An endogenous polypeptide, its preparation method and application

Abstract

The application discloses an endogenous polypeptide, a preparation method and application thereof. A structural formula of the polypeptide is shown in formula (I): R1-(AA1)m-AA2-AA3-(AA4)n-R2, formula (I); wherein m is independently 2 or 3; n is independently 1, 2 or 3; AA1 is independently K, R or H; AA2 is independently I or W; AA3 is independently I or W; AA4 is independently K, R or H; R1 is any one of -H, Y, LY, PLY or LLY; and R2 is any one of -OH, L, LL, E, EL, EW, W, EH or EHL. The polypeptide has removed a high-affinity heparin binding domain while retaining the targeted antioxidant and anti-inflammatory functions, thereby avoiding signal interference and potential biological safety risks possibly caused by full-length PF4, and meeting the safety requirements of cosmetic ingredients.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to an endogenous polypeptide, its preparation method, and its application. Background Technology

[0002] Platelet factor 4 (PF4) is a cytokine stored in platelet granules. During platelet activation or thrombosis, it is secreted in large quantities and released extracellularly. PF4 has a wide range of biological functions. In addition to participating in the regulation of platelet aggregation and exerting antibacterial and antiviral activities, PF4 also plays an important role in physiological processes such as inflammatory responses, immune regulation, and angiogenesis, and is crucial for maintaining homeostasis.

[0003] PF4 is also closely related to aging. In particular, PF4 has significant potential in reducing inflammation and improving age-related cognitive function. Activating PF4 can not only rebuild the neural structure of the hippocampus in the aging brain and restore its cognitive function, but also significantly reduce the systemic inflammatory response. Furthermore, PF4 exerts its anti-inflammatory effects through multiple pathways, including inhibiting inflammatory cell activation, reducing the secretion level of inflammatory mediators, and promoting inflammatory cell apoptosis. In addition, PF4 activation can effectively promote the proliferation of hippocampal precursor cells in the aging brain, and upregulating PF4 expression levels can significantly improve age-related aging phenotypes and cognitive impairment, thereby slowing brain aging and restoring normal brain physiological function. Summary of the Invention

[0004] In order to provide a polypeptide with antioxidant, anti-inflammatory, moisturizing, skin brightening and skin pigmentation improvement effects, this invention provides an endogenous polypeptide, its preparation method and application.

[0005] Based on the quantitative structure-activity relationship (QSAR) of PF4, this invention designs and screens a series of novel PF4-derived peptide sequences. Efficacy verification experiments show that this series of derived peptides not only maintains the inherent anti-inflammatory activity of PF4 but also endows it with excellent antioxidant capacity, significantly inhibiting melanin production and accumulation, thus better meeting the application requirements in the cosmetics field. Therefore, the PF4-derived peptides provided by this invention possess multiple effects, including anti-inflammatory, antioxidant, skin brightening, and improvement of dull skin, and exhibit excellent safety and stability. They can be used as active ingredients in the development of cosmetics or pharmaceutical products, demonstrating promising application prospects and industrialization potential.

[0006] The present invention solves the above-mentioned technical problems through the following technical means: This invention provides a polypeptide, its stereoisomer, or a salt thereof, the structural formula of which is shown in Formula (I): R1-(AA1)m-AA2-AA3-(AA4)n-R2, formula (I); Where m is independently 2 or 3; n can be 1, 2, or 3 independently; AA1 can be K, R, or H independently; AA2 can be either I or W independently; AA3 can be either I or W independently; AA4 can be K, R, or H independently; R1 is any one of -H, Y, LY, PLY, or LLY; R2 is any one of -OH, L, LL, E, EL, EW, W, EH, or EHL.

[0007] In this invention, -H and -OH represent hydrogen and hydroxyl groups, respectively, and H represents histidine or its amino acid residue.

[0008] In some embodiments of the present invention m is 2; n is 1; AA1 is K; AA4 is K; R1 is -H, Y, LY, or PLY; R2 is L, H, EL, EH, W, or EW; AA2 is I; AA3 is I; or, m is 2; n is 2; AA1 is K; AA4 is K; R1 is -H, Y, LLY or PLY; R2 is L, H, EL, EH, W or EW; AA2 is I; AA3 is I; or, m is 2; n is 1 or 2; AA1 is K; AA4 is K; R1 is -H, Y, LY, LLY or PLY; R2 is L, H, EL, EH, W or EW; AA2 is W or I; AA3 is W or I; and AA2 and AA3 are not both I; or, m is 2; n is 2; AA1 is R; AA4 is R; R1 is LLY; R2 is L; AA2 is I; AA3 is I; or, m is 2; n is 2; AA1 is H; AA4 is H; R1 is LY; R2 is L; AA2 is I; AA3 is I.

[0009] In some preferred embodiments of the present invention m is 2; n is 1; AA1 is K; AA4 is K; R1 is -H, Y, LY, or PLY; R2 is EH or EW; AA2 is I; AA3 is I; or, m is 2; n is 2; AA1 is K; AA4 is K; R1 is -H, Y, LLY or PLY; R2 is L or W; AA2 is I; AA3 is I; or, m is 2; n is 1 or 2; AA1 is K; AA4 is K; R1 is -H, Y, LY, LLY or PLY; R2 is L, H, EL, EH, W or EW; AA2 is W or I; AA3 is W or I; and AA2 and AA3 are not both I.

[0010] In some embodiments of the present invention m is 2; n is 2; AA1 is K; AA4 is K; R1 is -H, Y, or PLY; R2 is L or W; AA2 is W or I; AA3 is I; or, m is 2; n is 1; AA1 is K; AA4 is K; R1 is LLY; R2 is EH; AA2 is W; AA3 is I; or, m is 2; n is 1; AA1 is K; AA4 is K; R1 is -H, Y, LY, or PLY; R2 is EH or EW; AA2 is I; AA3 is I; or, m is 2; n is 1 or 2; AA1 is K; AA4 is K; R1 is Y, LY, LLY or PLY; R2 is L, H, EH or EW; AA2 is W; AA3 is I or W; or, m is 2; n is 1 or 2; AA1 is K; AA4 is K; R1 is -H, Y, LY, LLY or PLY; R2 is L, EL, EH, W or EW; AA2 is I; AA3 is W.

[0011] In some preferred embodiments of the present invention m is 2; n is 2; AA1 is K; AA4 is K; R1 is -H, Y, or PLY; R2 is L; AA2 is I; AA3 is I; or, m is 2; n is 2; AA1 is K; AA4 is K; R1 is -H or PLY; R2 is L or W; AA2 is W; AA3 is I.

[0012] In some embodiments of the present invention, the polypeptide comprises an amino acid sequence as shown in any one of SEQ ID NO: 1-36.

[0013] In some preferred embodiments of the present invention, the amino acid sequences of the polypeptides are shown in any one of SEQ ID NO: 1-36.

[0014] In some more preferred embodiments of the present invention, the amino acids of the stereoisomers are each independently L-type or D-type.

[0015] A second aspect of the present invention provides an isolated nucleic acid that encodes a polypeptide, a stereoisomer thereof, or a salt thereof as described in the first aspect of the present invention.

[0016] A third aspect of the present invention provides a recombinant expression vector comprising the isolated nucleic acid as described in the second aspect of the present invention.

[0017] A fourth aspect of the present invention provides a transformant comprising a nucleic acid as described in the second aspect of the present invention or a recombinant expression vector as described in the third aspect of the present invention; the transformant is a non-animal or non-plant variety.

[0018] The fifth aspect of the present invention provides a method for preparing polypeptides, stereoisomers thereof, or salts thereof as described in the first aspect of the present invention, said method being the Fmoc solid-phase polypeptide synthesis method or the liquid-phase polypeptide synthesis method.

[0019] In some preferred embodiments of the present invention, the method includes: (1) The first Fmoc-protected amino acid is immobilized on the resin, and then the deprotection and the next amino acid are cyclically linked to gradually extend the peptide chain to obtain the crude peptide. (2) Subsequently, the crude peptide was cleaved from the resin using a cleavage fluid; (3) The lysed polypeptide is collected by centrifugation and then purified by reversed-phase high-performance liquid chromatography to obtain the polypeptide.

[0020] A sixth aspect of the present invention provides a composition comprising one or more of the polypeptide, its stereoisomers or salts thereof as described in the first aspect of the present invention, and a pharmaceutically acceptable carrier.

[0021] In some preferred embodiments of the present invention, the carrier is selected from one or more of matrix raw materials, auxiliary raw materials and functional raw materials; the matrix raw materials include oily raw materials, powdery raw materials and solvent raw materials; the auxiliary raw materials include fragrances, pigments, preservatives, surfactants, thickeners and water-soluble polymers; the functional raw materials include anti-aging agents, moisturizers, whitening agents, sunscreens, nutritional additives, deodorants and hair removal agents.

[0022] In some preferred embodiments of the present invention, the oily raw material is selected from one or more of oils, waxes and higher fatty acids; the powdery raw material is selected from one or more of mica powder, talc powder, kaolin, silica, calcium carbonate and perlite; the solvent raw material is selected from water, alcohols, ketones, ethers and esters of aromatic organic compounds; and the thickener includes one or more of carrageenan and xanthan gum.

[0023] In some further preferred embodiments of the invention, the composition is selected from any one of toner, face mask, serum, lotion, cream, and freeze-dried powder.

[0024] In a specific embodiment of the present invention, the composition comprises 0.1% of the polypeptide, 0.55% of acrylate / C10-30 alkanol acrylate crosspolymer, 3% 1,3-butanediol, 0.4% p-hydroxyacetophenone, 0.60% 1,2-hexanediol, 0.2% L-arginine, and 0.08% sodium hydroxide.

[0025] The seventh aspect of the present invention provides the use of one or more of the polypeptides, stereoisomers thereof, or salts thereof as described in the first aspect of the present invention, or compositions as described in the fifth aspect of the present invention, in one or more products for skin anti-oxidation, anti-inflammatory, skin brightening, skin moisturizing, and skin barrier repair.

[0026] In some preferred embodiments of the present invention, the product is a cosmetic or a pharmaceutical.

[0027] In some more preferred embodiments of the present invention, the cosmetic is a skin care product.

[0028] The eighth aspect of the present invention provides a method for skin care that does not intervene in the human body or cause trauma, characterized in that the method comprises: applying to a subject in need any one or more of the polypeptide, its stereoisomer or salt thereof, or a composition as described in the fifth aspect of the present invention; the skin care includes any one or more of the following pathways: anti-oxidation, anti-inflammatory, skin brightening, skin moisturizing and skin barrier repair.

[0029] In some preferred embodiments of the present invention, the method is for non-therapeutic purposes.

[0030] In some more preferred embodiments of the present invention, the antioxidant effect is manifested in scavenging superoxide anion free radicals; the anti-inflammatory effect is manifested in inhibiting the expression of IL-6 and / or CXCL8; and the skin brightening effect is manifested in inhibiting the production and / or accumulation of melanin.

[0031] The ninth aspect of the present invention provides the use of one or more of the polypeptides, their stereoisomers or salts thereof as described in the first aspect of the present invention, or the compositions as described in the fifth aspect of the present invention, in skin care; said skin care includes one or more of the following pathways: anti-oxidation, anti-inflammatory, skin brightening, skin moisturizing and skin barrier repair.

[0032] In some preferred embodiments of the present invention, the application is for non-therapeutic purposes.

[0033] In some more preferred embodiments of the present invention, the antioxidant effect is manifested in scavenging superoxide anion free radicals; the anti-inflammatory effect is manifested in inhibiting the expression of IL-6 and / or CXCL8; and the skin brightening effect is manifested in inhibiting the production and / or accumulation of melanin.

[0034] In this invention, cosmetics refer to daily chemical industrial products applied to the surface of the human body, such as skin, hair, nails, and lips, by means of rubbing, spraying, or other similar methods, for the purpose of cleaning, protecting, beautifying, and modifying.

[0035] In this invention, skin care products are a type of cosmetic, which are daily care products used to protect and improve the condition of the skin, covering functional categories such as cleansing, moisturizing, hydrating, nourishing, sun protection, whitening, anti-inflammatory, and antioxidant.

[0036] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0037] The reagents and raw materials used in this invention are all commercially available.

[0038] The positive and progressive effects of this invention are as follows: While retaining its targeted antioxidant and anti-inflammatory functions, the peptide has removed the domain that binds to heparin with high affinity, thereby avoiding signal interference and potential biosafety risks that may be caused by the full-length PF4, and meeting the safety requirements for cosmetic ingredients.

[0039] The peptides not only directly scavenge free radicals, exhibiting excellent antioxidant activity, but also effectively inhibit the release of inflammation-related factors, achieving an anti-inflammatory effect. These two effects reinforce each other, working together on key pathways of abnormal skin pigmentation, providing a more comprehensive and efficient solution for brightening skin tone and improving dullness than single-function ingredients.

[0040] The peptides have excellent moisturizing properties, which help maintain a healthy skin barrier and further reduce the risk of inflammation and dull skin caused by external stimuli.

[0041] The peptide described has significant advantages: In existing technologies, it is usually necessary to physically combine multiple ingredients with different functions such as anti-oxidation, anti-inflammation, moisturizing, brightening skin tone, and improving abnormal skin pigmentation. This invention achieves the same or even better comprehensive effects with a single-structure peptide, simplifying the formulation process, improving product stability, and avoiding compatibility issues that may exist between multiple ingredients. This provides a superior technical means for developing highly effective and safe cosmetics or pharmaceuticals.

[0042] By systematically analyzing the functional domains of the PF4 amino acid sequence, peptides with independent antioxidant and anti-inflammatory activities were designed and screened. These peptides have a molecular weight much smaller than the full-length protein, exhibiting excellent skin permeability and effectively delivering to the target skin layer, overcoming the core obstacle of poor transdermal absorption of full-length PF4. Furthermore, their chemical structure is well-defined, easy to synthesize and purify, making them highly suitable for large-scale application as active ingredients in cosmetics, skincare products, or topical pharmaceuticals. Attached Figure Description

[0043] Figure 1 For polypeptide pairs IL6 The effect of mRNA expression inhibition is shown in the figure.

[0044] Figure 2 For polypeptide pairs CXCL8 The effect of mRNA expression inhibition is shown in the figure.

[0045] Figure 3 This is a diagram showing the inhibitory effect of the peptide on IL-6 expression.

[0046] Figure 4 This is a diagram showing the inhibitory effect of peptides on melanin production. Detailed Implementation

[0047] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0048] Example 1 Preparation of formulas (1) to (36)

[0049] The synthesis was carried out using 2-chlorotriphenylmethyl chloride resin (2-CL resin) based on the Fmoc solid-phase peptide synthesis method (Fmoc-SPPS); 0.15 mmol of resin (0.1 g) was weighed according to the resin loading. The main reagents used are shown in Table 1.

[0050] Table 1. Main reagents used in polypeptide synthesis (1) to (36)

[0051] ① Place the weighed resin in a reaction vessel and add DCM to swell for 20 min. Then, weigh out 0.15 mmol of Fmoc-Lys(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Leu-OH, or Fmoc-Pro-OH (each 1.5 eq) and add them to the reaction vessel. ② Add DIEA (3.0 eq) and DCM (5 times the volume of the resin) to the reaction vessel. After shaking and reacting at room temperature for 6 h, add an equal volume of a methanol and DIEA mixture and continue shaking for 30 min to quench the reaction. Then wash the resin three times with DMF solution (3 times the volume of the resin), 1 min each time. ③ Prepare a 20% piperidine / DMF solution (piperidine / DMF = 1:4), completely immerse the resin in the deprotection solution, and react with shaking at room temperature. The reaction is carried out in two steps: the first reaction is 5 min, the waste liquid is discarded, fresh deprotection solution is added, and the reaction continues for 15 min. After the reaction was complete, a small amount of resin was tested for ninhydrin. The resin was then washed twice each with DMF, methanol, and DMF, for 1 min each time. ④ Weigh 0.15 mmol of Fmoc-Lys(Boc)-OH, Fmoc-Tyr(tBu)-OH, or Fmoc-Leu-OH (all 3.0 eq), and dissolve them together with HBTU (2.9 eq), HOBt (3.0 eq), and DIEA (6.0 eq) in DMF (solvent volume 5 times that of resin). After pre-activation at room temperature for 3 min, the activation solution was added to the resin, and the reaction was carried out with shaking at room temperature for 1 h. After the reaction was complete, the resin was washed four times with DMF, for 1 min each time. A small amount of resin was tested for ninhydrin: if the resin was colorless, the reaction was complete; if it showed color, the condensation was incomplete, and the reaction needed to continue. Finally, the resin was washed twice each with DMF, methanol, and DMF, for 1 min each time. ⑤ Following the pre-defined amino acid sequence (as shown in Table 2), the subsequent amino acids were added sequentially. After each round of condensation reaction, ninhydrin was tested. If the result was positive, condensation reagent was added and the reaction continued for 30 min. ⑥ After the last amino acid lost its Fmoc protecting group, the resin was washed four times with DMF and methanol. The resin was filtered to dryness and then thoroughly dried with a blower to obtain resin carrying the target peptide. ⑦ The peptide was cleaved from the resin using a cleavage buffer to obtain crude peptide. The crude peptide was purified using a C18 reverse-phase preparative column, the main fraction was collected, and finally, it was freeze-dried under vacuum to obtain pure target peptide.Among them, the purity of formulas (2), (5), (11), (21), (22), (29) and (35) is greater than 95%, and the LCMS m / z are 943.64[M+H]+, 871.37[M+H]+, 1033.71[M+H]+, 622.98[M+2H]2+, 658.92[M+2H]2+, 696.04[M+2H]2+ and 1357.79[M+H]+, respectively.

[0052] Table 2. Related information of peptides in formulas (1) to (36)

[0053] Example 2: Determination of antioxidant activity

[0054] 1. Experimental Principle: Superoxide anion radicals are not only precursors to other highly reactive reactive oxygen species (ROS) in organisms, but can also directly oxidize proteins, damage DNA, and trigger lipid peroxidation, thus participating in various pathological processes such as aging and inflammation. Therefore, accurately assessing a sample's ability to scavenge superoxide anion radicals is crucial for evaluating its antioxidant activity and related biological effects. Under weakly alkaline conditions, pyrogallol undergoes an auto-oxidation reaction, continuously generating superoxide anion radicals and producing colored intermediates. This process exhibits characteristic absorption peaks at 325 nm or 420 nm wavelengths, with absorbance increasing linearly with reaction time. Adding a test sample with superoxide anion radical scavenging capabilities inhibits the auto-oxidation rate, resulting in a slower absorbance increase curve. By calculating the scavenging rate, the sample's ability to scavenge superoxide anion radicals can be evaluated.

[0055] 2. Experimental Procedure: The peptide test sample solutions (100 ppm) of formulas (2), (5), (11), (21), (22), (29), and (35) were mixed with Tris-HCl buffer at pH 8.0, and then freshly prepared pyrogallol solution was added to initiate the reaction. The mixture was incubated at room temperature for 30 minutes, and then the pH was adjusted to 3.0 to terminate the reaction. The absorbance of each group was measured at 420 nm using a UV-Vis spectrophotometer, and the superoxide anion scavenging rate was calculated using the following formula: Scavenging rate (%) = [1-(A S -A0) / A C ] × 100% (A S A0 is the absorbance of the sample group containing antioxidants, and A0 is the background absorbance of the antioxidant group without pyrogallol. C (Absorbance of the control group containing pyrogallol without antioxidants).

[0056] 3. Experimental Results: As shown in Table 3, peptides of formulas (2), (5), (11), (21), (22), (29), and (35) all exhibited significant inhibitory effects on superoxide anions at a concentration of 100 ppm, with scavenging rates ranging from 51.23% to 79.41%. The experimental results indicate that peptides of formulas (2), (5), (11), (21), (22), (29), and (35) possess significant antioxidant capabilities.

[0057] Table 3 Results of Superoxide Anion Suppression Test

[0058] Example 3 Anti-inflammatory activity analysis

[0059] 1. Detection of inflammation-related gene expression

[0060] 1) Experimental Principle: To assess the anti-inflammatory capacity of the test samples, this study established a UVB irradiation-induced keratinocyte inflammation model. This model can simulate the skin inflammation response induced by ultraviolet radiation. The anti-inflammatory effect of the samples can be reflected by detecting changes in the gene expression levels of two key inflammation-related factors—interleukin-6 (IL-6) and CXC motif chemokine 8 (CXCL8). Under UVB stimulation, the expression of IL-6 and CXCL8 is usually significantly upregulated, which are markers of inflammation activation. The anti-inflammatory capacity of the samples can be reflected by comparing the differences in the expression of the mRNA of these factors between the sample treatment group and the model control group.

[0061] 2) Research process: (1) Experimental groups: blank group (no UVB radiation); model group (UVB radiation); experimental group: each group of peptides of formula (2), formula (5), formula (11), formula (21), formula (22), formula (29) and formula (35) was treated with UVB radiation and 30 ppm aqueous solution of the corresponding compound, respectively.

[0062] (2) Experimental steps: ① When the keratinocyte deposition rate in the 24-well plate reached 40%–60%, the cells were divided into groups and administered the drug. Each group had 3 replicates, and 1 mL of cell culture medium containing the corresponding concentration of the test sample was added to each well. After drug administration, the 24-well plate was placed in an incubator (37℃, 5% CO2) for 24 hours. ② According to the test protocol, the groups requiring irradiation were subjected to UVB irradiation at a dose of 30 mJ / cm². 2After irradiation, the cells were incubated in an incubator (37℃, 5% CO2) for 24 h. ③ After 24 h of incubation, the cell supernatant was collected, and the cells were washed twice with 1 mL / well of PBS. 1 mL of RNAiso Plus was added to each well, and the cells were lysed by pipetting. ④ RNA was extracted, reverse transcribed into cDNA, and then detected by quantitative real-time PCR using a 2... -△△CT The method is used to calculate the results.

[0063] 3) Experimental Results: The experimental results are shown in Figure 1 And Table 4. Compared with the blank group, the model group IL6 The relative expression level of mRNA increased by 78%. Compared with the model group, the expression levels of equations (2), (5), (11), (21), (22), (29), and (35) were significantly higher. IL6 The relative expression levels of mRNA decreased by 30.21% to 73.17% (inhibition rate). The above results indicate that the peptides of formula (2), formula (5), formula (11), formula (21), formula (22), formula (29) and formula (35) have the ability to significantly inhibit inflammatory responses.

[0064] The experimental results are shown in Figure 2 And Table 5. Compared with the blank group, the model group CXCL8 The relative expression level of mRNA increased by 55%. Compared with the model group, the expression levels of equations (2), (5), (11), (21), (22), (29), and (35) were significantly higher. CXCL8 The relative expression level of mRNA decreased by 51.93% to 86.05% (inhibition rate). The above results indicate that the peptides of formula (2), formula (5), formula (11), formula (21), formula (22), formula (29) and formula (35) have the ability to significantly inhibit inflammatory response.

[0065] Table 4 IL6 Gene expression test results

[0066] Table 5 CXCL8 Gene expression test results

[0067] 2. Detection of expression of inflammation-related factor proteins

[0068] 1) Experimental principle: Under ultraviolet (UV) radiation, skin cells suffer direct damage to their DNA, proteins, and cell membranes, accompanied by the generation of large amounts of reactive oxygen species (ROS). Damaged cells then activate inflammatory signaling pathways, releasing various pro-inflammatory cytokines such as IL-1, IL-6, and TNF-α. Persistent inflammation can lead to collagen and elastin degradation, inhibited collagen synthesis, disordered pigment metabolism, and persistent oxidative stress, thereby exacerbating cell damage and disrupting the normal skin structure. UVB irradiation can induce damage to HaCaT cells, leading to the release of inflammatory factors. ELISA detection of IL-6 concentration in samples can objectively evaluate the anti-inflammatory efficacy of the tested samples.

[0069] 2) Research process: (1) Experimental groups: blank group (no UVB radiation); model group (UVB radiation); experimental group: each group of peptides of formula (2), formula (5), formula (11), formula (21), formula (22), formula (29) and formula (35) was treated with UVB radiation and 20 ppm aqueous solution of the corresponding compound, respectively.

[0070] (2) Experimental steps: ① When the keratinocyte deposition rate in the 24-well plate reached 40%–60%, the cells were divided into groups and administered the drug. Each group had 3 replicates, and 1 mL of cell culture medium containing the corresponding concentration of the test sample was added to each well. After drug administration, the 24-well plate was placed in an incubator (37 ℃, 5% CO2) and cultured for 24 h. ② According to the test protocol, the groups requiring irradiation were subjected to UVB irradiation at a dose of 30 mJ / cm². 2 After irradiation, the cells were placed in an incubator (37 ℃, 5% CO2) and cultured for another 24 h. ③ After 24 h of culture, the cell supernatant was collected and ELISA was performed according to the IL-6 ELISA kit instructions.

[0071] (3) Experimental results: The experimental results are shown in Figure 3 See Table 6. Compared with the model group, the IL-6 content in groups (2), (5), (11), (21), (22), (29), and (35) decreased by 29.12% to 64.41% (inhibition rate). The above results indicate that the peptides (2), (5), (11), (21), (22), (29), and (35) have a significant ability to inhibit inflammatory responses.

[0072] Table 6 IL6 content detection results

[0073] Example 4 Melanin Inhibition Ability Test

[0074] 1. Experimental Principle: B16 cells (mouse skin melanoma cells) can activate the intracellular melanin synthesis pathway under stimulation by specific inducers such as α-MSH, thereby upregulating tyrosinase activity and promoting melanin synthesis and accumulation. The inhibitory or promoting effect of the test substance on melanin production can be evaluated by quantitatively detecting melanin content or tyrosinase activity. In this experiment, melanin content was detected by measuring absorbance (OD) at a wavelength of 405 nm, and the melanin inhibition rate was calculated using the following formula: Melanin inhibition rate (%) = (Model group OD - OD of each well) / (Model group OD - Blank group OD) × 100%.

[0075] 2. Research Process: (1) Experimental groups: blank group (containing only culture medium); negative control group (B16 cells + culture medium); model group (B16 cells + culture medium containing 10 μM α-MSH); experimental group: each group was cultured with culture medium containing 10 μM α-MSH and 30 ppm peptide samples of formula (2), formula (5), formula (11), formula (21), formula (22), formula (29) and formula (35).

[0076] (2) Experimental steps: 1) Seed B16 cells into a six-well plate, with 1×10 cells seeded into each well. 5 1) Add 1 mL of complete culture medium to each cell. 2) When the cell confluence reaches 50%–60%, discard the original culture medium, gently wash the cell surface with PBS, and then process according to the experimental groups. 3) After processing, discard the culture medium, wash once with PBS, and add 1 mL of 0.25% trypsin to each well for digestion. When most cells become rounded but still adhere to the wall, immediately add complete culture medium to stop digestion, gently pipette to detach the cells, collect the cell suspension into a centrifuge tube, centrifuge, discard the supernatant, resuspend the cells with PBS, and centrifuge again. 4) Add an appropriate amount of 1 mol / L NaOH solution containing 10% DMSO to the cell pellet, heat in an 80 ℃ water bath to fully dissolve the melanin. After centrifuging the sample for 10 min, transfer the supernatant to an EP tube, vortex to mix, and add 200 μL to a 96-well plate. Measure the OD value at a wavelength of 405 nm.

[0077] 3. Experimental Results: The experimental results are shown in Figure 4 And Table 7. The melanin inhibition rates of formulas (2), (5), (11), (21), (22), (29) and (35) were 58.92% to 90.02%, respectively, indicating that the peptides of formulas (2), (5), (11), (21), (22), (29) and (35) have a significant ability to inhibit melanin production.

[0078] Table 7 Results of Melanin Inhibition Test

[0079] Example 5: Human Testing

[0080] 1. Experimental Procedure: The study employed a self-controlled pre- and post-treatment design with a duration of 4 weeks, requiring at least 10 valid participants to complete the entire trial process. Inclusion criteria for participants: age 30-60 years, gender not limited; no history of allergy to any of the test product's ingredients; and ability to strictly adhere to the trial requirements and cooperate fully throughout the study. The test sample was an essence containing 0.1% of the target active peptide (Formula (2), (5), (11), (21), (22), (29), or (35)). The main matrix components included: 0.55% acrylates / C10-30 alkyl acrylate crosspolymer, 3% 1,3-butanediol, 0.4% p-hydroxyacetophenone, 0.60% 1,2-hexanediol, 0.2% L-arginine, and 0.08% sodium hydroxide. Participants were required to strictly follow the trial usage guidelines, applying the test essence to their face daily for 4 consecutive weeks at home. During the testing period, switching to other functional skincare products is prohibited, and no facial aesthetic procedures are allowed to be performed to ensure stable and consistent testing conditions. Instrumental testing was conducted in a standardized environment with constant temperature and humidity before product use (baseline) and after 4 weeks of continuous use: skin L-colorimeter was used to measure skin L-colorimetry. a Color parameters were analyzed to assess changes in skin tone-related indicators; transepidermal water loss (TEWL) and stratum corneum moisture content were measured using the Dermalab skin analyzer to objectively evaluate the skin barrier and hydration status.

[0081] 2. Experimental Results

[0082] The results are shown in Table 8.

[0083] Table 8. Skin TEWL value, skin moisture content, and L after treatment with the target active peptide. value and a value

[0084] The results showed that, compared with before use, after using the serum containing the target peptide, the skin's TEWL value was significantly reduced, and the skin's moisture content was significantly increased. The value increased significantly, a The value decreased significantly. In summary, the target peptide has the effects of increasing the moisture content of the stratum corneum, enhancing the skin barrier's protective ability, and improving skin tone.

Claims

1. A polypeptide, its stereoisomer, or a salt thereof, characterized in that, The structural formula of the polypeptide is shown in formula (I): R1-(AA1)m-AA2-AA3-(AA4)n-R2, formula (I); Where m is independently 2 or 3; n can be 1, 2, or 3 independently; AA1 can be K, R, or H independently; AA2 can be either I or W independently; AA3 can be either I or W independently; AA4 can be K, R, or H independently; R1 is any one of -H, Y, LY, PLY, or LLY; R2 is any one of -OH, L, LL, E, EL, EW, W, EH, or EHL.

2. The polypeptide, its stereoisomer, or its salt as described in claim 1, characterized in that, m is 2; n is 1; AA1 is K; AA4 is K; R1 is -H, Y, LY, or PLY; R2 is L, H, EL, EH, W, or EW; AA2 is I; AA3 is I; or, m is 2; n is 2; AA1 is K; AA4 is K; R1 is -H, Y, LLY or PLY; R2 is L, H, EL, EH, W or EW; AA2 is I; AA3 is I; or, m is 2; n is 1 or 2; AA1 is K; AA4 is K; R1 is -H, Y, LY, LLY or PLY; R2 is L, H, EL, EH, W or EW; AA2 is W or I; AA3 is W or I; and AA2 and AA3 are not both I; or, m is 2; n is 2; AA1 is R; AA4 is R; R1 is LLY; R2 is L; AA2 is I; AA3 is I; or, m is 2; n is 2; AA1 is H; AA4 is H; R1 is LY; R2 is L; AA2 is I; AA3 is I; Preferably, m is 2; n is 1; AA1 is K; AA4 is K; R1 is -H, Y, LY or PLY; R2 is EH or EW; AA2 is I; AA3 is I; or, m is 2; n is 2; AA1 is K; AA4 is K; R1 is -H, Y, LLY or PLY; R2 is L or W; AA2 is I; AA3 is I; or, m is 2; n is 1 or 2; AA1 is K; AA4 is K; R1 is -H, Y, LY, LLY or PLY; R2 is L, H, EL, EH, W or EW; AA2 is W or I; AA3 is W or I; and AA2 and AA3 are not both I.

3. The polypeptide, its stereoisomer, or its salt as described in claim 1 or 2, characterized in that, m is 2; n is 2; AA1 is K; AA4 is K; R1 is -H, Y, or PLY; R2 is L or W; AA2 is W or I; AA3 is I; or, m is 2; n is 1; AA1 is K; AA4 is K; R1 is LLY; R2 is EH; AA2 is W; AA3 is I; or, m is 2; n is 1; AA1 is K; AA4 is K; R1 is -H, Y, LY, or PLY; R2 is EH or EW; AA2 is I; AA3 is I; or, m is 2; n is 1 or 2; AA1 is K; AA4 is K; R1 is Y, LY, LLY or PLY; R2 is L, H, EH or EW; AA2 is W; AA3 is I or W; or, m is 2; n is 1 or 2; AA1 is K; AA4 is K; R1 is -H, Y, LY, LLY or PLY; R2 is L, EL, EH, W or EW; AA2 is I; AA3 is W; Preferably, m is 2; n is 2; AA1 is K; AA4 is K; R1 is -H, Y, or PLY; R2 is L; AA2 is I; AA3 is I; or, m is 2; n is 2; AA1 is K; AA4 is K; R1 is -H or PLY; R2 is L or W; AA2 is W; AA3 is I.

4. The polypeptide, its stereoisomer, or its salt as described in claim 3, characterized in that, The polypeptide comprises an amino acid sequence as shown in any one of SEQ ID NO: 1-36; Preferably, the amino acid sequences of the polypeptides are as shown in any one of SEQ ID NO: 1-36; More preferably, the amino acids of the stereoisomers are each independently L-type or D-type.

5. An isolated nucleic acid encoding a polypeptide, a stereoisomer thereof, or a salt thereof as described in any one of claims 1-4.

6. A recombinant expression vector comprising the isolated nucleic acid as described in claim 5.

7. A transformant, characterized in that, The transformant comprises the nucleic acid as described in claim 5 or the recombinant expression vector as described in claim 6; the transformant is a non-animal or non-plant variety.

8. A method for preparing the polypeptide, its stereoisomer, or a salt thereof as described in any one of claims 1-4, characterized in that, The method is either the Fmoc solid-phase peptide synthesis method or the liquid-phase peptide synthesis method. Preferably, the method includes: (1) The first Fmoc-protected amino acid is immobilized on the resin, and then the deprotection and the next amino acid are cyclically linked to gradually extend the peptide chain to obtain the crude peptide. (2) Subsequently, the crude peptide was cleaved from the resin using a cleavage fluid; (3) The lysed polypeptide is collected by centrifugation and then purified by reversed-phase high-performance liquid chromatography to obtain the polypeptide.

9. A composition, characterized in that, The composition comprises one or more of the polypeptide, its stereoisomer or salt thereof as described in any one of claims 1-4, and a pharmaceutically acceptable carrier. Preferably, the carrier is selected from one or more of matrix raw materials, auxiliary raw materials, and functional raw materials; the matrix raw materials include oily raw materials, powdery raw materials, and solvent-based raw materials; the auxiliary raw materials include fragrances, pigments, preservatives, surfactants, thickeners, and water-soluble polymers; the functional raw materials include anti-aging agents, moisturizers, whitening agents, sunscreens, nutritional additives, deodorants, and hair removal agents; More preferably, the oily raw material is selected from one or more of oils, waxes, and higher fatty acids; the powdery raw material is selected from one or more of mica powder, talc powder, kaolin, silica, calcium carbonate, and perlite; the solvent raw material is selected from water, alcohols, ketones, ethers, and ester aromatic organic compounds; and the thickener includes one or more of carrageenan and xanthan gum. More preferably, the composition is selected from any one of toner, face mask, serum, lotion, cream, and freeze-dried powder.

10. The use of any one or more of the polypeptides, their stereoisomers or salts thereof as described in any one of claims 1-4, or the composition as described in claim 9, in products for skin anti-oxidation, anti-inflammatory, skin brightening, skin moisturizing and skin barrier repair; Preferably, the product is a cosmetic or a pharmaceutical product; More preferably, the cosmetic is a skin care product.

11. A skincare method that does not intervene in the human body or cause trauma, characterized in that, The method comprises: applying to a subject in need any one or more of the polypeptide, its stereoisomer, or salt thereof as described in any one of claims 1-4, or the composition as described in claim 9; the skin care comprises any one or more of the following pathways: anti-oxidation, anti-inflammatory, skin brightening, skin moisturizing, and skin barrier repair. Preferably, the method is for non-therapeutic purposes; More preferably, the antioxidant effect is manifested in scavenging superoxide anion free radicals; the anti-inflammatory effect is manifested in inhibiting the expression of IL-6 and / or CXCL8; and the skin brightening effect is manifested in inhibiting the production and / or accumulation of melanin.

12. The use of any one or more of the polypeptide, its stereoisomer, or salt thereof as described in any one of claims 1-4, or the composition as described in claim 9, in skin care; wherein the skin care includes any one or more of the following pathways: anti-oxidation, anti-inflammatory, skin brightening, skin moisturizing, and skin barrier repair; Preferably, the application is for non-therapeutic purposes; More preferably, the antioxidant effect is manifested in scavenging superoxide anion free radicals; the anti-inflammatory effect is manifested in inhibiting the expression of IL-6 and / or CXCL8; and the skin brightening effect is manifested in inhibiting the production and / or accumulation of melanin.

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