A polypeptide and uses thereof

Abstract

The present application relates to the field of polypeptides, in particular, to a polypeptide and use thereof. More particularly, to a polypeptide having at least one effect of anti-wrinkle, repair, whitening and use thereof. The polypeptide of the present application shows versatility in biological activity: in terms of anti-wrinkle, it can increase the content of intracellular type I collagen; in terms of repair, it can have a significant healing effect on keratin; in terms of whitening, it can inhibit melanin production. Therefore, the polypeptide of the present application has at least one effect of anti-wrinkle, repair and whitening, and can meet various application requirements in the field of cosmetics.

Description

Technical Field

[0001] This application relates to the field of peptides, specifically, to a peptide and its uses. More specifically, it relates to a peptide with anti-wrinkle, repairing, and whitening effects, and its uses. Background Technology

[0002] Skin aging occurs over time, influenced by both internal and external factors. In daily life, skin is easily damaged by external stimuli such as ultraviolet radiation, dryness, and stress, leading to wrinkles, pigmentation, and barrier dysfunction.

[0003] However, the selection of active ingredients that can repair related problems in existing technologies is limited. In order to meet the diversity of choices, it is necessary to develop a new ingredient with at least one of the effects of anti-wrinkle, repair, and whitening, so as to enrich the raw material library and meet the application needs of the cosmetics field. Summary of the Invention

[0004] This application is made in view of the above-mentioned issues, and its purpose is to provide a polypeptide product that has at least one of the effects of anti-wrinkle, repair, and whitening.

[0005] To achieve the above objectives, the first aspect of this application provides a polypeptide whose amino acid sequence is PGAPSMK (SEQ ID NO.1).

[0006] In several embodiments of this application, experimental results show that the polypeptides of this application exhibit multifaceted biological activities: In terms of anti-wrinkle effects, it can increase the content of type I collagen in cells; In terms of repair, it has a significant healing effect on the stratum corneum; In terms of skin whitening, it can inhibit melanin production.

[0007] Therefore, the peptides in this application have at least one of the effects of anti-wrinkle, repair, and whitening, which can meet the diverse application needs in the cosmetics field.

[0008] On another front, this application provides an isolated nucleic acid molecule, comprising: 1) A nucleotide sequence capable of encoding the polypeptide as described above, or 2) A sequence that can strictly hybridize with the nucleotide sequence encoding the polypeptide described in 1).

[0009] On the other hand, this application provides a carrier comprising the isolated nucleic acid molecules as described above.

[0010] On the other hand, this application provides a host cell comprising the isolated nucleic acid molecule as described above or the vector as described above.

[0011] In another aspect, this application provides a composition comprising the polypeptide as described above, the isolated nucleic acid molecule as described above, the vector as described above, or the host cell as described above.

[0012] Furthermore, this application provides the use of the above composition in the preparation of the following products: 1) Use in the preparation of skin care or cosmetic products having at least one of the following effects, said effects including repair, anti-wrinkle, or whitening; or 2) Used in the preparation of anti-aging skin care products or cosmetics.

[0013] In another aspect, this application provides a skin care product or cosmetic product comprising the above-described composition.

[0014] In some specific implementations, the concentration of peptides in skin care or cosmetic products is 0.001 mg / ml to 5 mg / ml.

[0015] In some specific implementations, skin care products or cosmetics may also include one or more of the following: emollients, fillers, colorants, synthetic waxes, oils, humectants, preservatives, binders, thickeners, or emulsifiers.

[0016] In some specific implementations, skincare or cosmetic products also include purified water, carbomer 940, butylene glycol, 1,2-hexanediol, p-hydroxyacetophenone, ethylhexyl palmitate, cetearyl alcohol, glyceryl stearate A165, stearyl alcohol (octadecyl alcohol), polyacrylate-13, polyisobutylene, polysorbate-20 (Sepiplus 400), and triethanolamine. Attached Figure Description

[0017] Figure 1 This is the mass spectrum of the polypeptide in this application.

[0018] Figure 2 This is the liquid phase spectrum of the polypeptide in this application.

[0019] Figure 3 This is a 24-hour scratch healing experiment for the control group.

[0020] Figure 4 This is a scratch healing experiment of the polypeptide sample in this application.

[0021] Figure 5 This is a graph showing the CAM test results of the polypeptide in this application.

[0022] Figure 6 This is a graph showing the CAM test results for the control group. Detailed Implementation

[0023] The "range" disclosed in this application is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of a particular range. Ranges defined in this way can include or exclude endpoints and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60-120 and 80-110 are listed for a specific parameter, it is expected that ranges of 60-110 and 80-120 are also included. Furthermore, if minimum range values ​​of 1 and 2 are listed, and if maximum range values ​​of 3, 4, and 5 are listed, then the following ranges are all expected: 1-3, 1-4, 1-5, 2-3, 2-4, and 2-5. In this application, unless otherwise stated, the numerical range "ab" represents a shortened representation of any combination of real numbers between a and b, where a and b are real numbers. For example, the numerical range "0-5" indicates that all real numbers between "0-5" have been listed in this article; "0-5" is simply a shortened representation of these numerical combinations. Furthermore, when a parameter is stated as an integer ≥2, it is equivalent to disclosing that the parameter is, for example, an integer such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0024] Unless otherwise specified, all embodiments and optional embodiments of this application can be combined to form new technical solutions.

[0025] Unless otherwise specified, all technical features and optional technical features of this application may be combined to form new technical solutions.

[0026] Unless otherwise specified, all steps in this application may be performed sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), indicating that the method may include steps (a) and (b) performed sequentially, or it may include steps (b) and (a) performed sequentially. For example, the method may also include step (c), indicating that step (c) may be added to the method in any order. For example, the method may include steps (a), (b), and (c), or it may include steps (a), (c), and (b), or it may include steps (c), (a), and (b), etc.

[0027] Unless otherwise specified, the terms "comprising" and "including" as used in this application can be open-ended or closed-ended. For example, "comprising" and "including" can mean that other components not listed may also be included, or that only the listed components may be included.

[0028] In this application, the peptide sequences were obtained as follows. Specifically, *Lilium sarmentosum* was pulverized and homogenized to obtain a homogenate. Sodium carbonate was added, and after standing, the mixture was centrifuged and filtered to obtain a filtrate. Glacial acetic acid was added to the filtrate to adjust the pH, and after standing and centrifugation, the precipitate obtained was *Lilium sarmentosum* protein. Neutral protease was used for enzymatic hydrolysis, followed by centrifugation and filtration. The filtrate was then subjected to ultrafiltration fractionation to obtain candidate peptide sequences. The candidate peptide sequence set was analyzed, yielding a total of 660 candidate peptide sequences. Subsequently, bioinformatics tools were used to predict the activity of the candidate peptides, including scoring each candidate peptide using Peptide Ranker. 94 peptide sequences with scores higher than a preset threshold (>0.5) were selected. Based on this, a comprehensive evaluation was further conducted combining the peptides' toxicity, sensitization, solubility, and resistance to enzymatic hydrolysis, resulting in the selection of 7 candidate peptides. Subsequently, the above 7 candidate peptides were further analyzed using Anti-skin-aging Peptide Predictor for anti-skin aging activity prediction. Finally, one target peptide sequence, PGAPSMK, was selected, exhibiting the best overall anti-skin aging activity.

[0029] Based on this, in a first aspect, this application provides a polypeptide whose amino acid sequence is PGAPSMK (SEQ ID NO.1).

[0030] In several embodiments of this application, experimental results show that the polypeptides of this application exhibit multifaceted biological activities: In terms of anti-wrinkle effects, it can increase the content of type I collagen in cells; In terms of repair, it has a significant healing effect on the stratum corneum; In terms of skin whitening, it can inhibit melanin production.

[0031] Therefore, the peptides in this application have at least one of the effects of anti-wrinkle, repair, and whitening, which can meet the diverse application needs in the cosmetics field.

[0032] In some specific embodiments, the polypeptide of this application may be a polypeptide having 80% or more identity with the amino acid sequence shown in SEQ ID NO.1, a polypeptide having 90% or more identity, a polypeptide having 95% or more identity, or a polypeptide having 99% or more identity, and having similar biological effects to the amino acid sequence shown in SEQ ID NO.1.

[0033] To determine sequence identity, SEQ ID NO.1 of this application is used as a reference sequence, and the sequence to be tested is compared with the reference sequence. Then, a sequence comparison algorithm is used to calculate the sequence homology of the test sequence relative to the reference sequence. Two examples of algorithms suitable for determining sequence homology are the BLAST and BLAST2.0 algorithms, described in Altschul et al. (1997) Nuc. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analysis is publicly available from NCBI.

[0034] Secondly, this application provides an isolated nucleic acid molecule comprising: 1) A nucleotide sequence capable of encoding the polypeptide as described above, 2) A sequence that can strictly hybridize with the nucleotide sequence encoding the polypeptide as described above.

[0035] The stringent conditions are moderately stringent, medium-high stringent, high stringent, or very high stringent.

[0036] In this application, "hybridization" means the ability of a polynucleotide or oligonucleotide to bind to a substantially complementary sequence under stringent conditions, without non-specific binding to non-complementary sequences. Preferably, the sequence is 90-100% complementary. This characteristic of complementary sequences capable of specific binding is applied, for example, in Northern or Southern blotting techniques, or in primer binding for PCR or RT-PCR. According to the invention, hybridization occurs under moderately stringent, moderately-highly stringent, highly stringent, or very highly stringent conditions. Such hybridization conditions are described in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6. For example, specific hybridization conditions are as follows: (1) Low-toughness hybridization conditions: 6× sodium chloride / sodium citrate (SSC) at about 45°C, then at at least 50°C, washed twice in 0.2× SSC, 0.1% SDS (for low-toughness conditions, the washing temperature can be increased to 55°C); (2) Medium-toughness hybridization conditions: 6× SSC at about 45°C, then at 60°C, washed once or more in 0.2× SSC, 0.1% SDS; (3) High-toughness hybridization conditions: 6× SSC at about 45°C, then at 65°C, washed once or more in 0.2× SSC, 0.1% SDS, preferably; (4) Very high-toughness hybridization conditions: 0.5M sodium phosphate, 7% SDS at 65°C, then at 65°C, washed once or more in 0.2× SSC, 1% SDS.

[0037] In some specific embodiments, the nucleotide sequence of the polypeptide of this application is shown in SEQ ID NO.2: 5'-CCGGGCGCACCGAGCATGAAA-3' (SEQ ID NO. 2).

[0038] In some specific embodiments, the nucleotide sequence of the polypeptide of this application is a completely complementary sequence to the nucleotide sequence shown in SEQ ID NO.2.

[0039] Thirdly, this application provides a vector (e.g., a cloning vector or an expression vector) comprising the nucleic acid molecules described above.

[0040] In some preferred embodiments, the vector of this application is, for example, a plasmid, granule, bacteriophage, virus, etc. In some preferred embodiments, the vector is capable of expressing the recombinant protein of this application in eukaryotic or prokaryotic cells. In some preferred embodiments, the vector is capable of expressing the recombinant protein of this application in a subject (e.g., a mammal, such as a human).

[0041] In some specific implementations, the vector may be pcDNA3.1 plasmid or pET-32a plasmid.

[0042] Fourthly, this application provides a host cell comprising the isolated nucleic acid molecules of this application, or the vector of this application.

[0043] Such host cells include, but are not limited to, prokaryotic cells such as Escherichia coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.).

[0044] In some preferred embodiments, the host cell of this application is a mammalian cell, such as CHO (e.g., CHO-K1, CHO-S, CHO DG44) or HEK293.

[0045] Fifthly, this application provides a method for preparing the polypeptide of this application, comprising culturing the host cell of this application under conditions that allow the expression of the polypeptide, and recovering the polypeptide from the cultured host cell culture, or synthesizing it using a solid-phase synthesis method.

[0046] In some preferred embodiments, the method includes: 1) Construct an expression vector containing the nucleotide sequence encoding the polypeptide of this application; 2) Transform the expression vector described in step 1) into host cells; 3) Culturing the host cells described in step 2) under conditions that allow for the expression of the polypeptides of this application; and 4) The polypeptide is recovered from the cultured host cell culture.

[0047] In a sixth aspect, this application provides a composition comprising the polypeptide and its derivatives as described above, or the isolated nucleic acid molecule as described above, or the carrier as described above, or the host cell as described above.

[0048] Understandably, the “derivative” can be a substance in various forms that is mainly a polypeptide and can perform similar biological functions, such as a stereoisomer of the polypeptide, a mixture of stereoisomers, with a terminal protecting group selected from any of the following: acetyl, palmitoyl, myristyl, stearyl and polyethylene glycol polypeptides, or a salt of the polypeptide.

[0049] In the second aspect, this application provides the use of preparing skin care products or cosmetics having at least one of the following effects, including repair, anti-wrinkle, or whitening.

[0050] Furthermore, this application provides the use of the above composition in the preparation of anti-aging skin care products or cosmetics.

[0051] Furthermore, the skincare or cosmetic product can be used on the face or the lips.

[0052] Eighthly, this application provides a skin care product or cosmetic product comprising the composition described above.

[0053] In some specific embodiments, the concentration of the polypeptide in the skincare or cosmetic product is 0.001 mg / ml to 5 mg / ml. For example, the concentration in the skincare or cosmetic product can be 0.001 mg / ml, 0.01 mg / ml, 0.1 mg / ml, 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, or a range between any two of the above values. Preferably, the concentration of the polypeptide in the skincare composition is 0.01 mg / ml to 0.1 mg / ml. For example, each 100 ml of the skincare composition of this application may contain 0.001 mg to 5 mg of polypeptide, preferably 0.01 mg to 0.1 mg of polypeptide.

[0054] In some specific implementations, the skin care or cosmetic products of this application further include one or more of the following: emollients, fillers, colorants, synthetic waxes, oils, humectants, preservatives, binders, thickeners, or emulsifiers.

[0055] The skincare or cosmetic products described in this application may contain any amount of water. The water used in cosmetics, pharmaceuticals, etc., may be, for example, ion-exchanged water, distilled water, ultrapure water, tap water, etc.

[0056] In some specific implementation schemes, the synthetic waxes described in this application may include microcrystalline wax, beeswax, candelilla wax, ceresin wax, cotton wax, carnauba wax, myrica wax, insect wax, whale wax, lignite wax, rice bran wax, lanolin, kapok wax, acetylated lanolin, lanolin oil, sugarcane wax, isopropyl lanolinate, hexyl laurate, hydrogenated lanolin, jojoba wax, hard lanolin, shellac wax, etc.

[0057] In some specific embodiments, the moisturizers described in this application may include polyethylene glycol, propylene glycol, glycerin, 1,3-butanediol, xylitol, sorbitol, maltitol, chondroitin sulfate, hyaluronic acid, mucin sulfate, cholesterol-12-hydroxystearate, sodium lactate, bile salts, pyrrolidone carboxylates, short-chain soluble collagen, diglyceride PO adduct, yarrow extract, lavender extract, etc.

[0058] In some specific implementations, the preservatives described in this application may include parabens such as methylparaben, ethylparaben, and butylparaben, as well as benzoic acid, salicylic acid, sorbic acid, p-chloro-m-cresol, hexachlorophenol, benzalkonium chloride, chlorhexidine, trichlorocarbonyl aniline, photosensitizer, phenoxyethanol, etc.

[0059] In some specific implementation schemes, the thickeners described in this application may include gum arabic, carrageenan, tragacanth gum, carob gum, casein, dextrin, gelatin, sodium pectate, sodium alginate, methylcellulose, ethylcellulose, CMC, hydroxyethylcellulose, hydroxypropylcellulose, polyvinyl alcohol (PVA), polyvinyl methyl ether (PVM), polyvinylpyrrolidone (PVP), sodium polyacrylate, carboxyvinyl polymer, locust bean gum, guar gum, tamarind gum, dialkyl dimethyl ammonium sulfate cellulose, xanthan gum, magnesium aluminum silicate, bentonite, lithium montmorillonite, propolis, silicic anhydride, etc.

[0060] In some specific embodiments, the emulsifiers described in this application may include nonionic surfactants, amphiphilic polymers such as (acrylate / stearyl methacrylate-20) copolymers, hydrophobically modified alkyl cellulose, hydrogenated lecithin, lauric acid sorbitol, palmitic acid sorbitol, oleic acid sorbitol, stearic acid sorbitol, isostearic acid sorbitol, sesquioleic acid sorbitol, trioleic acid sorbitol, tristearate sorbitol, and sesquiisostearate sorbitol.

[0061] In some specific embodiments, this application provides a face cream comprising: the polypeptide of this application, purified water, Carbopol® 940 polymer, butylene glycol, 1,2-hexanediol, p-hydroxyacetophenone, ethylhexyl palmitate, cetearyl alcohol, glyceryl stearate A165, stearyl alcohol (octadecyl alcohol), polyacrylate-13, polyisobutylene, polyacrylate-13 / polyisobutylene / polysorbate-20 (Sepiplus 400), and triethanolamine.

[0062] Specifically, the synthesis of the peptides, or stereoisomers thereof, or mixtures thereof, or salts thereof disclosed herein can be carried out according to conventional methods known in the prior art, such as solid-phase synthesis, liquid-phase synthesis, or a combination of solid and liquid methods. They can also be prepared by biotechnological methods aimed at producing a desired sequence, or by controlled hydrolysis of proteins of animal, fungal, or plant origin.

[0063] Example The following describes embodiments of this application. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0064] Example 1: Peptide Synthesis The polypeptide (PGAPSMK) in this application was synthesized by Jier Biotechnology Co., Ltd. The crude synthesized product was purified by high-performance liquid chromatography (HPLC) to obtain the purified polypeptide sample. Purity analysis was performed using a SHIMADZU LCMS-2020 mass spectrometer and HPLC. The results are shown below. Figure 1 and Figure 2 The analysis results confirm that the polypeptide of this application was synthesized, with an average molecular weight of 686.83 Da and a purity of 98.67%.

[0065] Example 2: Anti-wrinkle effects of peptides The relative content of type I collagen in cell supernatant was tested using enzyme-linked immunosorbent assay (ELISA). The rate at which the sample increased the relative content of type I collagen secreted by human skin fibroblasts was calculated, and the anti-wrinkle efficacy of the sample was evaluated.

[0066] Materials and methods 1. Cells HFF-1 human skin fibroblasts.

[0067] 2. Main reagents DMEM culture medium (Gibco), fetal bovine serum (Gibco), and Collagen I ELISA kit (Huamei Biotechnology).

[0068] 3. Main equipment Carbon dioxide incubator (Healforce), biosafety cabinet (Healforce), multi-functional microplate reader (Tecan).

[0069] 4. Experimental Methods 1) Seed cells in 96-well plates (1×10⁶ cells / well). 4 (each well), incubated overnight at 37°C in a 5% CO2 incubator.

[0070] 2) Drug administration: Remove the culture medium, and perform three replicates per group. Add 150 μL of culture medium to each well of the blank control group (BC), and add culture medium containing the corresponding concentration of the test sample to the sample group. Incubate for 24 h.

[0071] 3) Detection: After culturing for 24 hours, the supernatant was collected, centrifuged, and then detected using an enzyme-linked immunosorbent assay kit to calculate the average protein content.

[0072] 4) Calculation of improvement rate: Improvement rate (%) = (Sample group - Blank control group) / Blank control group × 100% 4.3 Statistical Analysis Calculate the mean and standard deviation (SD) of each group of experiments. P <0.05 is considered a significant difference. P <0.01 indicates a highly significant difference; P >0.05 indicates no significant difference, and the results are shown in Table 1.

[0073] Table 1

[0074] As shown in Table 1, compared with the control group, the average content of type I collagen in the peptide of this application at concentrations of 10 μg / mL and 100 μg / mL was 282.79 ng / mL, respectively. p <0.05) and 354.02 ( p <0.05). The peptides in this application significantly promote the secretion of type I collagen.

[0075] Example 3: Repairing effects of polypeptides The test sample was evaluated for its ability to heal scratches on the HaCaT immortalized keratinocyte scratch model, thus assessing the sample's repair efficacy.

[0076] Materials and methods 1. Experimental cells: HaCaT human immortalized keratinocytes.

[0077] 2. Main reagents: DMEM medium (Gibco), fetal bovine serum (Gibco).

[0078] 3. Main equipment: carbon dioxide incubator (healforce), biosafety cabinet (healforce), fluorescence inverted microscope (OLYMPUS).

[0079] 4. Experimental Methods 1) Cell plating: Dilute the cell suspension to a density of 6 × 10⁻⁶. 5 Inoculate 2 mL / well into 6-well plates and incubate at 37°C for 24 h after inoculation. 2) Scratching and Sample Addition: Remove the 6-well plate, discard the supernatant, and mark the bottom of each well with a marker by drawing horizontal lines on the back of each well, averaging 5 lines. Add 1 mL of PBS to each well, and use a pipette tip to draw a vertical line on the bottom of the 6-well plate. Wash slowly with PBS 3 times to remove detached cells. The blank control group (BC) is added with serum-free DMEM high-glucose medium, while the test sample groups are prepared with serum-free medium to the corresponding concentration. After drug addition, incubate at 37℃, 5% CO2 for 24h±2h.

[0080] 3) Photography: At 0 and 24 hours after drug administration, photograph the scratches in the 6-well plate under a microscope. The horizontal line on the back divides the scratches into 6 regions. Take a picture of the lower field of view of each region. The scratch position should be consistent in the field of view selected at different time points. Each group has 6 different fields of view at each time point, i.e., 6 pictures.

[0081] 4) The area A of the image was calculated using the microscope's built-in software.

[0082] The formula for calculating scratch healing rate is as follows:

[0083] The mean and standard deviation (SD) of each group of experiments were calculated. Statistical analysis of the data was performed using Graphpad Prism software, and one-way Anova analysis was used for data between multiple groups. P <0.05 is considered a significant difference. P <0.01 indicates a highly significant difference; P >0.05 indicates no significant difference. Experimental results are shown in Table 2. Figure 3 and 4 As shown.

[0084] Table 2

[0085] As shown in Table 2, compared with the blank group (BC), the scratch healing rate of the peptide in this application at a concentration of 100 μg / mL was 70.04%. p <0.05), which has a significant ability to promote scratch healing.

[0086] Example 4: Whitening effect of peptides The TYR gene is the most critical rate-limiting enzyme in the melanin synthesis pathway, encoding tyrosinase, which initiates melanin biosynthesis by catalyzing the hydroxylation of tyrosine to dopaquinone. qRT-PCR was used to detect the expression level of the TYR gene in B16-F10 cells, examining the effect of samples on the expression level of the TYR gene in B16-F10 cells.

[0087] Reagents and equipment DMEM medium (Gibco), fetal bovine serum (Gibco), Trizol (Takara), TYR gene primers (Cesofi), PrimeScript™ RT reagent Kit with gDNA Eraser (Takara), TB Green® Premix EXTaq™ (Takara), CO2 incubator (Healforce), biosafety cabinet (Healforce), multi-functional microplate reader (Tecan), qRT-PCR instrument (Thermo Fisher) Experimental methods 1) Seed cells in 6-well plates (3 × 10⁻⁶ cells / well). 5 (each well) was incubated overnight at 37°C in a 5% CO2 incubator.

[0088] 2) Drug administration: Remove the culture medium, and repeat the experiment in 3 replicates per group. Add 2 ml of culture medium to the blank control group, add α-MSH to the model group, and add culture medium containing the corresponding concentration of the test sample to the sample group. Incubate at 37℃ and 5% CO2 for 24 h.

[0089] 3) Detection: After 24 hours of culture, the supernatant was removed, and the cell surface was washed twice with PBS. 1 mL of Trizol, 200 μL of chloroform, isopropanol, and 75% ethanol were added sequentially for RNA extraction according to the RNA extraction procedure. Finally, 10 μL of RNase-free water was added to dissolve the RNA, and the total RNA concentration was measured using a multi-functional microplate reader. The RNA was reverse transcribed into cDNA, and the target gene and internal control gene were amplified using qRT-PCR according to the TBGreen® Premix EX Taq™ kit instructions. The experimental results are shown in Table 3.

[0090] Table 3

[0091] As shown in Table 3, compared with the blank control, the relative expression level of the TYR gene in the model group was 1.07±0.03 (p<0.05), indicating successful model establishment. The relative expression level of the TYR gene at a concentration of 100 μg / mL was 0.74±0.01 (p<0.05). p <0.01), significantly reducing the relative expression level of the TYR gene. It also showed a relatively significant effect in reducing the relative expression level of the TYR gene at a concentration of 10 μg / mL.

[0092] Example 5: Safety of the peptide 1. Detection of the cytotoxic effect of the peptides in this application on human fibroblasts 1) Cell plating: Dilute the cell suspension to a density of 1×10⁻⁶.5 Inoculate 100 μL / well into 96-well plates and incubate at 37°C for 24 h after inoculation. 2) Sample addition: Remove the 96-well plate, discard the supernatant, add culture medium to the normal control group, and add culture medium containing different concentrations of samples to the sample group, 100 μL / well, and incubate in a 37℃, 5% CO2 incubator for 24 h; 3) Cell viability assay: Remove the 96-well plate, let it equilibrate for about 10 minutes, add 10 μL of CCK8 assay reagent per well, incubate at 37℃ for 1 hour, and then measure the OD value; calculate cell viability: cell viability (%) = OD value of sample group / OD value of normal control group × 100%. The results are shown in Table 4. It can be seen that the peptides in this application have no effect on cell viability.

[0093] Table 4

[0094] 2. Detection of irritation by the polypeptide in this application. The chicken embryo chorioallantoic membrane (CAM) test is used to assess the ability of a test substance to cause changes in CAM toxicity, evaluate the potential eye irritation or corrosiveness of the substance being evaluated, and screen for non-irritating samples.

[0095] Materials and methods: 1. Experimental materials: SPF-grade fertilized chicken embryos of White Laihang chickens, sodium chloride (Solarbio), sodium hydroxide (Shanghai Maclean's Co., Ltd.) 2. Experimental Methods: Based on SN / T2329-2009 "Cosmetic Eye Irritation / Corrosivity Test: Chicken Embryo Villi Allantoic Membrane Test" Using the endpoint assessment method, 0.3 ml of sample was added to the CAM membrane. After 3 minutes of incubation, the sample was gently rinsed with physiological saline to observe for bleeding, coagulation, and angiogenesis, and photographs were taken. A negative control of 0.9% NaCl was also included. The images of the sample and negative control are shown below. Figure 5 and Figure 6 As shown. Compared with the negative control group, no bleeding, coagulation, or vascular fusion was observed in the CAM of the peptide of this application at a concentration of 5 mg / ml. Bleeding score: 0, coagulation score: 0, vascular fusion score: 0; therefore, the ES score of this sample at a concentration of 5 mg / ml was 0. Thus, under these test conditions, the peptide of this application is non-irritating at a concentration of 5 mg / ml.

[0096] Example 6: Preparation of skin care products or cosmetics A face cream is prepared through the following steps: 1. Inspect and clean the equipment to ensure it is clean, dry, and in good working order; check the raw material weighing; accurately weigh the required raw materials and verify them.

[0097] 2. Add raw materials (oil phase) 6-10 to the oil phase pot, heat and stir to about 85℃ until the materials are completely melted, and set aside. 3. Prepare fresh deionized water, weigh the exact amount into the emulsification pot, and start stirring at a speed of 20 r / min.

[0098] 4. Raise the emulsification pot lid, slowly sprinkle ingredient #2 CARBOPOL® 940 POLYMER close to the liquid surface, stir continuously for 10 minutes, homogenize for 2 minutes (speed 2000 rpm), stir and heat to 85±3℃, turn on homogenization, speed 2500 rpm, time 3-5 minutes.

[0099] 5. Maintain a stirring speed of 20-30 rpm and add the pre-mixed raw materials 6-10 (oil phase) to the emulsification tank. After the materials are completely drawn into the emulsification tank, homogenize under vacuum for 10-15 minutes at a homogenization speed of 2500-3500 rpm. After homogenization, stir and cool. When the temperature drops to about 60℃, add raw materials 3, 4, and 5 (raw material 5 is pre-dissolved in raw materials 3 and 4). Maintain stirring and cooling. When the temperature drops below 45℃, add raw materials 11, 12 (the peptides in this application), and 13. Continue to cool and stir for 5-10 minutes, then homogenize under vacuum for 2-5 minutes (2000 rpm). After homogenization, stir the paste under vacuum for 5-10 minutes until fully mixed and homogeneous before discharging. The specific formula is shown in Table 5 below.

[0100] Table 5

[0101] It should be noted that this application is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments with the same structure and effect as the technical concept within the scope of this application are included in the technical scope of this application. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of this application, are also included in the scope of this application.

Claims

1. A polypeptide, characterized in that, The amino acid sequence of the polypeptide is PGAPSMK (SEQ ID NO.1).

2. An isolated nucleic acid molecule, characterized in that, The isolated nucleic acid molecules include: 1) A nucleotide sequence capable of encoding the polypeptide as described in claim 1, or 2) A sequence that can strictly hybridize with the nucleotide sequence encoding the polypeptide described in 1).

3. A carrier, characterized in that, The carrier comprises the isolated nucleic acid molecule as described in claim 2.

4. A host cell, characterized in that, The host cell comprises the isolated nucleic acid molecule as described in claim 2, or the vector as described in claim 3.

5. A composition, characterized in that, The composition comprises the polypeptide or a derivative thereof as claimed in claim 1, or the isolated nucleic acid molecule as claimed in claim 2, or the carrier as claimed in claim 3, or the host cell as claimed in claim 4.

6. Use of the composition of claim 5 in the preparation of the following products: 1) Use in the preparation of skin care or cosmetic products having at least one of the following effects, said effects including repair, anti-wrinkle, or whitening; or 2) Used in the preparation of anti-aging skin care products or cosmetics.

7. A skincare or cosmetic product, characterized in that, The skin care or cosmetic product includes the composition as described in claim 5.

8. The skincare or cosmetic product according to claim 7, characterized in that, The concentration of the polypeptide in the skin care product or cosmetic product is 0.001 mg / ml to 5 mg / ml.

9. The skincare or cosmetic product according to claim 7 or 8, characterized in that, The skin care or cosmetic products also include one or more of the following: emollients, fillers, colorants, synthetic waxes, oils, humectants, preservatives, binders, thickeners, or emulsifiers.

10. The skincare or cosmetic product according to claim 9, characterized in that, The skincare or cosmetic products also include purified water, carbomer 940, butylene glycol, 1,2-hexanediol, p-hydroxyacetophenone, ethylhexyl palmitate, cetearyl alcohol, glyceryl stearate A165, stearyl alcohol, polyacrylate-13, polyisobutylene, polysorbate-20, and triethanolamine.

Images