combination of tryptophan, tyrosine and beta-alanine through an amide bond. The whitening polypeptide compound provided by the present application has excellent whitening activity, and is verified by B16-F10 cells induced by alpha-MSH. The whitening polypeptide compound can efficiently inhibit skin melanin synthesis, and has the effects of whitening and freckle lightening. Meanwhile, the whitening polypeptide compound has high whitening activity, good mild safety, a simple preparation process, high synthesis efficiency, low cost, strong stability in a cosmetic system, good skin permeability, can simultaneously realize efficient whitening, freckle lightening and skin barrier repair, can be widely applied to whitening cosmetics to prepare various dosage forms, improve product whitening durability and stability, has high application value, and meets the demand of the cosmetic industry for high-performance mild whitening active ingredients.

Description

Technical Field

[0001] This invention relates to the field of polypeptide compound technology, and in particular to a skin-whitening polypeptide compound, its preparation method, and its application. Background Technology

[0002] As the cosmetics industry rapidly develops towards "natural, efficient, and safe" products, skin whitening has become one of the core demands in the functional skincare field. Consumers are increasingly demanding higher standards for the safety, gentleness, and efficacy of whitening cosmetics. Among these, peptide-based active ingredients, due to their advantages such as good skin penetration, high biocompatibility, and low side effects, are more easily absorbed by the skin and less likely to cause irritation compared to traditional whitening ingredients (such as vitamin C, niacinamide, and arbutin). They are gradually becoming a research hotspot in the whitening cosmetics field and are being widely explored for application in whitening functional cosmetics.

[0003] Currently, there are relatively few types of skin-whitening peptides that have been explored and applied in cosmetics, and existing skin-whitening peptide compounds generally suffer from low skin-whitening activity, poor stability, complex preparation processes, low synthesis efficiency, and high costs.

[0004] Therefore, existing technologies still need to be improved and developed. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide a whitening polypeptide compound, its preparation method and application, in order to solve the problems of low whitening activity and poor stability of existing whitening polypeptide compounds.

[0006] The technical solution of the present invention is as follows: A skin-whitening polypeptide compound, wherein the skin-whitening polypeptide compound is obtained by free arrangement and combination of tryptophan, tyrosine, and β-alanine through amide bonds.

[0007] The whitening polypeptide compound, wherein the amino acid sequence of the whitening polypeptide compound is NH2-Bal-Tyr-Trp-OH, and its structural formula is: .

[0008] The whitening polypeptide compound wherein the C-terminus of the whitening polypeptide compound is modified with an amide group; and the N-terminus of the whitening polypeptide compound is modified with an acetamino group.

[0009] A method for preparing a skin-whitening polypeptide compound, comprising the following steps: Fmoc in the preloaded resin of the first amino acid was removed by using an eluent, and the first solid phase was obtained after solid-liquid separation. The first solid substance is subjected to a dehydration condensation reaction with the second amino acid to obtain the second solid substance; After the second solid phase substance and the third amino acid undergo a dehydration condensation reaction, the Fmoc in the third amino acid is removed by an eluent to obtain the third solid phase substance. The third solid phase substance is mixed with the lysis solution, and after the lysis reaction, it is precipitated to obtain the whitening polypeptide compound.

[0010] The method for preparing the whitening polypeptide compound, wherein the eluent includes at least one of diethylamine, 1,8-diazabicyclo[5.4.0]undec-7-ene, piperidine, and N,N-dimethylformamide.

[0011] The method for preparing the whitening polypeptide compound, wherein the concentration of the eluent is 5wt%-30wt%.

[0012] The method for preparing the whitening polypeptide compound, wherein the lysis buffer is one or more of trifluoroacetic acid, water, triisopropylsilane, and 2,2'-(1,2-ethylenedioxy)bis(ethyl mercaptan). Preferably, the pyrolysis reaction takes 1-3 hours.

[0013] The method for preparing the whitening polypeptide compound, wherein the molar ratio of the first amino acid preloaded resin, the second amino acid, and the third amino acid is 1:(1-3):(1-3).

[0014] Application of a whitening polypeptide compound in whitening cosmetics.

[0015] In the aforementioned application, the concentration of the whitening polypeptide compound in the whitening cosmetic is 0.1 mM-2 mM.

[0016] Beneficial Effects: This invention provides a skin-whitening polypeptide compound, its preparation method, and its applications. The skin-whitening polypeptide compound is obtained by the free arrangement and combination of tryptophan, tyrosine, and β-alanine through amide bonds. The skin-whitening polypeptide compound provided by this invention exhibits excellent skin-whitening activity. Verification using α-MSH (α-melanocyte-stimulating hormone)-induced B16-F10 cells (mouse skin melanoma cells) shows that this skin-whitening polypeptide compound can effectively inhibit skin melanin synthesis, thus possessing skin-whitening and spot-fading effects. Simultaneously, this skin-whitening polypeptide compound exhibits high skin-whitening activity, good gentleness and safety, a simple preparation process, high synthesis efficiency, and low cost. Furthermore, it demonstrates strong stability and excellent skin permeability in cosmetic systems, simultaneously achieving highly effective skin whitening, spot-fading and brightening, and skin barrier repair. Moreover, this skin-whitening polypeptide compound can be widely applied to skin-whitening cosmetics to prepare various dosage forms, improving the whitening durability and stability of products. It has high application value and meets the cosmetic industry's demand for high-performance, gentle skin-whitening active ingredients. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the process flow for preparing a skin-whitening polypeptide compound according to the present invention. Figure 2 The mass spectrum of the whitening polypeptide compound ZT-144 prepared in Example 1 is shown. Figure 3 The image shows the high-performance liquid chromatography (HPLC) chromatogram of the whitening polypeptide compound ZT-144 obtained in Example 1. Figure 4 The image shows the cytotoxicity test results of the skin-whitening polypeptide compound ZT-144. Figure 5 The image shows the experimental results of detecting melanin content in cells using the skin-whitening polypeptide compound ZT-144. Figure 6 The figure shows the results of an in vitro tyrosinase activity inhibition experiment of the skin-whitening polypeptide compound ZT-144. Detailed Implementation

[0018] This invention provides a skin-whitening polypeptide compound, its preparation method, and its application. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0019] It will be understood by those skilled in the art that, unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. It should also be understood that terms such as those defined in general dictionaries should be understood to have the same meaning as in the context of the prior art, and should not be interpreted in an idealized or overly formal sense unless specifically defined as herein.

[0020] Peptides are small molecule compounds formed by amino acids linked by peptide bonds. Their structure is similar to fragments of human proteins, allowing them to quickly penetrate the dermis and achieve whitening effects by regulating the production, transport, or metabolism of melanin. They can also help repair the skin barrier and alleviate the irritation that whitening ingredients may cause, thus providing both whitening and skincare benefits.

[0021] Currently, the types of skin-whitening peptides explored for application in cosmetics are relatively few, and existing skin-whitening peptide compounds generally suffer from the following technical challenges, limiting their further promotion and application in skin-whitening cosmetics: Firstly, the whitening activity of existing whitening peptides is limited, and their effects on core whitening needs such as inhibiting melanin, fading dark spots, and brightening skin tone are not significant enough. Some whitening peptides require higher concentrations to achieve the desired whitening effect, which not only increases the production cost of cosmetics but may also cause safety issues such as skin irritation and allergies due to high concentrations. It is difficult to meet the dual requirements of "highly effective whitening" and "gentle and safe" and cannot satisfy consumers' core demand for gentle and effective whitening.

[0022] Secondly, existing methods for preparing skin-whitening peptides have significant drawbacks. Traditional solid-phase and liquid-phase synthesis methods generally suffer from cumbersome reaction steps, harsh reaction conditions (such as requiring high temperature, high pressure, or expensive catalysts), low synthesis efficiency, low product purity, and numerous byproducts. Furthermore, the subsequent separation and purification processes are complex, resulting in high production costs for skin-whitening peptide products, which is not conducive to large-scale production and industrial application. At the same time, some preparation methods introduce harmful impurities, which not only affect the bioactivity of skin-whitening peptides but may also potentially irritate the skin, failing to meet the stringent requirements for purity and safety of skin-whitening active ingredients in the cosmetics industry.

[0023] Third, existing whitening peptides have poor stability in cosmetic systems and are easily affected by external conditions such as temperature, light, and pH, resulting in degradation and oxidation reactions, which reduce or even eliminate their whitening activity. This not only affects the longevity of the whitening effect of cosmetics but also shortens their shelf life. In addition, the skin permeability of some whitening peptides still has room for improvement, making it difficult for them to fully penetrate into the dermis to exert their melanin regulation function, further limiting their whitening efficacy and application value.

[0024] Fourth, there are very few peptide-based whitening compounds on the market that can directly inhibit melanin synthesis, which makes it difficult to meet the growing skincare needs of consumers.

[0025] Based on this, the present invention provides a skin-whitening polypeptide compound, wherein the skin-whitening polypeptide compound is obtained by freely arranging and combining tryptophan, tyrosine, and β-alanine through amide bonds.

[0026] In this embodiment, the provided whitening polypeptide compound exhibits excellent whitening activity. Verification using α-MSH (α-melanocyte-stimulating hormone)-induced B16-F10 cells (mouse skin melanoma cells) showed that this whitening polypeptide compound can effectively inhibit skin melanin synthesis, thus possessing whitening and spot-fading effects. Simultaneously, this whitening polypeptide compound exhibits high whitening activity, good gentleness and safety, a simple preparation process, high synthesis efficiency, and low cost. Furthermore, it demonstrates strong stability in cosmetic systems and excellent skin permeability, simultaneously achieving highly effective whitening, spot-fading and brightening, and skin barrier repair. Moreover, this whitening polypeptide compound can be widely applied in whitening cosmetics to prepare various dosage forms, improving the whitening durability and stability of products. It has high application value and meets the cosmetic industry's demand for high-performance, gentle whitening active ingredients.

[0027] Specifically, this invention utilizes a tryptophan, a tyrosine, and a β-alanine to form a tripeptide sequence through free arrangement and connection by amide bonds, thus eliminating the redundant amino acid combinations commonly used in existing skin-whitening peptide compounds. Compared to existing skin-whitening peptides, the skin-whitening peptide compound provided by this invention possesses multiple performance breakthroughs, including: 1) Excellent transdermal absorption: It is generally believed that a molecular weight of 500 Daltons is the dividing line for transdermal absorption of active ingredients in skin care products. The whitening polypeptide compound provided by this invention is a tripeptide compound with an extremely low molecular weight of only 439 Daltons, obtained by the free arrangement and combination of tryptophan, tyrosine and β-alanine through amide bonds. When used in the fields of biomedicine, medical reagents, cosmetic raw materials, etc., it can efficiently penetrate the stratum corneum of the skin without the need for additional penetration enhancers and reach melanocytes to take effect.

[0028] 2) Strong whitening activity: The whitening polypeptide compound provided by this invention can specifically bind to tyrosinase, thereby inhibiting tyrosinase activity, and can significantly inhibit melanin production even at low concentrations.

[0029] 3) High safety: The whitening polypeptide compound provided by this invention is composed of common natural amino acids, which is highly safe, non-cytotoxic, non-skin irritating, suitable for sensitive skin, and meets the safety standards for cosmetic raw materials.

[0030] In some embodiments, the amino acid sequence of the skin-whitening polypeptide compound is NH2-Bal-Tyr-Trp-OH (denoted as ZT-144), and its structural formula is: .

[0031] Specifically, the skin-whitening polypeptide compound ZT-144 is composed of β-alanine, tyrosine, and tryptophan linked sequentially by amide bonds. It effectively inhibits melanin synthesis in the skin, exhibiting skin-whitening and spot-fading effects. Furthermore, ZT-144 has a molecular weight of only 439 Daltons, allowing it to penetrate the stratum corneum efficiently without the need for additional penetration enhancers, directly reaching melanocytes to exert its effects. Simultaneously, this skin-whitening polypeptide compound can specifically bind to tyrosinase, inhibiting its activity and significantly suppressing melanin production even at low concentrations.

[0032] In some embodiments, the C-terminus of the skin-whitening polypeptide compound is modified with an amide group; the N-terminus of the skin-whitening polypeptide compound is modified with an acetamino group. Amidation is used to convert the free carboxyl group (-COOH) at the C-terminus of the skin-whitening polypeptide compound into an amide group (-CONH2), and acetylation is used to attach the acetamino group to the N-terminal amino group (-NH2) of the skin-whitening polypeptide compound, forming an acetamino group (-NHCOCH3). Specifically, N-terminal acetylation of the skin-whitening polypeptide compound neutralizes the positive charge of the α-amino group, preventing aminopeptidase cleavage; C-terminal amidation converts the carboxyl group into an amide group, avoiding degradation by carboxylpeptidase.

[0033] In addition, such as Figure 1 As shown, the present invention also provides a method for preparing a skin-whitening polypeptide compound, comprising the following steps: Step S10: Use an eluent to remove Fmoc from the first amino acid preloaded resin, and after solid-liquid separation, obtain the first solid phase substance; Step S20: The first solid phase substance and the second amino acid undergo a dehydration condensation reaction to obtain the second solid phase substance; Step S30: After the second solid phase substance and the third amino acid undergo a dehydration condensation reaction, the Fmoc in the third amino acid is removed using an eluent to obtain the third solid phase substance; Step S40: The third solid phase substance is mixed with the lysis solution, and after the lysis reaction, it is precipitated to obtain the whitening polypeptide compound.

[0034] In this embodiment, the preparation method employs an optimized solid-phase synthesis approach, simplifying steps, reducing reaction severity, improving synthesis efficiency and product purity, and lowering costs, making it suitable for large-scale production. Furthermore, the whitening peptide compound prepared using this method exhibits excellent whitening activity. Verification using α-MSH (α-melanocyte-stimulating hormone)-induced B16-F10 cells (mouse skin melanoma cells) demonstrates that this whitening peptide compound can effectively inhibit skin melanin synthesis, exhibiting whitening and spot-fading effects. Simultaneously, this whitening peptide compound possesses high whitening activity, good gentleness and safety, a simple preparation process, high synthesis efficiency, and low cost. It also exhibits strong stability in cosmetic systems and excellent skin permeability, simultaneously achieving highly effective whitening, spot-fading and brightening, and skin barrier repair. Moreover, this whitening peptide compound can be widely applied in whitening cosmetics to prepare various dosage forms, improving the whitening durability and stability of products, demonstrating high application value and meeting the cosmetic industry's demand for high-performance, gentle whitening active ingredients.

[0035] In some embodiments, the eluent includes at least one of diethylamine, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), piperidine, and N,N-dimethylformamide (DMF). The eluent can efficiently and selectively remove the Fmoc protecting group at the amino terminus of amino acids. DMF, as a polar aprotic inert solvent, can fully swell the solid resin support and improve the mass transfer efficiency within the resin channels. Simultaneously, it dissolves the organic base component, ensuring sufficient contact between the base molecules and the Fmoc group. The organic base in the system can precisely attack the fluorene carbonyl site of the Fmoc group, breaking the bond between the protecting group and the amino group through a β-elimination reaction, generating free amino groups, fluorene byproducts, and corresponding ammonium salts, thus completing the deprotection and activation process.

[0036] In some embodiments, the concentration of the eluent is 5wt%-30wt%, which can achieve a precise balance between the deprotection reaction rate, reaction selectivity, and product safety. When the concentration is maintained in this low range (around 5wt%), the system is mildly alkaline, which can slowly and steadily trigger the β-elimination cleavage reaction of the Fmoc group, effectively avoiding the problems of racemization of the chiral center of amino acids, side reactions of sensitive side chain functional groups, and peptide bond hydrolysis caused by high concentrations of strong base. At the same time, the low concentration of alkaline solution can reduce excessive swelling and damage of the resin carrier and reduce the loss of the solid-phase synthesis carrier. As the concentration is gradually increased to the optimal range of 15wt%~25wt%, the organic base molecules are evenly distributed in the DMF solvent, the mass transfer and penetration efficiency is greatly improved, and it can quickly penetrate into the interior of the resin micropores to fully contact the Fmoc protection sites, significantly accelerate the deprotection reaction process, shorten the single removal time, and avoid the generation of subsequent condensation peptide-deficient and mispeptide impurities due to residual Fmoc groups caused by incomplete reaction. At this concentration, it can also fully dissolve and remove fluorene byproducts and salt impurities, ensuring the high activity of free amino groups on the resin surface. Strictly controlling the concentration to not exceed the upper limit of 30wt% can eliminate the negative effects of excessive alkali concentration. This prevents the peptide chain from breaking and premature deprotection of side chains caused by excessive local alkalinity, and also reduces the problem of high concentration organic alkali residues being difficult to rinse clean. This reduces the risk of interference in subsequent coupling reactions. Ultimately, while ensuring complete removal of Fmoc, it can steadily improve the purity and synthesis yield of crude peptide products, taking into account both process safety and production controllability.

[0037] In some embodiments, the lysis solution is one or more of trifluoroacetic acid, water, triisopropylsilane, and 2,2'-(1,2-ethylenedioxy)bis(ethyl mercaptan).

[0038] In some embodiments, the pyrolysis reaction takes 1-3 hours.

[0039] In some embodiments, in step S40, the reaction residue after the pyrolysis reaction is settled using icy isopropyl ether to obtain the whitening polypeptide compound.

[0040] In some embodiments, steps S20 and S30, after the dehydration condensation reaction, further include solid-liquid separation.

[0041] In some embodiments, the solid-liquid separation step includes a washing process; the solvent used in the washing process includes one or more of methanol, ethanol, N,N-dimethylformamide, and dichloromethane.

[0042] In some embodiments, the molar ratio of the first amino acid preloaded resin, the second amino acid, and the third amino acid is 1:(1-3):(1-3).

[0043] In a preferred embodiment, the molar ratio of the first amino acid preloaded resin, the second amino acid, and the third amino acid is 1:2:2.

[0044] In some embodiments, taking the skin-whitening polypeptide compound NH2-Bal-Tyr-Trp-OH (denoted as ZT-144) as an example, its preparation process includes the following steps: Step 1: Remove Fmoc from Fmoc-Trp(Boc)-Wang Resin using an eluent, and obtain the first solid phase substance after solid-liquid separation; Step 2: After mixing and reacting the first solid phase with Fmoc-Tyr(tBu)-OH at room temperature, Fmoc is removed using an eluent, and after solid-liquid separation, the second solid phase is obtained. Step 3: After mixing and reacting the second solid phase with Fmoc-Bal-OH at room temperature, Fmoc is removed using an eluent, and after solid-liquid separation, the third solid phase is obtained. Step 4: The third solid phase substance is subjected to a pyrolysis reaction with the pyrolysis solution to obtain a reaction residue. The reaction residue is then settled using icy isopropyl ether to obtain the multifunctional short peptide ZT-144.

[0045] In addition, the present invention also provides an application of a whitening polypeptide compound in whitening cosmetics.

[0046] In this embodiment, a whitening polypeptide compound is applied to cosmetics, which can effectively inhibit melanin synthesis in the skin, thus exhibiting whitening and spot-fading effects. Furthermore, this whitening polypeptide compound has a molecular weight of only 439 Daltons, allowing it to efficiently penetrate the stratum corneum of the skin and reach melanocytes directly without the need for additional penetration enhancers. Simultaneously, this whitening polypeptide compound can specifically bind to tyrosinase, inhibiting its activity and significantly suppressing melanin production even at low concentrations.

[0047] In some embodiments, the concentration of the whitening polypeptide compound in the whitening cosmetic is 0.1 mM-2 mM. By controlling the concentration of the whitening polypeptide compound within this range in the whitening cosmetic, it can specifically bind to tyrosinase, thereby inhibiting tyrosinase activity, and significantly inhibiting melanin production even at low concentrations.

[0048] The following examples further illustrate the present invention in detail. It should also be understood that the following examples are only for further explanation of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above description of the present invention are within the scope of protection of the present invention.

[0049] Example 1 This embodiment provides a skin-whitening polypeptide compound with the amino acid sequence NH2-Bal-Tyr-Trp-OH (denoted as ZT-144), and its structural formula is as follows: The specific preparation process includes the following: Fmoc was removed from Fmoc-Trp(Boc)-Wang Resin (10 mmol) using an eluent (10 wt%). The reaction was carried out at room temperature for 30 minutes. After solid-liquid separation, the first solid phase was obtained. The eluent was obtained by dissolving diethylamine in N,N-dimethylformamide.

[0050] After mixing and reacting the first solid phase with Fmoc-Tyr(tBu)-OH (equivalent to 2 molar amounts of Fmoc-Trp(Boc)-Wang Resin) at room temperature for 1 hour, Fmoc was removed using an eluent (10wt%) and the reaction was carried out at room temperature for 30 minutes. After solid-liquid separation, the second solid phase was obtained. After mixing and reacting the second solid phase with Fmoc-Bal-OH (equivalent to 2 molar amounts of Fmoc-Trp(Boc)-Wang Resin) at room temperature for 1 hour, Fmoc was removed using an eluent (10wt%), and after solid-liquid separation, the third solid phase was obtained. The third solid phase was subjected to a pyrolysis reaction with trifluoroacetic acid (50 mL) at room temperature for 1 hour. The solid and liquid phases were separated, and the pyrolysis solution was concentrated by rotary evaporation to obtain the reaction residue. The reaction residue was precipitated at room temperature using ice-cold isopropyl ether (100 mL), purified by preparative liquid phase, concentrated by rotary evaporation, and lyophilized to obtain the whitening polypeptide compound ZT-144.

[0051] The purity of the skin-whitening peptide compound ZT-144 was analyzed using high-performance liquid chromatography (HPLC), and its molecular weight was determined using mass spectrometry. The mass spectrum of the skin-whitening peptide compound ZT-144 is shown below. Figure 2 As shown, the experiment proved the successful synthesis of ZT-144. The high-performance liquid chromatography (HPLC) detection chromatogram is shown below. Figure 3 As shown in the figure, the purity was determined to be 98.32% based on the peak area. Therefore, the whitening polypeptide compound ZT-144 was finally identified.

[0052] Example 2 In this embodiment, the whitening polypeptide compound ZT-144 prepared in Example 1 was used for cytotoxicity experiments. The effect on cell proliferation was detected using a CCK8 assay kit. The specific procedures are as follows: Mouse skin melanoma cells (B16-F10 cells) were cultured in 1640 medium + 10% fetal bovine serum + 1% penicillin & streptomycin in a cell culture incubator at 37°C and 5% CO2. Before grouping experiments, the medium was replaced with DMEM high-glucose medium + 10% fetal bovine serum + 1% penicillin & streptomycin complete medium. B16-F10 cells were seeded at 10,000 cells per well in 96-well plates for 24 hours. 100 μL of ZT-144 complete medium (0-16 mM) was added to each well, with 6 replicates per group. After 24 hours of incubation, the old medium was removed, and 110 μL of medium containing CCK8 solution (complete medium: CCK8 = 10:1) was added. The cells were incubated for 2 hours in a cell culture incubator at 37°C and 5% CO2. The absorbance of each well was measured at 450 nm using a microplate reader, and cell viability was calculated. The results of the ZT-144 cytotoxicity assay are shown in the figure below. Figure 4 As shown.

[0053] The results showed that ZT-144 had no effect on the proliferation of B16-F10 cells in the range of 0-1 mM.

[0054] Example 3 In this embodiment, the skin-whitening polypeptide compound ZT-144 prepared in Example 1 was used to detect melanin content in mouse skin melanoma cells (B16-F10 cells). The specific steps are as follows: Mouse skin melanoma cells (B16-F10 cells) were cultured in 1640 medium + 10% fetal bovine serum + 1% penicillin & streptomycin in a cell culture incubator at 37°C and 5% CO2. Before grouping experiments, the medium was replaced with DMEM high glucose medium + 10% fetal bovine serum + 1% penicillin & streptomycin complete medium. B16-F10 cells were seeded at 200,000 cells per well in 6-well plates for 24 hours. The cells were induced to form a melanocyte-stimulating hormone (MSH) model. The experimental groups were added 2 ml of complete medium containing 0.5 mM and 1 mM ZT-144, while the positive control group was added 2 ml of complete medium containing 0.5 mM and 1 mM kojic acid or α-arbutin. Each group was in triplicate. After 48 hours of incubation, the medium was aspirated, allowed to stand, and photographed. After photographing, 200 μL of medium from each group was added to 96-well plates, in triplicate, and the absorbance was measured at 405 nm using a microplate reader. The results of the ZT-144 melanin content detection experiment in cells are shown in the figure below. Figure 5 As shown, compared with the model group, ZT-144 significantly reduced the melanin content secreted into the culture medium, and its effect was similar to that of the positive control group. This indicates that ZT-144 has good whitening and freckle-removing effects.

[0055] Example 4 In this embodiment, the whitening polypeptide compound ZT-144 prepared in Example 1 was used to conduct a tyrosinase activity inhibition experiment. The specific steps are as follows: The test method for inhibiting tyrosinase activity of cosmetic raw materials (in vitro method) was conducted in accordance with the Guangdong Provincial Cosmetic Society Group Standard (T / GDCA 006—2021). The in vitro tyrosinase activity inhibition test results for ZT-144 are shown in the figure below. Figure 6 As shown.

[0056] Experimental results showed that, according to the comparison of kojic acid equivalent values ​​(unit: mM / mM), the in vitro tyrosinase activity results were as follows (the smaller the kojic acid equivalent value, the stronger the tyrosinase inhibition ability of the sample): kojic acid (1) > ZT-144 (7.22) > α-arbutin (30.81), indicating that ZT-144 has a significant tyrosinase activity inhibition effect.

[0057] In summary, this demonstrates that ZT-144 can inhibit melanin production by suppressing tyrosinase activity, thereby exerting its whitening and spot-removing effects.

[0058] In summary, this invention provides a skin-whitening polypeptide compound, its preparation method, and its applications. The skin-whitening polypeptide compound is obtained by the free arrangement and combination of tryptophan, tyrosine, and β-alanine through amide bonds. The skin-whitening polypeptide compound provided by this invention exhibits excellent skin-whitening activity. Verification using α-MSH (α-melanocyte-stimulating hormone)-induced B16-F10 cells (mouse skin melanoma cells) showed that this skin-whitening polypeptide compound can effectively inhibit skin melanin synthesis, thus possessing skin-whitening and spot-fading effects. Simultaneously, this skin-whitening polypeptide compound exhibits high skin-whitening activity, good gentleness and safety, a simple preparation process, high synthesis efficiency, and low cost. Furthermore, it demonstrates strong stability and excellent skin permeability in cosmetic systems, simultaneously achieving efficient skin whitening, spot fading and brightening, and skin barrier repair. Moreover, this skin-whitening polypeptide compound can be widely used in skin-whitening cosmetics to prepare various dosage forms, improving the whitening durability and stability of products. It has high application value and meets the cosmetic industry's demand for high-performance, gentle skin-whitening active ingredients.

[0059] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A skin-whitening polypeptide compound, characterized in that, The whitening polypeptide compound is obtained by freely arranging and combining tryptophan, tyrosine, and β-alanine through amide bonds.

2. The skin-whitening polypeptide compound according to claim 1, characterized in that, The amino acid sequence of the whitening polypeptide compound is NH2-Bal-Tyr-Trp-OH, and its structural formula is: 。 3. The skin-whitening polypeptide compound according to claim 1, characterized in that, The C-terminus of the whitening polypeptide compound is modified with an amide group; the N-terminus of the whitening polypeptide compound is modified with an acetamino group.

4. A method for preparing the skin-whitening polypeptide compound according to any one of claims 1-3, characterized in that, Including the following steps: Fmoc in the preloaded resin of the first amino acid was removed by using an eluent, and the first solid phase was obtained after solid-liquid separation. The first solid substance is subjected to a dehydration condensation reaction with the second amino acid to obtain the second solid substance; After the second solid phase substance and the third amino acid undergo a dehydration condensation reaction, the Fmoc in the third amino acid is removed by an eluent to obtain the third solid phase substance. The third solid phase substance is mixed with the lysis solution, and after the lysis reaction, it is precipitated to obtain the whitening polypeptide compound.

5. The method for preparing the skin-whitening polypeptide compound according to claim 4, characterized in that, The eluent includes at least one of diethylamine, 1,8-diazabicyclo[5.4.0]undec-7-ene, piperidine, and N,N-dimethylformamide.

6. The method for preparing the skin-whitening polypeptide compound according to claim 4, characterized in that, The concentration of the eluent is 5wt%-30wt%.

7. The method for preparing the skin-whitening polypeptide compound according to claim 4, characterized in that, The pyrolysis solution is one or more of trifluoroacetic acid, water, triisopropylsilane, and 2,2'-(1,2-ethylenedioxy)bis(ethyl mercaptan); Preferably, the pyrolysis reaction takes 1-3 hours.

8. The method for preparing the skin-whitening polypeptide compound according to claim 4, characterized in that, The molar ratio of the first amino acid preloaded resin, the second amino acid, and the third amino acid is 1:(1-3):(1-3).

9. The use of a whitening polypeptide compound as described in any one of claims 1-3 in whitening cosmetics.

10. The application according to claim 9, characterized in that, In the whitening cosmetic, the concentration of the whitening polypeptide compound is 0.1mM-2mM.

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