Tranexamic acid-muscle peptide conjugate and application thereof

Abstract

The application discloses antranilic acid-carnosine conjugate and application thereof, and the structure of the antranilic acid-carnosine conjugate is as follows: N-{[4-(aminomethyl)cyclohexyl]carbonyl}-beta-alanyl-L-histidine. After the antranilic acid-carnosine conjugate of the application enters the skin, an amide bond connecting antranilic acid and L-carnosine is recognized and slowly hydrolyzed by endogenous enzymes existing in the deep epidermis and dermis, so that controlled release of the two active ingredients, antranilic acid and L-carnosine, at the target site of the skin is realized. The targeted and slow release mechanism not only makes the effect of the antranilic acid-carnosine conjugate mild, solves the problem of greater skin irritation of natural free antranilic acid, but also enables the two active ingredients to simultaneously and locally exert effects, thereby ensuring the synergistic effect of the two ingredients and maximizing skin care efficacy.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a tranexamic acid-carnosine conjugate and its applications. Background Technology

[0002] Skin aging is a complex process, mainly manifested as pigmentation (such as age spots and melasma) and structural aging (such as wrinkles and decreased elasticity). Pigmentation is mainly related to the excessive production of melanin, while structural aging is closely related to the glycation and oxidative damage of extracellular matrix proteins such as collagen.

[0003] Tranexamic acid (TXA), chemically known as trans-4-(aminomethyl)cyclohexanecarboxylic acid, is a synthetic lysine derivative that has shown good efficacy in treating skin pigmentation disorders such as melasma and post-inflammatory hyperpigmentation. However, due to its small molecular weight and strong hydrophilicity, tranexamic acid diffuses extremely rapidly after penetrating the stratum corneum and entering the aqueous environment of the epidermis. The local concentration around nerve endings spikes from zero to a peak value in a very short time, triggering a stinging reaction.

[0004] L-Carnosine (β-alanyl-L-histidine) is a natural dipeptide composed of β-alanine and L-histidine, and is recognized in the skincare field as a highly effective anti-glycation and antioxidant agent. Despite its remarkable skincare benefits, carnosine's application in the body is severely limited. Carnosinase, present in human serum and tissues, rapidly hydrolyzes L-carnosine into amino acids, significantly reducing its bioavailability and the duration of its efficacy. Compared to carnosine itself, the antioxidant and anti-glycation effects of the amino acid monomers obtained after carnosine hydrolysis are significantly weakened. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a tranexamic acid-carnosine conjugate and its application, to overcome the problems of tranexamic acid being highly irritating to the skin and L-carnosine being easily hydrolyzed in the prior art.

[0006] In a first aspect, the present invention provides a tranexamic acid-carnosine conjugate, the structure of which is shown below: N-{[4-(aminomethyl)cyclohexyl]carbonyl}-β-alanyl-L-histidine.

[0007] Compared with existing technologies, the tranexamic acid-carnosine conjugate of the present invention, having the specific structure described above, allows the amide bond connecting tranexamic acid and L-carnosine to be recognized and slowly hydrolyzed by endogenous enzymes present in the deep epidermis and dermis after entering the skin. This achieves controlled release of the two active ingredients—tranexamic acid and L-carnosine—at the target site on the skin. The released tranexamic acid inhibits melanin production, while the released L-carnosine scavenges reactive carbonyl groups, inhibits AGEs formation, and scavenge free radicals. This targeted and slow release mechanism not only makes the effect of the tranexamic acid-carnosine conjugate gentle but also allows the two active ingredients to work simultaneously and at the same location, thus ensuring their synergistic effect and maximizing skincare efficacy.

[0008] Furthermore, compared to naturally occurring free tranexamic acid, which has a smaller molecular weight and diffuses rapidly into the skin after penetrating the stratum corneum, it can easily create a sudden surge in concentration, causing stimulation of nerve endings (redness, stinging). This invention couples tranexamic acid with L-carnosine to form a larger molecule, slowing down the diffusion rate of tranexamic acid and avoiding stimulation caused by sudden increases in local concentration. This allows the tranexamic acid molecule to diffuse and penetrate more smoothly and continuously, thereby significantly reducing the risk of irritation.

[0009] Furthermore, the tranexamic acid-carnosine conjugate is obtained by reacting tranexamic acid and L-carnosine to form an amide bond.

[0010] In the above technical solution, the tranexamic acid-carnosine conjugate is formed by an amide bond between the carboxyl group of tranexamic acid and the amino group of β-alanine at the N-terminus of L-carnosine. This amide bond is a non-α-amide bond, which naturally resists proteases commonly found on the skin surface and in vivo that specifically recognize and hydrolyze α-amide bonds. Furthermore, compared to natural L-carnosine, this invention, by coupling the carboxyl group of tranexamic acid to the amino group at the N-terminus of carnosine, essentially adds a large molecular "cap" (acylation modification) to the N-terminus of carnosine, completely disrupting the specific spatial recognition of the substrate by carnosinase, preventing it from binding and initiating hydrolytic catalysis. This solves the problem that the amide bond inside natural L-carnosine is easily decomposed by carnosinase, ensuring that the tranexamic acid-carnosine conjugate of this invention remains highly stable before reaching the skin target site.

[0011] Secondly, the present invention provides the application of the above-mentioned tranexamic acid-carnosine conjugate in the preparation of cosmetics.

[0012] Compared with the prior art, using the tranexamic acid-carnosine conjugate of the present invention to prepare cosmetics can enable the cosmetics to have the effects of anti-skin oxidation, anti-skin glycation, and inhibition of melanin formation, thereby playing a role in whitening, lightening spots, delaying the appearance of wrinkles, or anti-aging.

[0013] Thirdly, the present invention provides a cosmetic composition comprising the above-mentioned tranexamic acid-carnosine conjugate, and one or more cosmetic excipients.

[0014] Furthermore, the effective content of tranexamic acid-carnosine conjugate in the cosmetic composition is 0.01%-10% by mass percentage.

[0015] Furthermore, the effective content of tranexamic acid-carnosine conjugate in the cosmetic composition is 0.1%-5% by mass percentage.

[0016] Furthermore, excipients acceptable in the cosmetics field include any one or more of the following: solvents, dispersants, surfactants, preservatives, pigments, fragrances, lubricants, thickeners, penetration enhancers, humectants, sunscreens, antioxidants, chelating agents, film-forming agents, suspending agents, foaming agents, and pH adjusters.

[0017] Furthermore, the dosage form of the cosmetic composition includes aqueous, emulsion, gel, cream, powder, or sheet mask formulations.

[0018] Fourthly, the present invention provides the use of the above-mentioned tranexamic acid-carnosine conjugate or the above-mentioned cosmetic composition in improving the appearance of the skin.

[0019] Furthermore, improving skin appearance includes at least one of the following: a) Whitens and fades dark spots, inhibits melanin production; b) Inhibits skin glycation.

[0020] It should be noted that the above-mentioned improvements in skin appearance are for non-disease treatment areas, such as beauty and skincare. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the synthesis reaction of tranexamic acid-carnosine conjugate. Detailed Implementation

[0022] To make the technical problems to be solved, the technical solutions, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are only for explaining the present invention and are not intended to limit the present invention.

[0023] In a first aspect, embodiments of the present invention provide a tranexamic acid-carnosine conjugate, which, according to the nomenclature rules of the International Union of Pure and Applied Chemistry (IUPAC), is named N-{[4-(aminomethyl)cyclohexyl]carbonyl}-β-alanyl-L-histidine.

[0024] The structure of the tranexamic acid-carnosine conjugate of the present invention is shown in formula (I): Equation (I); The molecular formula of the tranexamic acid-carnosine conjugate of this invention is C 17 H 27 N5O4, with a precise molecular weight of 365.2066 g / mol, is formed by an amide bond between the carboxyl group (-COOH) of tranexamic acid and the amino group (-NH2) of β-alanine at the N-terminus of L-carnosine.

[0025] The tranexamic acid-carnosine conjugate of this invention is a "bifunctional prodrug" with a significantly higher molecular weight than the parent molecule tranexamic acid. This larger molecular weight significantly reduces the diffusion rate of tranexamic acid through the stratum corneum, preventing a sudden, high-concentration "shock" of tranexamic acid in the skin. The active ingredient, tranexamic acid, can penetrate smoothly and continuously, thus significantly reducing the risk of irritation. Furthermore, after entering the skin, the non-α-amide bond connecting tranexamic acid and L-carnosine in the tranexamic acid-carnosine conjugate exhibits natural resistance to common proteases that hydrolyze α-peptide bonds in vivo, and is not easily hydrolyzed. Once the tranexamic acid-carnosine conjugate reaches its target site deep within the skin, it is recognized and slowly hydrolyzed by endogenous enzymes present in the deep epidermis and dermis. This allows for the controlled release of the two active ingredients, tranexamic acid and L-carnosine, at the targeted sites (such as melanocytes in the deep epidermis and collagen fibers in the dermis). The released tranexamic acid inhibits melanin production, while the released L-carnosine scavenges reactive carbonyl groups, inhibits AGEs (advanced aging products), and eliminates free radicals. This targeted and slow-release mechanism not only enhances the gentleness of the tranexamic acid-carnosine conjugate but also ensures that the two active ingredients work synergistically to maximize skincare efficacy.

[0026] The tranexamic acid-carnosine conjugate of the present invention can be prepared by chemical methods or by biological methods. Both methods can use tranexamic acid and L-carnosine as reaction raw materials. Although the preparation methods are different, the resulting products have the same structure.

[0027] For example, the steps for preparing the above-mentioned tranexamic acid-carnosine conjugate by chemical method can be referred to as follows.

[0028] Step 1: Protection reaction of the N-terminal tert-butyloxycarbonyl (Boc) group of tranexamic acid Anhydrous methanol, tranexamic acid, and triethylamine were added to a reaction vessel. A methanol solution of di-tert-butyl dicarbonate was added dropwise at 0°C-5°C. After the addition was complete, the reaction was continued under sealed conditions for 12 hours at room temperature to obtain a reaction solution. The reaction solution was then concentrated under reduced pressure to remove methanol, yielding a concentrated solution. The concentrated solution was then solubilized using a saturated sodium bicarbonate aqueous solution, and extracted with ethyl acetate. The pH of the extracted aqueous phase was adjusted to 2-3 at 0°C-5°C, precipitating a white solid. The white solid was washed and dried to obtain Boc-tranexamic acid.

[0029] Step 2: Protective reaction of C-terminal methyl esterification (OMe) of carnosine L-carnosine and anhydrous methanol were added to a reaction vessel. Under ice bath stirring, thionyl chloride was added dropwise, and the dropping rate was controlled so that the temperature inside the system did not exceed 5°C. After the addition was complete, the system was heated to 65°C and reacted under total reflux at 65°C for 6 hours. After the reaction was terminated, the reaction solution was cooled to room temperature, and thionyl chloride and methanol were removed by vacuum concentration to obtain white viscous crystals. The white viscous crystals were washed and dried to obtain L-carnosine methyl ester hydrochloride.

[0030] Step 3: Condensation reaction Boc-tranexamic acid and L-carnosine methyl ester hydrochloride were dissolved together in dimethylformamide. At 0°C, 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were added sequentially. Finally, N,N-diisopropylethylamine was added to neutralize the hydrochloride and maintain the system's weak alkalinity. The reaction was carried out under closed stirring at 25°C for 18 hours. After the reaction was completed, the reaction solution was poured into ice water and then extracted with ethyl acetate. The extracted organic phase was washed sequentially with citric acid solution, saturated brine, and sodium bicarbonate solution. The washed organic phase was dried with anhydrous magnesium sulfate and then concentrated under reduced pressure to obtain crude intermediate Boc-TXA-carnosine methyl ester. The crude intermediate Boc-TXA-carnosine methyl ester was purified using a chromatographic column to obtain the final intermediate Boc-TXA-carnosine methyl ester.

[0031] Step 4: Deprotection reaction and purification The intermediate Boc-TXA-carnosine methyl ester was dissolved in a mixed solvent of tetrahydrofuran and water. Lithium hydroxide monohydrate was added to the solution at 0°C, and the reaction was carried out at room temperature for 4 hours. After the reaction was completed, the reaction solution was neutralized to neutral with hydrochloric acid, and the tetrahydrofuran was evaporated under reduced pressure to obtain a reaction concentrate. A dichloromethane solution of trifluoroacetic acid was added to the reaction concentrate, and the reaction was carried out at room temperature for 2 hours. The resulting reaction solution was flash evaporated under vacuum to remove trifluoroacetic acid and dichloromethane to obtain a hydrochloride product. Deionized water was added to the hydrochloride product for solubilization. The solubilized hydrochloride product was desalted and purified twice by high performance liquid chromatography. The target product was collected, freeze-dried, and the tranexamic acid-carnosine conjugate was obtained.

[0032] As another example, the steps for preparing the above-mentioned tranexamic acid-carnosine conjugate using biological methods can be referred to as follows: There is no enzyme in nature that can directly catalyze the reaction of tranexamic acid and L-carnosine to produce tranexamic acid-carnosine conjugates. The inventors mutated the adenylation domain of a nonribosomal peptide synthase (NRPS) derived from *Streptomyces cerevisiae*, and fused the mutated adenylation domain, its homologous thioesterification domain, and a condensation domain from a fengycin synthase cluster derived from *Bacillus* to construct a tranexamic acid-carnosine ligase. The amino acid sequence of this tranexamic acid-carnosine ligase is shown in SEQ ID NO:4.

[0033] The gene encoding tranexamic acid-carnosine ligase was named TCLigase. Based on the codon bias of Kluyveromyces lactis, the TCLigase gene was optimized. The optimized gene was cloned into the expression vector pKLAC2 to construct a recombinant plasmid, named pKLAC2-TCLigase.

[0034] The recombinant plasmid pKLAC2-TCLigase was transformed into K. lactis GG799 competent cells to construct a recombinant strain expressing tranexamic acid-carnosine ligase.

[0035] The recombinant strain was inoculated into a fermenter and fermented at 25℃-35℃ and pH 5.0-6.0. The initial culture medium in the fermenter could be YPD medium. When the glucose in the initial medium was depleted and the dissolved oxygen (DO) value suddenly increased (approximately 12-14 hours of fermentation), a 50% (w / v) glycerol aqueous solution (i.e., 50g glycerol per 100 mL of glycerol aqueous solution) was added as feed. DO-stat feedback control was used during feeding to maintain the DO value at approximately 30%. The average flow rate of the glycerol aqueous solution was 8-12 mL / h per liter of fermentation broth. Fermentation was continued until the OD value increased. 600After reaching 80-120, add galactose to a final concentration of 1.5%-2.5% (w / v) for induction culture (i.e., use 1.5g-2.5g of galactose per 100mL of fermentation broth for induction culture) to induce the recombinant strain to express tranexamic acid-carnosine ligase. Induce culture at 25℃-30℃ for 22-26 hours to obtain fermentation broth containing tranexamic acid-carnosine ligase.

[0036] A mixed aqueous solution of tranexamic acid and L-carnosine was added to the above fermentation broth. The concentrations of tranexamic acid and L-carnosine in the mixed solution were both 0.5 mol / L-1.5 mol / L. The amount of mixed aqueous solution added was 5%-30% of the volume of the fermentation broth. The flow rate of the mixed solution was 0.05 mL / min-1.0 mL / min per liter of fermentation broth. Under the conditions of a temperature of 25℃-37℃ and a pH of 6.0-7.0, the whole cells in the fermentation broth were used as catalysts to catalyze the reaction of tranexamic acid and L-carnosine to generate tranexamic acid-carnosine conjugates.

[0037] Secondly, embodiments of the present invention provide the application of the above-mentioned tranexamic acid-carnosine conjugate in the preparation of cosmetics.

[0038] Thirdly, embodiments of the present invention provide a cosmetic composition comprising the above-mentioned tranexamic acid-carnosine conjugate, and one or more cosmetically acceptable excipients.

[0039] When the tranexamic acid-carnosine conjugate of the present invention is added to a cosmetic composition, the tranexamic acid-carnosine conjugate simultaneously exerts antioxidant, anti-glycation, and melanin-inhibiting effects in the cosmetic composition, thereby giving the cosmetic composition whitening, spot-fading, and anti-aging effects.

[0040] Furthermore, based on the above embodiments, the embodiments of the present invention further select that the effective content of tranexamic acid-carnosine conjugate in the cosmetic composition is 0.01%-10% by mass percentage.

[0041] For example, the effective content of tranexamic acid-carnosine conjugate in the cosmetic composition may be 0.01%, 0.1%, 1%, 3%, 5%, 7% or 10% by mass percentage, preferably 0.1%-5%.

[0042] Furthermore, based on the above embodiments, the embodiments of the present invention further select excipients acceptable in the cosmetic field, including any one or more of solvents, dispersants, surfactants, preservatives, pigments, fragrances, lubricants, thickeners, penetration enhancers, humectants, sunscreens, antioxidants, chelating agents, film-forming agents, suspending agents, foaming agents, and pH adjusters.

[0043] Furthermore, based on the above embodiments, the dosage form of the cosmetic composition is further selected to include aqueous formulation, emulsion, gel, ointment, powder, or sheet mask.

[0044] Fourthly, embodiments of the present invention provide the application of the above-mentioned tranexamic acid-carnosine conjugate or the above-mentioned cosmetic composition in improving the appearance of the skin.

[0045] Furthermore, based on the above embodiments, the embodiments of the present invention further select at least one of the following to improve skin appearance: a) Whitens and fades dark spots, inhibits melanin production; b) Inhibits skin glycation.

[0046] It should be understood that, unless otherwise specified, all raw materials used in the following examples are commercially available.

[0047] Example 1 Chemical preparation of tranexamic acid-carnosine conjugates Step 1: Protection reaction of the N-terminal tert-butyloxycarbonyl (Boc) group of tranexamic acid Feeding and reaction: In a 500 mL three-necked flask, add 200 mL of anhydrous methanol, 0.10 mol of tranexamic acid (TXA, 15.72 g), and 0.20 mol of triethylamine (TEA, 20.24 g). Stir mechanically to completely suspend the materials. Under ice-water bath conditions (0°C-5°C), slowly add 50 mL of a methanol solution of di-tert-butyl dicarbonate (containing 0.12 mol of di-tert-butyl dicarbonate) in three batches, with the total adding time controlled at 40-45 minutes.

[0048] Post-treatment: After the addition was complete, the ice-water bath was removed, and the reaction was continued in a sealed environment at room temperature (25°C) for 12 hours. After the reaction was completed, the substrate was monitored for complete conversion by thin-layer chromatography (TLC). The methanol in the resulting reaction solution was evaporated under reduced pressure at 35°C and a vacuum of -0.085 MPa. Then, 150 mL of saturated sodium bicarbonate aqueous solution was added to the solution for solubilization. The solubilized reaction solution was extracted with 100 mL of ethyl acetate. The extraction was repeated twice to remove excess di-tert-butyl dicarbonate. The aqueous phases from the two extractions were combined, and the pH of the combined aqueous phase was adjusted to 2-3 with 2.0 mol / L dilute hydrochloric acid in an ice bath (0°C-5°C). A large amount of white solid was collected.

[0049] Drying and Yield: The white solid collected above was washed with ethyl acetate (150 mL) three times to remove residual impurities. The washed white solid was then vacuum dried at 40 °C and 90 Pa for 12 hours to obtain 22.68 g of white powdery solid Boc-tranexamic acid (Boc-TXA), with a yield of 88.2%.

[0050] Step 2: Protective reaction of C-terminal methyl esterification (OMe) of carnosine Feeding and Reaction: In a 500 mL dry flask equipped with a tail gas absorption device, 0.10 mol (22.62 g) of natural L-carnosine and 250 mL of anhydrous methanol were added. Under ice bath stirring conditions at -5°C, 0.12 mol (14.28 g) of highly active thionyl chloride was added dropwise extremely slowly through a constant pressure dropping funnel, with strict control over the dropping rate to ensure that the internal temperature of the system did not exceed 5°C.

[0051] Post-treatment: After the addition is complete, the system is heated steadily to the reflux temperature (65°C) and kept at 65°C for a total reflux reaction for 6 hours. During the reaction, a large amount of HCl gas is released. After the reaction is terminated, the material obtained from the reaction is cooled to room temperature, and then subjected to reduced pressure flash evaporation at a temperature of 45°C and a vacuum degree of -0.095MPa to completely remove excess thionyl chloride and methanol from the material until a white viscous crystal is obtained.

[0052] Purification and Yield: 80 mL of anhydrous diethyl ether was added to the above white viscous crystals for stirring and washing. The solid filter cake was collected by vacuum filtration and placed in a phosphorus pentoxide vacuum dryer for drying at 45°C and a vacuum of -0.095 MPa for 24 hours. Finally, 23.63 g of hygroscopic, off-white crystalline powder L-carnosine methyl ester hydrochloride (Carnosine-OMe·HCl) was obtained, with a yield of 85.4%.

[0053] Step 3: Condensation reaction Material preparation and reaction: Weigh 0.05 mol (12.87 g) of the prepared Boc-TXA and 0.05 mol (13.84 g) of L-carnosine methyl ester hydrochloride and dissolve them together in 150 mL of chromatographic grade dimethylformamide (DMF). Then, at 0°C, add 0.06 mol (HOBt, 8.11 g) of 1-hydroxybenzotriazole and 0.06 mol (EDCI, 11.50 g) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride sequentially. Finally, slowly inject 0.10 mol (DIEA, 12.93 g) of N,N-diisopropylethylamine through a syringe to neutralize the hydrochloride and maintain the weak alkalinity of the system.

[0054] Reaction and crude product extraction: The above materials were transferred to a sealed container at 25°C and stirred for 18 hours. After the reaction was terminated, the reaction solution was poured into 500 mL of ice water, and then extracted with ethyl acetate (200 mL). The extraction was repeated 4 times, and the organic phases were combined. The combined organic phases were then washed sequentially with 5 wt% citric acid solution, saturated brine, and 5 wt% sodium bicarbonate solution to remove residual condensation byproducts.

[0055] Silica gel column chromatography separation loss: The washed organic phase was dried with anhydrous magnesium sulfate and then concentrated under reduced pressure at 40°C and -0.095 MPa to obtain crude intermediate Boc-TXA-carnosine methyl ester. Due to the non-specific N-acylation side reaction of the imidazole ring of the carnosine side chain under strong reactive agents, the crude product contained about 14.2% of diacylated unknown impurities (identified by micro-LC-MS). The crude product was further separated by fine chromatography using a 200-mesh ordinary silica gel column. The sample loading amount was 25.0 g, and the sample loading flow rate was 1.0 BV / h. After loading, elution was performed using eluent (chloroform:methanol:water = 65:30:5, volume ratio) at a flow rate of 2.0 BV / h. The purified target component eluent was collected and concentrated until a large amount of solid precipitated. The solid was collected to obtain 12.51 g of the fully protected intermediate Boc-TXA-carnosine methyl ester. The actual separation yield of this step was only 52.1%.

[0056] Step 4: Deprotection reaction Step 1 (Demethylation of ester bonds): 24.0 mmol (11.50 g) of the above intermediate Boc-TXA-carnosine methyl ester was dissolved in a mixed solvent of tetrahydrofuran (THF) and water, with a volume of 80 mL and a volume ratio of THF to water of 3:1. Then, 36.0 mmol (1.51 g) of lithium hydroxide monohydrate was added at 0°C, and the mixture was stirred at 25°C for 4 hours to completely hydrolyze the C-terminal methyl ester of the above intermediate. After the reaction was completed, 1.0 mol / L hydrochloric acid was added to the reaction solution to neutralize it. Then, the THF was evaporated under reduced pressure at 35°C and a vacuum of -0.09 MPa to obtain the reaction concentrate.

[0057] Step 2 (De-tert-butoxycarbonyl Boc bond): 60 mL of a 30% (v / v) trifluoroacetic acid (TFA) dichloromethane (DCM) solution was directly injected into the above reaction concentrate. The mixture was stirred at room temperature for 2 hours to remove the Boc protecting group. After the reaction was completed, excess TFA and DCM were evaporated under vacuum at 40°C and -0.095 MPa. The remaining highly viscous hydrochloride product was dissolved in 30 mL of deionized water.

[0058] Step 5: Purification Preparative chromatographic purification: The solubilized hydrochloride product was desalted and purified twice using a reversed-phase C18 preparative high-performance liquid chromatography system. The sample loading volume was 35 mL, and the flow rate was 12 mL / min. After loading, gradient elution was performed: 0 min-5 min: 95% water (containing 0.05 wt% formic acid), 5% acetonitrile; 5 min-25 min: the water volume percentage decreased linearly from 95% to 60%, and the acetonitrile volume percentage increased linearly from 5% to 40%; 25 min-30 min: the water volume percentage decreased linearly from 60% to 5%, and the acetonitrile volume percentage increased linearly from 40% to 95%. The target elution peak was collected.

[0059] Endpoint yield and purity analysis: The collected target elution peaks were freeze-dried. First, they were pre-frozen at -60℃ for 6 hours, and then heated to -10℃ at a rate of 0.5℃ / min and maintained for 20 hours under a vacuum of 8 Pa to complete the main drying stage. Subsequently, the temperature was increased to 25℃ at a rate of 0.5℃ / min and maintained for 6 hours to complete the desorption drying stage, finally yielding 3.37 g of pure tranexamic acid-carnosine conjugate, which is a white, highly hygroscopic, fluffy solid.

[0060] Example 2 Biological methods for preparing tranexamic acid-carnosine conjugates Using bioinformatics methods, an adenylated domain of a wild-type nonribosomal peptide synthase (NRPS) derived from *Streptomyces coelicolor* was identified. This adenylated domain can activate cyclohexyl-related substrates and is named A-domain-ori. The amino acid sequence of the adenylated domain A-domain-ori is shown in SEQ ID NO:1.

[0061] Nine key amino acid residues within the substrate-binding pocket of the adenylated domain A-domain-ori were mutated as follows: amino acid position 303 was mutated from D to G, amino acid position 304 from A to V, amino acid position 307 from E to L, amino acid position 346 from R to F, amino acid position 367 from Q to L, amino acid position 390 from F to G, amino acid position 398 from I to V, amino acid position 399 from N to D, and amino acid position 404 from K to G. After mutation, the optimized adenylated domain was obtained and named TCL-A-domain. The amino acid sequence of this optimized adenylated domain TCL-A-domain is shown in SEQ ID NO:2.

[0062] The DNA sequence was deduced by reverse engineering based on the optimized amino acid sequence of the adenylated domain TCL-A-domain to obtain the coding gene for the TCL-A-domain. The coding gene for the TCL-A-domain, the coding gene for its homologous thioesterification domain (T-domain), and the coding gene for a condensation domain (C-domain) with good catalytic activity (derived from the fengycin synthase cluster of Bacillus) were tandem to obtain a complete coding gene for tranexamic acid-carnosine ligase, TCLigase.

[0063] The above-mentioned coding gene TCLigase was codon optimized according to the codon preference of Kluyveromyces lactis. The optimized coding gene was cloned into the expression vector pKLAC2 to obtain a recombinant plasmid, named pKLAC2-TCLigase.

[0064] The optimized encoding gene TCLigase sequence is shown in SEQ ID NO:3, and the amino acid sequence of the tranexamic acid-carnosine ligase it encodes is shown in SEQ ID NO:4.

[0065] The recombinant plasmid pKLAC2-TCLigase was digested with the restriction endonuclease SacII to obtain a linearized recombinant plasmid pKLAC2-TCLigase. The linearized recombinant plasmid pKLAC2-TCLigase was electroporated into K. lactis GG799 competent cells. The transformed strain was plated on YCB-Acetamide (acetamide as the sole nitrogen source) plates and cultured at 30°C for 72 h. The largest single colony on the plate was picked, and genomic DNA was extracted. The copy number of TCLigase was determined by qPCR. Strains with higher copy numbers generally had higher protein expression levels. The strain with the highest copy number was named K. lactis-TCL and used for subsequent fermentation.

[0066] The above-mentioned YCB-Acetamide medium formula is as follows: 1.17% (w / v) yeast carbon base (YCB), 5 mM acetamide, 2% (w / v) agar powder, with water as the solvent, and used after sterilization.

[0067] The above-mentioned strain K. lactis-TCL was inoculated into a 5L fermenter (containing 2.5L of initial medium) for high-density culture. The initial medium in the fermenter was YPD medium. Fermentation was carried out at 30℃ and pH 5.5. When the glucose in the initial medium was depleted and the dissolved oxygen (DO) suddenly increased (approximately 12-14 hours of fermentation), a 50% (w / v) glycerol aqueous solution (containing 50g of glycerol per 100 mL of glycerol aqueous solution) was added as a feedstock. DO-stat feedback control was used to maintain DO at 30%, and the average flow rate of the feedstock was 10 mL / h per liter of fermentation broth. Fermentation was continued to increase OD... 600 Once the concentration reaches 100, galactose at a final concentration of 2% (w / v) is added for induction culture (i.e., 2g of galactose is added per 100mL of fermentation broth for induction culture). The strain is induced to express tranexamic acid-carnosine ligase. The culture is then induced at 30℃ for 24 hours to obtain a fermentation broth containing tranexamic acid-carnosine ligase.

[0068] After fermentation, the pH of the fermentation broth was adjusted to 6.5 using a 10% NaOH solution. Then, a mixed aqueous solution of tranexamic acid and L-carnosine (pH 6.5) at a constant rate of 0.2 mL / min was pumped into the fermentation broth at 10% of the broth volume. The concentrations of tranexamic acid and L-carnosine in the mixed aqueous solution were both 1 mol / L. Biotransformation was carried out for 48 hours. Using whole cells in the fermentation broth as a catalyst, the reaction of tranexamic acid and L-carnosine was catalyzed to generate tranexamic acid-carnosine conjugates at a temperature of 30℃ and a pH of 6.5.

[0069] The reaction process of tranexamic acid-carnosine ligase catalyzing the reaction of substrate tranexamic acid and L-carnosine to form tranexamic acid-carnosine conjugates is as follows: Figure 1 As shown.

[0070] After the above biotransformation is completed, the tranexamic acid-carnosine conjugate in the transformation solution is purified. The purification steps are as follows: The conversion solution was filtered using a ceramic membrane with a pore size of 0.22 μm to remove bacterial cells, and the clear filtrate was collected. The filtrate was then loaded onto an Amberlite XAD-16 macroporous adsorption resin column at a loading volume of 1 BV and a loading flow rate of 1.5 BV / h. After loading, the sample was first washed with 4 BV of water to remove impurities, and then eluted with 4 BV of 20 vol% ethanol at a flow rate of 1 BV / h. The resin eluent was collected.

[0071] Anion exchange chromatography: The resin eluent was concentrated under reduced pressure at 45°C and -0.09 MPa until no ethanol odor remained (ethanol removal). The concentrate was diluted 2-fold with water, and the pH was adjusted to 8.0 using 10% NaOH solution to obtain the sample solution. The sample solution was loaded onto an anion exchange chromatography column packed with DEAE Sepharose Fast Flow packing material at a loading volume of 0.5 BV and a flow rate of 1.2 BV / h. The anion exchange chromatography column was pre-equilibrated with 20 mM Tris-HCl buffer at pH 8 before loading. After loading, the column was washed with equilibration buffer for 2 column volumes, followed by elution with 20 mM Tris-HCl buffer (pH 8.0) containing 0.2 M NaCl at a flow rate of 1.0 BV / h. The eluent containing the target product was collected.

[0072] The elution product was collected for RP-HPLC: a C18 column was used; mobile phase A was 0.1% (v / v) trifluoroacetic acid aqueous solution, and mobile phase B was 0.1% (v / v) trifluoroacetic acid acetonitrile solution; the flow rate was 10 mL / min, and the detection wavelength was 214 nm. The gradient elution program was as follows: 0 min ~ 5 min: maintain 5% volume of mobile phase B and 95% volume of mobile phase A; 5 min ~ 25 min: the volume percentage of mobile phase B linearly increased from 5% to 30%, and the volume percentage of mobile phase A linearly decreased from 95% to 70%; 25 min ~ 30 min: the volume percentage of mobile phase B linearly increased from 30% to 95%, and the volume percentage of mobile phase A linearly decreased from 70% to 5% (wash); 30 min ~ 35 min: maintain 95% volume of mobile phase B and 5% volume of mobile phase A. The fraction of the major absorption peak at 15 min ~ 20 min was collected.

[0073] Drying: After collecting the fraction by RP-HPLC, the collected fraction was subjected to vacuum freeze drying. First, it was pre-frozen at -70°C for 4 hours, and then dried under a vacuum of 8 Pa for 36 hours. During the drying process, the cold trap temperature was maintained at -80°C, and the shelf temperature was first increased from -30°C to 0°C at a heating rate of 0.5°C / min and maintained at the same temperature for 20 hours (completing the main drying stage); then, it was increased from 0°C to 25°C at a heating rate of 1.0°C / min and maintained at the same temperature for 16 hours (completing the secondary drying stage). After drying, a white powder of tranexamic acid-carnosine conjugate was obtained.

[0074] Finally, 25.5 g of a white powder containing tranexamic acid-carnosine conjugate with a purity of 99.1 wt% (HPLC detection) was obtained from 4 L of conversion buffer, with an overall yield of approximately 70% of the substrate conversion. Mass spectrometry analysis (ESI-MS) showed that the molecular ion peak of the tranexamic acid-carnosine conjugate was at m / z 366.21 [M+H]. + This is consistent with the theoretical value.

[0075] The tranexamic acid-carnosine conjugates used in Examples 3 to 9 below are all tranexamic acid-carnosine conjugates prepared by the biosynthesis method in Example 2.

[0076] Example 3 In vitro anti-glycation activity assay To establish an in vitro glycation model of bovine serum albumin (BSA)-methylglyoxal (MGO): BSA was dissolved in phosphate buffer (pH 7.4) to prepare a 20 mg / mL BSA solution, and MGO was dissolved in phosphate buffer (pH 7.4) to prepare a 5 mM MGO solution. The BSA and MGO solutions were mixed at a 1:1 volume ratio to obtain the model solution.

[0077] Four model solutions were established according to the above method. One model solution served as a blank control group. The other three model solutions were supplemented with tranexamic acid-carnosine conjugate (final concentration 1 mM), L-carnosine (final concentration 1 mM), and aminoguanidine (final concentration 1 mM), respectively (positive control). After incubation at 37°C in the dark for 24 hours, the characteristic fluorescence intensity of AGEs (advanced glycosylation end products) was measured using a fluorescence spectrophotometer at an excitation wavelength of 370 nm and an emission wavelength of 440 nm. The inhibition rate of AGEs characteristic fluorescence intensity relative to the blank control group was calculated. The results are shown in Table 1.

[0078] Inhibition rate = (characteristic fluorescence intensity of AGEs in blank control group - characteristic fluorescence intensity of target AGEs) ÷ characteristic fluorescence intensity of AGEs in blank control group × 100%.

[0079] Table 1 The results above show that the tranexamic acid-carnosine conjugate of the present invention inhibited the formation of AGEs by 85.2% at 1 mM, which is comparable to the effect of 1 mM L-carnosine (inhibition rate of 89.5%) and significantly better than the blank control group. This indicates that the compound of the present invention completely retains the core anti-glycation efficacy of L-carnosine.

[0080] Example 4 In vitro tyrosinase inhibitory activity assay Preparation of the reaction system: Add the tranexamic acid-carnosine conjugate to be tested and mushroom tyrosinase to a final concentration of 20 U / mL in a sodium phosphate buffer solution with a concentration of 50 mM and a pH of 6.8.

[0081] Five reaction systems were prepared according to the above method, with final concentrations of tranexamic acid-carnosine conjugate in the five reaction systems being 50 µM, 100 µM, 200 µM, 500 µM, and 1000 µM, respectively.

[0082] Preparation of blank control group: Add mushroom tyrosinase to a final concentration of 20 U / mL in sodium phosphate buffer with a concentration of 50 mM and a pH of 6.8.

[0083] After pre-incubating the above five reaction systems and the blank control group at 30℃ for 10 minutes, the substrate L-DOPA was added to each system to initiate the reaction at a final concentration of 0.5 mM. The product, dopachrome, exhibits characteristic absorption at 475 nm. The absorbance change at 475 nm for each reaction system was monitored, and the rate of absorbance change was used to characterize the enzyme activity of mushroom tyrosinase. Using the enzyme activity of mushroom tyrosinase in the blank control group as a baseline, the inhibition rate of different concentrations of tranexamic acid-carnosine conjugate on the enzyme activity of mushroom tyrosinase was calculated. A dose-response curve was constructed with the concentration of tranexamic acid-carnosine conjugate on the x-axis and the enzyme activity inhibition rate on the y-axis, thus obtaining the half-maximal inhibitory concentration (IC50) of tranexamic acid-carnosine conjugate on mushroom tyrosinase. 50 The results are shown in Table 2.

[0084] Using the same test method described above, the half-maximal inhibitory concentrations (IC50) of tranexamic acid and kojic acid against mushroom tyrosinase were calculated respectively. 50 The results are shown in Table 2.

[0085] Table 2 The results above show that the half-maximal inhibitory concentration (IC50) of the tranexamic acid-carnosine conjugate of this invention against mushroom tyrosinase is... 50 The concentration of 150 µM was significantly better than the half-maximal inhibitory concentration (IC50) of free tranexamic acid against mushroom tyrosinase. 50 This indicates that the coupled tranexamic acid-carnosine conjugate significantly enhances the direct inhibitory effect on tyrosinase.

[0086] Example 5 Cellular level melanin production inhibition detection Experiments were conducted using B16F10 mouse melanoma cells. B16F10 cells were seeded into four wells of a 6-well plate and divided into four groups (1-4). After culturing for 24 hours, α-melanocyte-stimulating hormone (α-MSH) at a final concentration of 100 nM was added to groups 1-4 to induce melanin production. Simultaneously, tranexamic acid-carnosine conjugate at a final concentration of 100 µM was added to group 1, tranexamic acid at a final concentration of 100 µM was added to group 2, kojic acid at a final concentration of 100 µM was added to group 3, and kojic acid at a final concentration of 100 µM was added to group 3. Group 4 served as a blank control group, receiving only α-MSH without the test sample.

[0087] After culturing for another 72 hours, B16F10 cells from each group were collected and lysed at 80°C using sodium hydroxide solution (1 mol / L)-dimethyl sulfoxide (10%, v / v) to dissolve melanin granules. The absorbance was measured at 405 nm. Total protein content was determined using the BCA method for standardization. With the melanin content of the blank control group as 100%, the relative melanin content and melanin inhibition rate of the other three groups were calculated. The results are shown in Table 3.

[0088] Melanin inhibition rate = (Melanin content of blank control group - Target melanin content) ÷ Melanin content of blank control group × 100%.

[0089] Table 3 The results above show that, compared to the blank control group, the tranexamic acid-carnosine conjugate of this invention reduced melanin production by 45%, tranexamic acid reduced melanin production by 30%, and kojic acid reduced melanin production by 50%. This indicates that, compared to tranexamic acid, the tranexamic acid-carnosine conjugate of this invention significantly enhances the inhibitory effect on melanin production, demonstrating that the tranexamic acid-carnosine conjugate of this invention has excellent skin whitening and spot-fading potential at the cellular level.

[0090] Example 6 In vitro skin irritation test Following OECD testing guideline 439, irritation testing was conducted using a commercially available reconstructed human epidermal model (EpiDerm™). Four reconstructed human epidermal models were established. The tissues of these four models were exposed to 5% (w / v) aqueous solution of the compound of this invention, tranexamic acid-carnosine conjugate (i.e., 5 g tranexamic acid-carnosine conjugate per 100 mL of solution), 5% (w / v) aqueous solution of tranexamic acid (i.e., 5 g tranexamic acid per 100 mL of solution), 5% (w / v) aqueous solution of SDS (i.e., 5 g SDS per 100 mL of solution), and PBS buffer adjusted to pH 6.5 (close to the physiological pH of skin), respectively. After treatment for 60 minutes, each model was rinsed and cultured for another 42 hours. Finally, cell viability was assessed using the MTT assay. The results are shown in Table 4.

[0091] Table 4 The results above show that the cell viability of the model tissue treated with tranexamic acid decreased to 45% (<50%), and it was classified as an irritant (GHS Category 2) according to OECD standards. In contrast, the cell viability of the model tissue treated with the tranexamic acid-carnosine conjugate of this invention reached 88% (>50%), and it was classified as a non-irritant. This demonstrates that the tranexamic acid-carnosine conjugate of this invention significantly improves skin mildness compared to tranexamic acid.

[0092] Example 7 Metabolic stability and prodrug activation validation in skin homogenates To verify the prodrug mechanism of the tranexamic acid-carnosine conjugate of this invention, an in vitro skin metabolism experiment was conducted. Fresh porcine back skin was homogenized, and the tranexamic acid-carnosine conjugate of this invention was added to a final concentration of 10 µM, followed by incubation at 37°C. Samples were taken at 0, 2, 4, and 8 hours after incubation, and proteins were precipitated with acetonitrile. The supernatant was then analyzed by LC-MS / MS to quantitatively determine the concentration of the tranexamic acid-carnosine conjugate and the concentration of free active substances (free tranexamic acid and free L-carnosine). The results are shown in Table 5.

[0093] Table 5 The results above show that the concentration of the tranexamic acid-carnosine conjugate of this invention decreases in a time-dependent manner with increasing incubation time, and approximately 60% is degraded after 8 hours. Simultaneously, the concentrations of the free active substances, namely free tranexamic acid and free L-carnosine, increase in a corresponding time-dependent manner. This result directly demonstrates that the tranexamic acid-carnosine conjugate of this invention can be hydrolyzed by skin enzymes, releasing free tranexamic acid and free L-carnosine, thus confirming its prodrug design mechanism of action.

[0094] Example 8 This invention provides a whitening and anti-glycation essence, comprising the following components by weight percentage: The mixture consists of 1.5% tranexamic acid-carnosine conjugate, 2.0% nicotinamide, 5% glycerol, 4% butylene glycol, 0.5% sodium hyaluronate, 0.2% xanthan gum, 0.8% phenoxyethanol, 0.2% ethylhexylglycerin, and a citric acid-sodium citrate solution to adjust the pH of the system to 6.0, with the remainder being water.

[0095] The preparation method of the whitening and anti-glycation essence in this embodiment is as follows: Phase A preparation: Add water, butylene glycol, glycerin, sodium hyaluronate, and xanthan gum to an emulsifying pot, heat to 80°C, stir until completely dissolved, and keep warm for 20 minutes. The first part of water is 90 wt% of the total water volume.

[0096] Cooling: Turn on the cooling water to cool phase A down to below 45°C.

[0097] Phase B preparation and addition: Add tranexamic acid-carnosine conjugate and nicotinamide to the remaining water and stir at room temperature until completely dissolved to form a clear and transparent aqueous solution of active ingredients. Add this solution to the cooled phase A and stir until homogeneous.

[0098] C phase addition: Add phenoxyethanol and ethylhexylglycerin, and stir until homogeneous.

[0099] pH adjustment: Adjust the pH of the system to 6.0 using a citric acid-sodium citrate solution.

[0100] Discharge: After stirring evenly, stop stirring, filter through a microporous membrane with a pore size of 0.22 μm, collect the filtrate, let it stand to defoam, and then fill it into containers.

[0101] Example 9 This invention provides a firming and brightening face cream (O / W type), comprising the following components by weight percentage: The ingredients are: 3% glycerol, 0.15% xanthan gum, 1.5% tranexamic acid-carnosine conjugate, 8% caprylic / capric triglyceride, 3% jojoba seed oil, 4% cetearyl alcohol / cetearyl glucoside, 0.8% phenoxyethanol, 0.2% ethylhexylglycerin, and the balance being water.

[0102] The preparation method of the firming and brightening face cream in this embodiment is as follows: Oil phase (A phase) preparation: Caprylic / capric triglyceride, jojoba seed oil, and cetearyl alcohol / cetearyl glucoside are added to the oil phase pot, heated to 85°C, and stirred until dissolved.

[0103] Preparation of aqueous phase (phase B): Add water, glycerol, and xanthan gum to the aqueous phase pot, heat to 85°C, and stir until dissolved.

[0104] Emulsification: Phase A is drawn into Phase B at a rate of 4 L / min. During the drawing process, the scraper agitator and the bottom homogenizer are turned on simultaneously. The scraper agitator speed is 60 rpm, and the bottom homogenizer speed is 10,000 rpm. Emulsification is carried out for 5 min.

[0105] Cooling: Turn on the cooling water to cool down, and reduce the stirring speed of the scraper from 60 rpm to 30 rpm as the temperature increases.

[0106] Addition of active ingredient: The tranexamic acid-carnosine conjugate is pre-dissolved in water at a mass ratio of 1:5 to obtain a tranexamic acid-carnosine conjugate solution. When the temperature drops below 45°C, the tranexamic acid-carnosine conjugate solution is added and stirred until homogeneous.

[0107] Preservation and discharge: Add phenoxyethanol and ethylhexylglycerin, stir evenly, cool to 38℃ and discharge, then fill to obtain the final product.

[0108] Test case Product efficacy verification: In order to verify the efficacy advantages of the tranexamic acid-carnosine conjugate of the present invention in the final cosmetic formulation, the following comparative experiment was designed.

[0109] 1. Experimental Samples Experimental group: The whitening and anti-glycation essence prepared in Example 8 above (containing 1.5 wt% tranexamic acid-carnosine conjugate).

[0110] Control group: A comparative essence was prepared, with the same formula and preparation method as in Example 8, except that the "tranexamic acid-carnosine conjugate" was replaced in equal amounts with a physical mixture of tranexamic acid and L-carnosine (molar ratio of 1:1).

[0111] 2. Experimental methods and results (1) Human skin patch test (mildness): According to the skin occlusive patch test method in the Cosmetic Safety Technical Specification, 30 healthy volunteers were selected and applied the experimental group and control group products to the inner side of the left and right arms for 24 hours. Professional dermatologists observed the skin of the test area and recorded the skin reaction 0.5 hours and 24 hours after the patch was removed.

[0112] Evaluation criteria: Erythema: 0 points: None; 1 point: Barely visible; 2 points: Obvious erythema; 3 points: Severe erythema; 4 points: Purplish-red with eschar.

[0113] Edema: 0 points: None; 1 point: Barely raised; 2 points: Raised outline clear; 3 points: Raised ≈ 1mm; 4 points: Raised > 1mm.

[0114] Based on the above evaluation criteria, symptoms such as redness and papules in the test area were graded as erythema and edema, and the mean skin irritation index (MII) was calculated. MII = (total score of erythema and edema) ÷ (total number of subjects × number of observations). MII < 0.10 was considered non-irritating, and 0.10 ≤ MII < 0.50 was considered slightly irritating.

[0115] Experimental results: Clinical statistics show that the average skin irritation index (MII) of the experimental group products was 0.00. None of the 30 volunteers experienced any redness, swelling, stinging or itching reactions, and their skin tolerance was excellent. The total score was 0, and the products were judged to be non-irritating.

[0116] The average skin irritation index (MII) of the control group product was 0.1, and 3 volunteers developed erythema (each scored 1 point). They were observed 0.5h and 24h after the patch was removed (a total of 2 times). The total score was (1 point × 2 times) × 3 people = 6 points. The MII was calculated as: 6 points ÷ (30 people × 2 times) = 0.10, which was judged to be non-irritating.

[0117] The above comparative data demonstrate that the tranexamic acid-carnosine conjugate of the present invention can significantly reduce the transient diffusion impact of small molecular weight tranexamic acid and improve skin tolerance.

[0118] (2) B16F10 cell melanin inhibition experiment (whitening effect): The experimental group essence diluted 100 times and the control group essence diluted 100 times were used to treat the two groups of B16F10 cells induced by α-MSH respectively. After culturing for 72 hours, the melanin content in each group of B16F10 cells was measured. The melanin content of the blank control group (only α-MSH was added to B16F10 cells to induce melanin production, without adding test samples) was taken as 100%. The inhibition rate of melanin production by the experimental group essence and the control group essence was calculated respectively (the experimental and calculation methods are referred to Example 4).

[0119] Experimental results: At the same dilution factor, the experimental group showed an inhibition rate of 48.5% on melanin production, which was better than the 32.1% inhibition rate of the control group.

[0120] As can be seen from the above results, in formulation applications, the tranexamic acid-carnosine conjugate of the present invention, compared with a simple physical mixture of tranexamic acid and L-carnosine, not only has a stronger ability to inhibit melanin production through synergistic effect, but also significantly reduces skin irritation, making it more suitable for use in high-efficacy skin care products.

[0121] 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 tranexamic acid-carnosine conjugate, characterized in that, The structure of the tranexamic acid-carnosine conjugate is shown below: N-{[4-(aminomethyl)cyclohexyl]carbonyl}-β-alanyl-L-histidine.

2. The tranexamic acid-carnosine conjugate according to claim 1, characterized in that, The tranexamic acid-carnosine conjugate is obtained by reacting tranexamic acid and L-carnosine to form an amide bond.

3. The use of the tranexamic acid-carnosine conjugate according to claim 1 or 2 in the preparation of cosmetics.

4. A cosmetic composition, characterized in that, The cosmetic composition comprises the tranexamic acid-carnosine conjugate as described in claim 1 or 2, and one or more cosmetic excipients.

5. The cosmetic composition according to claim 4, characterized in that, The effective content of the tranexamic acid-carnosine conjugate in the cosmetic composition is 0.01%-10% by mass percentage.

6. The cosmetic composition according to claim 5, characterized in that, The effective content of the tranexamic acid-carnosine conjugate in the cosmetic composition is 0.1%-5% by mass percentage.

7. The cosmetic composition according to claim 4, characterized in that, The excipients acceptable in the cosmetics field include any one or more of the following: solvents, dispersants, surfactants, preservatives, pigments, fragrances, lubricants, thickeners, penetration enhancers, humectants, sunscreens, antioxidants, chelating agents, film-forming agents, suspending agents, foaming agents, and pH adjusters.

8. The cosmetic composition according to claim 4, characterized in that, The dosage forms of the cosmetic compositions include aqueous solutions, emulsions, gels, ointments, powders, or sheet masks.

9. The use of the tranexamic acid-carnosine conjugate according to claim 1 or 2, or the cosmetic composition according to any one of claims 4-8, in improving skin appearance.

10. The application according to claim 9, characterized in that, The improvement in skin appearance includes at least one of the following: a) Whitens and fades dark spots, inhibits melanin production; b) Inhibits skin glycation.

Images