Anti-wrinkle complex polypeptide and preparation method thereof

Abstract

The invention relates to the technical field of polypeptide, in particular to an anti-wrinkle complex polypeptide and a preparation method thereof, the anti-wrinkle complex polypeptide comprises the following raw materials by weight: 4.5-5.5% of complex liposome, 24.5-28.5% of block polypeptide, 0.3-0.5% of palmitoyl tripeptide-1, 0.4-0.6% of chlorella growth factor, and the balance of water; through a gel structure formed by the composite liposome and the block polypeptide, anti-aging bioactive substances can be effectively stored, inactivation caused by external factors is avoided, and meanwhile, due to the unique biocompatibility characteristic, the good anti-wrinkle characteristic can be embodied at the low concentration.

Description

Technical Field

[0001] The invention relates to the technical field of polypeptides, and in particular to an anti-wrinkle polypeptide and a preparation method thereof. Background Art

[0002] With the increase of age and the influence of external factors, human skin gradually ages, the collagen in the dermis gradually decreases, the skin becomes loose and wrinkles appear. The commonly used anti-aging ingredients in cosmetics on the market currently include palmitoyl tripeptide-1, palmitoyl tetrapeptide-7, palmitoyl tripeptide-5, acetyl hexapeptide-3, carnosine, coenzyme Q10, chlorella growth factor and other bioactive substances. However, these substances have certain defects, such as poor water solubility, poor photostability, and high usage concentrations, which make them difficult to use and cannot meet market demand. Summary of the invention

[0003] The purpose of the present invention is to provide an anti-wrinkle complex peptide and a preparation method thereof. The gel structure formed by the composite liposome and the block peptide can effectively store anti-aging bioactive substances to avoid inactivation due to external factors. At the same time, due to its unique biocompatibility, it can show good anti-wrinkle properties at a lower concentration.

[0004] The object of the present invention can be achieved by the following technical scheme: a method for preparing an anti-wrinkle complex peptide, comprising the following steps: weighing the following raw materials in weight percentage: 4.5-5.5% of composite liposomes, 24.5-28.5% of block polypeptides, 0.3-0.5% of palmitoyl tripeptide-1 and 0.4-0.6% of chlorella growth factor, the balance being water, mixing the composite liposomes, block polypeptides, palmitoyl tripeptide-1, chlorella growth factor and water, and stirring at room temperature to prepare an anti-wrinkle complex peptide;

[0005] The palmitoyl tripeptide-1 is Purui Bio Palmitoyl Tripeptide-1;

[0006] The chlorella growth factor is peptide-safe chlorella growth factor;

[0007] The composite liposome is prepared by the following steps:

[0008] Step A1: Coenzyme Q10, soybean lecithin, cholesterol and 1,2-propylene glycol are mixed, stirred at a stirring rate of 120-140 rpm and a temperature of 75°C for 20 minutes to obtain an oil phase system, acetyl hexapeptide-3, glycerol and deionized water are mixed, stirred at a stirring rate of 120-140 rpm and a temperature of 45°C for 15 minutes to obtain an aqueous phase system, the aqueous phase system is added to the oil phase system, stirred at a stirring rate of 120-140 rpm and a temperature of 60°C for 30 minutes, and then emulsified at a stirring rate of 8000 rpm for 2 minutes, and then added to a high-pressure homogenizer, homogenized 5 times at 100 MPa, and cooled to room temperature to obtain loaded liposomes;

[0009] The dosage ratio of coenzyme Q10, soy lecithin, cholesterol, 1,2-propylene glycol, acetyl hexapeptide-3, glycerol and deionized water is 1.4-1.5g: 6.0-6.3g: 0.2-0.3g: 23-25mL: 0.6-0.8g: 11-13mL: 60-70mL;

[0010] During the reaction, the aqueous phase system was added to the oil phase system, and after emulsification, the loaded liposomes loaded with coenzyme Q10 and acetyl hexapeptide-3 were prepared by high pressure homogenization method;

[0011] Step A2: chitosan and acetic acid solution are mixed, and 3,4-dihydroxyphenylpropionic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide, ethanol and deionized water are added under stirring at a rate of 180-200 rpm and a temperature of 40°C, and the mixture is reacted for 12-14 hours to obtain modified chitosan. The modified chitosan, loaded liposomes and acetic acid solution are mixed, and sodium tripolyphosphate is added under stirring at a rate of 120-140 rpm and a temperature of room temperature, and the mixture is reacted for 4-6 hours to obtain composite liposomes.

[0012] The mass fraction of the acetic acid solution in the preparation process of the modified chitosan is 1%, and the amount ratio of chitosan, acetic acid solution, 3,4-dihydroxyphenylpropionic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide, ethanol and deionized water is 2.1-2.3g: 100-105mL: 0.01-0.011mol: 1.8-1.9g: 1.1-1.2g: 50mL: 45mL; the mass fraction of the acetic acid solution in the preparation process of the composite liposome is 5%, and the amount ratio of the modified chitosan, loaded liposome, acetic acid solution and sodium tripolyphosphate is 0.02-0.022g: 0.01-0.012g: 90-100mL: 0.035-0.036g;

[0013] During the reaction, under the action of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide, the amino group in chitosan reacts with the carboxyl group in 3,4-dihydroxyphenylpropionic acid to form an amide structure to obtain modified chitosan, and then the modified chitosan is aggregated on the surface of the loaded liposome through the ion interaction between the modified chitosan and sodium tripolyphosphate to form a chitosan layer to obtain a composite liposome;

[0014] The block polypeptide is prepared by the following steps:

[0015] Step B1: glutamic acid, copper sulfate and deionized water are mixed, sulfuric acid solution is added to adjust the pH value to 5, and the mixture is reacted for 40 minutes at a stirring rate of 160-180 rpm and a temperature of 70°C, and then 2-nitrobenzyl bromide, tetramethylguanidine and N,N-dimethylformamide are added, and the mixture is reacted for 12 hours at a stirring rate of 160-180 rpm and a temperature of 40°C to obtain intermediate a, and intermediate a, triphosgene and ethyl acetate are mixed, and the mixture is reacted for 4-6 hours at a stirring rate of 80-100 rpm and a temperature of 85°C to obtain intermediate b;

[0016] The dosage ratio of glutamic acid, copper sulfate, deionized water, 2-nitrobenzyl bromide, tetramethylguanidine and N,N-dimethylformamide is 0.012-0.014 mol: 3.1-3.3 g: 100-120 mL: 0.012-0.014 mol: 11-12 mL: 40 mL; the dosage ratio of intermediate a, triphosgene and ethyl acetate is 0.02-0.025 mol: 2.5-2.6 g: 150-160 mL;

[0017] During the reaction, in deionized water, glutamic acid and copper ions form a complex, and then under the action of tetramethylguanidine, the carboxyl group in the complex reacts with the bromine in 2-nitrobenzyl bromide to obtain intermediate a, and then under the action of triphosgene, the carboxyl group in intermediate a reacts with the amino group to form an N-internal carboxylic acid anhydride structure to obtain intermediate b;

[0018] Step B2: amino-terminated polyethylene glycol, tris(2-aminoethyl)amine, intermediate b, tetrahydrofuran and deionized water were mixed, and reacted for 12 h under nitrogen protection, stirring at a rate of 140-160 rpm and a temperature of 55° C. to obtain a block polypeptide;

[0019] The amount ratio of amino-terminated polyethylene glycol, tris(2-aminoethyl)amine, intermediate b, tetrahydrofuran and deionized water is 12-13 g: 0.001-0.0012 mol: 0.08-0.09 mol: 80 mL: 40 mL;

[0020] During the reaction, the intermediate b is ring-opened and reacts with the amino group in the amino-terminated polyethylene glycol and tris(2-aminoethyl)amine to form a polypeptide chain segment. At the same time, due to the presence of tris(2-aminoethyl)amine, a certain branched structure is formed to obtain a block polypeptide.

[0021] Beneficial effects of the invention: The invention discloses an anti-wrinkle composite peptide and a preparation method thereof. The gel structure formed by the composite liposome and the segmented polypeptide can effectively store anti-wrinkle bioactive substances and avoid inactivation due to external factors. At the same time, due to its unique biocompatibility, it can show good anti-wrinkle properties at a lower concentration. Since the segmented polypeptide itself contains a branched structure and a polypeptide chain segment, it can form hydrogen bonds with the modified chitosan on the composite liposome to form a gel structure, thereby storing anti-aging bioactive substances and avoiding inactivation due to external factors, thereby extending its service life. The modified chitosan loaded on the outside of the composite liposome also improves the stability of the liposome and promotes the liposome itself to penetrate deeper into the skin more easily, thereby releasing anti-aging bioactive factors deep in the skin to improve the anti-aging effect. DETAILED DESCRIPTION

[0022] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] Example 1 A method for preparing an anti-wrinkle complex peptide comprises the following steps: weighing the following raw materials in weight percentage: 4.5% of composite liposomes, 24.5% of block polypeptides, 0.3% of palmitoyl tripeptide-1 and 0.4% of chlorella growth factor, and the balance being water, mixing the composite liposomes, block polypeptides, palmitoyl tripeptide-1, chlorella growth factor and water, and stirring at room temperature to prepare an anti-wrinkle complex peptide;

[0024] The palmitoyl tripeptide-1 is Purui Bio Palmitoyl Tripeptide-1;

[0025] The chlorella growth factor is peptide-safe chlorella growth factor;

[0026] The composite liposome is prepared by the following steps:

[0027] Step A1: Coenzyme Q10, soybean lecithin, cholesterol and 1,2-propylene glycol were mixed, stirred at a stirring rate of 140 rpm and a temperature of 75°C for 20 minutes to obtain an oil phase system, acetyl hexapeptide-3, glycerol and deionized water were mixed, stirred at a stirring rate of 120 rpm and a temperature of 45°C for 15 minutes to obtain an aqueous phase system, the aqueous phase system was added to the oil phase system, stirred at a stirring rate of 140 rpm and a temperature of 60°C for 30 minutes, and then emulsified at a stirring rate of 8000 rpm for 2 minutes, and then added to a high-pressure homogenizer, homogenized 5 times at 100 MPa, and cooled to room temperature to obtain loaded liposomes;

[0028] The dosage ratio of coenzyme Q10, soy lecithin, cholesterol, 1,2-propylene glycol, acetyl hexapeptide-3, glycerol and deionized water is 1.5g:6.0g:0.2g:25mL:0.6g:13mL:60mL;

[0029] The coenzyme Q10 is Nanshi coenzyme Q10;

[0030] Step A2: chitosan and acetic acid solution are mixed, and 3,4-dihydroxyphenylpropionic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide, ethanol and deionized water are added under stirring at a rate of 200 rpm and a temperature of 40°C, and the reaction is carried out for 12 hours to obtain modified chitosan. The modified chitosan, loaded liposomes and acetic acid solution are mixed, and sodium tripolyphosphate is added under stirring at a rate of 140 rpm and a temperature of room temperature, and the reaction is carried out for 4 hours to obtain composite liposomes;

[0031] The mass fraction of acetic acid solution in the preparation process of modified chitosan is 1%, and the amount ratio of chitosan, acetic acid solution, 3,4-dihydroxyphenylpropionic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide, ethanol and deionized water is 2.3g:100mL:0.011mol:1.8g:1.2g:50mL:45mL; the mass fraction of acetic acid solution in the preparation process of composite liposomes is 5%, and the amount ratio of modified chitosan, loaded liposomes, acetic acid solution and sodium tripolyphosphate is 0.02g:0.012g:90mL:0.036g;

[0032] The chitosan is Runjie chitosan;

[0033] The block polypeptide is prepared by the following steps:

[0034] Step B1: glutamic acid, copper sulfate and deionized water are mixed, sulfuric acid solution is added to adjust the pH value to 5, and the mixture is reacted for 40 minutes at a stirring rate of 160 rpm and a temperature of 70°C. Then 2-nitrobenzyl bromide, tetramethylguanidine and N,N-dimethylformamide are added, and the mixture is reacted for 12 hours at a stirring rate of 180 rpm and a temperature of 40°C to obtain intermediate a. Intermediate a, triphosgene and ethyl acetate are mixed, and the mixture is reacted for 6 hours at a stirring rate of 80 rpm and a temperature of 85°C to obtain intermediate b;

[0035] The ratio of glutamic acid, copper sulfate, deionized water, 2-nitrobenzyl bromide, tetramethylguanidine and N,N-dimethylformamide is 0.012 mol: 3.3 g: 100 mL: 0.012 mol: 11 mL: 40 mL; the ratio of intermediate a, triphosgene and ethyl acetate is 0.025 mol: 2.5 g: 150 mL;

[0036] Step B2: amino-terminated polyethylene glycol, tris(2-aminoethyl)amine, intermediate b, tetrahydrofuran and deionized water were mixed, and reacted for 12 h under nitrogen protection, at a stirring rate of 160 rpm and a temperature of 55° C. to obtain a block polypeptide;

[0037] The amount ratio of amino-terminated polyethylene glycol, tris(2-aminoethyl)amine, intermediate b, tetrahydrofuran and deionized water is 12g:0.0012mol:0.08mol:80mL:40mL;

[0038] The amino-terminated polyethylene glycol is source leaf NH2-PEG300 amino-terminated polyethylene glycol.

[0039] Example 2 A method for preparing an anti-wrinkle complex peptide comprises the following steps: weighing the following raw materials in weight percentage: 4.5% of composite liposomes, 24.5% of block polypeptides, 0.3% of palmitoyl tripeptide-1 and 0.4% of chlorella growth factor, the balance being water, mixing the composite liposomes, block polypeptides, palmitoyl tripeptide-1, chlorella growth factor and water, and stirring at room temperature to prepare an anti-wrinkle complex polypeptide;

[0040] The palmitoyl tripeptide-1 is Purui Bio Palmitoyl Tripeptide-1;

[0041] The chlorella growth factor is peptide-safe chlorella growth factor;

[0042] The composite liposome is prepared by the following steps:

[0043] Step A1: Coenzyme Q10, soybean lecithin, cholesterol and 1,2-propylene glycol were mixed, stirred at a stirring rate of 120 rpm and a temperature of 75°C for 20 minutes to obtain an oil phase system, acetyl hexapeptide-3, glycerol and deionized water were mixed, stirred at a stirring rate of 120 rpm and a temperature of 45°C for 15 minutes to obtain an aqueous phase system, the aqueous phase system was added to the oil phase system, stirred at a stirring rate of 120 rpm and a temperature of 60°C for 30 minutes, and then emulsified at a stirring rate of 8000 rpm for 2 minutes, and then added to a high-pressure homogenizer, homogenized 5 times at 100 MPa, and cooled to room temperature to obtain loaded liposomes;

[0044] The dosage ratio of coenzyme Q10, soy lecithin, cholesterol, 1,2-propylene glycol, acetyl hexapeptide-3, glycerol and deionized water is 1.4g:6.0g:0.2g:23mL:0.6g:11mL:60mL;

[0045] The coenzyme Q10 is Nanshi coenzyme Q10;

[0046] Step A2: chitosan and acetic acid solution are mixed, and 3,4-dihydroxyphenylpropionic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide, ethanol and deionized water are added under stirring at a rate of 180 rpm and a temperature of 40°C, and the mixture is reacted for 12 hours to obtain modified chitosan. The modified chitosan, loaded liposomes and acetic acid solution are mixed, and sodium tripolyphosphate is added under stirring at a rate of 120 rpm and a temperature of room temperature, and the mixture is reacted for 4 hours to obtain composite liposomes.

[0047] The mass fraction of acetic acid solution in the preparation process of modified chitosan is 1%, and the amount ratio of chitosan, acetic acid solution, 3,4-dihydroxyphenylpropionic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide, ethanol and deionized water is 2.1g:100mL:0.01mol:1.8g:1.1g:50mL:45mL; the mass fraction of acetic acid solution in the preparation process of composite liposomes is 5%, and the amount ratio of modified chitosan, loaded liposomes, acetic acid solution and sodium tripolyphosphate is 0.02g:0.01g:90mL:0.035g;

[0048] The chitosan is Runjie chitosan;

[0049] The block polypeptide is prepared by the following steps:

[0050] Step B1: glutamic acid, copper sulfate and deionized water are mixed, sulfuric acid solution is added to adjust the pH value to 5, and the mixture is reacted for 40 minutes at a stirring rate of 160 rpm and a temperature of 70°C, and then 2-nitrobenzyl bromide, tetramethylguanidine and N,N-dimethylformamide are added, and the mixture is reacted for 12 hours at a stirring rate of 160 rpm and a temperature of 40°C to obtain intermediate a, and intermediate a, triphosgene and ethyl acetate are mixed, and the mixture is reacted for 4 hours at a stirring rate of 80 rpm and a temperature of 85°C to obtain intermediate b;

[0051] The ratio of glutamic acid, copper sulfate, deionized water, 2-nitrobenzyl bromide, tetramethylguanidine and N,N-dimethylformamide is 0.012 mol: 3.1 g: 100 mL: 0.012 mol: 11 mL: 40 mL; the ratio of intermediate a, triphosgene and ethyl acetate is 0.02 mol: 2.5 g: 150 mL;

[0052] Step B2: amino-terminated polyethylene glycol, tris(2-aminoethyl)amine, intermediate b, tetrahydrofuran and deionized water were mixed, and reacted for 12 h under nitrogen protection, stirring at a rate of 140 rpm and a temperature of 55° C. to obtain a block polypeptide;

[0053] The amount ratio of amino-terminated polyethylene glycol, tris(2-aminoethyl)amine, intermediate b, tetrahydrofuran and deionized water is 12 g: 0.001 mol: 0.08 mol: 80 mL: 40 mL;

[0054] The amino-terminated polyethylene glycol is source leaf NH2-PEG300 amino-terminated polyethylene glycol.

[0055] Example 3 A method for preparing an anti-wrinkle complex peptide, comprising the following steps: weighing the following raw materials in weight percentage: 5.5% of composite liposomes, 28.5% of block polypeptides, 0.5% of palmitoyl tripeptide-1 and 0.6% of chlorella growth factor, with the remainder being water, mixing the composite liposomes, block polypeptides, palmitoyl tripeptide-1, chlorella growth factor and water, and stirring at room temperature to prepare an anti-wrinkle complex peptide;

[0056] The palmitoyl tripeptide-1 is Purui Bio Palmitoyl Tripeptide-1;

[0057] The chlorella growth factor is peptide-safe chlorella growth factor;

[0058] The composite liposome is prepared by the following steps:

[0059] Step A1: Coenzyme Q10, soybean lecithin, cholesterol and 1,2-propylene glycol were mixed, stirred at a stirring rate of 140 rpm and a temperature of 75°C for 20 minutes to obtain an oil phase system, acetyl hexapeptide-3, glycerol and deionized water were mixed, stirred at a stirring rate of 140 rpm and a temperature of 45°C for 15 minutes to obtain an aqueous phase system, the aqueous phase system was added to the oil phase system, stirred at a stirring rate of 140 rpm and a temperature of 60°C for 30 minutes, and then emulsified at a stirring rate of 8000 rpm for 2 minutes, and then added to a high-pressure homogenizer, homogenized 5 times at 100 MPa, and cooled to room temperature to obtain loaded liposomes;

[0060] The dosage ratio of coenzyme Q10, soy lecithin, cholesterol, 1,2-propylene glycol, acetyl hexapeptide-3, glycerol and deionized water is 1.5g:6.3g:0.3g:25mL:0.8g:13mL:70mL;

[0061] The coenzyme Q10 is Nanshi coenzyme Q10;

[0062] Step A2: chitosan and acetic acid solution are mixed, and 3,4-dihydroxyphenylpropionic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide, ethanol and deionized water are added under stirring at a rate of 200 rpm and a temperature of 40°C, and the reaction is carried out for 14 hours to obtain modified chitosan. The modified chitosan, loaded liposomes and acetic acid solution are mixed, and sodium tripolyphosphate is added under stirring at a rate of 140 rpm and a temperature of room temperature, and the reaction is carried out for 6 hours to obtain composite liposomes;

[0063] The mass fraction of acetic acid solution in the preparation process of modified chitosan is 1%, and the amount ratio of chitosan, acetic acid solution, 3,4-dihydroxyphenylpropionic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide, ethanol and deionized water is 2.3g:105mL:0.011mol:1.9g:1.2g:50mL:45mL; the mass fraction of acetic acid solution in the preparation process of composite liposomes is 5%, and the amount ratio of modified chitosan, loaded liposomes, acetic acid solution and sodium tripolyphosphate is 0.022g:0.012g:100mL:0.036g;

[0064] The chitosan is Runjie chitosan;

[0065] The block polypeptide is prepared by the following steps:

[0066] Step B1: glutamic acid, copper sulfate and deionized water are mixed, sulfuric acid solution is added to adjust the pH value to 5, and the mixture is reacted for 40 minutes at a stirring rate of 180 rpm and a temperature of 70°C. Then 2-nitrobenzyl bromide, tetramethylguanidine and N,N-dimethylformamide are added, and the mixture is reacted for 12 hours at a stirring rate of 180 rpm and a temperature of 40°C to obtain intermediate a. Intermediate a, triphosgene and ethyl acetate are mixed, and the mixture is reacted for 6 hours at a stirring rate of 100 rpm and a temperature of 85°C to obtain intermediate b;

[0067] The ratio of glutamic acid, copper sulfate, deionized water, 2-nitrobenzyl bromide, tetramethylguanidine and N,N-dimethylformamide is 0.014 mol: 3.3 g: 120 mL: 0.014 mol: 12 mL: 40 mL; the ratio of intermediate a, triphosgene and ethyl acetate is 0.025 mol: 2.6 g: 160 mL;

[0068] Step B2: amino-terminated polyethylene glycol, tris(2-aminoethyl)amine, intermediate b, tetrahydrofuran and deionized water were mixed, and reacted for 12 h under nitrogen protection, at a stirring rate of 160 rpm and a temperature of 55° C. to obtain a block polypeptide;

[0069] The amount ratio of amino-terminated polyethylene glycol, tris(2-aminoethyl)amine, intermediate b, tetrahydrofuran and deionized water is 13 g: 0.0012 mol: 0.09 mol: 80 mL: 40 mL;

[0070] The amino-terminated polyethylene glycol is source leaf NH2-PEG300 amino-terminated polyethylene glycol.

[0071] Comparative Example 1 Compared with Example 3, this comparative example is replaced by chitosan in the preparation process of composite liposomes in Example 3, and the other steps are the same.

[0072] Comparative Example 2 Compared with Example 3, this comparative example is characterized in that tri(2-aminoethyl)amine in the preparation process of the block polypeptide in Example 3 is replaced by N-methyl-2,2-diaminodiethylamine, and the other steps are the same.

[0073] The anti-wrinkle complex peptides prepared in Example 1, Example 2, Example 3, Comparative Example 1 and Comparative Example 2 were tested for their cytotoxicity using the CCK8 method. The selected cells were NCTC clone 929 cells, and the culture medium solution was a culture medium solution containing 10% fetal bovine serum. The cell survival rate was calculated to assess its cytotoxicity. Referring to T / AHPCA 049-2023, the free radical scavenging method was used to measure its free radical scavenging rate to assess its anti-aging ability. The sample was placed at a temperature of 45°C and 30W, 254nm ultraviolet light for 120min, and then its free radical scavenging rate was measured, and its performance retention rate was calculated to assess its photothermal stability. The test results are shown in Table 1 below:

[0074] Table 1 Test results

[0075]

[0076] It can be seen from the test results in the table shown that when Example 1, Example 2 and Example 3 are compared with Comparative Example 1 and Comparative Example 2, Comparative Example 1 replaces the modified chitosan in the preparation process of the composite liposome in Example 3 with chitosan, and its performance is reduced due to the lack of modification. Comparative Example 2 replaces tri(2-aminoethyl)amine in the preparation process of the block polypeptide in Example 3 with N-methyl-2,2-diaminodiethylamine, and its performance is reduced due to the lack of a branched structure.

[0077] In the description of the specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0078] The above contents are merely examples and explanations of the concept of the present invention. Those skilled in the art may make various modifications or additions to the specific embodiments described or replace them in a similar manner. As long as they do not deviate from the concept of the invention, they should all fall within the protection scope of the present invention.

Claims

1. A method for preparing an anti-wrinkle polypeptide, characterized in that: The method comprises the following steps: weighing the following raw materials in weight percentage: 4.5-5.5% of composite liposomes, 24.5-28.5% of block polypeptides, 0.3-0.5% of palmitoyl tripeptide-1 and 0.4-0.6% of chlorella growth factor, and the balance being water; mixing the composite liposomes, block polypeptides, palmitoyl tripeptide-1, chlorella growth factor and water; and stirring at room temperature to prepare an anti-wrinkle composite polypeptide.

2. The method for preparing an anti-wrinkle complex peptide according to claim 1, characterized in that: The composite liposome is prepared by the following steps: Step A1: Coenzyme Q10, soybean lecithin, cholesterol and 1,2-propylene glycol are mixed, stirred at a stirring rate of 120-140 rpm and a temperature of 75°C for 20 minutes to obtain an oil phase system, acetyl hexapeptide-3, glycerol and deionized water are mixed, stirred at a stirring rate of 120-140 rpm and a temperature of 45°C for 15 minutes to obtain an aqueous phase system, the aqueous phase system is added to the oil phase system, stirred at a stirring rate of 120-140 rpm and a temperature of 60°C for 30 minutes, and then emulsified at a stirring rate of 8000 rpm for 2 minutes, and then added to a high-pressure homogenizer, homogenized 5 times at 100 MPa, and cooled to room temperature to obtain loaded liposomes; Step A2: chitosan and acetic acid solution are mixed, and 3,4-dihydroxyphenylpropionic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide, ethanol and deionized water are added under stirring at a rate of 180-200 rpm and a temperature of 40°C, and the reaction is carried out for 12-14 hours to obtain modified chitosan. The modified chitosan, loaded liposomes and acetic acid solution are mixed, and sodium tripolyphosphate is added under stirring at a rate of 120-140 rpm and a temperature of room temperature, and the reaction is carried out for 4-6 hours to obtain composite liposomes.

3. The method for preparing an anti-wrinkle polypeptide according to claim 2, characterized in that: In step A1: the usage ratio of coenzyme Q10, soy lecithin, cholesterol, 1,2-propylene glycol, acetyl hexapeptide-3, glycerol and deionized water is 1.4-1.5 g: 6.0-6.3 g: 0.2-0.3 g: 23-25 ​​mL: 0.6-0.8 g: 11-13 mL: 60-70 mL.

4. The method for preparing an anti-wrinkle complex peptide according to claim 2, characterized in that: In step A2: the mass fraction of the acetic acid solution in the preparation process of the modified chitosan is 1%, and the amount ratio of chitosan, acetic acid solution, 3,4-dihydroxyphenylpropionic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide, ethanol and deionized water is 2.1-2.3g: 100-105mL: 0.01-0.011mol: 1.8-1.9g: 1.1-1.2g: 50mL: 45mL; the mass fraction of the acetic acid solution in the preparation process of the composite liposome is 5%, and the amount ratio of the modified chitosan, the loaded liposome, the acetic acid solution and the sodium tripolyphosphate is 0.02-0.022g: 0.01-0.012g: 90-100mL: 0.035-0.036g.

5. The method for preparing an anti-wrinkle complex peptide according to claim 1, characterized in that: The block polypeptide is prepared by the following steps: Step B1: glutamic acid, copper sulfate and deionized water are mixed, sulfuric acid solution is added to adjust the pH value to 5, and the mixture is reacted for 40 minutes at a stirring rate of 160-180 rpm and a temperature of 70°C, and then 2-nitrobenzyl bromide, tetramethylguanidine and N,N-dimethylformamide are added, and the mixture is reacted for 12 hours at a stirring rate of 160-180 rpm and a temperature of 40°C to obtain intermediate a, and intermediate a, triphosgene and ethyl acetate are mixed, and the mixture is reacted for 4-6 hours at a stirring rate of 80-100 rpm and a temperature of 85°C to obtain intermediate b; Step B2: amino-terminated polyethylene glycol, tri(2-aminoethyl)amine, intermediate b, tetrahydrofuran and deionized water were mixed, and reacted for 12 h under nitrogen protection, at a stirring rate of 140-160 rpm and a temperature of 55° C. to obtain a block polypeptide.

6. The method for preparing an anti-wrinkle complex peptide according to claim 5, characterized in that: In step B1, the ratio of glutamic acid, copper sulfate, deionized water, 2-nitrobenzyl bromide, tetramethylguanidine and N,N-dimethylformamide is 0.012-0.014 mol: 3.1-3.3 g: 100-120 mL: 0.012-0.014 mol: 11-12 mL: 40 mL; the ratio of intermediate a, triphosgene and ethyl acetate is 0.02-0.025 mol: 2.5-2.6 g: 150-160 mL.

7. The method for preparing an anti-wrinkle complex peptide according to claim 5, characterized in that: In step B2, the amount ratio of amino-terminated polyethylene glycol, tris(2-aminoethyl)amine, intermediate b, tetrahydrofuran and deionized water is 12-13 g: 0.001-0.0012 mol: 0.08-0.09 mol: 80 mL: 40 mL.

8. An anti-wrinkle polypeptide, characterized in that: Prepared according to any one of claims 1 to 7.

Images