Polypeptides or salts thereof, and methods of making and using the same

Abstract

This invention discloses polypeptides or their salts, their preparation methods, and applications, belonging to the field of biotechnology. The polypeptide or its salt according to formula (I) has the following structure: L-methionyl-L-prolyl-D-phenylalanyl-L-arginyl-D-tryptophanyl-L-phenylalanyl-X-L-prolyl-L-valine (I); wherein X is a 4-(aminomethyl)cyclohexane carbonyl group. The polypeptide or its salt of this invention exhibits both antagonistic activity against MC1R receptors and inhibitory activity against tyrosinase, with perfect temporal and spatial synergy, thereby effectively inhibiting melanin production in the skin and improving hyperpigmentation.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to polypeptides or their salts, their preparation methods, and applications. Background Technology

[0002] Hyperpigmentation, characterized by uneven dark patches or spots on the skin, is a prevalent skin problem worldwide. Its root cause lies in the excessive production and uneven distribution of melanin in skin melanocytes. To address hyperpigmentation, the cosmetics and pharmaceutical industries have developed various active ingredients, each intervening in the melanin production process through different mechanisms. For example, nonapeptide-1 acts as an α-MSH antagonist, competitively binding to the MC1R receptor on the surface of melanocytes, blocking the binding of α-MSH to MC1R, and inhibiting the activation of the cAMP-MITF-tyrosinase signaling pathway induced by α-MSH at its source, thereby reducing melanin production. Tranexamic acid can inhibit plasminogen activation, thereby reducing plasmin production, weakening plasmin-mediated skin inflammation, and indirectly inhibiting tyrosinase activity induced by inflammatory factors (such as prostaglandins), thus reducing melanin production. Furthermore, tranexamic acid can also directly inhibit tyrosinase activity, blocking melanin production, thereby achieving a whitening effect.

[0003] However, both nonapeptide-1 and tranexamic acid have relatively simple mechanisms for inhibiting melanin production, failing to comprehensively cover the multiple key stages of melanin production, and thus have limited effects on improving excessive skin pigmentation. Furthermore, simply combining nonapeptide-1 and tranexamic acid through physical mixing fails to achieve true synergistic effects due to their different penetration, distribution, and metabolic characteristics in the skin. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a polypeptide or its salt, a method for its preparation and application, to overcome the problem that the single active ingredient nonapeptide-1 or tranexamic acid in the prior art is not effective in improving excessive skin pigmentation, and that it is difficult to achieve synergistic effect after simply mixing the two physically.

[0005] In a first aspect, the present invention provides a polypeptide or a salt thereof, the structure of which is shown in formula (I): L-methionyl-L-prolyl-D-phenylalanyl-L-arginyl-D-tryptophanyl-L-phenylalanyl-XL-prolyl-L-valineamide (I); wherein X is 4-(aminomethyl)cyclohexane carbonyl.

[0006] Compared with existing technologies, on the one hand, the sequence of the polypeptide or its salt of the present invention is carefully designed so that its spatial conformation can mimic α-MSH, bind to MC1R on the melanocyte membrane, occupy the binding site of MC1R, effectively block the binding of MC1R to endogenous α-MSH, and sever the classic melanin synthesis signaling pathway of cAMP-PKA-CREB-MITF, thus preventing melanin production at its source. On the other hand, the tranexamic acid moiety in the polypeptide or its salt of the present invention can inhibit the activity of tyrosinase that is already present in the cell or that is newly generated in small amounts, ensuring that melanin synthesis is effectively suppressed. Furthermore, the tranexamic acid moiety can also effectively reduce the generation of downstream inflammatory mediators by preventing the activation of plasmin, thereby alleviating the inflammatory response, avoiding the inflammatory mediator-induced increase in tyrosinase activity, and reducing melanin production. This invention integrates the receptor antagonistic effect of nonapeptide-1 and the enzyme inhibition and anti-inflammatory effects of tranexamic acid into a single, covalently linked molecular entity, ensuring that the two functional groups can reach the target cells simultaneously in a fixed 1:1 stoichiometric ratio, achieving perfect synergy in time and space, thereby achieving biological effects far exceeding those of nonapeptide-1 and tranexamic acid alone or a simple physical mixture.

[0007] Furthermore, this invention introduces a non-natural amino acid, tranexamic acid, into the peptide chain. This structural modification can effectively resist degradation by proteases in the skin, prolong its half-life in the skin, and thus improve bioavailability.

[0008] Furthermore, the salt is a pharmaceutically or cosmetically acceptable salt formed by polypeptides and inorganic or organic acids.

[0009] Furthermore, organic acids include acetic acid, citric acid, lactic acid, malonic acid, maleic acid, tartaric acid, fumaric acid, benzoic acid, aspartic acid, glutamic acid, succinic acid, oleic acid, trifluoroacetic acid, oxalic acid, pyric acid, or gluconic acid; inorganic acids include hydrochloric acid, sulfuric acid, boric acid, or carbonic acid.

[0010] Secondly, the present invention provides a method for preparing a polypeptide, comprising the following steps: Prepare a solid resin with C-terminal amide linkages; The Fmoc-protected amino acids Fmoc-L-Val-OH, Fmoc-L-Pro-OH, Fmoc-Trx-OH, Fmoc-L-Phe-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-L-Arg(Pbf)-OH, Fmoc-D-Phe-OH, Fmoc-L-Pro-OH, and Fmoc-L-Met-OH are sequentially linked to the resin through coupling and deprotection reactions to form peptide chains. The peptide chain is cleaved from the resin using a lysis buffer, and all protecting groups on the amino acid side chains are removed at the same time to obtain the polypeptide.

[0011] Compared with the prior art, the present invention uses a solid-phase peptide synthesis method with Fmoc (9-fluorenylmethoxycarbonyl) protection strategy to prepare the peptide of the present invention. The reaction conditions for deprotection of the Fmoc group are mild, which reduces peptide chain damage. Moreover, the operation process is relatively simple, and it is easy to monitor in real time during synthesis, thereby improving the success rate of synthesis.

[0012] Thirdly, the present invention provides a method for preparing a polypeptide salt, wherein the polypeptide is reacted with an inorganic acid or an organic acid to obtain a salt formed by the polypeptide and the inorganic acid, or a salt formed by the polypeptide and the organic acid.

[0013] Fourthly, the present invention provides a composition comprising the above-mentioned polypeptide or a salt thereof, and one or more carriers acceptable in cosmetic or pharmaceutical terms.

[0014] Compared with the prior art, the beneficial effects of the composition of the present invention are the same as those of the above-mentioned polypeptides or their salts, and will not be repeated here.

[0015] Furthermore, based on the mass of the composition, the content of the polypeptide or its salt in the composition is 0.001% to 5%.

[0016] Furthermore, the dosage form of the composition includes serum, lotion, cream, gel, mask, or lyophilized powder.

[0017] Fifthly, the present invention provides the use of the above-mentioned polypeptide or its salt, or the above-mentioned composition, in the preparation of products for treating hyperpigmentation of the skin or inhibiting the production of melanin in the skin.

[0018] Compared with the prior art, the peptides or their salts, or compositions of the present invention can simultaneously target the signaling pathways and key enzymes of melanin production, effectively inhibiting melanin production and improving hyperpigmentation of the skin through the synergistic effect of the dual action mechanism.

[0019] Furthermore, hyperpigmentation of the skin includes melasma, freckles, age spots, solar lentigines, or post-inflammatory hyperpigmentation. Detailed Implementation

[0020] To make the technical problems, technical solutions, and beneficial effects of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and are not intended to limit the invention.

[0021] In a first aspect, embodiments of the present invention provide a polypeptide of formula (I) or a salt thereof, the structure of which is shown below: L-methionyl-L-prolyl-D-phenylalanyl-L-arginyl-D-tryptophanyl-L-phenylalanyl-XL-prolyl-L-valineamide (I); wherein X is 4-(aminomethyl)cyclohexane carbonyl.

[0022] Alternatively, equation (I) can also be expressed as follows: Wherein, X is 4-(aminomethyl)cyclohexane carbonyl. It should be understood that 4-(aminomethyl)cyclohexane carbonyl is a residue formed by the loss of a hydrogen atom from the amino group and a hydroxyl group from the carboxyl group in tranexamic acid.

[0023] The aforementioned polypeptide was obtained by replacing the L-lysine residue (i.e., L-lysyl) at position 7 with a tranexamic acid residue based on the sequence of nonapeptide-1. The sequence of nonapeptide-1 is: L-methionyl-L-prolyl-D-phenylalanyl-L-arginyl-D-tryptophanyl-L-phenylalanyl-L-lysyl-L-prolyl-L-valineamide.

[0024] According to the nomenclature of the International Union of Pure and Applied Chemistry (IUPAC), peptides containing non-standard amino acid residues are systematically named. The systematic chemical name of the compound of this invention is: L-methionyl-L-prolyl-D-phenylalanyl-L-arginyl-D-tryptophanyl-L-phenylalanyl-[4-(aminomethyl)cyclohexanecarbonyl]-L-prolyl-L-valineamide.

[0025] In the above nomenclature, the peptide chain is listed sequentially from the N-terminus (L-methionine) to the C-terminus (L-valine). Each amino acid with a chiral center is labeled with its stereoconfiguration (L- or D-). The substituted seventh residue, a structural unit derived from tranexamic acid (chemically known as trans-4-(aminomethyl)cyclohexane-1-carboxylic acid), is named "4-(aminomethyl)cyclohexanecarbonyl" when integrated into the peptide backbone as an acyl group, and is enclosed in square brackets to indicate that it is a non-standard residue. The "amide" at the end of the name indicates that its C-terminus is in amide form, rather than a free carboxylic acid.

[0026] By precisely calculating the molecular weight of each amino acid residue, the molecular formula of the polypeptide of this invention can be determined as: C 71 H 99 N 15 O 10 S; its precise molecular weight is 1385.74 g / mol, which is consistent with the theoretical calculation value (1386.8 g / mol, average molecular weight) obtained from electrospray mass spectrometry analysis in Example 2.

[0027] The peptides in the above-mentioned technical solution retain the antagonistic function of nonapeptide-1 against melanocortin receptor 1 (MC1R), while the modification with tranexamic acid increases the inhibitory function on tyrosinase and the anti-inflammatory function. It can simultaneously target the signaling pathway and key enzymes of melanin production, achieving perfect synergy in time and space, and realizing biological effects far exceeding those of nonapeptide-1 and tranexamic acid alone or a simple physical mixture, further inhibiting melanin synthesis.

[0028] Furthermore, based on the above embodiments, the embodiments of the present invention further define the salt as a pharmaceutically or cosmetically acceptable salt formed by polypeptides and inorganic or organic acids.

[0029] Organic acids include acetic acid, citric acid, lactic acid, malonic acid, maleic acid, tartaric acid, fumaric acid, benzoic acid, aspartic acid, glutamic acid, succinic acid, oleic acid, trifluoroacetic acid, oxalic acid, pyric acid, or gluconic acid; inorganic acids include hydrochloric acid, sulfuric acid, boric acid, or carbonic acid.

[0030] Secondly, embodiments of the present invention provide a method for preparing a polypeptide, comprising the following steps: S1. Prepare a solid resin with C-terminal amide linkages. For example, RinkAmide AM resin purchased from Chempep can be selected.

[0031] S11. Resin swelling: Place 0.1 mmol of Rink Amide AM resin in a reactor, add 5 mL of DMF (dimethylformamide), and allow it to swell at room temperature for 1 hour before draining the DMF.

[0032] S12, Fmoc Deprotection: The swollen resin was deprotected using Fmoc. 5 mL of a 20% (v / v) piperidine / DMF solution was added each time, and the reaction was repeated twice, each time for 10 minutes, to remove the Fmoc protecting group from the resin linker arms. The resin was then washed several times alternately with DMF and IPA (isopropanol) until the eluent tested negative for ninhydrin.

[0033] S2. Following the order from C-terminus to N-terminus, the Fmoc-protected amino acids Fmoc-L-Val-OH, Fmoc-L-Pro-OH, Fmoc-Trx-OH, Fmoc-L-Phe-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-L-Arg(Pbf)-OH, Fmoc-D-Phe-OH, Fmoc-L-Pro-OH, and Fmoc-L-Met-OH are sequentially linked to the resin through coupling and deprotection reactions to form peptide chains.

[0034] The process of linking individual amino acids is as follows: S21. Based on the initial degree of substitution (in moles) of the solid-phase synthetic resin, the Fmoc-amino acid (3 equivalents), HATU (2.9 equivalents) and HOAt (3 equivalents) to be coupled are dissolved in 3 mL of DMF, and DIPEA (6 equivalents) is added to activate for 5 minutes to obtain the activation solution.

[0035] S22. Add the above activating solution to the resin that has had its Fmoc protecting group removed, and react at room temperature for 2 hours to couple amino acids onto the resin or to the ends of polypeptide chains on the resin. After the reaction is complete, confirm the reaction is complete using the ninhydrin assay.

[0036] S23. After coupling, wash the resin with DMF and IPA, twice with each solvent, for 3 minutes each time. After washing, deprotect the resin with Fmoc as described in step S12, and then couple the next amino acid.

[0037] S24. After the last amino acid (Fmoc-L-Met-OH) is coupled, the resin is deprotected by Fmoc according to the method described in step S12. Then, the resin is thoroughly washed with DMF and DCM, each solvent is used twice for 3 minutes each time. After washing, the resin is placed in a vacuum drying oven and dried at 40°C for 4 hours.

[0038] S3. The peptide chains are cleaved from the resin using a lysis buffer, and all protecting groups on the amino acid side chains are removed simultaneously to obtain the polypeptide, specifically including: S31. Place the dried resin in a reaction flask, add 10 mL of lysis buffer, and stir at 300 rpm for 3 hours at room temperature to release the synthesized polypeptide chain completely from the resin, while simultaneously removing the protecting groups on the amino acid side chains. The lysis buffer is freshly prepared Reagent K lysis buffer, composed of TFA, phenol, water, anisole, and EDT in a ratio of 82.5 mL: 5 g: 5 mL: 5 mL: 2.5 mL.

[0039] S32. Filter the above reaction product to obtain a filtrate containing peptides and solid resin residue. Wash the solid resin residue with 1-2 mL of TFA to recover the residual peptides, and combine the washings with the filtrate. Slowly add the combined filtrate dropwise at a rate of 1-2 drops / second to 10 times the combined filtrate volume in anhydrous diethyl ether pre-cooled to 0-4℃. A white precipitate is observed to form. Let it stand in an ice bath for 30 minutes to allow complete precipitation. This precipitate is the peptide.

[0040] Thirdly, embodiments of the present invention provide a method for preparing a polypeptide salt, which involves reacting the polypeptide with an inorganic acid or an organic acid to obtain a salt formed by the polypeptide and the inorganic acid, or a salt formed by the polypeptide and the organic acid.

[0041] For example, peptides react with inorganic acid salts to prepare peptide hydrochloride. The specific method is as follows: The lyophilized peptide powder was dissolved in water to obtain a solution of 10 mg / mL-20 mg / mL. This solution was carefully titrated with 1 M hydrochloric acid to a pH of 4.0-5.0, with the titration rate gradually decreasing from 1-2 drops / second initially to half a drop / 10-15 seconds at the final stage. The resulting solution was pre-frozen at -80°C for at least 4 hours until completely solidified, then transferred to a freeze dryer and dried for 24-48 hours under a vacuum below 10 Pa and a cold trap temperature below -50°C to obtain a loose, powdery solid, which is the peptide hydrochloride.

[0042] Another example is the reaction of peptides with the organic acid acetic acid to prepare peptide acetate. The specific method can be found below: The lyophilized peptide powder was dissolved in an aqueous solution containing 1% acetic acid by volume, and then directly freeze-dried. The solution was pre-frozen at -80°C for at least 4 hours until it was completely solidified. Then it was transferred to a freeze dryer and dried for 24-48 hours under conditions of vacuum degree below 10 Pa and cold trap temperature below -50°C to obtain a fluffy powdery solid, which is peptide acetate.

[0043] Fourthly, embodiments of the present invention provide a composition containing the above-mentioned polypeptide or its salt, and one or more carriers acceptable in cosmetics or pharmaceuticals.

[0044] The content of the polypeptide or its salt in the composition is 0.001% to 5% by weight.

[0045] Furthermore, based on the above embodiments, the embodiments of the present invention further specify that the dosage form of the composition can be selected as serum, lotion, cream, gel, mask or lyophilized powder.

[0046] Fifthly, embodiments of the present invention provide the use of the above-mentioned polypeptide or its salt, or the above-mentioned composition, in the preparation of products for treating hyperpigmentation of the skin or inhibiting the production of melanin in the skin.

[0047] Compared with the prior art, the peptides or their salts, or compositions of the present invention can simultaneously target the signaling pathways and key enzymes of melanin production, effectively inhibiting melanin production and improving hyperpigmentation of the skin through the synergistic effect of the dual action mechanism.

[0048] Furthermore, hyperpigmentation of the skin includes melasma, freckles, age spots, solar lentigines, or post-inflammatory hyperpigmentation.

[0049] It should be understood that, unless otherwise specified, all raw materials used in the following examples are commercially available.

[0050] Example 1 Polypeptide synthesis 1. Resin swelling: Take 0.1 mmol of Rink Amide AM resin (200 mesh, degree of substitution 0.5 mmol / g) and place it in a reactor. Add 5 mL of DMF (dimethylformamide). After swelling at room temperature for 1 hour, drain the DMF.

[0051] 2. Fmoc Deprotection: The swollen resin was deprotected with Fmoc by adding 5 mL of 20% (v / v) piperidine / DMF solution twice, each time for 10 minutes, to remove the Fmoc protecting group from the resin linker arms. The resin was then washed several times alternately with DMF and IPA (isopropanol) until the eluent tested negative for ninhydrin.

[0052] 3. Amino Acid Coupling Cycle: The following amino acids are coupled sequentially from C-terminus to N-terminus: Fmoc-L-Val-OH, Fmoc-L-Pro-OH, Fmoc-Trx-OH, Fmoc-L-Phe-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-L-Arg(Pbf)-OH, Fmoc-D-Phe-OH, Fmoc-L-Pro-OH, and Fmoc-L-Met-OH. The molar ratios of Fmoc-amino acids, coupling agents, and bases are maintained throughout each coupling cycle.

[0053] The specific process of single amino acid coupling is as follows: Based on the initial degree of substitution (in moles) of the solid-phase synthetic resin, the Fmoc-amino acid (3 equivalents), HATU (2.9 equivalents) and HOAt (3 equivalents) to be coupled were dissolved in 3 mL of DMF, and DIPEA (6 equivalents) was added to activate for 5 minutes to obtain the activation solution.

[0054] The above activating solution was added to the resin that had been deprotected from the Fmoc protecting group, and the reaction was carried out at room temperature for 2 hours to couple amino acids onto the resin or to the ends of polypeptide chains on the resin. After the reaction was completed, the ninhydrin assay was used to confirm that the reaction was complete.

[0055] After coupling, the resin was washed with DMF and IPA, twice with each solvent for 3 minutes each time. After washing, the resin was deprotected with Fmoc as in step 2, and then the next amino acid was coupled.

[0056] 4. End-of-line treatment: After the last amino acid (Fmoc-L-Met-OH) is coupled, the resin is deprotected by Fmoc according to the method described in step 2. Then, the resin is thoroughly washed with DMF and DCM, twice with each solvent for 3 minutes each time. After washing, the resin is placed in a vacuum drying oven and dried at 40°C for 4 hours.

[0057] 5. Place the dried resin in a reaction flask, add 10 mL of lysis buffer, and stir at 300 rpm for 3 hours at room temperature to release the synthesized polypeptide chain completely from the resin, while simultaneously removing the protecting groups on the amino acid side chains. The lysis buffer is freshly prepared Reagent K lysis buffer, composed of TFA, phenol, water, anisole, and EDT, in a ratio of 82.5 mL:5 g:5 mL:5 mL:2.5 mL.

[0058] 6. Crude Peptide Precipitation: Filter the above reaction product to obtain a filtrate containing peptides and solid resin residue. Wash the solid resin residue with 2 mL of TFA to recover the residual peptides, and combine the washings with the filtrate. Slowly add the combined filtrate dropwise at a rate of 1 drop / second to 10 times the combined filtrate volume in anhydrous diethyl ether pre-cooled to 0°C. A white precipitate is observed to form. Let it stand in an ice bath for 30 minutes to allow complete precipitation. This precipitate is the crude peptide.

[0059] Example 2 Polypeptide purification The precipitate from Example 1 was collected by centrifugation at 5000 x g for 5 minutes at 4°C. The precipitate was washed three times with cold diethyl ether, vortexing for 1 minute each time, followed by centrifugation and discarding the supernatant. The resulting solid precipitate was dried overnight at room temperature in a vacuum drying oven. The vacuum-dried crude peptide was dissolved in 5 mL of an aqueous solution containing 0.1% TFA and purified by reversed-phase high-performance liquid chromatography.

[0060] Chromatographic column: C18 column; Mobile phase: A phase is an aqueous solution containing 0.1% TFA, and B phase is acetonitrile containing 0.1% TFA.

[0061] The linear gradient elution program is shown in Table 1, and the elution time is 40 minutes.

[0062] Table 1 Collect the eluent corresponding to the main peak.

[0063] The collected eluent was freeze-dried. First, it was pre-frozen at -80°C for 4 hours, and then transferred to a freeze dryer and dried for 48 hours under a vacuum of 8 Pa and a cold trap temperature of -55°C to obtain the polypeptide freeze-dried powder.

[0064] The purity of the above lyophilized powder was determined by analytical RP-HPLC, and the results showed that the purity was greater than 98.6%.

[0065] Electrospray ionization mass spectrometry was used to analyze the lyophilized peptide powder and determine its molecular weight. The measured molecular weight was consistent with the theoretical calculation value (1386.8 g / mol, precise molecular weight), confirming the successful synthesis of the target product.

[0066] Example 3 Efficacy test Cell culture: Using the B16F10 mouse melanoma cell line, cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin (final concentration: 100 U / mL penicillin, 100 μg / mL streptomycin) at 37°C in a 5% CO2 incubator.

[0067] Melanin production induction and treatment: The above cells were treated with 5 × 10 4 The samples were seeded at a density of 100 samples per well in 24-well plates. After 24 hours of incubation, the original medium was replaced with fresh DMEM medium (containing 10% fetal bovine serum) containing different test samples, and α-MSH (final concentration 100 nM) was added to induce melanin production (except for the blank control group). The following experimental groups were set up: Group 1: Blank control group (no addition of α-MSH to induce melanin production).

[0068] Group 2: Model group, containing only α-MSH that induces melanin production.

[0069] Group 3: Peptide group, containing α-MSH and the peptide prepared in Example 2 (final concentration of 50 µM).

[0070] Group 4: Nonapeptide-1 group, containing α-MSH and nonapeptide-1 (final concentration of 50 µM).

[0071] Group 5: Tranexamic acid group, containing α-MSH and tranexamic acid (final concentration of 50 µM).

[0072] Group 6: Mixture group, containing a mixture of α-MSH and nonapeptide-1 with tranexamic acid in a 1:1 molar ratio (the final concentrations of both nonapeptide-1 and tranexamic acid are 50 µM).

[0073] Melanin content determination: After culturing the cells in each group for another 72 hours, the culture medium was discarded, and the cells were collected by trypsin digestion. The cells were then lysed with 1N NaOH solution at 60°C for 60 minutes to dissolve the melanin. The absorbance of the cell lysates was measured at 405 nm using a microplate reader. The relative melanin content of each group was calculated after correction with the total cellular protein content, and the half-maximal inhibitory concentration (IC50) of each test sample was also calculated. The results are shown in Table 1.

[0074] Intracellular tyrosinase activity assay: The cell lysates from each group were reacted with L-DOPA substrate (1 mg / mL) at 37°C for 1 hour. The reaction products were used to assess the intracellular tyrosinase activity by monitoring the dopachrome production rate at 475 nm. The results are shown in Table 2.

[0075] Table 2. Inhibitory effects of different test samples on melanin production in B16F10 cells The results above show that the IC50 of the polypeptide of the present invention inhibits melanin production and intracellular tyrosinase activity. 50 The values ​​were all significantly lower than those of nonapeptide-1, tranexamic acid, and a 1:1 physical mixture of the two, indicating that the covalent linkage of the two active groups into a single molecular entity in this invention has a significant synergistic effect, producing a "1+1>2" effect, and can significantly inhibit the production of melanin in cells. Simultaneously, the peptide of this invention can effectively inhibit tyrosinase activity in cells, while nonapeptide-1 has almost no direct inhibitory effect on intracellular tyrosinase activity, indicating that the peptide of this invention successfully integrates the tyrosinase-inhibiting function of tranexamic acid.

[0076] Example 4 Detection of expression levels of melanin production-related proteins and genes Cell culture: The B16F10 mouse melanoma cell line was used. The cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin at 37°C in a 5% CO2 incubator.

[0077] Melanin production induction and treatment: The above cells were treated with 5 × 10 4 The samples were seeded at a density of 100 samples per well in 24-well plates. After 24 hours of incubation, the original culture medium was replaced with fresh DMEM medium (containing 10% fetal bovine serum) containing different test samples, and α-MSH was added to a final concentration of 100 nM to induce melanin production (except for the blank control group). The following experimental groups were set up: Blank control group: No α-MSH was added to induce melanin production.

[0078] Model group: Contains only α-MSH that induces melanin production.

[0079] Group 1 of peptides: Contains α-MSH and peptides prepared in Example 2 (final concentration of 50 µM).

[0080] Group 2 of peptides: Contains α-MSH and peptides prepared in Example 2 (final concentration of 100 µM).

[0081] Positive control group: Contains α-MSH and kojic acid (final concentration 200µM).

[0082] 1. Western Blot: The expression levels of melanin production-related proteins MITF, TYR, TRP-1 and TRP-2 in cells were detected using specific antibodies. The specific detection methods are shown in Table 3, and the detection results are shown in Table 4.

[0083] Table 3 Western Blot analysis protocol for melanin production-related proteins Table 4. Relative expression levels of melanin production-related proteins The results above show that, compared with the α-MSH-stimulated model group, the protein expression levels of TYR, MITF, TRP-1, and TRP-2 in the peptide-treated group decreased in a dose-dependent manner, while the protein expression level of the internal reference β-actin remained basically consistent among the groups, indicating that the peptide of the present invention downregulated the melanin synthesis pathway at the protein level.

[0084] 2. RT-PCR: Appropriate primers were designed, and the mRNA transcription levels of melanin production-related genes Mitf, Tyr, Trp-1, and Trp-2 in cells were detected by real-time quantitative PCR. The specific detection methods are shown in Table 5, and the detection results are shown in Table 6.

[0085] Table 5. qRT-PCR analysis protocol for melanin production-related genes. Table 6. Relative expression levels of melanin production-related genes The results above show that, compared with the blank control group, the mRNA expression levels of Mitf, the main regulator of melanin production, and its downstream key enzyme genes Tyr, Trp-1, and Trp-2 were significantly upregulated in the α-MSH-stimulated model group, indicating that the in vitro cell model successfully simulated the melanin production process induced by signal stimulation. Meanwhile, compared with the model group, both peptide 1 and peptide 2 groups significantly downregulated the mRNA expression levels of Mitf, Tyr, Trp-1, and Trp-2, indicating that the peptides of this invention downregulated the melanin synthesis pathway at the gene level, inhibiting melanin production.

[0086] Example 5 1. A highly effective whitening essence containing peptides, the formula of which is shown in Table 7.

[0087] Table 7 The preparation method of the above-mentioned high-efficiency whitening essence is as follows: Deionized water, glycerol, and propylene glycol are mixed in proportion to obtain phase A.

[0088] Xanthan gum was slowly dispersed in phase A at a rate of 0.1 g / s under stirring at 800 rpm, and then stirred at a high speed of 1500 rpm until completely dissolved to form a gel matrix.

[0089] The polypeptide, nicotinamide, and sodium hyaluronate of the present invention were added to the above gel matrix in proportion and stirred evenly.

[0090] Finally, add phenoxyethanol and ethylhexylglycerin in the correct proportions and stir well.

[0091] The pH value was measured and adjusted to 6.0 using a buffer system prepared with citric acid and sodium citrate.

[0092] 2. A skin-brightening and repairing cream containing peptides, the formula of which is shown in Table 8.

[0093] Table 8 The preparation method of the above-mentioned brightening and repairing cream is as follows: Deionized water, glycerol, and butylene glycol were mixed in proportion and heated to 75°C to obtain phase A.

[0094] Caprylic / capric triglyceride, jojoba seed oil, glyceryl stearate, PEG-100 stearate, and cetearyl alcohol were mixed in proportion and heated to 75°C to obtain phase B.

[0095] Phase B was slowly added to phase A at a rate of 5 mL / min while homogenizing at 5000 rpm, and homogenized for 3 minutes to form a uniform emulsion.

[0096] Begin by slowly stirring and cooling at 200 rpm. When the temperature drops to 40°C, add the pre-dissolved peptide solution (10 mg / mL), tocopherol, phenoxyethanol, and ethylhexylglycerin. Continue stirring at 200 rpm until homogeneous, then cool to room temperature.

[0097] Example 6 Clinical tests on the skin whitening and blemish-removing effects on human skin Subjects: 40 healthy female volunteers aged 30-60 with obvious pigmentation on their faces (such as melasma and freckles) were recruited.

[0098] Product: The test product was the serum containing 2% peptides as described in Example 5 above, and the placebo was a serum with the same matrix but without peptides.

[0099] Methods: A self-comparison method was used, applying the test product and a placebo to designated areas on the left and right sides of the face, respectively, twice daily, morning and evening.

[0100] Evaluation: Efficacy was evaluated using professional skin testing instruments and by dermatologists at weeks 0, 2, 4, 6, and 8.

[0101] Instrument measurements: The melanin index of the skin was measured using the Mexameter® MX 18. The skin's lightness value was measured using the Chromameter® CR-400. ), red value ( ) and yellow value ( ).according to and The value is calculated using the formula: An increase in the ITA° value indicates that the skin tone has become brighter.

[0102] Doctor's assessment: An experienced dermatologist will visually score the size and color depth of the target pigmentation.

[0103] The results are shown in Table 9.

[0104] Table 9 The results above show that, compared to the placebo group, the melanin index in the test group was significantly reduced, while the brightness value L∗(Δ) and individual skin tone angle ITA° were significantly increased, indicating a reduction in melanin and an overall brighter skin tone. Dermatologist evaluations also showed that the peptide-containing serum had a fading effect on localized pigmentation, demonstrating that the peptides of this invention have excellent whitening and pigmentation-fading effects on human skin. Furthermore, no adverse skin reactions were observed throughout the entire trial.

[0105] Example 7 In vitro cytotoxicity test The cytotoxicity of the peptides of this invention against human immortalized keratinocytes (HaCaT) and B16F10 mouse melanoma cells was evaluated using the MTT assay. Both cell types were seeded into 96-well plates and cultured for 24 hours. The original culture medium was then replaced with medium containing different concentrations of the peptides of this invention, and the cells were cultured for another 48 hours. MTT solution was added, and after incubation for 4 hours, formazan crystals were dissolved in DMSO. The absorbance was measured at 570 nm, and cell viability was calculated. The results are shown in Table 10.

[0106] Table 10 The results above show that the invented peptide, at a concentration as high as 1000 µM, had no significant effect on the survival rate of HaCaT and B16F10 cells (cell survival rate >95%), and its calculated IC50 value was [not specified]. 50 The value is greater than 1000µM, which indicates that the peptide of the present invention does not have cytotoxicity to skin cells within its effective concentration range and has good safety.

[0107] Example 8 Human Repeatable Injury Patch Test (HRIPT) This test was conducted in strict accordance with international cosmetic safety assessment standards, aiming to evaluate the potential irritation and sensitization of the product under repeated use.

[0108] Subjects: 200 healthy volunteers were recruited, including individuals who self-reported as having sensitive skin.

[0109] Test sample: The serum containing 2% polypeptide from Example 5 above.

[0110] Test procedure: Induction Phase: A patch containing the test sample is applied to the skin on the subject's back and removed after 24 hours. This process is repeated three times a week for three consecutive weeks, for a total of nine applications.

[0111] Rest Phase: Stop applying the patch for 2 weeks to allow the body to develop possible immune memory.

[0112] Challenge Phase: A challenge patch was applied to a new skin site, and the skin reaction was observed and recorded by a dermatologist at 24 and 48 hours later. The results are shown in Table 11.

[0113] Table 11 The results above show that the essence containing the polypeptides of this invention is non-irritating and non-allergenic to human skin.

[0114] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A polypeptide or a salt thereof, characterized by, The structure of the polypeptide is shown in formula (I): L-methionyl-L-prolyl-D-phenylalanyl-L-arginyl-D-tryptophanyl-L-phenylalanyl-XL-prolyl-L-valineamide (I); Wherein, X is 4-(aminomethyl)cyclohexane carbonyl.

2. The polypeptide or salt thereof according to claim 1, characterized by The salt is a pharmaceutically or cosmetically acceptable salt formed by the polypeptide and an inorganic or organic acid.

3. The polypeptide or salt thereof according to claim 2, characterized by The organic acids include acetic acid, citric acid, lactic acid, malonic acid, maleic acid, tartaric acid, fumaric acid, benzoic acid, aspartic acid, glutamic acid, succinic acid, oleic acid, trifluoroacetic acid, oxalic acid, pyric acid, or gluconic acid; the inorganic acids include hydrochloric acid, sulfuric acid, boric acid, or carbonic acid.

4. A method for preparing a polypeptide, used to prepare the polypeptide according to any one of claims 1 to 3, characterized in that, Includes the following steps: Prepare a solid resin with C-terminal amide linkages; The Fmoc-protected amino acids Fmoc-L-Val-OH, Fmoc-L-Pro-OH, Fmoc-Trx-OH, Fmoc-L-Phe-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-L-Arg(Pbf)-OH, Fmoc-D-Phe-OH, Fmoc-L-Pro-OH, and Fmoc-L-Met-OH are sequentially linked to the resin through coupling and deprotection reactions to form peptide chains. The peptide chain is cleaved from the resin using a lysis buffer, and the protecting groups of all amino acid side chains are removed simultaneously to obtain the polypeptide.

5. A method for preparing a polypeptide salt, used to prepare the polypeptide salt according to any one of claims 1 to 3, characterized in that, The polypeptide prepared according to claim 4 is reacted with inorganic acid or organic acid to obtain salts formed by the polypeptide and inorganic acid or salts formed by the polypeptide and organic acid.

6. A composition, characterized in that, The composition contains the polypeptide or salt thereof as described in any one of claims 1 to 3, and one or more carriers that are acceptable in cosmetic or pharmaceutical applications.

7. The composition according to claim 6, characterized in that, The content of the polypeptide or its salt in the composition is 0.001% to 5% based on the mass of the composition.

8. The composition according to claim 6 or 7, characterized in that, The dosage forms of the composition include serums, lotions, creams, gels, masks, or lyophilized powders.

9. The use of the polypeptide or salt thereof according to any one of claims 1 to 3, or the composition according to any one of claims 6 to 8, in the preparation of products for treating hyperpigmentation of the skin or inhibiting the production of melanin in the skin.

10. The application according to claim 9, characterized in that, Hyperpigmentation of the skin includes melasma, freckles, age spots, solar lentigines, or post-inflammatory hyperpigmentation.

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