Synthesis method of GHK-CU

Abstract

The invention belongs to the technical field of polypeptide synthesis, and particularly relates to a synthesis method of GHK-CU. According to the synthesis method of the GHK-CU (blue copper peptide), the raw materials are pretreated, the reaction activity of the raw materials is improved, the reaction system is improved, the specially-made functionalized ionic liquid is added into the reaction system, and the selectivity and efficiency of the reaction can be remarkably improved by adding the ionic liquid; according to the method, the reaction is promoted by means of microwaves, ultrasonic waves and the like during the reaction, and local overheating or side reaction is avoided, so that the yield and the purity of the product are improved, and the synthesized blue copper peptide has very good effects of tightening, wrinkle resistance, wound healing and the like, and has very good application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of polypeptide synthesis, and particularly relates to a method for synthesizing GHK-CU. Background Art

[0002] GHK-Cu (blue copper peptide) is a bioactive tripeptide composed of a glycyl-histidyl-lysine tripeptide bound to a divalent copper ion. It occurs naturally in human blood, saliva, and urine. At the age of 20, the blood concentration of GHK is 200 ng / mL. By the age of 60, this concentration drops to 80 ng / mL due to a significant decrease in organ regeneration. In 1973, Pickart first isolated GHK-Cu from human plasma and found that the copper peptide was highly effective in treating wounds and skin damage. In 1986, ProCyte Corporation was founded in Washington, D.C., USA, dedicated to applying copper-bonded amino acids to therapies for tissue repair, anti-aging, wrinkle reduction, and hair growth. In 1999, the Neova brand was founded, officially introducing blue copper in anti-aging and wrinkle-reducing beauty products. This groundbreaking GHK-Cu technology, patented globally, boosts skin vitality without damaging or irritating the skin. It gradually restores lost collagen, tightens and strengthens subcutaneous tissue, and accelerates wound healing, ultimately achieving the desired wrinkle and anti-aging effects. GHK and its copper complex act as activators of tissue remodeling and signaling peptides, effectively promoting collagen and elastin production, increasing blood vessel growth and antioxidant capacity, and stimulating the production of glucosamines (GAGs), helping the skin restore its self-repair capabilities. GHK-Cu is also a key factor in the body's ability to combat inflammation in injured tissues, playing a crucial role in stimulating the production of normal proteins to replace damaged ones. Blue copper peptides have multiple functions, acting as both a messenger and a transporter. Furthermore, their gentleness, non-irritation, and safety make them highly valuable for topical skincare products.

[0003] The existing GHK-Cu synthesis technology has problems such as the traditional solvent system causing uneven dispersion of copper ions, forming metal clusters that affect the coordination reaction; poor thermodynamic control reaction selectivity, a large proportion of by-products, and conventional heating methods causing local overheating, triggering amino acid racemization reactions, which seriously limit the promotion and application of blue copper peptides.

[0004] Based on this, we proposed a synthesis method of GHK-CU, hoping to solve the shortcomings of the existing technology. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for synthesizing GHK-CU in response to the existing problems.

[0006] The present invention is achieved through the following technical solutions: A method for synthesizing GHK-CU comprises the following steps: S1. Glycine, histidine, and lysine are dissolved in an ethanol-water mixed solvent at a molar ratio of 1:1:1, and then subjected to a CO2 expansion fluid treatment for 30-40 minutes to precipitate crystals, which are then freeze-dried to obtain amino acid powder; S2. Prepare 0.1M copper sulfate solution and 0.2M reducing agent, add the prepared copper sulfate solution and reducing agent into the microchannel reactor simultaneously, react at 30-50°C and a flow rate of 10mL / min, and collect the Cu(OH)2@SiO2 nanoparticle suspension after the reaction is completed; S3, melt-reacting 1-butyl-3-methylimidazolium chloride and aspartic acid-modified β-cyclodextrin at a molar ratio of 1:1-2 at 80-90° C. to obtain a functionalized ionic liquid, then adding amino acid powder and Cu(OH)2@SiO2 nanoparticle suspension to the ionic liquid, stirring and mixing, adjusting the pH to 5-6, and then adding the mixture to a microwave-ultrasonic composite reactor for reaction; S4. After the reaction is completed, the mixture is cooled to room temperature, acetone is added to precipitate the ionic liquid, and the supernatant is collected by centrifugation. The supernatant is filtered and chromatographed on a reverse C18 column to collect the elution peak of the blue copper peptide, and the mixture is lyophilized.

[0007] Furthermore, the volume ratio of ethanol to water in the ethanol-water mixed solvent described in step S1 is 1:4-5; When treating CO2 expansion fluid, the set pressure is 15~20MPa, the temperature is 40~45℃, and the CO2 flow rate is 400~500mL / min.

[0008] Furthermore, the reducing agent in step S2 is ascorbic acid, and the reducing agent contains tetraethyl orthosilicate, which reacts with Cu in the reaction system. 2+ The molar ratio is 2~3:1; The inner diameter of the microchannel reactor is 500 μm.

[0009] Furthermore, the preparation of aspartic acid-modified β-cyclodextrin described in step S3 comprises the following steps: (1) Dissolve β-cyclodextrin in dimethyl sulfoxide, add sodium hydroxide and stir to mix, then add propargyl bromide dropwise and react at room temperature for 20-26 hours. After the reaction is completed, pour into ice water to precipitate, filter, wash with deionized water 4-6 times, and dry to obtain pretreated β-cyclodextrin; (2) Dissolve polyaspartic acid in dichloromethane, add N,N-dicyclohexylcarbodiimide at a molar amount of 1.3 to 1.4 times that of polyaspartic acid, stir and mix, then add sodium azide at a molar amount of 1 to 2 times that of polyaspartic acid, react at room temperature for 16 to 18 hours, filter after the reaction, concentrate the filtrate, precipitate with ether, filter and dry to obtain pretreated polyaspartic acid; (3) Pretreated β-cyclodextrin and aspartic acid were added to phosphate buffer at a molar ratio of 1:1~2, the pH was adjusted to 6~8, transglutaminase was added, and the mixture was stirred at 30~40°C for 8~10 hours. Pretreated aspartic acid was added and stirred, and copper sulfate and sodium ascorbate were added. The mixture was stirred at room temperature for 16~20 hours. After the reaction was completed, the unreacted raw materials were removed by chromatography to obtain aspartic acid-modified β-cyclodextrin.

[0010] Furthermore, the molar ratio of β-cyclodextrin to propargyl bromide in step (1) is 1:1-3; The amount of sodium hydroxide added is 0.6~1% of the mass of β-cyclodextrin.

[0011] Furthermore, the amount of transglutaminase added in step (3) is 3-4% of the total mass of β-cyclodextrin and aspartic acid; The molar ratio of pretreated β-cyclodextrin to pretreated aspartic acid is 1:1.3~1.7; The concentration of copper sulfate is 0.03~0.07mM, and the concentration of sodium ascorbate is 0.3~0.7mM.

[0012] Furthermore, when the amino acid powder and Cu(OH)2@SiO2 nanoparticle suspension are added to the ionic liquid in step S3, the total concentration of the amino acid is controlled to be 0.5~1M, and the Cu(OH)2@SiO2 nanoparticle suspension is controlled to be 0.5~1M. 2+ The molar ratio of amino acid is 1:1.2~1.4.

[0013] Furthermore, when the reaction is carried out in the microwave-ultrasonic composite reactor described in step S3, the microwave parameters are: dual-frequency alternating irradiation, 2.45 GHz and 5.8 GHz for 10 s each, and the initial power density is 50 W / cm 2 , increase by 50W / cm every 5 minutes 2 Up to 200W / cm 2 ; Ultrasonic parameters: frequency of 20-100 kHz, pulse mode of 30 s on and 10 s off.

[0014] Furthermore, the supernatant in step S4 is filtered using an ultrafiltration membrane with a pore size of 10 kDa; The mobile phase for reversed-phase C18 column chromatography was 2:8 acetonitrile and water containing 0.1% trifluoroacetic acid.

[0015] Compared with the prior art, the present invention has the following advantages: 1. The present invention provides a method for synthesizing GHK-CU (blue copper peptide). By pretreating the raw materials and improving the reaction system, the selectivity and efficiency of the reaction are significantly improved. During the reaction, microwaves, ultrasound and other means are used to promote the reaction while avoiding local overheating or side reactions, thereby improving the yield and purity of the product. The synthesized blue copper peptide has excellent firming, anti-wrinkle and wound healing effects.

[0016] 2. The present invention uses CO2 expansion fluid technology to recrystallize glycine, histidine, and lysine into small-sized crystals with a D90 less than 10 μm, thereby increasing their specific surface area, improving dissolution rate, and reaction activity. Copper sulfate reacts with a reducing agent in a microchannel reactor to generate 20-50 nm Cu(OH)2@SiO2 core-shell nanoparticles. The SiO2 shell can control the Cu 2+ The slow release rate can be improved to avoid agglomeration, thereby improving the activity and uniform dispersion of copper ions in subsequent reactions, thereby improving the reaction efficiency.

[0017] 3. The present invention adds a specially prepared functionalized ionic liquid to the reaction system. The cation of the ionic liquid (1-butyl-3-methylimidazole) provides a polar environment, and the anion (aspartic acid-modified β-cyclodextrin) has triple functions. The cavity of aspartic acid-modified β-cyclodextrin includes amino acids through molecular recognition and directional arrangement of their functional groups; the carboxyl group of aspartic acid and Cu 2+ Pre-coordination forms a copper ion-carboxyl intermediate, reducing the activation energy of the coordination reaction; aspartic acid-modified β-cyclodextrin can form a complex with the blue copper peptide, increasing the solubility of the blue copper peptide in the reaction system, enabling it to be better dispersed in the solution and fully contact with other reactants, which is conducive to the reaction.

[0018] 4. The present invention uses microwaves and ultrasound in combination, and microwave dual-frequency irradiation (2.45GHz and 5.8GHz alternately) generates a high-frequency alternating electromagnetic field to accelerate molecular motion and promote collision coordination between amino acids and copper ions. The power density gradient control (50~300W / cm 2 ), realize temperature gradient regulation, and at the same time use the effect of ultrasound to break the shell of nanoparticles and release Cu 2+ , further improving the efficiency of the reaction. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the phenotype diagram of the efficacy experiment of 0.5ppm blue copper peptide; Figure 2This is the phenotype diagram of the efficacy experiment of 20ppm blue copper peptide; Figure 3 This is a comparison chart of the elastase inhibition rate between the sample group and the blank group. DETAILED DESCRIPTION

[0020] In order to further explain the present invention, it is described below with reference to the following specific embodiments.

[0021] Example 1 A method for synthesizing GHK-CU comprises the following steps: S1. Glycine, histidine, and lysine were dissolved in an ethanol-water mixed solvent (the volume ratio of ethanol to water was 1:4) at a molar ratio of 1:1:1. The mixture was then subjected to a CO2 expansion fluid treatment at a set pressure of 15 MPa, a temperature of 40°C, and a CO2 flow rate of 400 mL / min. Crystals were precipitated after 30 minutes and freeze-dried to obtain amino acid powder. S2. Prepare 0.1M copper sulfate solution and 0.2M reducing agent, add the prepared copper sulfate solution and reducing agent simultaneously into a microchannel reactor (inner diameter 500μm), react at 30℃ and flow rate 10mL / min, and collect the Cu(OH)2@SiO2 nanoparticle suspension after the reaction. The reducing agent is ascorbic acid, which contains tetraethyl orthosilicate. Tetraethyl orthosilicate reacts with Cu in the reaction system. 2+ The molar ratio is 2:1; S3, 1-butyl-3-methylimidazolium chloride and aspartic acid modified β-cyclodextrin were melt-reacted at 80 ° C in a molar ratio of 1:1 to obtain a functionalized ionic liquid, and then amino acid powder and Cu(OH)2@SiO2 nanoparticle suspension were added to the ionic liquid to control the total concentration of amino acid to 0.5 M and Cu(OH)2@SiO ... 2+ The molar ratio of the amino acid is 1:1.2, the mixture is stirred and the pH is adjusted to 5, and then added into a microwave-ultrasonic composite reactor for reaction; Microwave parameters: Dual-frequency alternating irradiation, 2.45 GHz and 5.8 GHz, 10 s each cycle, initial power density 50 W / cm 2 , increase by 50W / cm every 5 minutes 2 Up to 200W / cm 2 ; Ultrasonic parameters: frequency of 20 kHz, pulse mode of 30 s on and 10 s off; The preparation of the aspartic acid-modified β-cyclodextrin comprises the following steps: (1) β-cyclodextrin was dissolved in dimethyl sulfoxide, sodium hydroxide was added and stirred, and propargyl bromide was added dropwise. The mixture was reacted at room temperature for 20 h. After the reaction, the mixture was poured into ice water to precipitate. The precipitate was filtered, washed with deionized water for 4 times, and dried to obtain the pretreated β-cyclodextrin. The molar ratio of β-cyclodextrin to propargyl bromide is 1:1; The amount of sodium hydroxide added was 0.6% of the mass of β-cyclodextrin; (2) Dissolve polyaspartic acid in dichloromethane, add N,N-dicyclohexylcarbodiimide (1.3 times the molar amount of polyaspartic acid), stir and mix, then add sodium azide (1 times the molar amount of polyaspartic acid), react at room temperature for 16 hours. After the reaction is completed, filter, concentrate the filtrate, precipitate with ether, filter and dry to obtain pretreated polyaspartic acid; (3) Pretreated β-cyclodextrin and aspartic acid were added to phosphate buffer at a molar ratio of 1:1, the pH was adjusted to 6, transglutaminase was added, and the mixture was stirred at 30°C for 8 h. Pretreated aspartic acid was added and stirred until uniformly mixed. Copper sulfate and sodium ascorbate were added and the mixture was stirred at room temperature for 16 h. After the reaction was completed, the unreacted raw materials were removed by chromatography to obtain aspartic acid-modified β-cyclodextrin. The amount of transglutaminase added is 3% of the total mass of β-cyclodextrin and aspartic acid; The molar ratio of pretreated β-cyclodextrin to pretreated aspartic acid was 1:1.3; The concentration of copper sulfate was 0.03 mM, and the concentration of sodium ascorbate was 0.3 mM; S4. After the reaction is completed, cool to room temperature, add acetone to precipitate the ionic liquid, collect the supernatant by centrifugation, filter the supernatant using an ultrafiltration membrane with a pore size of 10 kDa, and then pass it through reverse C18 column chromatography (the mobile phase is acetonitrile and water in a ratio of 2:8, containing 0.1% trifluoroacetic acid), collect the blue copper peptide elution peak, and freeze-dry.

[0022] Example 2 A method for synthesizing GHK-CU comprises the following steps: S1. Glycine, histidine, and lysine were dissolved in an ethanol-water mixed solvent (volume ratio of ethanol to water was 1:4.5) at a molar ratio of 1:1:1. The mixture was then subjected to a CO2 expansion fluid treatment at a set pressure of 17 MPa, a temperature of 43°C, and a CO2 flow rate of 450 mL / min. Crystals were precipitated after 35 minutes and lyophilized to obtain amino acid powder. S2. Prepare 0.1M copper sulfate solution and 0.2M reducing agent, add the prepared copper sulfate solution and reducing agent simultaneously into a microchannel reactor (inner diameter 500μm), react at 40℃ and flow rate 10mL / min, and collect the Cu(OH)2@SiO2 nanoparticle suspension after the reaction. The reducing agent is ascorbic acid, which contains tetraethyl orthosilicate. Tetraethyl orthosilicate reacts with Cu in the reaction system. 2+ The molar ratio is 2.5:1; S3, 1-butyl-3-methylimidazolium chloride and aspartic acid modified β-cyclodextrin were melt-reacted at 85 ° C in a molar ratio of 1:1.5 to obtain a functionalized ionic liquid, and then amino acid powder and Cu(OH)2@SiO2 nanoparticle suspension were added to the ionic liquid to control the total concentration of amino acid to 0.7 M and Cu(OH)2@SiO ... 2+ The molar ratio of the amino acid is 1:1.3, the pH is adjusted to 5.5 after stirring and mixing, and then added to a microwave-ultrasonic composite reactor for reaction; Microwave parameters: Dual-frequency alternating irradiation, 2.45 GHz and 5.8 GHz, 10 s each cycle, initial power density 50 W / cm 2 , increase by 50W / cm every 5 minutes 2 Up to 200W / cm 2 ; Ultrasonic parameters: frequency of 60 kHz, pulse mode of 30 s on and 10 s off; The preparation of the aspartic acid-modified β-cyclodextrin comprises the following steps: (1) β-cyclodextrin was dissolved in dimethyl sulfoxide, sodium hydroxide was added and stirred, and propargyl bromide was added dropwise. The mixture was reacted at room temperature for 23 hours. After the reaction, the mixture was poured into ice water to precipitate. The precipitate was filtered, washed with deionized water for 5 times, and dried to obtain the pretreated β-cyclodextrin. The molar ratio of β-cyclodextrin to propargyl bromide is 1:2; The amount of sodium hydroxide added was 0.8% of the mass of β-cyclodextrin; (2) Dissolve polyaspartic acid in dichloromethane, add N,N-dicyclohexylcarbodiimide (1.35 times the molar amount of polyaspartic acid), stir and mix, then add sodium azide (1.5 times the molar amount of polyaspartic acid), react at room temperature for 17 hours. After the reaction is completed, filter, concentrate the filtrate, precipitate with ether, filter and dry to obtain pretreated polyaspartic acid; (3) Pretreated β-cyclodextrin and aspartic acid were added to phosphate buffer at a molar ratio of 1:1.5, the pH was adjusted to 7, transglutaminase was added, and the mixture was stirred at 35°C for 9 hours. Pretreated aspartic acid was added and stirred until uniformly mixed. Copper sulfate and sodium ascorbate were added and the mixture was stirred at room temperature for 18 hours. After the reaction was completed, the unreacted raw materials were removed by chromatography to obtain aspartic acid-modified β-cyclodextrin. The amount of transglutaminase added is 3.5% of the total mass of β-cyclodextrin and aspartic acid; The molar ratio of pretreated β-cyclodextrin to pretreated aspartic acid was 1:1.5; The concentration of copper sulfate was 0.05 mM, and the concentration of sodium ascorbate was 0.5 mM; S4. After the reaction is completed, cool to room temperature, add acetone to precipitate the ionic liquid, collect the supernatant by centrifugation, filter the supernatant using an ultrafiltration membrane with a pore size of 10 kDa, and then pass it through reverse C18 column chromatography (the mobile phase is acetonitrile and water in a ratio of 2:8, containing 0.1% trifluoroacetic acid), collect the blue copper peptide elution peak, and freeze-dry.

[0023] Example 3 A method for synthesizing GHK-CU comprises the following steps: S1. Glycine, histidine, and lysine were dissolved in an ethanol-water mixed solvent (the volume ratio of ethanol to water was 1:5) at a molar ratio of 1:1:1. The mixture was then subjected to a CO2 expansion fluid treatment at a set pressure of 20 MPa, a temperature of 45°C, and a CO2 flow rate of 500 mL / min. Crystals were precipitated after 40 minutes and freeze-dried to obtain amino acid powder. S2. Prepare 0.1M copper sulfate solution and 0.2M reducing agent, add the prepared copper sulfate solution and reducing agent simultaneously into a microchannel reactor (inner diameter 500μm), react at 50℃ and flow rate 10mL / min, and collect the Cu(OH)2@SiO2 nanoparticle suspension after the reaction is completed; The reducing agent is ascorbic acid, which contains tetraethyl orthosilicate. Tetraethyl orthosilicate reacts with Cu in the reaction system. 2+ The molar ratio is 3:1; S3, 1-butyl-3-methylimidazolium chloride and aspartic acid modified β-cyclodextrin were melt-reacted at 90 ° C in a molar ratio of 1:2 to obtain a functionalized ionic liquid, and then amino acid powder and Cu(OH)2@SiO2 nanoparticle suspension were added to the ionic liquid to control the total concentration of amino acid to 1M and Cu(OH)2@SiO ... 2+ The molar ratio of the amino acid is 1:1.4, the mixture is stirred and the pH is adjusted to 6, and then added into a microwave-ultrasonic composite reactor for reaction; Microwave parameters: Dual-frequency alternating irradiation, 2.45 GHz and 5.8 GHz, 10 s each cycle, initial power density 50 W / cm 2 , increase by 50W / cm every 5 minutes 2 Up to 200W / cm 2 ; Ultrasonic parameters: frequency was 100 kHz, and the treatment was performed in a pulse mode of 30 s on and 10 s off; The preparation of the aspartic acid-modified β-cyclodextrin comprises the following steps: (1) β-cyclodextrin was dissolved in dimethyl sulfoxide, sodium hydroxide was added and stirred, and propargyl bromide was added dropwise. The mixture was reacted at room temperature for 26 hours. After the reaction, the mixture was poured into ice water to precipitate the precipitate, filtered, washed with deionized water for 6 times, and dried to obtain the pretreated β-cyclodextrin. The molar ratio of β-cyclodextrin to propargyl bromide is 1:3; The amount of sodium hydroxide added was 1% of the mass of β-cyclodextrin; (2) Dissolve polyaspartic acid in dichloromethane, add N,N-dicyclohexylcarbodiimide (1.4 times the molar amount of polyaspartic acid), stir and mix, then add sodium azide (2 times the molar amount of polyaspartic acid), react at room temperature for 18 hours. After the reaction is completed, filter, concentrate the filtrate, precipitate with ether, filter and dry to obtain pretreated polyaspartic acid; (3) Pretreated β-cyclodextrin and aspartic acid were added to phosphate buffer at a molar ratio of 1:2, the pH was adjusted to 8, transglutaminase was added, and the mixture was stirred at 40°C for 10 h. Pretreated aspartic acid was added and stirred until uniformly mixed. Copper sulfate and sodium ascorbate were added and the mixture was stirred at room temperature for 20 h. After the reaction was completed, the unreacted raw materials were removed by chromatography to obtain aspartic acid-modified β-cyclodextrin. The amount of transglutaminase added is 4% of the total mass of β-cyclodextrin and aspartic acid; The molar ratio of pretreated β-cyclodextrin to pretreated aspartic acid was 1:1.7; The concentration of copper sulfate was 0.07 mM, and the concentration of sodium ascorbate was 0.7 mM; S4. After the reaction is completed, cool to room temperature, add acetone to precipitate the ionic liquid, collect the supernatant by centrifugation, filter the supernatant using an ultrafiltration membrane with a pore size of 10 kDa, and then pass it through reverse C18 column chromatography (the mobile phase is acetonitrile and water in a ratio of 2:8, containing 0.1% trifluoroacetic acid), collect the blue copper peptide elution peak, and freeze-dry.

[0024] Comparative Example 1 On the basis of Example 2, CO2 expansion fluid treatment is not performed in step S1, and glycine, histidine and lysine are directly freeze-dried according to a molar ratio of 1:1:1. The rest of the technical scheme is consistent with the technical scheme of Example 2.

[0025] Comparative Example 2 On the basis of Example 2, the aspartic acid-modified β-cyclodextrin in step S3 was replaced with untreated β-cyclodextrin, and the rest of the technical scheme was consistent with that of Example 2.

[0026] Comparative Example 3 On the basis of Example 2, the step of adding the mixture into the microwave-ultrasonic composite reactor for reaction in step S3 is omitted and replaced by direct stirring treatment. The rest of the technical scheme is consistent with the technical scheme of Example 2.

[0027] 1. Yield and purity test Blue copper peptides were prepared using the methods of Examples 1 to 3 and Comparative Examples 1 to 3, respectively, and then characterized using HPLC. The experimental results are shown in Table 1 below.

[0028] Table 1

[0029] As can be seen from Table 1 above, the present invention significantly improves the yield and purity and shortens the reaction time through raw material activation, ionic liquid directional coordination, and energy field synergy.

[0030] 2. Blue copper peptide zebrafish maintenance effect Experimental system: Wild-type AB zebrafish.

[0031] Zebrafish age: 3 days post fertilization (3 dpf).

[0032] The sample size of each experimental group was 15 (N=10).

[0033] Adult fish rearing and breeding methods: The methods are in accordance with the applicant's laboratory standard rearing and breeding methods, which meet the requirements of the international AAALAC accreditation (certification number: 001458).

[0034] Testing agency: Guangzhou Huante Zhiyu Youjian Biotechnology Co., Ltd.

[0035] Zebrafish tail fin regeneration is divided into three processes: wound healing, blastema formation, and regeneration outcome, with blastema formation being the most core. Fin regeneration cells come from multiple sources, including epidermal cells, fibroblasts, and osteoblasts, all of which contribute to tail fin regeneration, and these cells are highly lineage-restricted. The regeneration of the zebrafish tail fin is similar to the repair mechanism of human skin, bones, and blood vessels. The zebrafish tail fin has become an important model for studying tissue regeneration processes due to its simple structure, ease of surgical operation, no impact on survival after surgery, and ease of observation. The tail fin is cut off perpendicular to the torso with a scalpel, and the repair efficacy of the sample is evaluated by quantifying the area of the regenerated zebrafish tail fin.

[0036] Experimental methods (1) Use a scalpel to remove the zebrafish tail fin to establish a zebrafish tail fin injury model; (2) Model zebrafish were randomly assigned to 6-well plates, with 15 zebrafish per well; (3) Water-soluble administration sample (Example 2), normal control group and model control group were set up at the same time, and the volume of each well was 3 mL; (4) Incubate in the dark at 28°C for 48 h; (5) Ten zebrafish were randomly selected from each experimental group and photographed under a dissecting microscope. Advanced image processing software was used to analyze and collect data. The tail fin area (A) of the zebrafish was analyzed and the formula was used to calculate and determine whether the sample had a repair effect.

[0037]

[0038] Statistical analysis: P < 0.05 was considered to be a significant difference.

[0039] The test results are shown in Table 2 and Figure 1 、 Figure 2 shown.

[0040] Table 2

[0041] From Table 1 and Figure 1 、 Figure 2 It can be seen that the tail fin area of the 0.5ppm and 20ppm blue copper peptide groups was significantly increased compared with the model control group, revealing that the sample has a repair effect.

[0042] 2. Anti-wrinkle and firming effects (elastase inhibition rate test) Experimental methods (1) Add 0.2 mL of the sample solution (Example 2) of the same concentration to the sample tube and the sample background, add 1.0 mL of pH 8.8 boric acid buffer to the sample background, add 0.2 mL of pH 8.8 boric acid buffer to the enzyme reaction tube, and add 1.0 mL of pH 8.8 boric acid buffer to the solvent background.

[0043] (2) Add 10 mg of orcein-elastin and 1.0 mL of elastase to the sample tube and enzyme reaction tube, respectively. Mix the mixture thoroughly and shake it in a constant temperature water bath for 20 minutes.

[0044] (3) Add 2 mL of pH 6.0 phosphate buffer to each of the sample tube, sample background, enzyme reaction tube, and solvent background. Then, add an equal amount of a mixture of pH 8.8 boric acid buffer and pH 6.0 phosphate buffer to make the volume 5 mL. Centrifuge for 5 minutes and collect 3 mL of the supernatant.

[0045] (4) Transfer each reaction solution into a 1 cm cuvette and measure the absorbance at 590 nm.

[0046] The experimental results are shown in Table 3 and Figure 3 shown.

[0047] Table 3

[0048] Note: The significance analysis of the sample group and the blank group was statistically significant when P < 0.05. The P value of the comparison between the two was 0.000, indicating a significant difference.

[0049] From Table 2 and Figure 3 It can be seen that under the experimental conditions, the elastase inhibition rate of the sample tripeptide-1 copper was tested. When the sample concentration was 0.1%, 0.2%, and 0.3%, the elastase inhibition rate was 6.95%, 11.47%, and 22.78%, respectively. Compared with the blank group, the elastase inhibition rate of each concentration in the sample group was better than that of the blank group, and the difference was significant (P < 0.05). This shows that tripeptide-1 copper has an inhibitory effect on elastase under the experimental conditions and has certain anti-wrinkle and firming effects.

[0050] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. A method for synthesizing GHK-CU, characterized in that: The steps include: S1. Glycine, histidine, and lysine are dissolved in an ethanol-water mixed solvent at a molar ratio of 1:1:1, and then subjected to a CO2 expansion fluid treatment for 30-40 minutes to precipitate crystals, which are then freeze-dried to obtain amino acid powder; S2. Prepare 0.1M copper sulfate solution and 0.2M reducing agent, add the prepared copper sulfate solution and reducing agent into the microchannel reactor simultaneously, react at 30-50°C and a flow rate of 10mL / min, and collect the Cu(OH)2@SiO2 nanoparticle suspension after the reaction is completed; S3, melt-reacting 1-butyl-3-methylimidazolium chloride and aspartic acid-modified β-cyclodextrin at a molar ratio of 1:1-2 at 80-90° C. to obtain a functionalized ionic liquid, then adding amino acid powder and Cu(OH)2@SiO2 nanoparticle suspension to the ionic liquid, stirring and mixing, adjusting the pH to 5-6, and then adding the mixture to a microwave-ultrasonic composite reactor for reaction; S4. After the reaction is completed, the mixture is cooled to room temperature, acetone is added to precipitate the ionic liquid, and the supernatant is collected by centrifugation. The supernatant is filtered and chromatographed on a reverse C18 column to collect the elution peak of the blue copper peptide, and the mixture is lyophilized.

2. The method for synthesizing GHK-CU according to claim 1, wherein: The volume ratio of ethanol to water in the ethanol-water mixed solvent described in step S1 is 1:4-5; When treating CO2 expansion fluid, the set pressure is 15~20MPa, the temperature is 40~45℃, and the CO2 flow rate is 400~500mL / min.

3. The method for synthesizing GHK-CU according to claim 1, wherein: The reducing agent in step S2 is ascorbic acid, which contains tetraethyl orthosilicate. Tetraethyl orthosilicate reacts with Cu in the reaction system. 2+ The molar ratio is 2~3:1; The inner diameter of the microchannel reactor is 500 μm.

4. The method for synthesizing GHK-CU according to claim 1, wherein: The preparation of aspartic acid-modified β-cyclodextrin described in step S3 comprises the following steps: (1) Dissolve β-cyclodextrin in dimethyl sulfoxide, add sodium hydroxide and stir to mix, then add propargyl bromide dropwise and react at room temperature for 20-26 hours. After the reaction is completed, pour into ice water to precipitate, filter, wash with deionized water 4-6 times, and dry to obtain pretreated β-cyclodextrin; (2) Dissolve polyaspartic acid in dichloromethane, add N,N-dicyclohexylcarbodiimide at a molar amount of 1.3 to 1.4 times that of polyaspartic acid, stir and mix, then add sodium azide at a molar amount of 1 to 2 times that of polyaspartic acid, react at room temperature for 16 to 18 hours, filter after the reaction, concentrate the filtrate, precipitate with ether, filter and dry to obtain pretreated polyaspartic acid; (3) Pretreated β-cyclodextrin and aspartic acid were added to phosphate buffer at a molar ratio of 1:1~2, the pH was adjusted to 6~8, transglutaminase was added, and the mixture was stirred at 30~40°C for 8~10 hours. Pretreated aspartic acid was added and stirred, and copper sulfate and sodium ascorbate were added. The mixture was stirred at room temperature for 16~20 hours. After the reaction was completed, the unreacted raw materials were removed by chromatography to obtain aspartic acid-modified β-cyclodextrin.

5. The method for synthesizing GHK-CU according to claim 4, wherein: The molar ratio of β-cyclodextrin to propargyl bromide in step (1) is 1:1-3; The amount of sodium hydroxide added is 0.6~1% of the mass of β-cyclodextrin.

6. The method for synthesizing GHK-CU according to claim 4, wherein: The amount of transglutaminase added in step (3) is 3-4% of the total mass of β-cyclodextrin and aspartic acid; The molar ratio of pretreated β-cyclodextrin to pretreated aspartic acid is 1:1.3~1.7; The concentration of copper sulfate is 0.03~0.07mM, and the concentration of sodium ascorbate is 0.3~0.7mM.

7. The method for synthesizing GHK-CU according to claim 1, wherein: When the amino acid powder and Cu(OH)2@SiO2 nanoparticle suspension are added to the ionic liquid in step S3, the total concentration of the amino acid is controlled to be 0.5~1M, and the Cu(OH)2@SiO2 nanoparticle suspension is controlled to be 0.5~1M. 2 + The molar ratio of amino acid is 1:1.2~1.

4.

8. The method for synthesizing GHK-CU according to claim 1, wherein: When the microwave-ultrasonic composite reactor is added to the reaction in step S3, the microwave parameters are: dual-frequency alternating irradiation, 2.45 GHz and 5.8 GHz, 10 s each cycle, initial power density of 50 W / cm 2 , increase by 50W / cm every 5 minutes 2 Up to 200W / cm 2 ; Ultrasonic parameters: frequency of 20-100 kHz, pulse mode of 30 s on and 10 s off.

9. The method for synthesizing GHK-CU according to claim 1, wherein: The supernatant in step S4 is filtered using an ultrafiltration membrane with a pore size of 10 kDa; The mobile phase for reversed-phase C18 column chromatography was 2:8 acetonitrile and water containing 0.1% trifluoroacetic acid.

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