Metal peptide compound as well as preparation method and application thereof

Abstract

The invention discloses a metal peptide compound as well as a preparation method and application thereof, and belongs to the technical field of biology. The metal peptide compound is formed by coordination of peptide ligands and bivalent copper ions, and the structural formula of the metal peptide compound is shown in the formula (I). The metal peptide compound can target two major pathways of'inflammation repair 'and'pigmentation' of PIH at the same time, so that post-inflammatory pigmentation is comprehensively and efficiently improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biotechnology, in particular to a metal peptide complex and a preparation method and application thereof. BACKGROUND

[0002] Tripeptide-1 copper complex (GHK-Cu, blue copper peptide, CAS No.: 89030-95-5) is a widely known repair ingredient in the field of cosmetics. In this complex, copper ions are coordinated with the imidazole ring nitrogen of the histidine side chain, the alpha-amino nitrogen of glycine, and the deprotonated amide nitrogen between the two, forming a stable planar tetragonal structure, which has strong anti-inflammatory, wound healing and tissue repair-promoting ability, however, GHK-Cu has limited direct inhibitory effect on melanogenesis.

[0003] Tranexamic acid (TXA) is a synthetic lysine derivative that can effectively block melanogenesis by inhibiting plasmin activity and directly inhibiting tyrosinase, and is a recognized highly effective whitening ingredient.

[0004] Post-inflammatory hyperpigmentation (PIH) is a common acquired skin disease, which is caused by excessive production or abnormal distribution of melanin after inflammation or injury, and is manifested as brown, dark brown or blue-gray patches. Its pathological mechanism is complex, and there are problems of low clearance rate, long treatment course and easy recurrence in treatment. SUMMARY

[0005] Therefore, the purpose of the present application is to provide a metal peptide complex and a preparation method and application thereof, which can overcome the problem of poor treatment effect on post-inflammatory hyperpigmentation in the prior art.

[0006] In a first aspect, the present application provides a metal peptide complex, which is formed by coordination of a peptide ligand and a divalent copper ion, and the structural formula of the metal peptide complex is shown as formula (I): Formula (I) Compared with the prior art, the lysine in the GHK peptide is replaced by tranexamic acid to obtain a new peptide ligand Gly-His-TXA, and the new peptide ligand is further chelated with divalent copper ions to form a brand new metal peptide complex Gly-His-TXA-Cu. The metal peptide complex of the present application retains the "Gly-His-Cu" core structure responsible for the biological activity in GHK-Cu, while introducing the tranexamic acid "functional tail" with whitening effect. Through the "integrated" molecular design, a new active substance capable of simultaneously targeting the "inflammation repair" and "pigment generation" two pathways of PIH through a single molecule is created, and the post-inflammatory pigmentation is comprehensively and efficiently improved.

[0007] Further, the structural formula of the peptide ligand is shown as formula (II): Formula (II).

[0008] In a second aspect, the present application provides a preparation method of a metal peptide complex for preparing the above metal peptide complex, comprising the following steps: using TXA-ligase to catalyze the reaction of glycyl-L-histidine ethyl ester and tranexamic acid to generate a peptide ligand; mixing the peptide ligand with a soluble divalent copper salt in an aqueous medium to generate a metal peptide complex by reaction.

[0009] Compared with the prior art, the present application obtains a brand new TXA-ligase through genetic engineering, which can specifically catalyze the connection of glycyl-L-histidine derivative and tranexamic acid to generate a peptide ligand. Then, the peptide ligand is chelated with copper ions in the soluble divalent copper salt in an aqueous medium to generate a metal peptide complex.

[0010] Further, using TXA-ligase to catalyze the reaction of glycyl-L-histidine ethyl ester and tranexamic acid to generate a peptide ligand comprises: fermenting the recombinant strain containing the TXA-ligase gene, and obtaining the fermentation liquor containing the TXA-ligase after the enzyme activity reaches the plateau stage; adding the substrates glycyl-L-histidine ethyl ester and tranexamic acid to the fermentation liquor containing the TXA-ligase to react, to obtain the fermentation liquor containing the peptide ligand Gly-His-TXA; wherein the concentration of glycyl-L-histidine ethyl ester in the fermentation liquor is 10mM~100mM, and the concentration of tranexamic acid is 10mM~100mM.

[0011] Further, mixing the peptide ligand with a soluble divalent copper salt in an aqueous medium to generate a metal peptide complex by reaction comprises: A soluble divalent copper salt was added to the fermentation broth containing the peptide ligand Gly-His-TXA, and the reaction was carried out to obtain a fermentation broth containing a metal peptide complex. The recombinant strain was removed to obtain a clear liquid containing the metal peptide complex. The molar ratio of the peptide ligand Gly-His-TXA to the copper ions in the soluble divalent copper salt was (1~1.3):1.

[0012] The above technical solution further defines the integrated biological synthesis method of metal peptide complexes. By expressing TXA-ligase through fermentation of recombinant strains and synthesizing peptide ligand Gly-His-TXA in the fermentation broth, the metal peptide complex Gly-His-TXA-Cu is obtained by in situ chelation, avoiding the complex steps and environmental pollution problems of traditional chemical synthesis.

[0013] In addition, the clear liquid containing the metal peptide complex not only contains the core active ingredient Gly-His-TXA-Cu, but is also rich in a variety of skin-beneficial byproducts produced by the metabolism of recombinant strains, which can further improve skin pigmentation.

[0014] Furthermore, the amino acid sequence of the TXA-ligase is shown in SEQ ID NO:3.

[0015] The above technical solution further defines the amino acid sequence of the TXA-ligase as shown in SEQ ID NO:3. Compared with wild-type Bacillus subtilis protease BPN' (amino acid sequence shown in SEQ ID NO:1), this invention first introduces two mutations, S220C and P224A, to modify it into a Bacillus subtilis ligase with ligase activity (amino acid sequence shown in SEQ ID NO:2). Then, the Bacillus subtilis ligase is further mutated and modified by introducing N76D, L126A, G166S, Y216K, and N217S mutation sites to obtain the TXA-ligase. These mutations together optimize the structure of the TXA-ligase, making the TXA-ligase more adaptable to the substrate tranexamic acid, and further improving the catalytic efficiency of the reaction between the substrate glycyl-L-histidine ethyl ester and tranexamic acid to generate peptide ligands.

[0016] Furthermore, soluble divalent copper salts include copper acetate or copper sulfate.

[0017] Furthermore, the fermentation broth containing the peptide ligand Gly-His-TXA was prepared at a reaction temperature of 25℃~40℃ and a pH value of 7.0~8.5; and / or, The reaction temperature for preparing the metal peptide complex is 25℃~45℃, and the pH value is 5.5~6.5.

[0018] Thirdly, the present invention provides a cosmetic composition containing the above-mentioned metal peptide complex, or a metal peptide complex prepared using the above-mentioned preparation method, and one or more cosmetically acceptable carriers or excipients.

[0019] Compared with the prior art, the beneficial effects of the cosmetic composition of the present invention are the same as those of the above-mentioned metal peptide complex, and will not be repeated here.

[0020] Fourthly, the present invention provides the use of the above-mentioned metal peptide complex or the above-mentioned cosmetic composition in the preparation of products that improve skin pigmentation.

[0021] Compared with the prior art, the metal peptide complex or cosmetic composition of the present invention can promote skin barrier repair, anti-inflammation and inhibit melanin production. Through the synergistic effect of anti-inflammatory, repair and whitening pathways, it can improve skin pigmentation, especially for post-inflammatory hyperpigmentation, and has excellent improvement effect. 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 metal peptide complex, which is formed by coordination of a peptide ligand with a divalent copper ion, and the structural formula of the metal peptide complex is shown in formula (I): Formula (I) The structural formula of the peptide ligand is shown in formula (II): Equation (II) The metal peptide complex in the above technical solution is a [glycyl-L-histyl-tranexamic acid]-copper complex, namely Gly-His-TXA-Cu. In this metal peptide complex, the copper ions coordinate with the imidazole ring nitrogen of the histidine side chain, the α-amino nitrogen of glycine, and the deprotonated amide nitrogen between them, forming a stable planar tetragonal structure. This structure gives the metal peptide complex of the present invention powerful anti-inflammatory and skin barrier repair-promoting functions. Simultaneously, the metal peptide complex of the present invention also contains tranexamic acid, the addition of which enables the metal peptide complex to effectively inhibit melanin production. The metal peptide complex of the present invention combines the dual mechanisms of "anti-inflammatory repair" and "pigmentation inhibition," effectively improving skin pigmentation, especially showing excellent improvement effects on post-inflammatory hyperpigmentation (PIH).

[0024] Secondly, embodiments of the present invention provide a method for preparing a metal peptide complex, comprising the following steps: S1. The reaction of glycyl-L-histidine ethyl ester and tranexamic acid is catalyzed by TXA-ligase to generate peptide ligands.

[0025] Furthermore, the specific steps include the following: S11. The recombinant strain containing the gene encoding TXA-ligase was fermented until the enzyme activity reached a plateau, yielding a fermentation broth containing TXA-ligase. After fermentation, the TXA-ligase activity in the broth was 500 U / mL to 5000 U / mL. The recombinant strain could be a Pichia pastoris recombinant strain.

[0026] The specific steps for fermentation culture of the recombinant strain containing the TXA-ligase gene are as follows: Seed activation: The recombinant strain preserved in glycerol tubes was streaked onto YPD solid medium and cultured at 30°C for 48 hours.

[0027] Primary seed culture: Select a single colony from the YPD solid medium and inoculate it into 50 mL of BMGY medium. Incubate at 30°C and 250 rpm for 24 hours. At this time, the recombinant strain will grow to the logarithmic growth phase, and the primary seed culture solution will be obtained.

[0028] High-density fermentation: The entire primary seed culture was inoculated into a 10L fermenter containing 5L of basal salt medium, to which an additional 40 g / L of glycerol was added as an initial carbon source. Fermentation was carried out at a temperature of 28℃-30℃, a pH of 5.5 (automatically controlled by adding ammonia), and dissolved oxygen (DO) maintained above 20% to accumulate cell biomass. Once the initial glycerol was depleted (manifested as a sharp rise in dissolved oxygen), a 50% (w / v) glycerol solution was added at a constant rate of 18 mL / h / L for 4 hours of feed starvation.

[0029] Methanol-induced expression: Stop adding glycerol and start slowly adding pure methanol to activate the AOX1 promoter, causing TXA-ligase to begin expression and secretion into the fermentation broth. Maintain the methanol concentration in the culture medium between 0.5% and 1.5% by volume and induce culture for 48 to 72 hours. During this period, sample enzyme activity is taken regularly until the enzyme activity reaches the plateau phase, yielding a fermentation broth containing TXA-ligase.

[0030] S12. Add the substrate glycyl-L-histidine ethyl ester and tranexamic acid to the fermentation broth containing TXA-ligase to obtain a fermentation broth containing the peptide ligand Gly-His-TXA; wherein the concentration of glycyl-L-histidine ethyl ester in the fermentation broth is 10 mM to 100 mM, and the concentration of tranexamic acid is 10 mM to 100 mM. For example, the concentration of glycyl-L-histidine ethyl ester can be 10 mM, 30 mM, 50 mM, 70 mM, or 100 mM; the concentration of tranexamic acid can be 10 mM, 30 mM, 50 mM, 70 mM, or 100 mM; or a range of values ​​from any of these points. It should be understood that after adding the substrate, the molar ratio of glycyl-L-histidine ethyl ester to tranexamic acid in the fermentation broth is 1:(1~1.5).

[0031] The above steps for preparing the fermentation broth containing the peptide ligand Gly-His-TXA involve a reaction temperature of 25℃~40℃ and a pH value of 7.0~8.5; for example, the reaction temperature can be 25℃, 30℃, 35℃ or 40℃, and the pH value can be 7.0, 7.5, 8.0 or 8.5; or a range of values ​​composed of any of these points.

[0032] In step S1 above, the amino acid sequence of the TXA-ligase is shown in SEQ ID NO:3. It is obtained by mutating wild-type subtilisin BPN' and can specifically and efficiently catalyze the reaction of glycyl-L-histidine ethyl ester and tranexamic acid to generate peptide ligands. Compared with the amino acid sequence of wild-type subtilisin BPN' (as shown in SEQ ID NO:1), it has the following mutations: The 220th amino acid is mutated from S to C; The amino acid at position 224 is mutated from P to A; The 76th amino acid is mutated from N to D; The 126th amino acid is mutated from L to A; The 166th amino acid was mutated from G to S; The 216th amino acid is mutated from Y to K; The 217th amino acid is mutated from N to S.

[0033] S2. The peptide ligand is mixed with a soluble divalent copper salt in an aqueous medium to generate a metal peptide complex.

[0034] Furthermore, after obtaining the fermentation broth containing the peptide ligand Gly-His-TXA in step S1, a soluble divalent copper salt is added to the fermentation broth containing the peptide ligand Gly-His-TXA, and the reaction yields a fermentation broth containing a metal peptide complex. The recombinant strain is removed to obtain a clear liquid containing the metal peptide complex; the concentration of the metal peptide complex in the clear liquid is 0.5 mM to 100 mM.

[0035] In the above steps, the molar ratio of the peptide ligand Gly-His-TXA to the copper ions in the soluble divalent copper salt is (1~1.3):1. For example, the molar ratio can be 1:1, 1.1:1, 1.2:1 or 1.3:1; or a range of values ​​consisting of any points therein.

[0036] The soluble divalent copper salt in the above steps can be copper acetate or copper sulfate; the reaction temperature is 25℃~45℃, and the pH value is 5.5~6.5. For example, the reaction temperature can be 25℃, 30℃, 35℃, 40℃ or 45℃; the pH value can be 5.5, 6.0 or 6.5; or a range of values ​​composed of any points therein.

[0037] Thirdly, embodiments of the present invention provide a cosmetic composition containing the above-mentioned metal peptide complex, or containing a metal peptide complex prepared using the above-mentioned preparation method, and one or more cosmetically acceptable carriers or excipients.

[0038] For example, the cosmetic composition described above can be a blemish repair serum containing the metal peptide complex Gly-His-TXA-Cu, the formulation of which is shown in Table 1.

[0039] Table 1 The preparation method of the above-mentioned serum is as follows: Mix all components of phase A according to the specified ratio and heat to 75℃-80℃ until dissolved and homogeneous. Mix all components of phase B according to the specified ratio until homogeneous. Add phase B to phase A at 300rpm-500rpm and stir until homogeneous. While stirring at 300rpm-500rpm, slowly add xanthan gum (phase C) at a rate of 0.1 g / s. After the xanthan gum is evenly dispersed, homogenize at 5000rpm-8000rpm for 1-3 minutes until a uniform light blue gel-like essence is formed. Cool to room temperature and check that the pH value is between 5.5 and 6.5.

[0040] Fourthly, the present invention provides the use of the above-mentioned metal peptide complex or the above-mentioned cosmetic composition in the preparation of products that improve skin pigmentation.

[0041] Compared with the prior art, the beneficial effects of the metal peptide complex or cosmetic composition of the present invention in the preparation of products that improve skin pigmentation are the same as the beneficial effects of the metal peptide complex described above, and will not be repeated here.

[0042] It should be understood that, unless otherwise specified, all raw materials used in the following examples are commercially available.

[0043] Example 1 Design and Enzyme Activity Assay of TXA-Ligase wild-type Bacillus subtilis protease derived from Bacillus amyloliquefaciens (Its amino acid sequence is shown in SEQ ID NO:1) Targeted mutagenesis was performed. First, through key mutations in S220C and P224A, the wild-type protease was... The modified Bacillus subtilis ligase has basic ligase activity, and its amino acid sequence is shown in SEQ ID NO:2.

[0044] Subsequently, through multiple rounds of random mutagenesis and high-throughput screening of residues in the substrate binding pockets (especially the S1' and S2' pockets that recognize nucleophilic substrates), further mutations were performed to obtain the final mutant with high specificity and catalytic efficiency of para-tranexamic acid as a nucleophilic substrate: TXA-ligase, whose amino acid sequence is shown in SEQ ID NO:3. Based on the amino acid sequence of SEQ ID NO:2, it further includes N76D, L126A, G166S, Y216K, and N217S mutations.

[0045] Kinetic analyses were performed on the wild-type Bacillus subtilis protease BPN', the Bacillus subtilis ligase containing S220C and P224A mutations, and the TXA-ligase. N-succinyl-Ala-Ala-Pro-Phe-p-nitroaniline (Suc-AAPF-pNA) was used as the proteolysis substrate, and glycyl-L-histidine ethyl ester (Gly-His-OEt) and tranexamic acid (TXA) were used as the ligation reaction substrates. The initial reaction rates at different substrate concentrations were measured, and the Michaelis-Menten equation was used for fitting the kinetic parameters of each enzyme. The results are shown in Table 2.

[0046] Table 2 The results above indicate that wild-type subtilisin It possesses extremely high proteolytic activity but lacks ligation activity. After undergoing an S220C / P224A mutation, the resulting *Bacillus subtilis* ligase acquired ligation activity, but with low ligation efficiency. This invention further mutates the *Bacillus subtilis* ligase to obtain the TXA-ligase. Value decrease Value and The increased values ​​indicate that the affinity of TXA-ligase for the substrates glycyl-L-histidine ethyl ester and tranexamic acid is significantly enhanced, resulting in a significant increase in catalytic rate.

[0047] That is, the TXA-ligase obtained after mutation in this invention has high catalytic activity for the reaction substrates glycyl-L-histidine ethyl ester and tranexamic acid, and can efficiently catalyze the reaction of glycyl-L-histidine ethyl ester and tranexamic acid to generate the peptide ligand Gly-His-TXA.

[0048] Example 2 Preparation of the metal-peptide complex Gly-His-TXA-Cu The codons of the TXA-ligase gene obtained above were optimized to obtain the optimized TXA-ligase gene, as shown in SEQ ID NO:4.

[0049] The optimized TXA-ligase gene was cloned into the expression vector pPICZαA to obtain the recombinant expression vector pPICZαA-TXA. This expression vector pPICZαA contains an α-mating factor secretion signal peptide gene (for protein secretion), a methanol-induced strong promoter AOX1, and a Zeocin™ resistance gene (Shble gene) for screening positive transformants. Originally developed by Invitrogen™, this vector is now owned by Thermo Fisher Scientific and has the trade name Cat. No. V195-20.

[0050] The specific construction steps of the recombinant expression vector pPICZαA-TXA are as follows: 1. Preparation of the target gene fragment: A DNA fragment was synthesized from the whole gene using chemical methods. The fragment contains an XhoI restriction site at the 5' end, an optimized TXA-ligase gene sequence (SEQ ID NO:4), a translation stop codon (TAA), and a NotI restriction site at the 3' end.

[0051] 2. Enzyme digestion of the vector and gene fragment: The pPICZαA expression vector was double-digested with restriction endonucleases XhoI and NotI to obtain a linearized pPICZαA vector. Simultaneously, the synthesized target gene fragment was double-digested with the same restriction endonucleases XhoI and NotI to obtain a DNA fragment with matching sticky ends.

[0052] 3. Ligation reaction: The linearized pPICZαA vector and the target gene fragment, after enzyme digestion, were separated by agarose gel electrophoresis and purified using a gel extraction kit. Then, the two were ligated overnight at 16°C using T4 DNA ligase to construct a circular recombinant plasmid.

[0053] 4. Transformation and Screening: The ligation product was transformed into *E. coli* DH5α competent cells and plated on low-salt LB agar containing 100 µg / mL Zeocin™. The cells were incubated overnight at 37°C. Positive clones were selected, plasmids were extracted, and the correct insertion of the target gene and sequence were confirmed by PCR, restriction enzyme digestion, and DNA sequencing. This confirmed the successful construction of the recombinant expression vector. .

[0054] Verify the correct recombinant expression vector Linearized with SacI enzyme, the cells were transformed into competent Pichia pastoris cells (X-33 strain) via electroporation. The transformed cells were plated on YPD solid medium containing 1M sorbitol and incubated at 30°C for 24 hours. The bacterial growth on the plates was then transferred to plates containing different concentrations of sorbitol. High copy number screening was performed on YPD solid medium at concentrations of 100 µg / mL, 500 µg / mL, and 1000 µg / mL.

[0055] High-copy-count screening: After the Zeocin™ resistance gene in the expression vector is co-integrated with the target gene, Pichia pastoris strains can grow on plates containing high concentrations of antibiotics. The higher the copy number of the target gene integrated into the Pichia pastoris genome and the higher the expression level, the stronger the resistance to antibiotics. Therefore, selection is preferentially performed on plates containing 1000 µg / mL high concentrations of antibiotics. Single colonies growing on a plate.

[0056] Small-scale expression validation: The selected highly resistant colonies were inoculated into BMGY medium and cultured until OD500. 600 After reaching level 4, the culture medium was transferred to BMMY (containing methanol) for small-scale induction expression. After 72 hours of induction, the fermentation supernatant was collected, and the expression level of TXA-ligase was detected by SDS-PAGE. The strain with the highest enzyme secretion level was screened and named recombinant strain X-33 / pPICZαA-TXA, which was stored in glycerol tubes for subsequent large-scale fermentation.

[0057] The above-mentioned recombinant strain was fermented on a large scale using a standard Pichia pastoris high-density fermentation protocol. The specific fermentation process is as follows: Seed activation: The recombinant strain X-33 / pPICZαA-TXA preserved in glycerol tubes was streaked onto YPD solid medium and incubated at 30°C for 48 hours.

[0058] Primary seed culture: Select a single colony from the YPD solid medium and inoculate it into 50 mL of BMGY medium. Incubate at 30°C and 250 rpm for 24 hours. At this time, the recombinant strain will grow to the logarithmic growth phase, and the primary seed culture solution will be obtained.

[0059] High-density fermentation: The entire primary seed culture was inoculated into a 10L fermenter containing 5L of basal salt medium pre-added with 40 g / L glycerol. Fermentation was carried out at 30°C, pH 5.5 (automatically controlled by adding ammonia), and dissolved oxygen (DO) maintained above 20% to accumulate cell biomass. Once the glycerol was depleted, a 50% (w / v) glycerol solution was added at a rate of 18 mL / h / L for 4 hours of feed starvation.

[0060] Methanol-induced expression: Stop adding glycerol and start slowly adding pure methanol to activate the AOX1 promoter, causing TXA-ligase to begin expression and secretion into the fermentation broth. Maintain the methanol concentration in the culture medium at 1% (v / v) and continue induction culture, taking samples regularly to detect enzyme activity until the enzyme activity reaches the plateau phase.

[0061] After the TXA-ligase activity reached a plateau, glycyl-L-histidine ethyl ester and tranexamic acid were added to the fermenter. The reaction was carried out at 30°C and pH 7.5 for 12 hours to synthesize the peptide ligand Gly-His-TXA in situ. At the start of peptide ligand synthesis, the TXA-ligase activity in the fermentation broth was 2000 U / mL, and the concentrations of glycyl-L-histidine ethyl ester and tranexamic acid in the fermentation broth were both 50 mM.

[0062] After the synthesis of the peptide ligand Gly-His-TXA reached a plateau, a sterile aqueous solution of copper acetate was added to the fermenter. Under mild conditions of 30°C and pH 6.0, the reaction was continued with stirring for 1.5 hours, allowing the peptide ligand Gly-His-TXA to chelate with copper ions in the copper acetate, forming the metal-peptide complex Gly-His-TXA-Cu. At the initial stage of metal-peptide complex formation, the concentration of copper acetate in the fermentation broth was 45.7 mM, the concentration of the peptide ligand Gly-His-TXA was 48 mM, and the molar ratio of copper ions to peptide ligand Gly-His-TXA in the fermentation broth was 1:1.05.

[0063] After the chelation reaction, the fermentation broth was collected and centrifuged at 8000 x g for 20 minutes at 4°C using a high-speed refrigerated centrifuge to precipitate yeast cells and most of the insoluble matter. The supernatant was then aspirated and pre-filtered through a 0.45 µm polyethersulfone (PES) membrane to remove fine particles, followed by sterilization through a 0.22 µm sterile PES membrane. Finally, a clear, sterile blue liquid was collected, which is the liquid containing the metal peptide complex Gly-His-TXA-Cu.

[0064] Example 3 Efficacy evaluation experiment 1. Anti-inflammatory test Model: A lipopolysaccharide (LPS)-induced mouse macrophage (RAW 264.7) inflammation model was used.

[0065] Test method: Mouse macrophages were divided into 7 groups and seeded into culture plates. After 24 hours of adherent growth, the culture medium of each group was replaced with serum-free culture medium containing different test samples. After 2 hours of pre-incubation, LPS (except for the control group) was added and cultured for another 24 hours.

[0066] The test samples contained in the serum-free culture medium of each group of cells are as follows: Control group: Serum-free culture medium containing no other components and without the addition of LPS for induction.

[0067] LPS group: Serum-free culture medium containing no other components.

[0068] LPS+Gly-His-TXA-Cu group: serum-free culture medium containing the metal peptide complex Gly-His-TXA-Cu of the present invention, with a final concentration of 10µM.

[0069] LPS+Gly-His-TXA group: Serum-free culture medium containing peptide ligand Gly-His-TXA, with a final concentration of 10µM.

[0070] LPS+GHK-Cu group: serum-free culture medium containing GHK-Cu, with a final concentration of 10µM.

[0071] LPS+TXA group: Serum-free culture medium containing tranexamic acid (TXA) at a final concentration of 10 µM.

[0072] LPS+TXA+GHK-Cu group: The serum-free culture medium contained both GHK-Cu and TXA, with a final concentration of 10µM for both GHK-Cu and TXA.

[0073] The amount of NO produced in the culture medium was detected using the Griess kit. The results are shown in Table 3.

[0074] Table 3 The results above show that both the metal peptide complexes Gly-His-TXA-Cu and GHK-Cu significantly inhibited NO production, while the copper-free peptide ligands Gly-His-TXA and TXA had a weaker inhibitory effect on NO production. This indicates that copper ions and their coordination structures are the core structures for exerting anti-inflammatory effects.

[0075] Furthermore, the metal peptide complex Gly-His-TXA-Cu of the present invention exhibits the best inhibitory effect on NO production, which is superior to GHK-Cu alone and also superior to the LPS+TXA+GHK-Cu group containing both TXA and GHK-Cu. This indicates that by replacing lysine in GHK-Cu with TXA, the anti-inflammatory efficacy of the present invention is further improved.

[0076] Furthermore, GHK-Cu alone showed better inhibitory effects on NO than the LPS+TXA+GHK-Cu group containing both TXA and GHK-Cu, indicating that simply physically mixing GHK-Cu and TXA may actually affect the anti-inflammatory efficacy of GHK-Cu.

[0077] 2. Test to promote skin barrier repair function Model: Human immortalized keratinocytes (HaCaT) were stimulated with TNF-α / IFN-γ to simulate skin barrier damage under inflammatory conditions.

[0078] Test method: HaCaT cells were divided into 7 groups and cultured in DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics, respectively, and placed in a constant temperature cell culture incubator at 37℃ and 5% CO2 for routine culture. After 24 hours of routine culture, the culture medium of each group was replaced with DMEM high-glucose medium containing different test samples, pre-incubated for 2 hours, and then TNF-α / IFN-γ (except for the control group) was added and cultured for another 24 hours.

[0079] The test samples contained in each group of cell culture media are as follows: Control group: The culture medium contained no other components and no TNF-α / IFN-γ stimulation was added.

[0080] TNF-α / IFN-γ group: The culture medium does not contain other components.

[0081] TNF-α / IFN-γ+Gly-His-TXA-Cu group: The culture medium contained the metal peptide complex Gly-His-TXA-Cu of the present invention, with a final concentration of 10µM.

[0082] TNF-α / IFN-γ+Gly-His-TXA group: The culture medium contained the peptide ligand Gly-His-TXA, with a final concentration of 10µM.

[0083] TNF-α / IFN-γ+GHK-Cu group: The culture medium contained GHK-Cu, with a final concentration of 10µM.

[0084] TNF-α / IFN-γ+TXA group: The culture medium contained tranexamic acid (TXA) at a final concentration of 10 µM.

[0085] TNF-α / IFN-γ+TXA+GHK-Cu group: The culture medium contained both GHK-Cu and TXA, with a final concentration of 10µM for both GHK-Cu and TXA.

[0086] The expression level of filaggrin (FLG), a key barrier protein, was detected by Western blotting. The results are shown in Table 4.

[0087] Table 4 The results above indicate that: in inflammatory factors Under stimulation, the expression of FLG protein was significantly downregulated, and the metallopeptide complex... GHK-Cu significantly reversed this downregulation, restoring protein expression to near-normal levels. In contrast, the copper-free peptide ligands Gly-His-TXA and TXA showed little effect, indicating that copper ions and their coordination structures are crucial for promoting skin barrier repair.

[0088] Furthermore, the metal peptide complex of the present invention The reversal effect on the relative downregulation of FLG protein expression was the best, which was superior to GHK-Cu alone, and also superior to the TNF-α / IFN-γ+TXA+GHK-Cu group containing both TXA and GHK-Cu. This indicates that after replacing lysine in GHK-Cu with TXA, the repair function of the skin barrier was further improved.

[0089] Furthermore, GHK-Cu alone is more effective than GHK-Cu in reversing the relative downregulation of FLG protein expression than GHK-Cu containing both TXA and Cu. of Group, the explanation is simply to Physically mixing GHK-Cu with TXA can actually impair its skin barrier repair function.

[0090] 3. Test for inhibiting melanin production Model: Adopted melanocyte-stimulating hormone (MSH) A mouse melanoma cell (B16F10) melanin production model induced by α.

[0091] Test method: B16F10 cells were divided into 7 groups and cultured in DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics, respectively, and placed in a constant temperature cell culture incubator at 37℃ and 5% CO2 for routine culture. After 24 hours of routine culture, the culture medium of each group was replaced with DMEM high-glucose medium containing different test samples, pre-incubated for 2 hours, and then α-MSH (except for the control group) was added and cultured for another 24 hours.

[0092] The test samples contained in each group of cell culture media are as follows: Control group: The culture medium contained no other components and no α-MSH was added for induction.

[0093] α-MSH group: The culture medium does not contain any other components.

[0094] α-MSH+Gly-His-TXA-Cu group: The culture medium contained the metal peptide complex Gly-His-TXA-Cu of the present invention, with a final concentration of 10µM.

[0095] α-MSH+Gly-His-TXA group: The culture medium contained the peptide ligand Gly-His-TXA, with a final concentration of 10µM.

[0096] α-MSH+GHK-Cu group: The culture medium contained GHK-Cu, with a final concentration of 10µM.

[0097] α-MSH+TXA group: The culture medium contained tranexamic acid (TXA) at a final concentration of 10 µM.

[0098] α-MSH+TXA+GHK-Cu group: The culture medium contained both GHK-Cu and TXA, with a final concentration of 10µM for both GHK-Cu and TXA.

[0099] Intracellular melanin content was measured, and the melanin inhibition rate relative to the α-MSH group was calculated. The results are shown in Table 5.

[0100] Table 5 The results above show that both the metal peptide complex Gly-His-TXA-Cu and the peptide ligand Gly-His-TXA exhibit strong melanin inhibition capabilities, with significantly better effects than GHK-Cu without TXA. This indicates that the TXA portion of the metal peptide complex successfully endows it with powerful whitening functions.

[0101] Furthermore, the metal peptide complex Gly-His-TXA-Cu of the present invention exhibits a better inhibitory effect on melanin than TXA alone, and is also better than the α-MSH+GHK-Cu group containing both TXA and GHK-Cu. This indicates that by replacing lysine in GHK-Cu with TXA, the metal peptide complex obtained in the present invention further improves the inhibitory effect on melanin.

[0102] Example 4 A blemish-repairing serum containing the metal peptide complex Gly-His-TXA-Cu has the following formula as shown in Table 6.

[0103] Table 6 The preparation method of the above-mentioned serum is as follows: Mix all components of phase A according to the specified ratio and heat to 80℃ until dissolved and homogeneous. Mix all components of phase B according to the specified ratio until homogeneous. Add phase B to phase A at 400 rpm and stir until homogeneous. While stirring at 400 rpm, slowly add xanthan gum (phase C) at a rate of 0.1 g / s. After the xanthan gum is evenly dispersed, homogenize at 7000 rpm for 2 minutes to form a uniform light blue gel-like essence. Cool to room temperature and check the pH of the essence to be 6.0.

[0104] Example 5 Efficacy evaluation at the skin testing level 1. Safety evaluation: Human repeated injury patch test (HRIPT) was conducted to evaluate the skin irritation and sensitization of the blemish repair essence prepared in Example 4.

[0105] Subjects: Fifty healthy adult volunteers aged 18 to 65 years, including 25 with normal skin and 25 who self-reported as having sensitive skin, with no skin diseases, lesions, or pigmentation abnormalities at the test sites. The trial followed the standard protocol of the Human Repeatable Injury Patch Test (HRIPT).

[0106] Induction Phase: 0.2 mL of the test sample (the serum from Example 4) is placed on a occlusive patch (Finn Chamber®) and applied to one side of the subject's back. The patch is left in place for 24 hours before being removed by a professional for skin assessment. This process is repeated three times a week (Monday, Wednesday, and Friday) for three consecutive weeks, for a total of nine applications.

[0107] Rest Phase: After the final induction patch is applied, the subject enters a 2-week rest phase during which no test sample is touched.

[0108] Challenge Phase: After the resting phase, a new patch containing the test sample is applied to a fresh skin location on the other side of the subject's back that has not been in contact with the sample, and left in place for 24 hours.

[0109] Outcome assessment: At 24 and 48 hours after the removal of the irritation patch, a dermatologist assessed and scored the skin response at the irritation site according to the following international standards: 0 points: No visible reaction; 1 point: Mild, patchy erythema; 2 points: Moderate, confluent erythema; 3 points: Severe erythema with edema; 4 points: Severe erythema with blisters or erosions.

[0110] The results are shown in Table 7.

[0111] Table 7: Summary of Human Repeated Injury Patch Test (HRIPT) Results Based on the HRIPT results of this study, which included 50 subjects (50% of whom had sensitive skin), it is demonstrated that the blemish repair essence prepared in Example 4 is non-irritating and non-sensitizing to human skin under the test conditions and has excellent skin compatibility.

[0112] 2. Evaluation of human efficacy: Subjects: 30 healthy subjects aged 18 to 40 years with post-inflammatory hyperpigmentation (PIH) (acne scars) on their face.

[0113] Trial Design: A randomized, double-blind, self-controlled split-face design was employed. Each participant's left and right sides were randomly assigned to receive either the test product or a placebo. The test product was the blemish-repairing serum from Example 4, while the placebo was a matrix containing no metal peptide complex of this invention, but with all other ingredients identical to the serum from Example 4.

[0114] Product Usage: After cleansing their face morning and evening, subjects should apply the designated product (test product or placebo) to one side of their assigned face, using 0.25 mL each time. Subjects should be instructed to use the designated gentle cleanser and sunscreen during the trial and to avoid using other functional skincare products.

[0115] Efficacy assessment: Before the start of the trial (D0), after 4 weeks of use (D28), and after 8 weeks of use (D56), professionals measured acne scars on the same area of ​​the subjects' faces using skin testing instruments in a temperature- and humidity-controlled laboratory.

[0116] Instrument measurements: The L value (lightness) and a value (redness) of the skin in the acne scar area were measured using a Konica Minolta chromameter. The melanin index and erythroglobulin index were measured using a Mexameter® MX 18. The results are shown in Table 8.

[0117] Table 8: Changes in skin parameters in acne scar areas after 8 weeks of use (D56 vs D0) Note: Data are expressed as mean ± standard deviation. Positive values ​​indicate an increase in the parameter, and negative values ​​indicate a decrease in the parameter. p < 0.05 indicates that the difference is statistically significant.

[0118] The results above indicate that, after an 8-week self-comparison test of the left and right sides of the face, compared with the placebo, the skin on one side of the face treated with the blemish-fading and repairing essence containing the metal peptide complex of this invention showed the following effects: Skin tone is noticeably brighter: The value (brightness) increased significantly, indicating that the darkening of the acne scar area has been significantly improved.

[0119] Red acne marks have significantly faded: The values ​​(redness) and hemoglobin index were significantly reduced, indicating that the repair serum can effectively improve post-inflammatory redness.

[0120] Brown acne marks are significantly faded: The melanin index is significantly reduced, indicating that the product can effectively inhibit and lighten existing pigmentation.

[0121] In summary, the product of this invention can comprehensively and significantly improve post-inflammatory hyperpigmentation (acne scars) through the synergistic effects of three major pathways: anti-inflammatory, repair, and whitening.

[0122] 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 metallopeptide complex, characterized in that, The metal peptide complex is formed by coordination of a peptide ligand with a divalent copper ion, and the structural formula of the metal peptide complex is shown as formula (I): Formula (I).

2. The metallopeptide complex of claim 1, wherein, The structural formula of the peptide ligand is shown as formula (II): Formula (II).

3. A method for preparing a metallopeptide complex according to claim 1 or 2, characterized in that, The method comprises the following steps: The peptide ligand is generated by using TXA-ligase to catalyze the reaction of glycyl-L-histidine ethyl ester and tranexamic acid; The peptide ligand is mixed with a soluble divalent copper salt in an aqueous medium to generate the metal peptide complex.

4. The production method according to claim 3, characterized by, The peptide ligand is generated by using TXA-ligase to catalyze the reaction of glycyl-L-histidine ethyl ester and tranexamic acid; The fermentation broth containing TXA-ligase is obtained by fermenting a recombinant strain containing a gene encoding TXA-ligase, and after the enzyme activity reaches a plateau, the fermentation broth containing TXA-ligase is obtained; The fermentation broth containing the peptide ligand Gly-His-TXA is obtained by adding substrates glycyl-L-histidine ethyl ester and tranexamic acid to the fermentation broth containing TXA-ligase; wherein the concentration of the glycyl-L-histidine ethyl ester in the fermentation broth is 10mM-100mM, and the concentration of the tranexamic acid is 10mM-100mM.

5. The preparation method according to claim 3, characterized in that, The peptide ligand is mixed with a soluble divalent copper salt in an aqueous medium to generate the metal peptide complex. The fermentation broth containing the metal peptide complex is obtained by adding the soluble divalent copper salt to the fermentation broth containing the peptide ligand Gly-His-TXA, and removing the recombinant strain to obtain the supernatant containing the metal peptide complex; wherein the molar ratio of the peptide ligand Gly-His-TXA to copper ions in the soluble divalent copper salt is (1-1.3):

1.

6. The method of any one of claims 3 to 5, wherein the compound is prepared by the method comprising the steps of: The amino acid sequence of the TXA-ligase is shown as SEQ ID NO:

3.

7. The method of any one of claims 3 to 5, wherein the compound is prepared by the method of any one of claims 1 to 2. The soluble divalent copper salt includes copper acetate or copper sulfate.

8. The preparation method according to any one of claims 3 to 5, characterized in that, The reaction temperature for preparing the fermentation broth containing the peptide ligand Gly-His-TXA is 25-40℃, and the pH value is 7.0-8.5; and / or The reaction temperature for preparing the metal peptide complex is 25-45℃, and the pH value is 5.5-6.

5.

9. A cosmetic composition characterized in that, The cosmetic composition contains the metal peptide complex of claim 1 or 2, or the metal peptide complex prepared by the preparation method of any one of claims 3-8, and one or more cosmetically acceptable carriers or excipients.

10. Use of the metal peptide complex of claim 1 or 2 or the cosmetic composition of claim 9 in the preparation of a product for improving skin pigmentation.

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